Shin-ichi Usami

Prevalent connexin 26 gene (GJB2) mutations in Japanese

The gene responsible for DNFB1 and DFNA3, connexin 26 (GJB2), was recently identified and more than 20 disease causing mutations have been reported so far. This paper presents mutation analysis for GJB2 in Japanese non-syndromic hearing loss patients compatible with recessive inheritance. It was confirmed that GJB2mutations are an important cause of hearing loss in this population, with three mutations, 235delC, Y136X, and R143W, especially frequent. Of these three mutations, 235delC was most prevalent at 73%. Surprisingly, the 35delG mutation, which is the most commonGJB2 mutation in white subjects, was not found in the present study. Our data indicated that specific combinations of GJB2 mutation exist in different populations.

Origins and frequencies of SLC26A4 (PDS) mutations in east and south Asians: global implications for the epidemiology of deafness

Recessive mutations of SLC26A4 (PDS) are a common cause of Pendred syndrome and non-syndromic deafness in western populations. Although south and east Asia contain nearly one half of the global population, the origins and frequencies of SLC26A4 mutations in these regions are unknown. We PCR amplified and sequenced seven exons of SLC26A4 to detect selected mutations in 274 deaf probands from Korea, China, and Mongolia. A total of nine different mutations of SLC26A4 were detected among 15 (5.5%) of the 274 probands. Five mutations were novel and the other four had seldom, if ever, been identified outside east Asia. To identify mutations in south Asians, 212 Pakistani and 106 Indian families with three or more affected offspring of consanguineous matings were analysed for cosegregation of recessive deafness with short tandem repeat markers linked to SLC26A4. All 21 SLC26A4 exons were PCR amplified and sequenced in families segregating SLC26A4 linked deafness. Eleven mutant alleles of SLC26A4 were identified among 17 (5.4%) of the 318 families, and all 11 alleles were novel. SLC26A4 linked haplotypes on chromosomes with recurrent mutations were consistent with founder effects. Our observation of a diverse allelic series unique to each ethnic group indicates that mutational events at SLC26A4 are common and account for approximately 5% of recessive deafness in south Asians and other populations.

Distribution and frequencies of PDS (SLC26A4) mutations in Pendred syndrome and nonsyndromic hearing loss associated with enlarged vestibular aqueduct: a unique spectrum of mutations in Japanese.

Molecular diagnosis makes a substantial contribution to precise diagnosis, subclassification, prognosis, and selection of therapy. Mutations in the PDS (SLC26A4) gene are known to be responsible for both Pendred syndrome and nonsyndromic hearing loss associated with enlarged vestibular aqueduct, and the molecular confirmation of the PDS gene has become important in the diagnosis of these conditions. In the present study, PDS mutation analysis confirmed that PDS mutations were present and significantly responsible in 90% of Pendred families, and in 78.1% of families with nonsyndromic hearing loss associated with enlarged vestibular aqueduct. Furthermore, variable phenotypic expression by the same combination of mutations indicated that these two conditions are part of a continuous category of disease. Interestingly, the PDS mutation spectrum in Japanese, including the seven novel mutations revealed by this study, is very different from that found in Caucasians. Of the novel mutations detected, 53% were the H723R mutation, suggesting a possible founder effect. Ethnic background is therefore presumably important and should be noted when genetic testing is being performed. The PDS gene mutation spectrum in Japanese may be representative of those in Eastern Asian populations and its elucidation is expected to facilitate the molecular diagnosis of a variety of diseases.

Prevalence of mitochondrial gene mutations among hearing impaired patients

The frequency of three mitochondrial point mutations, 1555A→G, 3243A→G, and 7445A→G, known to be associated with hearing impairment, was examined using restriction fragment length polymorphism (RFLP) analysis in two Japanese groups: (1) 319 unrelated SNHL outpatients (including 21 with aminoglycoside antibiotic injection history), and (2) 140 cochlear implantation patients (including 22 with aminoglycoside induced hearing loss). Approximately 3% of the outpatients and 10% of the cochlear implantation patients had the 1555A→G mutation. The frequency was higher in the patients with a history of aminoglycoside injection (outpatient group 33%, cochlear implantation group 59%). One outpatient (0.314%) had the 3243A→G mutation, but no outpatients had the 7445A→G mutation and neither were found in the cochlear implantation group. The significance of the 1555A→G mutation, the most prevalent mitochondrial mutation found in this study of a hearing impaired population in Japan, among subjects with specific backgrounds, such as aminoglycoside induced hearing loss, is evident.

A New Autosomal Recessive Form of Stickler Syndrome Is Caused by a Mutation in the COL9A1 Gene

Stickler syndrome is characterized by ophthalmic, articular, orofacial, and auditory manifestations. It has an autosomal dominant inheritance pattern and is caused by mutations in COL2A1, COL11A1, and COL11A2. We describe a family of Moroccan origin that consists of four children with Stickler syndrome, six unaffected children, and two unaffected parents who are distant relatives (fifth degree). All family members were clinically investigated for ear, nose, and throat; ophthalmologic; and radiological abnormalities. Four children showed symptoms characteristic of Stickler syndrome, including moderate-to-severe sensorineural hearing loss, moderate-to-high myopia with vitreoretinopathy, and epiphyseal dysplasia. We considered the COL9A1 gene, located on chromosome 6q13, to be a candidate gene on the basis of the structural association with collagen types II and XI and because of the high expression in the human inner ear indicated by cDNA microarray. Mutation analysis of the coding region of the COL9A1 gene showed a homozygous R295X mutation in the four affected children. The parents and four unaffected children were heterozygous carriers of the R295X mutation. Two unaffected children were homozygous for the wild-type allele. None of the family members except the homozygous R295X carriers had any signs of Stickler syndrome. Therefore, COL9A1 is the fourth identified gene that can cause Stickler syndrome. In contrast to the three previously reported Stickler syndrome-causing genes, this gene causes a form of Stickler syndrome with an autosomal recessive inheritance pattern. This finding will have a major impact on the genetic counseling of patients with Stickler syndrome and on the understanding of the pathophysiology of collagens. Mutation analysis of this gene is recommended in patients with Stickler syndrome with possible autosomal recessive inheritance.

