Hologenomics - "JunkDNA diseases"

Personalized Medicine in the Genome Based Economy

Hereditary Diseases: “Not all is in the genes”

this compilation is by the

International HoloGenomics Society

This website was originally established in 2006
Today, in 2010, Personalized Participatory Prevention became a possibility for some conditions by a Genome Computing Architecture:

Personally announced in Google Tech Talk (2008) : http://www.youtube.com/watch?v=WJMFuc75V_w

Announced in a Churchill Club Panel (2009) : http://www.youtube.com/watch?v=znowfg7WJ1Q

Demonstrated by HolGenTech PMWC (2010) : http://www.youtube.com/watch?v=mSRMCDCVg6Y

Thakker
NEW YORK TIMES, February 7, 2006
"There are about 30,000 genes in the human genome, but there are at least 150,000 different genetic disorders," Dr. Thakker said. "You can't just look at the genes that code for proteins, you've got to look at the surrounding regulatory regions, as well in the 'junk.' "

Mattick' Review
Non-coding RNAs may provide the key... Dysregulation of miRNAs..in a mouse knockout of presenilin (Alzheimer's), miR-175 implicated... in early-onset Parkinson's disease

Sharp
"It's a revolution in how we understand the genome and how the cell functions," says MIT Nobel laureate Phillip Sharp. "There's a whole new frontier there." Sharp and a few microRNA researchers have founded Alnylam Pharmaceuticals to invent RNA-based drugs to treat Parkinson's, cystic fibrosis and spinal injury. The firm has development deals with Merck and Novartis, and such rivals as San Francisco's Sirna Therapeutics are in pursuit.

Lesson
Going "Beyond Genes" can not be accomplished by individual effort - even workshops may not do justice to the challange.
Creation of an International HoloGenomics Society was needed.

A REPRESENTATIVE LIST OF PROVEN OR SUSPECTED "POSTGENE DISEASES"

AIDS
ALCOHOLISM
ALPORT SYNDROME
ALZHEIMER'S DISEASE
ANGELMAN SYNDROME
ASTHMA

ATAXIA TELANGIECTASIA
AUTISM
AUTOIMMUNE DISEASES
BIPOLAR DISORDER
BRUGADA SYNDROME - ARRYTHMIA
CANCER: BREAST
CANCER: CARTILAGE HAIR HYPOPLASIA
CANCER: LUNG
CANCER: LYMPHOMA
CANCER: MULIPLE MYELOMA
CANCER: NON-POLYPOSIS COLORECTAL
CANCER: PROSTATE
CANCER: THYROID
CARDIOVASCULAR DISEASE
CYSTIC FIBROSIS
DIABETES MELLITUS TYPE II
DiGEORGE SYNDROME
DUCHENNE MUSCULAR DYSTROPHY
DYSKERATOSIS CONGENITA
EPILEPSY
ESSENTIAL HYPERTENSION
FAMILIAL DYSAUTOSOMIA
FRAGILE X SYNDROME, AUTISM
FRIEDREICH CEREBELLAR ATAXIA
HIRSCHSPRUNG DISEASE
HYPERCHOLESTEROLEMIA, FAMILIAL
HYPER-FERRITINEMIA-CATARACT SYNDROME, FAMILIAL
HYPER-IgM SYNDROME TYPE 1
LUPUS ERYTHEMATOSUS
LYMPHOMA, B-CELL
MALARIA
MEASLES
MELANOMA

MENTAL RETARDATION
MULTIPLE SCLEROSIS
MYOCARDIAL INFARCTION
MYOTONIC DYSTROPHY
NEUROFIBROMATOSIS
PARKINSONS' DISEASE
PRADER-WILLI SYNDROME
PSEUDOXANTHOMA ELASTICUM (PXE, GROENBLAD-STRANDBERG SYNDROME)
PSORIASIS
RETT SYNDROME
RETINITIS PIGMENTOSA
SCLERODERMA
SPINOCEREBELLAR ATAXIA
STROKE
SCHIZOPHRENIA

TAY-SACHS DISEASE
TOURETTE SYNDROME
VON WILLEBRAND DISEASE

GO TO DETAILS
OF A REPRESENTATIVE LIST OF PROVEN OR SUSPECTED 
"POSTGENE DISEASES"

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Forbes, December issue of 2005:

"What genetic researchers used to call junk DNA may conceal the most important medical secrets of all"

"This will revolutionize human genetics over the next few decades, says David Haussler, a Howard Hughes investigator at UC, Santa Cruz who was on the government team that decoded the human genome. He predicts that most disease-causing genetic flaws will be found lurking in our junk DNA."

"It's a revolution in how we understand the genome and how the cell functions," says MIT Nobel laureate Phillip Sharp.

"JunkDNA Diseases"

A primary goal of IHGS is to elevate awareness of the fact that "some, if not all" hereditary diseases do not stop at the boundaries of "genes" (especially since the definition of "gene" over the Century of 1905-2005 became increasingly restrictive).

The so-called "'Junk' DNA diseases", now defined as "HoloGenomic Diseases", is a newly emerging field, and HoloGenomics evolves its proper terminology. The presently "umbrella" term should certainly not be taken too literally; it does *not* deny the existence of "Gene diseases". It merely opens up a field of "Genomic diseases beyond genes". The most important and now undeniable fact is that in the Second Century of Genomics (after 100 years of "Genetics") it is already established that hereditarty diseases do not stop at the boundaries of (artificial and conceptually ambiguous) old term "gene".

Those diseases for which it has already been documented that they don't, and those that are strongly suspected that they don't, are complied here on an ongoing bases - comment by Dr. Andras Pellionisz, originator of IHGS.

"Personalized Medicine" in the Genome Based Economy: focusing on Prevention of JunkDNA Disease


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Background

Prelude: M Ridley, Genome: The Autobiography of Species in 23 Chapters (1999) Fourth Estate, London, 55

"Open any catalogue of the human genome and you will be confronted not with a list of human potentialities, but a list of diseases, mostly named after pairs of obscure central-European doctors. This gene causes Niemann-Pick disease; that one causes Wolf-Hirschhorn syndrome. The impression given is that genes are there to cause diseases. .."

"Yet to define genes by the diseases they cause is about as absurd as defining organs of the body by the diseases they get: livers are there to cause cirrhosis, hearts to cause heart attacks and brains to cause strokes. It is a measure, not of our knowledge but of our ignorance, that this is the way the genome catalogues read. It is literally true that the only thing we know about some genes is that their malfunction causes a particular disease. This is a pitifully small thing to know about a gene, and a terribly misleading one. It leads to the dangerous shorthand that runs as follows: `X has got the Wolf-Hirschhorn gene'. Wrong. We all have the Wolf-Hirschhorn gene, except, ironically, people who have Wolf-Hirschhorn syndrome. Their sickness is caused by the fact that the gene is missing altogether. In the rest of us the gene is a positive, not a negative force. The sufferers have the mutation, not the gene."

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International PostGenetics Society (IPGS - among its Founders some pairs of "central-European doctors"...) calls attention to the well documented facts, that many dreadful diseases are caused not by "genes" but by errors in the "non-coding DNA" ("junkDNA"). The list is by no means is full - it is for representative purposes.

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No amount of $$$ spent on "Gene Discovery" will reveal the cause of a disease if it originates in the 98.7% of DNA - that are "non-genes". Many tens of millions of patients affected with the listed (and similar) diseases may wish their tax dollars put to work to eradicate "JunkDNA diseases".

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A PRELIMINARY SURVERY OF PROMINENT "JUNK DNA DISEASES"

The Big Picture

AS BIOLOGISTS SIFT more and more novel kinds of active RNA genes out of the long-neglected introns and intergenic stretches of DNA, they are realizing that science is still far from having a complete parts list for humans or any other higher species. Unlike protein-making genes, which have standard "start" and "stop" codes, RNA-only genes vary so much that computer programs cannot reliably pick them out of DNA sequences. To spur the technology on, the NHGRI is launching this autumn an ambitious $36-million project to produce an "Encyclopedia of DNA Elements." The goal is to catalogue every kind of RNA and protein made from a select 1 percent of the human genome in three years.

No one knows yet just what the big picture of genetics will look like once this hidden layer of information is made visible, "Indeed, what was damned as junk because it was not understood may, in fact, turn out to be the very basis of human complexity,'' Mattick suggests. Pseudogenes, riboswitches and all the rest aside, there is a good reason to suspect that is true. Active RNA, it is now coming out, helps to control the large-scale structure of the chromosomes and some crucial chemical modifications to them--an entirely different, epigenetic layer of information in the genome.

The exploration of that epigenetic layer is answering old conundrums: How do human beings survive with a genome horribly cluttered by seemingly useless, parasitic bits of DNA? Why is it so hard to clone an adult animal yet so easy to clone an embryo? Why do certain traits skip generations in an apparently unpredictable way?

DNA sequences long considered genomic garbage are finally getting a little respect. Researchers have figured out how short stretches of DNA that do not normally code for proteins worm their way into genes.

This can result in the production of abnormal proteins and lead to genetic diseases, such as Alport Syndrome, a rare kidney disease.

A growing number of hereditary neurodegenerative disorders have been found to be caused by expansion of trinucleotide repeats. A smaller number of diseases such as fragile X syndrome, myotonic dystrophy, and Friedreich's ataxia, have been found to be due to expansions in non-coding DNA.

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RNA regulation: a new genetics? (Mattick, 2004)

... Non-coding transcripts. An increasing number of ncRNA genes are being identified, several of which have links to human diseases such as B-cell lymphoma, lung cancer, prostate cancer, cartilage-hair hypoplasia, spinocerebellar ataxia type 8, DiGeorge syndrome, autism and schizophrenia, among others14,25,26,29,42–44.

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Med J Aust. 2003 Aug 18;179(4):212-6.

The human genome and the future of medicine.

Mattick JS. Institute for Molecular Bioscience, University of Queensland, St Lucia, QLD 4072, Australia. j.mattick@imb.uq.edu.au

The draft human genome sequence (about 3 billion base pairs) was completed in 2001. Humans have fewer protein-coding genes than expected, and most of these are highly conserved among animals. Humans and other complex organisms produce massive amounts of non-coding RNAs, which may form another level of genetic output that controls differentiation and development. Aside from classical monogenic diseases and other differences caused by mutations and polymorphisms in protein-coding genes, much of the variation between individuals, including that which may affect our predispositions to common diseases, is probably due to differences in the non-coding regions of the genome (ie, the control architecture of the system). Within 10 years we can expect to see: increased penetration of DNA diagnostic tests to assess risk of disease, to diagnose pathogens, to determine the best treatment regimens, and for individual identification; a range of new pharmaceuticals as well as new gene and cell therapies to repair damage, to optimise health and to minimise future disease risk; and medicine become increasingly personalised, with the knowledge of individual genetic make-up and lifestyle influences.

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Non-coding RNA in the nervous system

Mark F. Mehler and John S. Mattick


Abstract

Increasing evidence suggests that the development and function of the nervous system is heavily dependent on RNA editing and the intricate spatiotemporal expression of a wide repertoire of non-coding RNAs, including micro RNAs, small nucleolar RNAs and longer non-coding RNAs. Non-coding RNAs may provide the key to understanding the multi-tiered links between neural development, nervous system function, and neurological diseases.


Introduction

The nervous system is unique among organs in its precise and sophisticated patterns of regional cellular morphogenesis, cellular diversity, membrane electrical properties, responses to changing environmental inputs and perturbations, neural network connections, and dynamic activity-dependent alterations in synaptic strength underlying higher order cognitive functions including learning and memory (Abrous et al., 2005).

These functional properties are, in turn, orchestrated by a corresponding set of multilayered developmental mechanisms (Mehler, 2002a, b), including neural induction, neural patterning and axis formation within the evolving neural plate and neural tube, elaboration of stem cell generative zones throughout the neuraxis and the evolution of connections between specialized regional neuronal and glial cell types.

Alterations of specific components of these developmental stages and maturational processes result in a broad spectrum of neurodevelopmental disorders and predispose to an equally complex array of adult neurological and neuropsychiatric disorders of unknown aetiology, underscoring the levels of complexity in developmental and mature brain-behaviour relationships. However, we have little understanding of the genetic programs and molecular mechanisms that orchestrate nervous system development, plasticity and function, or how these programs and mechanisms are perturbed in disease.

Although only about 1.2% of the mammalian genome encodes proteins, most of the genome is transcribed, in complex patterns of interlacing and overlapping transcripts from both strands (Carninci et al., 2005; Cheng et al., 2005a; Frith et al., 2005;

Katayama et al., 2005; Engstrom et al., 2006; Mattick & Makunin, 2006), at least some of which are processed to form small regulatory RNAs such as microRNAs and small nucleolar RNAs (reviewed in (Mattick & Makunin, 2005). A range of evidence suggests that these RNAs form complex networks that direct the trajectories of differentiation and development, via regulation of chromatin modification, transcription, RNA modification, splicing, mRNA translation, and RNA stability (Mattick & Gagen, 2001; Mattick, 2003, 2004a) as well as other mechanisms (Prasanth et al., 2005; Willingham et al., 2005). It is also clear that multiple classes of non-coding RNAs (ncRNAs) are overly represented in the central and peripheral nervous system (Hsieh & Gage, 2004; Kim et al., 2004; Rogelj & Giese, 2004; Cheng et al., 2005b; Davies et al., 2005; Klein et al., 2005; Rogaev, 2005; Cao et al., 2006; Ravasi et al., 2006), underscoring the likelihood that nervous system development and function is heavily dependent on RNA regulatory networks, and that perturbations of these networks underlie many neurological diseases.


MicroRNAs

MicroRNAs (miRNAs) are short 21-23 nucleotide regulatory sequences that inhibit the translation or stability of target RNAs (reviewed in (Mattick & Makunin, 2005; Zamore & Haley, 2005). In mice, there are numerous brain-specific miRNAs (Krichevsky et al., 2003; Cheng et al., 2005b; Lim et al., 2005; Xie et al., 2005), a significant subset of which have been directly implicated in neural development and neural cell differentiation (Kawasaki & Taira, 2003; Smirnova et al., 2005). A wide variety of miRNAs are localized to neuronal subtypes with the highest concentration in the cerebral cortex and the cerebellum (Kosik & Krichevsky, 2005; Krichevsky et al., 2006). Additional miRNAs are present within glial cell subtypes with others exhibiting more ubiquitous or neural progenitor cell-specific patterns of expression (Krichevsky et al., 2003; Klein et al., 2005; Smirnova et al., 2005). In zebrafish, the miRNA miR-430 rescues defects of neurulation, neural tube formation, segmental morphogenesis, neural stem cell maintenance and axonal pathfinding observed in dicer mutants that are defective in miRNA processing - although not completely, indicating that that other miRNAs are involved in later stages of neural development (Giraldez et al., 2005).

miRNAs are also abundantly expressed in the adult brain and appear to regulate the maintenance of mature neural traits and synaptic plasticity (Krichevsky et al., 2003; Jin et al., 2004; Sempere et al., 2004; Cheng et al., 2005b; Kosik & Krichevsky, 2005; Smirnova et al., 2005; Conaco et al., 2006; Schratt et al., 2006). Numerous studies suggest that miRNAs are intimately involved in synaptic function and input specificity during memory formation (Martin & Kosik, 2002; Schaeffer et al., 2003; Kim et al., 2004; Lugli et al., 2005; Ashraf et al., 2006; Schratt et al., 2006). Moreover, transcripts encoding synapse-associated proteins also comprise the largest subgroup of predicted miRNA targets, including synapsin 1 and the fragile X mental retardation protein (FMRP) (John et al., 2004).

A novel RNA called dsNRSE (double-stranded neuron-restrictive silencing element) that resembles a miRNA in structure and length acts as a transcriptional activator of neuronal differentiation genes by converting the neuronal silencer factor (REST/NRSF) from a transcriptional repressor in undifferentiated and non-neuronal cells to a transcriptional activator during neuroblast differentiation (Kuwabara et al., 2004). Interestingly, recent studies have revealed that REST modulates the expression of a family of miRNAs including the CNS-specific miR-124a (Conaco et al., 2006).

Perturbations in miRNAs are associated with a number of neural diseases. Deletion of DGCR8, which encodes a component of the complex that processes miRNAs (Gregory et al., 2004; Landthaler et al., 2004), results in DiGeorge syndrome, a multi-system disorder associated with significant learning disabilities (Shiohama et al., 2003).

Dysregulation of miRNAs also occurs in a mouse knockout of presenilin 1, the gene mutated in a subset of early familial forms of Alzheimer’s disease (AD) (Krichevsky et al., 2003). Further, miR-175 has been implicated in a form of X-linked mental retardation (MRX3) and in a type of early-onset Parkinson’s disease (Waisman syndrome) (Dostie et al., 2003). Other studies have implicated miRNAs in diverse neuropsychiatric conditions, particularly those associated with developmental pathogenesis (Rogaev, 2005). In addition, predicted miRNA targets include numerous proteins implicated in neurodevelopmental and neurodegenerative diseases (Rogaev, 2005). Sequence variations in the binding site for miR-189 in the SLIT and Trk-like family member1 (SLITRK1) mRNA have been associated with Tourette’s syndrome (Abelson et al., 2005). SLITRK1 is essential for neuronal growth, guidance and neurite branching and is also differentially expressed in many different neural tumours (Aruga & Mikoshiba, 2003; Aruga et al., 2003). Profound over-expression of miR-21 is seen in glioblastoma multiforme, a highly malignant tumour of the brain, whereas less dramatic degrees of miR-21 over-expression are seen in other neural-specific tumour types (Chan et al., 2005).

In mammals, ADARs are differentially expressed during organogenesis with ADAR3 restricted to brain and ADAR2 preferentially expressed in the nervous system (Chen et al., 2000; Bass, 2002). RNA editing also exhibits precise CNS regional specificity and essential regulatory roles during neuronal maturation (Lai et al., 1997; Kohr et al., 1998; Bernard et al., 1999; Paupard et al., 2000). RNA editing can also affect multiple sites on the same RNA with diverse functional outcomes catalyzed by different ADARs (Valente & Nishikura, 2005). ADAR mutants exhibit complex behavioural defects in C. elegans, Drosophila and mice (Reenan, 2001; Tonkin et al., 2002). Moreover, abnormalities in RNA editing have been implicated in a spectrum of nervous system disorders including Alzheimer’s and Huntington’s diseases, amyotrophic lateral sclerosis, epilepsy, schizophrenia, depression, suicidal ideation, autosomal dominant episodic ataxia type I and Prader-Willi and Angelman syndromes (reviewed in (Valente & Nishikura, 2005).

Intriguingly, in humans, A-I editing occurs far more frequently in transcripts than had been previously appreciated, with the vast majority of the editing occurring in inverted Alu repeats predicted to form intra-molecular duplexes in non-coding RNA sequences in introns, intergenic transcripts and UTRs (Athanasiadis et al., 2004; Blow et al., 2004; Kim et al., 2004; Levanon et al., 2004). These observations raise the intriguing possibility that the predominance of Alu elements in the human genome (10.5% of which is comprised of Alu elements) may not be simply an accident of history, [anybody said it was? - AJP] but the result of positive selection for these sequences as a natural substrate for A-I editing, in turn driven by selection for increased cognitive capacity in the primate lineage (Mattick, 2004b).

It is also worth noting that other small brain-specific trans-acting RNAs such as the primate-specific dendritic BC200 RNA and the analogous rodent dendritic BC1 RNA are both descended from retrotransposed sequences (Martignetti & Brosius, 1993; Ohashi et al., 2000), and it appears likely that many, if not most, transposon-derived sequences in our genome have been exapted into function, primarily at the regulatory level (Brosius,1999).


Longer non-coding RNAs

There are tens of thousands of larger ncRNAs, both polyadenylated and nonpolyadenylated, that are transcribed from the mammalian genome (Carninci et al., 2005; Cheng et al., 2005a; Kapranov et al., 2005; Engstrom et al., 2006), many of which MBII-78 and MBII-85 (Cavaille et al., 2000; Huttenhofer et al., 2001; Rogelj & Giese, 2004). At least some of these miRNAs show differential expression in different areas of the brain, such as the hippocampus and amygdala, areas associated with learning and memory, and are transiently modulated during contextual memory consolidation (fear conditioning) (Rogelj et al., 2003). Human homologs of these snoRNAs are also highly enriched in brain (Cavaille et al., 2000). Certain snoRNAs (RBI-36) exhibit genusspecific functions in rat brain, further attesting to the potential complexity of nonhousekeeping snoRNA functions in the nervous system (Cavaille et al., 2001).

In addition it has recently been shown that HBII-52 modifies the A-I RNA editing and alternative splicing of the serotonin 5-HT (2C) receptor subunit (Kishore & Stamm, 2006). HBII-52 is not expressed in the Prader-Willi developmental syndrome and 5-HT (2C) receptor isoforms distinct from the normal expression pattern are present, suggesting that anomalous splicing may contribute to disease pathogenesis (Cavaille et al., 2000; Kishore & Stamm, 2006). In humans, HBII-13, HBII-52 and HBII-85 map to the Prader-Willi syndrome locus suggesting that snoRNAs may be involved in, or regulated by, genomic imprinting (Rogelj & Giese, 2004). Many larger non-coding RNAs are also imprinted and also implicated in the genetic transactions which underlie imprinting, which clearly affects brain development and function in a variety of ways (see below).


RNA editing

Adenosine to inosine (A-I) RNA editing catalyzed by ADARs is particularly active in the brain, especially in transcripts encoding proteins involved in nerve cell function (Bass, 2002), such as voltage-gated ion channels, ligand-gated receptors, intracellular transduction molecules, apoptosis and cell cycle arrest proteins and modulators of presynaptic terminal integrity (Morse et al., 2002; Hoopengardner et al., 2003; Maas et al., 2003; Athanasiadis et al., 2004; Gelbard, 2004; Levanon et al., 2004; Wang et al., 2004a; Valente & Nishikura, 2005). A-I editing has the capacity to change the coding capacity of mRNA (Bass, 2002), to modulate splice site choice (Laurencikiene et al., 2006), miRNA and miRNA target diversity (Blow et al., 2006), miRNA processing (Yang et al., 2006), and perhaps other targets including chromatin architecture (Fernandez et al., 2005; Valente & Nishikura, 2005), as well as to be inhibited by snoRNAs (Vitali et al., 2005), further evidence of the complexity of RNA regulatory networks.

Regulatory actions are only beginning to be understood (reviewed in (Korneev & O'Shea, 2005). These antisense RNAs exhibit dynamic developmentally regulated and spatially discrete expression profiles, and modulate the expression of genes involved in brain morphogenesis, stem cell renewal and proliferation, stress responses, cell polarity and cytoskeletal functions, and neuronal survival, maturation and synaptic plasticity (Korneev & O'Shea, 2005).

In both schizophrenia and bipolar illness, susceptibility loci are present within the disabled in schizophrenia 1 (DISC1) gene and in the large antisense DISC2 RNA that modulates its expression (Millar et al., 2000; Millar et al., 2004). DISC1 is involved in intracellular transport, cell polarity and neuronal migration and disruption of function during cortical developmental may, in part, underlie the developmental pathogenesis of these heterogeneous neuropsychiatric diseases (Kamiya et al., 2005).


Non-coding RNAs and brain imprinting

Imprinted genes have essential roles in both neural development and adult CNS functioning, and alterations in their expression profiles are associated with a spectrum of complex neurodevelopmental and neuropsychiatric disorders (Costa, 2005; Davies et al., 2005; Davies et al., 2006). These allele-selective genes exhibit preferential and exquisite cell-specific patterns of expression within the brain, and are frequently processed from larger transcriptional units encompassing multiple tandemly repeated snoRNAs and miRNAs (Sleutels et al., 2000; Seitz et al., 2004; Davies et al., 2005; Lewis & Reik, 2006). These imprinted loci also generate a complex spectrum of spliced and unspliced larger ncRNAs of presently unknown function (Sleutels et al., 2000; Davies et al., 2005; O'Neill, 2005; Furuno, 2006). Additional ncRNAs associated with imprinted loci include the production of antisense RNAs to reciprocally imprinted neighbouring protein-coding genes (Sleutels et al., 2000; Davies et al., 2005). The seminal role of imprinted genes in regulating distinct brain signalling systems and in mediating brain-behaviour relationships is illustrated by the spectrum of neurological diseases associated with parent of origin effects and caused by disruptions in imprinted loci: autism, schizophrenia, attention deficit hyperactivity disorder, bipolar disorder and Tourette’s syndrome (see Davies et al., 2004; Wang et al., 2004b; Davies et al., 2005; Davies et al., 2006).


Transfer RNAs and ribosomal RNAs

Transfer RNAs (tRNAs) and ribosomal RNAs (rRNAs) have recently been implicated in a broad array of neural developmental and mature CNS functions as indicated by the effects of mutations in these two classes of ncRNAs which underlie a range of neurodevelopmental, neurodegenerative and neuropsychiatric diseases, including chronic progressive external ophthalmoplegia (CPEO), Kearn-Sayre syndrome (KSS: CPEO with retinal degeneration), MELAS syndrome (mitochondrial encephalopathy with stroke-like syndromes and migraine headaches), MERRF syndrome (myoclonus epilepsy, mitochondrial myopathy, cerebellar ataxia and less commonly dementia, hearing loss and peripheral neuropathy) (reviewed in Dimauro, 2004; Dimauro & Davidzon, 2005; Fattal et al., 2006) and motor neuron disease (Borthwick et al., 2006). MELAS syndrome and other tRNA-mediated diseases are also associated with prominent neuropsychiatric diseases including schizophrenia, psychosis, delirium, personality disorders, major depressive disorders, and anxiety disorders (Fattal et al., 2006).


RNA trinucleotide expansions

A range of neurodevelopmental and neurodegenerative diseases associated with trinucleotide repeat expansion appear to be caused by RNA-mediated mechanisms (reviewed in Gallo et al., 2005; Gatchel & Zoghbi, 2005). These include fragile X syndrome which results from dramatically expanded (>200) CGG repeats in the 5’ UTR of the Fmr1 gene and the related disease associated with smaller (60-200) trinucleotide repeat expansions called FXTAS (fragile X tremor/ataxia syndrome) (FXTAS) associated with tremor, cerebellar ataxia, cognitive decline, peripheral neuropathy, Parkinson’s disease, autonomic dysfunction, proximal muscle weakness, multisystem atrophy and dementia (Hagerman et al., 2005; Van Esch, 2006). Trinucleotide repeat expansions also underlie myotonic dystrophy, which is predominantly a muscle disorder but exists in two forms with associated CNS pathology: DM1 with mental retardation, memory and visuo-spatial and executive dysfunction and DM2 with preferential executive dysfunction (D'Angelo & Bresolin, 2006). DM1 is associated with CTG expansion within the 3’ UTR of the dystrophia myotonica protein kinase gene, DMPK, and DM2 is linked to CCTG expansion in intron 1 of the zinc finger protein gene, ZNF9 (Brook et al., 1992; Fu et al., 1992; Mahadevan et al., 1992; Ranum et al., 1998; Liquori et al., 2001). These mutant RNAs orchestrate different forms of pathogenesis through the degree and type of repeat length expansion and their molecular interactions with RNAbinding proteins of the muscleblind-like (MBNL) family (Jiang et al., 2004; Pascual et al., 2006).

Several forms of spinocerebellar ataxia (SCA) may also be caused by different RNAmediated pathological mechanisms. SCA8 results from CTG expansion of the 3’ UTR of an untranslated antisense RNA with partial overlap with the Kelch-like 1 (KLHL1) gene (Koob et al., 1999; Nemes et al., 2000; Mutsuddi et al., 2004; Gatchel & Zoghbi, 2005).

Moreover, using SCA8 as a sensitized background in a modifier screen resulted in the identification of four novel ncRNAs with preferential neuronal expression (Mutsuddi et al., 2004). SCA10 is mediated by an unstable ATTCT repeat expansion in the 3’ end of a large intron of a gene of presently unknown function that may result in transcriptional silencing or in a different RNA-associated toxic mechanism (Matsuura et al., 2000). SCA12 is caused by CAG expansion in the non-coding 5’ promoter/5’ UTR of the PPP2R2B gene, which encodes a brain specific regulatory subunit of protein phosphatase 2A (Holmes et al., 1999). Depending on the precise location of the expanded trinucleotide repeat, disease pathogenesis may be mediated by distinct trans-dominant RNA or alternate toxic gain of function mechanisms (Holmes et al., 2003).


Conclusion

The known list of both small and large ncRNAs that are involved in the nervous system almost certainly represents only a tiny fraction of the total transcriptome devoted to RNA-mediated mechanisms underlying the development, functional complexity and plasticity of the mammalian brain. Indeed, it appears that the majority of human genomic programming is devoted to RNA-based regulatory circuitry (Mattick, 2003; Mattick & Makunin, 2006). It also appears that the traditional presumption that most genetic information is transacted by proteins has led to a fundamental misunderstanding of the genetic programming of human differentiation and development, both generally and specifically in the brain, where RNA transactions appear to be at their most complex.

