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Motivation: Only a few large systematic studies have evaluated the impact of copy number variants (CNVs) on common diseases. Several million individuals have been genotyped on single nucleotide variation arrays, which could be used for genome-wide CNVs association studies. However, CNV calls remain prone to false positives and only empirical filtering strategies exist in the literature. To overcome this issue, we defined a new quality score (QS) estimating the probability of a CNV called by PennCNV to be confirmed by other software. Results: Out-of-sample comparison showed that the correlation between the consensus CNV status and the QS is twice as high as it is for any previously proposed CNV filters. ROC curves displayed an AUC higher than 0.8 and simulations showed an increase up to 20% in statistical power when using QS in comparison to other filtering strategies. Superior performance was confirmed also for alternative consensus CNV definition and through improving known CNV-trait associations. Availability and Implementation: http://goo.gl/T6yuFM Contact: zoltan.kutalik@unil.ch or aurelien@mace@unil.ch Supplementary information: Supplementary data are available at Bioinformatics online.

Accumulation of fat in the liver increases the risk to develop fibrosis and cirrhosis and is associated with development of the metabolic syndrome. Here, to identify genes or gene pathways that may underlie the genetic susceptibility to fat accumulation in liver, we studied A/J and C57Bl/6 mice that are resistant and sensitive to diet-induced hepatosteatosis and obesity, respectively. We performed comparative transcriptomic and lipidomic analysis of the livers of both strains of mice fed a high fat diet for 2, 10, and 30 days. We found that resistance to steatosis in A/J mice was associated with the following: (i) a coordinated up-regulation of 10 genes controlling peroxisome biogenesis and β-oxidation; (ii) an increased expression of the elongase Elovl5 and desaturases Fads1 and Fads2. In agreement with these observations, peroxisomal β-oxidation was increased in livers of A/J mice, and lipidomic analysis showed increased concentrations of long chain fatty acid-containing triglycerides, arachidonic acid-containing lysophosphatidylcholine, and 2-arachidonylglycerol, a cannabinoid receptor agonist. We found that the anti-inflammatory CB2 receptor was the main hepatic cannabinoid receptor, which was highly expressed in Kupffer cells. We further found that A/J mice had a lower pro-inflammatory state as determined by lower plasma levels and IL-1β and granulocyte-CSF and reduced hepatic expression of their mRNAs, which were found only in Kupffer cells. This suggests that increased 2-arachidonylglycerol production may limit Kupffer cell activity. Collectively, our data suggest that genetic variations in the expression of peroxisomal β-oxidation genes and of genes controlling the production of an anti-inflammatory lipid may underlie the differential susceptibility to diet-induced hepatic steatosis and pro-inflammatory state.

Polysaccharide sidechains attached to proteins play important roles in cell-cell and receptor-ligand interactions. Variation in the carbohydrate component has been extensively studied for the iron transport protein transferrin, because serum levels of the transferrin isoforms asialotransferrin 1 disialotransferrin (carbohydrate-deficient transferrin, CDT) are used as biomarkers of excessive alcohol intake. We conducted a genome-wide association study to assess whether genetic factors affect CDT concentration in serum. CDT was measured in three population-based studies: one in Switzerland (CoLaus study, n = 5181) and two in Australia (n = 1509, n = 775). The first cohort was used as the discovery panel and the latter ones served as replication. Genome-wide single-nucleotide polymorphism (SNP) typing data were used to identify loci with significant associations with CDT as a percentage of total transferrin (CDT%). The top three SNPs in the discovery panel (rs2749097 near PGM1 on chromosome 1, and missense polymorphisms rs1049296, rs1799899 in TF on chromosome 3) were successfully replicated, yielding genome-wide significant combined association with CDT% (P = 1.9 × 10 -9 , 4 × 10 -39 , 5.5 × 10 -43 , respectively) and explain 5.8% of the variation in CDT%. These allelic effects are postulated to be caused by variation in availability of glucose-1-phosphate as a precursor of the glycan (PGM1), and variation in transferrin (TF) structure. Refereed/Peer-reviewed

In previous studies, we identified a locus for schizophrenia on 6q23.3 and proposed the Abelson helper integration site 1 (AHI1) as the candidate gene. AHI1 is expressed in the brain and plays a key role in neurodevelopment, is involved in Joubert syndrome, and has been recently associated with autism. The neurodevelopmental role of AHI1 fits with etiological hypotheses of schizophrenia. To definitively confirm our hypothesis, we searched for associations using a dense map of the region. Our strongest findings lay within the AHI1 gene: single-nucleotide polymorphisms rs11154801 and rs7759971 showed significant associations (P=6.23E-06; P=0.84E-06) and haplotypes gave P values in the 10E-8 to 10E-10 range. The second highest significant region maps close to AHI1 and includes the intergenic region between BC040979 and PDE7B (rs2038549 at P=9.70E-06 and rs1475069 at P=6.97E-06), and PDE7B and MAP7. Using a sample of Palestinian Arab families to confirm these findings, we found isolated signals. While these results did not retain their significance after correction for multiple testing, the joint analysis across the 2 samples supports the role of AHI1, despite the presence of heterogeneity. Given the hypothesis of positive selection of schizophrenia genes, we resequenced a 11 kb region within AHI1 in ethnically defined populations and found evidence for a selective sweep. Network analysis indicates 2 haplotype clades, with schizophrenia-susceptibility haplotypes clustering within the major clade. In conclusion, our data support the role of AHI1 as a susceptibility gene for schizophrenia and confirm it has been subjected to positive selection, also shedding light on new possible candidate genes, MAP7 and PDE7B.-Torri, F., Akelai, A., Lupoli, S., Sironi, M., Amann-Zalcenstein, D., Fumagalli, M., Dal Fiume, C., BenAsher, E., Kanyas, K., Cagliani, R., Cozzi, P., Trombetti, G., Lievers, L. S., Salvi, E., Orro, A., Beckmann, J. S., Lancet, D., Kohn, Y., Milanesi, L., Ebstein, R. B., Lerer, B., Macciardi, F. Fine mapping of AHI1 as a schizophrenia susceptibility gene: from association to evolutionary evidence.

