Generation of four induced pluripotent stem cells lines from PBMC of the DFNA58 family members: Two hearing-impaired duplication carriers (USPi006-A e USPi007-A) and two normal-hearing noncarriers (USPi004-A and USPi005-A).

The DFNA58 locus contains a genomic duplication involving three protein-coding genes (CNRIP1, PLEK, and PPP3R1's exon 1) and other uncharacterized lncRNA genes (LOC101927723, LOC107985892 and LOC102724389). To clarify the role of these genes in hearing and precisely determine their role in hearing loss, four iPSC lines were generated from two carriers and two noncarriers of the duplication.

Waardenburg Syndrome: The Contribution of Next-Generation Sequencing to the Identification of Novel Causative Variants.

Waardenburg syndrome (WS) is characterized by hearing loss and pigmentary abnormalities of the eyes, hair, and skin. The condition is genetically heterogeneous, and is classified into four clinical types differentiated by the presence of dystopia canthorum in type 1 and its absence in type 2. Additionally, limb musculoskeletal abnormalities and Hirschsprung disease differentiate types 3 and 4, respectively. Genes PAX3, MITF, SOX10, KITLG, EDNRB, and EDN3 are already known to be associated with WS. In WS, a certain degree of molecularly undetected patients remains, especially in type 2. This study aims to pinpoint causative variants using different NGS approaches in a cohort of 26 Brazilian probands with possible/probable diagnosis of WS1 (8) or WS2 (18). DNA from the patients was first analyzed by exome sequencing. Seven of these families were submitted to trio analysis. For inconclusive cases, we applied a targeted NGS panel targeting WS/neurocristopathies genes. Causative variants were detected in 20 of the 26 probands analyzed, these being five in PAX3, eight in MITF, two in SOX10, four in EDNRB, and one in ACTG1 (type 2 Baraitser-Winter syndrome, BWS2). In conclusion, in our cohort of patients, the detection rate of the causative variant was 77%, confirming the superior detection power of NGS in genetically heterogeneous diseases.

New Insights into the Identity of the DFNA58 Gene.

Hearing loss is the most common sensory deficit, affecting 466 million people worldwide. The vast and diverse genes involved reflect the complexity of auditory physiology, which requires the use of animal models in order to gain a fuller understanding. Among the loci with a yet-to-be validated gene is the DFNA58, in which ~200 Kb genomic duplication, including three protein-coding genes (PLEK, CNRIP1, and PPP3R1's exon1), was found to segregate with autosomal dominant hearing loss. Through whole genome sequencing, the duplication was found to be in tandem and inserted in an intergenic region, without the disruption of the topological domains. Reanalysis of transcriptomes data studies (zebrafish and mouse), and RT-qPCR analysis of adult zebrafish target organs, in order to access their orthologues expression, highlighted promising results with Cnrip1a, corroborated by zebrafish in situ hybridization and immunofluorescence. Mouse data also suggested Cnrip1 as the best candidate for a relevant role in auditory physiology, and its importance in hearing seems to have remained conserved but the cell type exerting its function might have changed, from hair cells to spiral ganglion neurons.

Hearing aid effectiveness on patients with chronic tinnitus and associated hearing loss

Abstract Objective: Our study aimed to measure the effectiveness of using HA in reducing the disturbance caused by tinnitus. Methods: Study was designed as a within-subjects clinical trial. Nineteen patients with chronic tinnitus and untreated sensorineural hearing loss were under counseling, HA fitting and 6 months follow-up. Tinnitus assessment was performed with Tinnitus Handicap Inventory (THI), Visual Analog Scale (VAS), pitch and loudness matching, and Minimum Masking Level measurements (MML). Results: following 6 months of HA use, a reduction in reported tinnitus and hearing handicap scales scores was observed both statistically and clinically. The pitch and loudness matching, as well as MML at the baseline and final evaluation were compared. MML's thresholds reduced significantly after 6 months of HA use. Conclusion: Our study has provided evidence that HA fitting is a valuable treatment strategy for chronic tinnitus relief and associated hearing loss subtype of patient. Level of evidence: 3.

Hearing aid effectiveness on patients with chronic tinnitus and associated hearing loss.

