Whole-exome sequencing links a variant in DHDDS to retinitis pigmentosa

Increasingly, mutations in genes causing Mendelian disease will be supported by individual and small families only; however, exome sequencing studies have thus far focused on syndromic phenotypes characterized by low locus heterogeneity. In contrast, retinitis pigmentosa (RP) is caused by >50 known genes, which still explain only half of the clinical cases. In a single, one-generation, nonsyndromic RP family, we have identified a gene, dehydrodolichol diphosphate synthase (DHDDS), demonstrating the power of combining whole-exome sequencing with rapid in vivo studies. DHDDS is a highly conserved essential enzyme for dolichol synthesis, permitting global N-linked glycosylation. Zebrafish studies showed virtually identical photoreceptor defects as observed with N-linked glycosylation-interfering mutations in the light-sensing protein rhodopsin. The identified Lys42Glu variant likely arose from an ancestral founder, because eight of the nine identified alleles in 27,174 control chromosomes were of confirmed Ashkenazi Jewish ethnicity. These findings demonstrate the power of exome sequencing linked to functional studies when faced with challenging study designs and, importantly, link RP to the pathways of N-linked glycosylation, which promise new avenues for therapeutic interventions.     

Networks of neuronal genes affected by common and rare variants in autism spectrum disorders

Autism spectrum disorders (ASD) are neurodevelopmental disorders with phenotypic and genetic heterogeneity. Recent studies have reported rare and de novo mutations in ASD, but the allelic architecture of ASD remains unclear. To assess the role of common and rare variations in ASD, we constructed a gene co-expression network based on a widespread survey of gene expression in the human brain. We identified modules associated with specific cell types and processes. By integrating known rare mutations and the results of an ASD genome-wide association study (GWAS), we identified two neuronal modules that are perturbed by both rare and common variations. These modules contain highly connected genes that are involved in synaptic and neuronal plasticity and that are expressed in areas associated with learning and memory and sensory perception. The enrichment of common risk variants was replicated in two additional samples which include both simplex and multiplex families. An analysis of the combined contribution of common variants in the neuronal modules revealed a polygenic component to the risk of ASD. The results of this study point toward contribution of minor and major perturbations in the two sub-networks of neuronal genes to ASD risk.     

High-throughput sequencing of mGluR signaling pathway genes reveals enrichment of rare variants in autism

Identification of common molecular pathways affected by genetic variation in autism is important for understanding disease pathogenesis and devising effective therapies. Here, we test the hypothesis that rare genetic variation in the metabotropic glutamate-receptor (mGluR) signaling pathway contributes to autism susceptibility. Single-nucleotide variants in genes encoding components of the mGluR signaling pathway were identified by high-throughput multiplex sequencing of pooled samples from 290 non-syndromic autism cases and 300 ethnically matched controls on two independent next-generation platforms. This analysis revealed significant enrichment of rare functional variants in the mGluR pathway in autism cases. Higher burdens of rare, potentially deleterious variants were identified in autism cases for three pathway genes previously implicated in syndromic autism spectrum disorder, TSC1, TSC2, and SHANK3, suggesting that genetic variation in these genes also contributes to risk for non-syndromic autism. In addition, our analysis identified HOMER1, which encodes a postsynaptic density-localized scaffolding protein that interacts with Shank3 to regulate mGluR activity, as a novel autism-risk gene. Rare, potentially deleterious HOMER1 variants identified uniquely in the autism population affected functionally important protein regions or regulatory sequences and co-segregated closely with autism among children of affected families. We also identified rare ASD-associated coding variants predicted to have damaging effects on components of the Ras/MAPK cascade. Collectively, these findings suggest that altered signaling downstream of mGluRs contributes to the pathogenesis of non-syndromic autism.     