Background Experimental evidences show that glutathione and its rate-limiting synthesizing enzyme, the glutamate-cysteine ligase (GCL), are involved in the pathogenesis of schizophrenia. Furthermore, genetic association has been previously reported between two single nucleotide polymorphisms lying in noncoding regions of glutamate cysteine ligase modifier (GCLM) gene, which specifies for the modifier subunit of GCL and schizophrenia. Objective We wanted to investigate the presence of GCLM true functional mutations, likely in linkage disequilibrium with the previously identified single nucleotide polymorphism alleles, in the same set of cases that allowed the detection of the original association signal. Methods We screened all the coding regions of GCLM and their intronic flanking vicinities in 353 patients with schizophrenia by direct DNA sequencing. Results Ten sequence variations were identified, five of which were not previously described. None of these DNA changes was within the GCLM coding sequence and in-silico analysis failed to indicate functional impairment induced by these variations. Furthermore, screening of normal controls and downstream statistical analyses revealed no significant relationship of any of these DNA variants with schizophrenia. Conclusion It is unlikely that functional mutations in the GCLM gene could play a major role in genetic predisposition to schizophrenia and further studies will be required to assess its etiological function in the disease.

The development of chemoresistance represents a major obstacle in the successful treatment of cancers such as neuroblastoma (NB), a particularly aggressive childhood solid tumour. The mechanisms underlying the chemoresistant phenotype in NB were addressed by gene expression profiling of two doxorubicin (DoxR)-resistant vs sensitive parental cell lines. Not surprisingly, the MDR1 gene was included in the identified upregulated genes, although the highest overexpressed transcript in both cell lines was the frizzled-1 Wnt receptor (FZD1) gene, an essential component of the Wnt/beta-catenin pathway. FZD1 upregulation in resistant variants was shown to mediate sustained activation of the Wnt/beta-catenin pathway as revealed by nuclear beta-catenin translocation and target genes transactivation. Interestingly, specific micro-adapted short hairpin RNA (shRNAmir)-mediated FZD1 silencing induced parallel strong decrease in the expression of MDR1, another beta-catenin target gene, revealing a complex, Wnt/beta-catenin-mediated implication of FZD1 in chemoresistance. The significant restoration of drug sensitivity in FZD1-silenced cells confirmed the FZD1-associated chemoresistance. RNA samples from 21 patient tumours (diagnosis and postchemotherapy), showed a highly significant FZD1 and/or MDR1 overexpression after treatment, underlining a role for FZD1-mediated Wnt/beta-catenin pathway in clinical chemoresistance. Our data represent the first implication of the Wnt/beta-catenin pathway in NB chemoresistance and identify potential new targets to treat aggressive and resistant NB.

Summary. The genes encoding bovine prolactin and rhodopsin were assigned to syntenic groups on the basis of hybridization of DNA from a panel of bovine-hamster hybrid somatic cell lines with cloned prolactin and rhodopsin gene probes. Prolactin was found to be syntenic with previously mapped glyoxalase, BoLA and 21-hydroxylase genes, establishing a syntenic conservation with human chromosome 6. The presence of bovine rhodopsin sequences among the various hybrid cell lines was not concordant with any gene previously assigned to one of the 23 defined autosomal syntenic groups. Thus, rhodopsin marks a new bovine syntenic group, U24, leaving only five cattle autosomes unmarked by at least one biochemical or molecular marker.

Two bovine populations, a Holstein-Friesian dairy stock and a synthetic (Baladi X Hereford X Simmental X Charolais) beef stock, were screened for restriction fragment length polymorphisms (RFLPs) at the growth hormone and prolactin genes. Most RFLPs at the growth hormone gene are apparently the consequence of an insertion/deletion event which was localized to a region downstream of the structural gene. The restriction map for the genomic region including the growth hormone gene was extended. Two HindIII RFLPs at the growth hormone locus, as well as several RFLPs at the prolactin gene, seemed to be the consequence of a series of point mutations. The results are discussed in terms of the possibility that minor genomic variability underlies quantitative genetic variation.

Loss-of-function mutations in calpain 3 have been shown to cause limb-girdle muscular dystrophy type 2A (LGMD2A), an autosomal recessive disorder that results in gradual wasting of the muscles of the hip and shoulder areas. Due to the inherent instability of calpain 3, recombinant expression of the full-length enzyme has not been possible, making in vitro analysis of specific LGMD2A-causing mutations difficult. However, because calpain 3 is highly similar in amino acid sequence to calpain 2, the recently solved crystal structure of full-length, Ca2+-bound, calpastatin-inhibited rat calpain 2 has allowed us to model calpain 3 as a Ca2+-bound homodimer. The model revealed three distinct areas of the enzyme that undergo a large conformational change upon Ca2+ binding. Located in these areas are several residues that undergo mutation to cause LGMD2A. We investigated the in vitro effects of six of these mutations by making the corresponding mutations in rat calpain 2. All six mutations examined in this study resulted in a decrease in enzyme activity. All but one of the mutations caused an increased rate of autoproteolytic degradation of the enzyme as witnessed by SDS-PAGE, indicating the decrease in enzyme activity is caused, at least in part, by an increase in the rate of autoproteolytic degradation. The putative in vivo effects of these mutations on calpain 3 activity are discussed with respect to their ability to cause LGMD2A.

Transfection with polyethylenimine (PEI) was evaluated as a method for the generation of recombinant Chinese hamster ovary (CHO DG44) cell lines by direct comparison with calcium phosphate-DNA coprecipitation (CaPO4) using both green fluorescent protein (GFP) and a monoclonal antibody as reporter proteins. Following transfection with a GFP expression vector, the proportion of GFP-positive cells as determined by flow cytometry was fourfold higher for the PEI transfection as compared to the CaPO4 transfection. However, the mean level of transient GFP expression for the cells with the highest level of fluorescence was twofold greater for the CaPO4 transfection. Fluorescence in situ hybridization on metaphase chromosomes from pools of cells grown under selective pressure demonstrated that plasmid integration always occurred at a single site regardless of the transfection method. Importantly, the copy number of integrated plasmids was measurably higher in cells transfected with CaPO4. The efficiency of recombinant cell line recovery under selective pressure was fivefold higher following PEI transfection, but the average specific productivity of a recombinant antibody was about twofold higher for the CaPO4-derived cell lines. Nevertheless, no difference between the two transfection methods was observed in terms of the stability of protein production. These results demonstrated the feasibility of generating recombinant CHO-derived cell lines by PEI transfection. However, this method appeared inferior to CaPO4 transfection with regard to the specific productivity of the recovered cell lines.