OBJECTIVE: Our study aimed to measure the effectiveness of using HA in reducing the disturbance caused by tinnitus. METHODS: Study was designed as a within-subjects clinical trial. Nineteen patients with chronic tinnitus and untreated sensorineural hearing loss were under counseling, HA fitting and 6 months follow-up. Tinnitus assessment was performed with Tinnitus Handicap Inventory (THI), Visual Analog Scale (VAS), pitch and loudness matching, and Minimum Masking Level measurements (MML). RESULTS: following 6 months of HA use, a reduction in reported tinnitus and hearing handicap scales scores was observed both statistically and clinically. The pitch and loudness matching, as well as MML at the baseline and final evaluation were compared. MML's thresholds reduced significantly after 6 months of HA use. CONCLUSION: Our study has provided evidence that HA fitting is a valuable treatment strategy for chronic tinnitus relief and associated hearing loss subtype of patient.

Genetic etiology of non-syndromic hearing loss in Latin America.

Latin America comprises all countries from South and Central America, in addition to Mexico. It is characterized by a complex mosaic of regions with heterogeneous genetic profiles regarding the geographical origin of the ancestors and proportions of admixture between the Native American, European and African components. In the first years following the findings of the role of the GJB2/GJB6 genes in the etiology of hearing loss, most scientific investigations about the genetics of hearing loss in Latin America focused on assessing the frequencies of pathogenic variants in these genes. More recently, modern techniques allowed researchers in Latin America to make exciting contributions to the finding of new candidate genes, novel mechanisms of inheritance in previously known genes, and characterize a wide diversity of variants, many of them unique to Latin America. This review aimed to provide a general landscape of the genetic studies about non-syndromic hearing loss in Latin America and their main scientific contributions. It allows the conclusion that, although there are similar contributions of some genes, such as GJB2/GJB6, when compared to European and North American countries, Latin American populations revealed some peculiarities that indicate the need for tailored strategies of screening and diagnosis to specific geographic regions.

Molecular and genetic characterization of a large Brazilian cohort presenting hearing loss.

Hearing loss is one of the most common sensory defects, affecting 5.5% of the worldwide population and significantly impacting health and social life. It is mainly attributed to genetic causes, but their relative contribution reflects the geographical region's socio-economic development. Extreme genetic heterogeneity with hundreds of deafness genes involved poses challenges for molecular diagnosis. Here we report the investigation of 542 hearing-impaired subjects from all Brazilian regions to search for genetic causes. Biallelic GJB2/GJB6 causative variants were identified in 12.9% (the lowest frequency was found in the Northern region, 7.7%), 0.4% carried GJB2 dominant variants, and 0.6% had the m.1555A > G variant (one aminoglycoside-related). In addition, other genetic screenings, employed in selected probands according to clinical presentation and presumptive inheritance patterns, identified causative variants in 2.4%. Ear malformations and auditory neuropathy were diagnosed in 10.8% and 3.5% of probands, respectively. In 3.8% of prelingual/perilingual cases, Waardenburg syndrome was clinically diagnosed, and in 71.4%, these diagnoses were confirmed with pathogenic variants revealed; seven out of them were novel, including one CNV. All these genetic screening strategies revealed causative variants in 16.2% of the cases. Based on causative variants in the molecular diagnosis and genealogy analyses, a probable genetic etiology was found in ~ 50% of the cases. The present study highlights the relevance of GJB2/GJB6 as a cause of hearing loss in all Brazilian regions and the importance of screening unselected samples for estimating frequencies. Moreover, when a comprehensive screening is not available, molecular diagnosis can be enhanced by selecting probands for specific screenings.

Frequency and origin of the c.2090T>G p.(Leu697Trp) MYO3A variant associated with autosomal dominant hearing loss.

