Han D

Loss-of-Function Mutations in the PRPS1 Gene Cause a Type of Nonsyndromic X-linked Sensorineural Deafness, DFN2

We report a large Chinese family with X-linked postlingual nonsyndromic hearing impairment in which the critical linkage interval spans a genetic distance of 5.41 cM and a physical distance of 15.1 Mb that overlaps the DFN2 locus. Mutation screening of the PRPS1 gene in this family and in the three previously reported DFN2 families identified four different missense mutations in PRPS1. These mutations result in a loss of phosphoribosyl pyrophosphate (PRPP) synthetase 1 activity, as was shown in silico by structural analysis and was shown in vitro by enzymatic activity assays in erythrocytes and fibroblasts from patients. By in situ hybridization, we demonstrate expression of Prps1 in murine vestibular and cochlea hair cells, with continuous expression in hair cells and postnatal expression in the spiral ganglion. Being the second identified gene associated with X-linked nonsyndromic deafness, PRPS1 will be a good candidate gene for genetic testing for X-linked nonsyndromic hearing loss.

Functional Mutation of SMAC/DIABLO, Encoding a Mitochondrial Proapoptotic Protein, Causes Human Progressive Hearing Loss DFNA64

SMAC/DIABLO is a mitochondrial proapoptotic protein that is released from mitochondria during apoptosis and counters the inhibitory activities of inhibitor of apoptosis proteins, IAPs. By linkage analysis and candidate screening, we identified a heterozygous SMAC/DIABLO mutation, c.377C>T (p.Ser126Leu, refers to p.Ser71Leu in the mature protein) in a six-generation Chinese kindred characterized by dominant progressive nonsyndromic hearing loss, designated as DFNA64. SMAC/DIABLO is highly expressed in human embryonic ears and is enriched in the developing mouse inner-ear hair cells, suggesting it has a role in the development and homeostasis of hair cells. We used a functional study to demonstrate that the SMAC/DIABLO(S71L) mutant, while retaining the proapoptotic function, triggers significant degradation of both wild-type and mutant SMAC/DIABLO and renders host mitochondria susceptible to calcium-induced loss of the membrane potential. Our work identifies DFNA64 as the human genetic disorder associated with SMAC/DIABLO malfunction and suggests that mutant SMAC/DIABLO(S71L) might cause mitochondrial dysfunction.

Mitochondrial haplotype and phenotype of 13 Chinese families may suggest multi-original evolution of mitochondrial C1494T mutation

Mutations in mitochondrial DNA (mtDNA) are associated with sensorineural hearing loss. In this study, we traced the origin of the 12S rRNA C1494T mutation through analysis of the clinical, genetic, and molecular characteristics of 13 Han Chinese pedigrees with aminoglycoside-induced and non-syndromic bilateral hearing loss that were selected by C1494T screening in 3133 subjects with non-syndromic hearing impairment from 27 regions of China (13/3133). Clinical evaluation revealed the variable phenotypes of hearing impairment including severity, age-of-onset, and audiometric configuration in these subjects. Through the whole mitochondrial genome DNA sequence analysis, we identified two evolutionarily conservative variants in protein-coding genes: tRNA(Ala) T 5628C and tRNA(Tyr) A5836G mutations. However, the pedigrees with these mutations did not have a higher or lower penetrance of deafness than in other pedigrees. These results suggested that both T 5628C and A5836G mutations might not significantly modify the manifestation of the C1494T mutation. Sequencing analysis of the whole mitochondrial genome of the probands showed that 13 pedigrees from seven different provinces were classified into 10 haplogroups by the distinct sets of mtDNA polymorphisms, including haplogroups A, B, D, D4, D4b2, F1, M, M7c, N9a1, and H2b. This result suggested that the C1494T mutation occurred sporadically with multi-origins through the evolution of the mtDNA in China, and these mtDNA haplogroup-specific variants may not play an important role in the phenotypic expression of the C1494T mutation in these Chinese families with different penetrance of hearing loss. In addition, the lack of a significant mutation in the GJB2 gene ruled out the possible involvement of GJB2 in the phenotypic expression of the C1494T mutation in those affected subjects. Therefore, the aminoglycosides is solo well-established factor to contribute to the deafness manifestation of the C1494T mutation, and prevention by avoiding the administration of aminoglycosides in individuals carrying C1494T mutation is the most effective way to protect their vulnerability to deafness.

