A rare haplotype of the GJD3 gene segregating in familial Meniere Disease interferes with connexin assembly

Familial Meniere Disease (FMD) is a rare polygenic disorder of the inner ear. Mutations in the connexin gene family, which encodes gap junction proteins, can also cause hearing loss, but their role in FMD is largely unknown. Here, we found an enrichment of rare missense variants in the GJD3 gene when comparing allelic frequencies in FMD (N=94) with the Spanish reference population (OR=3.9[1.92-7.91], FDR=2.36E-03). In the GJD3 sequence, we identified a rare haplotype (TGAGT) composed of two missense, two synonymous, and one downstream variants. This haplotype was found in five individuals with FMD, segregating in three unrelated families with a total of ten individuals; and in another eight Meniere Disease individuals. GJD3 encodes the gap junction protein delta 3, also known as human connexin 31.9 (CX31.9). The protein model predicted that the NP_689343.3:p.(His175Tyr) missense variant could modify the interaction between connexins and the connexon assembly, affecting the homotypic GJD3 gap junction between cells. Our studies in mice revealed that the mouse ortholog Gjd3 - encoding Gjd3 or mouse connexin 30.2 (Cx30.2) - was expressed in the organ of Corti and vestibular organs, particularly in the tectorial membrane, the base of inner and outer hair cells and the nerve fibers. The present results describe a novel association between GJD3 and familial FMD, providing evidence that FMD is related to changes in the inner ear channels; in addition, it supports a new role of tectorial membrane proteins in FMD. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement JALE has received funds to support research on genetics in Meniere's disease by The University of Sydney (K7013\_B3413 Grant), Asociacion Sindrome de Meniere Espana (ASMES), Meniere's Society, UK, and the European Union's Horizon 2020 Research and Innovation Programme, Grant Agreement Number 848261. AGM has received funds from the Andalusian Health Department (Grant PI-0266-2021) and by CuresWithinReach and the Knight Family. JALE and AGM have received funds from the Andalusian Government (CECEU 2020, grant code: DOC\_01677). IVN and SMC have received funds from PID2020-115274RB-I00 MCIN/AEI/10.13039/501100011033 and COST Action CA20113 PROTEOCURE. AEB and PRB are funded by the European Union's Horizon 2020 Research and Innovation Programme, Grant Agreement Number 848261. AMPP is supported by a predoctoral grant from the Regional Ministry of Economic Transformation, Industry, Knowledge and Universities of Junta de Andalucia (Grant number PREDOC2021/00343). The computations and data handling were enabled by resources provided by the Swedish National Infrastructure for Computing (SNIC) at SNIC/UPPMAX partially funded by the Swedish Research Council through grant agreement no. 2018-05973. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The Portal de Etica de la Investigacion Biomedica de Andalucia of Junta de Andalucia gave ethical approval for this work. The Landesamt fur Gesundheit und Soziales of Landes Berlin gave ethical approval for this work. The Direccion General de Agricultura, Ganaderia y Alimentacion of Comunidad de Madrid gave ethical approval for this work. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes Anonymized genetic raw dataset and family pedigrees used in this study are available from the corresponding author upon reasonable request.