Human Organoids for Rapid Validation of Gene Variants Linked to Cochlear Malformations

Author:

Zafeer Mohammad Faraz1,Ramzan Memoona1,Duman Duygu2,Mutlu Ahmet3,Seyhan Serhat4,Kalcioglu Tayyar3,Fitoz Suat5,DeRosa Brooke A.1,Guo Shengru1,Dykxhoorn Derek M.1,Tekin Mustafa1

Affiliation:

1. University of Miami Miller School of Medicine

2. Ankara University Faculty of Health Sciences

3. Istanbul Medeniyet University

4. Memorial Şişli Hospital

5. Ankara University School of Medicine

Abstract

Abstract

Developmental anomalies of the hearing organ, the cochlea, are diagnosed in approximately one-fourth of individuals with congenital deafness. Most patients with cochlear malformations remain etiologically undiagnosed due to insufficient knowledge about underlying genes or the inability to make conclusive interpretations of identified genetic variants. We used exome sequencing for genetic evaluation of hearing loss associated with cochlear malformations in three probands from unrelated families. We subsequently generated monoclonal induced pluripotent stem cell (iPSC) lines, bearing patient-specific knockins and knockouts using CRISPR/Cas9 to assess pathogenicity of candidate variants. We detected FGF3 (p.Arg165Gly) and GREB1L (p.Cys186Arg), variants of uncertain significance in two recognized genes for deafness, and PBXIP1(p.Trp574*) in a candidate gene. Upon differentiation of iPSCs towards inner ear organoids, we observed significant developmental aberrations in knockout lines compared to their isogenic controls. Patient-specific single nucleotide variants (SNVs) showed similar abnormalities as the knockout lines, functionally supporting their causality in the observed phenotype. Therefore, we present human inner ear organoids as a tool to rapidly validate the pathogenicity of DNA variants associated with cochlear malformations.

Publisher

Research Square Platform LLC

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