Suoqiang Zhai

A distinct spectrum of SLC26A4 mutations in patients with enlarged vestibular aqueduct in China.

There is a worldwide interest in studying SLC26A4 mutations that are responsible for enlarged vestibular aqueduct (EVA) in different ethnic background and populations. The spectrum of SLC26A4 mutations in Chinese population is yet to be fully characterized. In this study, all the 21 exons of SLC26A4 were screened in 107 Chinese patients with hearing loss associated with EVA or both EVA and Mondini dysplasia (MD), taken from six multiplex and 95 simplex families. The two types of control populations consisted of 84 normal‐hearing subjects and 46 sensorineural hearing loss subjects without inner ear malformations. Biallelic mutations were found in 12 patients from multiplex families and 84 patients (88.4%) from the simplex families. In addition, monoallelic variant was detected in nine patients in the remaining 11 simplex families. Overall, up to 97.9% patients were found having at least one possible pathogenic variant in SLC26A4, with most having biallelic variants consistent with recessive inheritance of this disorder. A total of 40 mutations including 25 novel mutations were identified in the Chinese patients but were not detected in all the controls except for one normal subject. For the Chinese mutation spectrum of SLC26A4 gene, IVS7‐2A>G mutation was the most common form accounting for 57.63% (102/177) of all the mutant alleles.

Cosegregation of the G7444A Mutation in the Mitochondrial COI/tRNASer(UCN) Genes with the 12S rRNA A1555G Mutation in a Chinese Family with Aminoglycoside-induced and Nonsyndromic Hearing Loss

We report here on the characterization of a three‐generation Chinese family with aminoglycoside‐induced and nonsyndromic hearing impairment. Ten of 17 matrilineal relatives exhibited bilateral and sensorineural hearing impairment. Of these, nine matrilineal relatives, who had a history of exposure to aminoglycosides, exhibited variable severity and audiometric configuration of hearing loss. The dose and age at the time of drug administration seemed to be correlated with the severity of the hearing loss experienced by affected individuals. Sequence analysis of the complete mitochondrial genome in the pedigree showed the presence of homoplasmic A1555G mutation and 37 variants belonging to haplogroup D4a. Of those variants, the G7444A mutation is of special interest as the mutation at this position results in a read‐through of the stop condon AGA of the COI message, thereby adding three amino acids (Lys–Gln–Lys) to the C‐terminal of the polypeptide. Alternatively, the G7444A mutation is adjacent to the site of 3′ end endonucleolytic processing of L‐strand RNA precursor, spanning tRNASer(UCN) and ND6 mRNA. Thus, the G7444A mutation, similar to the deafness‐associated A7445G mutation, may lead to a defect in the processing of the L‐strand RNA precursor, thus influencing the phenotypic expression of the A1555G mutation. These data also imply that nuclear background plays a role in the aminoglycoside ototoxicity associated with the A1555G mutation in this Chinese pedigree.

Variants in mitochondrial tRNAGlu, tRNAArg, and tRNAThr may influence the phenotypic manifestation of deafness-associated 12S rRNA A1555G mutation in three Han Chinese families with hearing loss†

We report here on the clinical, genetic, and molecular characterization of three Han Chinese pedigrees with aminoglycoside‐induced and nonsyndromic hearing loss. Clinical evaluation revealed the variable phenotype of hearing loss including severity, age‐at‐onset, audiometric configuration in these subjects. Penetrances of hearing loss in BJ107, BJ108, and BJ109 pedigrees are 35%, 63%, and 67%, respectively. Mutational analysis of the complete mitochondrial genomes in these pedigrees showed the identical homoplasmic A1555G mutation and distinct sets of mitochondrial DNA (mtDNA) variants belonging to haplogroups N, F, and M, respectively. Of these variants, the A14693G mutation in the tRNAGlu, the T15908C mutation in the tRNAThr, and the T10454C mutation in the tRNAArg are of special interest as these mutations occur at positions which are highly evolutionarily conserved nucleotides of corresponding tRNAs. These homoplasmic mtDNA mutations were absent among 156 unrelated Chinese controls. The A14693G and T10454C mutations occur at the highly conserved bases of the TψC‐loop of tRNAGlu and tRNAArg, respectively. Furthermore, the T15908C mutation in the tRNAThr disrupts a highly conserved A‐U base‐pairing at the D‐stem of this tRNA. The alteration of structure of these tRNAs by these mtDNA mutations may lead to a failure in tRNA metabolism, thereby causing impairment of mitochondrial translation. Thus, mitochondrial dysfunctions, caused by the A1555G mutation, would be worsened by these mtDNA mutations. Therefore, these mtDNA mutations may have a potential modifier role in increasing the penetrance and expressivity of the deafness‐associated 12S rRNA A1555G mutation in those Chinese pedigrees.

