Gap Junction Beta-2 p.Val84Met Can Cause Autosomal Dominant Syndromic Hearing Loss With Keratoderma.

In this study, we report a case of bilateral mild hearing loss and keratoderma caused by a gap junction beta-2 (GJB2) variant. The proband was a nine-year-old Japanese boy with bilateral mild hearing loss at birth. The proband's father, sister, paternal aunt, and cousins had mild sensorineural hearing loss. Further evaluation revealed keratoderma on the feet of the proband, father, sister, paternal aunt, and cousins. We identified a heterozygous c.250G>A (p.Val84Met) variant in GJB2 as the cause of the autosomal dominant syndromic hearing loss with the skin disorder in this Japanese family and delineated the pathological significance of the variant. The Val84Met variant in GJB2 contributes to the autosomal dominant form of syndromic hearing loss with keratoderma.

Loss of Pax3 causes reduction of melanocytes in the developing mouse cochlea.

Cochlear melanocytes are intermediate cells in the stria vascularis that generate endocochlear potentials required for auditory function. Human PAX3 mutations cause Waardenburg syndrome and abnormalities of skin and retinal melanocytes, manifested as congenital hearing loss (~ 70%) and hypopigmentation of skin, hair and eyes. However, the underlying mechanism of hearing loss remains unclear. Cochlear melanocytes in the stria vascularis originated from Pax3-traced melanoblasts and Plp1-traced Schwann cell precursors, both of which derive from neural crest cells. Here, using a Pax3-Cre knock-in mouse that allows lineage tracing of Pax3-expressing cells and disruption of Pax3, we found that Pax3 deficiency causes foreshortened cochlea, malformed vestibular apparatus, and neural tube defects. Lineage tracing and in situ hybridization show that Pax3+ derivatives contribute to S100+, Kir4.1+ and Dct+ melanocytes (intermediate cells) in the developing stria vascularis, all of which are significantly diminished in Pax3 mutant animals. Taken together, these results suggest that Pax3 is required for the development of neural crest cell-derived cochlear melanocytes, whose absence may contribute to congenital hearing loss of Waardenburg syndrome in humans.

Prediction of Cochlear Implant Effectiveness With Surface-Based Morphometry.

OBJECTIVE: This study aimed to determine whether surface-based morphometry of preoperative whole-brain three-dimensional T1-weighted magnetic resonance imaging (MRI) images can predict the clinical outcomes of cochlear implantation. STUDY DESIGN: This was an observational, multicenter study using preoperative MRI data. SETTING: The study was conducted at tertiary care referral centers. PATIENTS: Sixty-four patients with severe to profound hearing loss (≥70 dB bilaterally), who were scheduled for cochlear implant (CI) surgery, were enrolled. The patients included 19 with congenital hearing loss and 45 with acquired hearing loss. INTERVENTIONS: Participants underwent CI surgery. Before surgery, high-resolution three-dimensional T1-weighted brain MRI was performed, and the images were analyzed using FreeSurfer. MAIN OUTCOME MEASURES: The primary outcome was monosyllable audibility under quiet conditions 6 months after surgery. Cortical thickness residuals within 34 regions of interest (ROIs) as per the Desikan-Killiany cortical atlas were calculated based on age and healthy-hearing control regression lines. RESULTS: Rank logistic regression analysis detected significant associations between CI effectiveness and five right hemisphere ROIs and five left hemisphere ROIs. Predictive modeling using the cortical thickness of the right entorhinal cortex and left medial orbitofrontal cortex revealed a significant correlation with speech discrimination ability. This correlation was higher in patients with acquired hearing loss than in those with congenital hearing loss. CONCLUSIONS: Preoperative surface-based morphometry could potentially predict CI outcomes and assist in patient selection and clinical decision making. However, further research with larger, more diverse samples is necessary to confirm these findings and determine their generalizability.

Pax3 deficiency diminishes melanocytes in the developing mouse cochlea.

