Comprehensive genetic testing with ethnic‐specific filtering by allele frequency in a Japanese hearing‐loss population

Recent advances in targeted genomic enrichment with massively parallel sequencing (TGE+MPS) have made comprehensive genetic testing for non‐syndromic hearing loss (NSHL) possible. After excluding NSHL subjects with causative mutations in GJB2 and the MT‐RNR1 (1555A>G) variant by Sanger sequencing, we completed TGE+MPS on 194 probands with presumed NSHL identified across Japan. We used both publicly available minor allele frequency (MAF) datasets and ethnic‐specific MAF filtering against an in‐house database of 200 normal‐hearing Japanese controls. Ethnic‐specific MAF filtering allowed us to re‐categorize as common 203 variants otherwise annotated as rare or novel in non‐Japanese ethnicities. This step minimizes false‐positive results and improves the annotation of identified variants. Causative variants were identified in 27% of probands with solve rates of 35%, 35% and 19% for dominant, recessive and sporadic NSHL, respectively. Mutations in MYO15A and CDH23 follow GJB2 as the frequent causes of recessive NSHL; copy number variations in STRC are a major cause of mild‐to‐moderate NSHL. Ethnic‐specific filtering by allele frequency is essential to optimize the interpretation of genetic data.     

Genomic copy number alterations in non‐syndromic hearing loss

Genetic heterogeneity has made the identification of genes related to hearing impairment a challenge. In the absence of a clear phenotypic aetiology, recurrence risk estimates are often based on family segregation and may be imprecise. We profiled by oligonucleotide array‐CGH patients presenting non‐syndromic hearing loss with presumptive autosomal recessive (n = 50) or autosomal dominant (n = 50) patterns of inheritance. Rare copy number variants (CNVs) were detected in 12 probands; four of the detected CNVs comprised genes previously associated with hearing loss (POU4F3, EYA4, USH2A, and BCAP31) and were considered causative, stressing the contribution of genomic imbalance to non‐syndromic deafness. In six cases, segregation of the CNVs in pedigrees excluded them as causative. In one case, segregation could not be investigated, while in another case, a point mutation likely explains the phenotype. These findings show that the presumptive patterns of inheritance were incorrect in at least two cases, thereby impacting genetic counselling. In addition, we report the first duplication reciprocal to the rare ABCD1, BCAP31, and SLC6A8 contiguous deletion syndrome; as with most microduplication syndromes, the associated phenotype is much milder than the respective microdeletion and, in this case, was restricted to hearing impairment.     

Novel recessive PDZD7 biallelic mutations in two Chinese families with non‐syndromic hearing loss

Autosomal recessive non‐syndromic hearing loss (ARNSHL) is a highly heterogeneous genetic condition. PDZD7 has emerged as a new genetic etiology of ARNSHL. Biallelic mutations in the PDZD7 gene have been reported in two German families, four Iranian families, and a Pakistani family with ARNSHL. The effect of PDZD7 on ARNSHL in other population has yet to be elucidated. Two Chinese ARNSHL families, each of which had two affected siblings, were included in this study. The families underwent target region capture and high‐throughput sequencing to analyze the exonic, splice‐site, and intronic sequences of 128 genes. Furthermore, 1751 normal Chinese individuals served as controls, and 122 Chinese families segregating with apparent ARNSHL, who had been previously excluded for variants in the common deafness genes GJB2 and SLC26A4, were subjected to screening for candidate mutations. We identified a novel homozygous missense mutation (p.Arg66Leu) and novel compound heterozygous frameshift mutations (p.Arg56fsTer24 and p.His403fsTer36) in Chinese families with ARNSHL. This is the first report to identify PDZD7 as an ARNSHL‐associated gene in the Chinese population. Our finding could expand the pathogenic spectrum and strengthens the clinical diagnostic role of the PDZD7 gene in ARNSHL patients.     

“New” autosomal‐dominant infantile sensorineural non‐progressive high‐frequency hearing loss: Report on a Brazilian family.

We report on a three‐generation Brazilian family with seven patients affected with non‐progressive high‐frequency sensorineural hearing loss with no associated anomalies first noted in early infancy. To our knowledge this is the first report on this autosomal‐dominant condition. Clinical, audiological, and genetic aspects are discussed. Am. J. Med. Genet. 95:13–16, 2000. © 2000 Wiley‐Liss, Inc.     