A novel DFNA5 mutation, IVS8+4 A>G, in the splice donor site of intron 8 causes late-onset non-syndromic hearing loss in a Chinese family.

We report here the clinical, genetic, and molecular characteristics of a large Chinese family exhibiting non‐syndromic, late‐onset autosomal dominant sensorineural hearing loss. Clinical evaluation revealed variable phenotypes of hearing loss in terms of severity and age‐at‐onset of disease in these subjects. Genome‐wide linkage analysis mapped the disease gene to the DFNA5 locus with a maximum two‐point log odds score of 5.39 at [theta]u2003=u20030 for marker D7S2457. DNA sequencing of DFNA5 revealed a novel heterozygous IVS8+4 A>G substitution in the splice donor site of intron 8. Reverse transcriptase–polymerase chain reaction (RT–PCR) showed skipping of exon 8 in the mutant transcript. This mutation faithfully cosegregated with hearing loss in the family. In addition, the mutation was absent in 100 unrelated control DNA samples of Chinese origin. The IVS8+4 A>G mutation is predicted to create a shift in the reading frame and introduce a stop codon at position 372, thereby resulting in a prematurely truncated DFNA5 protein. Up to date, a total of four mutations in DFNA5 have been reported to lead to hearing impairment, all of them result in skipping of exon 8 at the mRNA level. Our findings provide further support for the hypothesis that DFNA5‐associated hearing loss is caused by a very specific gain‐of‐function mutation.

Novel mutations in the vWFA2 domain of COCH in two Chinese DFNA9 families.

To the Editor: The autosomal dominant sensorineural nonsyndromic hearing loss (HL) 9 (DFNA9) locus is known to be associated with vestibular dysfunction (1). The DFNA9 causative gene COCH localizes on chromosome 14q12-q13 (2) and encodes cochlin, an extracellular matrix protein. Cochlin contains a region homologous to a domain in factor C of Limulus, also known as LCCL domain, and two von Willebrand factor A-like domains (vWFA1 and 2). Interestingly, most of the previously described COCH mutations are missense point mutations located within exon 4 or 5 encoding the LCCL domain of cochlin (1, 3–10). In 2005, Street et al. (11) reported a p.C542F mutation in an American DFNA9 familywithHLaswell as vestibular andoculomotor disturbances. This mutation located within the second vWFA (vWFA2) domain of cochlin, representing the first reported DFNA9 mutation outside the LCCL domain. In the present study, we have ascertained a large Chinese family (SD-Z001) with late-onset autosomal dominant non-syndromic progressive sensorineural HL. The family included 113 members spanning six generations. Appropriate informed consent was obtained from participants in accordance with the Ethics Committee of Chinese PLA General Hospital. Thirty-six members from the family considered informative were selected for linkage analysis. A maximum two-point logarithm of odds ratio score of 6.69 at y 1⁄4 0 was obtained for marker D14S1040. Haplotype analysis placed the locus within a 7.6-cM genetic interval defined by markers D14S1021 and D14S70, overlapping with the DFNA9 locus on chromosome 14q12-q13. DNA sequencing of the coding regions as well as exon–intron boundaries of the COCH gene and subsequent confirmation by MfeI restriction endonuclease analysis revealed a c.1625G.A mutation (Fig. 1a,c) in exon 12 that cosegregates with auditory dysfunction in the pedigree. The mutation results in a predicted p.C542Y substitution at an evolutionarily conserved cysteine residue in the vWFA2 domain of cochlin (Fig. 1b,d). Furthermore, we screened COCH for mutations in 26 Chinese DFNA families as well as in 19 small families (the inheritance pattern could not be recognized) and 22 sporadic patients with lateonset progressive sensorineural HL. A heterozygous missense mutation (c.1535T.C) that converted an evolutionarily conserved methionine residue to a threonine residue (p.M512T) was also identified in the vWFA2 domain (Fig. 1 a,b,d) in a small family (HLJ-Z079), which had only two patients. Both mutations were absent in 100 unrelated control DNA samples of Chinese background, supporting the hypothesis that they represent a causative mutation and not a rare polymorphism. We did not detect any polymorphism in the COCH gene in the families during the sequence analysis. The age of onset of HL varied in the SD-Z001 family. It ranged from the second to the fifth decade of life. For generation III, the age at onset was at the fifth decade, generation IV at fourth, generation V at third and generation VI at second to third. The finding of a lower age at diagnosis among offspring compared with their parents suggests the presence of genetic anticipation. To the best of our knowledge, there are no earlier reports on the phenomenon in DFNA9 families. There are multiple mechanisms by which anticipation occurs including biological, environmental, and also statistical because of ascertainment or truncation bias (12). Therefore, in the absence of biological information, the intergenerational difference in age at diagnosis is difficult to interpret, and attention to observational biases is warranted. Tinnitus at the onset of HL was reported in 82% of the affected family members. The HL first affected the high frequencies and later involved all frequencies. Overall, the patients display a downward sloping audiogram contour. In the HLJ-Z079 family, the proband (III-5) began suffering bilateral HL at the age of 43 years. At the onset of HL, she

