Whole Exome Sequencing and Homozygosity Mapping Identify Mutation in the Cell Polarity Protein GPSM2 as the Cause of Nonsyndromic Hearing Loss DFNB82

Massively parallel sequencing of targeted regions, exomes, and complete genomes has begun to dramatically increase the pace of discovery of genes responsible for human disorders. Here we describe how exome sequencing in conjunction with homozygosity mapping led to rapid identification of the causative allele for nonsyndromic hearing loss DFNB82 in a consanguineous Palestinian family. After filtering out worldwide and population-specific polymorphisms from the whole exome sequence, only a single deleterious mutation remained in the homozygous region linked to DFNB82. The nonsense mutation leads to an early truncation of the G protein signaling modulator GPSM2, a protein that is essential for maintenance of cell polarity and spindle orientation. In the mouse inner ear, GPSM2 is localized to apical surfaces of hair cells and supporting cells and is most highly expressed during embryonic development. Identification of GPSM2 as essential to the development of normal hearing suggests dysregulation of cell polarity as a mechanism underlying hearing loss.     

A p.C343S missense mutation in PJVK causes progressive hearing loss

Mutations in PJVK, encoding Pejvakin, cause autosomal recessive nonsyndromic hearing loss in humans at the DFNB59 locus on chromosome 2q31.2. Pejvakin is involved in generating auditory and neural signals in the inner ear. We have identified a consanguineous Pakistani family segregating sensorineural progressive hearing loss as a recessive trait, consistent with linkage to DFNB59. We sequenced PJVK and identified a novel missense mutation, c.1028G>C in exon 7 (p.C343S) co-segregating with the phenotype in the family. The p.C343 residue is fully conserved among orthologs from different vertebrate species. We have also determined that mutations in PJVK are not a common cause of hearing loss in families with moderate to severe hearing loss in Pakistan. This is the first report of PJVK mutation in a Pakistani family and pinpoints an important residue for PJVK function.     

Mutations of GIPC3 cause nonsyndromic hearing loss DFNB72 but not DFNB81 that also maps to chromosome 19p

A missense mutation of Gipc3 was previously reported to cause age-related hearing loss in mice. Point mutations of human GIPC3 were found in two small families, but association with hearing loss was not statistically significant. Here, we describe one frameshift and six missense mutations in GIPC3 cosegregating with DFNB72 hearing loss in six large families that support statistically significant evidence for genetic linkage. However, GIPC3 is not the only nonsyndromic hearing impairment gene in this region; no GIPC3 mutations were found in a family cosegregating hearing loss with markers of chromosome 19p. Haplotype analysis excluded GIPC3 from the obligate linkage interval in this family and defined a novel locus spanning 4.08?Mb and 104 genes. This closely linked but distinct nonsyndromic hearing loss locus was designated DFNB81.     

DFNB89, a novel autosomal recessive nonsyndromic hearing impairment locus on chromosome 16q21-q23.2

DFNB89 is a novel autosomal recessive nonsyndromic hearing impairment (ARNSHI) locus that was mapped to 16q21-q23.2. Linkage to the region was established by carrying out genome-wide linkage scans in two unrelated, consanguineous Pakistani families segregating ARNSHI. The maximum multipoint LOD score is 9.7 for both families and for each family, a significant maximum LOD score of 6.0 and 3.7 were obtained. The 3-unit support interval and the region of homozygosity for the two families extend from rs717293 (chr16: 62.1?Mb) to rs728929 (chr16: 78.2?Mb) and contain 16.1?Mb of sequence. A total of 146 genes are within the DFNB89 interval. Eight candidate genes, CALB2, CDH1, CDH3, CDH11, HAS3, NOB1, PLEKHG4 and SMPD3, were sequenced, but no potentially causal variants were discovered. DFNB89 is the second ARNSHI locus mapped to chromosome 16.     