Cochlear melanocytes are intermediate cells in the stria vascularis that generate endocochlear potentials required for auditory function. Human PAX3 mutations cause Waardenburg syndrome and abnormalities of melanocytes, manifested as congenital hearing loss and hypopigmentation of skin, hair and eyes. However, the underlying mechanism of hearing loss remains unclear. During development, cochlear melanocytes in the stria vascularis are dually derived from Pax3-Cre+ melanoblasts migrating from neuroepithelial cells including neural crest cells and Plp1+ Schwann cell precursors originated from also neural crest cells, differentiating in a basal-apical manner. Here, using a Pax3-Cre mouse line, we found that Pax3 deficiency causes foreshortened cochlea, malformed vestibular apparatus, and neural tube defects. Lineage tracing and in situ hybridization show that Pax3-Cre derivatives contribute to S100+ , Kir4.1+ and Dct+ melanocytes (intermediate cells) in the developing stria vascularis, all significantly diminished in Pax3 mutant animals. Taken together, these results suggest that Pax3 is required for the development of neural crest cell-derived cochlear melanocytes, whose absence may contribute to congenital hearing loss of Waardenburg syndrome in human.

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.

The pathogenic role of the BRCA2 c.7847C>T (p.Ser2616Phe) variant in breast and ovarian cancer predisposition.

Substantial numbers of variants of unknown significance (VUSs) have been identified in BRCA1/2 through genetic testing, which poses a significant clinical challenge because the contribution of these VUSs to cancer predisposition has not yet been determined. Here, we report 10 Japanese patients from seven families with breast or ovarian cancer harboring the BRCA2 c.7847C>T (p.Ser2616Phe) variant that was interpreted as a VUS. This variant recurs only in families from Japan and has not been reported in the global general population databases. A Japanese patient with Fanconi anemia with compound heterozygous variants c.7847C>T (p.Ser2616Phe) and c.475+1G>A in BRCA2 was reported. In silico predictions and quantitative cosegregation analysis suggest a high probability of pathogenicity. The clinical features of the variant carriers were not specific to, but were consistent with, those of patients with hereditary breast and ovarian cancer. A validated functional assay, called the mixed-all-nominated-in-one-BRCA (MANO-B) method and the accurate BRCA companion diagnostic (ABCD) test, demonstrated the deleterious effects of the variant. Altogether, following the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP) guidelines, this variant satisfied the "PS3," "PM2," "PM3," and "PP3" criteria. We thus conclude that the BRCA2 c.7847C>T (p.Ser2616Phe) variant is a "likely pathogenic" variant that is specifically observed in the Japanese population, leading to a breast and ovarian cancer predisposition.

Apoptosis of type I spiral ganglion neuron cells in Otof-mutant mice.

Otof, which encodes otoferlin, knockout mice are considered model mice for auditory neuropathy spectrum disorder, which is characterized by an absent auditory brainstem response (ABR) despite preserved distortion product otoacoustic emission (DPOAE). Although otoferlin-deficient mice lack neurotransmitter release at the inner hair cell (IHC) synapse, it remains unclear how the Otof mutation affects spiral ganglions. Thus, we used Otof-mutant mice carrying the Otoftm1a(KOMP)Wtsi allele (Otoftm1a) and analyzed spiral ganglion neurons (SGNs) in Otoftm1a/tm1a mice by immunolabeling type Ⅰ SGNs (SGN-Ⅰ) and type II SGNs (SGN-II). We also examined apoptotic cells in SGNs. Four-week-old Otoftm1a/tm1a mice had an absent ABR but normal DPOAEs. The number of SGNs was significantly lower in Otoftm1a/tm1a mice on postnatal day 7 (P7), P14, and P28 compared with that of wild-type mice. Moreover, significantly more apoptotic SGNs were observed in Otoftm1a/tm1a mice than in wild-type mice on P7, P14, and P28. SGN-IIs were not significantly reduced in Otoftm1a/tm1a mice on P7, P14, and P28. No apoptotic SGN-IIs were observed under our experimental conditions. In summary, Otoftm1a/tm1a mice showed a reduction in SGNs accompanied by apoptosis of SGN-Ⅰs even before the onset of hearing. We speculate that the reduction in SGNs with apoptosis is a secondary defect caused by a lack of otoferlin in IHCs. Appropriate glutamatergic synaptic inputs may be important for the survival of SGNs.

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.

Whole exome analysis of patients in Japan with hearing loss reveals high heterogeneity among responsible and novel candidate genes.

