Comprehensive Analysis of Deafness Genes in Families with Autosomal Recessive Nonsyndromic Hearing Loss

Comprehensive genetic testing has the potential to become the standard of care for individuals with hearing loss. In this study, we investigated the genetic etiology of autosomal recessive nonsyndromic hearing loss (ARNSHL) in a Turkish cohort including individuals with cochlear implant, who had a pedigree suggestive of an autosomal recessive inheritance. A workflow including prescreening of GJB2 and a targeted next generation sequencing panel (Illumına TruSight^TM Exome) covering 2761 genes that we briefly called as mendelian exome sequencing was used. This panel includes 102 deafness genes and a number of genes causing Mendelian disorders. Using this approach, we identified causative variants in 21 of 29 families. Three different GJB2 variants were present in seven families. Remaining 14 families had 15 different variants in other known NSHL genes (MYO7A, MYO15A, MARVELD2, TMIE, DFNB31, LOXHD1, GPSM2, TMC1, USH1G, CDH23). Of these variants, eight are novel. Mutation detection rate of our workflow is 72.4%, confirming the usefulness of targeted sequencing approach in NSHL.     

Exome Sequencing Identifies Three Novel Candidate Genes Implicated in Intellectual Disability

Intellectual disability (ID) is a major health problem mostly with an unknown etiology. Recently exome sequencing of individuals with ID identified novel genes implicated in the disease. Therefore the purpose of the present study was to identify the genetic cause of ID in one syndromic and two non-syndromic Pakistani families. Whole exome of three ID probands was sequenced. Missense variations in two plausible novel genes implicated in autosomal recessive ID were identified: lysine (K)-specific methyltransferase 2B (KMT2B), zinc finger protein 589 (ZNF589), as well as hedgehog acyltransferase (HHAT) with a de novo mutation with autosomal dominant mode of inheritance. The KMT2B recessive variant is the first report of recessive Kleefstra syndrome-like phenotype. Identification of plausible causative mutations for two recessive and a dominant type of ID, in genes not previously implicated in disease, underscores the large genetic heterogeneity of ID. These results also support the viewpoint that large number of ID genes converge on limited number of common networks i.e. ZNF589 belongs to KRAB-domain zinc-finger proteins previously implicated in ID, HHAT is predicted to affect sonic hedgehog, which is involved in several disorders with ID, KMT2B associated with syndromic ID fits the epigenetic module underlying the Kleefstra syndromic spectrum. The association of these novel genes in three different Pakistani ID families highlights the importance of screening these genes in more families with similar phenotypes from different populations to confirm the involvement of these genes in pathogenesis of ID.     

Short-Flagella Mutants of Chlamydomonas reinhardtii

Six short-flagella mutants were isolated by screening clones of mutagenized Chlamydomonas for slow swimmers. The six mutants identify three unlinked Mendelian genes, with three mutations in gene shf-1, two in shf-2 and one in shf-3. shf-1 and shf-2 have been mapped to chromosomes VI and I, respectively. Two of the shf-1 mutations have temperature-sensitive flagellar-assembly phenotypes, and one shf-2 mutant has a cold-sensitive phenotype. shf shf double mutants were constructed; depending on the alleles present they showed either flagellaless or short-flagella phenotypes. Phenotypic revertants of shf-1 and shf-2 mutants were isolated, and certain of them were found to carry extragenic suppressors, some dominant and some recessive. We suspect that the shf mutations affect components of a specific flagellar size-control system, the existence of which has been suggested by a variety of physiological experiments.     

The Genetic Basis of Mendelian Phenotypes: Discoveries,Challenges, and Opportunities