We recently described a novel missense variant [c.2090T>G:p.(Leu697Trp)] in the MYO3A gene, found in two Brazilian families with late-onset autosomal dominant nonsyndromic hearing loss (ADNSHL). Since then, with the objective of evaluating its contribution to ADNSHL in Brazil, the variant was screened in additional 101 pedigrees with probable ADNSHL without conclusive molecular diagnosis. The variant was found in three additional families, explaining 3/101 (~3%) of cases with ADNSHL in our Brazilian pedigree collection. In order to identify the origin of the variant, 21 individuals from the five families were genotyped with a high-density SNP array (~600 K SNPs- Axiom Human Origins; ThermoFisher). The identity by descent (IBD) approach revealed that many pairs of individuals from the different families have a kinship coefficient equivalent to that of second cousins, and all share a minimum haplotype of ~607 kb which includes the c.2090T>G variant suggesting it probably arose in a common ancestor. We inferred that the mutation occurred in a chromosomal segment of European ancestry and the time since the most common ancestor was estimated in 1100 years (CI = 775-1425). This variant was also reported in a Dutch family, which shares a 87,121 bp haplotype with the Brazilian samples, suggesting that Dutch colonists may have brought it to Northeastern Brazil in the 17th century. Therefore, the present study opens new avenues to investigate this variant not only in Brazilians but also in European families with ADNSHL.

New Insights on the Effect of TNF Alpha Blockade by Gene Silencing in Noise-Induced Hearing Loss.

Noise exposure represents the second most common cause of acquired sensorineural hearing loss and we observed that tumor necrosis factor α (TNFα) was involved in this context. The effect of Tnfα gene silencing on the expression profile related to the TNFα metabolic pathway in an experimental model of noise-induced hearing loss had not previously been studied. METHODS: Single ears of Wistar rats were pretreated with Tnfα small interfering RNA (siRNA) by trans-tympanic administration 24 h before they were exposed to white noise (120 dBSPL for three hours). After 24 h of noise exposure, we analyzed the electrophysiological threshold and the amplitude of waves I, II, III, and IV in the auditory brain response click. In addition, qRT-PCR was performed to evaluate the TNFα metabolic pathway in the ears submitted or not to gene silencing. RESULTS: Preservation of the electrophysiological threshold and the amplitude of waves was observed in the ears submitted to gene silencing compared to the ears not treated. Increased anti-apoptotic gene expression and decreased pro-apoptotic gene expression were found in the treated ears. CONCLUSION: Our results allow us to suggest that the blockade of TNFα by gene silencing was useful to prevent noise-induced hearing loss.

A rare genomic duplication in 2p14 underlies autosomal dominant hearing loss DFNA58.

Here we define a ~200 Kb genomic duplication in 2p14 as the genetic signature that segregates with postlingual progressive sensorineural autosomal dominant hearing loss (HL) in 20 affected individuals from the DFNA58 family, first reported in 2009. The duplication includes two entire genes, PLEK and CNRIP1, and the first exon of PPP3R1 (protein coding), in addition to four uncharacterized long non-coding (lnc) RNA genes and part of a novel protein-coding gene. Quantitative analysis of mRNA expression in blood samples revealed selective overexpression of CNRIP1 and of two lncRNA genes (LOC107985892 and LOC102724389) in all affected members tested, but not in unaffected ones. Qualitative analysis of mRNA expression identified also fusion transcripts involving parts of PPP3R1, CNRIP1 and an intergenic region between PLEK and CNRIP1, in the blood of all carriers of the duplication, but were heterogeneous in nature. By in situ hybridization and immunofluorescence, we showed that Cnrip1, Plek and Ppp3r1 genes are all expressed in the adult mouse cochlea including the spiral ganglion neurons, suggesting changes in expression levels of these genes in the hearing organ could underlie the DFNA58 form of deafness. Our study highlights the value of studying rare genomic events leading to HL, such as copy number variations. Further studies will be required to determine which of these genes, either coding proteins or non-coding RNAs, is or are responsible for DFNA58 HL.

Further evidence for loss-of-function mutations in the CEACAM16 gene causing nonsyndromic autosomal recessive hearing loss in humans.

Mutations in the CEACAM6 gene were first described as causing autosomal dominant nonsyndromic hearing loss, but two splice-altering variants have been recently described as causing autosomal recessive nonsyndromic hearing loss. We describe the novel and extremely rare loss-of-function variant c.436 C > T/p.(Arg146Ter) in the CEACAM16 gene segregating with post-lingual progressive autosomal recessive hearing loss. This variant is predicted to significantly reduce the size of the wild type protein. Our results give additional support that loss-of-function variants in CEACAM16 cause autosomal recessive hearing loss in humans.