Variation in cellular gene expression levels has been shown to be inherited. Expression is controlled at transcriptional and post-transcriptional levels. Internal ribosome entry sites (IRES) are used by viruses to bypass inhibition of cap-dependent translation, and by eukaryotic cells to control translation under conditions when protein synthesis is inhibited. We aimed at identifying genomic determinants of variability in IRES-mediated translation of viral [Encephalomyocarditis virus (EMCV)] and cellular IRES [X-linked inhibitor-of-apoptosis (XIAP) and c-myc]. Bicistronic lentiviral constructs expressing two fluorescent reporters were used to transduce laboratory and B lymphoblastoid cell lines [15 CEPH pedigrees (n = 205) and 50 unrelated individuals]. IRES efficiency varied according to cell type and among individuals. Control of IRES activity has a significant genetic component (h(2) of 0.47 and 0.36 for EMCV and XIAP, respectively). Quantitative linkage analysis identified a suggestive locus (LOD 2.35) on chromosome 18q21.2, and genome-wide association analysis revealed of a cluster of SNPs on chromosome 3, intronic to the FHIT gene, marginally associated (P = 5.9E-7) with XIAP IRES function. This study illustrates the in vitro generation of intermediate phenotypes by using cell lines for the evaluation of genetic determinants of control of elements such as IRES.

In high hyperdiploid acute lymphoblastic leukemia (ALL), the concurrence of specific trisomies confers a more favorable outcome than hyperdiploidy alone. Interphase fluorescence in situ hybridization (FISH) complements conventional cytogenetics (CC) through its sensitivity and ability to detect chromosome aberrations in nondividing cells. To overcome the limits of manual I-FISH, we developed an automated four-color I-FISH approach and assessed its ability to detect concurrent aneuploidies in ALL. I-FISH was performed using centromeric probes for chromosomes 4, 6, 10, and 17. Parameters established for nucleus selection and signal detection were evaluated. Cutoff values were determined. Combinations of aneuploidies were considered relevant when each aneuploidy was individually significant. Results obtained in 10 patient samples were compared with those obtained with CC. Various combinations of aneuploidies were identified. All clones detected by CC were observed also by I-FISH, and I-FISH revealed numerous additional abnormal clones in all patients, ranging from ≤1% to 31.6% of cells analyzed. We conclude that four-color automated I-FISH permits the identification of concurrent aneuploidies of potential prognostic significance. Large numbers of cells can be analyzed rapidly. The large number of nuclei scored revealed a high level of chromosome variability both at diagnosis and relapse, the prognostic significance of which is of considerable clinical interest and merits further evaluation.

The extensive variability of individual human genomes contributes to phenotypic variability. Structural genomic variants, and copy number variants (CNVs) in particular, have recently been rediscovered as contributors to the genomic plasticity and evolution and as pathoetiologic elements for both monogenic and complex traits. Herein we review some of the consequences of CNVs in the context of human inherited diseases.

OBJECTIVE: The ABCB1 (MDR1) gene, encoding the transporter P-glycoprotein, is known to act on a broad range of prescription medicines. For this reason a large number of studies have assessed the functional consequences of variation in this gene. Particular attention has focused on the ABCB1_3435C>T polymorphism, an exonic variant resulting in a synonymous change. This variant has been associated with mRNA, protein and serum levels, and with responses to a number of medicines. The results of association studies have, however, been variable and it is not currently clear whether this polymorphism is functional or is in linkage disequilibrium with functionally distinct alleles. RESULTS: To identify functional variation in the ABCB1 gene we assessed allelic imbalance by pyrosequencing cDNA from 80 lymphoblastoid B cell lines from the Centre d'Etude du Polymorphisme Humain (CEPH) collection, including 74 individuals heterozygous for 3435C>T. We found that the degree of ABCB1 allelic imbalance differed among B-cell lines. In an effort to fine-map variants that influence the proportion of the two allelic mRNA species we genotyped representative common variations near the 3435C>T polymorphism by using a tagging single nucleotide polymorphism (SNP) approach. In one approach, we assessed in segregating families the impact of cis-acting variants on mRNA levels by using allelic imbalance as the phenotype in a regression analysis that distinguishes the coupling arrangements (phase) among alleles. In a second approach, we assessed allelic imbalance levels in lymphoblastoid B-cell lines from unrelated HapMap individuals, and performed an association using tagSNPs in a 5-Mb region surrounding the gene. Two potential cis-acting variants, a promoter rs28656907/rs28373093 dinucleotide polymorphism (P=0.007) and the rs10245483 SNP (P=0.0003) located 2 Mb upstream from the gene, were predictors of ABCB1 expression. CONCLUSIONS: The study outlines a general experimental approach for fine mapping gene variants that influence mRNA expression by using cultured cell lines.

Gene transfer in eukaryotic cells and organisms suffers from epigenetic effects that result in low or unstable transgene expression and high clonal variability. Use of epigenetic regulators such as matrix attachment regions (MARs) is a promising approach to alleviate such unwanted effects. Dissection of a known MAR allowed the identification of sequence motifs that mediate elevated transgene expression. Bioinformatics analysis implied that these motifs adopt a curved DNA structure that positions nucleosomes and binds specific transcription factors. From these observations, we computed putative MARs from the human genome. Cloning of several predicted MARs indicated that they are much more potent than the previously known element, boosting the expression of recombinant proteins from cultured cells as well as mediating high and sustained expression in mice. Thus we computationally identified potent epigenetic regulators, opening new strategies toward high and stable transgene expression for research, therapeutic production or gene-based therapies.

Genetic-based optimization of treatment prescription is becoming a central research focus in the management of chronic diseases, such as multiple sclerosis, which incur a prolonged drug-regimen adjustment. This study was aimed to identify genetic markers that can predict response to glatiramer acetate (Copaxone) immunotherapy for relapsing multiple sclerosis. For this purpose, we genotyped fractional cohorts of two glatiramer acetate clinical trials for HLA-DRB1*1501 and 61 single nucleotide polymorphisms within a total of 27 candidate genes. Statistical analyses included single nucleotide polymorphism-by-single nucleotide polymorphism and haplotype tests of drug-by-genotype effects in drug-treated versus placebo-treated groups. We report the detection of a statistically significant association between glatiramer acetate response and a single nucleotide polymorphism in a T-cell receptor beta (TRB@) variant replicated in the two independent cohorts (odds ratio=6.85). Findings in the Cathepsin S (CTSS) gene (P=0.049 corrected for all single nucleotide polymorphisms and definitions tested, odds ratio=11.59) in one of the cohorts indicate a possible association that needs to be further investigated. Additionally, we recorded nominally significant associations of response with five other genes, MBP, CD86, FAS, IL1R1 and IL12RB2, which are likely to be involved in glatiramer acetate's mode-of-action, both directly and indirectly. Each of these association signals in and of itself is consistent with the no-association null-hypothesis, but the number of detected associations is surprising vis-à-vis chance expectation. Moreover, the restriction of these associations to the glatiramer acetate-treated group, rather than the placebo group, clearly demonstrates drug-specific genetic effects. These findings provide additional progress toward development of pharmacogenetics-based personalized treatment for multiple sclerosis.