Rare de novo variants associated with autism implicate a large functional network of genes involved in formation and function of synapses

Identification of complex molecular networks underlying common human phenotypes is a major challenge of modern genetics. In this study, we develop a method for network-based analysis of genetic associations (NETBAG). We use NETBAG to identify a large biological network of genes affected by rare de novo CNVs in autism. The genes forming the network are primarily related to synapse development, axon targeting, and neuron motility. The identified network is strongly related to genes previously implicated in autism and intellectual disability phenotypes. Our results are also consistent with the hypothesis that significantly stronger functional perturbations are required to trigger the autistic phenotype in females compared to males. Overall, the presented analysis of de novo variants supports the hypothesis that perturbed synaptogenesis is at the heart of autism. More generally, our study provides proof of the principle that networks underlying complex human phenotypes can be identified by a network-based functional analysis of rare genetic variants.     

Whole-exome sequencing in Tricho-rhino-phalangeal syndrome (TRPS) type I in a Korean family

Tricho-rhino-phalangeal syndrome (TRPS) is a rare autosomal dominant and monogenic disease. Among three types of TRPS, it is known that TRPS type I and type III are caused by deletions or substitutions in the TRPS1 gene, located on chromosome 8 (8q23.3). Although the mutations in TRPS1 gene are responsible for human TRPS, some cases are not detected by the mutations of TRPS1 gene and several cases are presented with different genetic variations. The present case was a sporadic and without TRPS1 mutation. Therefore, we performed whole-exome sequencing (WES) with one patient and his family (father, mother, and brother) and validated novel mutations using PCR and Sanger sequencing. Through family-based WES, we found the two de novo mutations such as ZNF 134 and EXD 3 genes. Through functional effect prediction using disease association Ensembl database, we propose that the de novo mutation of ZNF134 (p.Ser207Arg) could be one of potential candidate genes for causing TRPS and develope the TRPS phenotype in the present case.     

Whole-exome sequencing identified compound heterozygous variants in MMKS in a Chinese pedigree with Bardet-Biedl syndrome

Bardet-Biedl syndrome(BBS) is a genetically heterogeneous disorder characterized by retinal dystrophy, polydactyly, obesity,developmental delay, and renal defects. At least 21 candidate BBS-associated genes(BBS1-19, NPHP1, and IFT172) have previously been identified, and all of them play important roles in ciliary function. Here, we collected a BBS pedigree with four members and performed whole-exome sequencing on the proband. The variants were analyzed and evaluated to confirm their pathogenicity. We found compound heterozygous variants(c.1192CT, p.Q398* and c.1175CT, p.T392M) in MKKS in both the siblings, and these were likely to be pathogenic variants. We also found a missense variant(c.2029GC, p.E677Q) in NPHP1 and a missense variant(c.2470CT, p.R824C) in BBS9 in the proband only, which are variants of uncertain significance. The compound heterozygous variants were probably responsible for the BBS phenotype in this Chinese pedigree and the missense mutations in NPHP1 and BBS9 might contribute to the mutation load.     

Whole-exome sequencing identifies novel pathogenic mutations and putative phenotype-influencing variants in Polish limb-girdle muscular dystrophy patients

Background

Limb girdle muscular dystrophies (LGMD) are a group of heterogeneous hereditary myopathies with similar clinical symptoms. Disease onset and progression are highly variable, with an elusive genetic background, and around 50% cases lacking molecular diagnosis.

Methods

Whole exome sequencing (WES) was performed in 73 patients with clinically diagnosed LGMD. A filtering strategy aimed at identification of variants related to the disease included integrative analysis of WES data and human phenotype ontology (HPO) terms, analysis of genes expressed in muscle, analysis of the disease-associated interactome and copy number variants analysis.

Results

Genetic diagnosis was possible in 68.5% of cases. On average, 36.3 rare variants in genes associated with various muscle diseases per patient were found that could relate to the clinical phenotype. The putative causative mutations were mostly in LGMD-associated genes, but also in genes not included in the current LGMD classification (DMD, COL6A2, and COL6A3). In three patients, mutations in two genes were suggested as the joint cause of the disease (CAPN3+MYH7, COL6A3+CACNA1S, DYSF+MYH7). Moreover, a variety of phenotype-influencing variants were postulated, including in patients with an identified already known primary pathogenic mutation.