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Molecule crucial for processing non-coding RNA identified
October 21, 2005

Long-standing scientific question resolved

PHILADELPHIA – The discovery in 1977 that the coding regions of a gene could appear in separate segments along the DNA won the 1993 Nobel Prize in Physiology or Medicine for Richard J. Roberts and Phillip A. Sharp. The active segments of a gene were termed exons, separated from each other within the gene by inactive introns.

The research suggested the necessary existence of a number of biological processes and active entities, many of which have since been tracked down by other scientists. Some, however, have resisted intensive inquiry. Now, researchers at The Wistar Institute and colleagues have resolved one of the important biological questions to which this earlier research pointed. A report on their findings appears in the October 21 issue of Cell.

Researchers who followed Roberts and Sharp discovered a molecular machine called a spliceosome, which was responsible for processing messenger RNA, or mRNA, the gene transcript from which proteins are produced. The spliceosome does this by snipping out the introns from the mRNA and then stitching together the exons into the finished mRNA. The activity takes place in the nucleus of the cell.

The spliceosome itself is composed of proteins and so-called small nuclear RNAs, or snRNAs. These snRNAs, as is the case with other forms of non-coding RNA in the nucleus, never produce proteins but play important roles in facilitating and regulating genetic activity. How these snRNAs were processed, however, remained a mystery for over twenty years. And because the spliceosome underlies the successful transcription of every single gene in the body, the question has been a vital one to answer.

In the new study, the Wistar-led research team identifies an entirely novel multi-protein complex called the Integrator that plays a central role in the processing of snRNAs. The Integrator appears to perform two important duties simultaneously. It binds a molecule called CTD, which is a component of the polymerase enzyme that transcribes snRNA genes, and it also binds to the specific genes that code for the snRNAs. With CTD as a platform, the Integrator forms a bridge between the genes and the polymerase components that transcribe them. Then, as the polymerase transcribes the genes into RNA, the Integrator processes the RNA into finished snRNAs ready for transport into the cytoplasm and incorporation into the spliceosome.

Interestingly, the Integrator contains at least 12 subunits, all of which were previously unknown to scientists. The Integrator also appears to be an evolutionarily conserved complex, appearing in animals as diverse as humans, worms, and flies.

“The Integrator complex appears to be completely new, previously undefined in any way, which is surprising in this era of the Human Genome Project,” says Ramin Shiekhattar, Ph.D., a professor at Wistar and senior author on the Cell study. “People had hypothesized that a complex of this kind must exist and had looked for it for many years, but until now it had eluded them.”

The lead author on the Cell study is David Baillat, Ph.D. Mohamed-Ali Hakimi, Anders M. Naar, Ali Shilatifard, and Neil Cooch are coauthors. Baillat and Cooch are both members of the Shiekhattar laboratory at Wistar. Hakimi is at the CNRS in France, Naar is affiliated with Harvard Medical School and the Massachusetts General Hospital Cancer Center, and Shilatifard is at the St. Louis University Health Sciences Center. Senior author Shiekhattar is a professor in two programs at Wistar, the Gene Expression and Regulation program and the Molecular and Cellular Oncogenesis program. Support for the research was provided by the National Institutes of Health.

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DETAILS OF A REPRESENTATIVE LIST OF PROVEN OR SUSPECTED "JUNKDNA DISEASES"

AIDS

32-nucleotide deletion, associated with defence against hiv/aids, is a predominant mutation of CCR5 gene in the population of Georgia.

Kamkamidze G, Capoulade-Metay C, Butsashvili M, Dudoit Y, Chubinishvili O, Debre P, Theodorou L.

There is a special interest to investigate genetic peculiarities in the populations with a low HIV seroprevalence. Despite of presence of high-risk conditions for rapid spread of HIV/AIDS epidemics in Georgia, the prevalence of this infection in the country remains very low. We studied polymorphisms of CCR5 gene in Georgians. Blood samples from 190 women randomly selected from the cohort of pregnant women involved in the program of prevention of mother-to-child HIV transmission in Georgia have been investigated. Two-step PCR was used to amplify the whole CCR5 genetic sequence. Detection of mutations and polymorphisms was done by dHPLC. All samples showing specific patterns by dHPLC, were sequenced to identify the exact nature of the mutation. It was shown that CCR5-delta32 mutation is a predominant alteration of CCR5 gene among Georgians. All subjects bearing this mutation were heterozygotes. Frequency of delta32 CCR5 allele in the population of Georgia was equal to 5%. Only one case of R223Q mutation and two cases of mutations in the non-coding region of CCR5 gene were also found. Our findings differ from the existing data showing the absence of the CCR5-delta32 mutation among Georgians and provide further support to the hypothesis on a Northeastern European origin of this mutation and North to South gradient of its distribution.

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ALCOHOLISM

Alternative splicing and promoter use in the human GABRA2 gene.

Tian H, Chen HJ, Cross TH, Edenberg HJ. Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, 635 Barnhill Drive, Room 4063E, Indianapolis, IN 46202-5122, USA.

GABA(A) receptors mediate the majority of the fast synaptic inhibition in the mammalian brain. They are the targets of several important drugs, including benzodiazepines, which are used as anxiolytics, sedatives, anti-convulsants, and in the treatment of alcohol withdrawal symptoms. Non-coding variations in GABRA2, the gene encoding the alpha2 subunit, are associated with the risk for alcoholism, suggesting that regulatory differences are important. GABRA2 mRNAs from whole human brain and from three brain regions were examined for evidence of alternative splicing using reverse transcription-PCR and DNA sequencing. A complex pattern of alternative splicing and alternative promoter use of the human GABRA2 mRNA was demonstrated. There are four major isoforms consisting of combinations of two alternative 5' and 3' exons, as well as minor isoforms lacking exon 4 or exon 8. The alternative 5' exons each lie downstream of a functional promoter sequence, as shown by transient transfection assays. The promoter activities of naturally occurring haplotypes differed, indicating genetic differences in gene expression.

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ALPORT SYNDROME

Unusual deep intronic mutations in the COL4A5 gene cause X linked Alport syndrome.

King K, Flinter FA, Nihalani V, Green PM.

Division of Medical and Molecular Genetics, 7th Floor Guy's Tower, GKT school of Medicine, King's College, SE1 9RT London, UK.

The X-linked form of Alport syndrome is caused by mutations in the COL4A5 gene in Xq22. This large multiexonic gene has, in the past, been difficult to screen, with several studies detecting only about 50% of mutations. We report three novel intronic mutations that may, in part, explain this poor success rate and demonstrate that single base changes deep within introns can, and do, cause disease: one mutation creates a new donor splice site within an intron resulting in the inclusion of a novel in-frame cryptic exon; a second mutation results in a new exon splice enhancer sequence (ESE) that promotes splicing of a cryptic exon containing a stop codon; a third patient exhibits exon skipping as a result of a base substitution within the polypyrimidine tract that precedes the acceptor splice site. All three cases would have been missed using an exon-by-exon DNA screening approach.

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ALZHEIMER'S DISEASE

The transcriptional factor LBP-1c/CP2/LSF gene on chromosome 12 is a genetic determinant of Alzheimer's disease.

Lambert JC, Goumidi L, Vrieze FW, Frigard B, Harris JM, Cummings A, Coates J, Pasquier F, Cottel D, Gaillac M, St Clair D, Mann DM, Hardy J, Lendon CL, Amouyel P, Chartier-Harlin MC.

INSERM U508, Institut Pasteur de Lille, 1 rue du Professeur Calmette, BP 245, 59019 Lille Cedex, France.

PubMed Abstract:

Although the varepsilon4 allele of the apolipoprotein E gene appears as an important biological marker for Alzheimer's disease (AD) susceptibility, other genetic determinants are clearly implicated in the AD process. Here, we propose that a genetic variation in the transcriptional factor LBP-1c/CP2/LSF gene, located close to the LRP locus, is a genetic susceptibility factor for AD. We report an association between a non-coding polymorphism (G-->A) in the 3'-untranslated region of this gene and sporadic AD in French and British populations and a similar trend in a North American population. The combined analysis of these three independent populations provides evidence of a protective effect of the A allele (OR = 0.58, 95% CI 0.44-0.75). We describe a potential biologically relevant role for the A allele whereby it reduces binding to nuclear protein(s). The absence of the A allele was associated with a lower LBP-1c/CP2/LSF gene expression in lymphocytes from AD cases compared with controls. Our data suggest that polymorphic variation in the implication of the LBP-1c/CP2/LSF gene may be important for the pathogenesis of AD, particularly since LBP-1c/CP2/LSF interacts with proteins such as GSKbeta, Fe65 and certain factors involved in the inflammatory response.

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Candidate gene association studies of genes involved in neuronal cholinergic transmission in Alzheimer's disease suggests choline acetyltransferase as a candidate deserving further study.

Am J Med Genet B Neuropsychiatr Genet. 2005 Jan 5;132(1):5-8

Cook LJ, Ho LW, Wang L, Terrenoire E, Brayne C, Evans JG, Xuereb J, Cairns NJ, Turic D, Hollingworth P, Moore PJ, Jehu L, Archer N, Walter S, Foy C, Edmondson A, Powell J, Lovestone S, Williams J, Rubinsztein DC. Department of Medical Genetics, Cambridge Institute for Medical Research, Addenbrooke's Hospital, Hills Road, Cambridge, CB2 2XY, UK

Consistent deficits in the cholinergic system are evident in the brains of Alzheimer's Disease (AD) patients, including reductions in the activities of acetylcholine, acetylcholinesterase (AChE), and choline acetyltransferase (ChAT), increased butyrylcholinesterase (BChE) activity, and a selective loss of nicotinic acetylcholine receptors (nAChRs). Accordingly, we have analyzed polymorphisms in the genes encoding AChE, ChAT, BChE, and several of the subunit genes from neuronal nAChRs, for genetic associations with late-onset AD. A significant association for disease was detected for a non-coding polymorphism in ChAT (allele chi(1) (2) = 12.84, P = 0.0003; genotype chi(2) (2) = 11.89, P = 0.0026). Although replication analysis did not confirm the significance of this finding when the replication samples were considered alone (allele chi(1) (2) = 1.02, P = 0.32; genotype chi(2) (2) = 1.101, P = 0.58) the trends were in the correct direction and a significant association remained when the two sample sets were pooled (allele chi(1) (2) = 12.37, P = 0.0004; genotype chi(2) (2) = 11.61, P = 0.003). Previous studies have reported significant disease associations for both the K-variant of BChE and the coding ChAT rs3810950 polymorphism with AD. Replication analyses of these two loci failed to detect any significant association for disease in our case-control samples.

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Med Hypotheses. 2006;66(6):1140-1. Epub 2006 Feb 14.

Transfection "Junk" DNA - A link to the pathogenesis of Alzheimer's disease?

Macdonald AB. St. Catherine of Siena Medical Center, Department of Pathology, 50 Rte 25 A, Smithtown, NY 11787, USA.

A transfection product incorporates in[to] one molecule of human DNA, an inserted segment of DNA from another species. This communication addresses the hypothesis that a novel variation of the theme of transfection, namely "junk transfection" for which no protein product and no RNA is transcribed, might offer insights into the pathogenesis of Alzheimer's disease. It is hypothesized that spirochetal DNA gains access to the intracellular compartment of nerve cells during the subclinical latency phase of neuroborreliosis and chemically combines with human DNA. A previously reported Molecular interrogation of Alzheimer's disease autopsy tissues has yielded novel DNA sequences containing the 11q human chromosome and a short piece of the Borrelia burgdorferi Flagellin B DNA. Although the usually encountered transfection product bundles an entire nonhuman gene within it, this model proposes that shorter inserts into the human genome constitute "junk transfection" because no protein is derived from them. Junk transfections would offer an important new cognitive model for the detection of occult infections as the root causes for the Tauopathies, which are degenerative neurological disorders, including Alzheimer's disease.

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Neurosci Lett. 2003 Oct 23;350(2):77-80.

Association analysis between anterior-pharynx defective-1 genes polymorphisms and Alzheimer's disease.

Poli M, Gatta LB, Archetti S, Padovani A, Albertini A, Finazzi D. Institute of Chemistry, Faculty of Medicine, University of Brescia, viale Europa 11, 25123 Brescia, Italy.

Recent biological studies indicate the importance of anterior-pharynx defective-1 (APH-1) proteins in Alzheimer's disease (AD) pathogenesis. We scanned APH-1 genes for the presence of sequence variations by denaturing high performance liquid chromatography and analyzed their distribution in an Italian sample of 113 AD patients and 132 controls. We found six different polymorphisms: three of them, all in APH-1b, predict an aminoacid substitution (T27I, V199L and F217L); the others are either silent or in non-coding regions. None of them is significantly associated with the disease; data stratification by the apolipoprotein E epsilon4 carrier status show a trend for coexistence of the transversion c+651T>G (F217L) with the epsilon4 allele. Our data suggest that polymorphisms in APH-1a/b coding regions are not linked with higher risk for sporadic AD in our Italian population sample.

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Neurosci Lett. 2004 Oct 14;369(2):104-7.

Association between a T/C polymorphism in intron 2 of cholesterol 24S-hydroxylase gene and Alzheimer's disease in Chinese.

Wang B, Zhang C, Zheng W, Lu Z, Zheng C, Yang Z, Wang L, Jin F. Center for Human and Animal Genetics, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China.

A polymorphism (T/C) in intron 2 of the cholesterol 24-hydroxylase (CYP46) gene has recently been reported to be associated with the risk for late-onset Alzheimer's disease (LOAD). To investigate possible involvement of the CYP46 gene and apolipoprotein E (APOE) gene polymorphisms in the manifestation of LOAD, we analyzed 99 sporadic LOAD patients and 113 healthy controls of China. We found an obvious association between CYP46 TT genotype and LOAD (OR = 2.98, 95% CI 1.64-5.44, P < 0.001). A clear increase of the risk to develop LOAD was also observed in subjects carrying both the CYP46 TT genotype and the APOE epsilon4-allele (OR = 12.94, 95% CI 4.26-39.32, P < 0.001). Our data reveal that the polymorphism of CYP46 intron 2 is implicated in the susceptibility to LOAD and a strong synergistic interaction between CYP46 TT homozoygots and APOE epsilon4 carrier status on the risk of LOAD.

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ANGELMAN SYNDROME

The increasing number of RNA polymease II transcripts without any apparent open reading frame has increased our awareness that gene functions can be selected for without involving a protein product. [By conventional "Genetics" a "non-coding gene" is a contradiction in terms. "Genes" are defined as the "protein coding sequences" of the DNA, as opposed to the "non-coding DNA", which used to be described as "non-genic Junk". The Angelman- and related Prader-Willi Syndromes are living examples of the breakdown of overy rigid definition of "Genetics" - giving a strong impetus for moving to PostGenetics. - comment by A. Pellionisz]

By using the H19 gene as a point of reference, we highlight here several common features among non-coding genes, such as their antisense position in subchromosomal expression domains which are often genomically imprinted. We also discuss the need to critically examine the translatability of transcripts which are considered non-coding. Finally, we present a model to explain the origin of non-coding genes.

The human chromosome region 15q13-15, which is associated with genetic and epigenetic disturbances that generate both Prader-Willi syndrome and Angelmann syndrome, harbours non-coding genes such as IPW/Ipw, PAR1, PAR5, PARSN and ASR1,2 (ref. 14). IPW/Ipw (imprinted gene in Prader-Willi syndrome) expresses a processed RNA with no significant ORFs, which is localized predominantly in the cytoplasm15. An antisense RNA is produced from the ZNF127 locus, which encodes a protein with RING zinc-finger and multiple zinc-finger motifs. The ZNF127 and ZNF127 AS RNA vary in their expression patterns as well as in size of the transcripts12. The biological functions of IPW (sense) and ZNF127 (antisense) transcripts, if any, are not clear at this moment. The SNRPN gene, which spans 360 kb of DNA produces both coding and non-coding imprinted transcripts16. The exons at the 3¢ part of the gene generate a coding mRNA when spliced with exon 1 and noncoding RNAs when spliced with the 5¢ BD exons16. BD transcripts, encoded by two alternative 5¢ exons, BD1B and BD1A, have two alternative start sites and are subject to alternative splicing16. They are expressed from the paternal chromosome only, as the BD exons are heavily methylated on the maternal chromosome (16). It has recently been shown that paternally imprinted antisense RNA is produced at the 3¢ UTR of Angelmann syndrome gene, UBE3A (11), which encodes ubiquitin protein ligase that functions in protein turnover17.

ASTHMA

Variation in conserved non-coding sequences on chromosome 5q and susceptibility to asthma and atopy.
Donfack J, Schneider DH, Tan Z, Kurz T, Dubchak I, Frazer KA, Ober C.

Evolutionarily conserved sequences likely have biological function. Methods: To determine whether variation in conserved sequences in non-coding DNA contributes to risk for human disease, we studied six conserved non-coding elements in the Th2 cytokine cluster on human chromosome 5q31 in a large Hutterite pedigree and in samples of outbred European American and African American asthma cases and controls. RESULTS: Among six conserved non-coding elements (>100 bp, >70% identity; human-mouse comparison), we identified one single nucleotide polymorphism (SNP) in each of two conserved elements and six SNPs in the flanking regions of three conserved elements. We genotyped our samples for four of these SNPs and an additional three SNPs each in the IL13 and IL4 genes. While there was only modest evidence for association with single SNPs in the Hutterite and European American samples (P <0.05), there were highly significant associations in European Americans between asthma and haplotypes comprised of SNPs in the IL4 gene (P <0.001), including a SNP in a conserved non-coding element. Furthermore, variation in the IL13 gene was strongly associated with total IgE (P = 0.00022) and allergic sensitization to mold allergens (P = 0.00076) in the Hutterites, and more modestly associated with sensitization to molds in the European Americans and African Americans (P < 0.01). CONCLUSIONS: These results indicate that there is overall little variation in the conserved non-coding elements on 5q31, but variation in IL4 and IL13, including possibly one SNP in a conserved element, influence asthma and atopic phenotypes in diverse populations.

Full text

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ATAXIA TELANGIECTASIA

Exon-scanning mutation analysis of the ATM gene in patients with ataxia-telangiectasia.
Vorechovsky I, Luo L, Prudente S, Chessa L, Russo G, Kanariou M, James M, Negrini M, Webster AD, Hammarstrom L.
Department of Bioscience, NOVUM, Karolinska Institute, Huddinge, Sweden.

Using a polymerase chain reaction single strand conformation polymorphism (PCR-SSCP) assay, which amplifies individually all coding exons of the ATM gene deficient ataxia-telangiectasia (A-T), we have analyzed 10 patients with A-T for ATM mutations. Mutation were detected in 9 patients. We describe the first ATM mutation in the splice junction found in the 5' splice site of intron 17, leading to exon skipping. However, most mutations were small deletions or insertions resulting in premature termination of the translation product. The development of DNA-based methods for detection of unknown mutations and further characterization of ATM mutation pattern will facilitate identification of A-T carriers and assessment of their cancer risk.

PostGenetic Medicine of Ataxia Telangiectasia:

Bone marrow transplantation restores immune system function and prevents lymphoma in Atm-deficient mice.

Blood. 2004 Jul 15;104(2):572-8. Epub 2004 Mar 25.

Bagley J, Cortes ML, Breakefield XO, Iacomini J. Transplantation Biology

Research Center, Massachusetts General Hospital, MGH-East, 149-5210 13th St,
Boston, MA 02129, USA.

Ataxia-telangiectasia (A-T) is a human autosomal recessive disease caused by mutations in the gene encoding ataxia-telangiectasia mutated (ATM). A-T is characterized by progressive cerebellar degeneration, variable immunodeficiency, and a high incidence of leukemia and lymphoma. Recurrent sino-pulmonary infections secondary to immunodeficiency and hematopoietic malignancies are major causes of morbidity and mortality in A-T patients. In mice, an introduced mutation in Atm leads to a phenotype that recapitulates many of the symptoms of A-T, including immune system abnormalities and susceptibility to malignancy. Here we show that the replacement of the bone marrow compartment in Atm knockout mice (Atm(-/-)) using a clinically relevant, nonmyeloablative host-conditioning regimen can be used to overcome the immune deficiencies and prevent the malignancies observed in these mice.

Therefore, bone marrow transplantation may prove to be of therapeutic benefit in A-T patients.

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AUTISM

See Fragile X / Autism

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AUTOIMMUNE DISEASES

Association of the T-cell regulatory gene CTLA4 with susceptibility to autoimmune disease.

Genes and mechanisms involved in common complex diseases, such as the autoimmune disorders that affect approximately 5% of the population, remain obscure. Here we identify polymorphisms of the cytotoxic T lymphocyte antigen 4 gene (CTLA4)--which encodes a vital negative regulatory molecule of the immune system--as candidates for primary determinants of risk of the common autoimmune disorders Graves' disease, autoimmune hypothyroidism and type 1 diabetes. In humans, disease susceptibility was mapped to a non-coding 6.1 kb 3' region of CTLA4, the common allelic variation of which was correlated with lower messenger RNA levels of the soluble alternative splice form of CTLA4. In the mouse model of type 1 diabetes, susceptibility was also associated with variation in CTLA-4 gene splicing with reduced production of a splice form encoding a molecule lacking the CD80/CD86 ligand-binding domain. Genetic mapping of variants conferring a small disease risk can identify pathways in complex disorders, as exemplified by our discovery of inherited, quantitative alterations of CTLA4 contributing to autoimmune tissue destruction.

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Nature. 2003 May 29;423(6939):506-11. Epub 2003 Apr 30.

Association of the T-cell regulatory gene CTLA4 with susceptibility to autoimmune disease.

Ueda H, Howson JM, Esposito L, Heward J, Snook H, Chamberlain G, Rainbow DB, Hunter KM, Smith AN, Di Genova G, Herr MH, Dahlman I, Payne F, Smyth D, Lowe C, Twells RC, Howlett S, Healy B, Nutland S, Rance HE, Everett V, Smink LJ, Lam AC, Cordell HJ, Walker NM, Bordin C, Hulme J, Motzo C, Cucca F, Hess JF, Metzker ML, Rogers J, Gregory S, Allahabadia A, Nithiyananthan R, Tuomilehto-Wolf E, Tuomilehto J, Bingley P, Gillespie KM, Undlien DE, Ronningen KS, Guja C, Ionescu-Tirgoviste C, Savage DA, Maxwell AP, Carson DJ, Patterson CC, Franklyn JA, Clayton DG, Peterson LB, Wicker LS, Todd JA, Gough SC. Juvenile Diabetes Research Foundation/Wellcome Trust Diabetes and Inflammation Laboratory, Cambridge Institute for Medical Research, University of Cambridge, Wellcome Trust/MRC Building, Cambridge, CB2 2XY, UK.

Genes and mechanisms involved in common complex diseases, such as the autoimmune disorders that affect approximately 5% of the population, remain obscure. Here we identify polymorphisms of the cytotoxic T lymphocyte antigen 4 gene (CTLA4)--which encodes a vital negative regulatory molecule of the immune system--as candidates for primary determinants of risk of the common autoimmune disorders Graves' disease, autoimmune hypothyroidism and type 1 diabetes. In humans, disease susceptibility was mapped to a non-coding 6.1 kb 3' region of CTLA4, the common allelic variation of which was correlated with lower messenger RNA levels of the soluble alternative splice form of CTLA4. In the mouse model of type 1 diabetes, susceptibility was also associated with variation in CTLA-4 gene splicing with reduced production of a splice form encoding a molecule lacking the CD80/CD86 ligand-binding domain. Genetic mapping of variants conferring a small disease risk can identify pathways in complex disorders, as exemplified by our discovery of inherited, quantitative alterations of CTLA4 contributing to autoimmune tissue destruction.

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Immunogenetics. 2002 Nov;54(8):591-5. Epub 2002 Oct 9.

Novel polymorphisms in HLA-DOA and HLA-DOB in B-cell malignancies.

van Lith M, van Ham M, Neefjes J. Division of Tumor Biology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands.

In B cells, HLA-DO controls HLA-DM-mediated peptide loading on MHC class II molecules. We analyzed whether HLA-DO mutations are associated with autoimmune diseases characterized by an autoantibody component and with a linkage to HLA-DR or HLA-DQ. These diseases include systemic lupus erythematosus, rheumatoid arthritis, celiac disease, and Graves' disease. In addition, several B-cell leukemias were screened for mutations in HLA-DO. A limited number of polymorphisms in DOA and DOB were found, most of which are non-coding changes or result in a conserved amino acid change. A novel non-conserved Arg to Cys mutation in DOA was found in a patient suffering from chronic lymphocytic leukemia. Further analysis did not reveal any effect on the function of HLA-DO. We conclude that HLA-DO variants are not critically involved in the autoimmune diseases and B-cell leukemias studied here.

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BIPOLAR DISORDER

G protein receptor kinase 3 (GRK3)

Further stronger evidence for the role of GRK3 in bipolar disorder came from experiments conducted by Tom Barrett in the lab. Tom identified families with the strongest evidence of linkage to the GRK3 locus who would be most likely to be transmitting a mutation in the GRK3 gene. He then sequenced much of the gene in an effort to find disease causing mutations or just anonymous markers. This was a big job! He sequenced all coding regions and most of the non-coding regions. He found no coding sequence variants, but he did find six sequence variants in the promoter of the gene. These SNPs were in such a position so as to possibly influence the regulation of when the gene was turned on and off. Tom then examined these SNPs in about 150 families from the UCSD/UBC/UC and NIMH sets using the transmission disequilibrium test (TDT). This analysis indicated genetic association to two of these SNPs. We were then fortunate to be able to collaborate with Jim Kennedy at the U. of Toronto and to study another set of 250 triad families with bipolar disorder. Our results for one of the SNPs (P-5) was replicated in this sample!

Together these data argue that a regulatory mutation in or near the GRK3 promoter causes this gene to fail to be expressed when dopamine stimulates receptors in the brain. These receptors then fail to be desensitized in the normal fashion. This results in an effective supersensitivity to dopamine. This is exciting because post-synaptic dopamine receptor supersensitivity has been hypothesized for many decades based on many other lines of research.

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BRUGADA SYNDROME - ARRYTHMIA

Cryptic 5' splice site activation in SCN5A associated with Brugada syndrome.

Hong K, Guerchicoff A, Pollevick GD, Oliva A, Dumaine R, de Zutter M, Burashnikov E, Wu YS, Brugada J, Brugada P, Brugada R.

Molecular Genetics, Masonic Medical Research Laboratory, 2150 Bleecker Street, Utica, NY 13501, USA.

The Brugada syndrome (BS) is characterized by ST segment elevation in the right precordial leads and sudden cardiac death. The disease is linked to mutations in SCN5A in approximately 20% of cases. We collected a large family with BS and have identified a novel intronic mutation. We performed the clinical, genetic, molecular and biophysical characterization of this disease-causing mutation. With direct sequencing we identified an intronic insertion of TGGG 5 bp from the end of the Exon 27 of SCN5A. For transcript analysis, we investigated Epstein-Barr-transformed lymphoblastoid cell lines from patients and controls. Total RNA was extracted and RT-PCR experiments were performed to analyze the splicing patterns in exon 27 and 28. We identified two bands, one of the expected size and the other which showed a 96 bp deletion in exon 27, leading to a 32 amino acid in-frame deletion involving segments 2 and 3 of Domain IV of the SCN5A protein. This finding indicates that the intronic mutation creates a cryptic splice site inside Exon 27. Biophysical analysis using whole-cell patch-clamp techniques showed a complete loss of function of the mutated channels when heterologously expressed. In summary, this is the first report of a dysfunctional sodium channel created by an intronic mutation giving rise to cryptic splice site activation in SCN5A in a family with the BS. The deletion of fragments of segments 2 and 3 of Domain IV leads to complete loss of function, consistent with the biophysical data found in several mutations causing BS.

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CANCER: BREAST

Associations between Single Nucleotide Polymorphisms in Double-Stranded DNA Repair Pathway Genes and Familial Breast Cancer.

Sehl ME, Langer LR, Papp JC, Kwan L, Seldon JL, Arellano G, Reiss J, Reed EF, Dandekar S, Korin Y, Sinsheimer JS, Zhang ZF, Ganz PA.

Authors' Affiliations: Divisions of Hematology and Oncology and Geriatrics, Departments of Medicine, Biomathematics, and Human Genetics, David Geffen School of Medicine at University of California at Los Angeles.