Noncoding Mutations of HGF Are Associated with Nonsyndromic Hearing Loss, DFNB39

A gene causing autosomal-recessive, nonsyndromic hearing loss, DFNB39, was previously mapped to an 18 Mb interval on chromosome 7q11.22-q21.12. We mapped an additional 40 consanguineous families segregating nonsyndromic hearing loss to the DFNB39 locus and refined the obligate interval to 1.2 Mb. The coding regions of all genes in this interval were sequenced, and no missense, nonsense, or frameshift mutations were found. We sequenced the noncoding sequences of genes, as well as noncoding genes, and found three mutations clustered in intron 4 and exon 5 in the hepatocyte growth factor gene (HGF). Two intron 4 deletions occur in a highly conserved sequence that is part of the 3′ untranslated region of a previously undescribed short isoform of HGF. The third mutation is a silent substitution, and we demonstrate that it affects splicing in vitro. HGF is involved in a wide variety of signaling pathways in many different tissues, yet these putative regulatory mutations cause a surprisingly specific phenotype, which is nonsydromic hearing loss. Two mouse models of Hgf dysregulation, one in which an Hgf transgene is ubiquitously overexpressed and the other a conditional knockout that deletes Hgf from a limited number of tissues, including the cochlea, result in deafness. Overexpression of HGF is associated with progressive degeneration of outer hair cells in the cochlea, whereas cochlear deletion of Hgf is associated with more general dysplasia.     

Nonsense mutations in SMPX, encoding a protein responsive to physical force, result in X-chromosomal hearing loss

The fact that hereditary hearing loss is the most common sensory disorder in humans is reflected by, among other things, an extraordinary allelic and nonallelic genetic heterogeneity. X-chromosomal hearing impairment represents only a minor fraction of all cases. In a study of a Spanish family the locus for one of the X-chromosomal forms was assigned to Xp22 (DFNX4). We mapped the disease locus in the same chromosomal region in a large German pedigree with X-chromosomal nonsyndromic hearing impairment by using genome-wide linkage analysis. Males presented with postlingual hearing loss and onset at ages 3-7, whereas onset in female carriers was in the second to third decades. Targeted DNA capture with high-throughput sequencing detected a nonsense mutation in the small muscle protein, X-linked (SMPX) of affected individuals. We identified another nonsense mutation in SMPX in patients from the Spanish family who were previously analyzed to map DFNX4. SMPX encodes an 88 amino acid, cytoskeleton-associated protein that is responsive to mechanical stress. The presence of Smpx in hair cells and supporting cells of the murine cochlea indicates its role in the inner ear. The nonsense mutations detected in the two families suggest a loss-of-function mechanism underlying this form of hearing impairment. Results obtained after heterologous overexpression of SMPX proteins were compatible with this assumption. Because responsivity to physical force is a characteristic feature of the protein, we propose that long-term maintenance of mechanically stressed inner-ear cells critically depends on SMPX function.     

Novel autosomal recessive nonsyndromic hearing impairment locus DFNB90 maps to 7p22.1-p15.3

A novel locus DFNB90 was mapped to 7p22.1-p15.3 by carrying out a genome scan in a multigenerational consanguineous family from Pakistan with autosomal recessive nonsyndromic hearing impairment (ARNSHI).DFNB90 is the eighth ARNSHI locus mapped to chromosome 7. A multipoint LOD score of 4.0 was obtained at a number of SNP marker loci spanning from rs1468996 (chromosome 7: 5.7 Mb) tors957960 (chromosome 7: 18.8 Mb). The 3-unit support interval and the region of homozygosity for DFNB90 spans from markers rs1553960 (chromosome 7: 4.9 Mb) to rs206198 (chromosome 7: 20.3 Mb). Candidate genes ACTB, BZW, OCM, MACC1, NXPH1, PRPS1L1, RAC1 and RPA3, which lie within the DFNB90 region, were sequenced and no potentially causal variants were identified.     

