Together with GTP and the initiator methionyl-tRNA, the translation initiation factor eIF2 forms a ternary complex that binds the 40S ribosome and then scans an mRNA to select the AUG start codon for protein synthesis. Here, we show that a human X-chromosomal neurological disorder characterized by intellectual disability and microcephaly is caused by a missense mutation in eIF2γ (encoded by EIF2S3), the core subunit of the heterotrimeric eIF2 complex. Biochemical studies of human cells overexpressing the eIF2γ mutant and of yeast eIF2γ with the analogous mutation revealed a defect in binding the eIF2β subunit to eIF2γ. Consistent with this loss of eIF2 integrity, the mutation in yeast eIF2γ impaired translation start codon selection and eIF2 function in vivo in a manner that was suppressed by overexpression of eIF2β. These findings directly link intellectual disability to impaired translation initiation, and provide a mechanistic basis for the human disease due to partial loss of eIF2 function.
Retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA) are major causes of blindness. They result from mutations in many genes which has long hampered comprehensive genetic analysis. Recently, targeted next-generation sequencing (NGS) has proven useful to overcome this limitation. To uncover “hidden mutations” such as copy number variations (CNVs) and mutations in non-coding regions, we extended the use of NGS data by quantitative readout for the exons of 55 RP and LCA genes in 126 patients, and by including non-coding 5′ exons. We detected several causative CNVs which were key to the diagnosis in hitherto unsolved constellations, e.g. hemizygous point mutations in consanguineous families, and CNVs complemented apparently monoallelic recessive alleles. Mutations of non-coding exon 1 of EYS revealed its contribution to disease. In view of the high carrier frequency for retinal disease gene mutations in the general population, we considered the overall variant load in each patient to assess if a mutation was causative or reflected accidental carriership in patients with mutations in several genes or with single recessive alleles. For example, truncating mutations in RP1, a gene implicated in both recessive and dominant RP, were causative in biallelic constellations, unrelated to disease when heterozygous on a biallelic mutation background of another gene, or even non-pathogenic if close to the C-terminus. Patients with mutations in several loci were common, but without evidence for di- or oligogenic inheritance. Although the number of targeted genes was low compared to previous studies, the mutation detection rate was highest (70%) which likely results from completeness and depth of coverage, and quantitative data analysis. CNV analysis should routinely be applied in targeted NGS, and mutations in non-coding exons give reason to systematically include 5′-UTRs in disease gene or exome panels. Consideration of all variants is indispensable because even truncating mutations may be misleading.
Primary ciliary dyskinesia (PCD) is characterized by dysfunction in respiratory and reproductive cilia/flagella and random determination of visceral asymmetry. Here, we identify the DRC1 subunit of the Nexin-Dynein Regulatory Complex (N-DRC), an axonemal structure critical for regulation of the dynein motors, and demonstrate that DRC1/CCDC164 mutations are involved in the pathogenesis of PCD. Loss-of-function DRC1/CCDC164 mutations result in severe defects in assembly of the N-DRC structure and defective ciliary movement in Chlamydomonas and humans. Our results highlight the role of N-DRC integrity for regulation of ciliary beating and provide the first direct evidence that drc mutations cause human disease.
cilia; dynein; primary ciliary dyskinesia (PCD); Nexin-Dynein Regulatory Complex (N-DRC)
Pathologic thrombosis is a major cause of mortality. Hemolytic-uremic syndrome (HUS) features episodes of small vessel thrombosis resulting in microangiopathic hemolytic anemia, thrombocytopenia and renal failure1. Atypical HUS (aHUS) can result from genetic or autoimmune factors2 that lead to pathologic complement cascade activation3. By exome sequencing we identify recessive mutations in DGKE (diacylglycerol kinase epsilon) that co-segregate with aHUS in 9 unrelated kindreds, defining a distinctive Mendelian disease. Affected patients present with aHUS before age 1, have persistent hypertension, hematuria and proteinuria (sometimes nephrotic range), and develop chronic kidney disease with age. DGKE is found in endothelium, platelets, and podocytes. Arachidonic acid-containing diacylglycerols (DAG) activate protein kinase C, which promotes thrombosis. DGKE normally inactivates DAG signaling. We infer that loss of DGKE function results in a pro-thrombotic state. These findings identify a new mechanism of pathologic thrombosis and kidney failure and have immediate implications for treatment of aHUS patients.