PURPOSE: DNA damage recognition and repair play a major role in risk for breast cancer. We investigated 104 single nucleotide polymorphisms (SNP) in 17 genes whose protein products are involved in double-stranded break repair (DSBR). EXPERIMENTAL DESIGN: We used a case-control design. Both the case individuals affected with breast cancer or with both breast and ovarian cancers and the controls had similar familial risk of breast cancer and were participants in a high-risk cancer registry. RESULTS: We found that 12 of the polymorphisms are associated with breast or breast and ovarian cancers, most notably rs16888927, rs16888997, and rs16889040, found in introns of RAD21, suggesting that SNPs in other genes in the DSBR pathway in addition to BRCA1 and BRCA2 may affect breast cancer risk. CONCLUSIONS: SNPs within or near several DSBR DNA repair pathway genes are associated with breast cancer in individuals from a high-risk population. In addition, our study reemphasizes the unique perspective that recruitment of cases and controls from family cancer registries has for gene discovery studies.

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[Single Nucleotide Polymorphism tests for 185delAG in BRCA1, 5382insC in BRCA1, and 6174delT in BRCA2 "point mutations" are already offered by 23andME:

[source of image and text below: 23andMe - AJP]

"The researchers analyzed the effects of only the three BRCA mutations that are most common in Ashkenazi Jewish people: 185delAG in BRCA1, 5382insC in BRCA1, and 6174delT in BRCA2. The mutations account for 80-90% of hereditary breast and ovarian cancer in this ethnic group. But there are hundreds of other BRCA1/2 mutations that have been associated with cancer, and the authors caution that further studies taking these other BRCA1/2 mutations into account and using study subjects from diverse groups will be needed to confirm their results".
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In addition to not only hundreds of exonic, but also intronic "glitches" of BRCA1, BRCA2 and the RAD21 genes, it is becoming evident (Sotirou, C and Pusztai, J, 2009) that beyond the three genes targeted above, there may be at least 76 genes (with their intronic- as well as intergenic regions) involved in breast cancer:

[Sotirou and Pusztai, 2009 - AJP]

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CANCER: CARTILAGE HAIR HYPOPLASIA

Doctors struggled for more than nine years, for example, to nail down the gene responsible for cartilage hair hypoplasia. This recessive disease was first identified in the Amish, one in 19 of whom carries a copy of the defective gene, which causes an unusual kind of dwarfism. People with CHH are not only small in stature but also at high risk for cancer and immune disorders. Geneticist Maaret Ridanpaa of the University of Helsinki tracked the gene to chromosome nine, sequenced a large region and then proceeded to check all 10 protein-making genes in the area, one by one. None caused the disease.

Finally, in 2001, Ridanpaa and his co-workers identified the culprit, an RNA-only gene called RMRP. The RNA transcribed from RMRP links up with proteins to form an enzyme that works inside a cell's energy generators, the mitochondria. A change to just a single base at a critical spot on this RNA can mean the difference between a full-size, healthy life and a short, abbreviated one (if the same mutation is inherited from both parents). Such "analog" RNAs, which fold up into complex shapes just as proteins do, have also been discovered recently to be essential to the function of enzymes that protect the chromosomes and that escort secreted protein signals out of cells' portholes.

Perhaps the most intriguing form of RNA yet discovered is the riboswitch, isolated last year by Ronald R. Breaker's lab at Yale. He and others have long wondered how, billions of years ago, the very earliest chemical precursors to life got along in the RNA world before DNA and proteins existed. They speculated that such proto-organisms would need to use RNA as sensors and switches to respond to changes in the environment and in their metabolism. To test the idea, they tried to create RNAs with such capabilities.

"Our laboratory successfully produced a number of synthetic RNA switches," Breaker recalls. Dubbed riboswitches, these long RNAs are both coding and noncoding at once. As the RNA folds up, the noncoding end becomes a sensitive receptor for a particular chemical target. A collision with the target flips the switch, causing the other end, which contains a standard blueprint for a protein, to change shape. The riboswitch thus gives rise to a protein, much like a normal gene does--but only when it senses its target. [Sounds like a "conceptual meltdown" of the orthodox view of "genes"... AJP]

Breaker's group started hunting for riboswitches in the wild and soon found them hiding in intergenic DNA. These precision genetic switches have been extracted now from species in all three kingdoms of life. "This implies that they were probably present in the last common ancestor," not long after the dawn of evolution, Breaker argues.

In August, Breaker and his co-workers reported that one family of riboswitches regulates the expression of no fewer than 26 genes in Bacillus subtilis, a common kitchen bacterium. These are not once-in-a-blue-moon genes, either, but genes that the bacterium relies on to metabolize such basic staples as sulfur and amino acids. Breaker estimates that B. subtilis has at least 68 genes, nearly 2 percent of its total, under the control of riboswitches. His lab has already begun engineering the hybrid digital-analog molecules to do useful things, such as selectively kill germs.

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Hum Mol Genet. 2005 Dec 1;14(23):3723-40. Epub 2005 Oct 27.

Consequences of mutations in the non-coding RMRP RNA in cartilage-hair hypoplasia.

Hermanns P, Bertuch AA, Bertin TK, Dawson B, Schmitt ME, Shaw C, Zabel B, Lee B. Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA.

Cartilage-hair hypoplasia (CHH), also known as metaphyseal chondrodysplasia McKusick type (OMIM no. 250250), is an autosomal recessive, multi-systemic disease characterized by disproportionate short stature, fine and sparse hair, deficient cellular immunity and a predisposition to malignancy. It is caused by mutations in RMRP, the RNA component of the ribonucleoprotein complex RNase MRP, and, thus, CHH represents one of few Mendelian disorders caused by mutations in a nuclear encoded, non-coding RNA. While studies in yeast indicate that RMRP contributes to diverse cellular functions, the pathogenesis of the human condition is unknown. Studies of our CHH patient cohort revealed mutations in both the promoter and the transcribed region of RMRP. While mutations in the promoter abolished transcription in vitro, RMRP RNA levels in patients with transcribed mutations were also decreased suggesting an unstable RNA. RMRP mutations introduced into the yeast ortholog, NME1, exhibited normal mitochondrial function, chromosomal segregation and cell cycle progression, while a CHH fibroblast cell line exhibited normal mitochondrial content. However, the most commonly found mutation in CHH patients, 70A>G, caused an alteration in ribosomal processing by altering the ratio of the short versus the long form of the 5.8S rRNA in yeast. Transcriptional profiling of CHH patient RNAs showed upregulation of several cytokines and cell cycle regulatory genes, one of which has been implicated in chondrocyte hypertrophy. These data suggest that alteration of ribosomal processing in CHH is associated with altered cytokine signalling and cell cycle progression in terminally differentiating cells in the lymphocytic and chondrocytic cell lineages.

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CANCER: LUNG

Wu X, Zhao H, Wei Q, Amos CI, Zhang K, Guo Z, Qiao Y, Hong WK, Spitz MR.

Department of Epidemiology, The University of Texas M. D. Anderson Cancer Center, Houston, TX 77030, USA. xwu@mail.mdanderson.org

XPA, a DNA binding protein in the nucleotide excision repair (NER) pathway, modulates damage recognition. Recently, a common single-nucleotide polymorphism (A --> G) of unknown function was identified in the 5' non-coding region of the XPA gene. Because a deficiency in NER is associated with an increased risk of lung cancer, we evaluated the role of this polymorphism in 695 lung cancer case patients and 695 age-, sex-, ethnicity- and smoking-matched control subjects. We also studied the effect of this polymorphism on NER capacity in a subset sample for whom the host cell reactivation data were available. The presence of one or two copies of the G allele was associated with a reduced lung cancer risk for Caucasians [adjusted odds ratio (ORadj) = 0.69 [95% confidence interval (CI) = 0.53-0.90]], Mexican-Americans [ORadj = 0.32 (95% CI = 0.12-0.83)] and African-Americans [ORadj = 0.45 (95% CI = 0.16-1.22)]. In Caucasians, ever smokers with one or more copies of the G allele were observed to have a significantly reduced risk of lung cancer. Control subjects with one or two copies of the G allele demonstrated more efficient DRC than did those with the homozygous A allele. Our data suggest that the XPA 5' non-coding region polymorphism modulates NER capacity and is associated with decreased lung cancer risk, especially in the presence of exposure to tobacco carcinogens.

Full text

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CANCER: LYMPHOMA

BIC and miR-155 are highly expressed in Hodgkin, primary mediastinal and diffuse large B cell lymphomas

Dok1 expression and mutation in Burkitt's lymphoma cell lines.

Lee S, Huang H, Niu Y, Tommasino M, Lenoir G, Sylla BS.

International Agency for Research on Cancer, 150 Cours Albert-Thomas, Lyon 69008, France.

Dok1 is an adaptor tyrosine kinase substrate with tumor-suppressive activity. The gene encoding Dok1 maps to human chromosome 2p13, which is frequently rearranged in human tumors. We have previously reported a frameshift mutation of this gene and the down-regulation of its expression in chronic lymphocytic leukemia. In this study, we have determined the expression levels of Dok1 in Burkitt's lymphoma (BL) cell lines, lymphoblastoid cell lines from patients with X-linked lymphoproliferative (XLP-LCL), or from control healthy donors. We have also screened for Dok1 gene mutations by heteroduplex analysis and direct sequencing. Dok1 expression was down-regulated in all BL and XLP-LCL cell lines in comparison to the control cells. No Dok1 mutation or polymorphism was found in the coding region of Dok1 in the three types of cells. However, DNA sequence analysis revealed the presence of four nucleotide changes in Dok1 gene, T(90172)C (intron 1), C(89487)T and (89433)InsCTCT (intron 2), and A(87714)G (3' UTR). T(90172)C and (89433)InsCTCT that were detected in about 7% of BL, 9% of XLP-LCL and 4% of normal samples may represent a common polymorphism. C(89487)T and A(87714)G changes were detected in 9 and 6% of analyzed BL lines, respectively, but never in the control and XLP-LCL cells, indicating that these nucleotide substitution occurred during tumor development. Interestingly, the C(89487)T variant is associated with a significantly lower level of Dok1 expression compared to the control samples. A positive association was also found between the presence of EBV in BL and the Dok1 genetic variation. Our data show that Dok1 expression and structure are affected in a subset of Burkitt's lymphoma samples, suggesting its possible role in this type of cancer.

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CANCER: MULTIPLE MYELOMA

The case of IPGS Honorary Chairman Dr. Malcolm J. Simons

Multiple myeloma (MM) is a malignant plasma cell neoplasm that often is preceded by a common pre-malignant monoclonal expansion of plasma cells called monoclonal gammopathy of undetermined significance (MGUS). MGUS is reported to be present in 1% of the adult population and to progress to MM at a rate of 1% per year. Despite intense efforts over the last 20 years, the 5-year survival rate reported in the SEER database remains unchanged at 28%, and there is no known way to identify those who will progress from MGUS to MM, or to delay or prevent this progression. A better understanding of the molecular pathogenesis of these conditions is fundamental to developing more effective prognostic, treatment and prevention approaches.

4p16.3 – FGFR3 and MMSET.

The t(4;14) is identified in approximately 15% of MM tumors.2,3,9 The breakpoints occur in the telomeric region of chromosome 4 and result in a karyotypically silent translocation that cannot be identified by either G-banding or spectral karyotype analysis (Figure 4A ). In the IgH locus the breakpoints all occur in a switch region and dissociate the intronic enhancer from the 3' enhancer (Fig. 4B ). The 4p16 breakpoints fall 50-100kb centromeric to FGFR3, which becomes associated with the 3' enhancer(s) on der 14. This region includes the 5' non-coding (largely) exons of MMSET/WHSC1, so that this gene becomes dysregulated by juxtaposition to the IgH intronic enhancer on der 4, resulting in formation of hybrid mRNA transcripts with the JH and I-mu exons. The hybrid transcripts provide a very specific and sensitive means of detecting this translocation by RT-PCR. We have not identified any variant translocations, nor any translocations into the JH region, suggesting that dysregulation of both MMSET and FGFR3 may be important in the pathogenesis of MM, a hypothesis that is supported by our inability to find HMCL or MM tumors that have lost der 4.

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CANCER: NON-POLYPOSIS COLORECTAL

Genetic instability in human mismatch repair deficient cancers.

Duval A, Hamelin R.
Inserm U434-CEPH, 27, rue Juliette Dodu, 75010, Paris, France. alex.duval@cephb.fr

Cancers showing microsatellite instability (MSI-H) are frequent tumors characterized by inactivating alterations of mismatch repair (MMR) genes that lead to an incapacity to recognize and repair errors that occur during DNA replication. These cancers can be inherited as in the human non-polyposis colorectal cancer syndrome, or can occur sporadically in 10-15% of colorectal, gastric and endometrial cancers. MSI-H tumors have different clinicopathological features compared to cancers without this phenotype, termed MSS, and the repertoire of genetic events involved in tumoral progression of both phenotypes is thought to be different. In MSI-H tumors, most of the genetic changes occur at both non-coding and coding microsatellites that are particularly prone to errors during replication due to their repetitive sequence. This mechanism appears to be the main "genetic pathway" by which functional changes with putative oncogenic effects are accumulated in these tumors.

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Evolution of instability at coding and non-coding repeat sequences in human MSI-H colorectal cancers.

Duval A, Rolland S, Compoint A, Tubacher E, Iacopetta B, Thomas G, Hamelin R. INSERM U434, CEPH, 27 rue Juliette Dodu, 75010 Paris, France.

A number of human genes containing coding mononucleotide repeat sequences are particularly prone to mutations in tumors with defects in mismatch repair (MMR) genes (MSI-H cancers). In a large series of MSI-H colorectal tumors, we looked for mutations in 25 coding repeats contained in eight genes already known to be mutated in these cancers or in 17 other genes with an expected role in carcinogenesis. Mutations were found in 19 of the 25 candidate genes. Using a maximum likelihood statistical method, they were separated into two different groups that differed significantly in their mutation frequencies, and were likely to represent mutations that do or do not provide selective pressures during MSI-H tumoral progression, respectively. Three new target genes were found (GRB-14, RHAMM, RAD50). Our results provide evidence that MSI-H tumoral progression involves the cumulative mutations of a large number of genes. For each MSI-H tumor we calculated indexes representing the number of mutations found in genes of these groups. We also evaluated a shortening index at both the Bat-25 and Bat-26 non-coding mononucleotide tracts that are known to be almost always unstable in MSI-H cancers. A significant correlation was observed between instability at both coding and non-coding repeats, suggesting that Bat-25 and Bat-26 could be used as simple phenotypical markers of the tumoral evolution. A preferential order of mutations was deduced. During this process, hMSH3 alterations, a target gene encoding for a MMR protein, was found to play an important role by increasing the instability phenomenon characterizing these cancers.

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CANCER: PROSTATE

As antisense RNA gets intronic.

Reis EM, Louro R, Nakaya HI, Verjovski-Almeida S.
Department of Biochemistry, Institute of Chemistry, University of Sao Paulo, Sao Paulo, Brazil.

Recent work describing the transcriptional output of the human genome points to the existence of a significant number of non-coding RNA transcripts coming from intronic regions, with a fraction of these being oriented antisense relative to the protein-coding mRNA of the known gene. In this article, we survey the main findings of the large-scale expression analysis projects that led to the identification of antisense intronic messages and which demonstrate their ubiquitous expression in the human genome. We review the current knowledge on long, unspliced, intronic antisense transcripts, a new class of non-coding RNAs, recently described by our group to be correlated with the degree of tumor differentiation in prostate cancer, which we postulate is involved in the fine tuning of gene expression in eukaryotes. Possible mechanisms of antisense intronic transcript biogenesis and function in gene expression regulation are discussed, as is their involvement in human diseases. While there is still no conclusive evidence demonstrating a functional role for these long, intronic antisense messages, the far-reaching implications of their existence for the mechanisms regulating gene expression certainly warrant further experimentation.

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CANCER: THYROID

Role Of MicroRNA Identified In Thyroid Cancer

The presence of only five tiny strands of RNA is enough to clearly distinguish cancerous thyroid tissue from otherwise normal tissue, scientists say.

The findings provide more evidence that an emerging set of RNA genes called microRNA (miRNA) is a powerful regulatory force in the development of cancer and other diseases. The study is published online in the Dec. 19 Proceedings of the National Academy of Sciences.

Scientists already know that some people inherit a predisposition to developing papillary thyroid cancer (PTC), the most common form of thyroid cancer, representing about 80 percent of all cases. Although changes in key cell-signaling systems and gene translocations are sometimes present in thyroid tumors, no specific gene mutations have yet been identified that are directly linked to the predisposition of this type of cancer.

That led researchers in The Ohio State University Comprehensive Cancer Center to conclude that while genetic mutations may indeed cause some people to be more likely to develop PTC than others, the mutations may not occur often enough to be readily detectable. They hypothesized that any predisposition to PTC might be more reasonably linked to a more subtle, complex interaction among several genes – suggesting a possible role for miRNAs.

MiRNAs are smidgens of genetic material no longer than 22 or so nucleotides in length. A gene, in comparison, can be tens of thousands of nucleotides long. Scientists used to think miRNAs were parts of long stretches of functionless, “junk” DNA in the genome. But Dr. Huiling He, a research scientist in the Human Cancer Genetics Program at Ohio State and the lead author of the study, says researchers are now beginning to understand how important they may be.

&#xThe identification of miRNA ‘signatures’ in cancer and other diseases has really changed the way we think about the process of malignant growth,” says He.

Old dogma held that a gene carries a recipe for a molecule of messenger RNA which, in turn, carries a blueprint for the creation of a particular protein. Any mutation in the gene could affect the production of the protein. But recent studies have shown that protein production can also be manipulated indirectly through miRNAs.

&#xMiRNAs can latch on to part of the messenger RNA and scramble its ability to properly carry out its original coding instructions,” says He.

Under the direction of Dr. Albert de la Chapelle, a professor in the department of molecular virology, immunology and medical genetics at Ohio State, He and other researchers examined samples of malignant tissue from 15 patients diagnosed with PTC and compared them with normal appearing tissue adjacent to the tumors.

They found 23 miRNAs that were significantly altered in the cancerous tissue when compared with the normal samples, with three of the miRs – miR-146, miR-221 and miR-222 – dramatically overexpressed, or “turned on,” registering 11-to-19-fold higher levels of expression in the tumors than in the unaffected tissue nearby.

Further investigation revealed that two additional miRs – miR-21 and miR-181a – when coupled with the three that showed dramatic overexpression, formed a “signature” that clearly predicted the presence of malignant tissue.

&#xWe also discovered miR-221 expression in all of the apparently normal tissue of the patients with PTC, but it was significantly overexpressed in a subset of three of the samples, suggesting that increased activity of miR-221 may be one of the earliest signs of carcinogenesis,” says de la Chapelle.

Some scientists believe miRNAs act like oncogenes, molecules that promote cell growth, and they also feel they may be tumor and tissue specific. For example, in many other forms of cancer, miRNA activity is suppressed, but in PTC, researchers found just the opposite: 17 of the 23 miRNAs they discovered were overexpressed.

According to the American Cancer Society, the incidence of thyroid cancer has been increasing slightly over the past several years. It estimates that about 25,000 new cases will be diagnosed in the United States this year.

&#xThis is just the beginning of our work identifying the role of miRNAs in thyroid cancer,” says He. “But we are encouraged by these findings. We feel that they help point the way toward new options in diagnosis and treatment for this disease.”

A grant from the National Institutes of Health supported the research team, which included Drs. Krystian Jazdzewski, Wei Li, Stefano Volinia, George Calin, Carlo Croce and Chang-gong Liu, all of the Ohio State Human Cancer Genetics Program; Dr. Saul Suster, from OSU’s department of pathology; Dr. Richard Kloos from OSU’s departments of internal medicine and radiology; Rebecca Nagy, a genetic counselor in the Human Cancer Genetics Program; Sandra Liyanarachchi, a biostatistician in the Ohio State Human Cancer Genetics Program; and Dr. Kaarle Franssila, from the department of pathology at Helsinki University Central Hospital, Finland.

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CARDIOVASCULAR DISEASE

Pharmacogenomics. 2003 Sep;4(5):571-82.

Comparative and functional analysis of cardiovascular-related genes.

Cheng JF, Pennacchio LA. Department of Genome Sciences, MS 84-171, One Cyclotron Road, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA. jfcheng@lbl.gov

The ability to detect putative cis-regulatory elements in cardiovascular-related genes has been accelerated by the availability of genomic sequence data from numerous vertebrate species and the recent development of comparative genomic tools. This improvement is anticipated to lead to a better understanding of the complex regulatory architecture of cardiovascular (CV) genes and how genetic variants in these non-coding regions can potentially play a role in cardiovascular disease. This manuscript reviews a recently established database dedicated to the comparative sequence analysis of 250 human CV genes of known importance, 37 of which currently contain sequence comparison data for organisms beyond those of human, mouse and rat. These data have provided a glimpse into the variety of possible insights from deep vertebrate sequence comparisons and the identification of putative gene regulatory elements.

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CYSTIC FIBROSIS

Micro-RNA-like effects of complete intronic sequences.

Hill AE, Hong JS, Wen H, Teng L, McPherson DT, McPherson SA, Levasseur DN, Sorscher EJ.
Pittman General Clinical Research Center, Department of Computer and Information, University of Alabama at Birmingham, Birmingham, Alabama 35294.

MicroRNAs (miRNAs) have been suggested as suppressors of numerous target genes in human cells. In this report, we present gene chip array data indicating that in the absence of miRNA sequences, complete human introns are similarly capable of coordinating expression of large numbers of gene products at spatially diverse sites within the genome. The expression of selected intronic sequences (6a, 14b and 23) derived from the cystic fibrosis transmembrane conductance regulator (CFTR) gene caused extensive and specific transcriptional changes in epithelial cells (HeLa) that do not normally express this gene product. Each intron initiated a distinctive pattern of gene transcription. Affected genes such as FOXF1, sucrase-isomaltase, collagen, interferon, complement and thrombospondin 1 have previously been linked to CFTR function or are known to contribute to the related processes of epithelial differentiation and repair. A possible regulatory function of this nature has not been demonstrated previously for non-coding sequences within eukaryotic DNA. The results are consistent with the observation that splicesomal introns are found only in eukaryotic organisms and that the number of introns increases with phylogenetic complexity.

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DIABETES MELLITUS TYPE II

Bell’s team discovered overwhelming evidence that the risk-increasing abnormality in the calpain 10 gene occurs not in a functional part of the gene but in an intron. Introns are pieces of non-coding DNA, sometimes referred to as “junk DNA.” Introns are edited out when the DNA is transcribed into RNA.

Haplotype structure and phylogenetic shadowing of a hypervariable region in the CAPN10 gene.
Hum Genet. 2005 Jul;117(2-3):258-66. Epub 2005 May 20.

Clark VJ, Cox NJ, Hammond M, Hanis CL, Di Rienzo A. Department of Human Genetics, University of Chicago, 507F CLSC, 920 E. 58th Street, Chicago, IL 60637, USA.

It has been proposed that variation in calpain 10 (CAPN10) contributes to the risk of type 2 diabetes (T2D). A previous survey of CAPN10 in ethnically diverse populations revealed an intronic region with a significant excess of polymorphism levels relative to inter-species sequence divergence, suggesting that this region was the target of long-standing balancing selection. Based on the thrifty genotype hypothesis, variation that increases risk to T2D in contemporary humans at one time conferred a survival advantage in ancestral populations. Thus, the signature of positive natural selection in a T2D candidate gene could identify a genomic region containing variation that influences disease susceptibility. Here, we investigate this hypothesis by re-sequencing the CAPN10 region with unusual polymorphism levels in T2D cases and controls (n=91) from a Mexican American (MA) population, and by using networks to infer the evolutionary relationships between the major haplotypes. Haplotype tag SNPs (htSNPs) were then selected in each population sample and in MA cases and controls. By placing the htSNPs on the haplotype network, we investigate how cross-population differences in CAPN10 genetic architecture may affect the detection of the disease association. Interestingly, despite the small scale of our case-control study, we observe a nearly significant signal of association between T2D and variation in the putative target of balancing selection. Finally, we use phylogenetic shadowing across 10 primate species to search for conserved non-coding elements that may affect the expression and function of CAPN10. These elements are postulated to be the targets of long-standing balancing selection.

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Mol Cell Biochem. 2004 Jun;261(1-2):161-7.

Role of calpains in diabetes mellitus: a mini review.

Harris F, Chatfield L, Singh J, Phoenix DA. Department of Forensic and Investigative Science, Faculty of Science, University of Central Lancashire, Preston, PRI 2HE, England, UK.

Type 2 diabetes mellitus (T2DM) is characterized by defects in haepatic glucose production, insulin action and insulin secretion, which can also lead to a variety of secondary disorders. The disease can lead to death without treatment and it has been predicted that T2DM will affect 215 million people world-wide by 2010. T2DM is a multifactorial condition whose precise genetic causes and biochemical defects have not been fully elucidated but at both levels, calpains appear to play a role. Positional cloning studies mapped T2DM susceptibility to CAPN10, the gene encoding the intracellular cysteine protease, calpain 10. Further studies have shown a number of non-coding polymorphisms in CAPN10 to be functionally associated with T2DM whilst the identification of coding polymorphisms, suggested that mutant calpain 10 proteins may also contribute to the disease. The presence of both calpain 10 and its mRNA have been demonstrated in tissues from several mammalian species whilst calpain 10 appears to be associated with pathways involved in glucose metabolism, insulin secretion and insulin action. It appears that other calpains may also participate in these pathways and here we present an overview of recent studies on calpains and their putative role in T2DM.

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Pharmacogenetics. 2004 Dec;14(12):823-9.

Response to micronized fenofibrate treatment is associated with the peroxisome-proliferator-activated receptors alpha G/C intron7 polymorphism in subjects with type 2 diabetes.

Foucher C, Rattier S, Flavell DM, Talmud PJ, Humphries SE, Kastelein JJ, Ayyobi A, Pimstone S, Frohlich J, Ansquer JC, Steiner G; DAIS investigators.

Laboratoires Fournier S.A. Centre de Recherches, Department of Clinical Research and Medical Affairs, Daix, France. c.foucher@fr.fournierpharma.com

OBJECTIVE: The association between polymorphisms in candidate genes related to lipoprotein metabolism and the reduction in plasma triglyceride (TG) in response to fenofibrate treatment was evaluated in subjects with type 2 diabetes treated with micronized fenofibrate (200 mg/day) for at least 3 years in the Diabetes Atherosclerosis Intervention Study. METHODS: The cholesteryl ester transfer protein Taq1B, LPL S447X, hepatic lipase -514 C-->T, peroxisome-proliferator-activated receptors alpha (PPARA) L162V and G/C intron 7 polymorphisms and the apolipoprotein E2/E3/E4 alleles were genotyped using PCR and restriction enzyme digestion. Subjects were divided into high TG-responders (with > 30% TG relative reduction after treatment) and low TG-responders. RESULTS: The frequency of the PPARA intron 7 G/G genotype was higher in high TG-responders than in low TG-responders (85% vs. 69%, P < 0.05). There was no significant difference between the percentage of high TG-responders and low TG-responders for any of the other genetic polymorphisms examined. In stepwise logistic regression, baseline TG and only the PPARA intron 7 polymorphism among the others were selected in the model as significant predictors of TG-response (odds ratio: 3.10, 95% CI: 1.28-7.52, P = 0.012 for PPARA polymorphism). With age, gender, body mass index, smoking status and HbA1c as additional factors, baseline TG (P< 0.0001), intron 7 (P = 0.013), body mass index (P = 0.040) and LPL-S447X (P = 0.084) were significant predictors of TG-response. CONCLUSION: These results indicate that elevated baseline TG levels and PPARA gene intron 7 G/G genotype were associated with TG reduction > 30% after fenofibrate treatment in patients with type 2 diabetes.


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DiGEORGE SYNDROME

Genomics. 2001 May 1;73(3):264-71.

Isolation and characterization of a novel gene containing WD40 repeats from the region deleted in velo-cardio-facial/DiGeorge syndrome on chromosome 22q11.

Funke B, Pandita RK, Morrow BE.

Department of Molecular Genetics, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461, USA.

Three congenital disorders, cat-eye syndrome (CES), der(22) syndrome, and velo-cardio-facial syndrome/DiGeorge syndrome (VCFS/DGS), result from tetrasomy, trisomy, and monosomy, respectively, of part of 22q11. They share a 1.5-Mb region of overlap, which contains 24 known genes. Although the region has been sequenced and extensively analyzed, it is expected to contain additional genes, which have thus far escaped identification. To understand completely the molecular etiology of VCFS/DGS, der(22) syndrome, and CES, it is essential to isolate all genes in the interval. We have identified and characterized a novel human gene, located within the 1.5-Mb region deleted in VCFS/DGS, trisomic in der(22) syndrome and tetrasomic in CES. The deduced amino acid sequence of the human gene and its mouse homologue contain several WD40 repeats, but lack homology to known proteins. We termed this gene WDR14 (WD40 repeat-containing gene deleted in VCFS). It is expressed in a variety of human and mouse adult and fetal tissues with substantial expression levels in the adult thymus, an organ hypoplastic in VCFS/DGS. Copyright 2001 Academic Press.