Mutations in a novel isoform of TRIOBP that encodes a filamentous-actin binding protein are responsible for DFNB28 recessive nonsyndromic hearing loss

In a large consanguineous Palestinian kindred, we previously mapped DFNB28--a locus associated with recessively inherited, prelingual, profound sensorineural hearing impairment--to chromosome 22q13.1. We report here that mutations in a novel 218-kDa isoform of TRIOBP (TRIO and filamentous actin [F-actin] binding protein) are associated with DFNB28 hearing loss in a total of nine Palestinian families. Two nonsense mutations (R347X and Q581X) truncate the protein, and a potentially deleterious missense mutation (G1019R) occurs in a conserved motif in a putative SH3-binding domain. In seven families, 27 deaf individuals are homozygous for one of the nonsense mutations; in two other families, 3 deaf individuals are compound heterozygous for the two nonsense mutations or for Q581X and G1019R. The novel long isoform of TRIOBP has a restricted expression profile, including cochlea, retina, and fetal brain, whereas the original short isoform is widely expressed. Antibodies to TRIOBP reveal expression in sensory cells of the inner ear and colocalization with F-actin along the length of the stereocilia.     

Mutations of ESRRB encoding estrogen-related receptor beta cause autosomal-recessive nonsyndromic hearing impairment DFNB35

In a large consanguineous family of Turkish origin, genome-wide homozygosity mapping revealed a locus for recessive nonsyndromic hearing impairment on chromosome 14q24.3-q34.12. Fine mapping with microsatellite markers defined the critical linkage interval to a 18.7 cM region flanked by markers D14S53 and D14S1015. This region partially overlapped with the DFNB35 locus. Mutation analysis of ESRRB, a candidate gene in the overlapping region, revealed a homozygous 7 bp duplication in exon 8 in all affected individuals. This duplication results in a frame shift and premature stop codon. Sequence analysis of the ESRRB gene in the affected individuals of the original DFNB35 family and in three other DFNB35-linked consanguineous families from Pakistan revealed four missense mutations. ESRRB encodes the estrogen-related receptor beta protein, and one of the substitutions (p.A110V) is located in the DNA-binding domain of ESRRB, whereas the other three are substitutions (p.L320P, p.V342L, and p.L347P) located within the ligand-binding domain. Molecular modeling of this nuclear receptor showed that the missense mutations are likely to affect the structure and stability of these domains. RNA in situ hybridization in mice revealed that Esrrb is expressed during inner-ear development, whereas immunohistochemical analysis showed that ESRRB is present postnatally in the cochlea. Our data indicate that ESRRB is essential for inner-ear development and function. To our knowledge, this is the first report of pathogenic mutations of an estrogen-related receptor gene.     

Next-generation sequencing identifies mutations of SMPX, which encodes the small muscle protein, X-linked, as a cause of progressive hearing impairment

In a Dutch family with an X-linked postlingual progressive hearing impairment, a critical linkage interval was determined to span a region of 12.9 Mb flanked by the markers DXS7108 and DXS7110. This interval overlaps with the previously described DFNX4 locus and contains 75 annotated genes. Subsequent next-generation sequencing (NGS) detected one variant within the linkage interval, a nonsense mutation in SMPX. SMPX encodes the small muscle protein, X-linked (SMPX). Further screening was performed on 26 index patients from small families for which X-linked inheritance of nonsyndromic hearing impairment (NSHI) was not excluded. We detected a frameshift mutation in SMPX in one of the patients. Segregation analysis of both mutations in the families in whom they were found revealed that the mutations cosegregated with hearing impairment. Although we show that SMPX is expressed in many different organs, including the human inner ear, no obvious symptoms other than hearing impairment were observed in the patients. SMPX had previously been demonstrated to be specifically expressed in striated muscle and, therefore, seemed an unlikely candidate gene for hearing impairment. We hypothesize that SMPX functions in inner ear development and/or maintenance in the IGF-1 pathway, the integrin pathway through Rac1, or both.     

