Búsqueda de nuevos genes, mecanismos moleculares y abordajes diagnósticos en hipoacusias hereditarias poslocutivas

Tesis inédita de la Universidad Complutense de Madrid, Facultad de Ciencias Biológicas, leída el 30/03/2023Las hipoacusias neurosensoriales no sindrómicas son el trastorno sensorial más común y presentan una alta heterogeneidad genética. La identificación de nuevos genes y variantes asociados a hipoacusia avanza a un ritmo muy rápido, lo que hace que el uso de herramientas de secuenciación masiva sea esencial para realizar un diagnóstico genético. En este trabajo, hemos utilizado OTO-NGS-panel V2, un panel de secuenciación masiva diseñado por nuestro grupo que contiene 117 genes asociados a hipoacusias para estudiar el espectro mutacional de las hipoacusias no sindrómicas de herencia autosómica dominante en la población española. OTO-NGS-panel V2 fue validado para la identificación de mutaciones puntuales, pequeñas inserciones y deleciones y variaciones del número de copias. Con esta herramienta, se analizaron 108 familias con hipoacusia no sindrómica e identificamos la variante causal en 39 casos, lo que constituye una tasa diagnóstica del 36,11%. Los genes con mayor prevalencia son WFS1 (7,41 %), seguido de MYO7A (5,56 %), MYO6 (4,63 %), TECTA (2,78 %) y ACTG1 (2,78 %). Para determinar la causa genética de la sordera en los casos que permanecieron sin diagnosticar tras el análisis con OTO-NGS-panel V2, diseñamos un panel de genes candidatos, que incluye genes asociados a sordera en ratón pero sin mutaciones reportadas en humanos hasta la fecha. Se identificaron variantes candidatas en los genes XIRP2, USP31, PLS1 y KCNK5...Non-syndromic sensorineural hearing loss (SNHL) is the most common sensory disorder, and it presents a high genetic heterogeneity. New genes and variants associated with hearing loss are described at a very rapid pace, making the use of next-generation sequencing (NGS) approaches essential to undertake a genetic diagnosis.We have used OTO-NGS-panel V2, a custom-designed NGS panel containing 117 genes associated with hearing loss to study the mutational spectrum of autosomal dominant non-syndromic hearing loss in the Spanish clinical population. OTO-NGS-panel V2 was validated for the identification of single nucleotide variants (SNVs), small insertions and deletions (Indels) and copy number variations (CNVs), and 108 Spanish families with autosomal dominant non-syndromic hearing loss (ADNSHL) were analysed. We identified the causative variant in 39 cases, constituting a diagnostic rate of 36.11%. The genes with the highest prevalence are WFS1 (7.41%) followed by MYO7A (5.56%), MYO6 (4.63%), TECTA (2.78%) and ACTG1 (2.78%). To identify the genetic cause of deafness in the cases that remained undiagnosed upon analysis with OTO-NGS-panel V2, we designed a candidate gene panel, including genes known to cause deafness in mice but with no human mutations reported so far. Candidate variants were found in the genes XIRP2, USP31, PLS1 and KCNK5...Fac. de Ciencias BiológicasTRUEunpu

A Novel Truncating Mutation in HOMER2 Causes Nonsyndromic Progressive DFNA68 Hearing Loss in a Spanish Family

Nonsyndromic hereditary hearing loss is a common sensory defect in humans that is clinically and genetically highly heterogeneous. So far, 122 genes have been associated with this disorder and 50 of them have been linked to autosomal dominant (DFNA) forms like DFNA68, a rare subtype of hearing impairment caused by disruption of a stereociliary scaffolding protein (HOMER2) that is essential for normal hearing in humans and mice. In this study, we report a novel HOMER2 variant (c.832_836delCCTCA) identified in a Spanish family by using a custom NGS targeted gene panel (OTO-NGS-v2). This frameshift mutation produces a premature stop codon that may lead in the absence of NMD to a shorter variant (p.Pro278Alafs*10) that truncates HOMER2 at the CDC42 binding domain (CBD) of the coiled-coil structure, a region that is essential for protein multimerization and HOMER2-CDC42 interaction. c.832_836delCCTCA mutation is placed close to the previously identified c.840_840dup mutation found in a Chinese family that truncates the protein (p.Met281Hisfs*9) at the CBD. Functional assessment of the Chinese mutant revealed decreased protein stability, reduced ability to multimerize, and altered distribution pattern in transfected cells when compared with wild-type HOMER2. Interestingly, the Spanish and Chinese frameshift mutations might exert a similar effect at the protein level, leading to truncated mutants with the same Ct aberrant protein tail, thus suggesting that they can share a common mechanism of pathogenesis. Indeed, age-matched patients in both families display quite similar hearing loss phenotypes consisting of early-onset, moderate-to-profound progressive hearing loss. In summary, we have identified the third variant in HOMER2, which is the first one identified in the Spanish population, thus contributing to expanding the mutational spectrum of this gene in other populations, and also to clarifying the genotype–phenotype correlations of DFNA68 hearing loss

