Deafness Locus Research Articles

Homozygosity mapping in a family presenting with schizophrenia, epilepsy and hearing impairment

Homozygosity mapping within consanguineous families is a powerful method of localising genes for autosomal recessive disease. We investigated a family from Punjab, Pakistan, a region where consanguineous marriages are frequent. The parents have no detectable clinical disorders. However, five out of six children present with schizophrenia, epilepsy or hearing impairment either alone or in combination. This unusual phenotype in several offspring of first cousins is strongly suggestive of a rare, Mendelian recessive disorder. Two genome-wide scans initially using low-density microsatellites, and subsequently high-density SNP markers were used to map homozygous-by-descent regions in affected individuals. Candidate genes within these loci were subsequently screened for mutations. Homozygosity analysis and inbreeding coefficients were investigated to give an estimate of consanguinity. Two putative disease loci were mapped to 22q12.3-q13.3 and 2p24.3. The candidate locus on chromosome 2p24 overlaps with a deafness locus, DFNB47, linked to autosomal recessive hearing impairment, while positive findings reported for affective psychosis and schizophrenia cluster in a region of 4-5 cM on 22q13.1 within our second candidate locus. Sequence analysis of three candidate genes (KCNF1 (2p); ATF4, CACNG2 (22q)) did not reveal any exonic mutations. Inbreeding coefficients calculated for each family member support a very high degree of ancestral and recent inbreeding. The screening of other candidate genes located within these newly identified disease intervals on Chr2p24.3 and 22q12.3-q13.3 may lead to the discovery of causative variants, and consequent disrupted molecular pathways associated with this rare phenotype.

Targeted knockout and lacZ reporter expression of the mouse Tmhs deafness gene and characterization of the hscy-2J mutation.

The Tmhs gene codes for a tetraspan transmembrane protein that is expressed in hair cell stereocilia. We previously showed that a spontaneous missense mutation of Tmhs underlies deafness and vestibular dysfunction in the hurry-scurry (hscy) mouse. Subsequently, mutations in the human TMHS gene were shown to be responsible for DFNB67, an autosomal recessive nonsyndromic deafness locus. Here we describe a genetically engineered null mutation of the mouse Tmhs gene (Tmhs ( tm1Kjn )) and show that its phenotype is identical to that of the hscy missense mutation, confirming the deleterious nature of the hscy cysteine-to-phenylalanine substitution. In the targeted null allele, the Tmhs promoter drives expression of a lacZ reporter gene. Visualization of beta-galactosidase activity in Tmhs ( tm1Kjn ) heterozygous mice indicates that Tmhs is highly expressed in the cochlear and vestibular hair cells of the inner ear. Expression is first detectable at E15.5, peaks around P0, decreases slightly at P6, and is absent by P15, a duration that supports the involvement of Tmhs in stereocilia development. Tmhs reporter gene expression also was detected in several cranial and cervical sensory ganglia, but not in the vestibular or spiral ganglia. We also describe a new nontargeted mutation of the Tmhs gene, hscy-2J, that causes abnormal splicing from a cryptic splice site within exon 2 and is predicted to produce a functionally null protein lacking 51 amino acids of the wild-type sequence.

Identities, frequencies and origins of TMC1 mutations causing DFNB7/B11 deafness in Pakistan

Non-syndromic deafness is genetically heterogeneous. We previously reported that mutations of transmembrane channel-like gene 1 (TMC1) cause non-syndromic recessive deafness at the DFNB7/B11 locus on chromosome 9q13-q21 in nine Pakistani families. The goal of this study was to define the identities, origins and frequencies of TMC1 mutations in an expanded cohort of 557 large Pakistani families segregating recessive deafness. We screened affected family members for homozygosity at short-tandem repeats flanking known autosomal recessive (DFNB) deafness loci, followed by TMC1 sequence analysis in families segregating deafness linked to DFNB7/B11. We identified 10 new families segregating DFNB7/B11 deafness and TMC1 mutations, including three novel alleles. Overall, 9 different TMC1 mutations account for deafness in 19 (3.4%) of the 557 Pakistani families. A single mutation, p.R34X, causes deafness in 10 (1.8%) of the families. Genotype analysis of p.R34X-linked markers indicates that it arose from a common founder. We also detected p.R34X among normal control samples of African-American and northern European origins, raising the possibility that p.R34X and other mutations of TMC1 are prevalent contributors to the genetic load of deafness across a variety of populations and continents.