Genetic Basis of Y-Linked Hearing Impairment

A single Mendelian trait has been mapped to the human Y chromosome: Y-linked hearing impairment. The molecular basis of this disorder is unknown. Here, we report the detailed characterization of the DFNY1 Y chromosome and its comparison with a closely related Y chromosome from an unaffected branch of the family. The DFNY1 chromosome carries a complex rearrangement, including duplication of several noncontiguous segments of the Y chromosome and insertion of ∼160 kb of DNA from chromosome 1, in the pericentric region of Yp. This segment of chromosome 1 is derived entirely from within a known hearing impairment locus, DFNA49. We suggest that a third copy of one or more genes from the shared segment of chromosome 1 might be responsible for the hearing-loss phenotype.

Novel missense mutations in MYO7A underlying postlingual high- or low-frequency non-syndromic hearing impairment in two large families from China.

The myosin VIIA (MYO7A) gene encodes a protein classified as an unconventional myosin. Mutations within MYO7A can lead to both syndromic and non-syndromic hearing impairment in humans. Among different mutations reported in MYO7A, only five led to non-syndromic sensorineural deafness autosomal dominant type 11 (DFNA11). Here, we present the clinical, genetic and molecular characteristics of two large Chinese DFNA11 families with either high- or low-frequency hearing loss. Affected individuals of family DX-J033 have a sloping audiogram at young ages with high frequency are most affected. With increasing age, all test frequencies are affected. Affected members of family HB-S037 present with an ascending audiogram affecting low frequencies at young ages, and then all frequencies are involved with increasing age. Genome-wide linkage analysis mapped the disease loci within the DFNA11 interval in both families. DNA sequencing of MYO7A revealed two novel nucleotide variations, c.652G>A (p.D218N) and c.2011G>A (p.G671S), in the two families. It is for the first time that the mutations identified in MYO7A in the present study are being implicated in DFNA11 in a Chinese population. For the first time, we tested electrocochleography (ECochG) in a DFNA11 family with low-frequency hearing loss. We speculate that the low-frequency sensorineural hearing loss in this DFNA11 family was not associated with endolymphatic hydrops.

