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.     

Loss-of-function mutations of ILDR1 cause autosomal-recessive hearing impairment DFNB42

By using homozygosity mapping in a consanguineous Pakistani family, we detected linkage of nonsyndromic hearing loss to a 7.6 Mb region on chromosome 3q13.31-q21.1 within the previously reported DFNB42 locus. Subsequent candidate gene sequencing identified a homozygous nonsense mutation (c.1135G>T [p.Glu379X]) in ILDR1 as the cause of hearing impairment. By analyzing additional consanguineous families with homozygosity at this locus, we detected ILDR1 mutations in the affected individuals of 10 more families from Pakistan and Iran. The identified ILDR1 variants include missense, nonsense, frameshift, and splice-site mutations as well as a start codon mutation in the family that originally defined the DFNB42 locus. ILDR1 encodes the evolutionarily conserved immunoglobulin-like domain containing receptor 1, a putative transmembrane receptor of unknown function. In situ hybridization detected expression of Ildr1, the murine ortholog, early in development in the vestibule and in hair cells and supporting cells of the cochlea. Expression in hair cell- and supporting cell-containing neurosensory organs is conserved in the zebrafish, in which the ildr1 ortholog is prominently expressed in the developing ear and neuromasts of the lateral line. These data identify loss-of-function mutations of ILDR1, a gene with a conserved expression pattern pointing to a conserved function in hearing in vertebrates, as underlying nonsyndromic prelingual sensorineural hearing impairment.     

Identification of a novel mutation in POU3F4 for prenatal diagnosis in a Chinese family with X-linked nonsyndromic hearing loss

We present the clinical and genetic findings for a Chinese family with X-linked non-syndromic hearing loss in which the affected males showed congenital profound sensorineural hearing impairment. In two affected brothers, the computer tomography of temporal bone showed bilateral dilation of the internal auditory canal with fistulous communication between the lateral canal and the basal cochlear turn, which is consistent with the typical DFNX2 phenotype. A missense mutation (c.647G→A) in the POU3F4 gene caused a substitu- tion from glycine to glutamic acid at position 216 (p.G216E), and this mutation was found to consistently cosegregate with the deafness phenotype in the family. The mutation resulted in the loss of function of the POU3F4 by decreasing the affinity between the protein and DNA, as shown in silico by the structural analysis. Prenatal diagnosis of pregnant proband of this family revealed the c.647G→A muta- tion in DNA extracted from the amniotic fluid surrounding the fetus. The appropriate use of genetic testing and prenatal diagnosis plays a key role in reducing the recurrence of genetic defects in high-risk families.     

A Second Family with Nonsyndromic Sensorineural Hearing Loss Linked to Xp21.2: Refinement of the DFN4 Locus within DMD

X-linked inherited hearing impairment is a group of heterogeneous disorders accounting for less than 2% of hereditary hearing loss. DFN4, a sex-linked hearing impairment associated with profound sensorineural hearing loss, has been previously mapped to Xp21.2, a region containing the DMD locus. We have identified a family from Turkey with deafness in which the disease maps to and refines the DFN4 locus. In contrast to the previous family, the crossover points are entirely within the DMD locus. Two-point lod score analysis for the markers DXS 997, DXS 1214, and DXS 1219 showed a lod score of 2.59. 5′ and 3′ crossovers were between DMD 44 and DXS 1219 and between DXS 1214 and DXS 985, respectively, suggesting that DFN4 is either an allele of DMD or a mutation in a DMD nested gene. The restriction of the DFN4 locus to DMD suggests that dystrophin may play an important role in hearing.     