Unexpected genetic heterogeneity in a large consanguineous Brazilian pedigree presenting deafness

Nonsyndromic autosomal recessive deafness accounts for 80% of hereditary deafness. To date, 52 loci responsible for autosomal recessive deafness have been mapped and 24 genes identified. Here, we report a large inbred Brazilian pedigree with 26 subjects affected by prelingual deafness. Given the extensive consanguinity found in this pedigree, the most probable pattern of inheritance is autosomal recessive. However, our linkage and mutational analysis revealed, instead of an expected homozygous mutation in a single gene, two different mutant alleles and a possible third undetected mutant allele in the MYO15A gene (DFNB3 locus), as well as evidence for other causes for deafness in the same pedigree. Among the 26 affected subjects, 15 were homozygous for the novel c.10573delA mutation in the MYO15A gene, 5 were compound heterozygous for the mutation c.10573delA and the novel deletion c.9957_9960delTGAC and one inherited only a single c.10573delA mutant allele, while the other one could not be identified. Given the extensive consanguinity of the pedigree, there might be at least one more deafness locus segregating to explain the condition in some of the subjects whose deafness is not clearly associated with MYO15A mutations, although overlooked environmental causes could not be ruled out. Our findings illustrate a high level of etiological heterogeneity for deafness in the family and highlight some of the pitfalls of genetic analysis of large genes in extended pedigrees, when homozygosity for a single mutant allele is expected.

The autosomal recessive nonsyndromic deafness locus DFNB72 is located on chromosome 19p13.3

We ascertained three consanguineous Pakistani families (PKDF291, PKDF335 and PKDF793) segregating nonsyndromic recessive hearing loss. The hearing loss segregating in PKDF335 and PKDF793 is moderate to severe, whereas it is profound in PKDF291. The maximum two-point LOD scores are 3.01 (D19S1034), 3.85 (D19S894) and 3.71 (D19S894) for PKDF291, PKDF335 and PKDF793, respectively. Haplotype analyses of the three families define a 1.16 Mb region of overlap of the homozygous linkage intervals bounded by markers D19S216 (20.01 cM) and D19S1034 (20.75 cM). These results define a novel locus, DFNB72, on chromosome 19p13.3. There are at least 22 genes in the 1.16 Mb interval, including PTPRS, ZNRF4 and CAPS. We identified no pathogenic variants in the exons and flanking intronic sequences of these three genes in affected members of the DFNB72 families. DFNB72 is telomeric to DFNB68, the only other known deafness locus with statistically significant support for linkage to chromosome 19p.

Audioprofiling identifies TECTA and GJB2‐related deafness segregating in a single extended pedigree

An audioprofile displays phenotypic data from several audiograms on a single graph that share a common genotype. In this report, we describe the application of audioprofiling to a large family in which a genome-wide screen failed to identify a deafness locus. Analysis of audiograms by audioprofiling suggested that two persons with hearing impairment had a different deafness genotype. On this basis, we reassigned affectation status and identified a p.Cys1837Arg autosomal dominant mutation in alpha-tectorin segregating in all family members except two persons, who segregated autosomal recessive deafness caused by p.Val37Ile and p.Leu90Pro mutations in Connexin 26. One nuclear family in the extended pedigree segregates both dominant and recessive non-syndromic hearing loss.

Analysis and functional evaluation of the hair-cell transcriptome

An understanding of the molecular bases of the morphogenesis, organization, and functioning of hair cells requires that the genes expressed in these cells be identified and their functions ascertained. After purifying zebrafish hair cells and detecting mRNAs with oligonucleotide microarrays, we developed a subtractive strategy that identified 1,037 hair cell-expressed genes whose cognate proteins subserve functions including membrane transport, synaptic transmission, transcriptional control, cellular adhesion and signal transduction, and cytoskeletal organization. To assess the validity of the subtracted hair-cell data set, we verified the presence of 11 transcripts in inner-ear tissue. Functional evaluation of two genes from the subtracted data set revealed their importance in hair bundles: zebrafish larvae bearing the seahorse and ift 172 mutations display specific kinociliary defects. Moreover, a search for candidate genes that underlie heritable deafness identified a human ortholog of a zebrafish hair-cell gene whose map location is bracketed by the markers of a deafness locus.