Cone-rod dystrophies (CORDs) represent a heterogeneous group of monogenic diseases leading to early impairment of vision. The majority of CORD entities show autosomal modes of inheritance and X-linked traits are comparably rare. So far, three X-chromosomal entities were reported (CORDX1, -X2 and -X3). In this study, we analysed a large family of German origin with solely affected males over three generations showing a CORDX-like phenotype. Due to the heterogeneity of cone-rod dystrophies, we performed a combined linkage and X-exome sequencing approach and identified a novel large intragenic in-frame deletion encompassing exons 18 to 26 within the CACNA1F gene. CACNA1F is described causative for CORDX3 in a single family originating from Finland and alterations in this gene have not yet been reported in other CORDX pedigrees. Our data independently confirm CACNA1F as the causative gene for CORDX3-like phenotypes and detailed clinical characterization of the family expands the knowledge about the phenotypic spectrum of deleterious CACNA1F alterations.
In excess of % of human cancer incidents have a viral cofactor. Epidemiological studies of idiopathic human cancers indicate that additional tumor viruses remain to be discovered. Recent advances in sequencing technology have enabled systematic screenings of human tumor transcriptomes for viral transcripts. However, technical problems such as low abundances of viral transcripts in large volumes of sequencing data, viral sequence divergence, and homology between viral and human factors significantly confound identification of tumor viruses. We have developed a novel computational approach for detecting viral transcripts in human cancers that takes the aforementioned confounding factors into account and is applicable to a wide variety of viruses and tumors. We apply the approach to conducting the first systematic search for viruses in neuroblastoma, the most common cancer in infancy. The diverse clinical progression of this disease as well as related epidemiological and virological findings are highly suggestive of a pathogenic cofactor. However, a viral etiology of neuroblastoma is currently contested. We mapped transcriptomes of neuroblastoma as well as positive and negative controls to the human and all known viral genomes in order to detect both known and unknown viruses. Analysis of controls, comparisons with related methods, and statistical estimates demonstrate the high sensitivity of our approach. Detailed investigation of putative viral transcripts within neuroblastoma samples did not provide evidence for the existence of any known human viruses. Likewise, de-novo assembly and analysis of chimeric transcripts did not result in expression signatures associated with novel human pathogens. While confounding factors such as sample dilution or viral clearance in progressed tumors may mask viral cofactors in the data, in principle, this is rendered less likely by the high sensitivity of our approach and the number of biological replicates analyzed. Therefore, our results suggest that frequent viral cofactors of metastatic neuroblastoma are unlikely.
Many human cancers are caused by infections with tumor viruses and identification of these pathogens is considered a critical contribution to cancer prevention. Deep sequencing enables us to systematically investigate viral nucleotide signatures in order to either verify or exclude the existence of viruses in idiopathic human cancers. We have developed Virana, a novel computational approach for identifying tumor viruses in human cancers that is applicable to a wide variety of tumors and viruses. Virana firstly addresses several important biological confounding factors that may hinder successful detection of these pathogens. We applied our approach in the first systematic search for cancer-causing viruses in metastatic neuroblastoma, the most common form of cancer in infancy. Although the heterogeneous clinical progression of this disease as well as epidemiological and virological findings are suggestive of a pathogenic cofactor, the viral etiology of neuroblastoma is currently contested. We conducted an analysis of experimental controls, comparisons with related approaches, as well as statistical analyses in order to validate our method. In spite of the high sensitivity of our approach, analyses of neuroblastoma transcriptomes did not provide evidence for the existence of any known or unknown human viruses. Our results therefore suggest that frequent viral cofactors of metastatic neuroblastoma are unlikely.