Identification of a novel mutation in WFS1 in a family affected by low-frequency hearing impairment

Previously we confirmed linkage of autosomal dominantly inherited low-frequency sensorineural hearing impairment (LFSNHI) in a German family to the genetic locus DFNA6/DFNA14 on chromosome 4p16.3 close to the markers D4S432 and D4S431. Analysis of data from the Human Genome Project, showed that WFS1 is located in this region. Mutations in WFS1 are known to be responsible for Wolfram syndrome (DIDMOAD, MIM #606201), which follows an autosomal recessive trait. Studies in low-frequency hearing loss families showed that mutations in WFS1 were responsible for the phenotype. In all affected family members analysed, we detected a missense mutation in WFS1 (K705N) and therefore confirm the finding that the majority of mutations responsible for LFSNHI are missense mutations which localise to the C-terminal domain of the protein.     

Alpha-tectorin involvement in hearing disabilities: one gene – two phenotypes

The human alpha-tectorin (TECTA) gene has recently been cloned and proposed to be involved in autosomal dominant non-syndromic hearing impairment (NSHI) in two families linked to the DFNA12 locus. We have studied a Swedish pedigree with autosomal dominant NSHI with possible digenic inheritance of the disease, involving locus DFNA12 in chromosome 11 and locus DFNA2 in chromosome 1. Mutation analysis of the TECTA gene in this family has identified eight nucleotide substitutions indicating that TECTA is highly polymorphic. One of the changes results in a cysteine to serine (C 1057 S) mutation, in the zonadhesin domain of TECTA; this segregates with the disease haplotype on chromosome 11 and is not present in a control population. The mutation results in the replacement of a cysteine in one of the repeats of the zonadhesin/Von Willebrand domain of the protein and might cause a change in the crosslinking of the polypeptide. These findings add support to the involvement of TECTA in hearing disabilities. However, the three families carrying different TECTA mutations also show phenotypic differences: the hearing loss ranges from prelingual to progressive with late onset. The explanation for the different phenotypes and some clues regarding the functions of TECTA may lie in the localization of the mutations in the different modules of the protein. Another possibility is that the phenotype in the Swedish family is the result of two defective genes.     

Novel ACTG1 mutation causing autosomal dominant non-syndromic hearing impairment in a Chinese family

Theγ-actin(ACTG1)gene is a cytoplasmic nonmuscle actin gene,which encodes a major cytoskeletal protein in the sensory hair cells of the cochlea.Mutations in ACTG1 were found to cause autosomal dominant,progressive,sensorineural hearing loss linked to the DFNA 20/26 locus on chromosome 17q25.3 in European and American families,respectively.In this study,a novel missense mutation (c.364A>G;p.I122V)co-segregated with the affected individuals in the family and did not exist in the unaffected family members and 150 unrelated normal controls.The alteration of residue I1e122 was predicted to damage its interaction with actin-binding proteins,which may cause disruption of hair cell organization and function.These findings strongly suggested that the I122V mutation in ACTG1 caused autosomal dominant non-syndromic hearing impairment in a Chinese family and expanded the spectrum of ACTG1 mutations causing hearing loss.     

A novel locus for autosomal dominant nonsyndromic hearing loss, DFNA13, maps to chromosome 6p.

Nonsyndromic hearing loss (NSHL) is the most common type of hearing impairment in the elderly. Environmental and hereditary factors play an etiologic role, although the relative contribution of each is unknown. To date, 39 NSHL genes have been localized. Twelve produce autosomal dominant hearing loss, most frequently postlingual in onset and progressive in nature. We have ascertained a large, multigenerational family in which a gene for autosomal dominant NSHL is segregating. Affected individuals experience progressive hearing loss beginning in the 2d-4th decades, eventually making the use of amplification mandatory. A novel locus, DFNA13, was identified on chromosome 6p; the disease gene maps to a 4-cM interval flanked by D6S1663 and D6S1691, with a maximum two-point LOD score of 6.409 at D6S299.     

Multiple mutations of MYO1A,a cochlear-expressed gene,in sensorineural hearing loss

Myosin I isozymes have been implicated in various motile processes, including organelle translocation, ion-channel gating, and cytoskeleton reorganization. Unconventional myosins were among the first family of proteins found to be associated with hearing loss in both humans and mice. Here, we report the identification of a nonsense mutation, of a trinucleotide insertion leading to an addition of an amino acid, and of six missense mutations in MYO1A cDNA sequence in a group of hearing-impaired patients from Italy. MYO1A, which is located within the DFNA48 locus, is the first myosin I family member found to be involved in causing deafness and may be a major contributor to autosomal dominant-hearing loss.     