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DUCHENNE MUSCULAR DYSTROPHY

A genomic DNA investigation was performed on a 7-year-old boy, who showed symptoms of Duchenne muscular dystrophy (DMD), congenital adrenal hypoplasia, sensorineural hearing loss, glycerol kinase deficiency, congenital hypotonia, severe psychomotor retardation, and hypothyroidism.

His creatine phosphokinase level was highly elevated, and a muscle biopsy revealed the absence of Dystrophin by histochemistry and Western blot assays; however a multiplex polymerase chain reaction (PCR) assay for DNA deletion was normal. Although his karyotype showed a perfectly normal X chromosome, a DNA investigation revealed a sizable deletion of 5.6 to 7.8 metabases at the proximal end of his short arm of X chromosome (Xp21.2-p21.3) between the marker DXS1202 at the distal end and the marker DXS1036 at the proximal end. The inclusion of the 3-prime end of the Dystrophin gene in the deletion explains the absence of Dystrophin and symptoms of DMD. The non-inclusion in the deletion of all seventeen DMD exons involved in the DMD deletion hotspots and usually tested by multiplex PCR, explained his normal multiplex result. His symptoms of sensorineural hearing loss (DFN4 gene), congenital adrenal hypoplasia (AHC gene), glycerol kinase deficiency (GK gene), mental retardation (MRX5, MRX29) can be explained by the inclusion of the respective gene in the deletion. This is known as a contiguous gene syndrome. Other symptoms such as congenital hypotonia and hypothyroidsm are not readily explainable by the deletion, but they are to be expected in association with a chromosomal deletion. The putative inclusion of RP6 gene in the deletion leads us to believe that the patient shall most likely also develop retinitis pigmentosa later in life. Int Pediatr.

2001;16(3):168-172.

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DYSKERATOSIS CONGENITA

Gene. 2002 Jun 26;293(1-2):9-19.

Basal transcription activity of the dyskeratosis congenita gene is mediated by Sp1 and Sp3 and a patient mutation in a Sp1 binding site is associated with decreased promoter activity.

Salowsky R, Heiss NS, Benner A, Wittig R, Poustka A.

Division of Molecular Genome Analysis, Deutsches Krebsforschungszentrum (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany.

The multisystem disorder dyskeratosis congenita (DKC) is caused by mutations in the DKC1 gene. The protein dyskerin is a component of the box H+ACA small nucleolar RNAs (snoRNAs) and is also functionally associated with the RNA component of the human telomerase. The majority of mutations are missense mutations, although single examples of non-coding mutations have been described. One of these is a point mutation in a putative Sp1 binding site in the 5'-upstream region of the DKC1 gene which presumably represents the promoter region of the gene. In this report, we compare the promoter sequences of both the human and mouse genes and provide a first functional characterisation of the human DKC1 promoter. This includes a characterisation of the disease-associated implications caused by the mutation identified in one patient. By reporter gene analysis, functional regions of the DKC1 promoter were delineated. The core promoter region critical for basal level of transcription was found to lie at -10 to -180. Bandshift- and supershift experiments clearly demonstrated a mutual binding of transcription factors Sp1 and Sp3 to two of five putative GC-box/Sp1-binding sites located within the core promoter region. An additional GC-box interacts only with the Sp1 transcription factor. Further, we provide evidence that the DKC1 mutation in one of the Sp1 binding sites results in reduced promoter activity.

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EPILEPSY

Hum Mol Genet. 2006 May 1;15(9):1497-512. Epub 2006 Mar 24.

A profile of alternative RNA splicing and transcript variation of CACNA1H, a human T-channel gene candidate for idiopathic generalized epilepsies.

Zhong X, Liu JR, Kyle JW, Hanck DA, Agnew WS.

Highly alternatively spliced genes may provide complex targets for disease mutations. Structural changes created by missense mutations may differentially affect the activity of alternative gene products, whereas missense, silent and non-coding mutations may alter developmental regulation of splice variant expression. CACNA1H is a human gene encoding Ca(v)3.2 low-voltage-activated, T-type calcium channels associated with bursting behavior in neurons and has been linked to more than 30 mutations apparently predisposing to childhood absence epilepsy (CAE) and other idiopathic generalized epilepsies (IGEs). Biophysical properties, including the effects of missense mutations, have been evaluated previously for a single splice form of Ca(v)3.2 expressed in transformed cell lines. We here show that CACNA1H is alternatively spliced at 12-14 sites, capable of generating both functional and non-functional transcripts. Variable cytoplasmic and extracellular protein domains point to likely differences in gating behavior, sensitivity to neuromodulation and interactions with extracellular matrix. Biophysical profiles of selected physiological Ca(v)3.2 forms reveal variations in kinetics and steady-state gating parameters, most likely to affect membrane firing. These were comparable to or larger than changes reported for previously studied mutations. Missense CAE and IGE mutations were clustered near segments associated with anomalous splicing. Missense and silent mutations were found to destroy, create or change the regulatory specificity of predicted exonic splicing enhancer sequences that may control splicing regulation. We discuss a paradigm for CACNA1H expression of Ca(v)3.2 subunits, which may influence future basic and clinical studies.

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FAMILIAL DYSAUTONOMIA

Familial dysautonomia (FD; also known as “Riley-Day syndrome”), an Ashkenazi Jewish disorder, is the best known and most frequent of a group of congenital sensory neuropathies and is characterized by widespread sensory and variable autonomic dysfunction. Previously, we had mapped the FD gene, DYS, to a 0.5-cM region on chromosome 9q31 and had shown that the ethnic bias is due to a founder effect, with >99.5% of disease alleles sharing a common ancestral haplotype. To investigate the molecular basis of FD, we sequenced the minimal candidate region and cloned and characterized its five genes. One of these, IKBKAP, harbors two mutations that can cause FD. The major haplotype mutation is located in the donor splice site of intron 20. This mutation can result in skipping of exon 20 in the mRNA of patients with FD, although they continue to express varying levels of wild-type message in a tissue-specific manner. RNA isolated from lymphoblasts of patients is primarily wild-type, whereas only the deleted message is seen in RNA isolated from brain. The mutation associated with the minor haplotype in four patients is a missense (R696P) mutation in exon 19, which is predicted to disrupt a potential phosphorylation site. Our findings indicate that almost all cases of FD are caused by an unusual splice defect that displays tissue-specific expression; and they also provide the basis for rapid carrier screening in the Ashkenazi Jewish population.

The normal donor splice sequence [of intron 20 of IKAP] is GTAAGTG; the FD sequence is GTAAGCG

ESSENTIAL HYPERTENSION

Common variations in noncoding regions of the human natriuretic peptide receptor A gene have quantitative effects.

Knowles JW, Erickson LM, Guy VK, Sigel CS, Wilder JC, Maeda N.
Department of Pathology and Laboratory Medicine, University of North Carolina, Chapel Hill, NC 27599-7525, USA.

Genetic susceptibility to common conditions, such as essential hypertension and cardiac hypertrophy, is probably determined by various combinations of small quantitative changes in the expression of many genes. NPR1, coding for natriuretic peptide receptor A (NPRA), is a potential candidate, because NPRA mediates natriuretic, diuretic, and vasorelaxing actions of the nariuretic peptides, and because genetically determined quantitative changes in the expression of this gene affect blood pressure and heart weight in a dose-dependent manner in mice. To determine whether there are common quantitative variants in human NPR1, we have sequenced the entire human NPR1 gene and identified 10 polymorphic sites in its non-coding sequence by using DNA from 34 unrelated human individuals. Five of the sites are single nucleotide polymorphisms; the remaining five are length polymorphisms, including a highly variable complex dinucleotide repeat in intron 19. There are three common haplotypes 5' to this dinucleotide repeat and three 3' to it, but the 5' haplotypes and 3' haplotypes appear to be randomly associated. Transient expression analysis in cultured cells of reporter plasmids with the proximal promoter sequences of NPR1 and its 3' untranslated regions showed that these polymorphisms have functional effects. We conclude that common NPR1 alleles can alter expression of the gene as much as two-fold and could therefore significantly affect genetic risks for essential hypertension and cardiac hypertrophy in humans.

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FRAGILE X SYNDROME, AUTISM

THE MOST COMMONLY INHERITED FORM OF MENTAL RETARDATION

Fragile X is a hereditary/genetic condition which can impact families in many ways. It includes fragile X syndrome (FXS), the most common cause of genetically-inherited mental impairment ranging from subtle learning disabilities and a normal IQ, to severe cognitive or intellectual challenges (often still referred to as mental retardation) including autism or "autistic-like" behavior. Symptoms often include unique physical characteristics, behavioral deficits and delays in speech and language development.

Several disorders in humans are caused by the inheritance of genes that have undergone insertions of a stretch of identical codons repeated over and over. A locus on the human X chromosome contains such a stretch of nucleotides in which the triplet CGG is repeated (CGGCGGCGGCGG, etc.). The number of CGGs may be as few as 5 or as many as 50 without causing a harmful phenotype (these repeated nucleotides are in a noncoding region of the gene). Even 100 repeats usually cause no harm. However, these longer repeats have a tendency to grow longer still from one generation to the next (to as many as 4000 repeats).

Examples: Fragile X syndrome - (CCG)n in the 5' untranslated region of the FMR gene on the X chromosome. Fragile X is the commonest type of inherited mental retardation in boys. The effect of an expansion of the CCG repeat is to stop the gene being transcribed.

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FRIEDREICH CEREBELLAR ATAXIA

New Type of Trinucleotide Mutation Found in Friedreich's Ataxia

Overview Scientists have identified a new type of trinucleotide repeat mutation that leads to Friedreich's ataxia (FA), a rare childhood neurodegenerative disease. The discovery allows accurate screening for carriers of the disease and may lead to the first effective treatments.

Scientists have identified a new type of trinucleotide repeat mutation that leads to Friedreich's ataxia (FA), a rare childhood neurodegenerative disease. The discovery allows accurate screening for carriers of the disease and may lead to the first effective treatments.

The mutation contains between 200 and 900 repeats of a normal three-base (trinucleotide) sequence -- guanine, adenine, and adenine, or GAA -- in a newly described gene called X25. The normal gene contains only seven to 22 repeats. Mutated X25 genes produce much less messenger RNA, an intermediate step in protein production, than normal genes.

X25 normally codes for a protein called frataxin that is concentrated in the organs affected by FA, says Dr. Massimo Pandolfo of Baylor College of Medicine in Houston. Frataxin is unlike any previously described protein, and scientists are just beginning to learn how it works. Pandolfo conducted the study in collaboration with scientists from France, Spain, and Italy. The work was funded by the National Institute of Neurological Disorders and Stroke (NINDS) and by the Muscular Dystrophy Association. It appears in the March 8, 1996, issue of Science.*

About 98 percent of FA carriers have the expanded GAA sequence, so scientists can now diagnose carriers very accurately, Pandolfo says. Studying how X25 and frataxin function also will help researchers understand the disease and design treatments for it, he says.

FA affects several thousand people in the United States and causes degeneration of the spinal cord and its brain connections, the heart, and the pancreas. Symptoms typically appear between the ages of five and 25 and include progressive loss of coordination (ataxia), lack of leg tendon reflexes, and speech slurring. Most patients also develop heart enlargement, and about 10 percent acquire diabetes mellitus. At present there is no treatment to slow progression of the disease, and patients eventually must rely on a wheelchair. They also have a shortened life expectancy, averaging 37 years.

Unlike mutations in other trinucleotide expansion diseases, including Huntington's disease, the FA mutation is genetically recessive. This means a child must inherit two mutated copies of the gene -- one from each parent -- to develop the disorder. Preliminary results suggest that, in contrast to other trinucleotide expansions, the number of repeats in the FA mutation does not correlate with disease severity or age of onset. People carrying only one altered copy of the gene do not have FA but can pass the mutation to their children. Previously described trinucleotide repeat mutations are dominant, meaning only one altered copy of the gene leads to disease.

Since carriers of FA are themselves unaffected by the mutation, they are usually unaware of it and tend to have the same number of children as other people. This accounts for the relatively high frequency of carriers in the population -- about one in 100 people, according to the National Ataxia Foundation.

The FA trinucleotide expansion is the first disease-causing mutation found to occur in an intron. Introns code for sequences that are cut out of messenger RNA before proteins are assembled. Scientists have long wondered what function introns serve. The FA mutation supports previous evidence that they influence gene expression.

Along with the trinucleotide expansion, Pandolfo and his colleagues identified three single-nucleotide "point" mutations that inactivate the X25 gene and lead to disease. These mutations affected only eight of the 184 FA patients examined. These patients all had the expanded GAA repeat on the X25 gene from their other parent. "Patients with FA either have both genes with the unstable expansion, or they have one gene with the unstable expansion and the other with a point mutation," says Dr. Giovanna Spinella, a pediatric neurologist at NINDS.

While frataxin's function is still unclear, it includes a 27 amino acid sequence that is nearly identical to sequences from a worm species (C. elegans) and yeast (S. cerevisiae), Pandolfo says. The similarity of this sequence in such diverse species, which are separated by millions of years of evolution, suggests that it plays a critical role in organisms' survival. The rare "point" mutations all affect this region of the protein, underscoring its importance.

Studying how frataxin works in other species may help scientists learn what it does in humans and design treatments for FA, Pandolfo says. He and his colleagues are now working to further define frataxin's function. This work may reveal new protein interactions that are important to all cells, not just those affected by FA.

FA is the eighth disease linked to trinucleotide repeat mutations. The others are Huntington's disease, spinocerebellar ataxia type 1 (SCA1), X-linked spinobulbar muscular atrophy (Kennedy's disease), dentatorubral and pallidoluysian atrophy (DRPLA), spinocerebellar ataxia type 3 (SCA3 or Machado-Joseph disease), fragile X syndrome, and myotonic dystrophy. FA is the first disease linked to an expanded GAA sequence.

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HIRSCHSPRUNG DISEASE

Nature. 2005 Apr 14;434(7035):857-63.

A common sex-dependent mutation in a RET enhancer underlies Hirschsprung disease risk.

Emison ES, McCallion AS, Kashuk CS, Bush RT, Grice E, Lin S, Portnoy ME, Cutler DJ, Green ED, Chakravarti A.
McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.

The identification of common variants that contribute to the genesis of human inherited disorders remains a significant challenge. Hirschsprung disease (HSCR) is a multifactorial, non-mendelian disorder in which rare high-penetrance coding sequence mutations in the receptor tyrosine kinase RET contribute to risk in combination with mutations at other genes. We have used family-based association studies to identify a disease interval, and integrated this with comparative and functional genomic analysis to prioritize conserved and functional elements within which mutations can be sought. We now show that a common non-coding RET variant within a conserved enhancer-like sequence in intron 1 is significantly associated with HSCR susceptibility and makes a 20-fold greater contribution to risk than rare alleles do. This mutation reduces in vitro enhancer activity markedly, has low penetrance, has different genetic effects in males and females, and explains several features of the complex inheritance pattern of HSCR. Thus, common low-penetrance variants, identified by association studies, can underlie both common and rare diseases.

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Hum Mol Genet. 2005 Dec 15;14(24):3837-45. Epub 2005 Nov 3.

Evaluation of the RET regulatory landscape reveals the biological relevance of a HSCR-implicated enhancer.

Grice EA, Rochelle ES, Green ED, Chakravarti A, McCallion AS. McKusick-Nathans Institute of Genetic Medicine, Department of Comparative Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.

Evolutionary sequence conservation is now a relatively common approach for the prediction of functional DNA sequences. However, the fraction of conserved non-coding sequences with regulatory potential is still unknown. In this study, we focus on elucidating the regulatory landscape of RET, a crucial developmental gene within which we have recently identified a regulatory Hirschsprung disease (HSCR) susceptibility variant. We report a systematic examination of conserved non-coding sequences (n=45) identified in a 220 kb interval encompassing RET. We demonstrate that most of these conserved elements are capable of enhancer or suppressor activity in vitro, and the majority of the elements exert cell type-dependent control. We show that discrete sequences within regulatory elements can bind nuclear protein in a cell type-dependent manner that is consistent with their identified in vitro regulatory control. Finally, we focused our attention on the enhancer implicated in HSCR to demonstrate that this element drives reporter expression in cell populations of the excretory system and central nervous system (CNS) and peripheral nervous system (PNS), consistent with expression of the endogenous RET protein. Importantly, this sequence also modulates expression in the enteric nervous system consistent with its proposed role in HSCR.

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Int J Mol Med. 2002 Oct;10(4):367-70.

Hirschsprung, RET-SOX and beyond: the challenge of examining non-mendelian traits (Review).

Pusch CM, Sasiadek MM, Blin N. Division of Molecular Genetics, University of Tubingen, 72074 Tubingen, Germany.

Hirschsprung disease (HSCR), or congenital intestinal aganglionosis, is a common hereditary disorder causing intestinal obstruction, thereby showing considerable phenotypic variation in conjunction with complex inheritance. Moreover, phenotypic assessment of the disease has been complicated since a subset of the observed mutations is also associated with several additional syndromic anomalies. Coding sequence mutations in e.g. RET, GDNF, EDNRB, EDN3, and SOX10 lead to long-segment (L-HSCR) as well as syndromic HSCR but fail to explain the transmission of the much more common short-segment form (S-HSCR). Furthermore, mutations in the RET gene are responsible for approximately half of the familial and some sporadic cases, strongly suggesting, on the one hand, the importance of non-coding variations and, on the other hand, that additional genes involved in the development of the enteric nervous system still await their discovery. For almost all of the identified HSCR genes incomplete penetrance of the HSCR phenotype has been reported, probably due to modifier loci. Therefore, HSCR has become a model for a complex oligo-/polygenic disorder in which the relationship between different genes creating a non-mendelian inheritance pattern still remains to be elucidated.

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HYPERCHOLESTEROLEMIA, FAMILIAL

Atherosclerosis. 2005 Oct;182(2):331-40.

Genetic screening protocol for familial hypercholesterolemia which includes splicing defects gives an improved mutation detection rate.

Graham CA, McIlhatton BP, Kirk CW, Beattie ED, Lyttle K, Hart P, Neely RD, Young IS, Nicholls DP. Regional Genetics Centre, Belfast City Hospital, Belfast BT9 7AB, Northern Ireland, UK. colin.graham@bll.n-i.nhs.uk

Familial hypercholesterolemia (FH) is a common single gene disorder, which predisposes to coronary artery disease. In a previous study, we have shown that in patients with definite FH around 20% had no identifiable gene defect after screening the entire exon coding area of the low density lipoprotein receptor (LDLR) and testing for the common Apolipoprotein B (ApoB) R3500Q mutation. In this study, we have extended the screen to additional families and have included the non-coding intron splice regions of the gene. In families with definite FH (tendon xanthoma present, n=68) the improved genetic screening protocol increased the detection rate of mutations to 87%. This high detection rate greatly enhances the potential value of this test as part of a clinical screening program for FH. In contrast, the use of a limited screen in patients with possible FH (n=130) resulted in a detection rate of 26%, but this is still of significant benefit in diagnosis of this genetic condition. We have also shown that 14% of LDLR defects are due to splice site mutations and that the most frequent splice mutation in our series (c.1845+11 c>g) is expressed at the RNA level. In addition, DNA samples from the patients in whom no LDLR or ApoB gene mutations were found, were sequenced for the NARC-1 gene. No mutations were identified which suggests that the role of NARC-1 in causing FH is minor. In a small proportion of families (<10%) the genetic cause of the high cholesterol remains unknown, and other genes are still to be identified that could cause the clinical phenotype FH.

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HYPER-FERRITINEMIA-CATARACT SYNDROME, FAMILIAL

Rev Esp Enferm Dig. 2004 Jul;96(7):507-9, 510-1.

Hereditary hyperferritinemia-cataract syndrome. Study of a new family in Spain.

Ladero JM, Balas A, Garcia-Sanchez F, Vicario JL, Diaz-Rubio M. Department of Gastroenterology, Hospital Clinico San Carlos, Universidad Complutense, Madrid, Spain. jladero.hcsc@salud.madrid.org

The hyperferritinemia-cataract syndrome, inherited as a Mendelian dominant trait, is due to mutations in the 5' non-coding region of the ferritin light chain gene that modifies the shape of the IRE (iron responsive element) region, which loses its normal function of regulating the synthesis of ferritin light chains. Excess of light chains results in complexes that accumulate into the lens giving rise to early cataracts. We present a Spanish family with seven affected members through three generations. A genetic study reveals a substitution of a single base (C-->T) at position 33 in the IRE sequence in the index case and in one affected brother, whereas a non-affected sister shows the normal sequence. The hyperferritinemia-cataract syndrome was identified in 1995 and is still poorly understood. Clinicians should suspect it when treating any subject with early cataracts, even more if they are familial, or in patients with very high levels of ferritinemia without evidence of iron overload. There are no known consequences of the syndrome other than cataracts, and its proper diagnosis carries a favorable prognosis and eliminates the risk of unnecessary phlebotomies.

Full text

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HYPER-IgM SYNDROME TYPE 1

Biochim Biophys Acta. 2006 Mar;1762(3):335-40. Epub 2005 Oct 28.

Female hyper IgM syndrome type 1 with a chromosomal translocation disrupting CD40LG.

Imai K, Shimadzu M, Kubota T, Morio T, Matsunaga T, Park YD, Yoshioka A, Nonoyama S. Department of Pediatrics, National Defense Medical College, 359-8513, 3-2, Namiki, Tokorozawa, Saitama, Japan.

Hyper-IgM syndrome type 1 (HIGM1) is a primary immunodeficiency characterized by recurrent bacterial and opportunistic infections, associated with normal or high serum level of IgM and decreased serum levels of IgG, IgA and IgE due to the defect of class switch recombination. CD40LG, located in Xq26, has been reported to be mutated in male HIGM1 patients. Here, we report the second case of a female HIGM1 with the defect of CD40 ligand (CD40L) expression and of soluble serum CD40L. Clinical course and HIGM phenotype was indistinguishable from that of male HIGM1 including severe neutropenia. High-resolution chromosome banding revealed that this patient's karyotype is 46, X, t(X;14)(q26.3;q13.1), and FISH analysis demonstrated that the break point of the chromosomal translocation is within CD40LG. Using four chimeric cDNA clones obtained by 3' RACE method, the break point was identified within the intron 4 of CD40LG on X chromosome and non-coding region of chromosome 14. We also found an extremely skewed X-chromosome inactivation pattern by methylation-specific PCR. Thus, the reciprocal translocation caused the disruption of CD40LG, resulting in defective CD40L expression in the female patient with an extremely skewed X-inactivation pattern in T cells leading to the HIGM1 phenotype.

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LI-FRAUMENI SYNDROME

Cancer Genet Cytogenet. 2001 Aug;129(1):85-7.

Significance of intron 6 sequence variations in the TP53 gene in Li-Fraumeni syndrome.

Varley JM, McGown G, Thorncroft M, Kelsey AM, Birch JM. CRC Cancer Genetics Group, Paterson Institute for Cancer Research, Wilmslow Road, M20 4BX, Manchester, UK. jvarley@picr.man.ac.uk

Many polymorphisms have been reported in the TP53 gene. Some of these are within the coding region, and may affect the function of the p53 protein, others are within introns or non-coding regions, and their significance is unclear. Recently, a number of publications have claimed that polymorphisms within intron 6 are responsible for inherited predisposition to childhood malignancies, familial breast cancer, and Li-Fraumeni syndrome (LFS). We find no evidence for intron 6 sequence variants predisposing to LFS in our cohort of families and, furthermore, we show that some of the conclusions of other groups cannot be supported by data from our analysis.

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LUPUS ERYTHEMATOSUS

Autoimmunogenic HLA-DRB1*0301 allele (DR3) may be distinguished at the DRB1 non-coding regions of HLA-B8,DR3,Dw24 and B18,DR3,Dw25 haplotypes.

Segurado OG, Iglesias-Casarrubios P, Martinez-Laso J, Corell A, Martin-Villa JM, Arnaiz-Villena A.
Department of Immunology, Hospital 12 de Octubre, Madrid, Spain.

A novel TaqI restriction fragment length polymorphism (RFLP) of 4.15 kb is reported using a DR beta probe (pRTV1). This fragment corresponds to the DRB1 locus and allows the subdivision at the DNA level of the DRB1*0301 allele (DR3 antigen), which had not previously been reported. Both splits also distinguish each of the two DR3-bearing extended haplotypes (HLA-B8,SCO1,DR3,DQw2,Dw24 and B18,F1C30,DR3,DQw2,Dw25) found associated to several autoimmune diseases as insulin-dependent diabetes mellitus (IDDM), systemic lupus erythematosus (SLE) and myasthenia gravis. The fact that no polymorphism in the DRB1*0301 coding DNA sequence has been detected indicates that DRB1*0301 intronic, regulatory of neighbouring sequences might also contribute to differential disease associations (and pathogenic mechanisms) found linked to each of the two DR3-bearing haplotypes, i.e. IDDM and B8,DR3,Dw24 in North European/American Caucasoids vs IDDM and B18,DR3,Dw25 in Mediterraneans; SLE and B8,DR3,Dw24 in children vs SLE and B18,DR3,Dw25 in Spanish adults.

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LYMPHOMA, B-CELL

J Pathol. 2005 Oct;207(2):243-9.

BIC and miR-155 are highly expressed in Hodgkin, primary mediastinal and diffuse large B cell lymphomas.

Kluiver J, Poppema S, de Jong D, Blokzijl T, Harms G, Jacobs S, Kroesen BJ, van den Berg A.
Department of Pathology and Laboratory Medicine, University Medical Centre Groningen, University of Groningen, Groningen, The Netherlands.

In a previous study we demonstrated high expression of the non-coding BIC gene in the vast majority of Hodgkin's lymphomas (HLs). Evidence suggesting that BIC is a primary microRNA transcript containing the mature microRNA-155 (miR-155) as part of a RNA hairpin is now accumulating. We therefore analysed HL cell lines and tissue samples to determine whether miR-155 is also expressed in HL. High levels of miR-155 could be demonstrated, indicating that BIC is processed into a microRNA in HL. Most non-HL subtypes were negative for BIC as determined by RNA-ISH. However, in diffuse large B cell lymphoma (DLBCL) and primary mediastinal B cell lymphoma (PMBL), significant percentages of positive tumour cells were observed in 12/18 and 8/8 cases. A higher proportion of tumour cells were positive for BIC in DLBCL with activated B cell-like phenotype than in DLBCL with germinal centre B cell-like phenotype. Differential BIC expression was confirmed by qRT-PCR analysis. Northern blot analysis showed expression of miR-155 in all DLBCL and PMBL derived cell lines and tissue samples analysed. In summary, we demonstrate expression of primary microRNA BIC and its derivative miR-155 in HL, PMBL and DLBCL.

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MALARIA

The role of host genetic variation in determining susceptibility to complex disease traits is the subject of much research effort, but it often remains unclear whether disease-associated genetic polymorphisms are themselves functionally relevant or acting only as markers within an extended haplotype. Experimental approaches to investigate the functional impact of polymorphisms in non-coding regulatory DNA sequences for gene expression are discussed, including the role of gel-shift assays, DNA footprinting and reporter gene analysis. The limitations of different experimental approaches are presented together with future prospects for in vivo analysis. The strategic application of these functional approaches is discussed and illustrated by analysis of the role of genetic variation in the tumour necrosis factor promoter region in determining susceptibility to severe malaria.

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MEASLES

The sequence of the 300 nucleotides region of the measles virus genome was determined that includes a part of the 3'-untranslated region of the matrix (M) gene, the intergenic region and a part of the 5'-untranslated region of the fusion (F) gene [M-F region] for vaccine strain Leningrad-16 and 14 wild-type isolates. The data obtained demonstrate the variability of this long non-coding M-F region.

MELANOMA

J Med Genet. 2006 Jan;43(1):39-47. Epub 2005 Jun 3.

Comprehensive analysis of CDKN2A (p16INK4A/p14ARF) and CDKN2B genes in 53 melanoma index cases considered to be at heightened risk of melanoma.

Laud K, Marian C, Avril MF, Barrois M, Chompret A, Goldstein AM, Tucker MA, Clark PA, Peters G, Chaudru V, Demenais F, Spatz A, Smith MW, Lenoir GM, Bressac-de Paillerets B; French Hereditary Melanoma Study Group.

Service de Genetique, Institut Gustave Roussy, 94800 Villejuif, France.