Utilizing Ethnic-Specific Differences in Minor Allele Frequency to Recategorize Reported Pathogenic Deafness Variants

Ethnic-specific differences in minor allele frequency impact variant categorization for genetic screening of nonsyndromic hearing loss (NSHL) and other genetic disorders. We sought to evaluate all previously reported pathogenic NSHL variants in the context of a large number of controls from ethnically distinct populations sequenced with orthogonal massively parallel sequencing methods. We used HGMD, ClinVar, and dbSNP to generate a comprehensive list of reported pathogenic NSHL variants and re-evaluated these variants in the context of 8,595 individuals from 12 populations and 6 ethnically distinct major human evolutionary phylogenetic groups from three sources (Exome Variant Server, 1000 Genomes project, and a control set of individuals created for this study, the OtoDB). Of the 2,197 reported pathogenic deafness variants, 325 (14.8%) were present in at least one of the 8,595 controls, indicating a minor allele frequency (MAF) > 0.00006. MAFs ranged as high as 0.72, a level incompatible with pathogenicity for a fully penetrant disease like NSHL. Based on these data, we established MAF thresholds of 0.005 for autosomal-recessive variants (excluding specific variants in GJB2) and 0.0005 for autosomal-dominant variants. Using these thresholds, we recategorized 93 (4.2%) of reported pathogenic variants as benign. Our data show that evaluation of reported pathogenic deafness variants using variant MAFs from multiple distinct ethnicities and sequenced by orthogonal methods provides a powerful filter for determining pathogenicity. The proposed MAF thresholds will facilitate clinical interpretation of variants identified in genetic testing for NSHL. All data are publicly available to facilitate interpretation of genetic variants causing deafness.

Connexin 26 gene (GJB2) mutation modulates the severity of hearing loss associated with the 1555A-->G mitochondrial mutation.

We report a high prevalence of GJB2 heterozygous mutations in patients bearing the 1555A-->G mitochondrial mutation, and describe a family in which potential interaction between GJB2 and a mitochondrial gene appears to be the cause of hearing impairment. Patients who are heterozygotes for the GJB2 mutant allele show hearing loss more severe than that seen in sibs lacking a mutant GJB2 allele, suggesting that heterozygous GJB2 mutations may synergistically cause hearing loss when in the presence of a 1555A-->G mutation. The present findings indicate that GJB2 mutations may sometimes be an aggravating factor, in addition to aminoglycoside antibiotics, in the phenotypic expression of the non-syndromic hearing loss associated with the 1555A-->G mitochondrial mutation.

Clinical features of patients with GJB2 (connexin 26) mutations: severity of hearing loss is correlated with genotypes and protein expression patterns.

AbstractMutations in the GJB2 (connexin 26, Cx26) gene are the major cause of nonsyndromic hearing impairment in many populations. Genetic testing offers opportunities to determine the cause of deafness and predict the course of hearing, enabling the prognostication of language development. In the current study, we compared severity of hearing impairment in 60 patients associated with biallelic GJB2 mutations and assessed the correlation of genotypes and phenotypes. Within a spectrum of GJB2 mutations found in the Japanese population, the phenotype of the most prevalent mutation, 235delC, was found to show more severe hearing impairment than that of V37I, which is the second most frequent mutation. The results of the present study, taken together with phenotypes caused by other types of mutations, support the general rule that phenotypes caused by the truncating GJB2 mutations are more severe than those caused by missense mutations. The present in vitro study further confirmed that differences in phenotypes could be explained by the protein expression pattern.

Mutations in the COCH gene are a frequent cause of autosomal dominant progressive cochleo-vestibular dysfunction, but not of Meniere's disease

The COCH gene is the only gene identified in man that causes autosomal dominantly inherited hearing loss associated with vestibular dysfunction. The condition is rare and only five mutations have been reported worldwide. All affected families showed a similar progressive hearing loss and vestibular dysfunction. Since Menieres disease-like symptoms have also been described in some families, it was suggested that COCH mutations might be present in some patients diagnosed with Menieres disease. In this study, using a Japanese population, we performed a COCH mutation analysis in 23 patients from independent families with autosomal dominant hearing impairment, four of whom reported vestibular symptoms, and also in 20 Menieres patients. While a new point mutation, A119 T, was found in a patient with autosomal dominant hearing loss and vestibular symptoms, no mutations were found in the Menieres patients. Like all other previously identified COCH mutations, the mutation identified here is a missense mutation located in the FCH domain of the protein. The current mutation is located in close spatial proximity to W117, in which a mutation (W117R) had previously been associated with autosomal dominant hearing loss. Model building suggests that, like the W117R mutation, the A119 T mutation does not affect the structural integrity of the FCH domain, but may interfere with the interaction with a yet unknown binding partner. We conclude that mutations in the COCH gene are responsible for a significant fraction of patients with autosomal dominantly inherited hearing loss accompanied by vestibular symptoms, but not for dominant hearing loss without vestibular dysfunction, or sporadic Menieres disease.

A large cohort study of GJB2 mutations in Japanese hearing loss patients

Tsukada K, Nishio S, Usami S, and the Deafness Gene Study Consortium. A large cohort study of GJB2 mutations in Japanese hearing loss patients.