Discovering the genetic basis of a Mendelian phenotype establishes a causal link between genotype and phenotype, making possible carrier and population screening and direct diagnosis. Such discoveries also contribute to our knowledge of gene function, gene regulation, development, and biological mechanisms that can be used for developing new therapeutics. As of February 2015, 2,937 genes underlying 4,163 Mendelian phenotypes have been discovered, but the genes underlying ∼50% (i.e., 3,152) of all known Mendelian phenotypes are still unknown, and many more Mendelian conditions have yet to be recognized. This is a formidable gap in biomedical knowledge. Accordingly, in December 2011, the NIH established the Centers for Mendelian Genomics (CMGs) to provide the collaborative framework and infrastructure necessary for undertaking large-scale whole-exome sequencing and discovery of the genetic variants responsible for Mendelian phenotypes. In partnership with 529 investigators from 261 institutions in 36 countries, the CMGs assessed 18,863 samples from 8,838 families representing 579 known and 470 novel Mendelian phenotypes as of January 2015. This collaborative effort has identified 956 genes, including 375 not previously associated with human health, that underlie a Mendelian phenotype. These results provide insight into study design and analytical strategies, identify novel mechanisms of disease, and reveal the extensive clinical variability of Mendelian phenotypes. Discovering the gene underlying every Mendelian phenotype will require tackling challenges such as worldwide ascertainment and phenotypic characterization of families affected by Mendelian conditions, improvement in sequencing and analytical techniques, and pervasive sharing of phenotypic and genomic data among researchers, clinicians, and families.     

An N-ethyl-N-nitrosourea mutagenesis recessive screen identifies two candidate regions for murine cardiomyopathy that map to chromosomes 1 and 15

N-ethyl-N-nitrosourea (ENU) mutagenesis screens have been successful for identifying genes that affect important biological processes and diseases. However, for heart-related phenotypes, these screens have been employed exclusively for developmental phenotypes, and to date no adult cardiomyopathy-causing genes have been discovered through a mutagenesis screen. To identify novel disease-causing and disease-modifying genes for cardiomyopathy, we performed an ENU recessive mutagenesis screen in adult mice. Using noninvasive echocardiography to screen for abnormalities in cardiac function, we identified a heritable cardiomyopathic phenotype in two families. To identify the chromosomal regions where the mutations are localized, we used a single nucleotide polymorphism (SNP) panel for genetic mapping of mouse mutations. This panel provided whole-genome linkage information and identified the mutagenized candidate regions at the proximal end of chromosome 1 (family EN1), and at the distal end of chromosome 15 (family EN25). We have identified 94 affected mice in family EN1 and have narrowed the candidate interval to 1 Mb. We have identified 20 affected mice in family EN25 and have narrowed the candidate interval to 12 Mb. The identification of the genes responsible for the observed phenotype in these families will be strong candidates for disease-causing or disease-modifying genes in patients with heart failure. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Exome Sequencing of Index Patients with Retinal Dystrophies as a Tool for Molecular Diagnosis

Background

Retinal dystrophies (RD) are a group of hereditary diseases that lead to debilitating visual impairment and are usually transmitted as a Mendelian trait. Pathogenic mutations can occur in any of the 100 or more disease genes identified so far, making molecular diagnosis a rather laborious process. In this work we explored the use of whole exome sequencing (WES) as a tool for identification of RD mutations, with the aim of assessing its applicability in a diagnostic context.

Methodology/Principal Findings

We ascertained 12 Spanish families with seemingly recessive RD. All of the index patients underwent mutational pre-screening by chip-based sequence hybridization and resulted to be negative for known RD mutations. With the exception of one pedigree, to simulate a standard diagnostic scenario we processed by WES only the DNA from the index patient of each family, followed by in silico data analysis. We successfully identified causative mutations in patients from 10 different families, which were later verified by Sanger sequencing and co-segregation analyses. Specifically, we detected pathogenic DNA variants (∼50% novel mutations) in the genes RP1, USH2A, CNGB3, NMNAT1, CHM, and ABCA4, responsible for retinitis pigmentosa, Usher syndrome, achromatopsia, Leber congenital amaurosis, choroideremia, or recessive Stargardt/cone-rod dystrophy cases.