Partial deletions of the gene encoding the neuronal splicing regulator RBFOX1 have been reported in a range of neurodevelopmental diseases, including idiopathic generalized epilepsy. The RBFOX1 protein and its homologues (RBFOX2 and RBFOX3) regulate alternative splicing of many neuronal transcripts involved in the homeostatic control of neuronal excitability. In this study, we explored if structural microdeletions and exonic sequence variations in RBFOX1, RBFOX2, RBFOX3 confer susceptibility to rolandic epilepsy (RE), a common idiopathic focal childhood epilepsy. By high-density SNP array screening of 289 unrelated RE patients, we identified two hemizygous deletions, a 365 kb deletion affecting two untranslated 5′-terminal exons of RBFOX1 and a 43 kb deletion spanning exon 3 of RBFOX3. Exome sequencing of 242 RE patients revealed two novel probably deleterious variants in RBFOX1, a frameshift mutation (p.A233Vfs*74) and a hexanucleotide deletion (p.A299_A300del), and a novel nonsense mutation in RBFOX3 (p.Y287*). Although the three variants were inherited from unaffected parents, they were present in all family members exhibiting the RE trait clinically or electroencephalographically with only one exception. In contrast, no deleterious mutations of RBFOX1 and RBFOX3 were found in the exomes of 6503 non-RE subjects deposited in the Exome Variant Server database. The observed RBFOX3 exon 3 deletion and nonsense mutation suggest that RBFOX3 represents a novel risk factor for RE, indicating that exon deletions and truncating mutations of RBFOX1 and RBFOX3 contribute to the genetic variance of partial and generalized idiopathic epilepsy syndromes.
A complex network of genes determines sex in mammals. Here, we studied a European roe deer with an intersex phenotype that was consistent with a XY genotype with incomplete male-determination. Whole genome sequencing and quantitative real-time PCR analyses revealed a triple dose of the SOX9 gene, allowing insights into a new genetic defect in a wild animal.
Aging-related kidney diseases are a major health concern. Currently, models to study renal aging are lacking. Due to a reduced life-span progeroid models hold the promise to facilitate aging studies and allow examination of tissue-specific changes. Defects in genome maintenance in the Ercc1-/Δ progeroid mouse model result in premature aging and typical age-related pathologies. Here, we compared the glomerular transcriptome of young and aged Ercc1-deficient mice to young and aged WT mice in order to establish a novel model for research of aging-related kidney disease.
In a principal component analysis, age and genotype emerged as first and second principal components. Hierarchical clustering of all 521 genes differentially regulated between young and old WT and young and old Ercc1-/Δ mice showed cluster formation between young WT and Ercc1-/Δ as well as old WT and Ercc1-/Δ samples. An unexpectedly high number of 77 genes were differentially regulated in both WT and Ercc1-/Δ mice (p < 0.0001). GO term enrichment analysis revealed these genes to be involved in immune and inflammatory response, cell death, and chemotaxis. In a network analysis, these genes were part of insulin signaling, chemokine and cytokine signaling and extracellular matrix pathways.
Beyond insulin signaling, we find chemokine and cytokine signaling as well as modifiers of extracellular matrix composition to be subject to major changes in the aging glomerulus. At the level of the transcriptome, the pattern of gene activities is similar in the progeroid Ercc1-/Δ mouse model constituting a valuable tool for future studies of aging-associated glomerular pathologies.