Comprehensive analysis of the 9p21 locus including the CDKN2A, ARF, and CDKN2B genes in 53 individuals from melanoma index cases considered to be at heightened risk of melanoma. Using a combination of DNA sequencing, gene copy number by real time quantitative PCR, linkage analysis, and transcript analysis in haploid somatic cell hybrids, we found no evidence for germline alteration in either coding or non-coding domains of CDKN2A and CDKN2B. However, we identified a p14ARF exon 1beta missense germline mutation (G16D) in a melanoma-neural system tumour syndrome (CMM+NST) family and a 8474 bp germline deletion from 196 bp upstream of p14ARF exon 1beta initiation codon to 11233 bp upstream of exon 1alpha of p16(INK4A) in a family with five melanoma cases. For three out of 10 families with at least three melanoma cases, the disease gene was unlinked to the 9p21 region, while linkage analysis was not fully conclusive for seven families. CONCLUSIONS: These data reinforce the hypothesis that ARF is a melanoma susceptibility gene and suggest that germline deletions specifically affecting p14ARF may not be solely responsible for NST susceptibility. Predisposition to CMM+NST could either be due to complete disruption of the CDKN2A locus or be the result of more complex genetic inheritance. In addition, the absence of any genetic alteration in 50 melanoma prone families or patients suggests the presence of additional tumour suppressor genes possibly in the 9p21 region, and on other chromosomes.

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MENTAL RETARDATION

Respir Res. 2006 Feb 6;7(1):21
Epigenetics and airways disease.

Adcock IM, Ford P, Barnes PJ, Ito K.

Epigenetics is the term used to describe heritable changes in gene expression that are not coded in the DNA sequence itself but by post-translational modifications in DNA and histone proteins. These modifications include

• histone acetylation,
• methylation,
• ubiquitination,
• sumoylation and
• phosphorylation.

Epigenetic regulation is not only critical for generating diversity of cell types during mammalian development, but it is also important for maintaining the stability and integrity of the expression profiles of different cell types. Until recently, the study of human disease has focused on genetic mechanisms rather than on non-coding events. However, it is becoming increasingly clear that disruption of epigenetic processes can lead to several major pathologies, including cancer, syndromes involving chromosomal instabilities, and mental retardation. Furthermore, the expression and activity of enzymes that regulate these epigenetic modifications have been reported to be abnormal in the airways of patients with respiratory disease. The development of new diagnostic tools might reveal other diseases that are caused by epigenetic alterations. These changes, despite being heritable and stably maintained, are also potentially reversible and there is scope for the development of 'epigenetic therapies' for disease.

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Neurosci Lett. 2005 Apr 29;379(1):13-6. Epub 2005 Jan 12.

The A140V mutation in the MECP2 gene is not a common etiological factor among Brazilian mentally retarded males.

dos Santos JM, Abdalla CB, Campos M Jr, Santos-Reboucas CB, Pimentel MM.

Departamento de Biologia Celular e Genetica, Instituto de Biologia Roberto Alcantara Gomes, Universidade do Estado do Rio de Janeiro, Rua Sao Francisco Xavier, 524 PHLC, sala 218, Rio de Janeiro RJ 20550-013, Brazil.

In mammals, methyl-CpG binding proteins play a significant role in the control of gene expression through their association with chromatin-remodeling complexes. Mutations in the gene coding for methyl-CpG-binding protein 2 (MECP2) cause Rett syndrome and have also been reported in a number of X-linked mental retardation diseases. In this study, DNA samples from 363 male individuals with syndromic and non-syndromic mental retardation and other psychiatric diseases were screened for A140V (419C>T) mutation in the MECP2 gene, considered the most frequent MECP2 mutation in males. No 419C>T was found suggesting that the A140V mutation in the MECP2 gene is not a common cause of mental retardation in males. Recently, a new and abundant isoform of MECP2 was described, which has an alternative N-terminus, translated from exon 1, that was previously thought to be non-coding and has been excluded from many mutational screening, as well, the 5' and 3' UTR regions. We consider essential proceeding further screening in the whole extension of the MECP2 gene using clinically well-documented and larger sized sample to assure the overall contribution of MECP2 to mental retardation.

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MULTIPLE SCLEROSIS

Comparative analysis of the mouse and human peptidylarginine deiminase gene clusters reveals highly conserved non-coding segments and a new human gene, PADI6.

Chavanas S, Mechin MC, Takahara H, Kawada A, Nachat R, Serre G, Simon M.

UMR 5165 CNRS-UPS, Epidermis differentiation and rheumatoid autoimmunity, Institut Federatif de Recherche 30, Faculte de Medecine, (INSERM, CNRS, CHU Toulouse-Purpan, Universite Paul Sabatier), 37 allees Jules Guesde, 31073 Toulouse cedex 7, France.

Peptidylarginine deiminases (PADs) convert arginine residues in proteins into citrullines. They are suspected to be involved in multiple sclerosis and rheumatoid arthritis pathophysiology, and they play a role in epidermis homeostasis and possibly in regulation of gene expression through histone modification. In humans, four isoforms encoded by the genes PADI1-4 are known so far. We here report the characterization and comparative analysis of the human (355 kb) and mouse (240 kb) PAD gene clusters on chromosomes 1p35-36 and 4E1, respectively. We characterized an as yet unknown human PADI6 gene, and cloned the corresponding cDNA encoding a 694-amino-acid protein. RT-PCR analysis showed a rather restricted pattern of tissue-specific expression, mainly in ovary, testis and peripheral blood leukocytes. Nucleotide substitution rates suggest that PADI genes are under purifying selection. Comparative analysis of the human and mouse sequences identified 251 conserved non-coding segments predominantly clustered within the promoter regions, the large (>10 kb) first intron of each of the genes PADI1-3, and an 8 kb PADI1-2 intergenic region. The presence of numerous transcription factor binding sites suggests the segments are putative regulatory elements. This study is the first description of the human PADI6 gene and encoded protein, and the first step towards a better understanding of the coordinated regulation of PADI gene expression.

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MYOCARDIAL INFARCTION

DIABETES AND HEART DISEASE

Gene hunters at Johns Hopkins have discovered a common genetic mutation that increases the risk of inheriting a particular birth defect not by the usual route of disrupting the gene’s protein-making instructions, but by altering a regulatory ["junkDNA" - AJP] region of the gene. Although the condition, called Hirschsprung disease, is rare, its complex genetics mimics that of more common diseases, such as diabetes and heart disease.

"It’s a funny mutation in a funny place," says study leader Aravinda Chakravarti, Ph.D., director of the McKusick-Nathans Institute of Genetic Medicine. "But I think the majority of mutations found in major diseases are going to be funny mutations in funny places."

Far from being a problem, the finding is good news, he suggests. "Mutations in the protein-coding sequence can’t really be fixed, but those outside the protein-coding regions -- perhaps we can fiddle with them, perhaps they are ’tunable.’ The protein should be fine if we can just get the cells to make the right amount," he says.

"Our finding really underscores the fact that health and disease can be affected by all regions of a gene," he continues. "For diseases like diabetes and heart disease, just as for Hirschsprung disease, multiple inherited factors contribute to the disease, and these factors are not just going to be in protein-coding regions."

The researchers’ discovery, described in the April 14 issue of Nature, adds to growing evidence that problems with the amount of protein made from a gene’s instructions are likely to be just as important as - and perhaps more important than -- changes in the proteins themselves, they say.

"But finding important mutations outside of protein-coding sequences is a challenge because of the amount of genetic material to sort through," notes postdoctoral fellow Eileen Emison, Ph.D., the study’s first author. "Only 1.5 percent of the roughly 3 billion building blocks in our genetic material carry instructions for proteins."

Fortunately, about twice that much has stood the tests of time and evolution and remains the same, or very similar, among various species, indicating the regions’ biologic importance. By comparing the genetic sequences of humans and other species to find these regions, and then combining those results with traditional genetic studies of disease in families, the hunt for disease-related mutations in so-called non-coding sequences can be successful, the researchers show.

In fact, the researchers used this combined approach to discover the risk-increasing mutation in the RET gene in individuals with Hirschsprung disease. In this birth defect, the effects of multiple genetic mutations -- many still unknown -- combine to prevent proper development of the nerves that control intestinal function. Only 30 percent of Hirschsprung cases have been tied to a specific protein-changing mutation, even though protein-encoding regions of eight genes already are known to be involved in the disease.

The new risk-confirming mutation confirms Chakravarti’s long-held suspicion that some of Hirschsprung’s unknowns might be due to mutations in non-coding regions, which usually are not included in the hunt for disease-related mutations. A gene’s non-coding regions -- which don’t have to be adjacent to or even near a gene’s protein-coding sequences -- contain the gene’s on-switch (the promoter), areas that tweak whether, when and how the gene is used to make proteins (enhancers or suppressors) and other expanses that still just seem to be filler. The new mutation is in a gene called RET, whose protein-coding sequence had already been tied to the disease.

To hunt for Hirschsprung-related mutations in the largely uncharted non-coding regions, Chakravarti and his team first determined the identities of 28 specific genetic building blocks, or markers, in a large region surrounding the RET gene in samples from 126 people with Hirschsprung disease and their parents. (Earlier work had tied the disease in these families to a large region that includes RET, but no protein-changing mutations had been found in affected individuals.)

The genetic markers’ identities act as a sort of signature the researchers can track. Computer analysis identified three large regions of DNA, one including the RET gene, that were passed from parents to affected children (but not unaffected children) more often than one would expect by chance alone. One particular eight-marker signature around RET was most tightly associated with the disease, the researchers found.

Rather than sequencing the entire region in all the families, the researchers turned to comparative genomics to focus the search. Colleague Eric Green, Ph.D., and others at the National Institutes of Health determined the genetic sequences of a large region surrounding the equivalent of RET in 12 nonhuman vertebrates, including the chimpanzee, cow, mouse, dog, chicken and blowfish, for comparison to the human sequence (determined by the Human Genome Project).

"We found 84 areas within the region that were highly conserved, almost half of which were protein-coding areas of the RET gene and two other genes," says Emison. "That left us with 47 areas that didn’t carry instructions for proteins, but that were likely to be both biologically important and involved in the disease."

By overlapping the disease-linked regions and the smaller, highly conserved genetic snippets, the researchers uncovered five short areas within the overall RET gene on which to focus. Sequencing these five areas in patients revealed the culprit -- a genetic sequence that was identical in all mammals studied and in all unaffected individuals. In those with Hirschsprung, however, the sequence contained a single change.

In laboratory studies, Andrew McCallion, Ph.D., an assistant professor in Hopkins’ Institute of Genetic Medicine, and graduate student Elizabeth Grice determined that this region of RET normally enhances the gene’s activity. The mutation diminished that effect.

"Not everyone who has the mutation has the disease, but our analysis shows that the mutation clearly contributes to the risk of disease," says Emison. "Interestingly, the frequency of the mutation in different world populations mirrors that of the disease."

The frequency of the mutation, ranging from almost absent in Africa to 50 percent in Asia, is much higher than the incidence of the disease, which affects roughly 1 in 5,000 births, on average. The mutation is almost twice as common in Asians as in Europeans, and a study in the 1980s showed that Asian Americans in California were twice as likely to have a child with Hirschsprung disease than mothers of European descent. The mutation’s distribution also mirrors the greater incidence of the disease in boys, the researchers report.

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MYOTONIC DYSTROPHY: THE MOST COMMON OF MUSCULAR DYSTROPHIES

We know that the standard Myotonic Dystrophy is among the most common of Muscular Dystrophies with an incident of 1 in 7000 or 1 in 8000 (from MDA USA). In the Saquenay region of Quebec, myotonic dystrophy incident is 1 in 500. It is the most frequent diagnosis of the MDA of Canada.

Growing genes cause neurological diseases

Johansson J, Holmgren G, Forsgren L, Holmberg M.
Klinisk genetik/institutionen for cell- och molekylarbiologi, Umea Universitet.

A growing number of hereditary neurodegenerative disorders have been found to be caused by expansion of trinucleotide repeats. A smaller number of diseases such as fragile X syndrome, myotonic dystrophy, and Friedreich's ataxia, have been found to be due to expansions in non-coding DNA. In a large group of diseases, the expansion consists of CAG repeats in the coding region of the gene, producing an expanded polyglutamine sequence in the protein. Nine diseases have so far been identified as belonging to this group: Huntington's disease, spinobulbar muscular atrophy (SBMA), dentatorubral pallidoluysian atrophy (DRPLA), autosomal dominant "pure" spastic paraplegia (ADPSP), and five forms of spinocerebellar ataxia (SCA 1,2,3,6 and 7). Except for SBMA, all of the CAG repeat disorders are characterised by autosomal dominant heredity and anticipation (i.e., earlier onset age and increasing severity in successive generations). The mutated protein causes disease via an as yet unidentified gain-of-function mechanism in specific subsets of neurones. Today, DNA analysis permits the diagnosis of a trinucleotide disease in individual cases.

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J Cell Sci. 2005 Jul 1;118(Pt 13):2923-33. Epub 2005 Jun 16.

Colocalization of muscleblind with RNA foci is separable from mis-regulation of alternative splicing in myotonic dystrophy.

Ho TH, Savkur RS, Poulos MG, Mancini MA, Swanson MS, Cooper TA.

Department of Molecular and Cellular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA.

Myotonic dystrophy type I (DM1), which is caused by a non-coding CTG-repeat expansion in the dystrophia myotonica-protein kinase (DMPK) gene, is an RNA-mediated disease. Expanded CUG repeats in transcripts of mutant DMPK form nuclear foci that recruit muscleblind-like (MBNL) proteins, a family of alternative splicing factors. Although transcripts of mutant DMPK and MBNL proteins accumulate in nuclear RNA foci, it is not clear whether foci formation is required for splicing mis-regulation. Here, we use a co-transfection strategy to show that both CUG and CAG repeats form RNA foci that colocalize with green fluorescent protein (GFP)-MBNL1 and endogenous MBNL1. However, only CUG repeats alter splicing of the two tested pre-mRNAs, cardiac troponin T (cTNT) and insulin receptor (IR). Using FRAP, we demonstrate that GFP-MBNL1 in CUG and CAG foci have similar half-times of recovery and fractions of immobile molecules, suggesting that GFP-MBNL1 is bound by both CUG and CAG repeats. We also find an immobile fraction of GFP-MBNL1 in DM1 fibroblasts and a similar rapid exchange in endogenous CUG RNA foci. Therefore, formation of RNA foci and disruption of MBNL1-regulated splicing are separable events.

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Hum Mol Genet. 2005 Jun 1;14(11):1539-47. Epub 2005 Apr 20.

Transgenic mice expressing CUG-BP1 reproduce splicing mis-regulation observed in myotonic dystrophy.

Ho TH, Bundman D, Armstrong DL, Cooper TA. Department of Pathology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA.

Myotonic dystrophy type I (DM1) is an RNA-mediated disease caused by a non-coding CTG repeat expansion. A key feature of the RNA-mediated pathogenesis model for DM is the disrupted splicing of specific pre-mRNA targets. A link has been established between splicing regulation by CUG-BP1, a member of the CELF family of proteins, and DM1 pathogenesis. To determine whether increased CUG-BP1 function was sufficient to model DM, transgenic mice overexpressing CUG-BP1 (MCKCUG-BP1) in heart and skeletal muscle, two tissues affected in DM1, were generated. Histological and electron microscopic analyses of skeletal muscle reveal common pathological features with DM tissues: chains of central nuclei, degenerating fibers and centralized NADH reactivity. MCKCUG-BP1 mice have disrupted splicing of three CELF target pre-mRNAs, cardiac troponin T (Tnnt2), myotubularin-related 1 gene (Mtmr1) and the muscle-specific chloride channel (Clcn1), consistent with that observed in DM heart and skeletal muscle. The results are consistent with a mechanism for DM pathogenesis in which expanded repeats result in increased CUG-BP1 activity and/or other CELF family members and have trans-dominant effects on specific pre-mRNA targets.


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NEUROFIBROMATOSIS

Mouse neurofibromatosis type 1 cDNA sequence reveals high degree of conservation of both coding and non-coding mRNA segments.

Bernards A, Snijders AJ, Hannigan GE, Murthy AE, Gusella JF.

Molecular Neurogenetics Unit, Massachusetts General Hospital, Boston 02129.

To identify evolutionary conserved domains and facilitate the recognition of potentially significant mutations in NF1 patients or tumors, we have determined the complete approximately 12 kb sequence of mouse neurofibromatosis type 1 mRNA. The sequence predicts a 2841 amino acid protein that is more than 98% identical to human neurofibromin. All but 9 of the 45 amino acid differences between mouse and human neurofibromin occur in the N-terminal half of the protein, with 16 changes clustered just upstream of the IRA-related segment. Given the high degree of sequence identity, virtually any sequence alteration in NF1 patients or tumors is potentially significant. We have also found that the 3' untranslated segment of NF1 mRNA is highly conserved, suggesting that this region may also be a target for mutations in NF1 patients.

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PARKINSONS' DISEASE

RNA knockdown as a potential therapeutic strategy in Parkinson's disease

F P Manfredsson, A S Lewin and R J Mandel

Abstract

Parkinson's disease is a prevalent progressive degenerative disorder of the elderly. There is a current need for novel therapeutic strategies because the standard levodopa pharmacotherapy is only temporarily efficacious. Recently, there have been some high-profile successful preclinical results obtained in animal models of neurological disorders using small interfering RNAs delivered by viral vectors. RNA interference can theoretically be applied to Parkinson's disease since over-expression of various proteins is known to kill the dopamine neurons of the substantia nigra in animal models and in familial forms of Parkinson's disease. Potential RNA interfering strategies and caveats are discussed in this review.

Keywords: adeno-associated virus, ribozyme, interfering RNA

Introduction

Parkinson's disease (PD) is one of the most common neurological disorders, second only to Alzheimer's disease, affecting approximately 2% of adults. Although the cause of the idiopathic form of the disease is yet to be established, recent advances in the study of certain familial forms of the disease have shed some light on the overall process of neurodegeneration in PD. Still, much remains to be understood about the disease. The recent development of new techniques such as the use of short RNA molecules like small interfering RNAs (siRNA) have gained increasing popularity as tools to knockdown certain cellular mRNAs, and these tools have been valuable in elucidating molecular pathways and players involved in PD. Still, the ultimate goal is to treat the disease, and the current understanding of the potential etiology of PD makes it an attractive target for a siRNA therapy. One common feature amongst the various forms of the disease is protein accumulation and aberrant protein clearance. Hence, a reduction in translation of specific proteins may suffice to relieve the cellular stresses. In addition, a number of cell death signaling events likely to be involved in all forms of PD are also potential siRNA targets.

RNA interference

RNAi is a response to double-stranded RNA that has been identified in numerous organisms in all three eukaryotic kingdoms. The major enzymes involved are highly conserved amongst species, indicating that the response is an important cellular mechanism. Work into the world of naturally occurring siRNA has shown that this response is not only involved in protecting the genome from threats such as viruses,1 but is also mechanistically related to microRNA (miRNA) pathways that provide crucial regulation of temporal control of development. The understanding of the mechanisms involved in RNA interference has progressed dramatically in the past several years. Briefly, long double-stranded RNA are processed by an RNase III enzyme named dicer to shorter (21–23 nt) siRNA complexes. The actual gene-silencing is processed by a group of proteins collectively termed the RISC (RNA-induced silencing complex), which serves to bring the siRNA to the vicinity of a complimentary mRNA and provides the catalytic enzymes involved in the cleavage of the mRNA.2, 3, 4 miRNA works along a similar pathway, however, naturally occurring miRNA precursors are short (70 bp) non-coding hairpin sequences that are also processed by dicer into short stretches of single-stranded RNA. Unlike siRNAs, miRNAs contain short mismatches in the sequences complementary to their target mRNAs, and miRNAs are thought to inhibit translation rather than lead to the degradation of mRNA. miRNAs are extremely important in gene regulation, and certain neuronal disorders such as fragile X are linked to the miRNA pathway.5

The siRNA system has been commandeered to create a valuable tool in order to study the effects of knocking down certain transcripts. Experimentally, one scans a target gene for suitable target sequences 21–23 nt long and designs double-stranded RNAs to target these sites. Scanning is typically assisted by one of several public-access computer programs that incorporate experimental 'rules' for designing effective siRNAs. These rules include avoiding runs of four or more guanosines or adenosines, keeping the base composition between 30 and 55% G+C, and identifying sites with asymmetric thermal stability so that the antisense strand is preferentially incorporated into RISC.6, 7, 8, 9 As an alternative for rational design of siRNAs, several groups have developed strategies for creating and screening siRNA libraries targeted to a gene of interest.10, 11, 12, 13 This approach is valid because some of the most effective siRNAs that have been described in the recent literature would have been avoided according to the current design algorithms.

Delivery of siRNA

Short interfering RNAs can be introduced to mammalian cells and experimental animals either as RNA or as DNA clones that code for the double-stranded RNA. When DNA clones are used, they are often designed to produce short hairpin RNA (shRNA) that is processed to form siRNA in the cell. For cultured cells and for temporary dosing to tissue, direct delivery of siRNA has distinct advantages: short RNA molecules are commercially available in pure form. Double-stranded RNA can be stabilized by chemical modifications in both strands of the duplex.14, 15, 16 Certain ribose modifications in the sense strand have the added advantage of increasing the incorporation of the antisense strand of the duplex into RISC, therefore increasing cleavage of the intended mRNA. Using RNA also permits accurate dosing of the cells, since a known quantity of RNA is added. The disadvantage of direct RNA treatment is that a large amount of siRNA is required (typically 10–100 nM) and the effect is temporary (3–4 cell generations). Using a high dose of siRNA may saturate the nuclear-cytoplasmic transport of miRNAs and thus lead to non-specific effects.

Plasmids and viral vectors can be used to deliver shRNAs, which are usually produced under the control of RNA polymerase III (pol III) promoters. Pol III promoters generate large amounts of transcript, but much of it is sequestered in the nucleus away from the cytoplasmic RISC. Consequently, some investigators have switched to pol II promoter systems for the expression of shRNA17 or have disguised their siRNA hairpins as miRNA precursors to facilitate transport to the cytoplasm.18 Delivery with viral vectors has the advantages that hard-to-transfect primary cells can often be infected and that expression of the RNA hairpin may remain stable in nondividing cells or for many generations in dividing cells, if an integrating virus is employed.

While the delivery options are plentiful in the laboratory, bringing siRNA to the clinic reduces these options. Obvious problems arise from RNA stability as well as access to target organs. Nonviral delivery methods to the mammalian brain have included both non-specific lipid and polyethylenimine RNA complexes and cell-specific methods. For example, receptor-specific pegylated immunoliposomes (PIL) have proved successful when injected directly into tissue. PILs are 'nano-containers' with the ability to store plasmid DNA and are heavily coated with 2000-Da polyethyleneglycol (PEG). Tethered to a fraction of the PEG are antibodies specific for those cell types to be targeted. For instance, in a mouse model for intracranial human brain cancer, plasmid-encoded siRNA-targeting human epidermal growth factor receptor was injected with this vehicle, significantly increasing the survival of the animal.19 As an alternative to siRNA lipid complexes, electroporation of 'naked' RNA has been shown to be effective in delivering siRNA to a limited number of target organs, including the visual cortex and the CA1 region of the hippocampus.20 As with lipid complexes and RNA-nanoparticles, however, electroporation leads to only a transient reduction of gene expression, and the prospect of sequential re-administration to the brain is unattractive in a therapeutic setting.

Viral vectors provide the most suitable means of delivering and expressing siRNA for long-term therapy. Several recombinant viruses have been used for gene therapy in the brain, but the short duration of transduction and the proinflammatory properties of vectors based on adenovirus and herpes simplex virus have restricted their use to antitumor applications. Lentivirus vectors and recombinant adeno-associated virus (rAAV) vectors have been used for long-term transduction of the brain and particularly for gene therapy in animal models of neurodegenerative disorders such as Huntington's disease (HD),21, 22 spinocerebellar ataxia23 and PD.24, 25 Both virus types can infect nondividing cells and lead to long-term gene expression. Lentiviral vectors give a more localized response and can contain a larger 'payload' in terms of passenger genes, but delivery of siRNA does not exceed the 4.7 kb carrying capacity of AAV. While rAAV does not provoke an inflammatory response, infection does stimulate humoral immunity, and most people have circulating antibodies to AAV.26, 27 Indeed, pre-immunization of rats with AAV2 prevents striatal transduction with rAAV2 vectors.28 Several rAAV serotypes have been used to deliver marker genes or therapeutic genes to the midbrain, but to date, rAAV1 and rAAV5 appear to provide the widest area of transduction, while gene expression mediated by rAAV2 infection is more precisely located near the site of injection.29 siRNA therapeutics will almost certainly need to specifically target the SN. rAAV2 has the serendipitous feature that it transduces the nigral DA neurons with higher efficiency than other neurons in the anatomical vicinity providing specific nigrostriatal transduction.29, 30, 31

RNA interference holds a valuable potential as a future therapeutic, it could be used to simply knockdown a specific disease gene. siRNA is selective for fully complementary mRNA targets, and short mismatches has been shown to significantly lower the efficacy of target mRNA degradation.32 Nevertheless, as few as 11 sequential matches between the 3' end of the target RNA and the 5' end of the antisense strand are sufficient to mediate cleavage by RISC, so that allele specificity is thought to require three or more mismatches with nontarget RNAs. Allele specificity would prove valuable as it would enable treatment of dominant mutations by targeting only the mutant sequence. If specificity could not be achieved by targeting the degradation of an RNA bearing a point mutation, RNA replacement might be achieved by concomitantly expressing a 'hardened' mRNA, in which several silent nucleotide substitutions have been introduced. Thus, this approach essentially replaces the defective gene with a healthy one.

Recent work on an animal model for spinocerebellar ataxia type 1, a polyglutamine expansion disease characterized by progressive neurodegeneration, demonstrates the potential for pre-translational silencing of a gene.23 Polyglutamine diseases are characterized by the presence of intracellular inclusions containing the expanded protein as well as numerous components of the machinery involved in ubiquitin-mediated proteasomal processing. It has been shown in vitro that lengthier repeats cannot be processed through ubiquitin-mediated proteasomal degradation. These uncleaved stretches of amino acids are thought to further inhibit the proteasome and are also very prone to aggregation which may be toxic to the cell.33 However, when Davidson et al.23 injected a rAAV expressing a siRNA targeting the ataxin-1 mRNA into the cerebellum, they demonstrated that transduced cells had a lower level of inclusions and that the animals displayed a significant improvement in motor performance. Similar positive results were obtained delivering shRNAs to huntingtin in polyglutamine-expanded HD mice.21, 22

Similar to spino-cerebellar ataxia, several other neurodegenerative diseases are also characterized by the accumulation and aggregation of protein. In certain familial forms of Amyotrophic Lateral Sclerosis, a mutation in the gene encoding the antioxidant enzyme Cu,Zn superoxide dismutase, is thought to cause protein misfolding, oligomerization and/or proteasome inhibition.34, 35 siRNA treatment in transgenic animals carrying these mutations targeting the proapoptotic gene prostate apoptosis response-4, a protein shown to be upregulated in the lumbar spinal cord samples of ALS patients, protected from caspase 3-mediated apoptosis and preserved mitochondrial function in a staurosporine regimen.36 RNAi treatment has shown some promise in transmissible spongiform encephalopathies where the transmittable conformational change of an aberrant isoform of the prion protein causes neuronal cell death. When transfected into a scrapie-infected neuroblastoma cell line, siRNA targeting the prion mRNA reduced the levels of the protease-resistant isoform of the protein.37

In PD, several familial forms have been identified and linked to disease genes, several of which are thought to be inhibitory to proteasomal processing, or prone to accumulation and aggregation. It is also likely that this common theme of protein accumulation and aberrant protein degradation also share common downstream effectors, such as common cell death triggers which also become attractive targets in a siRNA-based therapy (Figure 2).

The average age of onset of the idiopathic form of the disease is at the age of 60 years, and the disease symptoms usually appear on one side of the body with a subsequent bilateral progression. Noticeable motor dysfunctions include a highly characteristic resting tremor, rigidity, bradykinesia, loss of postural reflexes and a distinguishable freezing phenomenon.38 Diagnosis cannot be complete without establishing a responsiveness to levodopa, a neurotransmitter replacement strategy, and currently the standard pharmacotherapy for PD.39 This treatment is limited, however, by the appearance of side-effects including reduced duration of therapeutic response to a single dose and peak-dose dyskinesias 3–5 years after the commencement of therapy.40 There are numerous experimental treatments that are currently undergoing clinical evaluation.24, 41, 42 Nonetheless, there is no protective treatment or a cure for PD.