Acoustic neuroma surgery for preservation of hearing: technique and experience in the Chinese PLA General Hospital

Abstract Conclusions: Good hearing preservation can be achieved during acoustic neuroma surgery assisted by real-time dynamic auditory monitoring and ear endoscopic techniques. Preservation of the arachnoid and its blood supply are important for hearing preservation, and injury to the internal auditory artery is the most important cause of hearing loss. Objective: To explore techniques to preserve hearing during acoustic neuroma resection. Methods: This was a retrospective case review in a hospital setting. From July 2003 to July 2007, intraoperative auditory monitoring using auditory brainstem response (ABR) and electrocochleography (EcochG) was conducted in 18 of 138 patients undergoing surgery for acoustic neuroma who had preoperative hearing. The retrosigmoid approach was used for 16 complete resections and 2 patients underwent partial resections. Assisted endoscopic surgery was conducted for 10 ears. The main outcome measure was preservation of hearing, assessed using the classification method of the American Institute of Otolaryngology-Head and Neck Surgery. Results: Hearing was preserved in 11 of the 18 patients (61.1%): 2 of 5 patients whose tumors were larger than 20 mm (40%), and 9 of 13 patients with smaller tumors (69.2%). Among the 10 cases of ear endoscope-assisted surgery, hearing was preserved in 8 (80%). Intraoperative monitoring revealed that the waveform was influenced when the posterior labium of the internal acoustic meatus was ground and drilled, or when traction or electrocoagulation was performed near the opening of the internal acoustic meatus, especially when the internal auditory artery was clamped, the tumor in the internal acoustic meatus was treated, and the arachnoid vessels in the inner-most layer of the tumor surface were clamped or electrocoagulated.

Resolving the genetic heterogeneity of prelingual hearing loss within one family: Performance comparison and application of two targeted next generation sequencing approaches.

Here, we report an unconventional Chinese pedigree consisting of three branches all segregating prelingual hearing loss (HL) with unclear inheritance pattern. After identifying the cause of one branch as maternally inherited aminoglycoside-induced HL, targeted next generation sequencing (NGS) was applied to identify the genetic causes for the other two branches. One affected subject from each branch was subject to targeted NGS whose genomic DNA was enriched either by whole-exome capture (Agilent SureSelect All Exon 50u2009Mb) or by candidate genes capture (Agilent SureSelect custom kit). By NGS analysis, we identified that patients from Branch A were compound heterozygous for p.E1006K and p.D1663V in the CDH23 (DFNB12) gene; and patients from Branch B were homozygous for IVS7-2A>G in the SLC26A4 (DFNB4) gene. Both CDH23 mutations altered conserved calcium binding sites of the extracellular cadherin domains. The co-occurrence of three different genetic causes in this family was exceedingly rare but fully compatible with the mutation spectrum of HL. Our study has also raised several technical and analytical issues when applying the NGS technique to genetic testing.

Exome Sequencing Identifies a Novel Frameshift Mutation of MYO6 as the Cause of Autosomal Dominant Nonsyndromic Hearing Loss in a Chinese Family

Autosomal dominant types of nonsyndromic hearing loss (ADNSHL) are typically postlingual in onset and progressive. High genetic heterogeneity, late onset age, and possible confounding due to nongenetic factors hinder the timely molecular diagnoses for most patients. In this study, exome sequencing was applied to investigate a large Chinese family segregating ADNSHL in which we initially failed to find strong evidence of linkage to any locus by whole‐genome linkage analysis. Two affected family members were selected for sequencing. We identified two novel mutations disrupting known ADNSHL genes and shared by the sequenced samples: c.328C>A in COCH (DFNA9) resulting in a p.Q110K substitution and a deletion c. 2814_2815delAA in MYO6 (DFNA22) causing a frameshift alteration p.R939Tfs*2. The pathogenicity of novel coding variants in ADNSHL genes was carefully evaluated by analysis of co‐segregation with phenotype in the pedigree and in light of established genotype–phenotype correlations. The frameshift deletion in MYO6 was confirmed as the causative variant for this pedigree, whereas the missense mutation in COCH had no clinical significance. The results allowed us to retrospectively identify the phenocopy in one patient that contributed to the negative finding in the linkage scan. Our clinical data also supported the emerging genotype–phenotype correlation for DFNA22.