Conclusions/Significance

Despite the absence of genetic information from other family members that could help excluding nonpathogenic DNA variants, we could detect causative mutations in a variety of genes known to represent a wide spectrum of clinical phenotypes in 83% of the patients analyzed. Considering the constant drop in costs for human exome sequencing and the relative simplicity of the analyses made, this technique could represent a valuable tool for molecular diagnostics or genetic research, even in cases for which no genotypes from family members are available.     

bfb,a Novel ENU-Induced blebs Mutant Resulting from a Missense Mutation in Fras1

Fras1 is an extracellular matrix associated protein with essential roles in adhesion of epithelia and mesenchyme during early embryonic development. The adhesive function of Fras1 is achieved through interaction with a group of related proteins, Frem 1–3, and a cytoplasmic adaptor protein Grip1. Mutation of each of these proteins results in characteristic epithelial blistering and have therefore become known as “blebs” proteins. Human Fraser syndrome presents with a similar phenotype and the blebs mice have been instrumental in identification of the genetic basis of Fraser syndrome. We have identified a new ENU-induced blebs allele resulting from a novel missense mutation in Fras1. The resulting mouse strain, blood filled blisters (bfb), presents with a classic blebs phenotype but does not exhibit embryonic lethality typical of other blebs mutants and in addition, we report novel palate and sternal defects. Analysis of the bfb phenotype confirms the presence of epithelial-mesenchymal adhesion defects but also supports the emerging role of blebs proteins in regulating signalling during organogenesis. The bfb strain provides new opportunities to investigate the role of Fras1 in development.     

Identification of a Recognizable Progressive Skeletal Dysplasia Caused by RSPRY1 Mutations

Skeletal dysplasias are highly variable Mendelian phenotypes. Molecular diagnosis of skeletal dysplasias is complicated by their extreme clinical and genetic heterogeneity. We describe a clinically recognizable autosomal-recessive disorder in four affected siblings from a consanguineous Saudi family, comprising progressive spondyloepimetaphyseal dysplasia, short stature, facial dysmorphism, short fourth metatarsals, and intellectual disability. Combined autozygome/exome analysis identified a homozygous frameshift mutation in RSPRY1 with resulting nonsense-mediated decay. Using a gene-centric “matchmaking” system, we were able to identify a Peruvian simplex case subject whose phenotype is strikingly similar to the original Saudi family and whose exome sequencing had revealed a likely pathogenic homozygous missense variant in the same gene. RSPRY1 encodes a hypothetical RING and SPRY domain-containing protein of unknown physiological function. However, we detect strong RSPRY1 protein localization in murine embryonic osteoblasts and periosteal cells during primary endochondral ossification, consistent with a role in bone development. This study highlights the role of gene-centric matchmaking tools to establish causal links to genes, especially for rare or previously undescribed clinical entities.Keyword: matchmaking, autozygome, exome, skeletal dysplasia, mucopolysaccharidosis, craniosynostosis     

Genotype–phenotype correlation in long QT syndrome families

Heterogeneity in clinical manifestations is a well-known feature in Long QT Syndrome (LQTS). The extent of this phenomenon became evident in families wherein both symptomatic and asymptomatic family members are reported. The study hence warrants genetic testing and/or screening of family members of LQTS probands for risk stratification and prediction.Of the 46 families screened, 18 probands revealed novel variations/compound heterozygosity in the gene/s screened. Families 1–4 revealed probands carrying novel variations in KCNQ1 gene along with compound heterozygosity of risk genotypes of the SCN5A, KCNE1 and NPPA gene/s polymorphisms screened. It was also observed that families- 5, 6 and 7 were typical cases of “anticipation” in which both mother and child were diagnosed with congenital LQTS (cLQTS). Families- 16 and 17 represented aLQTS probands with variations in IKs and INa encoding genes. First degree relatives (FDRs) carrying the same haplotype as the proband were also identified which may help in predictive testing and management of LQTS. Most of the probands exhibiting a family history were found to be genetic compounds which clearly points to the role of cardiac genes and their modifiers in a recessive fashion in LQTS manifestation.     

Compound mutations of PEO1 and TYMP in a progressive external ophthalmoplegia patient with incomplete mitochondrial neurogastrointestinal encephalomyopathy phenotype