Neuropathologically, the disease is defined as a progressive depletion of striatal dopamine (DA) with the concomitant loss of the dopaminergic neurons of the substantia nigra pars compacta (SN). Similar to the polyglutamine repeat disorders mentioned previously, a hallmark feature of PD is the presence of intracytoplasmic inclusions, termed Lewy Bodies, which are highly immunoreactive to components of the proteasomal machinery such as ubiquitin and proteasomal subunits.43

The cause of the idiopathic form of the disease is still unknown and is probably multifactorial. Numerous hypotheses have been put forth claiming that environmental factors such as pesticides or metals may be exogenous factors in the disease.44, 45 However, it appears likely that subsequent steps in the disease process include mitochondrial impairment and increased oxidative stress.46 At the point in time when motor functions are impaired, approximately 80% of striatal DA is already gone, and the actual mechanism leading to cell death in the idiopathic form of the disease is still unknown. Therefore, the need for a more complete understanding of the disease progression is obvious. Additional clues to the disease etiology have come with the discovery of familial forms of PD and the genes involved in these forms.

The first gene to be linked to PD was PARK1 that was identified in 1997 by Polymeropoulos et al.47 by studying several families in southern Europe. The mutation identified in the -synuclein gene was a single point mutation (A53T), and subsequently, several other mutations were also identified. In addition, a gene triplication was discovered in a US family,48 suggesting that overexpression of the gene is detrimental as well. -synuclein-familial PD differs from the sporadic disease in that it is generally earlier in onset, and has a greater component of dementia. The parkinsonism is often considered a subset in a more general disorder termed diffuse Lewy body disease. The function of -synuclein is still unknown, although there are many indications that it is involved in the maintenance of synaptic DA vesicles and pre-synaptic function.

Alpha-synuclein is considered a natively unfolded protein with a high propensity to aggregate, with the end product being a highly insoluble polymer termed fibrils. It is still unclear which is the toxic event involved in -synuclein-associated disease, but what all mutations have in common is that they enhance the formation of an oligomer intermediate (protofibril) to fibril formation, and this protofibril is thought to possibly have the ability to damage cellular membranes.49

Alpha-synuclein is a major component of LBs, the presence of misfolded -synuclein and proteasomal machinery led to the idea that LBs are, in fact, neuroprotective, a way for the cell to harness damaging misfolded, insoluble proteins into a contained 'compartment'.

The Alpha-synuclein deficient mouse is relatively absent of pathology and also experiences an attenuated response to certain neurotoxic stimuli such as a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) regimen, a common model toxin for nigro-striatal degeneration. The MPTP metabolite 1-methyl-4-phenylpyridinium (MPP+) is inhibitory to mitochondria leading to decrease in respiration and increase in the production of oxidative free radicals, subsequently leading to cell death through apoptosis or necrosis.50 Considering the tendency for -synuclein to aggregate the animal model data, and the toxicity involved in overexpression, targeting this gene with siRNA could hold therapeutic potential.

The PARK2 gene was identified in 1998 to be involved in an early onset form of PD; autosomal recessive juvenile PD (AR-JP).51 A distinguishable feature of this form of PD is that onset happens as early as in the teens. However, following the identification of this gene, several researchers identified PARK2 polymorphisms as a potential risk-factor in sporadic PD.52 Interestingly, patients with this form of the disease lack LBs, suggesting that parkin is required for their formation/maintenance. The gene product parkin is an E3 ligase involved in targeting certain substrate proteins for degradation by the proteasome.53 Briefly, ubiquitin is activated through an ATP-dependent process by an E1 ubiquitin-activating enzyme. The activated ubiquitin is then transferred to an E2 ubiquitin-conjugating enzyme. E2 enzymes act in combination with E3 accessory subunits that bind to specific degradation signals in protein substrates.

Parkin has two RING (really interesting new gene) domains in the carboxy-terminus of the protein which serves as a binding region for the E2. There is a unique parkin domain adjacent to the ring domains which is thought to serve as a substrate recognition region. Thus, parkin serves to facilitate the transfer of activated ubiquitin from the E2 to the substrate creating a substrate 'tagged' with a poly-ubiquitin chain, thereby targeting it for degradation in the 26S proteasome. The substrates that have been identified for parkin are CDC-rel 1 and CDC-rel 2 (septin family, associated with synaptic vesicles), cyclin E, Pael-Receptor (G protein-coupled receptor), p38 tRNA synthase, synaptotagmin XI, synphillin-1, as well as parkin itself.49 The cyclin E substrate is unique in that parkin is part of a multiprotein complex which includes Hsel-10 and Cullin-1.54 siRNA was used to evaluate the toxicity of some of these substrates. In Drosophila, Pael-R overexpression causes a selective degeneration of dopaminergic neurons, a condition that can be repressed through coexpression of a fly homologue of parkin (dparkin). However, when expressing a siRNA designed against dparkin, the Pael-R mediated degradation is accelerated, indicating that the endogenous dparkin is involved in the degradation of Pael-R.55 The inverse experiment would obviously be a natural extension of this test; using siRNA to target Pael-R in a parkin-deficient animal should inhibit cell death due to Pael-R accumulation. However, parkin knockout mice display an incomplete pathology with little connection to the human disease, and no accumulation of substrates.56

Similarly, Staropoli and co-workers used a siRNA against parkin to study the effects on cyclin E accumulation. Cyclin E is involved in the regulation of G1/S cell-cycle transition, and when upregulated in mitotic cells would lead to cell division, but in post-mitotic neurons increased cyclin activity is believed to trigger apoptosis.57 Cyclins have been shown to accumulate in response to proapoptotic stimuli such the excitotoxin, kainate. Furthermore, cyclin E has been shown to accumulate in parkin-deficient cells and tissue, and more importantly, in the brains of AR-PD patients. Parkin overexpression in cerebellar granule cells was shown to relieve toxicity and apoptosis induced by kainite. However, siRNA-mediated parkin reduction was shown to sensitize the cells to, and enhanced the effects of, kainite toxicity.54

Additional early onset familial forms of PD were discovered recently with the identification of two disease genes involved in mitochondrial function. The first one to be identified was the gene encoding DJ-1.58 The function of this gene remains unknown, and numerous hypotheses have been put forth. Probably, the most interesting proposed function in terms of PD pathology is the suggested role of DJ-1 in improved protection against oxidative stresses, perhaps acting as a cellular sensor for oxidative stress and modulator of gene expression.49 This was demonstrated in vitro when a siRNA targeting DJ-1 was expressed in the SH-SY5Y neuroblastoma cell line. These cells became more susceptible to the MPP+ and 6-OHDA toxins, similar to cells overexpressing the disease mutation.59

The second new familial PD gene identified was PINK-1, a mitochondrial kinase, which was shown to protect cells against proteolytic stresses, although it is unsure by which mechanism this protection occurs.60

An additional gene that has been linked to PD is PARK5 coding for ubiquitin C-terminal hydroxylase L1 (UCHL1).61 As the name suggests, this protein is also involved in ubiquitin-dependent protein degradation by acting as a deubiquitination enzyme removing ubiquitin from processed amino-acid fragments, and, as such, fits with the general idea of impaired protein degradation. However, only a single family has ever been identified with this gene allele, and more work needs to be done to assess its relevance to PD.

The genetic linkages that have been made in PD provide valuable insight and clues to the pathology involved in familial forms as well as in sporadic disease; after identifying the -synuclein form of the disease the field soon realized that -synuclein may also play a role in idiopathic disease. The identification of parkin mutations and the lack of LBs hints to the idea that proteasomal processing play an integral part of the molecular pathology, and the DJ-1 and PINK mutations also involves the mitochondria and oxidative stress in the disease. Although these are distinct molecular processes, they are likely to be interconnected and not necessarily exclusive. Given these ideas it is also likely that the end result of these various forms of the disease converge on a common path of cell death. It is inherently difficult to study cell death in the human PD brain, given the slow rate of cellular death occurring over an extended period of time (decades).

Even so, there are numerous reports of TUNEL-positive nigral neurons from patients with PD, suggesting that cell death is mediated through apoptosis, which may be a consequence of mitochondrial failure and oxidative stress. Further support for this idea comes from reports that show that dopaminergic neurons from PD brains show an upregulation of proapoptotic genes, and the fraction of neurons containing activated caspase 3 is significantly higher than in healthy individuals.62 This increase in apoptosis immediately raises the possibility to target proapoptotic markers and activators such as caspase 3 using RNA knockdown techniques. When expressed in hippocampal cultures, ribozymes designed against pro-caspase 3 successfully protected against staurosporine-induced apoptosis.63 Similarly, in an animal model for apoptosis; rat optic nerve transection, pretreatment of siRNAs targeting c-jun and apoptotic protease-activating factor-1 (Apaf-1) resulted in a significantly improved survival of retinal ganglion cells.64 Blockade of apoptotic enzymes has been shown to be beneficial in other model systems as well. For example, siRNA designed against FAS-ligand expressed in a basal cell carcinoma, was shown to reduce the level of apoptosis of infiltrating immune cells and led to a more efficient clearing of the tumor cells.65

Animal models of PD that completely recapitulate the human disease are not available. Toxins such as MPTP, 6-hydroxydopamine, and rotenone have been used to create an acute level of oxidative stress in the SN, causing DA neurons to die. Models such as these have mainly been used in cell-replacement/survival studies. Other models include -synuclein transgenic animals, which display a relatively wide array of pathology, but not resembling that of PD. In addition, overexpression of wt- synuclein or A53T using nigral-specific rAAV vectors induces over 50% nigral cell death and approximately an equal amount of striatal DA depletion in rats and monkeys.66, 67

Parkin knockout mice have been created, but again they do not mimic the real disease. As a matter of fact, parkin-deficient animals display no significant pathology at all, which is surprising for a protein that is so ubiquitously expressed throughout the body. It is possible that utilizing siRNA to regulate certain genes may actually provide us with better models to study PD. For instance, in the case of parkin, this would allow one to study cellular impairment with spatio-temporal precision, and also allow one to avoid any 'genomic compensation' that may have occurred throughout the development of an organism.

Previous studies in our laboratory tried to test this hypothesis. We designed a number of ribozymes against parkin. Ribozymes are enzymatic RNAs that detect certain sequences in the mRNA and cleave them. These ribozymes were then packaged into rAAV and unilaterally injected into the SNc of adult rats. Immunohistochemistry, 4 weeks following the injections, clearly shows transduced cells (as detected by green fluorescent protein), with a significant loss of parkin expression (Figure 1a–g). However, attempts to identify accumulation of substrates and quantifying levels of parkin expression were unsuccessful. This was the result of an inherent difficulty with RNA and protein levels specifically in the SN. Proteins such as parkin are ubiquitously expressed in the brain, but the rAAV transduction pattern that we experience is highly specific for the SNc; thus it becomes very difficult to isolate these nigral neurons, and to analyze their content without an overwhelming background from surrounding tissue. Whereas studying other gene products such as -synuclein, which is highly upregulated in the SNc as compared to surrounding cells, is less difficult (Figure 1j–m).

RNA interference has been and will continue to play an important role in PD research. However, the ultimate goal of researchers is to bring treatments and cures to the clinics. Given the relative ease by which one can design a siRNA to target a certain gene, there is no need for lengthy pharmacological analyses, and drug targeting issues. One can simply target the specific circuits in the brain using viral vectors expressing an siRNA. The potential targets in PD are numerous. First of all, one could target alpha-synuclein, which seems to be intimately tied to the disease. The substrates of parkin are also attractive targets. Blocking the production of various apoptotic activators also become potential treatment strategies, but this approach also raises some concern about the risk for cancer if the siRNA is expressed in glia.

For example, can a delivery system be refined enough, to target only dopaminergic cells? What happens if an antiapoptotic siRNA is expressed in the surrounding glia? What if there is a gain of function mutation of an otherwise required protein? Lessons learned from ribozyme studies demonstrate that one can target the mutated sequence specifically, but if there is extensive allelic heterogeneity among disease mutations, this approach becomes less feasible. In the event that the mutant allele cannot be targeted, one can target a common wild-type sequence, and attempt to replace the gene with a siRNA or ribozyme-resistant copy. Another concern is the efficacy with which you can knockdown a certain mRNA. Even in the most favorable circumstances 100% reduction is not feasible. Thus, experiments must be designed to determine if residual expression of the gene is still therapeutic.

For instance, one of the earliest successful in vivo siRNA experiments targeted tyrosine hydroxylase (TH) expression in the SNc of mice using rAAV-mediated expression. TH is the rate limiting enzyme in the production of DA, the main signaling molecule in this circuit. Although, the experiment showed a significant reduction in TH levels, the behavioral testing was not entirely consistent with complete DA depletion in the nigrostriatal tract.68 These results emphasize the point that in some instances siRNA may not be enough to achieve the intended biochemical result. An additional concern comes with that of 'off-targeting'. Although great care is practiced when designing the siRNA, you may still be affecting other genes, effects that may or may not have detrimental results.

Although in its infancy, applied siRNA technology has had great success in the laboratory, both to study pathology and to effect a cure. However, as of yet, only one clinical trial is underway, targeting vascular Endothelial Growth Factor Receptor-1 in age-related macular degeneration. In PD, there are numerous potential targets for RNA knockdown that when reduced could slow or even halt the progression of the disease (Figure 2).

Given the recent high-profile successes of virally delivered siRNA, this strategy is clearly poised to burst onto the PD research scene. All of the PD strategies to impact survival of DA neurons require nigral DA neuronal expression of the siRNA. Currently, the rAAV2 vector system has shown the most specificity for nigral DA neurons. In this review, we have pointed out some caveats when dealing with ubiquitously expressed proteins in the nigra (Figure 1). These potential problems in demonstrating knockdown must be taken into account in future studies.

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Acknowledgements

This work was supported by a Fast-track grant from the Michael J Fox Foundation to RJM and ASL.

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Molecular genetic analysis of the alpha-synuclein and the parkin gene in Parkinson's disease in Finland.

Autere JM, Hiltunen MJ, Mannermaa AJ, Jakala PA, Hartikainen PH, Majamaa K, Alafuzoff I, Soininen HS.

Department of Neurology, University Hospital and University of Kuopio, Finland.

Two mutations in the alpha-synuclein gene and various mutations in the parkin gene are associated with familial Parkinson's disease (PD). The present study was performed to analyse if mutations in these genes could be detected in Finnish patients with familial PD. The subjects comprised 22 unrelated patients with familial PD. The molecular genetic analysis consisted of sequence analysis of the non-coding and coding exons of the alpha-synuclein gene and screening of eight point mutations in the parkin gene. In addition, a total of 67 controls and 45 patients with sporadic PD were included in the association analysis on polymorphism of the alpha-synuclein gene. Screened point mutations in the parkin gene were not detected. Sequencing of the coding exons 2-6 of the alpha-synuclein gene did not reveal any mutations or polymorphisms. However, three novel alterations in the T10A7 sequence at the 5' end of the non-coding exon 1' of the alpha-synuclein gene were found. The frequencies of the exon 1' polymorphic genotypes or alleles between familial PD patients and control subjects revealed no statistically significant differences. No association for sporadic PD was observed. The results do not support a role for the alpha-synuclein gene or point mutations of the parkin gene in familial PD in our sample.

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Pharmacogenet Genomics. 2005 Sep;15(9):659-68.

Dopamine transporter (SLC6A3) 5' region haplotypes significantly affect transcriptional activity in vitro but are not associated with Parkinson's disease.

Kelada SN, Costa-Mallen P, Checkoway H, Carlson CS, Weller TS, Swanson PD, Franklin GM, Longstreth WT Jr, Afsharinejad Z, Costa LG. Department of Environmental and Occupational Health Sciences, University of Washington, Seattle, Washington 98105, USA. skelada@u.washington.edu

The dopamine transporter (DAT) plays a critical role in dopaminergic neurotransmission and is also the major site of action for some drugs of abuse. The coding region of the DAT gene, SLC6A3, is well conserved, but non-coding regions are more variable, most notably a variable number of tandem repeats (VNTR) polymorphism in the 3' untranslated region, which has been studied in a number of dopamine-related neurological disorders, including Parkinson's disease (PD). We aimed to characterize variation in the 5' region of SLC6A3 because little is known about the extent of variation in this region and potential consequences of such variation on gene expression. We identified multiple single nucleotide polymorphisms (SNPs) covering approximately 5000 bp 5' of exon 1 through the start of exon 2 (+2106). These SNPs segregated as eight haplotypes, six of which were common. These haplotypes differed significantly in activity in a reporter gene activity assay. However, we did not observe associations between common SNPs or haplotypes and PD in a case-control study of 261 incident cases and 376 age- and gender-matched unrelated controls. By contrast, we did observe a modest association of the 3' VNTR 9-repeat allele with PD (odds ratio=1.45; 95% confidence interval=1.04-2.03). This association was limited to subjects 60 years of age and greater versus those less than 60 years of age. We conclude that although DAT 5' region SNPs haplotypes significantly alter in vitro transcriptional activity, they are not related to PD risk. In addition, our findings provide further evidence supporting an association of PD with the VNTR polymorphism.

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Brain Res. 2004 Mar 12;1000(1-2):156-73.

Characterization of genomic organization of the adenosine A2A receptor gene by molecular and bioinformatics analyses.

Yu L, Frith MC, Suzuki Y, Peterfreund RA, Gearan T, Sugano S, Schwarzschild MA, Weng Z, Fink JS, Chen JF. Department of Neurology, Boston University School of Medicine, 715 Albany Street, Boston, MA 02118, USA.

The adenosine A(2A) receptor (A(2A)R) is abundantly expressed in brain and emerging as an important therapeutic target for Parkinson's disease and potentially other neuropsychiatric disorders. To understand the molecular mechanisms of A(2A)R gene expression, we have characterized the genomic organization of the mouse and human A(2A)R genes by molecular and bioinformatic analyses. Three new exons (m1A, m1B and m1C) encoding the 5' untranslated regions (5'-UTRs) of mouse A(2A)R mRNA were identified by rapid amplification of 5' cDNA end (5' RACE), RT-PCR analysis and genome sequence analyses. Similar bioinformatics analysis also suggested six variants of the non-coding "exon 1" (h1A, h1B, h1C, h1D, h1E and h1F) in the human A(2A)R gene, which were confirmed by RT-PCR analysis, while three of the human exon 1 variants (h1D, h1E and h1F) were likewise verified by 5' oligonucleotide capping analysis suggesting multiple transcription start sites. Importantly, RT-PCR and quantitative PCR analysis demonstrated that the A(2A)R transcripts with different exon 1 variants displayed tissue-specific expression patterns. For instance, the mouse exon m1A mRNA was detected only in brain (specifically striatum) and the human exon h1D mRNA in lymphoreticular system. Furthermore, the determination of the three new transcription start sites of human A(2A)R gene by 5' oligonucleotide capping and bioinformatics analyses led to the identification of three corresponding promoter regions which contain several important cis elements, providing additional target for further molecular dissection of A(2A)R gene expression. Finally, our analysis indicates that A(2A)R mRNA and a novel transcript partially overlapping with the 3' exon h3, but in opposite orientation to the A(2A)R gene, could conceivably form duplexes to mutually regulate transcript expression. Thus, combined molecular and bioinformatics analyses revealed a new A(2A)R genomic structure, with conserved coding exons 2 and 3 and divergent, tissue-specific exon 1 variants encoding for 5'-UTR. This raises the possibility of generating multiple tissue-specific A(2A)R mRNA species by alternative promoters with varying regulatory susceptibility.


Am. J. Hum. Genet. 77:685–693, 2005

[4 Independent laboratories attempted to reproduce the SNP results of Dr. Maraganore, reported below. No confirmation is known to date, July 16, 2006 AJP]

High-Resolution Whole-Genome Association Study of Parkinson Disease

Demetrius M. Maraganore,1 Mariza de Andrade,2 Timothy G. Lesnick,2 Kari J. Strain,2, Matthew J. Farrer,3 Walter A. Rocca,1,2 P. V. Krishna Pant,4 Kelly A. Frazer,4 David R. Cox,4 and Dennis G. Ballinger4

Departments of 1Neurology and 2Health Sciences Research, Mayo Clinic College of Medicine, Rochester, MN; 3Department of Neuroscience, Mayo Clinic College of Medicine, Jacksonville, FL; and 4Perlegen Sciences, Mountain View, CA

We performed a two-tiered, whole-genome association study of Parkinson disease (PD). For tier 1, we individually genotyped 198,345 uniformly spaced and informative single-nucleotide polymorphisms (SNPs) in 443 sibling pairs discordant for PD. For tier 2a, we individually genotyped 1,793 PD-associated SNPs ( in tier 1) P ! .01 and 300 genomic control SNPs in 332 matched case–unrelated control pairs. We identified 11 SNPs that were associated with PD (P ! .01) in both tier 1 and tier 2 samples and had the same direction of effect. For these SNPs, we combined data from the case–unaffected sibling pair (tier 1) and case–unrelated control pair (tier 2) samples and employed a liberalization of the sibling transmission/disequilibrium test to calculate odds ratios, 95% confidence intervals, and P values. A SNP within the semaphorin 5A gene (SEMA5A) had the lowest combined P value (P p 7.62# 1056). The protein encoded by this gene plays an important role in neurogenesis and in neuronal apoptosis, which is consistent with existing hypotheses regarding PD pathogenesis. A second SNP tagged the PARK11 late-onset PD susceptibility locus (P p 1.70#1055). In tier 2b, we also selected for genotyping additional SNPs that were borderline significant (P ! .05) in tier 1 but that tested a priori biological and genetic hypotheses regarding susceptibility to PD (n p 941 SNPs). In analysis of the combined tier 1 and tier 2b data, the two SNPs with the lowest P values (P p 9.07#1056; P p 2.96#1055) tagged the PARK10 late-onset PD susceptibility locus. Independent replication across populations will clarify the role of the genomic loci tagged by these SNPs in conferring PD susceptibility.

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PRADER-WILLI SYNDROME

The human chromosome region 15q13-15, which is associated with genetic and epigenetic disturbances that generate both Prader-Willi syndrome and Angelmann syndrome, harbours non-coding genes such as IPW/Ipw, PAR1, PAR5, PARSN and ASR1,2 (ref. 14). IPW/Ipw (imprinted gene in Prader-Willi syndrome) expresses a processed RNA with no significant ORFs, which is localized predominantly in the cytoplasm15. An antisense RNA is produced from the ZNF127 locus, which encodes a protein with RING zinc-finger and multiple zinc-finger motifs. The ZNF127 and ZNF127 AS RNA vary in their expression patterns as well as in size of the transcripts12. The biological functions of IPW (sense) and ZNF127 (antisense) transcripts, if any, are not clear at this moment. The SNRPN gene, which spans 360 kb of DNA produces both coding and non-coding imprinted transcripts16. The exons at the 3¢ part of the gene generate a coding mRNA when spliced with exon 1 and noncoding RNAs when spliced with the 5¢ BD exons16. BD transcripts, encoded by two alternative 5¢ exons, BD1B and BD1A, have two alternative start sites and are subject to alternative splicing16. They are expressed from the paternal chromosome only, as the BD exons are heavily methylated on the maternal chromosome (16). It has recently been shown that paternally imprinted antisense RNA is produced at the 3¢ UTR of Angelmann syndrome gene, UBE3A (11), which encodes ubiquitin protein ligase that functions in protein turnover17.

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Dev Biol. 2005 Oct 15;286(2):587-600. Epub 2005 Aug 29.

Dynamic developmental regulation of the large non-coding RNA associated with the mouse 7C imprinted chromosomal region.

Le Meur E, Watrin F, Landers M, Sturny R, Lalande M, Muscatelli F.

NMDA/IBDM, Campus de Luminy Case 907 13288 Marseille Cedex 09, France.

The mouse ortholog of the Prader-Willi/Angelman syndrome imprinted domain contains several paternal-specific transcripts and the maternally expressed gene encoding ubiquitin protein ligase E3A (Ube3a). A Large paternal Non-Coding RNA, encompassing Snurf-Snrpn exons and the Ube3a Antisense Transcript (Ube3a-ATS), has been recently characterized and named here LNCAT. Potential roles of LNCAT in imprinting, gene regulation, and disease are likely but have not been investigated. In order to establish the function(s) of LNCAT, we first determined its in vivo spatio-temporal expression pattern at the cellular level during development and in different adult brain tissues. We show here that LNCAT is developmentally regulated, with transcript variants being specifically expressed through neuronal differentiation in postmitotic neurons. We demonstrate that the LNCAT and Snurf-Snrpn transcripts are independent although they share common exons. We show an absence of expression of LNCAT through gametogenesis and in early embryo excluding a role of LNCAT in the imprint establishment. We also report a range of observations that challenges the widely accepted model of imprinted gene silencing of Ube3a. Although these last data do not completely exclude that the LNCAT variants including "Ube3a-ATS"exons could repress the paternal allele of Ube3a, they do allow us to propose an alternative and consistent model.


PSEUDOXANTHOMA ELASTICUM (PXE, GROENBLAD-STRANDBERG SYNDROME)

ABCC6 encodes MRP6, a member of the ABC protein family with an unknown physiological role. The human ABCC6 and its two pseudogenes share 99% identical DNA sequence. Loss-of-function mutations of ABCC6 are associated with the development of pseudoxanthoma elasticum (PXE), a recessive hereditary disorder affecting the elastic tissues. Various disease-causing mutations were found in the coding region; however, the mutation detection rate in the ABCC6 coding region of bona fide PXE patients is only 80%. This suggests that polymorphisms or mutations in the regulatory regions may contribute to the development of the disease. Here, we report the first characterization of the ABCC6 gene promoter. Phylogenetic in silico analysis of the 5' regulatory regions revealed the presence of two evolutionarily conserved sequence elements embedded in CpG islands. The study of DNA methylation of ABCC6 and the pseudogenes identified a correlation between the methylation of the CpG island in the proximal promoter and the ABCC6 expression level in cell lines. Both activator and repressor sequences were uncovered in the proximal promoter by reporter gene assays. The most potent activator sequence was one of the conserved elements protected by DNA methylation on the endogenous gene in non-expressing cells. Finally, in vitro methylation of this sequence inhibits the transcriptional activity of the luciferase promoter constructs. Altogether these results identify a DNA methylation-dependent activator sequence in the ABCC6 promoter.

[Detailed documentation of ABCC6, implicated in PXE, is available (Sharon Terry, Genetic Alliance). The thesis of PostGenetics (Genetics beyond Genes) maintains that "it is not all in the genes". PXE is a towering candidate of a "rare hereditary disease" to make it to the "small league" to which the new "whole genome funds" of NIH might focus (since an all-out analysis all of the 150,000+ "junk DNA diseases" can not be economically sustained when medicine is still "barking on the wrong tree" with an overwhelming portion of funds are spent on "Gene Discovery", unjustly neglecting spending taxpayer dollars on "PostGene Discovery"). PXE is a spectacular target of "PostGene Discovery" in PostGenetics. Why? Because in PXE "we have it all". Both a particular Gene and its PostGenes in the regulatory region, moreover evolutionarily conserved sequence elements are identified - and we also have an understanding of the disease caused by particular polymorphisms or mutations affecting coding AND non-coding regions. PostGenetics must corroborate the above by a novel, meaningful concept as well as technology. FractoGene does precisely that. With its first ("Fugu") prediction now supported by experimental facts published in peer-reviewed journal, there is the "Methylation Prediction of FractoGene". PXE is a unique target of validation the time-line of "PostGene Methylation", thereby opening an entirely new avenue for understanding "whole genome function" - towards "PostGenetic Medicine" - comment by A. Pellionisz]

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PSORIASIS

Sequence and Haplotype Analysis Supports HLA-C as the Psoriasis Susceptibility 1 Gene.

Nair RP, Stuart PE, Nistor I, Hiremagalore R, Chia NV, Jenisch S, Weichenthal M, Abecasis GR, Lim HW, Christophers E, Voorhees JJ and Elder JT

Am J Hum Genet (2006) 78:827-51


Previous studies have narrowed the interval containing PSORS1, the psoriasis-susceptibility locus in the major histocompatibility complex (MHC), to an ~300-kb region containing HLA-C and at least 10 other genes. In an effort to identify the PSORS1 gene, we cloned and completely sequenced this region from both chromosomes of five individuals. Two of the sequenced haplotypes were associated with psoriasis (risk), and the other eight were clearly unassociated (nonrisk). Comparison of sequence of the two risk haplotypes identified a 298-kb region of homology, extending from just telomeric of HLA-B to the HCG22 gene, which was flanked by clearly nonhomologous regions. Similar haplotypes cloned from unrelated individuals had nearly identical sequence. Combinatorial analysis of exonic variations in the known genes of the candidate interval revealed that HCG27, PSORS1C3, OTF3, TCF19, HCR, STG, and HCG22 bore no alleles unique to risk haplotypes among the 10 sequenced haplotypes. SPR1 and SEEK1 both had messenger RNA alleles specific to risk haplotypes, but only HLA-C and CDSN yielded protein alleles unique to risk. The risk alleles of HLA-C and CDSN (HLA-Cw6 and CDSN*TTC) were genotyped in 678 families with early-onset psoriasis; 620 of these families were also typed for 34 microsatellite markers spanning the PSORS1 interval. Recombinant haplotypes retaining HLA-Cw6 but lacking CDSN*TTC were significantly associated with psoriasis, whereas recombinants retaining CDSN*TTC but lacking HLA-Cw6 were not associated, despite good statistical power. By grouping recombinants with similar breakpoints, the most telomeric quarter of the 298-kb candidate interval could be excluded with high confidence. These results strongly suggest that HLA-Cw6 is the PSORS1 risk allele that confers susceptibility to early-onset psoriasis.