Coexistence of mitochondrial 12S rRNA C1494T and CO1/tRNASer(UCN) G7444A mutations in two Han Chinese pedigrees with aminoglycoside-induced and non-syndromic hearing loss

Mutations in mitochondrial DNA are one of the important causes of hearing loss. We report here the clinical, genetic, and molecular characterization of two Han Chinese pedigrees with maternally transmitted aminoglycoside-induced and nonsyndromic bilateral hearing loss. Clinical evaluation revealed the wide range of severity, age-at-onset, and audiometric configuration of hearing impairment in matrilineal relatives in these families. The penetrances of hearing loss in these pedigrees were 20% and 18%, when aminoglycoside-induced deafness was included. When the effect of aminoglycosides was excluded, the penetrances of hearing loss in these seven pedigrees were 10% and 15%. Sequence analysis of the complete mitochondrial genomes in these pedigrees showed the presence of the deafness-associated 12S rRNA C1494T and CO1/tRNA(Ser(UCN)) G7444A mutations. Their distinct sets of mtDNA polymorphism belonged to Eastern Asian haplogroup C4a1, while other previously identified six Chinese mitochondrial genomes harboring the C1494T mutation belong to haplogroups D5a2, D, R, and F1, respectively. This suggested that the C1494T or G7444A mutation occurred sporadically and multiplied through evolution of the mitochondrial DNA (mtDNA). The absence of functionally significant mutations in tRNA and rRNAs or secondary LHON mutations in their mtDNA suggest that these mtDNA haplogroup-specific variants may not play an important role in the phenotypic expression of the 12S rRNA C1494T and CO1/tRNA(Ser(UCN)) G7444A mutations in those Chinese families. However, aminoglycosides and other nuclear modifier genes play a modifying role in the phenotypic manifestation of the C1494T mutation in these Chinese families.

Basic fibroblast growth factor protects auditory neurons and hair cells from glutamate neurotoxicity and noise exposure

Objective To determine the protective effects of basic fibroblast growth factor (bFGF) on cochlear neurons and hair cells in vitro and in vivo. Material and Methods In Experiment I, cultured spiral ganglion neurons (SGNs) prepared from postnatal Day 3 mice were exposed to 20 mM glutamate for 2 h before the culture medium was replaced with fresh medium containing 0, 25, 50 or 100 ng/ml bFGF. Fourteen days later, all cultures were fixed with 4% paraformaldehyde and stained with 1% toluidine blue. The number of surviving SGNs was counted and the length of the neurites of the SGNs was measured. In Experiment II, in vivo studies were carried out with guinea pigs in which bFGF or normal saline was injected intramuscularly to assess possible protective effects of bFGF on cochlear hair cells and to accelerate the recovery of the auditory brainstem response (ABR). The ABRs were measured before, immediately after and 2 and 4 weeks after exposure to noise. Results Exposure to 20 mM glutamate for 2 h resulted in an inhibition of neurite outgrowth of SGNs and an increase in cell death. Treatment of the cultures with bFGF led to promotion of neurite outgrowth and an increase in the number of surviving SGNs. In Experiment II, significant (p<0.05) differences in ABR thresholds were observed between the groups injected with bFGF and saline (t=2.689) at 4 weeks after noise exposure. Cochleae were removed and hair cell loss analyzed in surface preparations prepared from all experimental animals. Acoustic trauma caused loss of 240 and 2160 inner hair cells in the groups injected with bFGF and saline, respectively. Similarly, more outer hair cells were lost in the normal saline injection group (99 291) than in the group treated with bFGF (70 377). Conclusions Our results demonstrate that bFGF protects SGNs against glutamate neurotoxicity in vitro. In addition, treatment with bFGF protects hair cells from acoustic trauma.

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] = 0 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.

The differentiation of mesenchymal stem cells into inner ear hair cell-like cells in vitro

Abstract Conclusion. Bone marrow mesenchymal stem cells (MSCs) have the ability to differentiate into hair cells, and this method of culturing MSCs provides a useful tool for studies on mammalian cochlear hair cell regeneration. Objective: To investigate a method to induce bone marrow MSCs to differentiate into inner ear hair cells. Methods: Rat bone marrow MSCs were isolated from healthy rats and cultured in vitro. To make sure that the cultured cells were bone marrow MSCs, the expression of MSC markers such as SH2, CD31, CD34, and CD44 genes on the cultured cells was assessed by RT-PCR. Adipogenic cells and osteogenic cells were induced by the differentiation of the cultured cells, respectively, suggesting that the cultured cells have the characteristic of pluripotent differentiation. Then they were induced to differentiate into neural stem cells and hair cell progenitor cells. Immunohistochemistry experiments were carried out to detect the expression of molecular markers. Scanning electron microscope samples were prepared for observation of the morphology of the cells. Results: Rat bone marrow MSCs were successfully isolated, purified, cultured, and identified in vitro. They were also successfully induced to differentiate into neural progenitor cells and then hair cell-like cells that expressed myosin VIIa.

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