Renal aging; Glomerular aging; Gene expression profiling; Microarray analysis; DNA damage; Nucleotide excision repair
Indian hedgehog (Ihh) signaling is a major determinant of various processes during embryonic development and has a pivotal role in embryonic skeletal development. A specific spatial and temporal expression of Ihh within the developing limb buds is essential for accurate digit outgrowth and correct digit number. Although missense mutations in IHH cause brachydactyly type A1, small tandem duplications involving the IHH locus have recently been described in patients with mild syndactyly and craniosynostosis. In contrast, a ∼600-kb deletion 5′ of IHH in the doublefoot mouse mutant (Dbf) leads to severe polydactyly without craniosynostosis, but with craniofacial dysmorphism. We now present a patient resembling acrocallosal syndrome (ACS) with extensive polysyndactyly of the hands and feet, craniofacial abnormalities including macrocephaly, agenesis of the corpus callosum, dysplastic and low-set ears, severe hypertelorism and profound psychomotor delay. Single-nucleotide polymorphism (SNP) array copy number analysis identified a ∼900-kb duplication of the IHH locus, which was confirmed by an independent quantitative method. A fetus from a second pregnancy of the mother by a different spouse showed similar craniofacial and limb malformations and the same duplication of the IHH-locus. We defined the exact breakpoints and showed that the duplications are identical tandem duplications in both sibs. No copy number changes were observed in the healthy mother. To our knowledge, this is the first report of a human phenotype similar to the Dbf mutant and strikingly overlapping with ACS that is caused by a copy number variation involving the IHH locus on chromosome 2q35.
Indian hedgehog; duplication; acrocallosal syndrome; polydactyly
The presence of a southeast to northwest gradient across Europe in human genetic diversity is a well-established observation and has recently been confirmed by genome-wide single nucleotide polymorphism (SNP) data. This pattern is traditionally explained by major prehistoric human migration events in Palaeolithic and Neolithic times. Here, we investigate whether (similar) spatial patterns in human genomic diversity also occur on a micro-geographic scale within Europe, such as in the Netherlands, and if so, whether these patterns could also be explained by more recent demographic events, such as those that occurred in Dutch population history.
We newly collected data on a total of 999 Dutch individuals sampled at 54 sites across the country at 443,816 autosomal SNPs using the Genome-Wide Human SNP Array 5.0 (Affymetrix). We studied the individual genetic relationships by means of classical multidimensional scaling (MDS) using different genetic distance matrices, spatial ancestry analysis (SPA), and ADMIXTURE software. We further performed dedicated analyses to search for spatial patterns in the genomic variation and conducted simulations (SPLATCHE2) to provide a historical interpretation of the observed spatial patterns.
We detected a subtle but clearly noticeable genomic population substructure in the Dutch population, allowing differentiation of a north-eastern, central-western, central-northern and a southern group. Furthermore, we observed a statistically significant southeast to northwest cline in the distribution of genomic diversity across the Netherlands, similar to earlier findings from across Europe. Simulation analyses indicate that this genomic gradient could similarly be caused by ancient as well as by the more recent events in Dutch history.
Considering the strong archaeological evidence for genetic discontinuity in the Netherlands, we interpret the observed clinal pattern of genomic diversity as being caused by recent rather than ancient events in Dutch population history. We therefore suggest that future human population genetic studies pay more attention to recent demographic history in interpreting genetic clines. Furthermore, our study demonstrates that genetic population substructure is detectable on a small geographic scale in Europe despite recent demographic events, a finding we consider potentially relevant for future epidemiological and forensic studies.