Extension of the clinical and molecular phenotype of DIAPH1‐associated autosomal dominant hearing loss (DFNA1)

In about 20% of non‐syndromic hearing loss (NSHL) cases, inheritance is autosomal dominant (ADNSHL). DIAPH1 mutations define the ADNSHL locus DFNA1. We identified two new families with heterozygous truncating DIAPH1 mutations (p.Ala1210Serfs*31 and p.Arg1213*). In contrast to the extensively studied original DFNA1 family, hearing loss was not confined to low frequencies, but congenital manifestation and rapid progression were confirmed. In line with a recent unrelated study, we identified an association with thrombocytopenia, reclassifying DFNA1 as a syndrome. Consequently, we suggest to include the blood count into the initial clinical workup of patients with autosomal dominant hearing loss to guide the genetic diagnosis. We provide the first data on DIAPH1 expression in the organ of Corti, where it localizes to the inner pillar cells, at the base of the outer hair cells. Homozygous truncating DIAPH1 mutations located N‐terminally to the DFNA1 mutations have recently been identified in autosomal recessive microcephaly. It is therefore noteworthy that we found DIAPH1 expression also in spiral ganglion neurons and in the barrier between the myelinating glia of the peripheral nervous system and oligodendrocytes that form the myelinating glia of the central nervous system (CNS).     

A founder TMIE mutation is a frequent cause of hearing loss in southeastern Anatolia

Using Affymetrix 10K arrays, we searched for regions of homozygosity in 51 Turkish families including at least three members with either congenital or prelingual autosomal recessive non-syndromic sensorineural hearing loss (ARNSSNHL), and identified four families whose deafness mapped to the DFNB6 locus on 3p21 containing the TMIE gene. Mutation analysis revealed the p.R84W mutation in all four families. Screening of this mutation in 254 families with ARNSSNHL, without GJB2 mutations, revealed four additional affected families. A novel mutation was found in a non-complementary marriage between a deaf couple who were homozygous for p.R84W and p.W57X, respectively with two affected children who were compound heterozygotes. Six of the TMIE families originated from southeastern Anatolia, making p.R84W a common cause of hearing loss in that region with a relative frequency of 10.3% (95% CI is 2.5–18.1%). The overall prevalence of the p.R84W mutation in ARNSSNHL in Turkey is 2.4% (95% CI is 0.7–4.0%). Genotyping of single-nucleotide polymorphisms flanking the TMIE gene revealed a conserved haplotype, suggesting a single origin for p.R84W from a common ancestor 1250 years ago (95% CI is 650–2500 years). We conclude that p.R84W could be a common mutation in other Middle Eastern populations and should be included in mutation screening offered to individuals with ARNSSNHL.     

Novel CRISPR/Cas12a-based genetic diagnostic approach for SLC26A4 mutation-related hereditary hearing loss

Hereditary hearing loss is a common defect of the auditory nervous system with high-incidence, seriously affecting the quality of life of the patients. The clinical manifestations of SLC26A4 mutation-related hearing loss are congenital sensorineural or mixed deafness. Sensitive and specific SLC26A4 mutation detection in the early clinical stage is key for the early indication of potential hearing loss in the lack of effective treatment. Using clustered regularly interspaced short palindromic repeats (CRISPR)-based nucleic acid detection technology, we designed a fast and sensitive detection system for SLC26A4 pathogenic mutations (c.919-2A > G, c.2168A > G and c.1229C > T). This recombinase-aided amplification-based detection system allows rapid target gene amplification and, in combination with the CRISPR-based nucleic acid testing (NAT) system, mutation site detection. Moreover, mismatches were introduced in CRISPR-derived RNA (crRNA) to increase signal differences between the wild-type genes and mutant genes. A total of 64 samples were examined using this approach and all results were verified using Sanger sequencing. The detection results were consistent with the polymerase chain reaction-Sanger sequencing results. Overall, this CRISPR-based NAT technology provides a sensitive and fast new approach for the detection of hereditary deafness and provides a crRNA optimization strategy for single-nucleotide polymorphism detection, which could be helpful for the clinical diagnosis of SLC26A4 mutation-related hereditary hearing loss.     