Pathological mechanisms and candidate therapeutic approaches in the hearing loss of mice carrying human MIR96 mutations

Background: Progressive hearing loss is a common problem in the human population with no effective therapeutics currently available. However, it has a strong genetic contribution, and investigating the genes and regulatory interactions underlying hearing loss offers the possibility of identifying therapeutic candidates. Mutations in regulatory genes are particularly useful for this, and an example is the microRNA miR-96, a post-transcriptional regulator which controls hair cell maturation. Mice and humans carrying mutations in miR-96 all exhibit hearing impairment, in homozygosis if not in heterozygosis, but different mutations result in different physiological, structural and transcriptional phenotypes. Methods: Here we present our characterisation of two lines of mice carrying different human mutations knocked-in to Mir96. We have carried out auditory brainstem response tests to examine their hearing with age and after noise exposure and have used confocal and scanning electron microscopy to examine the ultrastructure of the organ of Corti and hair cell synapses. Bulk RNA-seq was carried out on the organs of Corti of postnatal mice, followed by bioinformatic analyses to identify candidate targets. Results: While mice homozygous for either mutation are profoundly deaf from 2 weeks old, the heterozygous phenotypes differ markedly, with only one mutation resulting in hearing impairment in heterozygosis. Investigations of the structural phenotype showed that one mutation appears to lead to synaptic defects, while the other has a much more severe effect on the hair cell stereociliary bundles. Transcriptome analyses revealed a wide range of misregulated genes in both mutants which were notably dissimilar. We used the transcriptome analyses to investigate candidate therapeutics, and tested one, finding that it delayed the progression of hearing loss in heterozygous mice. Conclusions: Our work adds further support for the importance of the gain of novel targets in microRNA mutants and offers a proof of concept for the identification of pharmacological interventions to maintain hearing.</p

Mutations in <i>MINAR2</i> encoding membrane integral NOTCH2-associated receptor 2 cause deafness in humans and mice

Discovery of deafness genes and elucidating their functions have substantially contributed to our understanding of hearing physiology and its pathologies. Here we report on DNA variants in MINAR2 , encoding membrane integral NOTCH2-associated receptor 2, in four families underlying autosomal recessive nonsyndromic deafness. Neurologic evaluation of affected individuals at ages ranging from 4 to 80 y old does not show additional abnormalities. MINAR2 is a recently annotated gene with limited functional understanding. We detected three MINAR2 variants, c.144G > A (p.Trp48*), c.412_419delCGGTTTTG (p.Arg138Valfs*10), and c.393G > T, in 13 individuals with congenital- or prelingual-onset severe-to-profound sensorineural hearing loss (HL). The c.393G > T variant is shown to disrupt a splice donor site. We show that Minar2 is expressed in the mouse inner ear, with the protein localizing mainly in the hair cells, spiral ganglia, the spiral limbus, and the stria vascularis. Mice with loss of function of the Minar2 protein ( Minar2 tm1b/tm1b ) present with rapidly progressive sensorineural HL associated with a reduction in outer hair cell stereocilia in the shortest row and degeneration of hair cells at a later age. We conclude that MINAR2 is essential for hearing in humans and mice and its disruption leads to sensorineural HL. Progressive HL observed in mice and in some affected individuals and as well as relative preservation of hair cells provides an opportunity to interfere with HL using genetic therapies