BACKGROUND: Heterogeneous genetic loci contribute to hereditary hearing loss; more than 100 deafness genes have been identified, and the number is increasing. To detect pathogenic variants in multiple deafness genes, in addition to novel candidate genes associated with hearing loss, whole exome sequencing (WES), followed by analysis prioritizing genes categorized in four tiers, were applied. RESULTS: Trios from families with non-syndromic or syndromic hearing loss (n = 72) were subjected to WES. After segregation analysis and interpretation according to American College of Medical Genetics and Genomics guidelines, candidate pathogenic variants in 11 previously reported deafness genes (STRC, MYO15A, CDH23, PDZD7, PTPN11, SOX10, EYA1, MYO6, OTOF, OTOG, and ZNF335) were identified in 21 families. Discrepancy between pedigree inheritance and genetic inheritance was present in one family. In addition, eight genes (SLC12A2, BAIAP2L2, HKDC1, SVEP1, CACNG1, GTPBP4, PCNX2, and TBC1D8) were screened as single candidate genes in 10 families. CONCLUSIONS: Our findings demonstrate that four-tier assessment of WES data is efficient and can detect novel candidate genes associated with hearing loss, in addition to pathogenic variants of known deafness genes.

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 and genetic analysis of children with hearing loss and bilateral enlarged vestibular aqueducts.

OBJECTIVES: To evaluate the clinical and genetic features of children with hearing loss associated with one of the most common malformations of the inner ear: bilateral enlargement of vestibular aqueducts (EVA). METHODS: Clinical and genetic features were investigated in 28 children with hearing loss diagnosed with bilateral EVA by computed tomography from January 2008 to September 2019. RESULTS: Fourteen subjects had undergone newborn hearing screening (NHS). Nine subjects (64.3%) were referred in both ears, 4 subjects (28.6%) were referred in one ear, and one subject (7.1%) passed in both ears. Nineteen of 26 subjects (73.1%) who were followed for more than 3 years had hearing fluctuations, while 17 (65.4%) had hearing loss progression. Eleven of 28 subjects (39.2%) had vertigo attacks. Pathogenic variants were identified in two alleles of the SLC26A4 gene in 24 of 27 subjects (88.9%) by sequencing of all exons and flanking introns, leading to genetic diagnosis of Pendred syndrome/DFNB4. Our results indicate that genetic screening for specific SLC26A4 variants using a commercial clinical laboratory test in Japan would have achieved genetic diagnoses in 13 of the 27 subjects (54.2%). Although there was no statistically significance in the frequency of hearing fluctuation or progression depending on the presence or absence of the gene variant, mean hearing level was severe in subjects with two pathogenic variants in SLC26A4 gene. The most common variant detected in our subjects was p.His723Arg (13 alleles, 27.1%), followed by c. 919-2A > G (four alleles, 8.3%). Two novel variants were detected in this study: c.1544+1G > T and c.1614+5G > A. CONCLUSIONS: Our data suggest that some subjects may present with bilateral EVA that cannot be detected by NHS. We estimated that genetic diagnosis for SLC264 gene would not have been made in almost half subjects with the commercial genetic screening approach used in the present study in Japan. Although there were some limitations in this study, the subjects with pathogenic variants in two alleles of the SLC26A4 gene could have more severe hearing loss.

A Japanese boy with double diagnoses of 2p15p16.1 microdeletion syndrome and RP2-associated retinal disorder.

2p15p16.1 microdeletion syndrome is a recently recognized congenital disorder characterized by developmental delay and dysmorphic features. RP2-associated retinal disorder (RP2-RD) is an X-linked inherited retinal disease with a childhood onset caused by a loss-of-function variant in the RP2 gene. Here, we describe a 14-year-old boy with double diagnoses of 2p15p16.1 microdeletion syndrome and RP2-RD. The recurrence risk of each condition and the indication for potential therapeutic options for RP2-RD are discussed.

A High Risk of Missing Congenital Cytomegalovirus-Associated Hearing Loss through Newborn Hearing Screening in Japan.

