Preservation of Distortion Product Otoacoustic Emissions in OTOF -Related Hearing Impairment.

OBJECTIVES: Attenuation of otoacoustic emissions over time has been reported for many patients with hearing impairment harboring mutations in the OTOF gene. In this study, the time course of changes of distortion product otoacoustic emissions (DPOAEs) has been analyzed in a cohort of patients in the light of tympanometry results. DESIGN: The changes of DPOAEs in 16 patients with OTOF -related hearing impairment were retrospectively analyzed. RESULTS: All but one subject showed DPOAEs bilaterally at the time of diagnosis. Three patients diagnosed as adults still had DPOAEs at ages of 27, 31, and 47 years, respectively. Follow-up was available for 7 children diagnosed at the age of 1 to 3 years, who still showed preservation of DPOAEs at ages of 5 to 16 years. The responses were absent or attenuated in amplitude at some follow-up appointments in association with type B or C tympanograms. CONCLUSIONS: DPOAEs are preserved much longer than expected in a cohort of patients with OTOF -related hearing impairment. The previously reported loss of DPOAEs may have been caused in some children by increased middle ear impedance due to otitis media.

Cochlear Synaptopathy due to Mutations in OTOF Gene May Result in Stable Mild Hearing Loss and Severe Impairment of Speech Perception.

OBJECTIVES: Congenital profound hearing loss with preserved cochlear outer hair cell activity (otoacoustic emissions and cochlear microphonic) is the most common phenotype associated with mutations in the OTOF gene. The aim of this study was to investigate the pathophysiological mechanisms behind the auditory dysfunction in five patients (2 adults and 3 children) carrying biallelic mutations in OTOF, who showed an uncommon phenotype of mild hearing impairment associated with severe difficulties in speech perception and delay of language development. DESIGN: Patients underwent audiometric assessment with pure-tone and speech perception evaluation, and otoacoustic emissions and auditory brainstem response recording. Cochlear potentials were recorded in all subjects through transtympanic electrocochleography in response to clicks delivered in the free field from 120 to 60 dB peak equivalent SPL and were compared to recordings obtained from 20 normally hearing controls and from eight children with profound deafness due to mutations in the OTOF gene. Three patients out of five underwent unilateral cochlear implantation. Speech perception measures and electrically evoked auditory nerve potentials were obtained within 1 year of cochlear implant use. RESULTS: Pathogenic mutations in the two alleles of OTOF were found in all five patients, and five novel mutations were identified. Hearing thresholds indicated mild hearing loss in four patients and moderate hearing loss in one. Distortion product otoacoustic emissions were recorded in all subjects, whereas auditory brainstem responses were absent in all but two patients, who showed a delayed wave V in one ear. In electrocochleography recordings, cochlear microphonics and summating potentials showed normal latency and peak amplitude, consistently with preservation of both outer and inner hair cell activity. In contrast, the neural compound action potential recorded in normally hearing controls was replaced by a prolonged, low-amplitude negative response. No differences in cochlear potentials were found between OTOF subjects showing mild or profound hearing loss. Electrical stimulation through the cochlear implant improved speech perception and restored synchronized auditory nerve responses in all cochlear implant recipients. CONCLUSIONS: These findings indicate that disordered synchrony in auditory fiber activity underlies the impairment of speech perception in patients carrying biallelic mutations in OTOF gene who show a stable phenotype of mild hearing loss. Abnormal nerve synchrony with preservation of hearing sensitivity is consistent with selective impairment of vesicle replenishment at the ribbon synapses with relative preservation of synaptic exocytosis. Cochlear implants are effective in restoring speech perception and synchronous activation of the auditory pathway by directly stimulating auditory fibers.

Tryptophan-rich basic protein (WRB) mediates insertion of the tail-anchored protein otoferlin and is required for hair cell exocytosis and hearing.

