Jing Cheng

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.

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.

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.

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.

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

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 substitution 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 mutation 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.

Identification of two novel missense WFS1 mutations, H696Y and R703H, in patients with non-syndromic low-frequency sensorineural hearing loss.

Non-syndromic low-frequency sensorineural hearing loss (LFSNHL) is an unusual type of hearing loss in which frequencies ≤2000 Hz predominantly are affected. To date, different mutations in two genes, DIAPH1 and WFS1, have been found to be associated with LFSNHL. Here, we report a five-generation Chinese family with postlingual and progressive LFSNHL. We mapped the disease locus to a 2.5 Mb region on chromosome 4p16 between markers SNP_A-2167174 and D4S431, overlapping with the DFNA6/14/38 locus. Sequencing of candidate gene revealed a heterozygous c.2086C>T substitution in exon 8 of WFS1, leading to p.H696Y substitution at the C-terminus of Wolframin (WFS1). In addition, we performed mutational screening of WFS1 in 37 sporadic patients, 7-50 years of age, with LFSNHL. We detected a heterozygous c.2108G>A substitution in exon 8 of WFS1, leading to p.R703H substitution in a patient. The H696 and R703 in WFS1 are highly conserved across species, including human, orangutan, rat, mouse, and frog (Xenopus). Sequence analysis demonstrated the absence of c.2086C>T or c.2108G>A substitutions in the WFS1 genes among 200 unrelated control subjects of Chinese background, supporting the hypothesis that they represent causative mutations, and not rare polymorphisms. Our data provide additional molecular and clinical information for establishing a better genotype-phenotype correlation for LFSNHL.

Preimplantation Genetic Diagnosis for a Chinese Family with Autosomal Recessive Meckel-Gruber Syndrome Type 3 (MKS3)

Meckel-Gruber syndrome type 3 is an autosomal recessive genetic defect caused by mutations in TMEM67 gene. In our previous study, we have identified a homozygous TMEM67 mutation in a Chinese family exhibiting clinical characteristics of MKS3, which provided a ground for further PGD procedure. Here we report the development and the first clinical application of the PGD for this MKS3 family. Molecular analysis protocol for clinical PGD procedure was established using 50 single cells in pre-clinical set-up. After whole genomic amplification by multiple displacement amplification with the DNA from single cells, three techniques were applied simultaneously to increase the accuracy and reliability of genetic diagnosis in single blastomere, including real-time PCR with Taq Man-MGB probe, haplotype analysis with polymorphic STR markers and Sanger sequencing. In the clinical PGD cycle, nine embryos at cleavage-stage were biopsied and subjected to genetic diagnosis. Two embryos diagnosed as free of TMEM67 mutation were transferred and one achieving normal pregnancy. Non-invasive prenatal assessment of trisomy 13, 18 and 21 by multiplex DNA sequencing at 18 weeks’ gestation excluded the aneuploidy of the analyzed chromosomes. A healthy boy was delivered by cesarean section at 39 weeks’ gestation. DNA sequencing from his cord blood confirmed the result of genetic analysis in the PGD cycle. The protocol developed in this study was proved to be rapid and safe for the detection of monogenic mutations in clinical PGD cycle.

Identification of a Novel TECTA mutation in a Chinese DFNA8/12 family with prelingual progressive sensorineural hearing impairment.

Tectorial membrane, an extracellular matrix of the cochlea, plays a crucial role in the transmission of sound to the sensory hair cells. Alpha-tectorin is the most important noncollagenous component of the tectorial membrane and the otolith membrane in the maculae of the vestibular system. Defects in TECTA, the gene encodes alpha-tectorin, are cause of both dominant (DFNA8/12) and recessive (DFNB21) forms of deafness. Here, we report a three-generation Chinese family characterized by prelingual progressive sensorineural hearing impairment. We mapped the disease locus to chromosome 11q23-24 region, overlapping with the DFNA8/12 locus. Sequencing of candidate gene TECTA revealed a heterozygous c.5945C>A substitution in exon 19, causing amino acid substitution of Ala to Asp at a conservative position 1982. The A1982D substitution is consistent with hearing loss in this Chinese family and has not been found in 200 random control chromosomes. To our knowledge, this is the first TECTA mutation identified in Chinese population. Our data provides additional molecular and clinical information for establishing a better genotype–phenotype understanding of DFNA8/12.

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.