Update of spectrum c.35delG and c.‐23+1G>A mutations on the GJB2 gene in individuals with autosomal recessive nonsyndromic hearing loss

Hearing loss (HL) is the most common birth defect and the most prevalent sensorineural condition worldwide. It is associated with more than 1,000 mutations in at least 90 genes. Mutations of the gap junction beta‐2 protein (GJB2) gene located in the nonsyndromic hearing loss and deafness (DFNB1) locus (chromosome 13q11‐12) are the main causes of autosomal recessive nonsyndromic hearing loss worldwide, but important differences exist between various populations. In the present article, two common mutations of the GJB2 gene are compared for ethnic‐specific allele frequency, their function, and their contribution to genetic HL in different populations. The results indicated that mutations of the GJB2 gene could have arisen during human migration. Updates on the spectrum of mutations clearly show that frequent mutations in the GJB2 gene are consistent with the founder mutation hypothesis.     

Chondrocyte‐specific Smad4 gene conditional knockout results in hearing loss and inner ear malformation in mice

Smad4 is the central intracellular mediator of transforming growth factor‐β (TGF‐β) signaling, which plays crucial roles in tissue regeneration, cell differentiation, embryonic development, and regulation of the immune system. Conventional Smad4 gene knockout results in embryonic lethality, precluding its use in studies of the role of Smad4 in inner ear development. We used chondrocyte‐specific Smad4 knockout mice (Smad4^Co/Co) to investigate the function of Smad4 in inner ear development. Smad4^Co/Co mice were characterized by a smaller cochlear volume, bone malformation, and abnormalities of the osseous spiral lamina and basilar membrane. The development of the hair cells was also abnormal, as evidenced by the disorganized stereocilia and reduced density of the neuronal processes beneath the hair cells. Auditory function tests revealed the homozygous Smad4^Co/Co mice suffered from severe sensorineural hearing loss. Our results suggest that Smad4 is required for inner ear development and normal auditory function in mammals. Developmental Dynamics, 2009. © 2009 Wiley‐Liss, Inc.     

Speech‐perception training for older adults with hearing loss impacts word recognition and effort

The current pupillometry study examined the impact of speech‐perception training on word recognition and cognitive effort in older adults with hearing loss. Trainees identified more words at the follow‐up than at the baseline session. Training also resulted in an overall larger and faster peaking pupillary response, even when controlling for performance and reaction time. Perceptual and cognitive capacities affected the peak amplitude of the pupil response across participants but did not diminish the impact of training on the other pupil metrics. Thus, we demonstrated that pupillometry can be used to characterize training‐related and individual differences in effort during a challenging listening task. Importantly, the results indicate that speech‐perception training not only affects overall word recognition, but also a physiological metric of cognitive effort, which has the potential to be a biomarker of hearing loss intervention outcome.     

Mutations in KARS cause early‐onset hearing loss and leukoencephalopathy: Potential pathogenic mechanism

Leukoencephalopathies are a broad class of common neurologic deterioration for which the etiology remains unsolved in many cases. In a Chinese Han family segregated with sensorineural hearing loss and leukoencephalopathy, candidate pathogenic variants were identified by targeted next‐generation sequencing of 144 genes associated with deafness and 108 genes with leukoencephalopathy. Novel compound heterozygous mutations p.R477H and p.P505S were identified in KARS, which encodes lysyl‐tRNA synthetase (LysRS), as the only candidate causative variants. These two mutations were functionally characterized by enzymatic assays, immunofluorescence, circular dichroism analysis, and gel filtration chromatography. Despite no alteration in the dimer‐tetramer oligomerization and cellular distribution by either mutation, the protein structure was notably influenced by the R477H mutation, which subsequently released the protein from the multiple‐synthetase complex (MSC). Mutant LysRSs with the R477H and P505S mutations had decreased tRNA^Lys aminoacylation and displayed a cumulative effect when introduced simultaneously. Our studies showed that mutations in KARS lead to a newly defined subtype of leukoencephalopathy associated with sensorineural hearing impairment. The combined effect of reduced aminoacylation and release of LysRS from the MSC likely underlies the pathogenesis of the KARS mutations identified in this study.     

