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.

Association of ISL1 polymorphisms and eosinophilic levels among otitis media patients.

BACKGROUND: Otitis media (OM) is a middle ear inflammatory complex disorder involving genetic and environmental factors. It onsets during childhood and often recurs and perplexes in genetically susceptible patients. Previously, murine models had shown the association of ISL LIM homeobox 1 (ISL1) gene with otitis media with effusion. AIM: To investigate the association of ISL1 genetic variants with otitis media. SUBJECTS AND METHODS: A total of 285 cases and 277 controls were recruited for the study. The entire coding region of ISL1 gene was genotyped using Sanger sequencing or single-strand conformation polymorphism methods. Genotype, haplotype, in silico analysis, and linkage disequilibrium analysis were performed. RESULTS: The variants rs2303751 (c.504A>G) and rs121913540 (c.513G>A) were associated with OM, and the OR (95%CI) was 0.74 (0.57-0.95) and 0.43 (0.20-0.91), respectively. Besides, the rs2303751 AA genotype was associated with elevated eosinophil numbers in OM when compared to controls. The 5 SNP haplotype analysis of SNPs c.-492A>G, c.504A>G, c.513G>A, c.576C>T, and c.*651A>T revealed A-A-G-C-A to be a risk haplotype in females whereas the 3 SNP haplotype analysis of SNPs c.504A>G, c.513G>A, and c.567C>T suggested G-A-C as protective and A-G-C to be a risk haplotype for otitis media. CONCLUSION: Ours is the first report which shows a significant association of ISL1 variants (rs2303751 and rs121913540) with hearing-related disorder like otitis media in humans. These results implicate the possible role of ISL1 gene in the etiopathology of otitis media. The replication of the study in other ethnic populations may strengthen our findings.

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.

Otosclerosis Associated with a De Novo Mutation -832G > A in the TGFB1 Gene Promoter Causing a Decreased Expression Level.

Otosclerosis (OTSC) is defined by abnormal bone remodeling in the otic capsule of middle ear which leads to conductive hearing loss. In our previous study, we have identified a de novo heterozygous mutation -832G > A in the promoter of TGFB1 in an otosclerosis patient. In the present study, we progressively screened this mutation in a cohort of 254 cases and 262 controls. The family members of the patient positive for -832G > A variation were also screened and found inheritance of this variation only to her daughter. Interestingly, this variation is associated with a decreased level of the TGFB1 transcript in the patient compared to her parents and controls. In silico analysis of this mutation predicted the altered binding of two transcription factors v-Myb and MZF1 in the mutated promoter sequence. Further, functional analysis of this mutation using in vitro luciferase and electrophoretic mobility shift assays revealed that this variation is associated with decreased gene expression. In conclusion, this study established the fact that TGFB1 mutation -832G > A altered the TGFB1 promoter activity, which could affect the susceptibility to otosclerosis development. Further, systemic analysis of TGFB1 gene sequence and expression analysis of this gene might reveal its precise role in the pathogenesis of otosclerosis.

Genetic association and altered gene expression of osteoprotegerin in otosclerosis patients.

Otosclerosis (OTSC) is a late-onset hearing disorder characterized by increased bone turnover in the otic capsule. Disturbed osteoprotegerin expression has been found in the otosclerotic foci which may have an important role in the pathogenesis of OTSC. To identify the genetic risk factors, we sequenced the coding region and exon-intron boundaries of the OPG gene in 254 OTSC patients and 262 controls. Sequence analysis identified five known polymorphisms c.9C>G, c.30+15C>T, c.400+4C>T, c.768A>G, and c.817+8A>C. Testing of these SNPs revealed sex specific association with c.9C>G in males and c.30+15C>T in females after multiple correction. Furthermore, meta-analysis provided evidence of association of the c.9C>G polymorphism with OTSC. In secondary analysis, we investigated the mRNA expression of OPG and associated genes RANK and RANKL in otosclerotic tissues compared to controls. Expression analysis revealed significantly missing/reduced OPG expression only in otosclerotic tissues. However, the signal sequence polymorphism c.9C>G has shown no effect on OPG mRNA expression. In conclusion, our results suggest that the risk of OTSC is influenced by variations in the OPG gene along with other factors which might regulate its altered expression in otosclerotic tissues. Further research is warranted to elucidate the mechanisms underlying these observations.

Genetic association and gene expression profiles of TGFB1 and the contribution of TGFB1 to otosclerosis susceptibility.

Otosclerosis (OTSC) is a common form of acquired hearing loss resulting from disturbed bone remodeling in the otic capsule of the middle ear. Transforming growth factor-beta1 (TGFB1) produced by osteoblasts is the most abundant growth factor in human bone. Previous studies have shown the contribution of single-nucleotide polymorphisms (SNPs) in TGFB1 toward the risk of developing OTSC in some ethnic populations. The present study was aimed at investigating the genetic association and expression profiles of TGFB1 in OTSC patients. Two SNPs (c.-800G > A and c.-509C > T) in the promoter region and three SNPs (c.29T > C, c.74G > C, and c.788C > T) in the coding region were genotyped in 170 cases and 170 controls. The genetic association analysis revealed the significant association between c.-509C > T (p = 0.0067; odds ratio [OR] = 1.562; 95% confidence interval [CI], 1.140-2.139) and OTSC. The increased minor allele "T" frequency in cases (0.42) compared to controls (0.31) indicates its possible role in the etiology of the disease. The minor allele frequencies for the SNPs c.-800G > A, c.29T > C, and c.74G >C were similar among the cases (0.04, 0.47, and 0.08, respectively) and controls (0.05, 0.42, 0.07, respectively). We found that c.788C > T was monomorphic in this population. Interestingly, a four-locus haplotype (G-T-T-G) from these SNPs was found to be significantly associated with OTSC (p = 0.0077). We identified a de novo heterozygous mutation c.-832G > A in the promoter region of TGFB1 in 1 patient. In a secondary analysis, we investigated the possibility of abnormal TGFB1 expression and irregular bone growth in OTSC by expression analysis of TGFB1 mRNA in disease tissue compared to control. We found relatively increased expression of TGFB1 mRNA in the stapes tissues of cases compared to controls (p = 0.0057). In conclusion, this study identified a risk variant c.-509C > T and a risk haplotype G-T-T-G in the TGFB1 gene that contribute toward the susceptibility to OTSC.