The transmembrane recognition complex (TRC40) pathway mediates the insertion of tail-anchored (TA) proteins into membranes. Here, we demonstrate that otoferlin, a TA protein essential for hair cell exocytosis, is inserted into the endoplasmic reticulum (ER) via the TRC40 pathway. We mutated the TRC40 receptor tryptophan-rich basic protein (Wrb) in hair cells of zebrafish and mice and studied the impact of defective TA protein insertion. Wrb disruption reduced otoferlin levels in hair cells and impaired hearing, which could be restored in zebrafish by transgenic Wrb rescue and otoferlin overexpression. Wrb-deficient mouse inner hair cells (IHCs) displayed normal numbers of afferent synapses, Ca2+ channels, and membrane-proximal vesicles, but contained fewer ribbon-associated vesicles. Patch-clamp of IHCs revealed impaired synaptic vesicle replenishment. In vivo recordings from postsynaptic spiral ganglion neurons showed a use-dependent reduction in sound-evoked spiking, corroborating the notion of impaired IHC vesicle replenishment. A human mutation affecting the transmembrane domain of otoferlin impaired its ER targeting and caused an auditory synaptopathy. We conclude that the TRC40 pathway is critical for hearing and propose that otoferlin is an essential substrate of this pathway in hair cells.

Phenotype and genotype analysis of a French cohort of 119 patients with CHARGE syndrome.

CHARGE syndrome (CS) is a genetic disorder whose first description included Coloboma, Heart disease, Atresia of choanae, Retarded growth and development, Genital hypoplasia, and Ear anomalies and deafness, most often caused by a genetic mutation in the CHD7 gene. Two features were then added: semicircular canal anomalies and arhinencephaly/olfactory bulb agenesis, with classification of typical, partial, or atypical forms on the basis of major and minor clinical criteria. The detection rate of a pathogenic variant in the CHD7 gene varies from 67% to 90%. To try to have an overview of this heterogenous clinical condition and specify a genotype-phenotype relation, we conducted a national study of phenotype and genotype in 119 patients with CS. Selected clinical diagnostic criteria were from Verloes (2005), updated by Blake & Prasad (). Besides obtaining a detailed clinical description, when possible, patients underwent a full ophthalmologic examination, audiometry, temporal bone CT scan, gonadotropin analysis, and olfactory-bulb MRI. All patients underwent CHD7 sequencing and MLPA analysis. We found a pathogenic CHD7 variant in 83% of typical CS cases and 58% of atypical cases. Pathogenic variants in the CHD7 gene were classified by the expected impact on the protein. In all, 90% of patients had a typical form of CS and 10% an atypical form. The most frequent features were deafness/semicircular canal hypoplasia (94%), pituitary defect/hypogonadism (89%), external ear anomalies (87%), square-shaped face (81%), and arhinencephaly/anosmia (80%). Coloboma (73%), heart defects (65%), and choanal atresia (43%) were less frequent.

Novel Pathogenic Variants in PJVK, the Gene Encoding Pejvakin, in Subjects with Autosomal Recessive Non-Syndromic Hearing Impairment and Auditory Neuropathy Spectrum Disorder.

Pathogenic variants in the PJVK gene cause the DFNB59 type of autosomal recessive non-syndromic hearing impairment (AR-NSHI). Phenotypes are not homogeneous, as a few subjects show auditory neuropathy spectrum disorder (ANSD), while others show cochlear hearing loss. The numbers of reported cases and pathogenic variants are still small to establish accurate genotype-phenotype correlations. We investigated a cohort of 77 Spanish familial cases of AR-NSHI, in whom DFNB1 had been excluded, and a cohort of 84 simplex cases with isolated ANSD in whom OTOF variants had been excluded. All seven exons and exon-intron boundaries of the PJVK gene were sequenced. We report three novel DFNB59 cases, one from the AR-NSHI cohort and two from the ANSD cohort, with stable, severe to profound NSHI. Two of the subjects received unilateral cochlear implantation, with apparent good outcomes. Our study expands the spectrum of PJVK mutations, as we report four novel pathogenic variants: p.Leu224Arg, p.His294Ilefs*43, p.His294Asp and p.Phe317Serfs*20. We review the reported cases of DFNB59, summarize the clinical features of this rare subtype of AR-NSHI and discuss the involvement of PJVK in ANSD.