Population substructure; Genetic cline; Genome-wide diversity; SNP; Europe; Netherlands
ClC-2 is a voltage-dependent chloride channel that activates slowly at voltages negative to the chloride reversal potential. Adenosine triphosphate (ATP) and other nucleotides have been shown to bind to carboxy-terminal cystathionine-ß-synthase (CBS) domains of ClC-2, but the functional consequences of binding are not sufficiently understood. We here studied the effect of nucleotides on channel gating using single-channel and whole-cell patch clamp recordings on transfected mammalian cells. ATP slowed down macroscopic activation and deactivation time courses in a dose-dependent manner. Removal of the complete carboxy-terminus abolishes the effect of ATP, suggesting that CBS domains are necessary for ATP regulation of ClC-2 gating. Single-channel recordings identified long-lasting closed states of ATP-bound channels as basis of this gating deceleration. ClC-2 channel dimers exhibit two largely independent protopores that are opened and closed individually as well as by a common gating process. A seven-state model of common gating with altered voltage dependencies of opening and closing transitions for ATP-bound states correctly describes the effects of ATP on macroscopic and microscopic ClC-2 currents. To test for a potential pathophysiological impact of ClC-2 regulation by ATP, we studied ClC-2 channels carrying naturally occurring sequence variants found in patients with idiopathic generalized epilepsy, G715E, R577Q, and R653T. All naturally occurring sequence variants accelerate common gating in the presence but not in the absence of ATP. We propose that ClC-2 uses ATP as a co-factor to slow down common gating for sufficient electrical stability of neurons under physiological conditions.
Electronic supplementary material
The online version of this article (doi:10.1007/s00424-013-1286-0) contains supplementary material, which is available to authorized users.
Chloride channel; Single-channel recording; Channel gating; Epilepsy
Maximizing gains during probabilistic reinforcement learning requires the updating of choice – outcome expectations at the time when the feedback about a specific choice or action is given. Extant theories and evidence suggest that dopaminergic modulation plays a crucial role in reinforcement learning and the updating of choice – outcome expectations. Furthermore, recently a positive component of the event-related potential about 200 ms (P2) after feedback has been suggested to reflect such updating. The efficacy of dopaminergic modulation changes across the life span. However, to date investigations of age-related differences in feedback-related P2 during reinforcement learning are still scarce. The present study thus aims to investigate whether individual differences in the feedback-related P2 would be associated with polymorphic variations in a dopamine relevant gene PPP1R1B (also known as DARPP-32) and whether the genetic effect may differ between age groups. We observed larger P2 amplitudes in individuals carrying the genotype associated with higher dopamine receptor efficacy, i.e., a allele homozygotes of a single nucleotide polymorphism (rs907094) of the PPP1R1B gene. Moreover, this effect was more pronounced in children and older adults in comparison to adolescents and younger adults. Together, our findings indicate that polymorphic variations in a dopamine relevant gene are associated with individual differences in brain-evoked potentials of outcome updating and hint at the possibility that genotype effects on neurocognitive phenotypes may vary as a function of brain maturation and aging.
PPP1R1B (DARPP-32); dopamine; reward; reinforcement learning; child development; aging
Despite the high number of genes identified in hereditary polyneuropathies/Charcot–Marie–Tooth (CMT) disease, the genetic defect in many families is still unknown. Here we report the identification of a new gene for autosomal dominant axonal neuropathy in a large three-generation family. Linkage analysis identified a 5 Mb region on 9q33–34 with a LOD score of 5.12. Sequence capture and next-generation sequencing of the region of interest identified five previously unreported non-synonymous heterozygous single nucleotide changes or indels, four of which were confirmed by Sanger sequencing. Two sequence variants co-segregated with the disease, and one, a 2 bp insertion in the last exon of LRSAM1, was also absent in 676 ethnicity-matched control chromosomes. This frameshift mutation (p.Leu708Argfx28) is located in the C-terminal RING finger motif of the encoded protein. Ubiquitin ligase activity in transfected cells with constructs carrying the patient mutation was affected as measured by a higher level of abundance of TSG101, the only reported target of LRSAM1. Injections of morpholino oligonucleotides in zebrafish embryos directed against the ATG or last splice site of zebrafish Lrsam1 disturbed neurodevelopment, showing a less organized neural structure and, in addition, affected tail formation and movement. LRSAM1 is highly expressed in adult spinal cord motoneurons as well as in fetal spinal cord and muscle tissue. Recently, a homozygous mutation in LRSAM1 was proposed as a strong candidate for the disease in a family with recessive axonal polyneuropathy. Our data strongly support the hypothesis that LRSAM1 mutations can cause both dominant and recessive forms of CMT.