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.

An integrated genetic and functional analysis of the role of type II transmembrane serine proteases (TMPRSSs) in hearing loss

Building on our discovery that mutations in the transmembrane serine protease, TMPRSS3, cause nonsyndromic deafness, we have investigated the contribution of other TMPRSS family members to the auditory function. To identify which of the 16 known TMPRSS genes had a strong likelihood of involvement in hearing function, three types of biological evidence were examined: 1) expression in inner ear tissues; 2) location in a genomic interval that contains a yet unidentified gene for deafness; and 3) evaluation of hearing status of any available Tmprss knockout mouse strains. This analysis demonstrated that, besides TMPRSS3, another TMPRSS gene was essential for hearing and, indeed, mice deficient for Hepsin (Hpn) also known as Tmprss1 exhibited profound hearing loss. In addition, TMPRSS2, TMPRSS5, and CORIN, also named TMPRSS10, showed strong likelihood of involvement based on their inner ear expression and mapping position within deafness loci PKSR7, DFNB24, and DFNB25, respectively. These four TMPRSS genes were then screened for mutations in affected members of the DFNB24 and DFNB25 deafness families, and in a cohort of 362 sporadic deaf cases. This large mutation screen revealed numerous novel sequence variations including three potential pathogenic mutations in the TMPRSS5 gene. The mutant forms of TMPRSS5 showed reduced or absent proteolytic activity. Subsequently, TMPRSS genes with evidence of involvement in deafness were further characterized, and their sites of expression were determined. Tmprss1, 3, and 5 proteins were detected in spiral ganglion neurons. Tmprss3 was also present in the organ of Corti. TMPRSS1 and 3 proteins appeared stably anchored to the endoplasmic reticulum membranes, whereas TMPRSS5 was also detected at the plasma membrane. Collectively, these results provide evidence that TMPRSS1 and TMPRSS3 play and TMPRSS5 may play important and specific roles in hearing.

Predicting candidate genes for human deafness disorders: a bioinformatics approach.

BackgroundThere are more than 50 genes for autosomal dominant and autosomal recessive nonsyndromic hereditary deafness that are yet to be cloned. The human genome sequence and expression profiles of transcripts in the inner ear have aided positional cloning approaches. The knowledge of protein interactions offers additional advantages in selecting candidate genes within a mapped region.ResultsWe have employed a bioinformatic approach to assemble the genes encoded by genomic regions that harbor various deafness loci. The genes were then in silico analyzed for their candidacy by expression pattern and ability to interact with other proteins. Such analyses have narrowed a list of 2400 genes from suspected regions of the genome to a manageable number of about 140 for further analysis.ConclusionWe have established a list of strong candidate genes encoded by the regions linked to various nonsyndromic hereditary hearing loss phenotypes by using a novel bioinformatic approach. The candidates presented here provide a starting point for mutational analysis in well-characterized families along with genetic linkage to refine the loci. The advantages and shortcomings of this bioinformatic approach are discussed.

A novel G21R mutation of the GJB2 gene causes autosomal dominant non-syndromic congenital deafness in a Cuban family

Deafness is a complex disorder affecting 1/1000 infants. In developed countries, more than 50% of deafness cases are thought to have a genetic cause. At least 40 loci for dominant non-syndromic deafness and another 30 for recessive non-syndromic deafness have been described. Mutations in the GJB2 gene are the cause of an important number of cases of non-syndromic recessive deafness but are not as common in non-syndromic dominant deafness cases. We describe here a new dominant mutation (G21R) in the GJB2 gene which causes deafness and has been identified in a three generation Cuban family with dominant non-syndromic congenital sensorineural profound deafness.

Mutations in TRIOBP, Which Encodes a Putative Cytoskeletal-Organizing Protein, Are Associated with Nonsyndromic Recessive Deafness

In seven families, six different mutant alleles of TRIOBP on chromosome 22q13 cosegregate with autosomal recessive nonsyndromic deafness. These alleles include four nonsense (Q297X, R788X, R1068X, and R1117X) and two frameshift (D1069fsX1082 and R1078fsX1083) mutations, all located in exon 6 of TRIOBP. There are several alternative splice isoforms of this gene, the longest of which, TRIOBP-6, comprises 23 exons. The linkage interval for the deafness segregating in these families includes DFNB28. Genetic heterogeneity at this locus is suggested by three additional families that show significant evidence of linkage of deafness to markers on chromosome 22q13 but that apparently have no mutations in the TRIOBP gene.