Mutations in KARS,Encoding Lysyl-tRNA Synthetase,Cause Autosomal-Recessive Nonsyndromic Hearing Impairment DFNB89

Previously, DFNB89, a locus associated with autosomal-recessive nonsyndromic hearing impairment (ARNSHI), was mapped to chromosomal region 16q21–q23.2 in three unrelated, consanguineous Pakistani families. Through whole-exome sequencing of a hearing-impaired individual from each family, missense mutations were identified at highly conserved residues of lysyl-tRNA synthetase (KARS): the c.1129G>A (p.Asp377Asn) variant was found in one family, and the c.517T>C (p.Tyr173His) variant was found in the other two families. Both variants were predicted to be damaging by multiple bioinformatics tools. The two variants both segregated with the nonsyndromic-hearing-impairment phenotype within the three families, and neither mutation was identified in ethnically matched controls or within variant databases. Individuals homozygous for KARS mutations had symmetric, severe hearing impairment across all frequencies but did not show evidence of auditory or limb neuropathy. It has been demonstrated that KARS is expressed in hair cells of zebrafish, chickens, and mice. Moreover, KARS has strong localization to the spiral ligament region of the cochlea, as well as to Deiters’ cells, the sulcus epithelium, the basilar membrane, and the surface of the spiral limbus. It is hypothesized that KARS variants affect aminoacylation in inner-ear cells by interfering with binding activity to tRNA or p38 and with tetramer formation. The identification of rare KARS variants in ARNSHI-affected families defines a gene that is associated with ARNSHI.     

Targeted Capture and Next-Generation Sequencing Identifies C9orf75, Encoding Taperin, as the Mutated Gene in Nonsyndromic Deafness DFNB79

Targeted genome capture combined with next-generation sequencing was used to analyze 2.9 Mb of the DFNB79 interval on chromosome 9q34.3, which includes 108 candidate genes. Genomic DNA from an affected member of a consanguineous family segregating recessive, nonsyndromic hearing loss was used to make a library of fragments covering the DFNB79 linkage interval defined by genetic analyses of four pedigrees. Homozygosity for eight previously unreported variants in transcribed sequences was detected by evaluating a library of 402,554 sequencing reads and was later confirmed by Sanger sequencing. Of these variants, six were determined to be polymorphisms in the Pakistani population, and one was in a noncoding gene that was subsequently excluded genetically from the DFNB79 linkage interval. The remaining variant was a nonsense mutation in a predicted gene, C9orf75, renamed TPRN. Evaluation of the other three DFNB79-linked families identified three additional frameshift mutations, for a total of four truncating alleles of this gene. Although TPRN is expressed in many tissues, immunolocalization of the protein product in the mouse cochlea shows prominent expression in the taper region of hair cell stereocilia. Consequently, we named the protein taperin.     

Novel mutations in the connexin 26 gene (GJB2) that cause autosomal recessive (DFNB1) hearing loss.

Mutations in the connexin 26 (Cx26) gene (GJB2) are associated with the type of autosomal recessive nonsyndromic neurosensory deafness known as "DFNB1." Studies indicate that DFNB1 (13q11-12) causes 20% of all childhood deafness and may have a carrier rate as high as 2. 8%. This study describes the analysis of 58 multiplex families each having at least two affected children diagnosed with autosomal recessive nonsyndromic deafness. Twenty of the 58 families were observed to have mutations in both alleles of Cx26. Thirty-three of 116 chromosomes contained a 30delG allele, for a frequency of .284. This mutation was observed in 2 of 192 control chromosomes, for an estimated gene frequency of .01+/-.007. The homozygous frequency of the 30delG allele is then estimated at .0001, or 1/10,000. Given that the frequency of all childhood hearing impairment is 1/1,000 and that half of that is genetic, the specific mutation 30delG is responsible for 10% of all childhood hearing loss and for 20% of all childhood hereditary hearing loss. Six novel mutations were also observed in the affected population. The deletions detected cause frameshifts that would severely disrupt the protein structure. Three novel missense mutations, Val84Met, Val95Met, and Ser113Pro, were observed. The missense mutation 101T-->C has been reported to be a dominant allele of DFNA3, a dominant nonsyndromic hearing loss. Data further supporting the finding that this mutation does not cause dominant hearing loss are presented. This allele was found in a recessive family segregating independently from the hearing-loss phenotype and in 3 of 192 control chromosomes. These results indicate that 101T-->C is not sufficient to cause hearing loss.     

