Genetic Heterogeneity in Hereditary Hearing Loss: Potential Role of Kinociliary Protein TOGARAM2

Basic Medical Sciences Life Sciences Medicine Cell Biology Biochemistry Genetics
Genetic Heterogeneity Hereditary Hearing Loss TOGARAM2 Candidate Gene Bioinformatics Analysis Cochlear Hair Cells Dual Hearing Loss

Genetic Diversity in Hereditary Hearing Loss: The Potential Role of KINOCILIARY Protein TOGARAM2

Background

Hearing Loss (HL) is a feature with multiple causes, and currently, research has identified pathogenic variants in over 200 genes associated with HL. Despite extensive research, the causative factor remains unidentified in more than one-third of families, posing significant challenges for genetic analysis, particularly in multi-gene families. Multiple different gene variants may lead to different pathogenic causes in different individuals, even among members of the same family. Therefore, it is crucial to search for more potential deafness-causing variants.

Paper Source

This research was conducted by scholars including Memoona Ramzan, Mohammad Faraz Zafeer, Clemer Abad, Shengru Guo, and others, from institutions including the University of Miami in the USA and the University of Göttingen in Germany. The paper was published on February 19, 2024, in the “European Journal of Human Genetics”, with the detailed DOI: 10.1038/s41431-024-01562-6.

Research Objective

The aim of this study was to identify other variants hidden in known or new candidate deafness genes through exome or whole genome sequencing of four multi-gene families. Specifically, to investigate the role of TOGARAM2 variants as a potential cause of autosomal recessive non-syndromic HL by demonstrating their presence in families, expression in the cochlea, and protein localization in cochlear hair cells to clarify the pathogenic cause of these variants.

Research Methods

Ethics Statement and Study Participants

Four families became part of an international collaborative cohort study aimed at identifying genes associated with hearing loss. The study was approved by ethics committees of multiple institutions, and all participants provided written informed consent. For minor participants, consent was provided by their parents. Hearing tests for adults were conducted in soundproof rooms, while tests for minors followed standard operating procedures.

Genetic Analysis

The study first screened all affected members of each family for biallelic variants in GJB2 (MIM 121011). Exome Sequencing (ES) was performed on probands from each family, and Genome Sequencing (GS) was conducted on some affected individuals. Genomic data was aligned with the human GRCh37/hg19 genome assembly, and variant calling was performed using the GATK software package. Single nucleotide variants, insertions/deletions, and Copy Number Variants (CNVs) in known deafness genes were analyzed using in-house software. Some unresolved family members were further analyzed through GS.

Generation of TOGARAM2 Variants in HEK293 Cells

To generate the specific TOGARAM2 variant c.1543C>T (p.Gln515Ter), specific gRNA and repair donors were purchased from IDT and edited in HEK293 cells. Cells were electroporated using the 4D-Nucleofector system, and successfully generated TOGARAM2 variant cells were confirmed and verified by Sanger sequencing.

Quantitative Reverse Transcription PCR (qRT-PCR)

RNA was extracted and reverse transcribed to cDNA from three monoclonal homozygous normal control and three monoclonal TOGARAM2 (c.1543C>T) knock-in cells. mRNA expression analysis was performed using SYBR Green dye, with HPRT1 as an internal control.

Main Results

All members of the four families exhibited congenital or prelingual persistent bilateral severe to profound HL. In family 1596, three affected siblings were found to be homozygous for the c.1331+2 T>C variant in the MARVELD2 gene, while the remaining members without this variant were identified with a novel c.569 C>T (p.Thr190Met) variant in the TECTA gene through whole genome sequencing.

In family 2503, the c.1334 T>G (p.Leu445Trp) variant in the SLC26A4 gene could not explain the HL symptoms in all affected members. Further exome sequencing suggested that c.89G>T (p.Ser30Ter) in the SOX10 gene might also be a pathogenic cause.

Additionally, in family 2450, the c.4441 T>C (p.Ser1481Pro) variant in the MYO15A gene was found in one affected sibling. Whole genome sequencing of the sibling without this variant identified the c.1543 C>T (p.Gln515Ter) variant in the TOGARAM2 gene, which was predicted to potentially lead to nonsense-mediated mRNA decay (NMD).

Research Conclusions

This study demonstrates that individual analysis of each affected member in multi-gene families is an effective approach to identify pathogenic variants in known and new candidate genes. Notably, the discovery of TOGARAM2 gene variants and their localization and function in cochlear hair cells may play an important role in the pathogenesis of deafness, highlighting the potential of this gene in future research and clinical applications.

Research Highlights

The highlights of this research include: 1. Identification of multiple deafness gene variants associated with HL, advancing the understanding of hereditary hearing loss. 2. Demonstration of the potential of the TOGARAM2 gene as a candidate gene for deafness. 3. Provision of new evidence supporting the localization of TOGARAM2 in cochlear hair cells.

Significance

This research not only provides new insights in scientific research, aiding in the exploration of hereditary deafness mechanisms, but also offers valuable information for clinical genetic diagnosis and treatment. These findings are expected to promote the development of personalized medicine and improve the clinical management and counseling for hereditary hearing loss.