It remains unclear to what extent newborn hearing screening (NHS) detects congenital cytomegalovirus (cCMV)-associated sensorineural hearing loss (SNHL) in Japan. This study aimed to clarify the NHS results and audiological characteristics of patients with cCMV-associated SNHL. A total of 541 individuals with unilateral or bilateral hearing loss of unknown etiology were examined for cCMV infection. cCMV infection was defined by the presence of CMV DNA in the dried umbilical cord detected using real-time quantitative PCR. NHS results and audiological data were retrospectively obtained from medical records. Forty-four cases (8.1%) were positive for cCMV infection. Of them, 33 cases underwent NHS and 13 cases (39.4%) passed NHS bilaterally. The pure-tone audiograms of 21 patients were obtained. There were seven cases of unilateral SNHL, five cases of asymmetric bilateral SNHL, and nine cases of symmetric bilateral SNHL. cCMV-related hearing loss is highly heterogeneous, and there is a high risk of missing this condition through NHS.

Effects on cervical vestibular-evoked myogenic potentials of four clinically used head and neck measurement positions in healthy subjects.

BACKGROUND: The most reliable head and neck position for cervical vestibular-evoked myogenic potentials (cVEMPs) measurements yet to be determined. AIMS/OBJECTIVES: To assess how four body positions used during clinical recordings of cVEMPs affect cVEMP parameters. MATERIAL AND METHOD: cVEMPs of 10 healthy subjects (26-50 years old) were recorded in four body positions: A. sitting/head rotated; B. supine/head rotated; C. semi-recumbent/head rotated and elevated; D. supine/head elevated. RESULTS: Mean background sternocleidomastoid muscle (SCM) electrical activity was significantly higher in positions C and D than in positions A and B. The latencies of p13 and n23 differed significantly among the four positions. Raw p13-n23 complex amplitude was significantly greater in positions C and D than in A and B. These differences were reduced when amplitudes were corrected by SCM activity. For positions A and B, one and two subjects, respectively, had an abnormal raw asymmetry ratio (AR). After correction, all subjects had normal ARs in all positions. CONCLUSIONS AND SIGNIFICANCE: Body positions in which the head is elevated produce a quicker and larger cVEMP response compared to positions in which the head is not elevated. The difference in ARs among positions can be ignored as long as the correction is made.

Investigation of the hearing levels of siblings affected by a single GJB2 variant: Possibility of genetic modifiers.

OBJECTIVE: Variants in GJB2 can cause autosomal recessive deafness (DFNB1). There is evidence for genotype-phenotype correlations of GJB2 variants; however, several genotypes can cause varying levels of hearing loss likely attributable to differences in genetic or environmental background. As siblings share approximately 50% of their genetic background and usually have a common environmental background, analysis of phenotypes of siblings with a specific GJB2 variant may reveal factors relevant to phenotypic variation. There have been no previous analyses of differences in hearing among siblings carrying a single GJB2 genotype. Here, we investigated hearing differences between siblings with a single GJB2 variant, which can cause various levels of hearing loss. METHODS: We examined hearing levels in 16 pairs of siblings homozygous for the c.235delC variant of GJB2. Differences in hearing acuity between sibling pairs were detected by auditory evaluation. RESULTS: Average differences in acoustic threshold >30 dB were observed between five pairs of siblings, whereas the remaining 11 pairs had average threshold values within approximately 10 dB of one another. Hearing loss varied from moderate to profound. CONCLUSION: Our results indicate that auditory acuity associated with homozygosity for GJB2 c.235delC can vary in degree; however, in approximately 70% of younger siblings, it was approximately the same as that in the first child, despite a diverse spectrum of hearing loss among different families. These results suggest that differences in genetic background may modify the phenotype associated with homozygous GJB2 c.235delC.

Loss of imprinting of the human-specific imprinted gene ZNF597 causes prenatal growth retardation and dysmorphic features: implications for phenotypic overlap with Silver-Russell syndrome.

BACKGROUND: ZNF597, encoding a zinc-finger protein, is the human-specific maternally expressed imprinted gene located on 16p13.3. The parent-of-origin expression of ZNF597 is regulated by the ZNF597:TSS-DMR, of which only the paternal allele acquires methylation during postimplantation period. Overexpression of ZNF597 may contribute to some of the phenotypes associated with maternal uniparental disomy of chromosome 16 (UPD(16)mat), and some patients with UPD(16)mat presenting with Silver-Russell syndrome (SRS) phenotype have recently been reported. METHODS: A 6-year-old boy presented with prenatal growth restriction, macrocephaly at birth, forehead protrusion in infancy and clinodactyly of the fifth finger. Methylation, expression, microsatellite marker, single nucleotide polymorphism array and trio whole-exome sequencing analyses were conducted. RESULTS: Isolated hypomethylation of the ZNF597:TSS-DMR and subsequent loss of imprinting and overexpression of ZNF597 were confirmed in the patient. Epigenetic alterations, such as UPD including UPD(16)mat and other methylation defects, were excluded. Pathogenic sequence or copy number variants affecting his phenotypes were not identified, indicating that primary epimutation occurred postzygotically. CONCLUSION: We report the first case of isolated ZNF597 imprinting defect, showing phenotypic overlap with SRS despite not satisfying the clinical SRS criteria. A novel imprinting disorder entity involving the ZNF597 imprinted domain can be speculated.

