Characterization of a novel <i>GRHL2</i> mutation reveals molecular mechanisms underlying autosomal dominant hearing loss (DFNA28): insights from structural and functional studies

The GRHL2 gene, encoding the Grainyhead-like 2 transcription factor, is essential for various biological processes. While GRHL2 has a complex role in cancer biology, its genetic variants have been also implicated in different forms of hearing loss (HL), including autosomal dominant non-syndromic hearing loss (DFNA28). Here, we report a novel c.1061C>T, p.(Ala354Val) mutation within the DNA binding domain (DBD) of GRHL2 that was identified in a three-generation HL family using a targeted multi-gene panel covering 237 HL-related genes. Unlike the previously reported DFNA28-causing variants that result in protein truncation, the impact of the p.(Ala354Val) missense change cannot be attributed to GRHL2 transcript level or composition, but to an alteration in protein function. Molecular dynamics simulations revealed destabilization of the p.(Ala354Val) mutant GRHL2 dimer interface and an altered DNA binding dynamics, leading to chaotic interaction patterns despite increased binding affinity to DNA. Functional assays demonstrated that the p.(Ala354Val) mutation and other DFNA28-related mutations in the DBD lead to loss of GRHL2 transcriptional transactivation activity, while the p.(Arg537Profs*11) mutation in the dimerization domain results in a gain-of-function effect. The findings indicate that both GRHL2 haploinsufficiency and gain-of-function contribute to HL and underscore the complex regulatory role of GRHL2 in maintaining proper function of the auditory system. Our study emphasizes the need to consider structural and functional aspects of gene variants to better understand their pathogenic potential. As GRHL2 is involved in a multitude of cellular processes, the data gathered here can be also applicable to other conditions.