Xiaomei Ouyang

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.

Antioxidant enzymes, presbycusis, and ethnic variability.

OBJECTIVE: A proposed mechanism for presbycusis is a significant increase in oxidative stress in the cochlea. The enzymes glutathione S-transferase (GST) and N-acetyltransferase (NAT) are two classes of antioxidant enzymes active in the cochlea. In this work, we sought to investigate the association of different polymorphisms of GSTM1, GSTT1, and NAT2 and presbycusis and analyze whether ethnicity has an effect in the genotype-phenotype associations. STUDY DESIGN: Case-control study of 134 DNA samples. SETTING: University-based tertiary care center. SUBJECTS AND METHODS: Clinical, audiometric, and DNA testing of 55 adults with presbycusis and 79 control patients with normal hearing. RESULTS: The GSTM1 null genotype was present in 77 percent of white Hispanics and 51 percent of white non-Hispanics (Fishers exact test, 2-tail, P = 0.0262). The GSTT1 null genotype was present in 34 percent of control patients and in 60 percent of white presbycusis subjects (P = 0.0067, odds ratio [OR] = 2.843, 95% confidence interval [95% CI] = 1.379–5.860). The GSTM1 null genotype was more frequent in presbycusis subjects, i.e., 48 percent of control patients and 69 percent of white subjects carried this deletion (P = 0.0198, OR = 2.43, 95% CI = 1.163–5.067). The NAT2∗6A mutant genotype was more frequent among subjects with presbycusis (60%) than in control patients (34%; P = 0.0086, OR = 2.88, 95% CI = 1.355–6.141). CONCLUSION: We showed an increased risk of presbycusis among white subjects carrying the GSTM1 and the GSTT1 null genotype and the NAT∗6A mutant allele. Subjects with the GSTT1 null genotypes are almost three times more likely to develop presbycusis than those with the wild type. The GSTM1 null genotype was more prevalent in white Hispanics than in white non-Hispanics, but the GSTT1 and NAT2 polymorphisms were equally represented in the two groups.

Absence of GJB2 gene mutations, the GJB6 deletion (GJB6-D13S1830) and four common mitochondrial mutations in nonsyndromic genetic hearing loss in a South African population.

OBJECTIVE The purpose of this study was to determine the prevalence of mutations in the GJB2 gene, the GJB6-D13S1830 deletion and the four common mitochondrial mutations (A1555G, A3243G, A7511C and A7445G) in a South African population. METHODS Using single-strand conformation polymorphism and direct sequencing for screening GJB2 mutation; Multiplex PCR Amplification for GJB6-D13S1830 deletion and Restriction Fragment-Length Polymorphism (PCR-RFLP) analysis for the four common mtDNA mutations. We screened 182 hearing impaired students to determine the frequency of these mutations in the population. RESULTS None of the reported disease causing mutations in GJB2 nor any novel pathogenic mutations in the coding region were detected, in contrast to the findings among Caucasians. The GJB6-D13S1830 deletion and the mitochondrial mutations were not observed in this group. CONCLUSION These results suggest that GJB2 may not be a significant deafness gene among sub-Saharan Africans, pointing to other unidentified genes as responsible for nonsyndromic hearing loss in these populations.

Mutation analysis in the long isoform of USH2A in American patients with Usher Syndrome type II.

Usher syndrome type II (USH2) is an autosomal recessive disorder characterized by moderate to severe hearing impairment and progressive visual loss due to retinitis pigmentosa (RP). To identify novel mutations and determine the frequency of USH2A mutations as a cause of USH2, we have carried out mutation screening of all 72 coding exons and exon–intron splice sites of the USH2A gene. A total of 20 USH2 American probands of European descent were analyzed using single strand conformational polymorphism (SSCP) and direct sequencing methods. Ten different USH2A mutations were identified in 55% of the probands, five of which were novel mutations. The detected mutations include three missense, three frameshifts and four nonsense mutations, with c.2299delG/p.E767fs mutation, accounting for 38.9% of the pathological alleles. Two cases were homozygotes, two cases were compound heterozygotes and one case had complex allele with three variants. In seven probands, only one USH2A mutation was detected and no pathological mutation was found in the remaining eight individuals. Altogether, our data support the fact that c.2299delG/p.E767fs is indeed the most common USH2A mutation found in USH2 patients of European Caucasian background. Thus, if screening for mutations in USH2A is considered, it is reasonable to screen for the c.2299delG mutation first.