A mutation in CCDC50, a gene encoding an effector of epidermal growth factor-mediated cell signaling, causes progressive hearing loss

We previously mapped a novel autosomal dominant deafness locus, DFNA44, by studying a family with postlingual, progressive, nonsyndromic hearing loss. We report here on the identification of a mutation in CCDC50 as the cause of hearing loss in the family. CCDC50 encodes Ymer, an effector of epidermal growth factor (EGF)-mediated cell signaling that is ubiquitously expressed in different organs and has been suggested to inhibit down-regulation of the EGF receptor. We have examined its expression pattern in mouse inner ear. Western blotting and cell transfection results indicate that Ymer is a soluble, cytoplasmic protein, and immunostaining shows that Ymer is expressed in a complex spatiotemporal pattern during inner ear development. In adult inner ear, the expression of Ymer is restricted to the pillar cells of the cochlea, the stria vascularis, and the vestibular sensory epithelia, where it shows spatial overlap with the microtubule-based cytoskeleton. In dividing cells, Ymer colocalizes with microtubules of the mitotic apparatus. We suggest that DFNA44 hearing loss may result from a time-dependent disorganization of the microtubule-based cytoskeleton in the pillar cells and stria vascularis of the adult auditory system.     

Targeted next-generation sequencing identifies a homozygous nonsense mutation in ABHD12, the gene underlying PHARC, in a family clinically diagnosed with Usher syndrome type 3

ABSTRACT: BACKGROUND: 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. METHODS: Candidate loci were identified using genomewide SNP-array-based homozygosity mapping followed by targeted enrichment and next-generation sequencing. RESULTS: 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. CONCLUSION: 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.     

Maternally inherited non-syndromic hearing loss associated with mitochondrial 12S rRNA A827G mutation in a Chinese family

We explored the clinical and molecular characterization of a Chinese family with non-syndromic hearing impairment. Clinical evaluations revealed a possible maternal inheritance pattern, and showed an extremely similar phenotype of hearing loss including the age of onset, severity, and audiometric configuration. Sequence analysis of the mitochondrial 12S rRNA and tRNA(Ser(UCN)) genes led to the identification of a homoplasmic A827G mutation in all maternal relatives, which was absent in other family members and 40 Chinese controls. This mutation has previously been reported sporadically in a few individuals with aminoglycoside-induced and non-syndromic hearing loss. The A827G mutation is located at the A-site of the mitochondrial 12S rRNA gene which is highly evolutionarily conserved in mammals. The occurrence of the A827G mutation in these genetically unrelated subjects strongly suggests that this mutation is involved in the pathogenesis of hearing impairment. However, incomplete penetrance of hearing loss indicates that the A827G mutation alone is not sufficient to produce clinical phenotype but requires the involvement of modifier factors for the phenotypic expression, even though aminoglycosides and GJB2 gene may not contribute to the penetrance of the A827G mutation in this Chinese family. In contrast with the variable phenotype of hearing loss associated with other mitochondrial mutations, all of the patients in our family exhibited strikingly similar clinical features. This discrepancy likely reflects the difference of genetic backgrounds between this pedigree and others.     

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.     