A considerable and unanticipated plasticity of the human genome, manifested as inter-individual copy number variation, has been discovered. These structural changes constitute a major source of inter-individual genetic variation that could explain variable penetrance of inherited (Mendelian and polygenic) diseases and variation in the phenotypic expression of aneuploidies and sporadic traits, and might represent a major factor in the aetiology of complex, multifactorial traits. For these reasons, an effort should be made to discover all common and rare copy number variants (CNVs) in the human population. This will also enable systematic exploration of both SNPs and CNVs in association studies to identify the genomic contributors to the common disorders and complex traits.

Limb girdle muscular dystrophy type 2A results from mutations in the gene encoding the calpain 3 protease. Mutations in this disease are inherited in an autosomal recessive fashion and result in progressive proximal skeletal muscle wasting but no cardiac abnormalities. Calpain 3 has been shown to proteolytically cleave a wide variety of cytoskeletal and myofibrillar proteins and to act upstream of the ubiquitin-proteasome pathway. In this review, we summarize the known biochemical and physiological features of calpain 3 and hypothesize why mutations result in disease.

Our previous work demonstrated that the sterol response element binding proteins (SREBP)-1 and SREBP-2, which are the key regulators of storage lipid and cholesterol metabolism respectively, are highly expressed in Schwann cells of adult peripheral nerves. In order to evaluate the role of Schwann cell SREBPs in myelination and functioning of peripheral nerves we have determined their expression during development, after fasting and refeeding, and in a rodent model of diabetes. Our results show that SREBP-1c and SREBP-2, unlike SREBP-1a, are the major forms of SREBPs present in peripheral nerves. The expression profile of SREBP-2 follows the expression of genes involved in cholesterol biosynthesis, while SREBP-1c is co-expressed with genes involved in storage lipid metabolism. In addition, the expression of SREBP-1c in the endoneurial compartment of peripheral nerves depends on nutritional status and is disturbed in type 1 diabetes. In line with this, insulin elevates the expression of SREBP-1c in primary cultured Schwann cells by activating the SREBP-1c promoter. Taken together, these findings reveal that SREBP-1c expression in Schwann cells responds to metabolic stimuli including insulin and that this response is affected in type 1 diabetes mellitus. This suggests that disturbed SREBP-1c regulated lipid metabolism may contribute to the pathophysiology of diabetic peripheral neuropathy. © 2007 Elsevier Inc. All rights reserved.

The study of inherited monogenic diseases has contributed greatly to our mechanistic understanding of pathogenic mutations and gene regulation, and to the development of effective diagnostic tools. But interest has gradually shifted away from monogenic diseases, which collectively affect only a small fraction of the world's population, towards multifactorial, common diseases. The quest for the genetic variability associated with common traits should not be done at the expense of Mendelian disorders, because the latter could still contribute greatly to understanding the aetiology of complex traits.

Islet-brain 1 (IB1 or JIP-1) is a scaffold protein that interacts with components of the c-Jun N-terminal kinase (JNK) signal-transduction pathway. IB1 is expressed at high levels in neurons and in pancreatic beta-cells, where it controls expression of several insulin-secretory components and secretion. IB1 has been shown to homodimerize, but neither the molecular mechanisms nor the function of dimerization have yet been characterized. Here, we show that IB1 homodimerizes through a novel and unique set of Src homology 3 (SH3)-SH3 interactions. X-ray crystallography studies show that the dimer interface covers a region usually engaged in PxxP-mediated ligand recognition, even though the IB1 SH3 domain lacks this motif. The highly stable IB1 homodimer can be significantly destabilized in vitro by three individual point mutations directed against key residues involved in dimerization. Each mutation reduces IB1-dependent basal JNK activity in 293T cells. Impaired dimerization also results in a reduction in glucose transporter type 2 expression and in glucose-dependent insulin secretion in pancreatic beta-cells. Taken together, these results indicate that IB1 homodimerization through its SH3 domain has pleiotropic effects including regulation of the insulin secretion process.

The genetic mapping of drug-response traits is often characterised by a poor signal-to-noise ratio that is placebo related and which distinguishes pharmacogenetic association studies from classical case-control studies for disease susceptibility. The goal of this study was to evaluate the statistical power of candidate gene association studies under different pharmacogenetic scenarios, with special emphasis on the placebo effect. Genotype/phenotype data were simulated, mimicking samples from clinical trials, and response to the drug was modelled as a binary trait. Association was evaluated by a logistic regression model. Statistical power was estimated as a function of the number of single nucleotide polymorphisms (SNPs) genotyped, the frequency of the placebo 'response', the genotype relative risk (GRR) of the response polymorphism, the strategy for selecting SNPs for genotyping, the number of individuals in the trial and the ratio of placebo-treated to drugtreated patients. We show that: (i) the placebo 'response' strongly affects the statistical power of association studies -- even a highly penetrant drug-response allele requires at least a 500-patient trial in order to reach 80 per cent power, several-fold more than the value estimated by standard tools that are not calibrated to pharmacogenetics; (ii) the power of a pharmacogenetic association study depends primarily on the penetrance of the response genotype and, when this penetrance is fixed, power decreases for larger placebo effects; (iii) power is dramatically increased when adding markers; (iv) an optimal study design includes a similar number of placebo- and drugtreated patients; and (v) in this setting, straightforward haplotype analysis does not seem to have an advantage over single marker analysis.

Both transcription factors albumin site d-binding protein (DBP) and thyrotroph embryonic factor (TEF) are elements of the "cell-clock". Their circadian accumulation in suprachiasmatic nucleus (SCN) and peripheral tissues such as liver, kidney and lung is thought to participate in controlling circadian regulation of downstream genes. TEF and DBP control elements have never been investigated in the insulin-secreting cells, but impairment of the circadian rhythm of the beta-cells might be involved in the development of diabetic state as type 2 diabetics have lost daily temporal variations of insulin secretion. We investigated the expression pattern of TEF and DBP in insulin-secreting cells. TEF and DBP transcripts are expressed at extremely high levels in human pancreatic islets compared to other tissues, suggesting a potentially important circadian regulation of these cells. Both TEF and DPB accumulate in a circadian way in insulin-secreting cells after a serum shock known to restore circadian rhythms in cultured cells. In addition, the expression of islet-specific genes involved in glucose sensing (glucose transporter 2 (Glut2), glucokinase), insulin production (insulin) and secretion (migration inhibitory factor (MIF), somatostatin and syntaxin 1A) were modulated in the same daily rhythm as well. The circadian deregulation of these genes could therefore participate in the diabetic state development.