Audiological and genetic features of the mtDNA mutations

Conclusions. Significant difference in the incidence of mitochondrial DNA (mtDNA) mutations was found between the Chinese and USA populations. The identification of the mtDNA A1555G mutation in a large proportion of Chinese probands with nonsyndromic sensorineural hearing loss (NSHL) provides a molecular explanation for the high prevalence of aminoglycoside-induced deafness in China. Objective. The aim was to characterize the audiological and genetic features of NSHL due to mutations in mtDNA. Subjects and methods. The mtDNA and audiogram analyses were performed in 498 NSHL patients (290 from China and 208 from the USA) with and without history of aminoglycoside exposure. A PCR and restriction enzyme digestion protocol was used for mutational screening and the European Workshop on Genetic Hearing Loss criteria were applied for audiological classification. Results. All Chinese probands (15.5%) with mtDNA mutation were found to carry the homoplasmic mtDNA A1555G mutation, whereas four probands (1.9%) from the USA were found to carry the mtDNA A1555G and two (1%) had mtDNA G7444A. Approximately 63% of the probands with mtDNA mutations had post-lingual hearing loss and 56.8% of them had a medical history of exposure to aminoglycosides. Hearing losses are bilateral, sensorineural, and symmetric. The main audiogram shapes found were sloping.

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.

Mutational spectrum in Usher syndrome type II.

Usher syndrome type II is an autosomal recessive disorder characterized by moderate to severe hearing impairment and progressive visual loss due to retinitis pigmentosa (RP). We carried out a mutation screening of the USH2A gene in 88 probands with Usher syndrome type II to determine the frequency of USH2A mutations as a cause for USH2. Six mutations, including 2299delG, 921‐922insCAGC, R334W, N346H, R626X, and N357T were identified, with 2299delG mutation being the most frequent (16.5% of alleles), accounting for 77.5% of the pathologic alleles. Thirty‐five percent (31/88) of the probands had a USH2A mutation. Nine of them carried two pathogenic mutations: six cases were homozygotes and three were compound heterozygotes. Twenty‐two probands (25%) were found to carry only single USH2A mutations. One new missense mutation (N357T) occuring within the laminin N‐terminal (type VI) domain of usherin was identified. Eight polymorphisms were found, five of which are novel. Our data support the view that the 2299delG is the most common mutation in USH2A.

USH1C: a rare cause of USH1 in a non-Acadian population and a founder effect of the Acadian allele.

Usher syndrome (USH) is characterized by the associated findings of hearing loss and retinitis pigmentosa (RP), leading to progressive loss of vision. Three forms of USH can be distinguished clinically. In the most severe form, USH1, profound congenital deafness is associated with vestibular dysfunction and RP. To determine the frequency of USH1C mutations as a cause for USH1, 128 probands with Usher syndrome type 1 including seven from Acadian and 121 from non‐Acadian populations were systematically screened for mutations in USH1C using a combined single‐strand conformational polymorphisms (SSCP)/heteroduplex and sequencing method. All seven Acadian USH1 patients were found to be homozygous for both the 216G>A mutation and the 9‐repeat VNTR which characterizes the Acadian allele, confirming previous evidence for a founder effect by haplotype analysis. However, USH1C mutations were identified in only two non‐Acadian USH1 probands (1.65%) including one from Pakistan who was homozygous for a 238‐239insC mutation and one from Canada was also homozygous for the Acadian allele. The low prevalence of USH1C mutations in the present study suggests that the high prevalence of the 238‐239insC in Germany may reflect a founder effect. Comparison of the affected haplotypes in the Canadian patient with the Acadian USH1 patients yielded evidence for a founder effect. Our data suggest that USH1C is a relatively rare form of USH1 in non‐Acadian populations and that in addition to the 216G>A Acadian mutation, the 238‐239insC mutation appears to be common in some populations.

Mutation Analysis of Slc26a4 in Mainland Chinese Patients with Enlarged Vestibular Aqueduct

OBJECTIVE: We have characterized the spectrum of SLC26A4 mutations and clinical features in a population of mainland Chinese patients with nonsyndromic sensorineural hearing loss (SNHL) and enlarged vestibular aqueduct (EVA). STUDY DESIGN: Cross-sectional clinical genetic study. SETTING: Tertiary care outpatient otolaryngology clinic. METHODS: A total of 32 subjects identified with bilateral EVA using high-resolution CT were screened for mutations in SLC26A4 by denaturing high-performance liquid chromatography and direct sequencing methods. RESULTS: A total of 13 different mutations were identified in the SLC26A4 gene, five of which are novel. A total of 88 percent of the patients harbored biallelic mutations, 11 patients were homozygotes, and 17 were compound heterozygotes. Four patients were found to carry a single SLC26A4 mutation. The IVS7-2A>G mutation was the most frequent, accounting for 60 percent of the mutant alleles. We have not found any correlations between the type of SLC26A4 mutations and the type, degree, and progression of hearing loss. There are significant proportions of patients with asymmetric (26%), progressive (32%), or fluctuating hearing loss (21%). CONCLUSION: Our data confirm the high prevalence of SLC26A4 mutations in Chinese patients with SNHL and EVA. We could not establish any relationship between genotype and pheno-type. However, the high incidence of asymmetric, progressive, and fluctuating hearing loss found in the current study indicates that patients with those features should be routinely screened for SLC26A4 mutation in addition to diagnosis of EVA using CT or MRI.

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