Syndromic hearing loss molecular diagnosis: Application of massive parallel sequencing.

Syndromic hearing loss accounts for approximately 30% of all cases of hearing loss due to genetic causes. Mutation screening in known genes is important because it potentially sheds light on the genetic etiology of hearing loss and helps in genetic counseling of families. In this study, we describe a customized Ion AmpliSeq Panel, specifically designed for the investigation of syndromic hearing loss. The Ion AmpliSeq Panel was customized to cover the coding sequences of 52 genes. Twenty-four patients were recruited: 17 patients with a clinical diagnosis of a known syndrome, and seven whose clinical signs did not allow identification of a syndrome. Of 24 patients sequenced, potentially causative mutations were found in nine, all of which belonged to the group with a previous clinical diagnostic and none in the group not clinically diagnosed. We were able to provide conclusive molecular diagnosis to six patients, constituting a diagnostic rate of 25% (6/24). In the group of patients with a suspected clinical diagnosis, the diagnostic rate was 35% (6/17). Of the nine different mutations identified, three are novel, and were found in patients with Waardenburg, Treacher Collins and CHARGE syndromes. Since all patients with a conclusive molecular diagnosis through this panel had a previous suspected clinical diagnosis, our results suggest that this panel was more effective in diagnosing this group of patients. Therefore, the panel demonstrated effectiveness in molecular diagnosis when compared to others in the literature, especially for patients with a defined clinical diagnosis.

Characterization of a novel MYO3A missense mutation associated with a dominant form of late onset hearing loss.

Whole-exome sequencing of samples from affected members of two unrelated families with late-onset non-syndromic hearing loss revealed a novel mutation (c.2090 T > G; NM_017433) in MYO3A. The mutation was confirmed in 36 affected individuals, showing autosomal dominant inheritance. The mutation alters a single residue (L697W or p.Leu697Trp) in the motor domain of the stereocilia protein MYO3A, leading to a reduction in ATPase activity, motility, and an increase in actin affinity. MYO3A-L697W showed reduced filopodial actin protrusion initiation in COS7 cells, and a predominant tipward accumulation at filopodia and stereocilia when coexpressed with wild-type MYO3A and espin-1, an actin-regulatory MYO3A cargo. The combined higher actin affinity and duty ratio of the mutant myosin cause increased retention time at stereocilia tips, resulting in the displacement of the wild-type MYO3A protein, which may impact cargo transport, stereocilia length, and mechanotransduction. The dominant negative effect of the altered myosin function explains the dominant inheritance of deafness.

Mutation analysis of SLC26A4 (Pendrin) gene in a Brazilian sample of hearing-impaired subjects.

BACKGROUND: Mutations in the SLC26A4 gene are associated with Pendred syndrome and autosomal recessive non-syndromic deafness (DFNB4). Both disorders have similar audiologic characteristics: bilateral hearing loss, often severe or profound, which may be associated with abnormalities of the inner ear, such as dilatation of the vestibular aqueduct or Mondini dysplasia. But, in Pendred syndrome (OMIM #274600), with autosomal recessive inheritance, besides congenital sensorineural deafness, goiter or thyroid dysfunctions are frequently present. The aim of this study was to determine whether mutations in SLC26A4 are a frequent cause of hereditary deafness in Brazilian patients. METHODS: Microsatellite haplotypes linked to SLC26A4 were investigated in 68 families presenting autosomal recessive non-syndromic deafness. In the probands of the 16 families presenting segregation consistent with linkage to SLC26A4, Sanger sequencing of the 20 coding exons was performed. In an additional sample of 15 individuals with suspected Pendred syndrome, because of the presence of hypothyroidism or cochleovestibular malformations, the SLC26A4 gene coding region was also sequenced. RESULTS: In two of the 16 families with indication of linkage to SLC26A4, the probands were found to be compound heterozygotes for probably pathogenic different mutations: three novel (c.1003 T > G (p. F335 V), c.1553G > A (p.W518X), c.2235 + 2 T > C (IVS19 + 2 T > C), and one already described, c.84C > A (p.S28R). Two of the 15 individuals with suspected Pendred syndrome because of hypothyreoidism or cochleovestibular malformations were monoallelic for likely pathogenic mutations: a splice mutation (IVS7 + 2 T > C) and the previously described c.1246A > C (p.T416P). Pathogenic copy number variations were excluded in the monoallelic cases and in those with normal results after Sanger sequencing. Additional mutations in the SLC26A4 gene or other definite molecular cause for deafness were not identified in the monoallelic patients, after exome sequencing. CONCLUSIONS: Biallelic pathogenic mutations in SLC26A4 explained ~ 3% of cases selected because of autosomal recessive deafness. Monoallelic mutations were present in ~ 13% of isolated cases of deafness with cochleovestibular malformations or suspected Pendred syndrome. These data reinforce the importance of mutation screening of SLC26A4 in Brazilian subjects and highlight the elevated frequency of monoallelic patients.