In models of type 1 diabetes, cytokines induce pancreatic beta-cell death by apoptosis. This process seems to be facilitated by a reduction in the amount of the islet-brain 1/JNK interacting protein 1 (IB1/JIP1), a JNK-scaffold with an anti-apoptotic effect. A point mutation S59N at the N terminus of the scaffold, which segregates in diabetic patients, has the functional consequence of sensitizing cells to apoptotic stimuli. Neither the mechanisms leading to IB1/JIP1 down-regulation by cytokines nor the mechanisms leading to the decreased capacity of the S59N mutation to protect cells from apoptosis are understood. Here, we show that IB1/JIP1 stability is modulated by intracellular calcium. The effect of calcium depends upon JNK activation, which primes the scaffold for ubiquitination-mediated degradation via the proteasome machinery. Furthermore, we observe that the S59N mutation decreases IB1/JIP1 stability by sensitizing IB1/JIP1 to calcium- and proteasome-dependent degradation. These data indicate that calcium influx initiated by cytokines mediates ubiquitination and degradation of IB1/JIP1 and may, therefore, provide a link between calcium influx and JNK-mediated apoptosis in pancreatic beta-cells.

This report describes the characterisation of the expression profile of several myogenic determination genes during human embryogenesis. The data were obtained from axial structures and limb buds of human embryos aged between 3 and 8 weeks of development. Using in situ hybridisation to detect Pax3 and MyoD gene family mRNAs, and immunochemistry to follow Six and Eya protein accumulation, we have been able to establish the chronology of accumulation of these gene products. As in mouse, the first transcripts detected in myotomes of 3 week-old embryos are Pax3 and Myf5, followed by the expression of myogenin. MyoD appears to be activated well after Myf5, myogenin and MRF4 in the early myotome, whereas, in limb bud muscles, the presence of all four of these mRNAs is concomitant from 6 weeks. Six1, Six4 and Six5 homeoproteins are detected later than Myf5 activation. These Six homeoproteins are first observed in the cytoplasm of myogenin expressing cells. At later stages of development, Six1 and Six5, but not Six4, are translocated into the nuclei of myogenic cells, concomitantly with MyHCemb expression. Eya1 and Eya2 proteins, potential Six cofactors, were also detected in myogenin positive cells, but their accumulation was delayed and was mainly cytoplasmic. These results preclude that early activation of Myf5, myogenin and MRF4 is under the control of Six and Eya proteins, while Six and Eya proteins would be involved in later steps of myogenic differentiation.

Rolandic epilepsy (RE) is the most common idiopathic focal childhood epilepsy. Its molecular basis is largely unknown and a complex genetic etiology is assumed in the majority of affected individuals. The present study tested whether six large recurrent copy number variants at 1q21, 15q11.2, 15q13.3, 16p11.2, 16p13.11 and 22q11.2 previously associated with neurodevelopmental disorders also increase risk of RE. Our association analyses revealed a significant excess of the 600 kb genomic duplication at the 16p11.2 locus (chr16: 29.5-30.1 Mb) in 393 unrelated patients with typical (n = 339) and atypical (ARE; n = 54) RE compared with the prevalence in 65,046 European population controls (5/393 cases versus 32/65,046 controls; Fisher's exact test P = 2.83 × 10(-6), odds ratio = 26.2, 95% confidence interval: 7.9-68.2). In contrast, the 16p11.2 duplication was not detected in 1738 European epilepsy patients with either temporal lobe epilepsy (n = 330) and genetic generalized epilepsies (n = 1408), suggesting a selective enrichment of the 16p11.2 duplication in idiopathic focal childhood epilepsies (Fisher's exact test P = 2.1 × 10(-4)). In a subsequent screen among children carrying the 16p11.2 600 kb rearrangement we identified three patients with RE-spectrum epilepsies in 117 duplication carriers (2.6%) but none in 202 carriers of the reciprocal deletion. Our results suggest that the 16p11.2 duplication represents a significant genetic risk factor for typical and atypical RE.

Human perception of bitterness displays pronounced interindividual variation. This phenotypic variation is mirrored by equally pronounced genetic variation in the family of bitter taste receptor genes. To better understand the effects of common genetic variations on human bitter taste perception, we conducted a genome-wide association study on a discovery panel of 504 subjects and a validation panel of 104 subjects from the general population of São Paulo in Brazil. Correction for general taste-sensitivity allowed us to identify a SNP in the cluster of bitter taste receptors on chr12 (10.88- 11.24 Mb, build 36.1) significantly associated (best SNP: rs2708377, P = 5.31 × 10(-13), r(2) = 8.9%, β = -0.12, s.e. = 0.016) with the perceived bitterness of caffeine. This association overlaps with-but is statistically distinct from-the previously identified SNP rs10772420 influencing the perception of quinine bitterness that falls in the same bitter taste cluster. We replicated this association to quinine perception (P = 4.97 × 10(-37), r(2) = 23.2%, β = 0.25, s.e. = 0.020) and additionally found the effect of this genetic locus to be concentration specific with a strong impact on the perception of low, but no impact on the perception of high concentrations of quinine. Our study, thus, furthers our understanding of the complex genetic architecture of bitter taste perception.

Calpain 3 is known as the skeletal muscle–specific member of the calpains, a family of intracellular nonlysosomal cysteine proteases. It was previously shown that defects in the human calpain 3 gene are responsible for limb girdle muscular dystrophy type 2A (LGMD2A), an inherited disease affecting predominantly the proximal limb muscles. To better understand the function of calpain 3 and the pathophysiological mechanisms of LGMD2A and also to develop an adequate model for therapy research, we generated capn3-deficient mice by gene targeting. capn3-deficient mice are fully fertile and viable. Allele transmission in intercross progeny demonstrated a statistically significant departure from Mendel's law. capn3-deficient mice show a mild progressive muscular dystrophy that affects a specific group of muscles. The age of appearance of myopathic features varies with the genetic background, suggesting the involvement of modifier genes. Affected muscles manifest a similar apoptosis-associated perturbation of the IκBα/nuclear factor κB pathway as seen in LGMD2A patients. In addition, Evans blue staining of muscle fibers reveals that the pathological process due to calpain 3 deficiency is associated with membrane alterations.