Downsloping High-Frequency Hearing Loss Due to Inner Ear Tricellular Tight Junction Disruption by a Novel ILDR1 Mutation in the Ig-Like Domain

The immunoglobulin (Ig)-like domain containing receptor 1 (ILDR1) gene encodes angulin-2/ILDR1, a recently discovered tight junction protein, which forms tricellular tight junction (tTJ) structures with tricellulin and lipolysis-stimulated lipoprotein receptor (LSR) at tricellular contacts (TCs) in the inner ear. Previously reported recessive mutations within ILDR1 have been shown to cause severe to profound nonsyndromic sensorineural hearing loss (SNHL), DFNB42. Whole-exome sequencing of a Korean multiplex family segregating partial deafness identified a novel homozygous ILDR1 variant (p.P69H) within the Ig-like domain. To address the pathogenicity of p.P69H, the angulin-2/ILDR1 p.P69H variant protein, along with the previously reported pathogenic ILDR1 mutations, was expressed in angulin-1/LSR knockdown epithelial cells. Interestingly, partial mislocalization of the p.P69H variant protein and tricellulin at TCs was observed, in contrast to a severe mislocalization and complete failure of tricellulin recruitment of the other reported ILDR1 mutations. Additionally, three-dimensional protein modeling revealed that angulin-2/ILDR1 contributed to tTJ by forming a homo-trimer structure through its Ig-like domain, and the p.P69H variant was predicted to disturb homo-trimer formation. In this study, we propose a possible role of angulin-2/ILDR1 in tTJ formation in the inner ear and a wider audiologic phenotypic spectrum of DFNB42 caused by mutations within ILDR1.     

Localization of a novel autosomal recessive non-syndromic hearing impairment locus (DFNB38) to 6q26-q27 in a consanguineous kindred from Pakistan

For autosomal recessive nonsyndromic hearing impairment over 30 loci have been mapped and 19 genes have been identified. DFNB38, a novel locus for autosomal recessive nonsyndromic hearing impairment, was localized in a consanguineous Pakistani kindred to 6q26-q27. The affected family members present with profound prelingual sensorineural hearing impairment and use sign language for communications. Linkage was established to microsatellite markers located on chromosome 6q26-q27 (Multipoint lod score 3.6). The genetic region for DFNB38 spans 10.1 cM according to the Marshfield genetic map and is bounded by markers D6S980 and D6S1719. This genetic region corresponds to 3.4 MB on the sequence-based physical map.     

Mutations of the Gene Encoding Otogelin Are a Cause of Autosomal-Recessive Nonsyndromic Moderate Hearing Impairment

Already 40 genes have been identified for autosomal-recessive nonsyndromic hearing impairment (arNSHI); however, many more genes are still to be identified. In a Dutch family segregating arNSHI, homozygosity mapping revealed a 2.4 Mb homozygous region on chromosome 11 in p15.1-15.2, which partially overlapped with the previously described DFNB18 locus. However, no putative pathogenic variants were found in USH1C, the gene mutated in DFNB18 hearing impairment. The homozygous region contained 12 additional annotated genes including OTOG, the gene encoding otogelin, a component of the tectorial membrane. It is thought that otogelin contributes to the stability and strength of this membrane through interaction or stabilization of its constituent fibers. The murine orthologous gene was already known to cause hearing loss when defective. Analysis of OTOG in the Dutch family revealed a homozygous 1 bp deletion, c.5508delC, which leads to a shift in the reading frame and a premature stop codon, p.Ala1838ProfsX31. Further screening of 60 unrelated probands from Spanish arNSHI families detected compound heterozygous OTOG mutations in one family, c.6347C>T (p.Pro2116Leu) and c. 6559C>T (p.Arg2187X). The missense mutation p.Pro2116Leu affects a highly conserved residue in the fourth von Willebrand factor type D domain of otogelin. The subjects with OTOG mutations have a moderate hearing impairment, which can be associated with vestibular dysfunction. The flat to shallow “U” or slightly downsloping shaped audiograms closely resembled audiograms of individuals with recessive mutations in the gene encoding α-tectorin, another component of the tectorial membrane. This distinctive phenotype may represent a clue to orientate the molecular diagnosis.     