The Mendelian inherited progressive external ophthalmoplegia (PEO) and mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) are genetically heterogeneous mitochondrial diseases caused by nuclear-mitochondrial intergenomic defects. The PEO1 and TYMP nuclear genes are closely related in the machinery of the mitochondrial DNA (mtDNA) replication. Mutations in PEO1 and TYMP genes usually cause autosomal dominant PEO, and autosomal recessive MNGIE. We identified a PEO family of Korean origin with additional phenotype of incomplete MNGIE symptom (Family ID: MT16). The entire mitochondrial genome and all coding exons of PEO1, TYMP, ANT1, POLG1, POLG2, DGUOK, and TK2 nuclear genes were sequenced. Clinical information was obtained through history taking, physical examinations, clinical observations, and electrophysiological investigations. Muscle biopsy of left biceps brachii and shoulder magnetic resonance imaging (MRI) were undertaken. We found two heterozygous mutations, Arg374Gln in PEO1 and Glu106Gln in TYMP from the proband who showed complex phenotypes of a typical PEO and late-onset incomplete MNGIE. The PEO1 Arg374Gln has been reported in several PEO patients, but TYMP Glu106Gln has not been reported. Neither large deletion nor causative point mutations were observed in the mtDNA. We suggest that the heterozygous TYMP mutation might affect complex phenotypes as a secondary genetic cause in the co-presence of PEO1 mutation.     

Gene Dosage Effects of the Imprinted Delta-Like Homologue 1 (Dlk1/Pref1) in Development: Implications for the Evolution of Imprinting

Genomic imprinting is a normal process that causes genes to be expressed according to parental origin. The selective advantage conferred by imprinting is not understood but is hypothesised to act on dosage-critical genes. Here, we report a unique model in which the consequences of a single, double, and triple dosage of the imprinted Dlk1/Pref1, normally repressed on the maternally inherited chromosome, can be assessed in the growing embryo. BAC-transgenic mice were generated that over-express Dlk1 from endogenous regulators at all sites of embryonic activity. Triple dosage causes lethality associated with major organ abnormalities. Embryos expressing a double dose of Dlk1, recapitulating loss of imprinting, are growth enhanced but fail to thrive in early life, despite the early growth advantage. Thus, any benefit conferred by increased embryonic size is offset by postnatal lethality. We propose a negative correlation between gene dosage and survival that fixes an upper limit on growth promotion by Dlk1, and we hypothesize that trade-off between growth and lethality might have driven imprinting at this locus.     

Soluble THSD7A is an N-glycoprotein that promotes endothelial cell migration and tube formation in angiogenesis

Background

Thrombospondin type I domain containing 7A (THSD7A) is a novel neural protein that is known to affect endothelial migration and vascular patterning during development. To further understand the role of THSD7A in angiogenesis, we investigated the post-translational modification scheme of THS7DA and to reveal the underlying mechanisms by which this protein regulates blood vessel growth.

Methodology/Principal Findings

Full-length THSD7A was overexpressed in human embryonic kidney 293T (HEK293T) cells and was found to be membrane associated and N-glycosylated. The soluble form of THSD7A, which is released into the cultured medium, was harvested for further angiogenic assays. We found that soluble THSD7A promotes human umbilical vein endothelial cell (HUVEC) migration and tube formation. HUVEC sprouts and zebrafish subintestinal vessel (SIV) angiogenic assays further revealed that soluble THSD7A increases the number of branching points of new vessels. Interestingly, we found that soluble THSD7A increased the formation of filopodia in HUVEC. The distribution patterns of vinculin and phosphorylated focal adhesion kinase (FAK) were also affected, which implies a role for THSD7A in focal adhesion assembly. Moreover, soluble THSD7A increased FAK phosphorylation in HUVEC, suggesting that THSD7A is involved in regulating cytoskeleton reorganization.

Conclusions/Significance

Taken together, our results indicate that THSD7A is a membrane-associated N-glycoprotein with a soluble form. Soluble THSD7A promotes endothelial cell migration during angiogenesis via a FAK-dependent mechanism and thus may be a novel neuroangiogenic factor.     

