Correlation between genotype and phenotype with special attention to hearing in 14 Japanese cases of NF2-related schwannomatosis.

NF2-related schwannomatosis (NF2) is an autosomal dominant genetic disorder caused by variants in the NF2 gene. Approximately 50% of NF2 patients inherit pathogenic variants, and the remainder acquire de novo variants. NF2 is characterized by development of bilateral vestibular schwannomas. The genetic background of Japanese NF2 cases has not been fully investigated, and the present report performed a genetic analysis of 14 Japanese NF2 cases and examined genotype-phenotype correlations. DNA samples collected from peripheral blood were analyzed by next-generation sequencing, multiplex ligation-dependent probe amplification analysis, and in vitro electrophoresis. Ten cases had pathogenic or likely pathogenic variants in the NF2 gene, with seven truncating variants and three non-truncating variants. The age of onset in all seven cases with truncating variants was < 20 years. The age of onset significantly differed among cases with truncating NF2 variants, non-truncating NF2 variants, and no NF2 variants. However, the clinical course of tumor growth and hearing deterioration were not predicted only by germline pathogenic NF2 variants. The rate of truncating variants was higher in the present study than that of previous reports. Genotype-phenotype correlations in the age of onset were present in the analyzed Japanese NF2 cases.

Molecular basis of carotid body tumor and associated clinical features in Japan identified by genomic, immunohistochemical, and clinical analyses.

Carotid body tumor (CBT) is classified as a paraganglioma (PGL). Here, we report the genetic background, protein expression pattern, and clinical findings of 30 Japanese CBT cases. Germline pathogenic or likely pathogenic (P/LP) variants of genes encoding succinate dehydrogenase subunits (SDHs) were detected in 15 of 30 cases (50%). The SDHB variants were the most frequently detected, followed by SDHA and SDHD variants. One case with SDHAF2 variant was bilateral CBT, and other two multiple PGL cases were not detected P/LP variants. The three cases with germline variants that could be tested did not have somatic P/LP variants of the same genes. Immunohistochemical analysis showed negative SDHB signals in CBT tissues in five cases with germline P/LP variants of SDHB, SDHD, or SDHA. In addition, SDHB signals in CBT tissues were negative in four of nine cases without germline P/LP variants of SDHs. These findings suggest the involvement of unidentified molecular mechanisms affecting SDHs.

Phenotype-genotype correlation in patients with typical and atypical branchio-oto-renal syndrome.

Some patients have an atypical form of branchio-oto-renal (BOR) syndrome, which does not satisfy the diagnostic criteria, despite carrying a pathogenic variant (P variant) or a likely pathogenic variant (LP variant) of a causative gene. P/LP variants phenotypic indices have yet to be determined in patients with typical and atypical BOR syndrome. We hypothesized that determining phenotypic and genetic differences between patients with typical and atypical BOR syndrome could inform such indices. Subjects were selected from among patients who underwent genetic testing to identify the cause of hearing loss. Patients were considered atypical when they had two major BOR diagnostic criteria, or two major criteria and one minor criterion; 22 typical and 16 atypical patients from 35 families were included. Genetic analysis of EYA1, SIX1, and SIX5 was conducted by direct sequencing and multiplex ligation-dependent probe amplification. EYA1 P/LP variants were detected in 25% and 86% of atypical and typical patients, respectively. Four EYA1 P/LP variants were novel. Branchial anomaly, inner ear anomaly, and mixed hearing loss were correlated with P/LP variants. Development of refined diagnostic criteria and phenotypic indices for atypical BOR syndrome will assist in effective detection of patients with P/LP variants among those with suspected BOR syndrome.

Clinical Profiles of DFNA11 at Diverse Stages of Development and Aging in a Large Family Identified by Linkage Analysis.

HYPOTHESIS: The phenotype of DFNA11 consists of specific features at diverse developmental and age stages. BACKGROUND: Only eight mutations have been identified for autosomal dominant non-syndromic hearing loss related to MYO7A (DFNA11), and the onset and progression of DFNA11 are poorly understood. METHODS: After linkage analysis and following Sanger sequencing in a family suspected to have autosomal dominant hereditary hearing loss, we analyzed the audiometric and vestibular functions and their long-term changes in the subjects carrying the variant. RESULTS: A reported variant of uncertain significance, NP_000251.3:p.Arg853His, in MYO7A was detected and cosegregation data of this large family provided evidence that the variant was likely pathogenic for DFNA11. Family members with the variant had no other symptoms associated with hearing loss and were confirmed to have autosomal dominant non-syndromic sensorineural hearing loss. Audiograms tended to show gently sloping configuration in childhood and flat configuration after the age of 30 years. Hearing loss at high frequencies progressed slowly, while hearing at low frequencies started to deteriorate later but progressed more rapidly. Some subjects showed partly abnormal results in the distortion products of otoacoustic emissions before the elevation of hearing thresholds. Vestibular function was within the normal range in all the subjects tested. CONCLUSION: We revealed that hearing loss at high frequencies was mainly noted in early developmental stages and that thresholds increased more rapidly in the low frequency range, resulting in changes in audiometric configuration. Deterioration of distortion product otoacoustic emissions (DPOAE) before the elevation of hearing thresholds was considered as a clinical feature of DFNA11.