A multicenter study on the prevalence and spectrum of mutations in the otoferlin gene (OTOF) in subjects with nonsyndromic hearing impairment and auditory neuropathy.

Autosomal recessive nonsyndromic hearing impairment (NSHI) is a heterogeneous condition, for which 53 genetic loci have been reported, and 29 genes have been identified to date. One of these, OTOF, encodes otoferlin, a membrane-anchored calcium-binding protein that plays a role in the exocytosis of synaptic vesicles at the auditory inner hair cell ribbon synapse. We have investigated the prevalence and spectrum of deafness-causing mutations in the OTOF gene. Cohorts of 708 Spanish, 83 Colombian, and 30 Argentinean unrelated subjects with autosomal recessive NSHI were screened for the common p.Gln829X mutation. In compound heterozygotes, the second mutant allele was identified by DNA sequencing. In total, 23 Spanish, two Colombian and two Argentinean subjects were shown to carry two mutant alleles of OTOF. Of these, one Colombian and 13 Spanish subjects presented with auditory neuropathy. In addition, a cohort of 20 unrelated subjects with a diagnosis of auditory neuropathy, from several countries, was screened for mutations in OTOF by DNA sequencing. A total of 11 of these subjects were shown to carry two mutant alleles of OTOF. In total, 18 pathogenic and four neutral novel alleles of the OTOF gene were identified. Haplotype analysis for markers close to OTOF suggests a common founder for the novel c.2905_2923delinsCTCCGAGCGCA mutation, frequently found in Argentina. Our results confirm that mutation of the OTOF gene correlates with a phenotype of prelingual, profound NSHI, and indicate that OTOF mutations are a major cause of inherited auditory neuropathy.

CHARGE syndrome: a recurrent hotspot of mutations in CHD7 IVS25 analyzed by bioinformatic tools and minigene assays.

CHARGE syndrome is a rare genetic disorder mainly due to de novo and private truncating mutations of CHD7 gene. Here we report an intriguing hot spot of intronic mutations (c.5405-7G > A, c.5405-13G > A, c.5405-17G > A and c.5405-18C > A) located in CHD7 IVS25. Combining computational in silico analysis, experimental branch-point determination and in vitro minigene assays, our study explains this mutation hot spot by a particular genomic context, including the weakness of the IVS25 natural acceptor-site and an unconventional lariat sequence localized outside the common 40 bp upstream the acceptor splice site. For each of the mutations reported here, bioinformatic tools indicated a newly created 3' splice site, of which the existence was confirmed using pSpliceExpress, an easy-to-use and reliable splicing reporter tool. Our study emphasizes the idea that combining these two complementary approaches could increase the efficiency of routine molecular diagnosis.

Interaction between BDNF Polymorphism and Physical Activity on Inhibitory Performance in the Elderly without Cognitive Impairment.

Background: In the elderly, physical activity (PA) enhances cognitive performances, increases brain plasticity and improves brain health. The neurotrophic hypothesis is that the release of brain-derived neurotrophic factor (BDNF), which is implicated in brain plasticity and cognition, is triggered by PA because motoneurons secrete BDNF into the bloodstream during exercise. Individual differences in cognitive performance may be explained by individual differences in genetic predisposition. A single nucleotide polymorphism on the BDNF gene, BDNFVal66Met, affects activity-dependent BDNF secretion. This study investigated the influence of the BDNFVal66Met polymorphism on the relationship between PA and controlled inhibition performance in older adults. Methods: A total of 114 healthy elderly volunteers (mean age = 71.53 years old) were evaluated. Participants were genotyped for the BDNFVal66Met polymorphism. We evaluated inhibitory performance using choice reaction times (RT) and error rates from a Simon-like task and estimated their PA using two self-reported questionnaires. We established four groups according to PA level (active vs. inactive) and BDNFVal66Met genotype (Met carriers vs. Val-homozygous). The results were analyzed using ANOVA and ANCOVA, including age, gender and body mass index as covariates. Results: The BDNFVal66Met polymorphism interacted with PA on controlled inhibition performance. More specifically, inactive Val-homozygous participants exhibited a lower inhibition performance than active Val homozygotes and inactive Met carriers; the former had a higher error rate without differences in RT. Conclusion: Differences between individuals on inhibitory performance may be partially understood by the interaction between genetic influence in BDNF secretion and PA level. The results of this study clearly support the neurotrophic hypothesis that BDNF synthesis is an important mechanism underlying the influence of physical activity on brain structure and functions.