A Synthetic Lethal Screen Identifies a Role for Lin-44/Wnt in C. elegans Embryogenesis

BackgroundThe C. elegans proteins PTP-3/LAR-RPTP and SDN-1/Syndecan are conserved cell adhesion molecules. Loss-of-function (LOF) mutations in either ptp-3 or sdn-1 result in low penetrance embryonic developmental defects. Work from other systems has shown that syndecans can function as ligands for LAR receptors in vivo. We used double mutant analysis to test whether ptp-3 and sdn-1 function in a linear genetic pathway during C. elegans embryogenesis.ResultsWe found animals with LOF in both sdn-1 and ptp-3 exhibited a highly penetrant synthetic lethality (SynLet), with only a small percentage of animals surviving to adulthood. Analysis of the survivors demonstrated that these animals had a synergistic increase in the penetrance of embryonic developmental defects. Together, these data strongly suggested PTP-3 and SDN-1 function in parallel during embryogenesis. We subsequently used RNAi to knockdown ~3,600 genes predicted to encode secreted and/or transmembrane molecules to identify genes that interacted with ptp-3 or sdn-1. We found that the Wnt ligand, lin-44, was SynLet with sdn-1, but not ptp-3. We used 4-dimensional time-lapse analysis to characterize the interaction between lin-44 and sdn-1. We found evidence that loss of lin-44 caused defects in the polarization and migration of endodermal precursors during gastrulation, a previously undescribed role for lin-44 that is strongly enhanced by the loss of sdn-1.ConclusionsPTP-3 and SDN-1 function in compensatory pathways during C. elegans embryonic and larval development, as simultaneous loss of both genes has dire consequences for organismal survival. The Wnt ligand lin-44 contributes to the early stages of gastrulation in parallel to sdn-1, but in a genetic pathway with ptp-3. Overall, the SynLet phenotype provides a robust platform to identify ptp-3 and sdn-1 interacting genes, as well as other genes that function in development, yet might be missed in traditional forward genetic screens.     

Characterization of α1(IV) Collagen Mutations in Caenorhabditis elegans and the Effects of α1 and α2(IV) Mutations on Type IV Collagen Distribution

Type IV collagen is a major component of basement membranes. We have characterized 11 mutations in emb-9, the α1(IV) collagen gene of Caenorhabditis elegans, that result in a spectrum of phenotypes. Five are substitutions of glycines in the Gly-X-Y domain and cause semidominant, temperature-sensitive lethality at the twofold stage of embryogenesis. One is a glycine substitution that causes recessive, non–temperature-sensitive larval lethality. Three putative null alleles, two nonsense mutations and a deletion, all cause recessive, non–temperature-sensitive lethality at the threefold stage of embryogenesis. The less severe null phenotype indicates that glycine substitution containing mutant chains dominantly interfere with the function of other molecules. The emb-9 null mutants do not stain with anti–EMB-9 antisera and show intracellular accumulation of the α2(IV) chain, LET-2, indicating that LET-2 assembly and/or secretion requires EMB-9. Glycine substitutions in either EMB-9 or LET-2 cause intracellular accumulation of both chains. The degree of intracellular accumulation differs depending on the allele and temperature and correlates with the severity of the phenotype. Temperature sensitivity appears to result from reduced assembly/secretion of type IV collagen, not defective function in the basement membrane. Because the dominant interference of glycine substitution mutations is maximal when type IV collagen secretion is totally blocked, this interference appears to occur intracellularly, rather than in the basement membrane. We suggest that the nature of dominant interference caused by mutations in type IV collagen is different than that caused by mutations in fibrillar collagens.     

Inheritance of glabrous plants in <Emphasis Type="Italic">Helianthemum oelandicum</Emphasis> var. <Emphasis Type="Italic">oelandicum</Emphasis> and spatial patterns of allele frequencies in local populations

Helianthemum oelandicum var. oelandicum is an endemic taxon on the Baltic island of Öland, SE Sweden. Plants can be classified into two morphs: the bristled morph (with bristles and with or without scattered stellate hairs) and the glabrous morph (without bristles and stellate hairs). In crosses between plants assumed to be homozygous for the trait that characterises the phenotypes of the two morphs, offspring in F1 could not be distinguished from the bristled morph. Segregation in F2 did not deviate from the expected 3:1 ratio (bristled morphs/glabrous morphs), indicating one major gene with a dominant allele for the phenotype of the bristled morph and a recessive allele for the phenotype of the glabrous morph. Besides the Mendelian inheritance of presence/absence of hairs, the density of hairs appeared to be further modified by quantitative genes. The frequency of the recessive allele for the phenotype of the glabrous morph varied among local populations and showed a geographical structure, both on local and regional scales. Possible mechanisms behind the spatial variation in indumentum are discussed.