, and Iris Schrijver, MD 22 Purpose: The aim of the study was to determine the actual GJB2 and GJB6 mutation frequencies in North America after several years of generalized testing for autosomal recessive nonsyndromic sensorineural hearing loss to help guide diagnostic testing algorithms, especially in light of molecular diagnostic follow-up to universal newborn hearing screening. Methods:Mutation types, frequencies, ethnic distributions, and genotype-phenotype correlations for GJB2 and GJB6 were assessed in a very large North American cohort. Results: GJB2 variants were identified in 1796 (24.3%) of the 7401 individuals examined, with 399 (5.4%) homozygous and 429 (5.8%) compound heterozygous. GJB6 deletion testing was performed in 12.0% (888/7401) of all cases. The Ͼ300-kb deletion was identified in only nine individuals (1.0%), all of whom were compound heterozygous for mutations in GJB2 and GJB6. Among a total of 139 GJB2 variants identified, 53 (38.1%) were previously unreported, presumably representing novel pathogenic or benign variants. Conclusions: The frequency and distribution of sequence changes in GJB2 and GJB6 in North America differ from those previously reported, suggesting a considerable role for loci other than GJB2 and GJB6 in the etiology of autosomal recessive nonsyndromic sensorineural hearing loss, with minimal prevalence of the GJB6 deletion. Genet Med 2007:9(7):413-426.

DEAD box proteins are putative RNA helicases that function in all aspects of RNA metabolism, including translation, ribosome biogenesis, and pre-mRNA splicing. Because many processes involving RNA metabolism are spatially organized within the cell, we examined the subcellular distribution of a human DEAD box protein, DDX1, to identify possible biological functions. Immunofluorescence labeling of DDX1 demonstrated that in addition to widespread punctate nucleoplasmic labeling, DDX1 is found in discrete nuclear foci ϳ0.5 m in diameter. Costaining with anti-Sm and anti-promyelocytic leukemia (PML) antibodies indicates that DDX1 foci are frequently located next to Cajal (coiled) bodies and less frequently, to PML bodies. Most importantly, costaining with anti-CstF-64 antibody indicates that DDX1 foci colocalize with cleavage bodies. By microscopic fluorescence resonance energy transfer, we show that labeled DDX1 resides within a Fö rster distance of 10 nm of labeled CstF-64 protein in both the nucleoplasm and within cleavage bodies. Coimmunoprecipitation analysis indicates that a proportion of CstF-64 protein resides in the same complex as DDX1. These studies are the first to identify a DEAD box protein associating with factors involved in 3Ј-end cleavage and polyadenylation of pre-mRNAs. INTRODUCTIONDEAD box proteins are a family of putative RNA helicases found in all cellular organisms and in some viruses. They are characterized by eight conserved amino acid motifs, including the core DEAD (Asp-Glu-Ala-Asp) motif involved in ATP hydrolysis and coupling of ATPase and RNA helicase activity (Pause and Sonenberg, 1992). At least 14 human DEAD box proteins have been identified to date, summarized in the DExH/D protein family database (Jankowsky and Jankowsky, 2000). DEAD box proteins are thought to modulate RNA secondary structure in all cellular processes involving RNA, including transcription, pre-mRNA processing, ribosome biogenesis, RNA export, translation initiation, and RNA degradation (Schmid and Linder, 1992;de la Cruz et al., 1999). Although many of the biological functions of the prokaryotic and lower eukaryotic DEAD box proteins have been identified, DEAD box proteins in higher eukaryotes remain largely uncharacterized.DDX1 is a human DEAD box protein that was identified by differential screening of a cDNA library enriched in transcripts present in two retinoblastoma (RB) cell lines: Y79 and RB522A (Godbout and Squire, 1993). The 2.7-kb DDX1 transcript encodes a protein with a predicted molecular mass of 82.4 kDa (Godbout et al., 1998). In addition to the eight conserved DEAD box family motifs, DDX1 also contains a region with homology to heterogeneous nuclear ribonucleoprotein U (hnRNP U) (Godbout et al., 1994). HnRNP U or scaffold attachment factor A, a protein located in the nuclear matrix, has recently been shown to function as a repressor of RNA polymerase II elongation by inhibiting transcription factor TFIIH-mediated carboxyl-terminal domain phosphorylation (Kim and Nikodem, 1999). Interestingly, the ...