Dystroglycanopathies are a clinically and genetically diverse group of recessively inherited conditions ranging from the most severe of the congenital muscular dystrophies, Walker–Warburg syndrome, to mild forms of adult-onset limb-girdle muscular dystrophy. Their hallmark is a reduction in the functional glycosylation of α-dystroglycan, which can be detected in muscle biopsies. An important part of this glycosylation is a unique O-mannosylation, essential for the interaction of α-dystroglycan with extracellular matrix proteins such as laminin-α2. Mutations in eight genes coding for proteins in the glycosylation pathway are responsible for ∼50% of dystroglycanopathy cases. Despite multiple efforts using traditional positional cloning, the causative genes for unsolved dystroglycanopathy cases have escaped discovery for several years. In a recent collaborative study, we discovered that loss-of-function recessive mutations in a novel gene, called isoprenoid synthase domain containing (ISPD), are a relatively common cause of Walker–Warburg syndrome. In this article, we report the involvement of the ISPD gene in milder dystroglycanopathy phenotypes ranging from congenital muscular dystrophy to limb-girdle muscular dystrophy and identified allelic ISPD variants in nine cases belonging to seven families. In two ambulant cases, there was evidence of structural brain involvement, whereas in seven, the clinical manifestation was restricted to a dystrophic skeletal muscle phenotype. Although the function of ISPD in mammals is not yet known, mutations in this gene clearly lead to a reduction in the functional glycosylation of α-dystroglycan, which not only causes the severe Walker–Warburg syndrome but is also a common cause of the milder forms of dystroglycanopathy.
congenital muscular dystrophy; limb-girdle muscular dystrophy; dystroglycan; laminin; isoprenoid synthase
Using targeted exome sequencing we identified mutations in NNT, an antioxidant defence gene, in patients with familial glucocorticoid deficiency. In mice with Nnt loss, higher levels of adrenocortical cell apoptosis and impaired glucocorticoid production were observed. NNT knockdown in a human adrenocortical cell line resulted in impaired redox potential and increased ROS levels. Our results suggest that NNT may have a role in ROS detoxification in human adrenal glands.
adrenal insufficiency; oxidative stress; reactive oxygen species; familial glucocorticoid deficiency
Frank-ter Haar syndrome is a rare disorder associated with skeletal, cardiac, ocular and craniofacial features including hypertelorism and brachycephaly. The most common underlying genetic defect in Frank-ter Haar syndrome appears to be a mutation in the SH3PXD2B gene on chromosome 5q35.1. Craniosynostosis, or premature fusion of the calvarial sutures, has not previously been described in Frank-ter Haar syndrome.
We present a family of three affected siblings born to consanguineous parents with clinical features in keeping with a diagnosis of Frank-ter Haar syndrome. All three siblings have a novel mutation caused by the deletion of exon 13 of the SH3PXD2B gene. Two of the three siblings also have non-scaphocephalic sagittal synostosis associated with raised intracranial pressure.
The clinical features of craniosynostosis and raised intracranial pressure in this family with a confirmed diagnosis of Frank-ter Haar syndrome expand the clinical spectrum of the disease. The abnormal cranial proportions in a mouse model of the disease suggests that the association is not coincidental. The possibility of craniosynostosis should be considered in individuals with a suspected diagnosis of Frank-ter Haar syndrome.
Frank-ter Haar syndrome; Craniosynostosis; Sagittal synostosis; Intracranial pressure
Usher syndrome (USH) is an autosomal recessive genetically heterogeneous disorder with congenital sensorineural hearing impairment and retinitis pigmentosa (RP). We have identified a consanguineous Lebanese family with two affected members displaying progressive hearing loss, RP and cataracts, therefore clinically diagnosed as USH type 3 (USH3). Our study was aimed at the identification of the causative mutation in this USH3-like family.