IMSindel: An accurate intermediate-size indel detection tool incorporating de novo assembly and gapped global-local alignment with split read analysis.

Insertions and deletions (indels) have been implicated in dozens of human diseases through the radical alteration of gene function by short frameshift indels as well as long indels. However, the accurate detection of these indels from next-generation sequencing data is still challenging. This is particularly true for intermediate-size indels (≥50 bp), due to the short DNA sequencing reads. Here, we developed a new method that predicts intermediate-size indels using BWA soft-clipped fragments (unmatched fragments in partially mapped reads) and unmapped reads. We report the performance comparison of our method, GATK, PINDEL and ScanIndel, using whole exome sequencing data from the same samples. False positive and false negative counts were determined through Sanger sequencing of all predicted indels across these four methods. The harmonic mean of the recall and precision, F-measure, was used to measure the performance of each method. Our method achieved the highest F-measure of 0.84 in one sample, compared to 0.56 for GATK, 0.52 for PINDEL and 0.46 for ScanIndel. Similar results were obtained in additional samples, demonstrating that our method was superior to the other methods for detecting intermediate-size indels. We believe that this methodology will contribute to the discovery of intermediate-size indels associated with human disease.

The first sporadic case of DFNA11 identified by next-generation sequencing.

We report the first sporadic case of nonsyndromic autosomal dominant hearing loss (DFNA11). The patient was a 5-year-old boy with moderate bilateral hearing loss. Targeted next-generation sequencing analysis of patient DNA identified a known heterozygous DFNA11 mutation, c.689C > T, in MYO7A, encoding p.Ala230Val. The mutation was not detected in the parents of the patient and is considered to be de novo. This mutation is identical to the one reported previously in an Italian family. Accumulation of mutation data increases the feasibility of identifying autosomal dominant mutations in sporadic sensorineural hearing loss.

Microbiomes of the normal middle ear and ears with chronic otitis media.

OBJECTIVE: The aim of this study was to profile and compare the middle ear microbiomes of human subjects with and without chronic otitis media. STUDY DESIGN: Prospective multicenter cohort study. METHODS: All consecutive patients undergoing tympanoplasty surgery for chronic otitis media or ear surgery for conditions other than otitis media were recruited. Sterile swab samples were collected from the middle ear mucosa during surgery. The variable region 4 of the 16S rRNA gene in each sample were amplified using region-specific primers adapted for the Illumina MiSeq sequencer (Illumina, CA, USA)). The sequences were subjected to local blast and classified using Metagenome@KIN (World Fusion, Tokyo, Japan). RESULTS: In total, 155 participants were recruited from seven medical centers. Of these, 88 and 67 had chronic otitis media and normal middle ears, respectively. The most abundant bacterial phyla on the mucosal surfaces of the normal middle ears were Proteobacteria, followed by Actinobacteria, Firmicutes, and Bacteroidetes. The children and adults with normal middle ears differed significantly in terms of middle ear microbiomes. Subjects with chronic otitis media without active inflammation (dry ear) had similar middle ear microbiomes as the normal middle ears group. Subjects with chronic otitis media with active inflammation (wet ear) had a lower prevalence of Proteobacteria and a higher prevalence of Firmicutes than the normal middle ears. CONCLUSION: The human middle ear is inhabited by more diverse microbial communities than was previously thought. Alteration of the middle ear microbiome may contribute to the pathogenesis of chronic otitis media with active inflammation. LEVEL OF EVIDENCE: 2b. Laryngoscope, 127:E371-E377, 2017.

A novel nonsense mutation in the NOG gene causes familial NOG-related symphalangism spectrum disorder.