Effects of BDNF polymorphism and physical activity on episodic memory in the elderly: a cross sectional study.

BACKGROUND: The brain-derived neurotrophic factor (BDNF) concentration is highest in the hippocampus compared with that in other brain structures and affects episodic memory, a cognitive function that is impaired in older adults. According to the neurotrophic hypothesis, BDNF released during physical activity enhances brain plasticity and consequently brain health. However, even if the physical activity level is involved in the secretion of neurotrophin, this protein is also under the control of a specific gene. The aim of the present study was to examine the effect of the interaction between physical activity and BDNF Val66Met (rs6265), a genetic polymorphism, on episodic memory. METHODS: Two hundred and five volunteers aged 55 and older with a Mini Mental State Examination score ≥ 24 participated in this study. Four groups of participants were established according to their physical activity level and polymorphism BDNF profile (Active Val homozygous, Inactive Val homozygous, Active Met carriers, Inactive Met carriers). Episodic memory was evaluated based on the delayed recall of the Logical Memory test of the MEM III battery. RESULTS: As expected, the physical activity level interacted with BDNF polymorphism to affect episodic memory performance (p < .05). The active Val homozygous participants significantly outperformed the active Met carriers and inactive Val homozygous participants. CONCLUSION: This study clearly demonstrates an interaction between physical activity and BDNF Val66Met polymorphism that affects episodic memory in the elderly and confirms that physical activity contributes to the neurotrophic mechanism implicated in cognitive health. The interaction shows that only participants with Val/Val polymorphism benefited from physical activity.

Functional and splicing defect analysis of 23 ACVRL1 mutations in a cohort of patients affected by Hereditary Hemorrhagic Telangiectasia.

Hereditary Hemorrhagic Telangiectasia syndrome (HHT) or Rendu-Osler-Weber (ROW) syndrome is an autosomal dominant vascular disorder. Two most common forms of HHT, HHT1 and HHT2, have been linked to mutations in the endoglin (ENG) and activin receptor-like kinase 1 (ACVRL1or ALK1) genes respectively. This work was designed to examine the pathogenicity of 23 nucleotide variations in ACVRL1 gene detected in more than 400 patients. Among them, 14 missense mutations and one intronic variant were novels, and 8 missense mutations were previously identified with questionable implication in HHT2. The functionality of missense mutations was analyzed in response to BMP9 (specific ligand of ALK1), the maturation of the protein products and their localization were analyzed by western blot and fluorescence microscopy. The splicing impairment of the intronic and of two missense mutations was examined by minigene assay. Functional analysis showed that 18 out of 22 missense mutations were defective. Splicing analysis revealed that one missense mutation (c.733A>G, p.Ile245Val) affects the splicing of the harboring exon 6. Similarly, the intronic mutation outside the consensus splicing sites (c.1048+5G>A in intron 7) was seen pathogenic by splicing study. Both mutations induce a frame shift creating a premature stop codon likely resulting in mRNA degradation by NMD surveillance mechanism. Our results confirm the haploinsufficiency model proposed for HHT2. The affected allele of ACVRL1 induces mRNA degradation or the synthesis of a protein lacking the receptor activity. Furthermore, our data demonstrate that functional and splicing analyses together, represent two robust diagnostic tools to be used by geneticists confronted with novel or conflicted ACVRL1 mutations.

Auditory neuropathy in patients carrying mutations in the otoferlin gene (OTOF).