Recently, a single gene, DYSF, has been identified whichis mutated in patients with limb-girdle muscular dys-trophy type 2B (LGMD2B) and with Miyoshi myopathy(MM). This is of interest because these diseases havebeen considered as two distinct clinical conditions sincedifferent muscle groups are the initial targets. Dysferlin,the protein product of the gene, is a novel molecule with-out homology to any known mammalian protein. Wehave now raised a monoclonal antibody to dysferlin andreport on the expression of this new protein: immunola-belling with the antibody (designated NCL-hamlet) dem-onstrated a polypeptide of ∼230 kDa on western blots ofskeletal muscle, with localization to the muscle fibremembrane by microscopy at both the light and electronmicroscopic level. A specific loss of dysferlin labellingwas observed in patients with mutations in the LGMD2B/MM gene. Furthermore, patients with two different frame-shifting mutations demonstrated very low levels ofimmunoreactive protein in a manner reminiscent of thedystrophin expressed in many Duchenne patients.Analysis of human fetal tissue showed that dysferlin wasexpressed at the earliest stages of development exam-ined, at Carnegie stage 15 or 16 (embryonic age 5–6weeks). Dysferlin is present, therefore, at a time when thelimbs start to show regional differentiation. Lack of dys-ferlin at this critical time may contribute to the pattern ofmuscle involvement that develops later, with the onset ofa muscular dystrophy primarily affecting proximal or dis-tal muscles. INTRODUCTIONTo date, at least eight forms of autosomal recessive musculardystrophy have appeared under the general heading of limb-girdle muscular dystrophy (LGMD). These are in two groups:those with abnormal expression of the dystrophin–glycoproteincomplex ( 1) and those where labelling of proteins in this complexis unaffected. Thus, the sarcoglycanopathies (sometimes knownas LGMD types 2C, 2D, 2E and 2F) are caused by defects in thegenes for γ-, α-, β- or δ-sarcoglycan on chromosomes 13q12,17q12, 4q12 and 5q33, respectively (2–5). Among the dys-trophies where expression of the sarcoglycans is normal, the generesponsible for LGMD2A has been identified as the chromosome15q15-encoded muscle-specific protease calpain 3 (), and the6gene for LGMD2B was identified recently as the 2p13-locatedDYSF (7,8). Genes for LGMD2G and LGMD2H have beenlocalized to 17q11–q12 (9) and 9q31–q33 (10), and furtherLGMD genes are inferred ( 9,11). Although clinically heterogen-eous in terms of age of onset and rate of progression, the featurethat all these conditions share is weakness that starts with theproximal limb-girdle muscles. Unexpectedly, a disease character-ized by the early involvement of distal calf muscles, Miyoshimyopathy (MM), was shown to be caused by mutations in thesame gene as LGMD2B ( 8). Dysferlin, the protein product of theDYSF gene, is a novel molecule without homology to any knownmammalian protein. However, the gene does have significanthomology, throughout its length, to a nematode spermatogenesisfactor (fer-1), a fact that contributed to the name it was given(7,8). Here we present the first description of dysferlin proteinexpression.RESULTSDysferlin antibody generationA monoclonal antibody was developed that recognizes an epitopenear the C-terminus of dysferlin, within amino acids 1999–2016,just before the predicted transmembrane domain (7,8). Adsorp-tion with the immunizing peptide removed all immunoreactivity.Since the intention was to raise a diagnostically useful antibodythat would enable patients with LGMD2B to be identified, the

Homozygous targeted disruption of the mouse Caspase 8 (Casp8) gene was found to be lethal in utero. The Caspase 8 null embryos exhibited impaired heart muscle development and congested accumulation of erythrocytes. Recovery of hematopoietic colony-forming cells from the embryos was very low. In fibroblast strains derived from these embryos, the TNF receptors, Fas/Apo1, and DR3 were able to activate the Jun N-terminal kinase and to trigger IkappaB alpha phosphorylation and degradation. They failed, however, to induce cell death, while doing so effectively in wild-type fibroblasts. These findings indicate that Caspase 8 plays a necessary and nonredundant role in death induction by several receptors of the TNF/NGF family and serves a vital role in embryonal development.

Several candidate genes for non-insulin-dependent diabetes mellitus (NIDDM) map on chromosome 20, including the phosphoenolpyruvate carboxykinase gene (PCK1) and one of the maturity onset diabetes of the young genes (MODY1). Thus, we have investigated the entire long arm of chromosome 20. Linkage analyses were conducted in a total sample of 148 NIDDM families (301 NIDDM sib pairs) and in a subset of 42 early onset NIDDM families, where genetic components are likely to play a more important role (55 NIDDM sib pairs diagnosed at or before 45 years of age), using 10 highly polymorphic markers with an average map density of 7.5 cM. Using affected sib pair methods (two-point linkage and multipoint linkage analyses), significant results were obtained with the 20q13 region, in the vicinity of the PCK1 locus, only in the subset of 55 early onset NIDDM sib pairs (multipoint MLS = 2.74, P = 0.0004; MLS = 2.34, P = 0.0009 when using a conservative weighting procedure). Moreover, another region spanning the ribophorin II (RPNII, phospholipase C (PLC1) and adenosine deaminase (ADA) loci suggested linkage with NIDDM (multipoint MLS of 1.81 in all NIDDM sib pairs, P = 0.003; MLS = 1.31, P = 0.012 when using a conservative weighting procedure). Whereas our study suggests the location of a susceptibility locus for early onset NIDDM in the PCK1 gene region, further investigation in larger data sets is required to confirm these results and assess the role of other regions on chromosome 20q in human NIDDM.

The trace amine receptor 4 gene is not associated with schizophrenia in a sample linked to chromosome 6q23

We investigated the molecular basis of a severe form of early onset autosomal recessive muscular dystrophy with sarcoglycan (SG) deficiency in seven large Gypsy families living in different parts of Western Europe and apparently not closely related. They were linked to the LGMD2C locus (13q12) suggesting a primary defect in the gamma-SG gene coding for the 35 kDa dystrophin-associated glycoprotein. All of the 18 investigated patients were homozygous for the same G-->A transition in codon 283 producing the replacement of a conserved cysteine of the extra-cellular domain of the protein by a tyrosine. All affected chromosomes in homozygous and heterozygous relatives carried the same allele 5 of the intragenic marker D13S232. Flanking markers were studied to delineate a common ancestral haplotype, the size of which was used to compute the date of the founding mutation. We found evidence that the mutation occurred between 60 and 200 generations ago, therefore possibly predating the commonly accepted date of migration of the Gypsy ancestors out of India.

A series of patients affected by a muscular dystrophy, similar to the original description of a juvenile scapulo-humeral form by Erb in 1884 and fitting with the criteria used to define limb-girdle muscular dystrophies, was discovered in a small community living in the southern part of Reunion Island in the Indian Ocean. A detailed clinical analysis was conducted over 5 years on a cohort of 20 patients. This community presented a high degree of consanguinity as it was segregated from the majority of the island population for more than a century. In previous molecular genetic studies, the disease locus has been mapped to chromosome 15p. Mutations were recently identified in a gene located in this region encoding for muscle-specific calcium activated neutral protease (CANP3). Clinical, pathological, genetic and complete identification of the mutations are presented here, establishing, for the first time, precise clinico-genetic correlations in this form of autosomal recessive, juvenile, limb-girdle muscular dystrophy (LGMD).