Conclusions

We hypothesize that LGMD could be better described as oligogenic disorders in which dominant clinical presentation can result from the combined effect of mutations in a set of genes. In this view, the inter- and intrafamilial variability could reflect a specific genetic background and the presence of sets of phenotype-influencing or co-causative mutations in genes that either interact with the known LGMD-associated genes or are a part of the same pathways or structures.

     

Whole-exome sequencing reveals genetic risks of early-onset sporadic dilated cardiomyopathy in the Chinese Han population

To reveal genetic risks of early-onset sporadic dilated cardiomyopathy (DCM) patients in the Chinese Han population, we enlisted 363 DCM cases and 414 healthy controls. Whole-exome sequencing and phenotypic characterization were conducted. In total, we identified 26 loss-of-function (LOF) candidates and 66 pathogenic variants from 33 genes, most of which were novel. The deleterious variants can account for 25.07% (91/363) of all patients. Furthermore, rare missense variants in 21 genes were found to be significantly associated with DCM in burden tests. Other than rare variants, twelve common SNPs were significantly associated with an increased risk of DCM in allele-based genetic model association analysis. Of note, in the cumulative risk model, high-risk subjects had a 3.113-fold higher risk of developing DCM than low-risk subjects. Also, DCM in the high-risk group had a younger age of onset than that in the low-risk group. In terms of cardiac function, the mean left ventricular ejection fraction of patients with the deleterious variants was lower than those without (27.73%±10.02% vs. 30.61%±10.85%, P=0.026). To conclude, we mapped a comprehensive atlas of genetic risks in Chinese patients with DCM that might lead to new insights into the mechanisms and risk stratification for DCM.

     

Whole-exome sequencing identifies mutations in GPR179 leading to autosomal-recessive complete congenital stationary night blindness

Congenital stationary night blindness (CSNB) is a heterogeneous retinal disorder characterized by visual impairment under low light conditions. This disorder is due to a signal transmission defect from rod photoreceptors to adjacent bipolar cells in the retina. Two forms can be distinguished clinically, complete CSNB (cCSNB) or incomplete CSNB; the two forms are distinguished on the basis of the affected signaling pathway. Mutations in NYX, GRM6, and TRPM1, expressed in the outer plexiform layer (OPL) lead to disruption of the ON-bipolar cell response and have been seen in patients with cCSNB. Whole-exome sequencing in cCSNB patients lacking mutations in the known genes led to the identification of a homozygous missense mutation (c.1807C>T [p.His603Tyr]) in one consanguineous autosomal-recessive cCSNB family and a homozygous frameshift mutation in GPR179 (c.278delC [p.Pro93Glnfs^∗57]) in a simplex male cCSNB patient. Additional screening with Sanger sequencing of 40 patients identified three other cCSNB patients harboring additional allelic mutations in GPR179. Although, immunhistological studies revealed Gpr179 in the OPL in wild-type mouse retina, Gpr179 did not colocalize with specific ON-bipolar markers. Interestingly, Gpr179 was highly concentrated in horizontal cells and Müller cell endfeet. The involvement of these cells in cCSNB and the specific function of GPR179 remain to be elucidated.     