Inherited hearing impairment affects one in 2,000 newborns. Nonsyndromic prelingual forms are inherited mainly as autosomal recessive traits, for which 16 genes are currently known. Mutations in the genes encoding connexins 26 and 30 account for up to 50% of these cases. However, the individual contribution of the remaining genes to the whole remains undetermined. In addition, for most of the genes there is a need for studies on genotype-phenotype correlations, to identify distinctive clinical features which may direct the molecular diagnosis to specific genes. Here we present a mutation analysis and a genotype-phenotype correlation study on the gene encoding otoferlin (OTOF), responsible for the DFNB9 subtype of prelingual hearing impairment. Four novel mutations were identified: c.2122C>T (p.Arg708Ter), c.4275G>A (p.Trp1425Ter), c.4362+2T>G, and c.5860_5862delATC (p.Ile1954del). A total of 37 subjects with mutations in OTOF were studied clinically. They were phenotypically homogeneous, having profound hearing impairment with very early onset, as shown by pure-tone audiometry and auditory brainstem responses. Magnetic resonance imaging and computed tomography did not reveal any inner ear malformation. Unexpectedly, transient evoked otoacoustic emissions (TEOAEs) were present, either bilaterally or unilaterally in 11 subjects. Altogether, clinical data of these subjects met the diagnostic criteria of auditory neuropathy. A total of 10 subjects had been successfully provided with cochlear implants. The results of our study indicate that genetic diagnosis of subjects with auditory neuropathy and profound hearing impairment should be directed to the otoferlin gene. Our data are of concern to universal screening programs which use TEOAEs as the first detection test for hearing impairment in newborns, since this technique may overlook a nonnegligible proportion of cases.

[Genetic study of hearing loss in families from Argentina]. / Estudio genético de hipoacusia en familias de Argentina.

Recent advances in molecular genetics as well as improved strategies for the prevention and control of non-syndromic hearing loss (NSHL) have contributed to the rising importance of their inherited causes. In this study we report 32 families from Argentine with one (sporadic) or more (familial) individuals affected. All the families were initially screened for mutations in three autosomal nuclear genes and one mutation in mitochondrial DNA. These genes have been found in a great number of familial or sporadic cases of congenital deafness in Caucasians. The mutant allele 35 del G of connexin 26 (GJB2, locus DFNB1 on 13q12) was present in three families. We have investigated the gene encoding otoferlin (OTOF, locus DFNB9 on 2p22-p23) and we found the Q829X mutation in heterocigosity in two families. We have also identified in heterocigosity the 342-kb deletion of connexin 30 (GJB6, locus DFNB1 on 13q12) in one family. On the other hand, we have not found any patient with mitochondrial mutation. Since the screening for other mutations is very expensive, our main goal is to investigate the most frequent mutations in each separate gene in the argentine population and to develop simple and specific tests for each frequent mutations.

Abnormal cochlear potentials from deaf patients with mutations in the otoferlin gene.

Otoferlin is involved in neurotransmitter release at the synapse between inner hair cells (IHCs) and auditory nerve fibres, and mutations in the OTOF gene result in severe to profound hearing loss. Abnormal sound-evoked cochlear potentials were recorded with transtympanic electrocochleography from four children with otoferlin (OTOF) mutations to evaluate physiological effects in humans of abnormal neurotransmitter release from IHCs. The subjects were profoundly deaf with absent auditory brainstem responses and preserved otoacoustic emissions consistent with auditory neuropathy. Two children were compound heterozygotes for mutations c.2732_2735dupAGCT and p.Ala964Glu; one subject was homozygous for mutation p.Phe1795Cys, and one was compound heterozygote for two novel mutations c.1609delG in exon 16 and c.1966delC in exon 18. Cochlear potentials evoked by clicks from 60 to 120 dB peak equivalent sound pressure level were compared to recordings obtained from 16 normally hearing children. Cochlear microphonic (CM) was recorded with normal amplitudes from all but one ear. After cancelling CM, cochlear potentials were of negative polarity with reduced amplitude and prolonged duration compared to controls. These cochlear potentials were recorded as low as 50-90 dB below behavioural thresholds in contrast to the close correlation in controls between cochlear potentials and behavioural threshold. Summating potential was identified in five out of eight ears with normal latency whilst auditory nerve compound action potentials were either absent or of low amplitude. Stimulation at high rates reduced amplitude and duration of the prolonged potentials, consistent with neural generation. This study suggests that mechano-electrical transduction and cochlear amplification are normal in patients with OTOF mutations. The low-amplitude prolonged negative potentials are consistent with decreased neurotransmitter release resulting in abnormal dendritic activation and impairment of auditory nerve firing.

