Genetic variants and altered expression of SERPINF1 confer disease susceptibility in patients with otosclerosis.

Otosclerosis (OTSC) is a focal and diffuse bone disorder of the human middle ear characterized by abnormal bone growth and deposition at the stapes' footplate. This hinders the transmission of acoustic waves to the inner ear leading to subsequent conductive hearing loss. The plausible convections for the disease are genetic and environmental factors with yet an unraveled root cause. Recently, exome sequencing of European individuals with OTSC revealed rare pathogenic variants in the Serpin Peptidase Inhibitor, Clade F (SERPINF1) gene. Here, we sought to investigate the causal variants of SERPINF1 in the Indian population. The gene and protein expression was also evaluated in otosclerotic stapes to ameliorate our understanding of the potential effect of this gene in OTSC. A total of 230 OTSC patients and 230 healthy controls were genotyped by single-strand conformational polymorphism and Sanger sequencing methods. By comparing the case controls, we identified five rare variants (c.72 C > T, c.151 G > A, c.242 C > G, c.823 A > T, and c.826 T > A) only in patients. Four variants c.390 T > C (p = 0.048), c.440-39 C > T (p = 0.007), c.643 + 9 G > A (p = 0.035), and c.643 + 82 T > C (p = 0.005) were found to be significantly associated with the disease. Down-regulation of SERPINF1 transcript level in otosclerotic stapes was quantified by qRT-PCR, ddPCR and further validated by in situ hybridization. Similarly, reduced protein expression was observed by immunohistochemistry and immunofluorescence in otosclerotic stapes that corroborate with immunoblotting of patients' plasma samples. Our findings identified that SERPINF1 variants are associated with the disease. Furthermore, reduced expression of SERPINF1 in otosclerotic stapes might contribute to OTSC pathophysiology.

The risks of RELN polymorphisms and its expression in the development of otosclerosis.

Otosclerosis (OTSC) is the primary form of conductive hearing loss characterized by abnormal bone remodelling within the otic capsule of the human middle ear. A genetic association of the RELN SNP rs3914132 with OTSC has been identified in European population. Previously, we showed a trend towards association of this polymorphism with OTSC and identified a rare variant rs74503667 in a familial case. Here, we genotyped these variants in an Indian cohort composed of 254 OTSC cases and 262 controls. We detected a significant association of rs3914132 with OTSC (OR = 0.569, 95%CI = 0.386-0.838, p = 0.0041). To confirm this finding, we completed a meta-analysis which revealed a significant association of the rs3914132 polymorphism with OTSC (Z = 6.707, p<0.0001) across different ethnic populations. Linkage analysis found the evidence of linkage at RELN locus (LOD score 2.1059) in the OTSC family which has shown the transmission of rare variant rs74503667 in the affected individuals. To understand the role of RELN and its receptors in the development of OTSC, we went further to perform a functional analysis of RELN/reelin. Here we detected a reduced RELN (p = 0.0068) and VLDLR (p = 0.0348) mRNA levels in the otosclerotic stapes tissues. Furthermore, a reduced reelin protein expression by immunohistochemistry was confirmed in the otosclerotic tissues. Electrophoretic mobility shift assays for rs3914132 and rs74503667 variants revealed an altered binding of transcription factors in the mutated sequences which indicates the regulatory role of these variations in the RELN gene regulation. Subsequently, we showed by scanning electron microscopy a change in stapes bone morphology of otosclerotic patients. In conclusion, this study evidenced that the rare variation rs74503667 and the common polymorphism rs3914132 in the RELN gene and its reduced expressions that were associated with OTSC.

Evaluation of the Genetic Association and mRNA Expression of the COL1A1, BMP2, and BMP4 Genes in the Development of Otosclerosis.

Background: Otosclerosis (OTSC) is a genetically heterogeneous disorder, characterized by abnormal bone growth in the middle ear, affecting the stapes bone. Previous studies have shown that single nucleotide polymorphisms (SNPs) of the COL1A1, BMP2, and BMP4 genes are linked to susceptibility of OTSC, musculoskeletal degenerative diseases, and bone remodeling. Aims: To evaluate the genetic association and expression levels of COL1A1, BMP2, and BMP4 genes with OTSC in the Indian population. Methods: A total of 320 otosclerotic and 320 control samples were screened for four SNPs (rs1107946, rs11327935, rs2269336, and rs1800012) of the COL1A1 gene; rs3178250 of the BMP2 gene; and rs17563 of the BMP4 gene using single-strand conformation polymorphism analysis, and restriction fragment length polymorphism analyses. Genotypic, haplotypic, and linkage disequilibrium analyses were performed to assess the potential associations of these SNPs with OTSC. COL1A1, BMP2, and BMP4 mRNA expression levels were analyzed by semiquantitative RT-PCR and real-time PCR. Results: Genotypes of two SNPs, rs1800012 and rs17563, were found to be associated with OTSC (the rs1800012 GT genotype, p = 0.0022, OR = 0.481; and the rs17563 TC genotype, p = 0.0225, OR = 1.471). Haplotypic analyses revealed that the COL1A1 haplotype G-T-C-T (p = 0.021) was significantly increased among controls. Functional studies revealed an unexpected decrease in mRNA expression of COL1A1 but an increased expression of the BMP2 and BMP4 genes in otosclerotic stapes tissues. Conclusions: Our findings suggest that OTSC is a heterogeneous disorder, but that the GT genotype of the rs1800012 locus is protective and that the TC genotype at the rs17563 locus is a risk factor. In addition, our studies indicate that changes in the expression of the COL1A1, BMP2, and BMP4 genes may contribute to the genetic susceptibility of OTSC by regulating their mRNA levels.