Clinical genetics, practice, and research of deafblindness: From uncollected experiences to the national registry in Japan.

Deafblindness is a condition of combined vision and hearing loss that is extremely rare in children and young adults, as well as being a highly heterogeneous condition, with over 70 specific etiologies. Due to these features, sporadic clinical experiences have not been collated, which has hampered medical progress. Genetics plays a major role in the pathogenesis of deafblindness in children and young adults, with more than 50 hereditary syndromes and disorders associated with the condition, including CHARGE, Usher, Down, Stickler, and Dandy-Walker syndromes, which are the most common. Clinical diagnosis of deafblindness is often difficult, and a significant proportion of patients are undiagnosed. No curative therapy is currently available for the majority of patients with hereditary deafblindness; however, experimental studies using animal models have shown promising results by targeting specific genes that cause vision or hearing loss. In Japan, the Rare Disease Data Registry of Japan (RADDAR-J) has been established as a national registry of rare and intractable diseases. Diseases of deafblindness have been elected as a disease category in RADDAR-J. Currently, clinical and genomic data are being collected and analyzed using this system, with the aim of generating an overview of deafblindness to improve medical practice.

Single nucleotide polymorphisms in tinnitus patients exhibiting severe distress.

The association between distress caused by tinnitus and psychological factors such as depression and anxiety has been examined and reported. However, prognostic factors remain poorly understood because there are only a few reports on genetic associations. We theorized there might be an association between the grade of tinnitus distress and the genetic background related to psychological factors which might lead us to identify prognostic markers. We enrolled 138 patients who had suffered from tinnitus for over 3 months. Using Tinnitus Handicap Inventory (THI) scores, we examined the association between tinnitus distress and a genetic background related to depression or anxiety. A significant association between single nucleotide polymorphism rs131702 of the Breakpoint Cluster Region (BCR) gene and the severe THI score was identified. In addition, there was an association with the severity of the State-Trait Anxiety Inventory, an index of state anxiety severity. No association was found with the Self-Rating Depression Scale, an index of depression severity. It is reported that rs131702 of BCR in Japanese patients are related to bipolar II depression characterized by fluctuation between abnormal mood states of mania and depression. Our results indicate that rs131702 of BCR is independent of depression in this study and is, therefore, a prognostic factor unique to tinnitus. We conclude that the severity of tinnitus is associated with genes related to depression.

Differences in hearing levels between siblings with hearing loss caused by GJB2 mutations.

OBJECTIVE: Hearing loss caused by GJB2 mutations is inherited in an autosomal recessive manner (DFNB1); thus siblings of an affected child have a 25% chance of also being affected. Hearing loss among subsequent siblings carrying the same GJB2 mutation is a concern for parents and a frequent topic of enquiry during genetic counseling. Evidence exists for genotype-phenotype correlations of GJB2 mutations; however, no analysis of differences in hearing among siblings, in whom the common genetic background may decrease variation, has been reported. The purpose of the present study was to investigate hearing differences between siblings with identical GJB2 mutations. METHODS: We examined the hearing levels of 12 pairs of siblings; each pair had the same pathogenic GJB2 mutations. Differences in hearing acuity between sibling pairs detected by auditory evaluation. RESULTS: No significant correlation was detected between the average hearing levels of first and second affected siblings. Average differences in acoustic threshold >30 dB were observed between four pairs of siblings, whereas the remaining eight pairs had average threshold values within 20 dB of one another. CONCLUSION: Our results indicate that auditory acuity would be expected to approximate that found in the first child in approximately 70% of subsequent children with GJB2-mediated hearing loss, whereas 30% of subsequent siblings would have average differences of >30 dB.