Idiopathic nephrotic syndrome (INS) in childhood is characterized by massive proteinuria and minimal glomerular changes. Most patients with INS respond to steroid therapy. INS is generally regarded as a sporadic disease with favorable outcome. We investigated a distinct subgroup of nephrosis--the familial form of steroid resistant INS (SRN). These patients always progress to end-stage renal failure within a few years and show absence of recurrence of the disease after renal transplantation. The occurrence of the disorder in siblings and the high incidence of inbreeding in these families made an autosomal recessive mode of inheritance very likely. We performed whole genome linkage analysis in nine multiplex families of European or Northern African origin. Our results allowed us to assign a disease locus (SRN1) to a defined chromosomal region on 1q25-1q31, thus confirming the existence of a distinct entity of autosomal recessive nephrosis. Exclusion of linkage to the entire region in one family proves genetic heterogeneity.

Previous genetic and physical studies of LGMD2A, an autosomal recessive form of limb-girdle muscular dystrophy, have led to the establishment of a 10-12 Mb YAC contig encompassing the morbid locus. In order to progress toward the identification of the gene involved in LGMD2A, a primary transcription map of this genomic region was generated. The direct cDNA selection strategy was used with three YACs covering the candidate region and two different muscle cDNA libraries. Seventeen transcription units were identified among 171 cDNA fragments analysed. Five sequences corresponded to known genes, and twelve to new ones. They were characterized for their sequences, physical positions within the YAC contig, and expression patterns. Among those specifically transcribed in muscle, the calpain gene is a good positional and functional candidate for LGMD2A.

Item does not contain fulltext The male-to-female sex ratio at birth is constant across world populations with an average of 1.06 (106 male to 100 female live births) for populations of European descent. The sex ratio is considered to be affected by numerous biological and environmental factors and to have a heritable component. The aim of this study was to investigate the presence of common allele modest effects at autosomal and chromosome X variants that could explain the observed sex ratio at birth. We conducted a large-scale genome-wide association scan (GWAS) meta-analysis across 51 studies, comprising overall 114 863 individuals (61 094 women and 53 769 men) of European ancestry and 2 623 828 common (minor allele frequency >0.05) single-nucleotide polymorphisms (SNPs). Allele frequencies were compared between men and women for directly-typed and imputed variants within each study. Forward-time simulations for unlinked, neutral, autosomal, common loci were performed under the demographic model for European populations with a fixed sex ratio and a random mating scheme to assess the probability of detecting significant allele frequency differences. We do not detect any genome-wide significant (P < 5 x 10(-8)) common SNP differences between men and women in this well-powered meta-analysis. The simulated data provided results entirely consistent with these findings. This large-scale investigation across approximately 115 000 individuals shows no detectable contribution from common genetic variants to the observed skew in the sex ratio. The absence of sex-specific differences is useful in guiding genetic association study design, for example when using mixed controls for sex-biased traits.

The 15q24.1 locus, including CYP1A2, is associated with blood pressure (BP). The CYP1A2 rs762551 C allele is associated with lower CYP1A2 enzyme activity. CYP1A2 metabolizes caffeine and is induced by smoking. The association of caffeine consumption with hypertension remains controversial. We explored the effects of CYP1A2 variants and CYP1A2 enzyme activity on BP, focusing on caffeine as the potential mediator of CYP1A2 effects. Four observational (n = 16 719) and one quasi-experimental studies (n = 106) including European adults were conducted. Outcome measures were BP, caffeine intake, CYP1A2 activity and polymorphisms rs762551, rs1133323 and rs1378942. CYP1A2 variants were associated with hypertension in non-smokers, but not in smokers (CYP1A2-smoking interaction P = 0.01). Odds ratios (95% CIs) for hypertension for rs762551 CC, CA and AA genotypes were 1 (reference), 0.78 (0.59-1.02) and 0.66 (0.50-0.86), respectively, P = 0.004. Results were similar for the other variants. Higher CYP1A2 activity was linearly associated with lower BP after quitting smoking (P = 0.049 and P = 0.02 for systolic and diastolic BP, respectively), but not while smoking. In non-smokers, the CYP1A2 variants were associated with higher reported caffeine intake, which in turn was associated with lower odds of hypertension and lower BP (P = 0.01). In Mendelian randomization analyses using rs1133323 as instrument, each cup of caffeinated beverage was negatively associated with systolic BP [-9.57 (-16.22, -2.91) mmHg]. The associations of CYP1A2 variants with BP were modified by reported caffeine intake. These observational and quasi-experimental results strongly support a causal role of CYP1A2 in BP control via caffeine intake.

Family studies suggest a genetic component to the etiology of chronic kidney disease (CKD) and end stage renal disease (ESRD). Previously, we identified 16 loci for eGFR in genome-wide association studies, but the associations of these single nucleotide polymorphisms (SNPs) for incident CKD or ESRD are unknown. We thus investigated the association of these loci with incident CKD in 26,308 individuals of European ancestry free of CKD at baseline drawn from eight population-based cohorts followed for a median of 7.2 years (including 2,122 incident CKD cases defined as eGFR, Author Summary Chronic kidney disease (CKD) affects about 6%–11% of the general population, and progression to end stage renal disease (ESRD) has a significant public health impact. Family studies suggest that the risk for CKD and ESRD is heritable. Unraveling the genetic underpinning of risk for these diseases may lead to the identification of novel mechanisms and thus diagnostic and therapeutic tools. We have previously identified 16 genetic markers in association with kidney function and prevalent CKD in general population studies. However, little is known about the relevance of these SNPs to the initial development of CKD or to ESRD risk. Therefore, we have now analyzed the association of these markers with the initiation of CKD in more than 26,000 individuals from the general population using serial estimations of kidney function, and with ESRD in four case-control studies in subjects of European ancestry (3,775 cases, 4,577 controls). We show that many of the 16 markers are also associated or show a strong trend towards association with initiation of CKD, while only 2 markers are nominally associated with ESRD. Further work is required to characterize the association of genetic determinants of different stages of CKD progression.

