Next Generation Sequencing and Animal Models Reveal SLC9A3R1 as a New Gene Involved in Human Age-Related Hearing Loss

Age-related hearing loss (ARHL) is the most common sensory impairment in the elderly affecting millions of people worldwide. To shed light on the genetics of ARHL, a large cohort of 464 Italian patients has been deeply characterized at clinical and molecular level. In particular, 46 candidate genes, selected on the basis of genome-wide association studies (GWAS), animal models and literature updates, were analyzed by targeted re-sequencing. After filtering and prioritization steps, SLC9A3R1 has been identified as a strong candidate and then validated by “in vitro” and “in vivo” studies. Briefly, a rare (MAF: 2.886e-5) missense variant c.539G > A, p.(R180Q) was detected in two unrelated male patients affected by ARHL characterized by a severe to profound high-frequency hearing loss. The variant, predicted as damaging, was not present in healthy matched controls. Protein modeling confirmed the pathogenic effect of p.(R180Q) variant on protein’s structure leading to a change in the total number of hydrogen bonds. In situ hybridization showed slc9a3r1 expression in zebrafish inner ear. A zebrafish knock-in model, generated by CRISPR-Cas9 technology, revealed a reduced auditory response at all frequencies in slc9a3r1R180Q/R180Q mutants compared to slc9a3r1+/+ and slc9a3r1+/R180Q animals. Moreover, a significant reduction (5.8%) in the total volume of the saccular otolith (which is responsible for sound detection) was observed in slc9a3r1R180Q/R180Q compared to slc9a3r1+/+ (P = 0.0014), while the utricular otolith, necessary for balance, was not affected in agreement with the human phenotype. Overall, these data strongly support the role of SLC9A3R1 gene in the pathogenesis of ARHL opening new perspectives in terms of diagnosis, prevention and treatment

Missense variants in ANO4 cause sporadic encephalopathic or familial epilepsy with evidence for a dominant-negative effect

Anoctamins are a family of Ca$^{2+}$-activated proteins that may act as ion channels and/or phospholipid scramblases with limited understanding of function and disease association. Here, we identified five de novo and two inherited missense variants in ANO4 (alias TMEM16D) as a cause of fever-sensitive developmental and epileptic or epileptic encephalopathy (DEE/EE) and generalized epilepsy with febrile seizures plus (GEFS+) or temporal lobe epilepsy. In silico modeling of the ANO4 structure predicted that all identified variants lead to destabilization of the ANO4 structure. Four variants are localized close to the Ca$^{2+}$ binding sites of ANO4, suggesting impaired protein function. Variant mapping to the protein topology suggests a preliminary genotype-phenotype correlation. Moreover, the observation of a heterozygous ANO4 deletion in a healthy individual suggests a dysfunctional protein as disease mechanism rather than haploinsufficiency. To test this hypothesis, we examined mutant ANO4 functional properties in a heterologous expression system by patchclamp recordings, immunocytochemistry, and surface expression of annexin A5 as a measure of phosphatidylserine scramblase activity. All ANO4 variants showed severe loss of ion channel function and DEE/EE associated variants presented mild loss of surface expression due to impaired plasma membrane trafficking. Increased levels of Ca$^{2+}$-independent annexin A5 at the cell surface suggested an increased apoptosis rate in DEE-mutant expressing cells, but no changes in Ca$^{2+}$-dependent scramblase activity were observed. Co-transfection with ANO4 wild-type suggested a dominant-negative effect. In summary, we expand the genetic base for both encephalopathic sporadic and inherited fever-sensitive epilepsies and link germline variants in ANO4 to a hereditary disease

Identificazione e caratterizzazione funzionale di nuovi geni candidati e varianti associate a Sordit\ue0 Ereditaria e Presbiacusia