Construction of a multiplex allele-specific PCR-based universal array (ASPUA) and its application to hearing loss screening

We demonstrate a new method, using a universal array approach termed multiplex allele-specific PCR-based universal array (ASPUA), and applied it to the mutation detection of hereditary hearing loss. Mutations in many different genes may be the cause of hereditary hearing loss, a sensory defect disorder. Effective methods for genetic diagnosis are clearly needed to provide clinical management. Owing to the broad genetic basis of this condition, clinical assay of such a highly heterogeneous disorder is expensive and time consuming. In ASPUA, the allele discrimination reaction is carried out in solution by multiplex allele-specific PCR and a universal solid phase array with different tag probes is used to display the PCR result. The purpose of developing the ASPUA platform was to utilize the rapidity and simplicity of the amplification refractory mutation system (ARMS) with the detection power of microarray hybridization. This is the first report of the combination of these two technologies, which allow for the completion of allele-specific detection of 11 of the most frequent mutations causing hereditary hearing loss in under 5 hr. The ASPUA platform was validated by accurately analyzing 141 patient samples that had been previously genotyped for GJB2, GJB3, SLC26A4, and MTRNR1. In addition, we also developed a simplified assay by using streptavidin-coated magnetic beads instead of fluorescence for signal display that can be assessed through a conventional light microscope. We demonstrate that the ASPUA platform is rapid, cost-effective, and easily-used, and is especially appropriate for mutation detection in clinical genetic diagnostics.     

Haplogroup analysis supports a pathogenic role for the 7510T>C mutation of mitochondrial tRNA(Ser(UCN)) in sensorineural hearing loss

We ascertained a large North American family, LMG309, with matrilineal transmission of non-syndromic, progressive sensorineural hearing loss (SNHL). There was no history of aminoglycoside exposure, and penetrance was complete. We sequenced the entire mitochondrial genome and identified the previously reported 7510T>C transition in the tRNA^Ser(UCN) gene. The 7510T>C was homoplasmic in all affected members. The LMG309 mitochondrial sequence belongs to an unnamed subgroup of mitochondrial haplogroup H. We demonstrate that the previously reported Spanish family S258 carries 7510T>C on a different mitochondrial sub-haplogroup, H1. We did not detect 7510T>C among 79 Caucasian haplogroup H control samples, including 11 from sub-haplogroup H1 and one from the same sub-haplogroup as LMG309. Our results provide strong genetic evidence that 7510T>C is a pathogenic mutation that causes non-syndromic SNHL.     

Clinical update on sensorineural hearing loss in Turner syndrome and the X‐chromosome

Hearing loss is one of the major medical concerns in girls and women with Turner syndrome (TS) and has a negative effect on well‐being and quality of everyday life. Sensorineural hearing loss is the most common type of hearing loss, affecting more than half of adults with TS. Karyotypes with a loss of the short p‐arm on the X‐chromosome are more prone to ear and hearing problems. The importance of detecting, investigating, and treating hearing loss with hearing aids cannot be emphasized enough. The pathophysiology of the sensorineural hearing loss in TS is not known, but theories regarding estrogen deficiency, the cell cycle delay hypothesis, IGF‐1 deficiency and the possible role of the KDM6A gene are discussed. Due to the diversity of symptoms and conditions within the same karyotype, a combination of genetic factors altered by epigenetic and/or hormonal effects is probable. Further research is needed regarding the pathophysiology of ear and hearing problems in TS to develop new treatment methods.     

A connexin 26 mutation causes a syndrome of sensorineural hearing loss and palmoplantar hyperkeratosis (MIM 148350)

We report a missense mutation in the connexin 26 gene (GJB2) in a family with an autosomal dominant syndrome of hearing loss and hyperkeratosis. The affected family members have high frequency, slowly progressive, bilateral, sensorineural hearing loss and palmoplantar hyperkeratosis. The mutation causes an amino acid substitution (G59A), which may disrupt a reverse turn in the first extracellular loop of connexin 26. Connexin 26 mutations have been reported in syndromes of deafness and palmoplantar keratoderma. These data provide additional evidence for the role of connexin 26 in syndromes of this type.     