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 novel locus (DFNA23) for prelingual autosomal dominant nonsyndromic hearing loss maps to 14q21-q22 in a Swiss German kindred

DFNA23, a novel locus for autosomal dominant nonsyndromic hearing loss, was identified in a Swiss German kindred. DNA samples were obtained from 22 family members in three generations: 10 with hearing impairment caused by the DFNA23 locus, 8 unaffected offspring, and 4 spouses of hearing-impaired pedigree members. In this kindred, the hearing-impaired family members have prelingual bilateral symmetrical hearing loss. All audiograms from hearing-impaired individuals displayed sloping curves, with hearing ability ranging from normal hearing to mild hearing loss in low frequencies, normal hearing to profound hearing loss in mid frequencies, and moderate to profound hearing loss in high frequencies. A conductive component existed for 50% of the hearing-impaired family members. The majority of the hearing-impaired family members did not display progression of hearing loss. The DFNA23 locus maps to 14q21-q22. Linkage analysis was carried out under a fully penetrant autosomal dominant mode of inheritance with no phenocopies. A maximum multipoint LOD score of 5.1 occurred at Marker D14S290. The 3.0-LOD unit support interval is 9.4 cM and ranged from marker D14S980 to marker D14S1046.     

Mitochondrial 12S rRNA A827G mutation is involved in the genetic susceptibility to aminoglycoside ototoxicity

We have analyzed the clinical and molecular characterization of a Chinese family with aminoglycoside-induced and non-syndromic hearing impairment. Clinical evaluations revealed that only those family members who had a history of exposure to aminoglycoside antibiotics subsequently developed hearing loss, suggesting mitochondrial genome involvement. Sequence analysis of the mitochondrial 12S rRNA and tRNA(Ser(UCN)) genes led to the identification of a homoplasmic A827G mutation in all maternal relatives, a mutation that was identified previously in a few sporadic patients and in another Chinese family with non-syndromic deafness. The pathogenicity of the A827G mutation is strongly supported by the occurrence of the same mutation in two independent families and several genetically unrelated subjects. The A827G mutation is located at the A-site of the mitochondrial 12S rRNA gene which is highly conserved in mammals. It is possible that the alteration of the tertiary or quaternary structure of this rRNA by the A827G mutation may lead to mitochondrial dysfunction, thereby playing a role in the pathogenesis of hearing loss and aminoglycoside hypersensitivity. However, incomplete penetrance of hearing impairment indicates that the A827G mutation itself is not sufficient to produce clinical phenotype but requires the involvement of modifier factors for the phenotypic expression. Indeed, aminoglycosides may contribute to the phenotypic manifestation of the A827G mutation in this family. In contrast with the congenital or early-onset hearing impairment in another Chinese family carrying the A827G mutation, three patients in this pedigree developed hearing loss only after use of aminoglycosides. This discrepancy likely reflects the difference of genetic backgrounds, either mitochondrial haplotypes or nuclear modifier genes, between two families.     

An Alteration in ELMOD3, an Arl2 GTPase-Activating Protein,Is Associated with Hearing Impairment in Humans

Exome sequencing coupled with homozygosity mapping was used to identify a transition mutation (c.794T>C; p.Leu265Ser) in ELMOD3 at the DFNB88 locus that is associated with nonsyndromic deafness in a large Pakistani family, PKDF468. The affected individuals of this family exhibited pre-lingual, severe-to-profound degrees of mixed hearing loss. ELMOD3 belongs to the engulfment and cell motility (ELMO) family, which consists of six paralogs in mammals. Several members of the ELMO family have been shown to regulate a subset of GTPases within the Ras superfamily. However, ELMOD3 is a largely uncharacterized protein that has no previously known biochemical activities. We found that in rodents, within the sensory epithelia of the inner ear, ELMOD3 appears most pronounced in the stereocilia of cochlear hair cells. Fluorescently tagged ELMOD3 co-localized with the actin cytoskeleton in MDCK cells and actin-based microvilli of LLC-PK1-CL4 epithelial cells. The p.Leu265Ser mutation in the ELMO domain impaired each of these activities. Super-resolution imaging revealed instances of close association of ELMOD3 with actin at the plasma membrane of MDCK cells. Furthermore, recombinant human GST-ELMOD3 exhibited GTPase activating protein (GAP) activity against the Arl2 GTPase, which was completely abolished by the p.Leu265Ser mutation. Collectively, our data provide the first insights into the expression and biochemical properties of ELMOD3 and highlight its functional links to sound perception and actin cytoskeleton.