La perdita dell'udito \ue8 la pi\uf9 comune patologia sensoriale che colpisce oltre mezzo milione di persone. In questo studio abbiamo analizzato due forme di sordit\ue0 (monogenica e multifattoriale): l\u2019ipoacusia ereditaria non sindromica e la presbiacusia. La sordit\ue0 ereditaria non sindromica \ue8 una patologia neurosensoriale con elevata eterogeneit\ue0 genetica e oltre 115 geni gi\ue0 associati. Attraverso il sequenziamento dell'intero esoma abbiamo studiato quattro famiglie affette da sordit\ue0 ereditaria non sindromica (sei pazienti italiani, due pazienti olandesi ed una paziente francese), nelle quali abbiamo identificato delle varianti missenso predette patogenetiche a carico del gene USP48. USP48 codifica per un enzima con funzione di de-ubiquitinasi ed \ue8 conservato in diverse specie animali. Abbiamo dimostrato la patogenicit\ue0 delle varianti missenso identificate mediante modelli 3D delle proteine e saggi funzionali in-vitro. Gli esperimenti di immunoistologia hanno dimostrato che USP48 \ue8 espresso in specifiche strutture dell'orecchio interno umano in via di sviluppo. Successivamente, abbiamo ingegnerizzato dei modelli knock-down di zebrafish per il gene ortologo usp48 i quali hanno manifestato un ritardo nello sviluppo dei moto-neuroni primari, ridotto sviluppo e disorganizzazione dei neuroni statoacustici, essenziali per la funzione uditiva, una riduzione della velocit\ue0 di locomozione ed una capacit\ue0 natatoria alterata, nello specifico di nuoto in circolo, indicativo di disfunzione vestibolare e deficit uditivo.I test per l\u2019analisi della capacit\ue0 uditiva hanno rivelato una riduzione significativa della risposta uditiva degli zebrafish ingegnerizzati, alle lunghezze d'onda di 600 Hz e 800 Hz. In conclusione, abbiamo identificato un nuovo gene, USP48 associato a sordit\ue0 ereditaria autosomica dominante non sindromica mediante un approccio multi-step che associa il sequenziamento dell'esoma, l\u2019ingegnerizzazione di un modello animale, l\u2019immunoistologia di tessuti umani e saggi molecolari. Per quanto riguarda la presbiacusia, questa \ue8 considerata il disturbo sensoriale pi\uf9 comune nella popolazione anziana. Nonostante un'incidenza di oltre il 30% negli individui di et\ue0 superiore ai 65 anni, finora sono stati identificati solo pochi geni potenzialmente correlati allo sviluppo di tale malattia. In questo studio, \ue8 stato analizzato un set di 46 geni candidati in una coorte di 464 pazienti italiani affetti da presbiacusia. L\u2019analisi ha consentito l'identificazione di una serie di varianti potenzialmente patogenetiche ultra-rare nei geni DCLK1, SLC28A3, CEP104 e PCDH20. L\u2019effetto di queste varianti \ue8 stato valutato attraverso studi funzionali in-silico e in-vitro, mentre l'espressione dei geni nelle strutture deputate alla funzione uditiva \ue8 stata valutata attraverso l\u2019analisi dei geni ortologhi in topi e zebrafish. Questi risultati forniscono nuove importanti informazioni sulle basi genetiche e molecolari della perdita dell\u2019udito che \ue8 un disturbo molto comune, complesso ed eterogeneo.Hearing loss is the most common sensory disorder affecting over half a million of people. In this study we will consider two forms of deafness, Non-Syndromic Hereditary Hearing Loss and Age-Related Hearing Loss. Non-Syndromic Hereditary Hearing Loss (NSHHL) is a sensorineural disorder with high genetic heterogeneity and over 115 genes already associated. Through whole exome sequencing and data aggregation we identified an Italian family with six affected individuals and two unrelated Dutch patients displaying NSHHL and carrying predicted-to-be deleterious missense variants in USP48. We uncovered a ninth French patient presenting unilateral cochlear nerve aplasia and presenting a de-novo splice variant in the same gene. USP48 encodes for a de-ubiquitinating enzyme under evolutionary constraint. Pathogenicity of the missense variants is supported by 3D protein modeling and in-vitro functional assays. Immunohistology experiments showed that USP48 is expressed in specific structures of the developing human inner ear. Engineered zebrafish knocked-down models for the USP48 orthologue presented with a delayed in development of primary motoneurons, less developed and less organized statoacoustic neurons innervating the ears, decreased swimming velocity and circling swimming behavior indicative of vestibular dysfunction and hearing impairment. Acoustic startle response assays revealed a significant decrease of auditory response of usp48 knocked-down zebrafish at 600Hz and 800Hz wavelengths. In conclusion, we describe a novel autosomal dominant NSHHL gene through a multipronged approach combining exome sequencing, animal modeling, immunohistology and molecular assays. Age-related hearing loss (ARHL) or prebycusis is the most common sensory disorder in aging individuals. In spite of an incidence of more than 30% in individuals over 65 years, only few susceptibility genes have been identified so far. The analysis of a set of 46 ARHL candidate genes in a cohort of 464 Italian ARHL patients allowed the identification of a series of ultra-rare likely pathogenic variants in DCLK1, SLC28A3, CEP104, and PCDH20 genes. The potentially causative role of these variants has been tested through functional in-silico and in-vitro studies. While, the expression of these genes in the structures responsible for the auditory function has been evaluated through the study of the Mouse or Zebrafish orthologs. These results provide novel insights in the genetic and molecular bases of this complex and heterogeneous common disorder