[Note the 100:3 ratio of polymorphism in non-coding versus coding DNA - comment by AJP]

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Contribution by IPGS Founders Dr. Kemeny and Dr. Szell:

Identification and Characterization of a Novel, Psoriasis Susceptibility-related Noncoding RNA gene, PRINS*

Eniko Sonkoly, Zsuzsanna Bata-Csorgo¶, Andor Pivarcsi, Hilda Polyanka, Anna Kenderessy-Szabo, Gergely Molnar||, Karoly Szentpali**, Lilla Bari, Klara Megyeri, Yvette Mandi, Attila Dobozy, Lajos Kemeny, and Marta Szell

From the Departments of Dermatology and Allergology, **Surgery, and Medical Microbiology and Immunobiology, University of Szeged, Szeged 6720 and the Dermatological Research Group and the ||Institute of Plant Biology, Biological Research Center, Hungarian Academy of Sciences, Szeged 6701, Hungary

To identify genetic factors contributing to psoriasis susceptibility, gene expression profiles of uninvolved epidermis from psoriatic patients and epidermis from healthy individuals were compared. Besides already characterized genes, we identified a cDNA with yet unknown functions, which we further characterized and named PRINS (Psoriasis susceptibility-related RNA Gene Induced by Stress). In silico structural and homology studies suggested that PRINS may function as a noncoding RNA. PRINS harbors two Alu elements, it is transcribed by RNA polymerase II, and it is expressed at different levels in various human tissues. Real time reverse transcription-PCR analysis showed that PRINS was expressed higher in the uninvolved epidermis of psoriatic patients compared with both psoriatic lesional and healthy epidermis, suggesting a role for PRINS in psoriasis susceptibility. PRINS is regulated by the proliferation and differentiation state of keratinocytes. Treatment with T-lymphokines, known to precipitate psoriatic symptoms, decreased PRINS expression in the uninvolved psoriatic but not in healthy epidermis. Real time reverse transcription-PCR analysis showed that stress signals such as ultraviolet-B irradiation, viral infection (herpes simplex virus), and translational inhibition increased the RNA level of PRINS. Gene-specific silencing of PRINS by RNA interference revealed that down-regulation of PRINS impairs cell viability after serum starvation but not under normal serum conditions. Our findings suggest that PRINS functions as a noncoding regulatory RNA, playing a protective role in cells exposed to stress. Furthermore, elevated PRINS expression in the epidermis may contribute to psoriasis susceptibility

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J Med Genet. 2005 Dec 9;

Evidence for susceptibility determinant(s) to psoriasis vulgaris in or near PTPN22 in German patients

Huffmeier U, Steffens M, Burkhardt H, Lascorz J, Schurmeier-Horst F, Stander M, Kelsch R, Baumann C, Kuster W, Mossner R, Reich K, Wienker TF, Traupe H, Reis A. Institute of Human Genetics, University of Erlangen, Germany.

INTRODUCTION: Variant R620W of protein tyrosine phosphatase non-receptor type 22 (PTPN22) has consistently been reported as susceptibility factor for several autoimmune diseases. We were interested in its role in susceptibility to psoriasis, furthermore whether other disease-causing variants within PTPN22 might exist and whether they act independently from the major risk factor for psoriasis at HLA-C / PSORS1. METHODS: R620W was tested in a case control study with an exploratory set of 375 independent German patients and an enlarged sample of 418 additional patients. Analyses were extended to linkage disequilibrium (LD) based haplotypes. Potential interaction between one risk haplotype encompassing PTPN22 and the PSORS1 associated risk allele was tested by regression analysis. PTPN22 coding sequence was determined in 20 patients carrying the risk haplotype. Regression analysis as well as analysis of the strongest associated risk haplotypes were also performed in the extended case control study. RESULTS: R620W was not associated in both case control studies (375/793 patients) while significant association (corrected for multiple testing) with one haplotype (C-4) of the LD block encompassing PTPN22 as well with another haplotype (B-3) within an adjacent telomeric LD block was detected. No evidence for interaction between risk haplotype C-4 and the HLA-C associated risk allele was found. Sequencing excluded other coding variants within PTPN22 as basis for association findings. Analysis of the extended study group confirmed association for haplotypes B-3 and C-4 and independence of risk haplotypes C-4 and PSORS1. DISCUSSION: We exclude a major role of *620W in German psoriasis patients but suggest that [an]other susceptibility determinant[s] within the non-coding regions of PTPN22 or its proximity might exist that act independently from the major PSORS1 risk factor.

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RETT SYNDROME

[...] In the first talk Jeffrey Neul of Baylor College of Medicine presented a comprehensive description of the clinical features of Rett Syndrome. The videos of children with RTT embedded in his presentation were very helpful to the audience comprised mostly of basic scientists with limited clinical knowledge of RTT.

Michael Greenberg of ChildrenÍs Hospital Boston and Harvard Medical School discussed the importance of the interplay between certain enzymes and neuronal activity in the healthy development of the nervous system. He focused on the formation of synapses and how they are maintained and/or changed by neural activity. Of particular relevance to RTT is the possibility that stimuli may influence the release of MeCP2 from brain derived neurotrophic factor (BDNF) thereby allowing its production. Dr. GreenbergÍs talk provided some insights on how synapse development may be misregulated in Rett Syndrome.

Yi Sun of UCLA discussed potential roles of DNA methylation and methyl-DNA binding proteins in neuronal function and plasticity in early embryonic development in mouse. She touched on several transcription factors and one signaling pathway, and described how DNA methylation at these loci would affect the outcome of early neuron/glia differentiation.

Lisa Monteggia of University of Texas Southwestern Medical Center presented behavioral analysis of Mecp2 conditional mutant mice including locomotion test, open field test, fear conditioning, pain sensitivity, and social interaction test. She showed that these mice have RTT-like symptoms and can be used as an animal model to study the behavior abnormalities. [...]

Gail Mandel of Stonybrook and Howard Hughes Institute is investigating the repressor REST and its function in neuronal development. REST was shown to bind RE1 elements and recruit transcriptional co-repressors coREST and Sin3A. During ES cell differentiation REST is down-regulated in a post-transcriptional way by digestion with proteases. REST represses calbindin and synaptogonin genes, however the treatment with de-methylating agent (aza-C) and deacetylase inhibitor (TSA) relieved transcriptional repression. Methylated regions were identified near the calbindin promoter, which is bound by MeCP2. The proposed model suggests that MeCP2 and REST interact by a common co-repressor, Sin3A, and the presence of MeCP2 is necessary to maintain repression when REST is absent. SACO (Serial Analysis of Chromatin Occupancy) was introduced as a global method to identify protein binding sites of REST. Surprisingly, for some of the genes the RE1 site is not in close proximity to the promoter, suggesting that there may be long-range repression events. [...]

Denis Jugloff of the University of Toronto over-expressed MeCP2 in neurons along with GFP and found that neurons had longer axons and dendrites and were overall more complex than control neurons. No change in the short-term survival of the neurons was observed. This data supports the hypothesis that MeCP2 plays a role in the growth and branching pattern of neurons as they develop. It corroborates the striking finding that in human RTT brains dendritic arborizations are dramatically simplified. [...]

Mutations in MeCP2 have now been identified in the majority of girls diagnosed with RTT, along with some patients with atypical RTT, Angelman Syndrome or other features. However, too many girls remain for whom no cause has been identified. Dr. Eva Gak has found that patients with C-terminal deletions of MeCP2 have lower gene expression levels. At the Danek Gertner Institute of Human Genetics at the Sheba Medical Center, she has developed a screen using peripheral blood to identify individuals with lower MeCP2 levels, and has found that 3 of 10 patients with no identified MeCP2 coding mutations have decreased MeCP2 expression. Dr. Gak has identified a single nucleotide change in the non-coding end of MeCP2 in one of these patients, and suggests that the C-terminus of MeCP2 may be an important factor for RNA stability or transcription rate.

Carl Wu of NIH, is one of the foremost experts in the world on chromatin. MeCP2 is believed to control chromatin structure, and therefore RTT may be a disorder in which some poorly understood aspect of chromatin biology goes awry. Dr. Wu described recent important discoveries from his lab on how chromatin structure is changed during gene regulation by highly intricate molecular machines known as "chromatin remodeling ATPases." He drew the attention of the audience to possible effects of MeCP2 on dynamic changes in chromatin folding and organization. This is an insufficiently investigated subject, and careful study of this problem may provide insights on how to restore that process to normalcy in children with RTT.

Megumi Adachi of the Neurosciences Institute described her work on how the MeCP2 gene itself is controlled. Adachi discovered a mechanism that, in her words, provide a "link between physiological stimuli (such as stress and neurotrophic factors) and MeCP2 production by the brain." Further study of this mechanism may help develop strategies to overcome the lack of MeCP2 function in RTT.

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RETINITIS PIGMENTOSA

Zhonghua Yi Xue Za Zhi. 2005 Jun 22;85(23):1613-7.

[Digenic association of RHO and RP1 genes with retinitis pigmentosa among Chinese population in Hong Kong]
[Article in Chinese]

Wang DY, Fan BJ, Chan WM, Tam OS, Chiang WY, Lam SC, Pang CP.
Department of Ophthalmology & Visual Sciences, the Chinese University of Hong Kong, Hong Kong, China.

OBJECTIVE: To identify the mutation patterns of RHO and RP1 genes in the Chinese patients with retinitis pigmentosa (RP) and to explore their potential interactions in the pathogenesis of RP. METHODS: Sequence alterations in the entire coding region and splice sites of RHO and RP1 gene were screened in 151 RP affected probands and 150 unrelated controls who were all Hong Kong Chinese. Additional 46 relatives of 12 RP probands carrying possible mutations in RHO or RP1 were recruited for segregation analysis. Univariate analysis, multivariate analysis and genotype-pedigree disequilibrium test were used to examine the associations of polymorphisms in these two genes with RP. RESULTS: Two mutations in the RHO gene, 5211delC and P347L, were identified each in one proband from the 151 probands, accounting for 1.3% of the RP patients. Two mutations in the RP1 gene, R677X and D984G, were identified each in one proband from the 151 probands, also accounting for 1.3% of the RP patients. In univariate analysis, non-coding sequence variants in the RHO gene, -26G > A, was found to increase the risk of RP, while R872H in the RP1 gene was likely to be a protective factor for RP. Multivariable logistic regression analysis and haplotype analysis confirmed these associations. CONCLUSION: The prevalences of RHO and RP1 mutations among the RP patients in Chinese population are both less than reported in other populations. Besides the disease-causing mutations, non-coding sequence alterations may also be a modifier for RP. The potential interactions between RHO and RP1 suggest a digenic etiology for RP.

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SCHIZOPHRENIA

Biol Psychiatry. 2006 Jan 24;

Neuregulin 1 and Schizophrenia: Genetics, Gene Expression, and Neurobiology.

Harrison PJ, Law AJ. Department of Psychiatry (PJH, AJL), University of Oxford, Warneford Hospital, Oxford, United Kingdom.

Neuregulin 1 (NRG1) is a leading schizophrenia susceptibility gene. The NRG1 locus on chromosome 8p shows linkage to the disorder, and genetic association has been found between schizophrenia and various non-coding polymorphisms and haplotypes [=coding alleles], especially at the 5' end of the NRG1 gene, in many but not all case-control and family studies. NRG1 is a pleiotropic growth factor, important in nervous system development and functioning; roles include the modulation of neuronal migration, synaptogenesis, gliogenesis, neuron-glia communication, myelination, and neurotransmission. Understanding the neurobiology of NRG1 and its involvement in schizophrenia is challenged by the complexity of the gene, which gives rise to multiple functionally distinct isoforms, including six "types" of NRG1 defined by 5' exon usage. Type IV and type I NRG1 may be particularly relevant to schizophrenia, with initial data showing altered expression of these isoforms in the disorder or in association with NRG1 risk alleles. We review the structure and functions of NRG1, consider the evidence for and against it being a schizophrenia susceptibility gene, and discuss mechanisms that might underlie the contribution of NRG1 to disease pathophysiology.

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SCLERODERMA

Gene. 2002 Sep 4;297(1-2):61-7.

Association of 5'-untranslated region of the Fibrillin-1 gene with Japanese scleroderma.

Kodera T, Tan FK, Sasaki T, Arnett FC, Bona CA. Department of Microbiology, Mount Sinai School of Medicine, New York, NY 10029, USA. tkodera@yahoo.com

Excessive production of extracellular matrix (ECM) constituents is a hallmark scleroderma or systemic sclerosis (SSc). Fibrillin-1, a major component of microfibrils in the ECM, may play a role in the pathogenesis of SSc. The TSK1 mouse model of SSc bears an in-frame duplication of the Fibrillin-1 gene (FBN1) which results in a larger than normal protein that is more susceptible to proteolysis. Metabolic labeling studies of Fibrillin-1 in human SSc dermal fibroblasts demonstrated that while normal amounts of Fibrillin-1 are synthesized, the protein itself appears to be unstable. Moreover, autoantibodies specific for Fibrillin-1 have been demonstrated in serum from SSc patients and TSK1 mice. In particular, a high frequency of anti-Fibrillin-1 was observed in Japanese patients with diffuse and limited scleroderma or CREST (calcinosis, Raynaud phenomenon, esophageal dysmotility, sclerodactyly, telangiectasia) syndrome. Genetic studies in a Native American population with high prevalence of using microsatellite marker showed strong association between FBN1 haplotypes and SSc. Subsequently, studies of FBN1 single nucleotide polymorphisms (SNPs) demonstrated that certain FBN1 haplotypes were associated with SSc in both Native American and Japanese patients with limited scleroderma. Thus, FBN1 was sequenced in 22 Japanese SSc patients to ascertain the presence of any relevant mutations or SNPs. Sequence analysis revealed eight coding and 14 non-coding SNPs and other polymorphisms. Among them, a CT insertion in the 5'-untranslated region of exon A had a significant negative association with disease.

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SPINOCEREBELLAR ATAXIA

See Friedreich Spinocerebellar Ataxia, Ataxia Telangiectasia

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STRANDBERG SYNDROME

See PSEUDOXANTHOMA ELASTICUM (PXE, GROENBLAD-STRANDBERG SYNDROME)

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STROKE

Institute of Histology and General Embriology, School of Medicine, University La Sapienza, Rome, Italy.

In the Sertoli cell, FSH stimulates transcription of a cAMP-phosphodiesterase (PDE) gene (PDE4D) and accumulation of corresponding mRNA and PDE protein. The regulation of this PDE gene is an important component of the desensitization state induced by this hormone. Given the ubiquitous nature of this regulation controlling cAMP levels, the molecular basis for the PDE4D induction was further investigated. FSH stimulation of the Sertoli cell causes the accumulation of only two of the four known PDE4D mRNAs (PDE4D1 and PDE4D2). The promoter controlling the expression of these two messages was identified and characterized. An EcoRI fragment containing a coding exon as well as 5'-upstream sequence of the PDE4D1/2 mRNA was isolated from rat genomic libraries and sequenced. No TATA box was identified, but GC-rich regions were present upstream of the putative translation start site. RNAse protection and PCR analysis indicated the presence of at least two distinct cap sites. This genomic region had promoter activity when transfected both in Sertoli and MA-10 cells. Deletion mutation indicated that basal promoter activity was contributed by regions upstream of both cap sites. Transcription from this promoter was activated by FSH and (Bu)2cAMP, and elements responsible for cAMP regulation were present upstream from the second cap site. These data demonstrate that an intronic promoter that is cAMP- and hormone-inducible directs the expression of these truncated PDE proteins.

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TAY-SACHS DISEASE

A G to C mutation in the splice site of intron 12 has also been identified. This mutation creates a recognition site for the restriction enzyme Ddel resulting in abnormal splicing and the production of aberrant mRNA species.

Ten novel mutations in the HEXA gene in non-Jewish Tay-Sachs patients [published erratum appears in Hum Mol Genet 1993 Apr;2(4):496]

S Akli, JC Chomel, JM Lacorte, L Bachner, A Kahn and L Poenaru

Institut Cochin de Genetique Moleculaire (ICGM), Institut National de la Sante et de la Recherche Medicale, U129, Paris, France.

The heterogeneity of mutations causing Tay-Sachs disease in non-Jewish populations requires efficient techniques allowing the simultaneous screening for both known and novel mutations. beta-hexosaminidase mRNA isolated from cultured fibroblasts of 19 Tay-Sachs patients (7 with adult or late onset form of the disease and 12 with infantile Tay-Sachs disease) was amplified by cDNA-PCR in two overlapping segments spanning the entire coding sequence. We used chemical mismatch cleavage (CMC), denaturing gradient gel electrophoresis (DGGE) and direct sequencing of amplified fragments displaying a cleaved product or an altered melting behavior to screen the HEX A gene for mutations and to determine their distribution and frequency in the non-Jewish Tay-Sachs patients. These methods allowed us to identify 31 out of 38 alleles studied (82%). In addition to 9 previously described mutations (the 4 bp insertion in exon 11, G to A transitions at codons 170, 269, 482, 499 and 504, C to T transition at codon 499 and 504 and a GT to AT transition at the donor site of intron 9), we have identified 10 novel mutations. These include 1 donor splice site defect in intron 6, 8 missense mutations at non-randomly distributed conserved residues and a 2 bp deletion in exon 4. These results confirm the extreme molecular heterogeneity of mutations causing Tay-Sachs disease in non-Jewish population. The strategy used should be profitable for identifying mutations in large genes and for diagnostic purposes.

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TOURETTE SYNDROME

[...] The researchers published their findings in the October 14, 2005, issue of the journal Science. Matthew W. State of the Yale University School of Medicine was senior author of the paper. His research was supported by a Howard Hughes Medical Institute institutional award to Yale that was used to support early research by promising scientists at Yale.

Other co-authors at Yale included HHMI investigator Richard P. Lifton, and neurobiologists Nenad Sestan and Angeliki Louvi from the Yale Child Study Center. The Yale scientists collaborated with researchers from the University of California San Diego, Harvard Medical School, University of Missouri-Kansas City, University of Alabama at Birmingham, Johns Hopkins University School of Medicine and Cincinnati Children's Hospital Medical Center.

According to State, early theories suggesting that a single gene mutation causes TS have proven incorrect. “There has been an evolving hypothesis about Tourette syndrome being a much more complex disorder,” State said. “I think there is general consensus at this point that there are likely to be multiple genes, likely interacting, and probably different sets of genes in different people, that contribute to TS.” The notion of multiple genes is borne out by the complex phenotype of the syndrome, which is often associated with obsessive-compulsive disorder, attention deficit hyperactivity disorder, or depression, said State. [...]

In two people with TS, the researchers found another variant sequence within SLITRK1, in a region of the gene that was not part of the direct blueprint for the SLITRK1 protein. “This was the strongest piece of genetic evidence in the research paper,” said State. “We found two examples of exactly the same rare sequence change in a regulatory, non-coding region of the gene. And it was in two unrelated individuals with TS.” Furthermore, the researchers did not find the alteration, which they called variant 321, in 4,200 chromosomes from individuals without TS.

Variant 321 was located in a region of the genome predicted to be involved in regulating SLITRK1 activity by interacting with molecules called microRNAs. In further studies, the researchers found that a specific microRNA that regulates SLITRK1 binds to the genetic site of variant 321. They also found that SLITRK1 and the regulatory microRNA are both expressed in regions of the brain believed to be involved in TS. Finally, when they studied the function of SLITRK1 in cultures of neurons, they found that those with the normal gene showed longer connective branches, called dendrites, than did those with the mutation.

According to Lifton, “Matt's approach of looking for genetic outliers that contribute to the pathogenesis of Tourette's represents a new approach that has great potential to provide an avenue into the pathways that underlie this disease.”

Lifton pointed out that State's approach is somewhat different than his strategy of analyzing rare genetic abnormalities that tend to run in families. These types of genetic studies are complicated in Tourette syndrome, in part because people with TS tend to marry one another, Lifton noted. [...]

VON WILLEBRAND DISEASE

Comparison of the 5'-flanking sequences of the human and bovine von Willebrand factor-encoding genes reveals alternation of highly homologous domains with species-specific Alu-type repeats.

Janel N, Schwachtgen JL, Bakhshi MR, Barek L, Meyer D, Kerbiriou-Nabias D.

Unite de Recherches sur l'Hemostase et la Thrombose, INSERM U 143, Hopital de Bicetre, France.

von Willebrand factor (vWF), a multimeric glycoprotein important for hemostasis, is specifically synthesized in endothelial cells and in platelet precursors (megakaryocytes). Recent studies from two laboratories, including ours, were published regarding the cell-specific transcription of reporter genes controlled by the human (hu) vWF promoter in transfected bovine (bo) endothelial cells and cells of non-endothelial origins. In order to verify that the regulatory domains previously characterized in the 5' region of hu vWF are also present in bo vWF, we have sequenced 1.9 kb upstream from the cap site, plus five exons. The comparison of human and bovine exons two to five shows homology of 83% at the nucleotide (nt) level and 78% at the deduced amino-acid sequence level. The bovine and human exons one, which are non-coding and span 233 and 250 bp, respectively, are only 64% homologous. In the first exon, potentially involved in endothelial-cell-specific transcription, the binding site for factor Sp1 is present in bo vWF, whereas the GATA sequence is replaced by a GACA sequence. The sequence corresponding to the human basal promoter, located between nt -89 and +19, is well conserved with 82% homology. However, the human TAATTA sequence (at nt -32) considered to be a TATA box, is replaced by TCATTA, and the CCAAT element at nt -18 is replaced by CCTGT. Among domains involved in transcription, the negative regulatory domain located 5' from the core promoter is highly conserved. The bovine sequence upstream from the first intron can be aligned with the human sequence up to nt -656 which is located in a polymorphic poly(GT)18-26 sequence. At this site, the bovine DNA contains an insertion of 523 bp which corresponds to a bovine Alu-type art2 repeat of 331 bp flanked by bovine microsatellites. The art2 sequence is an Alu-type repeat in artiodactyls with at least 100,000 copies in the bovine genome. Upstream from this insertion, 368 bp of the bovine sequence can be aligned with the human counterpart up to a 9-bp element which flanks an human Alu repeat which is absent from the bovine DNA. Upstream of the human Alu insertion and a duplicate of the 9-bp element, the two sequences are again homologous.

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FURTHER READING (PROCESSED)

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Bioinformatics. 2005 Mar;21(6):811-6. Epub 2004 Oct 12.

TRbase: a database relating tandem repeats to disease genes for the human genome.

Boby T, Patch AM, Aves SJ. School of Biological and Chemical Sciences, Washington Singer Laboratories, University of Exeter, Perry Road, Exeter EX4 4QG, UK.

MOTIVATION: Tandem repeats are associated with disease genes, play an important role in evolution and are important in genomic organization and function. Although much research has been done on short perfect patterns of repeats, there has been less focus on imperfect repeats. Thus, there is an acute need for a tandem repeats database that provides reliable and up to date information on both perfect and imperfect tandem repeats in the human genome and relates these to disease genes. RESULTS: This paper presents a web-accessible relational tandem repeats database that relates tandem repeats to gene locations and disease genes of the human genome. In contrast to other available databases, this database identifies both perfect and imperfect repeats of 1-2000 bp unit lengths. The utility of this database has been illustrated by analysing these repeats for their distribution and frequencies across chromosomes and genomic locations and between protein-coding and non-coding regions. The applicability of this database to identify diseases associated with previously uncharacterized tandem repeats is demonstrated.

It has been proposed that variation in calpain 10 (CAPN10) contributes to the risk of type 2 diabetes (T2D). A previous survey of CAPN10 in ethnically diverse populations revealed an intronic region with a significant excess of polymorphism levels relative to inter-species sequence divergence, suggesting that this region was the target of long-standing balancing selection. Based on the thrifty genotype hypothesis, variation that increases risk to T2D in contemporary humans at one time conferred a survival advantage in ancestral populations. Thus, the signature of positive natural selection in a T2D candidate gene could identify a genomic region containing variation that influences disease susceptibility. Here, we investigate this hypothesis by re-sequencing the CAPN10 region with unusual polymorphism levels in T2D cases and controls (n=91) from a Mexican American (MA) population, and by using networks to infer the evolutionary relationships between the major haplotypes. Haplotype tag SNPs (htSNPs) were then selected in each population sample and in MA cases and controls. By placing the htSNPs on the haplotype network, we investigate how cross-population differences in CAPN10 genetic architecture may affect the detection of the disease association. Interestingly, despite the small scale of our case-control study, we observe a nearly significant signal of association between T2D and variation in the putative target of balancing selection. Finally, we use phylogenetic shadowing across 10 primate species to search for conserved non-coding elements that may affect the expression and function of CAPN10. These elements are postulated to be the targets of long-standing balancing selection.

BACKGROUND: Evolutionarily conserved sequences likely have biological function. METHODS: To determine whether variation in conserved sequences in non-coding DNA contributes to risk for human disease, we studied six conserved non-coding elements in the Th2 cytokine cluster on human chromosome 5q31 in a large Hutterite pedigree and in samples of outbred European American and African American asthma cases and controls. RESULTS: Among six conserved non-coding elements (> 100 bp, > 70% identity; human-mouse comparison), we identified one single nucleotide polymorphism (SNP) in each of two conserved elements and six SNPs in the flanking regions of three conserved elements. We genotyped our samples for four of these SNPs and an additional three SNPs each in the IL13 and IL4 genes. While there was only modest evidence for association with single SNPs in the Hutterite and European American samples (P < 0.05), there were highly significant associations in European Americans between asthma and haplotypes comprised of SNPs in the IL4 gene (P < 0.001), including a SNP in a conserved non-coding element. Furthermore, variation in the IL13 gene was strongly associated with total IgE (P = 0.00022) and allergic sensitization to mold allergens (P = 0.00076) in the Hutterites, and more modestly associated with sensitization to molds in the European Americans and African Americans (P < 0.01). CONCLUSION: These results indicate that there is overall little variation in the conserved non-coding elements on 5q31, but variation in IL4 and IL13, including possibly one SNP in a conserved element, influence asthma and atopic phenotypes in diverse populations.

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Pharmacogenomics. 2004 Oct;5(7):803-17.
Pharmacogenomics of adrenoceptors.

Flordellis C, Paris H, Karabinis A, Lymperopoulos A. University of Patras, Department of Pharmacology, School of Medicine, 26504 Rio Patras, Greece. flordell@med.upatras.gr.

Adrenoceptors (ARs) consist of nine subtypes (alpha(1A)-, alpha(1B)-, alpha(1D)-, beta(1)-, beta(2)-, beta(3)-, alpha(2A)-, alpha(2B)- and alpha(2C)-AR), which are involved in a wide spectrum of physiological functions and are the site of action for a considerable percentage of currently prescribed therapeutics. With the exception of alpha(1D), all AR subtypes are polymorphic with genetic variations in the coding and non-coding regions. This review discusses the biochemical consequences of these genetic variations and their impact in receptor function, disease pathophysiology, and drug response. Pharmacogenomic principles that have been discovered are also discussed.
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FEBS Lett. 2004 Jun 1;567(1):42-8.

Viroids: the minimal non-coding RNAs with autonomous replication.
Flores R, Delgado S, Gas ME, Carbonell A, Molina D, Gago S, De la Pena M.

Instituto de Biologia Molecular y Celular de Plantas (UPV-CSIC), Universidad Politecnica de Valencia, 46022 Valencia, Spain. rflores@ibmcp.upv.es

Viroids are small (246-401 nucleotides), non-coding, circular RNAs able to replicate autonomously in certain plants. Viroids are classified into the families Pospiviroidae and Avsunviroidae, whose members replicate in the nucleus and chloroplast, respectively. Replication occurs by an RNA-based rolling-circle mechanism in three steps:

(1). synthesis of longer-than-unit strands catalyzed by host DNA-dependent RNA polymerases forced to transcribe RNA templates, (2). processing to unit-length, which in family Avsunviroidae is mediated by hammerhead ribozymes, and

(3). circularization either through an RNA ligase or autocatalytically. Disease induction might result from the accumulation of viroid-specific small interfering RNAs that, via RNA silencing, could interfere with normal developmental pathways.
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Hematol Oncol. 2005 Jun;23(2):49-53.

New perspectives on neoplasia and the RNA world.