Polyethylene glycol fusion associated with antioxidants: A new promise in the treatment of traumatic facial paralysis.

BACKGROUND: Recent studies in invertebrates have taught us that early cell membrane regeneration is determinant for axonal recovery and survival after trauma. Many authors obtained extraordinary results in neural regeneration using polyethylene glycol fusion protocols, which also involved microsutures and antioxidants. METHODS: Sixty rats were evaluated with functional and histological protocol after facial nerve neurotmesis. Groups A and B had their stumps coapted with microsuture after 24 hours of neurotmesis and groups C and D after 72 hours. In addition to the microstructure, groups B and D used the polyethylene glycol-fusion protocol for the modulation of the Ca+2 . RESULTS: At the sixth week, the latency of group D and duration of group B was lower than groups A and C (P = .011). The axonal diameter of the groups that used polyethylene glycol-fusion was higher than those who did not use polyethylene glycol-fusion (P ≤ .001). CONCLUSION: Although not providing a functional improvement, polyethylene glycol-fusion slowed down demyelination.

Exome Sequencing Identifies a Novel Nonsense Mutation of MYO6 as the Cause of Deafness in a Brazilian Family.

We investigated 313 unrelated subjects who presented with hearing loss to identify the novel genetic causes of this condition in Brazil. Causative GJB2/GJB6 mutations were found in 12.7% of the patients. Among the familial cases (100/313), four were selected for exome sequencing. In one case, two novel heterozygous variants were found and were predicted to be pathogenic based on bioinformatics tools, that is, p.Ser906* (MYO6) and p.Arg42Cys (GJB3). We confirmed that this nonsense MYO6 mutation segregated with deafness in this family. Only the proband and her unaffected mother exhibited the GJB3 mutation, which is in the same amino acid of a known Erythrokeratodermia variabilis mutation. None of the patients exhibited this skin disease, but the proband exhibited a more severe hearing loss. Hence, the GJB3 mutation was considered to be a variant of uncertain significance. In conclusion, we described a novel nonsense MYO6 mutation that was responsible for the hearing loss in a Brazilian family. This mutation resides in the neck domain of myosin-VI after the motor domain. Thus, our data give further support for genotype-phenotype correlations, which state that when the motor domain of the protein is functioning, the hearing loss is milder and has a later onset. The three remaining families without mutations in the known genes suggest that there are still deafness genes to be revealed.

Evidence of progenitor cells in the adult human cochlea: sphere formation and identification of ABCG2.

OBJECTIVES: The aim of this study was to search for evidence of stem or progenitor cells in the adult human cochlea by testing for sphere formation capacity and the presence of the stem cell marker ABCG2. METHODS: Cochleas removed from patients undergoing vestibular schwannoma resection (n=2) and from brain-dead organ donors (n=4) were dissociated for either flow cytometry analysis for the stem cell marker ABCG2 or a sphere formation assay that is widely used to test the sphere-forming capacity of cells from mouse inner ear tissue. RESULTS: Spheres were identified after 2-5 days in vitro, and the stem cell marker ABCG2 was detected using flow cytometric analysis after cochlear dissociation. CONCLUSIONS: Evidence suggests that there may be progenitor cells in the adult human cochlea, although further studies are required.