The human noggin (NOG) gene is responsible for a broad spectrum of clinical manifestations of NOG-related symphalangism spectrum disorder (NOG-SSD), which include proximal symphalangism, multiple synostoses, stapes ankylosis with broad thumbs (SABTT), tarsal-carpal coalition syndrome, and brachydactyly type B2. Some of these disorders exhibit phenotypes associated with congenital stapes ankylosis. In the present study, we describe a Japanese pedigree with dactylosymphysis and conductive hearing loss due to congenital stapes ankylosis. The range of motion in her elbow joint was also restricted. The family showed multiple clinical features and was diagnosed with SABTT. Sanger sequencing analysis of the NOG gene in the family members revealed a novel heterozygous nonsense mutation (c.397A>T; p.K133*). In the family, the prevalence of dactylosymphysis and hyperopia was 100% while that of stapes ankylosis was less than 100%. Stapes surgery using a CO2 laser led to a significant improvement of the conductive hearing loss. This novel mutation expands our understanding of NOG-SSD from clinical and genetic perspectives.

A mutation in the heparin-binding site of noggin as a novel mechanism of proximal symphalangism and conductive hearing loss.

The access of bone morphogenetic protein (BMP) to the BMP receptors on the cell surface is regulated by its antagonist noggin, which binds to heparan-sulfate proteoglycans on the cell surface. Noggin is encoded by NOG and mutations in the gene are associated with aberrant skeletal formation, such as in the autosomal dominant disorders proximal symphalangism (SYM1), multiple synostoses syndrome, Teunissen-Cremers syndrome, and tarsal-carpal coalition syndrome. NOG mutations affecting a specific function may produce a distinct phenotype. In this study, we investigated a Japanese pedigree with SYM1 and conductive hearing loss and found that it carried a novel heterozygous missense mutation of NOG (c.406C>T; p.R136C) affecting the heparin-binding site of noggin. As no mutations of the heparin-binding site of noggin have previously been reported, we investigated the crystal structure of wild-type noggin to investigate molecular mechanism of the p.R136C mutation. We found that the positively charged arginine at position 136 was predicted to be important for binding to the negatively charged heparan-sulfate proteoglycan (HSPG). An in silico docking analysis showed that one of the salt bridges between noggin and heparin disappeared following the replacement of the arginine with a non-charged cysteine. We propose that the decreased binding affinity of NOG with the p.R136C mutation to HSPG leads to an excess of BMP signaling and underlies the SYM1 and conductive hearing loss phenotype of carriers.

Diverse spectrum of rare deafness genes underlies early-childhood hearing loss in Japanese patients: a cross-sectional, multi-center next-generation sequencing study.

BACKGROUND: Genetic tests for hereditary hearing loss inform clinical management of patients and can provide the first step in the development of therapeutics. However, comprehensive genetic tests for deafness genes by Sanger sequencing is extremely expensive and time-consuming. Next-generation sequencing (NGS) technology is advantageous for genetic diagnosis of heterogeneous diseases that involve numerous causative genes. METHODS: Genomic DNA samples from 58 subjects with hearing loss from 15 unrelated Japanese families were subjected to NGS to identify the genetic causes of hearing loss. Subjects did not have pathogenic GJB2 mutations (the gene most often associated with inherited hearing loss), mitochondrial m.1555A>G or 3243A>G mutations, enlarged vestibular aqueduct, or auditory neuropathy. Clinical features of subjects were obtained from medical records. Genomic DNA was subjected to a custom-designed SureSelect Target Enrichment System to capture coding exons and proximal flanking intronic sequences of 84 genes responsible for nonsyndromic or syndromic hearing loss, and DNA was sequenced by Illumina GAIIx (paired-end read). The sequences were mapped and quality-checked using the programs BWA, Novoalign, Picard, and GATK, and analyzed by Avadis NGS. RESULTS: Candidate genes were identified in 7 of the 15 families. These genes were ACTG1, DFNA5, POU4F3, SLC26A5, SIX1, MYO7A, CDH23, PCDH15, and USH2A, suggesting that a variety of genes underlie early-childhood hearing loss in Japanese patients. Mutations in Usher syndrome-related genes were detected in three families, including one double heterozygous mutation of CDH23 and PCDH15. CONCLUSION: Targeted NGS analysis revealed a diverse spectrum of rare deafness genes in Japanese subjects and underscores implications for efficient genetic testing.

GJB2-associated hearing loss undetected by hearing screening of newborns.