Candidate loci were identified using genomewide SNP-array-based homozygosity mapping followed by targeted enrichment and next-generation sequencing.
Using a capture array targeting the three identified homozygosity-by-descent regions on chromosomes 1q43-q44, 20p13-p12.2 and 20p11.23-q12, we identified a homozygous nonsense mutation, p.Arg65X, in ABHD12 segregating with the phenotype.
Mutations of ABHD12, an enzyme hydrolyzing an endocannabinoid lipid transmitter, cause PHARC (polyneuropathy, hearing loss, ataxia, retinitis pigmentosa, and early-onset cataract). After the identification of the ABHD12 mutation in this family, one patient underwent neurological examination which revealed ataxia, but no polyneuropathy. ABHD12 is not known to be related to the USH protein interactome. The phenotype of our patient represents a variant of PHARC, an entity that should be taken into account as differential diagnosis for USH3. Our study demonstrates the potential of comprehensive genetic analysis for improving the clinical diagnosis.
Usher syndrome; Deafness; Retinitis pigmentosa; ABHD12; PHARC
Functional impairment of DNA damage response pathways leads to increased genomic instability. Here we describe the centrosomal protein CEP152 as a new regulator of genomic integrity and cellular response to DNA damage. Using homozygosity mapping and exome sequencing, we identified CEP152 mutations in Seckel syndrome and showed that impaired CEP152 function leads to accumulation of genomic defects resulting from replicative stress through enhanced activation of ATM signaling and increased H2AX phosphorylation.
Genetic and nongenetic factors contribute to development of pseudoexfoliation (PEX) syndrome, a complex, age-related, generalized matrix process frequently associated with glaucoma. To identify specific genetic variants underlying its etiology, we performed a genome-wide association study (GWAS) using a DNA-pooling approach. Therefore, equimolar amounts of DNA samples of 80 subjects with PEX syndrome, 80 with PEX glaucoma (PEXG) and 80 controls were combined into separate pools and hybridized to 500K SNP arrays (Affymetrix). Array probe intensity data were analyzed and visualized with expressly developed software tools GPFrontend and GPGraphics in combination with GenePool software. For replication, independent German cohorts of 610 unrelated patients with PEX/PEXG and 364 controls as well as Italian cohorts of 249 patients and 190 controls were used. Of 19, 17 SNPs showing significant allele frequency difference in DNA pools were confirmed by individual genotyping. Further single genotyping at CNTNAP2 locus revealed association between PEX/PEXG for two SNPs, which was confirmed in an independent German but not the Italian cohort. Both SNPs remained significant in the combined German cohorts even after Bonferroni correction (rs2107856: Pc=0.0108, rs2141388: Pc=0.0072). CNTNAP2 was found to be ubiquitously expressed in all human ocular tissues, particularly in retina, and localized to cell membranes of epithelial, endothelial, smooth muscle, glial and neuronal cells. Confirming efficiency of GWAS with DNA-pooling approach by detection of the known LOXL1 locus, our study data show evidence for association of CNTNAP2 with PEX syndrome and PEXG in German patients.
pseudoexfoliation syndrome; DNA pooling; association study
Genome-wide association studies have identified three chromosomal regions at 15q25, 5p15, and 6p21 as being associated with the risk of lung cancer. To confirm these associations in independent studies and investigate heterogeneity of these associations within specific subgroups, we conducted a coordinated genotyping study within the International Lung Cancer Consortium based on independent studies that were not included in previous genome-wide association studies.
Genotype data for single-nucleotide polymorphisms at chromosomes 15q25 (rs16969968, rs8034191), 5p15 (rs2736100, rs402710), and 6p21 (rs2256543, rs4324798) from 21 case–control studies for 11 645 lung cancer case patients and 14 954 control subjects, of whom 85% were white and 15% were Asian, were pooled. Associations between the variants and the risk of lung cancer were estimated by logistic regression models. All statistical tests were two-sided.
