The GJB2 gene was the first known cause of deafness in the world and Iran [7, 21]. This gene is responsible for about 16-18% of ARNSHL in Iran. However, there is a large gap in the recognition of other genes responsible . Considering the high frequency of consanguineous marriages in Iran and high prevalence of hearing loss and the heterogeneity of NSHL, many genes are involved [22, 23]. In this study, targeted genomic capturing was applied to identify an inherited homozygous variation.
The ESRRB gene consists of 11 exons and codes the estrogen-related receptor beta protein, which is associated with hearing loss in mice . ESRRB is a member of orphan nuclear receptors and is related to estrogen receptors (ER), although does not bind to estrogen. The coded protein by ESRRB gene has two important domains including ligand-binding domain (LBD) and DNA-binding domain (DBD) (Figure 3), which are recruited as transcriptional regulation of target genes of estrogen . ESRRB gene is the only known gene that acts as a transcription factor and is associated with ARNSHL, while other transcription factors are related to autosomal dominant NSHL .
ESRRB genes are responsible for the recessive DFNB35 deafness forms. The locus DFNB35 has been identified in 2003 by studying a large Pakistani family with NSHL, so far more than 12 variations have been identified in DFNB35 which more than half of identified mutations are accrued in Pakistan ethnicity (p.A110V, p.L320P, p.E340del, p.V342L, p.L347P)[24, 27] and the rest of theme belongs to Czech Republic(p.R291L) , Tunisia(p.Y305H) , Turkey (p.V342GfsX44)  and China (p.R382C)  and UAE (c.1058‑3 C>A) and our reported variation (p.G167R) Iran population. With exception of c.1156C>T  and c.1237G>A  which are reported as polymorphism, other variations are considered functional mutations (table 2), and most of them are accumulated in the exon 8 ESRRB gene. As shown in figure 3, the majority of the pathogenic variation occurred in LBD with exception of p. A110V and our reported variation in this study, p.Gly167Arg.
In this report, sequence analysis of the ESRRB gene showed c.499G>A mutation as the first variation of ESRRB gene in Iranian ethnicity with ARNSHL and as a very rare cause of ARNSHL in this population which has been submitted by our group with a number rs1555342141. c.499G>A is a missense mutation in exon 5 of the ESRRB gene that causes substitution of glycine by arginine in 167 amino acids of ESRRB coded protein. The 167th amino acid is located in the highly conserved domain at DBD and may have deleterious effects on the function of ESRRB. In addition to in silico analysis of c.499G>A variant that is introduced as a damaging variant, based on ACMG Guidelines  This variation can be categorized as pathogenic because it is a novel missense variant (PM5) located in a functional domain (PM1), in addition, it is absent in controls (PM2) and is co-segregating in the pedigree (PP1). Molecular modeling prediction revealed that the substitution of an aspartic acid by a glycine residue leads to a large side chain which may increase inappropriate interaction between residues and it seems possibly decreased stability and disrupted the 3D structure of the DBD, therefore abnormal protein in the cells might result in not function properly. Furthermore, multiple sequence alignment of human ESRRB protein across the other species showed that the downstream residues are conserved. Different clinical symptoms as a result of frame-shift and the missense mutations suggest that the molecular mechanism underlying the hearing loss possibly results from a loss of function mutation. This idea supported by Collin .et al study in which in conditional Esrrb/ mice, malfunction hearing and balance coincide with ion homeostasis and disturbed endolymph production, and it is possibly that ESRRB of human is essential for these processes as well . This protein is considered a key factor for the function and development of the inner ear by mediating the effects of estrogens, glucocorticoid hormone, and thyroid hormone, which all are essential for inner ear cells. Also, it might be crucial for the preservation of hearing with aging in human adults, in other hand an animal study has shown that ESRRB/knock-out mice cause hearing impairment; however, it is not in accordance with the suggested role of estrogen in protecting hearing in DFNB35 patients .