Clinical efficacy of a next‐generation sequencing gene panel for primary immunodeficiency diagnostics

Primary immunodeficiencies (PIDs) are rare monogenic inborn errors of immunity that result in impairment of functions of the human immune system. PIDs have a broad phenotype with increased morbidity and mortality, and treatment choices are often complex. With increased accessibility of next‐generation sequencing (NGS), the rate of discovery of genetic causes for PID has increased exponentially. Identification of an underlying monogenic diagnosis provides important clinical benefits for patients with the potential to alter treatments, facilitate genetic counselling, and pre‐implantation diagnostics. We investigated a NGS PID panel of 242 genes within clinical care across a range of PID phenotypes. We also evaluated Phenomizer to predict causal genes from human phenotype ontology (HPO) terms. Twenty‐seven participants were recruited, and a total of 15 reportable variants were identified in 48% (13/27) of the participants. The panel results had implications for treatment in 37% (10/27) of participants. Phenomizer identified the genes harbouring variants from HPO terms in 33% (9/27) of participants. This study shows the clinical efficacy that genetic testing has in the care of PID. However, it also highlights some of the disadvantages of gene panels in the rapidly moving field of PID genomics and current challenges in HPO term assignment for PID.     

High performance of a new PCR‐based urine assay for HPV‐DNA detection and genotyping

Human papillomavirus (HPV) testing has been proposed as a means of replacing or supporting conventional cervical screening (Pap test). However, both methods require the collection of cervical samples. Urine sample is easier and more acceptable to collect and could be helpful in facilitating cervical cancer screening. The aim of this study was to evaluate the sensitivity and specificity of urine testing compared to conventional cervical smear testing using a PCR‐based method with a new, designed specifically primer set. Paired cervical and first voided urine samples collected from 107 women infected with HIV were subjected to HPV‐DNA detection and genotyping using a PCR‐based assay and a restriction fragment length polymorphism method. Sensitivity, specificity, Positive Predictive Value (PPV), and Negative Predictive Value (NPV) were calculated using the McNemar's test for differences. Concordance between tests was assessed using the Cohen's unweighted Kappa (k). HPV DNA was detected in 64.5% (95% CI: 55.1–73.1%) of both cytobrush and urine samples. High concordance rates of HPV‐DNA detection (k = 0.96; 95% CI: 0.90–1.0) and of high risk‐clade and low‐risk genotyping in paired samples (k = 0.80; 95% CI: 0.67–0.92 and k = 0.74; 95% CI: 0.60–0.88, respectively) were observed. HPV‐DNA detection in urine versus cervix testing revealed a sensitivity of 98.6% (95% CI: 93.1–99.9%) and a specificity of 97.4% (95% CI: 87.7–99.9%), with a very high NPV (97.4%; 95% CI: 87.7–99.9%). The PCR‐based assay utilized in this study proved highly sensitive and specific for HPV‐DNA detection and genotyping in urine samples. These data suggest that a urine‐based assay would be a suitable and effective tool for epidemiological surveillance and, most of all, screening programs. J. Med. Virol. 85:91–98, 2012. © 2012 Wiley Periodicals, Inc.     

A new amplicon‐based gene panel for next generation sequencing characterization of meningiomas

Meningiomas are the most frequent primary intracranial tumors. The considerable variety of histological subtypes has been expanded by the definition of molecular alterations, which can improve both diagnostic accuracy and determination of individual patient''s outcome. According to the upcoming WHO classification of brain tumors, the in‐time analysis of frequent molecular events in meningiomas may become mandatory to define meningioma subtypes. We have compiled a custom‐made amplicon‐based next generation sequencing (NGS) meningioma panel covering the most frequent known recurrent mutations in 15 different genes. In an unselected consecutive meningioma cohort (109 patients) analyzed over a period of 12 months, we detected mutations in 11 different genes, with most frequent alterations in NF2 (43%), AKT1 ^E17K (15%), and TRAF7 (13%). In 39 tumors (36%), two different mutations were detected, with NF2 and SUFU (n = 5) and KLF4 and TRAF7 (n = 5) being the most frequent combinations. No alterations were found in POLR2A, CDKN2A, CDKN2B, and BAP1, and no homozygous CDKN2A/B deletion was detected. NF2 mutations were found in tumors of all WHO grades, whereas mutations in KLF4, TRAF7, and SMO were restricted to WHO grade I meningiomas. In contrast, SMARCE1 and TERT mutations were associated with WHO grade II meningiomas (according to the WHO classification 2016). The distribution of mutations across histological subtypes or tumor localization was in line with the existing literature, with typical combinations like KLF4K^409Q/TRAF7 for secretory meningiomas and preferential skull base localization of meningiomas harboring SMO and AKT1 ^E17K mutations. Thus, we present a custom‐made NGS meningioma panel providing a time and cost‐efficient reliable detection of relevant somatic molecular alterations in meningiomas suitable for daily routine.