Fast homozygosity mapping and identification of a zebrafish ENU-induced mutation by whole-genome sequencing

Forward genetics using zebrafish is a powerful tool for studying vertebrate development through large-scale mutagenesis. Nonetheless, the identification of the molecular lesion is still laborious and involves time-consuming genetic mapping. Here, we show that high-throughput sequencing of the whole zebrafish genome can directly locate the interval carrying the causative mutation and at the same time pinpoint the molecular lesion. The feasibility of this approach was validated by sequencing the m1045 mutant line that displays a severe hypoplasia of the exocrine pancreas. We generated 13 Gb of sequence, equivalent to an eightfold genomic coverage, from a pool of 50 mutant embryos obtained from a map-cross between the AB mutant carrier and the WIK polymorphic strain. The chromosomal region carrying the causal mutation was localized based on its unique property to display high levels of homozygosity among sequence reads as it derives exclusively from the initial AB mutated allele. We developed an algorithm identifying such a region by calculating a homozygosity score along all chromosomes. This highlighted an 8-Mb window on chromosome 5 with a score close to 1 in the m1045 mutants. The sequence analysis of all genes within this interval revealed a nonsense mutation in the snapc4 gene. Knockdown experiments confirmed the assertion that snapc4 is the gene whose mutation leads to exocrine pancreas hypoplasia. In conclusion, this study constitutes a proof-of-concept that whole-genome sequencing is a fast and effective alternative to the classical positional cloning strategies in zebrafish.     

Localization of multiple melanoma tumor-suppressor genes on chromosome 11 by use of homozygosity mapping-of-deletions analysis

Loss-of-heterozygosity (LOH) studies have implicated one or more chromosome 11 tumor-suppressor gene(s) in the development of cutaneous melanoma as well as a variety of other forms of human cancer. In the present study, we have identified multiple independent critical regions on this chromosome by use of homozygosity mapping of deletions (HOMOD) analysis. This method of analysis involved the use of highly polymorphic microsatellite markers and statistics to identify regions of hemizygous deletion in unmatched melanoma cell line DNAs. Regions of loss were defined by the presence of an extended region of homozygosity (ERH) at > or =5 adjacent markers and having a statistical probability of < or =.001. Significant ERHs were similar in nature to deletions identified by LOH analyses performed on uncultured melanomas, although a higher frequency of loss (24 [60%] of 40 vs. 16 [34%] of 47) was observed in the cell lines. Overall, six small regions of overlapping deletions (SROs) were identified on chromosome 11 flanked by the markers D11S1338/D11S907 (11p13-15.5 [SRO1]), D11S1344/D11S11385 (11p11.2 [SRO2]), D11S917/D11S1886 (11q21-22.3 [SRO3]), D11S927/D11S4094 (11q23 [SRO4]), AFM210ve3/D11S990 (11q24 [SRO5]), and D11S1351/D11S4123 (11q24-25 [SRO6]). We propose that HOMOD analysis can be used as an adjunct to LOH analysis in the localization of tumor-suppressor genes.     

Whole-exome sequencing identifies mutations of KIF22 in spondyloepimetaphyseal dysplasia with joint laxity, leptodactylic type

Spondyloepimetaphyseal dysplasia with joint laxity (SEMDJL), leptodactylic (lepto-SEMDJL) or Hall type, is an autosomal-dominant skeletal dysplasia manifesting with short stature, joint laxity with dislocation(s), limb malalignment, and spinal deformity. Its causative gene mutation has not yet been discovered. We captured and sequenced the exomes of eight affected individuals in six unrelated kindreds (three individuals in a family and five simplex individuals). Five novel sequence variants in KIF22, which encodes a member of the kinesin-like protein family, were identified in seven individuals. Sanger sequencing of KIF22 confirmed that c.443C>T (p.Pro148Ser) cosegregated with the phenotype in the affected individuals in the family; c.442C>T (p.Pro148Leu) or c.446G>A (p.Arg149Gln) was present in four of five simplex individuals, but was absent in unaffected individuals in their family and 505 normal cohorts. KIF22 mRNA was detected in human bone, cartilage, joint capsule, ligament, skin, and primary cultured chondrocytes. In silico analysis of KIF22 protein structure indicates that Pro148 and Arg149 are important in maintaining hydrogen bonds in the ATP binding and motor domains of KIF22. We conclude that these mutations in KIF22 cause lepto-SEMDJL.