CDH23 mutation and phenotype heterogeneity: a profile of 107 diverse families with Usher syndrome and nonsyndromic deafness

Usher syndrome type I is characterized by congenital hearing loss, retinitis pigmentosa (RP), and variable vestibular areflexia. Usher syndrome type ID, one of seven Usher syndrome type I genetic localizations, have been mapped to a chromosomal interval that overlaps with a nonsyndromic-deafness localization, DFNB12. Mutations in CDH23, a gene that encodes a putative cell-adhesion protein with multiple cadherin-like domains, are responsible for both Usher syndrome and DFNB12 nonsyndromic deafness. Specific CDH23 mutational defects have been identified that differentiate these two phenotypes. Only missense mutations of CDH23 have been observed in families with nonsyndromic deafness, whereas nonsense, frameshift, splice-site, and missense mutations have been identified in families with Usher syndrome. In the present study, a panel of 69 probands with Usher syndrome and 38 probands with recessive nonsyndromic deafness were screened for the presence of mutations in the entire coding region of CDH23, by heteroduplex, single-strand conformation polymorphism, and direct sequence analyses. A total of 36 different CDH23 mutations were detected in 45 families; 33 of these mutations were novel, including 18 missense, 3 nonsense, 5 splicing defects, 5 microdeletions, and 2 insertions. A total of seven mutations were common to more than one family. Numerous exonic and intronic polymorphisms also were detected. Results of ophthalmologic examinations of the patients with nonsyndromic deafness have found asymptomatic RP-like manifestations, indicating that missense mutations may have a subtle effect in the retina. Furthermore, patients with mutations in CDH23 display a wide range of hearing loss and RP phenotypes, differing in severity, age at onset, type, and the presence or absence of vestibular areflexia.     

Deficiency of Angulin-2/ILDR1, a Tricellular Tight Junction-Associated Membrane Protein,Causes Deafness with Cochlear Hair Cell Degeneration in Mice

Tricellular tight junctions seal the extracellular spaces of tricellular contacts, where the vertices of three epithelial cells meet, and are required for the establishment of a strong barrier function of the epithelial cellular sheet. Angulins and tricellulin are known as specific protein components of tricellular tight junctions, where angulins recruit tricellulin. Mutations in the genes encoding angulin-2/ILDR1 and tricellulin have been reported to cause human hereditary deafness DFNB42 and DFNB49, respectively. To investigate the pathogenesis of DFNB42, we analyzed mice with a targeted disruption of Ildr1, which encodes angulin-2/ILDR1. Ildr1 null mice exhibited profound deafness. Hair cells in the cochlea of Ildr1 null mice develop normally, but begin to degenerate by two weeks after birth. Tricellulin localization at tricellular contacts of the organ of Corti in the cochlea was retained in Ildr1 null mice, but its distribution along the depth of tricellular contacts was affected. Interestingly, compensatory tricellular contact localization of angulin-1/LSR was observed in the organ of Corti in Ildr1 null mice although it was hardly detected in the organ of Corti in wild-type mice. The onset of hair cell degeneration in Ildr1 null mice was earlier than that in the reported Tric mutant mice, which mimic one of the tricellulin mutations in DFNB49 deafness. These results indicate that the angulin-2/ILDR1 deficiency causes the postnatal degenerative loss of hair cells in the cochlea, leading to human deafness DFNB42. Our data also suggest that angulin family proteins have distinct functions in addition to their common roles of tricellulin recruitment and that the function of angulin-2/ILDR1 for hearing cannot be substituted by angulin-1/LSR.