Associations between 15q25 and the risk of lung cancer were replicated in white ever-smokers (rs16969968: odds ratio [OR] = 1.26, 95% confidence interval [CI] = 1.21 to 1.32, Ptrend = 2 × 10−26), and this association was stronger for those diagnosed at younger ages. There was no association in never-smokers or in Asians between either of the 15q25 variants and the risk of lung cancer. For the chromosome 5p15 region, we confirmed statistically significant associations in whites for both rs2736100 (OR = 1.15, 95% CI = 1.10 to 1.20, Ptrend = 1 × 10−10) and rs402710 (OR = 1.14, 95% CI = 1.09 to 1.19, Ptrend = 5 × 10−8) and identified similar associations in Asians (rs2736100: OR = 1.23, 95% CI = 1.12 to 1.35, Ptrend = 2 × 10−5; rs402710: OR = 1.15, 95% CI = 1.04 to 1.27, Ptrend = .007). The associations between the 5p15 variants and lung cancer differed by histology; odds ratios for rs2736100 were highest in adenocarcinoma and for rs402710 were highest in adenocarcinoma and squamous cell carcinomas. This pattern was observed in both ethnic groups. Neither of the two variants on chromosome 6p21 was associated with the risk of lung cancer.
In this international genetic association study of lung cancer, previous associations found in white populations were replicated and new associations were identified in Asian populations. Future genetic studies of lung cancer should include detailed stratification by histology.
Cytosine methylation provides an epigenetic level of cellular plasticity that is important for development, differentiation and cancerogenesis. We adopted microdroplet PCR to bisulfite treated target DNA in combination with second generation sequencing to simultaneously assess DNA sequence and methylation. We show measurement of methylation status in a wide range of target sequences (total 34 kb) with an average coverage of 95% (median 100%) and good correlation to the opposite strand (rho = 0.96) and to pyrosequencing (rho = 0.87). Data from lymphoma and colorectal cancer samples for SNRPN (imprinted gene), FGF6 (demethylated in the cancer samples) and HS3ST2 (methylated in the cancer samples) serve as a proof of principle showing the integration of SNP data and phased DNA-methylation information into “hepitypes” and thus the analysis of DNA methylation phylogeny in the somatic evolution of cancer.
Gerodermia osteodysplastica (GO) is an autosomal recessive disorder characterized by wrinkly skin and osteoporosis. Here we demonstrate that GO is caused by loss-of-function mutations in SCYL1BP1, which is expressed at high levels in skin and osteoblasts. The protein localizes to the Golgi apparatus and interacts with Rab6, identifying SCYL1BP1 as a novel golgin. These results associate abnormalities of the secretory pathway with age-related changes in connective tissues.
Joubert syndrome (JBTS) is characterized by a specific brain malformation with various additional pathologies. It results from mutations in any one of at least 10 different genes, including NPHP1, which encodes nephrocystin-1. JBTS has been linked to dysfunction of primary cilia, since the gene products known to be associated with the disorder localize to this evolutionarily ancient organelle. Here we report the identification of a disease locus, JBTS12, with mutations in the KIF7 gene, an ortholog of the Drosophila kinesin Costal2, in a consanguineous JBTS family and subsequently in other JBTS patients. Interestingly, KIF7 is a known regulator of Hedgehog signaling and a putative ciliary motor protein. We found that KIF7 co-precipitated with nephrocystin-1. Further, knockdown of KIF7 expression in cell lines caused defects in cilia formation and induced abnormal centrosomal duplication and fragmentation of the Golgi network. These cellular phenotypes likely resulted from abnormal tubulin acetylation and microtubular dynamics. Thus, we suggest that modified microtubule stability and growth direction caused by loss of KIF7 function may be an underlying disease mechanism contributing to JBTS.