Estudio genético de hipoacusia en familias de Argentina / Genetic study of hearing loss in families from Argentina

Los últimos avances en genética molecular como así también el desarrollo de estrategias para la prevención y control de las hipoacusias no sindrómicas (HNS), han contribuido al esclarecimiento de las causas hereditarias de las mismas. En este estudio, se seleccionaron 32 familias argentinas con uno (esporádico) o más (familiar) individuos afectados. El análisis genético consistió en la búsqueda de tres genes autosómicos nucleares y uno en el ADN mitocondrial. Estos genes se localizaron en un gran número de casos familiares o esporádicos de sorderas congénitas en Caucásicos. El alelo mutado 35 del G de la conexina 26 (GJB2, locus DFNB1 en 13g12) se presentó en tres familias. Además se investigó el gen que codifica otoferlina (OTOF locus DFNB9 en 2p22-23) encontrándose en dos familias la mutación Q829X en heterocigocidad. También se identificó en una familia portadora heterocigota la deleción de 342 Kb en la conexina 30 (GJB6, locus DFNB1 en 13g12). Por otro lado, no encontramos ningún paciente con la mutación mitocondrial. Debido a que la búsqueda de otras mutaciones es demasiado costosa, nuestro principal objetivo es investigar aquellas mas frecuentes en la población argentina, a fin de desarrollar test simples y específicos para cada una de ellas.

Estudio genético de hipoacusia en familias de Argentina. / [Genetic study of hearing loss in families from Argentina]

Recent advances in molecular genetics as well as improved strategies for the prevention and control of non-syndromic hearing loss (NSHL) have contributed to the rising importance of their inherited causes. In this study we report 32 families from Argentine with one (sporadic) or more (familial) individuals affected. All the families were initially screened for mutations in three autosomal nuclear genes and one mutation in mitochondrial DNA. These genes have been found in a great number of familial or sporadic cases of congenital deafness in Caucasians. The mutant allele 35 del G of connexin 26 (GJB2, locus DFNB1 on 13q12) was present in three families. We have investigated the gene encoding otoferlin (OTOF, locus DFNB9 on 2p22-p23) and we found the Q829X mutation in heterocigosity in two families. We have also identified in heterocigosity the 342-kb deletion of connexin 30 (GJB6, locus DFNB1 on 13q12) in one family. On the other hand, we have not found any patient with mitochondrial mutation. Since the screening for other mutations is very expensive, our main goal is to investigate the most frequent mutations in each separate gene in the argentine population and to develop simple and specific tests for each frequent mutations.

Estudio genético de hipoacusia en familias de Argentina / Genetic study of hearing loss in families from Argentina

Los últimos avances en genética molecular como así también el desarrollo de estrategias para la prevención y control de las hipoacusias no sindrómicas (HNS), han contribuido al esclarecimiento de las causas hereditarias de las mismas. En este estudio, se seleccionaron 32 familias argentinas con uno (esporádico) o más (familiar) individuos afectados. El análisis genético consistió en la búsqueda de tres genes autosómicos nucleares y uno en el ADN mitocondrial. Estos genes se localizaron en un gran número de casos familiares o esporádicos de sorderas congénitas en Caucásicos. El alelo mutado 35 del G de la conexina 26 (GJB2, locus DFNB1 en 13g12) se presentó en tres familias. Además se investigó el gen que codifica otoferlina (OTOF locus DFNB9 en 2p22-23) encontrándose en dos familias la mutación Q829X en heterocigocidad. También se identificó en una familia portadora heterocigota la deleción de 342 Kb en la conexina 30 (GJB6, locus DFNB1 en 13g12). Por otro lado, no encontramos ningún paciente con la mutación mitocondrial. Debido a que la búsqueda de otras mutaciones es demasiado costosa, nuestro principal objetivo es investigar aquellas mas frecuentes en la población argentina, a fin de desarrollar test simples y específicos para cada una de ellas. (AU)