Pseudodominanz zweier rezessiver Connexin-Mutationen bei nicht-syndromaler Hörstörung?

Hitherto more than hundred genes and gene loci for non-syndromic or syndromic deafness have been identified. Mutations in the connexin 26 gene (GJB2) account for up to 50 % of the cases of autosomal recessive hearing loss. The genes GJB2 (Connexin 26), GJB3 (connexin 31) and GJB6 (connexin 31) are located on chromosome 13q11-12. In the inner ear up to four different connexins are expressed. Connexins appertain to a group of gap junction proteins. These proteins can oligomerize to form single-membrane channels called connexons. Each connexon is composed of six subunits, that allow communication between adjacent cells by providing a channel for diffusion of ions, metabolites and second messengers. Each of the exons and flanking splice regions of the connexin 26, 30, and 31 genes (GJB2, GJB3, and GJB6) have been analysed by direct sequencing. In the involved families three heterozygous mutations could be detected in the connexin 26 (GJB2) and connexin 30 (GJB6) genes. If a combination of two of those mutations occurs, 35DeltaG with 146/147DeltaC and 35DeltaG with GJB6-D13S1830 it results in hearing loss and deafness. By evidences of a familial background of hearing loss it is reasonable to analyse the connexin genes (GJB2, GJB3 and GJB6) for mutations, additionally to a specific hearing diagnostic, in order to enhance linguistic development through hearing aid or CI-implantation at an early stage.

Differential Expression of Genes within the Cochlea as Defined by a Custom Mouse Inner Ear Microarray

Microarray analyses have contributed greatly to the rapid understanding of functional genomics through the identification of gene networks as well as gene discovery. To facilitate functional genomics of the inner ear, we have developed a mouse inner-ear-pertinent custom microarray chip (CMA-IE1). Nonredundant cDNA clones were obtained from two cDNA library resources: the RIKEN subtracted inner ear set and the NIH organ of Corti library. At least 2000 cDNAs unique to the inner ear were present on the chip. Comparisons were performed to examine the relative expression levels of these unique cDNAs within the organ of Corti, lateral wall, and spiral ganglion. Total RNA samples were obtained from the three cochlear-dissected fractions from adult CF-1 mice. The total RNA was linearly amplified, and a dendrimer-based system was utilized to enhance the hybridization signal. Differentially expressed genes were verified by comparison to known gene expression patterns in the cochlea or by correlation with genes and gene families deduced to be present in the three tissue types. Approximately 22-25% of the genes on the array had significant levels of expression. A number of differentially expressed genes were detected in each tissue fraction. These included genes with known functional roles, hypothetical genes, and various unknown or uncharacterized genes. Four of the differentially expressed genes found in the organ of Corti are linked to deafness loci. None of these are hypothetical or unknown genes.

DFNB48, a new nonsyndromic recessive deafness locus, maps to chromosome 15q23-q25.1

Nonsyndromic deafness locus (DFNB48) segregating as an autosomal recessive trait has been mapped to the long arm of chromosome 15 in bands q23-q25.1 in five large Pakistani families. The deafness phenotype in one of these five families (PKDF245) is linked to D15S1005 with a lod score of 8.6 at theta=0, and there is a critical linkage interval of approximately 7 cM on the Marshfield human genetic map, bounded by microsatellite markers D15S216 (70.73 cM) and D15S1041 (77.69 cM). MYO9A, NR2E3, BBS4, and TMC3 are among the candidate genes in the DFNB48 region. The identification of another novel nonsyndromic recessive deafness locus demonstrates the high degree of locus heterogeneity for hearing impairment, particularly in the Pakistani population.

A Novel Autosomal Recessive Non-Syndromic Deafness Locus, DFNB66, Maps to Chromosome 6p21.2-22.3 in a Large Tunisian Consanguineous Family

Hereditary non-syndromic deafness is extremely heterogeneous. Autosomal recessive forms account for approximately 80% of genetic cases. Autosomal recessive non-syndromic sensorineural deafness segregating in a large consanguineous Tunisian family was mapped to chromosome 6p21.2-22.3. A maximum lod score of 5.36 at Θ = 0 was obtained for the polymorphic microsatellite marker IR2/IR4. Haplotype analysis defined a 16.5-Mb critical region between microsatellite markers D6S1602 and D6S1665. The screening of 3 candidate genes, COL11A2, BAK1 and TMHS, did not reveal any disease causing mutation, suggesting that this is a novel deafness locus, which has been named DFNB66. A search in the Human Cochlear EST Library for ESTs located in this critical interval allowed us to identify several candidates. Further investigations on these candidates are needed in order to identify the deafness-causing gene in this Tunisian family.