DDX1 bodies, cleavage bodies, Cajal bodies (CBs), and gems are nuclear suborganelles that contain factors involved in RNA transcription and/or processing. Although all four nuclear bodies can exist as distinct entities, they often colocalize or overlap with each other. To better understand the relationship between these four nuclear bodies, we examined their spatial distribution as a function of the cell cycle. Here, we report that whereas DDX1 bodies, CBs and gems are present throughout interphase, CPSF-100-containing cleavage bodies are predominantly found during S and G2 phases, whereas CstF-64-containing cleavage bodies are primarily observed during S phase. All four nuclear bodies associate with each other during S phase, with cleavage bodies colocalizing with DDX1 bodies, and cleavage bodies/DDX1 bodies residing adjacent to gems and CBs. Although inhibitors of RNA transcription had no effect on DDX1 bodies or cleavage bodies, inhibitors of DNA replication resulted in loss of CstF-64-containing cleavage bodies. A striking effect on nuclear structures was observed with latrunculin B, an inhibitor of actin polymerization, resulting in the formation of needlelike nuclear spicules made up of CstF-64, CPSF-100, RNA, and RNA polymerase II. Our results suggest that cleavage body components are highly dynamic in nature.

The remarkable progress in characterizing the human genome sequence, exemplified by the Human Genome Project and the HapMap Consortium, has led to the perception that knowledge and the tools (e.g., microarrays) are sufficient for many if not most biomedical research efforts. A large amount of data from diverse studies proves this perception inaccurate at best, and at worst, an impediment for further efforts to characterize the variation in the human genome. Since variation in genotype and environment are the fundamental basis to understand phenotypic variability and heritability at the population level, identifying the range of human genetic variation is crucial to the development of personalized nutrition and medicine. The Human Variome Project (HVP; http://www.humanvariomeproject.org/) was proposed initially to systematically collect mutations that cause human disease and create a cyber infrastructure to link locus specific databases (LSDB). We report here the discussions and recommendations from the 2008 HVP planning meeting held in San Feliu de Guixols, Spain, in May 2008.

The spinocerebellar ataxias (SCAs) are a large group of neurological disorders which may be hereditary or sporadic. The core clinical features of gait and limb ataxia are manifestations of degenerations of the cerebellum and its connections. Other neurological systems are variably involved producing features such as extraocular movement abnormalities, pyramidal tract dysfunction, sensory loss, bulbar dysfunction, and movement disorders such as parkinsonism, dystonia and tremor. The dominantly inherited SCAs were classified in the past according to a scheme suggested by Harding.1 In this system, the autosomal dominant cerebellar ataxias (ADCA) were separated clinically into three types. In addition to cerebellar ataxia, ADCA I patients had variable degrees of dementia, supranuclear ABSTRACT: Background: The spinocerebellar ataxias (SCAs) are a genetically and clinically heterogeneous group of neurodegenerative disorders. Relative frequencies vary within different ethnic groups and geographical locations. Objectives: 1) To determine the frequencies of hereditary and sporadic adult onset SCAs in the Movement Disorders population; 2) to assess if the fragile X mental retardation gene 1 (FMR1) premutation is found in this population. Methods: A retrospective chart review of individuals with a diagnosis of adult onset SCA was carried out. Testing for SCA types 1, 2, 3, 6, 7, and 8, Dentatorubral-pallidoluysian atrophy (DRPLA), Friedreich ataxia and the FMR1 expansion was performed. Results: A total of 69 patients in 60 families were identified. Twenty-one (35%) of the families displayed autosomal dominant and two (3.3%) showed autosomal recessive (AR) pattern of inheritance. A positive but undefined family history was noted in nine (15%). The disorder appeared sporadic in 26 patients (43.3%). In the AD families, the most common mutation was SCA3 (23.8%) followed by SCA2 (14.3%) and SCA6 (14.3%). The SCA1 and SCA8 were each identified in 4.8%. FA was found in a pseudodominant pedigree, and one autosomal recessive pedigree. One sporadic patient had a positive test (SCA3).Dentatorubral-pallidoluysian atrophy and FMR1 testing was negative. Conclusion: A positive family history was present in 53.3% of our adult onset SCA patients. A specific genetic diagnosis could be given in 61.9% of dominant pedigrees with SCA3 being the most common mutation, followed by SCA2 and SCA6. The yield in sporadic cases was low. The fragile X premutation was not found to be responsible for SCA.RÉSUMÉ: Ataxie spinocérébelleuse débutant chez l'adulte dans une clinique de désordres du mouvement au Canada. Contexte: Les ataxies spinocérébelleuses (ASCs) constituent un groupe hétérogène de maladies neurodégénératives tant au point de vue génétique qu'au point de vue clinique. Leur fréquence relative est très variable selon le groupe ethnique et le lieu géographique. Objectifs: 1) déterminer la fréquence d'ASCs héréditaires et sporadiques débutant chez l'adulte chez des patients fréquentant une clinique de désordres du mouvement ; 2) évaluer si on r...