The hearing loss caused by GJB2 mutations is usually congenital in onset, moderate to profound in degree, and non-progressive. The objective of this study was to study genotype/phenotype correlations and to document 14 children with biallelic GJB2 mutations who passed newborn hearing screening (NHS). Genetic testing for GJB2 mutations by direct sequencing was performed on 924 individuals (810 families) with hearing loss, and 204 patients (175 families) were found to carry biallelic GJB2 mutations. NHS results were obtained through medical records. A total of 18 pathological mutations were identified, which were subclassified as eight inactivating and 10 non-inactivating mutations. p.I128M and p.H73Y were identified as novel missense GJB2 mutations. Of the 14 children with biallelic GJB2 mutations who passed NHS, eight were compound heterozygotes and 3 were homozygous for the c.235delC mutation in GJB2, and the other three combinations of non-c.235delC mutations identified were p.Y136X-p.G45E/p.V37I heterozygous, c.512ins4/p.R143W heterozygous, and p.V37I/p.R143W heterozygous. These 14 cases demonstrate that the current NHS does not identify all infants with biallelic GJB2 mutations. They suggest that the frequency of non-penetrance at birth is approximately 6.9% or higher in DFNB1 patients and provide further evidence that GJB2 hearing loss may not always be congenital in onset.

Late-phase recovery in the cochlear lateral wall following severe degeneration by acute energy failure.

We previously reported a model of acute cochlear energy failure using a mitochondrial toxin, 3-nitropropionic acid (3-NP), to study mechanisms of inner ear disorders such as inner ear ischemia. In this model, the main cause of hearing loss is apoptosis of fibrocytes in the cochlear lateral wall. Here, we analyzed the time course of structural and hearing level changes in the cochlea from the acute phase to the chronic phase up to 2 months after surgery. Hearing levels as determined by auditory brainstem response (ABR) thresholds exceeded the maximum acoustic output (>87 dBSPL) of the system at all frequencies 1 day after 3-NP treatment. Histology showed nearly complete loss of fibrocytes 2 weeks after 3-NP treatment. However, after 2 months, ABR showed significant recovery at low frequency (8 kHz) in four of five rats treated with 3-NP. ABR thresholds at 20 kHz occasionally showed some recovery. At 40 kHz, recovery of ABR thresholds was not observed. Histology of 3-NP-treated rats revealed partial recovery of the lateral wall and the regenerated fibrocytes in the spiral ligament expressed Na/K-ATPase in the cochlear basal turn 2 months after 3-NP treatment. These results indicate that ABR recovery is caused by regeneration of the cochlear lateral wall. Our findings demonstrate the recoverable capacity of the cochlear lateral wall that leads to functional recovery after severe damage.

Promoted cell proliferation by connexin 30 gene transfection to head-and-neck cancer cell line.

BACKGROUND: There have been inconsistent results regarding the contribution of the connexin family of genes to tumor cell proliferation. MATERIALS AND METHODS: We aimed to clarify the role of connexin 30 (Cx30), by transfecting three kinds of vectors that express either full length Cx30 (Cx30-Full), Cx30 devoid of C-terminal region (Cx30-DelC) or Cx30 C-terminal region (Cx30-CT), in HSC-4, a head-and-neck cancer cell line. RESULTS: Transfected Cx30-Full was localized on the plasma membrane, while Cx30-DelC and Cx30-CT was expressed in the cytoplasm or circumnuclear sites. We studied the effect on the growth rate followed by immunostaining with anti-Ki-67 (MIB-1). The MIB indices of HSC-4 cells transfected with Cx30-Full and Cx30-DelC, but not Cx30-CT were shown to be significantly higher than that of the controls. CONCLUSION: Our results demonstrated that Cx30 enhanced the proliferation of HSC-4 cells and the proliferating activity was considered to be achieved without the transport of the protein onto the plasma membrane.

TMC and EVER genes belong to a larger novel family, the TMC gene family encoding transmembrane proteins.

BACKGROUND: Mutations in the transmembrane cochlear expressed gene 1 (TMC1) cause deafness in human and mouse. Mutations in two homologous genes, EVER1 and EVER2 increase the susceptibility to infection with certain human papillomaviruses resulting in high risk of skin carcinoma. Here we report that TMC1, EVER1 and EVER2 (now TMC6 and TMC8) belong to a larger novel gene family, which is named TMC for trans membrane channel-like gene family. RESULTS: Using a combination of iterative database searches and reverse transcriptase-polymerase chain reaction (RT-PCR) experiments we assembled contigs for cDNA encoding human, murine, puffer fish, and invertebrate TMC proteins. TMC proteins of individual species can be grouped into three subfamilies A, B, and C. Vertebrates have eight TMC genes. The majority of murine TMC transcripts are expressed in most organs; some transcripts, however, in particular the three subfamily A members are rare and more restrictively expressed. CONCLUSION: The eight vertebrate TMC genes are evolutionary conserved and encode proteins that form three subfamilies. Invertebrate TMC proteins can also be categorized into these three subfamilies. All TMC genes encode transmembrane proteins with intracellular amino- and carboxyl-termini and at least eight membrane-spanning domains. We speculate that the TMC proteins constitute a novel group of ion channels, transporters, or modifiers of such.