A novel missense mutation in a C2 domain ofOTOF results in autosomal recessive auditory neuropathy

Screening of 12 Turkish families with apparently autosomal recessive nonsyndromic sensorineural deafness without GJB2 and mtDNA m.1555A > G mutations for 11 previously mapped recessive deafness loci showed a family in which hearing loss cosegregated with the DFNB9 (OTOF) locus. Three affected children were later found to carry a novel homozygous c.3032T > C (p.Leu1011Pro) mutation in the OTOF gene. Both parents were heterozygous for the mutation. p.Leu1011Pro alters a conserved leucine residue in the C2D domain of otoferlin. Pure tone audiometry of the family showed severe to profound sensorineural hearing loss (with U-shape audiograms) in children, and normal hearing in the parents. Otoacoustic emissions and auditory brainstem response (ABR) suggested the presence of auditory neuropathy in affected individuals.

Mapping of a novel autosomal recessive nonsyndromic deafness locus (DFNB46) to chromosome 18p11.32-p11.31

Hereditary nonsyndromic deafness (NSD) is extremely heterogeneous. Autosomal recessive (AR) forms account for approximately 75% of genetic cases. To date, over 40 ARNSD loci have been mapped. A novel locus (DFNB46) for ARNSD was mapped to chromosome 18p11.32-p11.31 in a five-generation Pakistani family. A 10 cM genome-wide scan and fine mapping was carried out using microsatellite markers. A maximum multipoint LOD score of 3.8 was obtained at two markers, D18S481 and D18S1370. The three-unit support interval is flanked by markers D18S59 and D18S391, corresponds to a 17.6 cM region according to the deCode genetic map and spans 5.8 Mb on the sequence-based physical map.

A new locus for nonsyndromic deafness DFNB49 maps to chromosome 5q12.3-q14.1

Cosegregation of markers on chromosome 5q12.3-q14.1 with profound congenital deafness in two Pakistani families (PKDF041 and PKDF141) defines a new recessive deafness locus, DFNB49. A maximum two-point lod score of 4.44 and 5.94 at recombination fraction theta=0 was obtained for markers D5S2055 and D5S424 in families PKDF041 and PKDF141, respectively. Haplotype analysis revealed an 11 cM linkage region flanked by markers D5S647 (74.07 cM) and D5S1501 (85.25 cM). Candidate deafness genes in this region include SLC30A5, OCLN, GTF2H2, and BTF3, encoding solute carrier family 30 (zinc transporter) member 5, occludin, RNA polymerase II transcription initiation factor, and basic transcription factor 3, respectively. Sequence analysis of the coding exons of SLC30A5 in DNA samples from two affected individuals of families PKDF041 and PKDF141 revealed no mutation. The mapping of DFNB49 further confirms the heterogeneity underlying autosomal recessive forms of nonsyndromic deafness.

Characterization of Two Transgene Insertional Mutations at Pirouette, a Mouse Deafness Locus

The mouse mutant ‘pirouette’ (pi) exhibits profound hearing loss and vestibular defects due to inheritance of a recessive mutation on chromosome 5. Dysfunction has been correlated with defects during maturation of sensory cells in the inner ear. As an initial step in characterizing pirouette at the genetic level, we have localized the candidate interval to a small region on central chromosome 5 by analysis of a congenic strain of pirouette mice. This region exhibits conserved synteny with human chromosome 4 and suggests that pirouette may be a genetic model of the human nonsyndromic deafness disorder DFNB25, which has been localized to 4p15.3–q12. In addition to the original spontaneous pirouette strain, we have identified and characterized 2 additional mouse strains with allelic mutations at the same locus. Analysis of the morphology in each of the 3 pirouette alleles indicated very similar early postnatal alterations in maturation of stereocilia and suggests that the gene affected in pirouette normally plays a role in building or maintaining these structures that are critical for sensory mechanotransduction.