Accumulation of fat in the liver increases the risk to develop fibrosis and cirrhosis and is associated with development of the metabolic syndrome. Here, to identify genes or gene pathways that may underlie the genetic susceptibility to fat accumulation in liver, we studied A/J and C57Bl/6 mice that are resistant and sensitive to diet-induced hepatosteatosis and obesity, respectively. We performed comparative transcriptomic and lipidomic analysis of the livers of both strains of mice fed a high fat diet for 2, 10, and 30 days. We found that resistance to steatosis in A/J mice was associated with the following: (i) a coordinated up-regulation of 10 genes controlling peroxisome biogenesis and β-oxidation; (ii) an increased expression of the elongase Elovl5 and desaturases Fads1 and Fads2. In agreement with these observations, peroxisomal β-oxidation was increased in livers of A/J mice, and lipidomic analysis showed increased concentrations of long chain fatty acid-containing triglycerides, arachidonic acid-containing lysophosphatidylcholine, and 2-arachidonylglycerol, a cannabinoid receptor agonist. We found that the anti-inflammatory CB2 receptor was the main hepatic cannabinoid receptor, which was highly expressed in Kupffer cells. We further found that A/J mice had a lower pro-inflammatory state as determined by lower plasma levels and IL-1β and granulocyte-CSF and reduced hepatic expression of their mRNAs, which were found only in Kupffer cells. This suggests that increased 2-arachidonylglycerol production may limit Kupffer cell activity. Collectively, our data suggest that genetic variations in the expression of peroxisomal β-oxidation genes and of genes controlling the production of an anti-inflammatory lipid may underlie the differential susceptibility to diet-induced hepatic steatosis and pro-inflammatory state.

Calcium has a pivotal role in biological functions, and serum calcium levels have been associated with numerous disorders of bone and mineral metabolism, as well as with cardiovascular mortality. Here we report results from a genome-wide association study of serum calcium, integrating data from four independent cohorts including a total of 12,865 individuals of European and Indian Asian descent. Our meta-analysis shows that serum calcium is associated with SNPs in or near the calcium-sensing receptor (CASR) gene on 3q13. The top hit with a p-value of 6.3×10-37 is rs1801725, a missense variant, explaining 1.26% of the variance in serum calcium. This SNP had the strongest association in individuals of European descent, while for individuals of Indian Asian descent the top hit was rs17251221 (p = 1.1×10-21), a SNP in strong linkage disequilibrium with rs1801725. The strongest locus in CASR was shown to replicate in an independent Icelandic cohort of 4,126 individuals (p = 1.02×10-4). This genome-wide meta-analysis shows that common CASR variants modulate serum calcium levels in the adult general population, which confirms previous results in some candidate gene studies of the CASR locus. This study highlights the key role of CASR in calcium regulation., Author Summary Calcium levels in blood serum play an important role in many biological processes. The regulation of serum calcium is under strong genetic control. This study describes the first meta-analysis of a genome-wide association study from four cohorts totaling 12,865 participants of European and Indian Asian descent. Confirming previous results in some candidate gene studies, we find that common polymorphisms at the calcium-sensing receptor (CASR) gene locus are associated with serum calcium concentrations. We show that CASR variants give rise to the strongest signals associated with serum calcium levels in both European and Indian Asian populations, while no other locus reaches genome-wide significance. Our results show that CASR is a key player in genetic regulation of serum calcium in the adult general population.

To extend the understanding of host genetic determinants of HIV-1 control, we performed a genome-wide association study in a cohort of 2,554 infected Caucasian subjects. The study was powered to detect common genetic variants explaining down to 1.3% of the variability in viral load at set point. We provide overwhelming confirmation of three associations previously reported in a genome-wide study and show further independent effects of both common and rare variants in the Major Histocompatibility Complex region (MHC). We also examined the polymorphisms reported in previous candidate gene studies and fail to support a role for any variant outside of the MHC or the chemokine receptor cluster on chromosome 3. In addition, we evaluated functional variants, copy-number polymorphisms, epistatic interactions, and biological pathways. This study thus represents a comprehensive assessment of common human genetic variation in HIV-1 control in Caucasians., Author Summary The ability to spontaneously control HIV-1 upon infection is highly variable between individuals. To evaluate the contribution of variation in human genes to differences in plasma viral load and in disease progression rates, we performed a genome-wide association study in >2,500 HIV–infected individuals. This study achieved two goals: it completed the analysis of common variation influencing viral control, and it re-assessed the majority of previously reported genetic associations. We show that genetic variants located near the HLA-B and HLA-C genes are the strongest determinants of viral control, and that other independent associations exist in the same region of chromosome 6, the Major Histocompatibility Complex, known to contain a large number of genes involved in immune defense. We could not replicate most of the previously published associations with HIV candidate genes in this large, well-characterized cohort. Overall, common human genetic variation, together with demographic variables, explains up to 22% of the variability in viral load in the Caucasian population.

Telomeres are associated with chromatin-mediated silencing of genes in their vicinity. However, how epigenetic markers mediate mammalian telomeric silencing and whether specific proteins may counteract this effect are not known. We evaluated the ability of CTF1, a DNA- and histone-binding transcription factor, to prevent transgene silencing at human telomeres. CTF1 was found to protect a gene from silencing when its DNA-binding sites were interposed between the gene and the telomeric extremity, while it did not affect a gene adjacent to the telomere. Protein fusions containing the CTF1 histone-binding domain displayed similar activities, while mutants impaired in their ability to interact with the histone did not. Chromatin immunoprecipitation indicated the propagation of a hypoacetylated histone structure to various extents depending on the telomere. The CTF1 fusion protein was found to recruit the H2A.Z histone variant at the telomeric locus and to restore high histone acetylation levels to the insulated telomeric transgene. Histone lysine trimethylations were also increased on the insulated transgene, indicating that these modifications may mediate expression rather than silencing at human telomeres. Overall, these results indicate that transcription factors can act to delimit chromatin domain boundaries at mammalian telomeres, thereby blocking the propagation of a silent chromatin structure.

We report the study of a large American family displaying autosomal dominant retinitis pigmentosa with reduced penetrance, a form of progressive and hereditary retinal degeneration. Although the particular inheritance pattern and previous linkage mapping incriminated the PRPF31 gene, extensive screening of all its exons and their boundaries failed in the past to reveal any mutation. In this work, we sequenced the entire PRPF31 genomic region by both the classical Sanger method and ultra-high throughput (UHT) sequencing. Among the many variants identified, a single-base substitution (c.1374+654C>G) located deep within intron 13 and inside a repetitive DNA element was common to all patients and obligate asymptomatic carriers. This change created a new splice donor site leading to the synthesis of two mutant PRPF31 isoforms, degraded by nonsense-mediated mRNA decay. As a consequence, we observed an overall reduction in full-length PRPF31 mRNA and protein levels, with no evidence of mutant proteins being synthesized. Our results indicate that c.1374+654C>G causes retinitis pigmentosa via haploinsufficiency, similar to the vast majority of PRPF31 mutations described so far. We discuss the potential of UHT sequencing technologies in mutation screening and the continued identification of pathogenic splicing mutations buried deep within intronic regions.