Hall PA, Russell SH. Centre for Cancer Research and Cell Biology, Queen's University Belfast, U Floor, Belfast City Hospital, Belfast, BT9 7AB, Northern Ireland, UK. peter.hall@qub.ac.uk

Key tenets of modern biology are the central place of protein in cell regulation and the flow of genetic information from DNA to RNA to protein. However, it is becoming increasingly apparent that genomes are much more complex than hitherto thought with remarkably complex regulatory systems. The notion that the fraction of the genome involved in coding protein is all that matters is increasingly being questioned as the roles of non-coding RNA (ncRNA) in cellular systems becomes recognised. The RNA world, including microRNA (miRNA), small inhibitory RNA (siRNA) and other RNA species, are now recognised as being crucial for the regulation of chromatin structure, gene expression, mRNA processing and splicing, mRNA stability and translational control. Furthermore such ncRNA systems may be perturbed in disease states and most notably in neoplasia, including in haematological malignancies. Here the burgeoning evidence for a role of miRNA in neoplasia is reviewed and the importance of understanding the RNA world emphasised.
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Curr Opin Genet Dev. 2006 Feb;16(1):4-9. Epub 2005 Dec 19.

MicroRNAs as oncogenes.

Hammond SM. Department of Cell and Developmental Biology, Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC 27599, USA. hammond@med.unc.edu

MicroRNAs (miRNAs) are a class of non-coding RNAs that function as endogenous triggers of the RNA interference pathway. Originally discovered in Caenorhabditis elegans, this group of tiny RNAs has moved to the forefront of biology. With over 300 miRNA genes identified in the human genome, and a plethora of predicted mRNA targets, it is believed that these small RNAs have a central role in diverse cellular and developmental processes. Concordant with this, aberrant expression of miRNA genes could lead to human disease, including cancer. Although the connection of miRNAs with cancer has been suspected for several years, four recent studies have confirmed the suspicion that miRNAs regulate cell proliferation and apoptosis, and play a role in cancer.
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Nature. 2005 Jun 9;435(7043):828-33.

Comment in: Cell. 2005 Jul 15;122(1):6-7. Nature. 2005 Jun 9;435(7043):745-6.

A microRNA polycistron as a potential human oncogene.

He L, Thomson JM, Hemann MT, Hernando-Monge E, Mu D, Goodson S, Powers S, Cordon-Cardo C, Lowe SW, Hannon GJ, Hammond SM. Cold Spring Harbor Laboratory, Watson School of Biological Sciences, 1 Bungtown Road, Cold Spring Harbor, New York 11724, USA.

To date, more than 200 microRNAs have been described in humans; however, the precise functions of these regulatory, non-coding RNAs remains largely obscure. One cluster of microRNAs, the mir-17-92 polycistron, is located in a region of DNA that is amplified in human B-cell lymphomas. Here we compared B-cell lymphoma samples and cell lines to normal tissues, and found that the levels of the primary or mature microRNAs derived from the mir-17-92 locus are often substantially increased in these cancers. Enforced expression of the mir-17-92 cluster acted with c-myc expression to accelerate tumour development in a mouse B-cell lymphoma model. Tumours derived from haematopoietic stem cells expressing a subset of the mir-17-92 cluster and c-myc could be distinguished by an absence of apoptosis that was otherwise prevalent in c-myc-induced lymphomas. Together, these studies indicate that non-coding RNAs, specifically microRNAs, can modulate tumour formation, and implicate the mir-17-92 cluster as a potential human oncogene.
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Intern Med J. 2004 Mar;34(3):79-90.

Systematic genome-wide approach to positional candidate cloning for identification of novel human disease genes.

Kiyosawa H, Kawashima T, Silva D, Petrovsky N, Hasegawa Y, Sakai K, Hayashizaki Y. Technology and Development team for Mammalian Cellular Dynamics, Bioresource Center, RIKEN Tsukuba Institute, Tsukuba, Ibaraki, Japan.

BACKGROUND: Recent large-scale genome projects afford a unique opportunity to identify many novel disease genes and thereby better understand the genetic basis of human disease. Functional Annotation of Mouse (FANTOM) 2, the largest mouse transcriptome project yet, provides a wealth of data on novel genes, splice variants and non-coding RNA, and provides a unique opportunity to identify novel human disease genes. AIMS: To demonstrate the power of combining the FANTOM 2 cDNA dataset with a positional candidate approach and bioinformatics analysis to identify genes underlying human genetic disease. RESULTS: By mapping all FANTOM 2 cDNA to the human genome, we were able to identify mouse clones that co-localised on the human genome with mapped but uncloned human disease loci. By this method we identified mouse and corresponding human genes mapping within the loci of 100 different human genetic diseases (mapped interval of <5 cM). Of particular interest was the elucidation through FANTOM 2 novel mouse gene data of candidate human genes for the following:

(i) developmental -disorders: neural tube defect, Meckel syndrome, Wolf--Hirschhorn syndrome and keratosis follicularis spinulosa decalvans cum ophiasi;

(ii) neurological disorders: benign familial infantile convulsions 3, early-onset cerebellar ataxia with retained tendon reflexes, infantile-onset spinocerebellar ataxia and vacuolar neuro-myopathy and

(iii) cancer-related syndromes: tylosis with oesophageal cancer and low-grade B-cell chronic lymphatic leukaemia.

CONCLUSIONS: The FANTOM 2 data will dramatically accelerate efforts to identify genes underlying human disease. It will also facilitate the creation of transgenic mouse models to help elucidate the function of potential human disease genes.
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J Mol Med. 2005 Feb;83(2):97-109. Epub 2004 Dec 9.

Regulatory polymorphisms underlying complex disease traits.

Knight JC. Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, OX3 7BN, UK. julian@well.ox.ac.uk

There is growing evidence that genetic variation plays an important role in the determination of individual susceptibility to complex disease traits. In contrast to coding sequence polymorphisms, where the consequences of non-synonymous variation may be resolved at the level of the protein phenotype, defining specific functional regulatory polymorphisms has proved problematic. This has arisen for a number of reasons, including difficulties with fine mapping due to linkage disequilibrium, together with a paucity of experimental tools to resolve the effects of non-coding sequence variation on gene expression. Recent studies have shown that variation in gene expression is heritable and can be mapped as a quantitative trait. Allele-specific effects on gene expression appear relatively common, typically of modest magnitude and context specific. The role of regulatory polymorphisms in determining susceptibility to a number of complex disease traits is discussed, including variation at the VNTR of INS, encoding insulin, in type 1 diabetes and polymorphism of CTLA4, encoding cytotoxic T lymphocyte antigen, in autoimmune disease. Examples where regulatory polymorphisms have been found to play a role in mongenic traits such as factor VII deficiency are discussed, and contrasted with those polymorphisms associated with ischaemic heart disease at the same gene locus. Molecular mechanisms operating in an allele-specific manner at the level of transcription are illustrated, with examples including the role of Duffy binding protein in malaria. The difficulty of resolving specific functional regulatory variants arising from linkage disequilibrium is demonstrated using a number of examples including polymorphism of CCR5, encoding CC chemokine receptor 5, and HIV-1 infection. The importance of understanding haplotypic structure to the design and interpretation of functional assays of putative regulatory variation is highlighted, together with discussion of the strategic use of experimental tools to resolve regulatory polymorphisms at a transcriptional level. A number of examples are discussed including work on the TNF locus which demonstrate biological and experimental context specificity. Regulatory variation may also operate at other levels of control of gene expression and the modulation of splicing at PTPRC, encoding protein tyrosine phosphatase receptor-type C, and of translational efficiency at F12, encoding factor XII, are discussed.

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Clin Sci (Lond). 2003 May;104(5):493-501.

Functional implications of genetic variation in non-coding DNA for disease susceptibility and gene regulation.

Knight JC. Wellcome Trust Centre for Human Genetics, Roosevelt Drive, Headington, Oxford OX3 7BN, UK. julian@well.ox.ac.uk

The role of host genetic variation in determining susceptibility to complex disease traits is the subject of much research effort, but it often remains unclear whether disease-associated genetic polymorphisms are themselves functionally relevant or acting only as markers within an extended haplotype. Experimental approaches to investigate the functional impact of polymorphisms in non-coding regulatory DNA sequences for gene expression are discussed, including the role of gel-shift assays, DNA footprinting and reporter gene analysis. The limitations of different experimental approaches are presented together with future prospects for in vivo analysis. The strategic application of these functional approaches is discussed and illustrated by analysis of the role of genetic variation in the tumour necrosis factor promoter region in determining susceptibility to severe malaria.

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Int Immunol. 2005 Nov;17(11):1513-24. Epub 2005 Sep 30.

Chromatin-based regulation of cytokine transcription in Th2 cells and mast cells.

Monticelli S, Lee DU, Nardone J, Bolton DL, Rao A. Department of Pathology, Harvard Medical School, and CBR Institute for Biomedical Research, Boston, MA 02115, USA.

Th2 cells and mast cells are major sources of IL4, IL5 and IL13, cytokines that mediate immunity against parasites and are also central players in the pathophysiology of asthma, allergy and atopic disease. We asked whether Th2 cells and mast cells, which belong to the lymphoid and myeloid lineages, respectively, use different cis-acting regulatory regions to transcribe the cytokine genes. Comparison of DNase I hypersensitivity patterns at the RAD50/IL4/IL13 locus revealed that most hypersensitive sites (HSs) are common to Th2 and mast cells, but two regions [conserved non-coding sequence (CNS) 1 and mast cell HSs] show cell type-specific differences. CNS-1, one of the most highly conserved CNS regions in the RAD50/IL13/IL4 locus, displays two strong DNase I HSs in Th2 cells but is not DNase I hypersensitive in mast cells, explaining a previous finding that deletion of CNS-1 impairs cytokine expression in Th2 cells but not in mast cells. Conversely, two constitutive HSs (mast cell HSs) in the first intron of the IL13 gene are present in mast cells but not in Th2 cells; these sites develop early during mast cell differentiation and may have a role in maintaining accessibility of the IL13 locus to high-level transcription in stimulated cells.

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Oncogene. 2005 Dec 5;

Comprehensive analysis of microRNA expression patterns in hepatocellular carcinoma and non-tumorous tissues.

Murakami Y, Yasuda T, Saigo K, Urashima T, Toyoda H, Okanoue T, Shimotohno K. 1Laboratory of Human Tumor Virus, Institute for Viral Research, Kyoto University, Kyoto, Japan.

MicroRNAs (miRNAs) are a non-coding family of genes involved in post-transcriptional gene regulation. These transcripts are associated with

• cell proliferation,

• cell differentiation,

• cell death and

• carcinogenesis.

We analysed the miRNA expression profiles in 25 pairs of hepatocellular carcinoma (HCC) and adjacent non-tumorous tissue (NT) and nine additional chronic hepatitis (CH) specimens using a human miRNA microarray. Targets and references samples were co-hybridized to a microarray containing whole human mature and precursor miRNA sequences. Whereas three miRNAs exhibited higher expression in the HCC samples than that in the NT samples, five miRNAs demonstrated lower expression in the HCC samples than in the NT samples (P<0.0001). Classification of samples as HCC or NT by using support vector machine algorithms based on these data provided an overall prediction accuracy of 97.8% (45/46). In addition, the expression levels of four miRNAs were inversely correlated with the degree of HCC differentiation (P<0.01). A comparison of CH and liver cirrhosis samples revealed significantly different pattern of miRNA expression (P<0.01). There were no differences, however, between hepatitis B-positive and hepatitis C-positive samples. This information may help clarify the molecular mechanisms involved in the progression of liver disease, potentially serving as a diagnostic tool of HCC.

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Clin Genet. 2006 Feb;69(2):118-119.

Jewels in the rough - the study of non-coding genomic sequences as a conduit to understanding human disease.

Pouladi MA.

Department of Medical Genetics, Center for Molecular Medicine and Therapeutics, University of British Columbia, 980 West 28th Avenue, Vancouver, BC, Canada, V5Z 4H4. Tel.: +1 604 875 3809; fax: +1 604 875 3819; E-mail: pouladi@cmmt.ubc.ca.

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Expert Rev Neurother. 2006 Feb;6(2):223-33.

Silencing neurodegenerative disease: bringing RNA interference to the clinic.

Rodriguez-Lebron E, Gonzalez-Alegre P. Department of Neurology, Carver College of Medicine, University of Iowa, Iowa City, IA 52242-1101, USA. edgardo-rodriguez@mail.medicine.uiowa.edu

RNA interference (RNAi) is a recently described conserved biological pathway where non-coding RNAs suppress the expression of specific genes. Research efforts in the RNAi field aim to gain a better understanding of how its underlying machinery is orchestrated, to define the biological role of this conserved pathway, determine how to effectively manipulate RNAi in the laboratory and to integrate all this knowledge to develop novel therapies for human disease. This review summarizes the advances in the design of therapeutic RNAi for neurodegenerative diseases and discusses some of the experimental steps required to bring this therapy to human clinical trials.

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Curr Opin Genet Dev. 2002 Jun;12(3):266-71.

Dominantly inherited, non-coding microsatellite expansion disorders.

Ranum LP, Day JW. Institute of Human Genetics, University of Minnesota, MMC 206, 420 Delaware Street SE, Minneapolis, Minnesota 55455, USA. ranum001@umn.edu

Dominantly inherited diseases are generally caused by mutations resulting in gain of function protein alterations. However, a CTG expansion located in the 3' untranslated portion of a kinase gene was found to cause myotonic dystrophy type 1, a multisystemic dominantly inherited disorder. The recent discovery that an untranslated CCTG expansion causes the same constellation of clinical features in myotonic dystrophy type 2 (DM2), along with other recent discoveries on DM1 pathogenesis, have led to the understanding that both DM1 and DM2 mutations are pathogenic at the RNA level. These findings indicate the existence of a new category of disease wherein repeat expansions in RNA alter cellular function. Pathogenic repeat expansions in RNA may also be involved in spinocerebellar ataxia types 8, 10 and 12, and Huntington's disease-like type 2.

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J Hum Genet. 2006;51(3):209-16. Epub 2006 Feb 4.

Mutational screening of the RB1 gene in Italian patients with retinoblastoma reveals 11 novel mutations.

Sampieri K, Hadjistilianou T, Mari F, Speciale C, Mencarelli MA, Cetta F, Manoukian S, Peissel B, Giachino D, Pasini B, Acquaviva A, Caporossi A, Frezzotti R, Renieri A, Bruttini M. Medical Genetics, Molecular Biology Department, University of Siena, Policlinico Le Scotte, viale Bracci 2, 53100 Siena, Italy.

Retinoblastoma (RB, OMIM#180200) is the most common intraocular tumour in infancy and early childhood. Constituent mutations in the RB1 gene predispose individuals to RB development. We performed a mutational screening of the RB1 gene in Italian patients affected by RB referred to the Medical Genetics of the University of Siena. In 35 unrelated patients, we identified germline RB1 mutations in 6 out of 9 familial cases (66%) and in 7 out of 26 with no family history of RB (27%). Using the single-strand conformational polymorphism (SSCP) technique, 11 novel mutations were detected, including 3 nonsense, 5 frameshift and 4 splice-site mutations. Only two of these mutations (1 splice site and 1 missense) were previously reported. The mutation spectrum reflects the published literature, encompassing predominately nonsense or frameshift and splicing mutations. RB1 germline mutation was detected in 37% of our cases. Gross rearrangements outside the investigated region, altered DNA methylation, or mutations in non-coding regions, may be the cause of disease in the remainder of the patients. Some cases, e.g. a case of incomplete penetrance, or variable expressivity ranging from retinoma to multiple tumours, are discussed in detail. In addition, a case of pre-conception genetic counselling resolved by rescue of banked cordonal blood of the affected deceased child is described.

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Novartis Found Symp. 2003;254:177-88; discussion 189-92, 216-22, 250-2.

From immunogenetics to immunomics: functional prospecting of genes and transcripts.

Schonbach C. Biomedical Knowledge Discovery Team, Bioinformatics Group, RIKEN Genomic Sciences Center (GSC), 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan.

Human and mouse genome and transcriptome projects have expanded the field of 'immunogenetics' beyond the traditional study of the genetics and evolution of MHC, TCR and Ig loci into the new interdisciplinary area of 'immunomics'. Immunomics is the study of the molecular functions associated with all immune-related coding and non-coding mRNA transcripts. To unravel the function, regulation and diversity of the immunome requires that we identify and correctly categorize all immune-related transcripts. The importance of intercalated genes, antisense transcripts and non-coding RNAs and their potential role in regulation of immune development and function are only just starting to be appreciated. To better understand immune function and regulation, transcriptome projects (e.g. Functional Annotation of the Mouse, FANTOM), that focus on sequencing full-length transcripts from multiple tissue sources, ideally should include specific immune cells (e.g. T cell, B cells, macrophages, dendritic cells) at various states of development, in activated and unactivated states and in different disease contexts. Progress in deciphering immune regulatory networks will require the cooperative efforts of immunologists, immunogeneticists, molecular biologists and bioinformaticians. Although primary sequence analysis remains useful for annotation of new transcripts it is less useful for identifying novel functions of known transcripts in a new context (protein interaction network or pathway). The most efficient approach to mine useful information from the vast a priori knowledge contained in biological databases and the scientific literature, is to use a combination of computational and expert-driven knowledge discovery strategies. This paper will illustrate the challenges posed in attempts to functionally infer transcriptional regulation and interaction of immune-related genes from text and sequence-based data sources.

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Hum Mol Genet. 2005 Sep 1;14(17):2533-46. Epub 2005 Jul 21.

Analysis of intronic conserved elements indicates that functional complexity might represent a major source of negative selection on non-coding sequences.

Sironi M, Menozzi G, Comi GP, Cagliani R, Bresolin N, Pozzoli U. Scientific Institute IRCCS E. Medea, 23842 Bosisio Parini (LC), Italy.

The non-coding portion of human genome is punctuated by a large number of multispecies conserved sequence (MCS) elements with largely unknown function. We demonstrate that MCSs are unevenly distributed in human introns with the majority of relatively short introns (< 9 kb long) displaying no or a few MCSs and that MCS density reaching up to 10% of total size in longer introns. After correction for intron length, MCSs were found to be enriched within genes involved in development and transcription, whereas depleted in immune response loci. Moreover, many central nervous system tissues show a preferential expression of MCS-rich genes and MCS enrichment significantly correlates with gene functional complexity in terms of distinct protein domains. Analysis of human-mouse orthologous pairs indicated a significant association between intronic MCS density and conservation of protein sequence, promoter regions and untranslated sequences. Moreover, MCS density correlates with the predicted occurrence of human-mouse conserved alternative splicing events. These observations suggest that evolution acts on human genes as integrated units of coding and regulatory capacity and that functional complexity might represent a major source of negative selection on non-coding sequences. To substantiate our result, we also searched previously experimentally identified intronic regulatory elements and indicate that about half of these [intronic regulatory elements] sequences map to an MCS; in particular, support to the notion whereby mutations in MCSs can result in human genetic diseases is provided, because three previously identified intronic pathological variations were found to occur within MCSs, and human disease and cancer genes were found significantly enriched in MCSs.

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PLoS Genet. 2005 Dec 16;1(6):e78

Genome-Wide Associations of Gene Expression Variation in Humans.

Stranger BE, Forrest MS, Clark AG, Minichiello MJ, Deutsch S, Lyle R, Hunt S, Kahl B, Antonarakis SE, Tavare S, Deloukas P, Dermitzakis ET. Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, United Kingdom.

The exploration of quantitative variation in human populations has become one of the major priorities for medical genetics. The successful identification of variants that contribute to complex traits is highly dependent on reliable assays and genetic maps. We have performed a genome-wide quantitative trait analysis of 630 genes in 60 unrelated Utah residents with ancestry from Northern and Western Europe using the publicly available phase I data of the International HapMap project. The genes are located in regions of the human genome with elevated functional annotation and disease interest including the ENCODE regions spanning 1% of the genome, Chromosome 21 and Chromosome 20q12-13.2. We apply three different methods of multiple test correction, including Bonferroni, false discovery rate, and permutations. For the 374 expressed genes, we find many regions with statistically significant association of single nucleotide polymorphisms (SNPs) with expression variation in lymphoblastoid cell lines after correcting for multiple tests. Based on our analyses, the signal proximal (cis-) to the genes of interest is more abundant and more stable than distal and trans across statistical methodologies. Our results suggest that regulatory polymorphism is widespread in the human genome and show that the 5-kb (phase I) HapMap has sufficient density to enable linkage disequilibrium mapping in humans. Such studies will significantly enhance our ability to annotate the non-coding part of the genome and interpret functional variation. In addition, we demonstrate that the HapMap cell lines themselves may serve as a useful resource for quantitative measurements at the cellular level.

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Genome Biol. 2003;4(2):R13. Epub 2003 Jan 23.

Genome-wide analysis of microsatellite repeats in humans: their abundance and density in specific genomic regions.

Subramanian S, Mishra RK, Singh L. Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500 007, India.

BACKGROUND: Simple sequence repeats (SSRs) are found in most organisms, and occupy about 3% of the human genome. Although it is becoming clear that such repeats are important in genomic organization and function and may be associated with disease conditions, their systematic analysis has not been reported. This is the first report examining the distribution and density of simple sequence repeats (1-6 base-pairs (bp)) in the entire human genome. RESULTS: The densities of SSRs across the human chromosomes were found to be relatively uniform. However, the overall density of SSR was found to be high in chromosome 19. Triplets and hexamers were more predominant in exonic regions compared to intronic and intergenic regions, except for chromosome Y. Comparison of densities of various SSRs revealed that whereas trimers and pentamers showed a similar pattern (500-1,000 bp/Mb) across the chromosomes, di- tetra- and hexa-nucleotide repeats showed patterns of higher (2,000-3,000 bp/Mb) density. Repeats of the same nucleotide were found to be higher than other repeat types. Repeats of A, AT, AC, AAT, AAC, AAG, AGC, AAAC, AAAT, AAAG, AAGG, AGAT predominate, whereas repeats of C, CG, ACT, ACG, AACC, AACG, AACT, AAGC, AAGT, ACCC, ACCG, ACCT, CCCG and CCGG are rare. CONCLUSIONS: The overall SSR density was comparable in all chromosomes. The density of different repeats, however, showed significant variation. Tri- and hexa-nucleotide repeats are more abundant in exons, whereas other repeats are more abundant in non-coding regions.

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J Neurochem. 2003 Nov;87(4):809-19.

Human catecholamine sulfotransferase (SULT1A3) pharmacogenetics: functional genetic polymorphism.

Thomae BA, Rifki OF, Theobald MA, Eckloff BW, Wieben ED, Weinshilboum RM. Department of Molecular Pharmacology & Experimental Therapeutics, Mayo Medical School-Mayo Clinic, Rochester, Minnesota 55905, USA.

Sulfotransferase (SULT) 1A3 catalyzes the sulfate conjugation of catecholamines and structurally related drugs. As a step toward studies of the possible contribution of inherited variation in SULT1A3 to the pathophysiology of human disease and/or variation in response to drugs related to catecholamines, we have resequenced all seven coding exons, three upstream non-coding exons, exon-intron splice junctions and the 5'-flanking region of SULT1A3 using DNA samples from 60 African-American (AA) and 60 Caucasian-American (CA) subjects. Eight single nucleotide polymorphisms (SNPs) were observed in AA and five in CA subjects, including one non-synonymous cSNP (Lys234Asn) that was observed only in AA subjects with an allele frequency of 4.2%. This change in amino acid sequence resulted in only 28 +/- 4.5% (mean +/- SEM) of the enzyme activity of the wild-type (WT) sequence after transient expression in COS-1 cells, with a parallel decrease (54 +/- 2.2% of WT) in level of SULT1A3 immunoreactive protein. Substrate kinetic studies failed to show significant differences in apparent Km values of the two allozymes for either dopamine (10.5 versus 10.2 micro m for WT and variant, respectively) or the cosubstrate 3'-phosphoadenosine 5'-phosphosulfate (0.114 versus 0.122 micro m, respectively). The decrease in level of immunoreactive protein in response to this single change in amino acid sequence was due, at least in part, to accelerated SULT1A3 degradation through a proteasome-mediated process. These observations raise the possibility of ethnic-specific inherited alterations in catecholamine sulfation in humans.

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Brain Res Mol Brain Res. 2005 Jun 13;137(1-2):174-83. Epub 2005 Apr 8.

Alternative splicing and promoter use in the human GABRA2 gene.

Tian H, Chen HJ, Cross TH, Edenberg HJ. Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, 635 Barnhill Drive, Room 4063E, Indianapolis, IN 46202-5122, USA.

GABA(A) receptors mediate the majority of the fast synaptic inhibition in the mammalian brain. They are the targets of several important drugs, including benzodiazepines, which are used as anxiolytics, sedatives, anti-convulsants, and in the treatment of alcohol withdrawal symptoms. Non-coding variations in GABRA2, the gene encoding the alpha2 subunit, are associated with the risk for alcoholism, suggesting that regulatory differences are important. GABRA2 mRNAs from whole human brain and from three brain regions were examined for evidence of alternative splicing using reverse transcription-PCR and DNA sequencing. A complex pattern of alternative splicing and alternative promoter use of the human GABRA2 mRNA was demonstrated. There are four major isoforms consisting of combinations of two alternative 5' and 3' exons, as well as minor isoforms lacking exon 4 or exon 8. The alternative 5' exons each lie downstream of a functional promoter sequence, as shown by transient transfection assays. The promoter activities of naturally occurring haplotypes differed, indicating genetic differences in gene expression.

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Hum Mutat. 2002 Nov;20(5):368-74.

Different molecular mechanisms underlie genomic deletions in the MLH1 Gene.

Viel A, Petronzelli F, Della Puppa L, Lucci-Cordisco E, Fornasarig M, Pucciarelli S, Rovella V, Quaia M, Ponz de Leon M, Boiocchi M, Genuardi M.

Divisione di Oncologia Sperimentale 1, Centro di Riferimento Oncologico-IRCCS, Aviano, Italy.

In this study we examined a series of 52 patients belonging to hereditary nonpolyposis colorectal cancer (HNPCC) or HNPCC-related families, all who had previously tested negative for mismatch repair (MMR) gene point mutations. Southern blot mutational screening of MLH1 and MSH2 genes was carried out with the aim of detecting large genomic rearrangements and of identifying the molecular mechanisms underlying the inactivation of the MMR genes. Three patients had abnormal restriction patterns and were found to carry distinct MLH1 internal deletions. Long-range PCRs identified the loss of DNA tracts spanning exon 6 (about 2.4 kb in proband A-AV20 and 0.8 kb in proband A-PD5) and exon 3 (about 2.5 kb in proband R-RM2). In A-AV20 the breakpoints occurred into identical 33-bp regions in introns 5 and 6 and a mechanism of classical Alu-mediated homologous recombination was evident. Also, in patient A-PD5 the breakpoints were located in these introns, but without direct involvement of repetitive sequences. In patient R-RM2 the breakpoints were located within repetitive L1 elements with poor homology in intron 2 and 3 and the rearranged allele was characterized by a complex insertion deletion (delCCinsACATAGTA), giving rise to a palindromic CTTAACATAGTATGTTAAG sequence in proximity of the fusion site. This study confirms that genomic rearrangements are an important component of the spectrum of MMR mutations. Although Alu repeats are likely to be implicated in the majority of cases, different molecular mechanisms may also be responsible for the observed MLH1 intragenic deletions. In particular, HNPCC resulting from L1-mediated recombination has been identified as a novel mechanism for MMR inactivating mutation.

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Yi Chuan. 2005 Jul;27(4):539-43.

[Polymorphisms screening of PON gene cluster][Article in Chinese]

Wang XL, Fan ZJ, Huang JF, Su SY, Zhao JG, Gu DF. Division of Population Genetics and Prevention, Fuwai Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100037, China. wangxl_74@yahoo.com

To identify all putative functional polymorphisms of PON gene cluster in Chinese Han population. Common polymorphisms of PON1, PON2 and PON3 gene were identified by directly sequencing of genomic DNAs derived from 48 randomly selected patients with coronary heart disease. We designed PCR arrays to amplify regions up to about 1kb upstream from transcription-initiation sites, i.e., putative promoter regions, all exons and adjacent non-coding regions. In a total length of 13.9 kb explored, we identified thirty-one SNPs, of which, 17 were first reported. A new coding polymorphism was detected in PON1 gene, which gives rise to amino acid substitutions of arginine (R) for glycine (G) at codon 160, whereas L54M polymorphism, which is common in white population, was not detected in our Han population. Among the five polymorphisms identified in PON3 gene, one in the promoter regions at position -133 (C/A) was located in a potential binding site for transcription factor LF-A1. Allele frequencies of some polymorphisms are significantly different from those reported in Caucasian populations. Complete or nearly complete association between polymorphisms was frequently observed. The identified multiple putative functional polymorphisms in PON gene cluster and their linkage disequilibrium patterns in combination with the population specific frequencies are of values for further association studies of PON gene cluster with cardiovascular disease.

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