Abstract
Objective
The possible genetic relationship between otosclerosis and Vitamin D Receptor (VDR) gene polymorphism is uncertain. The aim of this study is to assess association between otosclerosis and VDR gene polymorphisms.
Study Design
Case–control Studies.
Setting
Tertiary referral center.
Subjects and Methods
Clinical diagnosis of stapes fixation was based on otoscopic, audiometric, tympanometric and surgical findings. We identified 25 eligible patient and 60 controls to investigate the association of the VDR gene polymorphisms FokI , BsmI , ApaI , and Taq I with otosclerosis. The patient and control DNA was genotyped for; VDR Bsm I (rs1544410), VDR Apa I (rs7975232), VDR Taq I (rs731236) and VDR Fok I (rs2228570) gene. Primer, simple probe sequences was genotyped by RT-PCR restriction fragment length polymorphism.
Results
There was a statistically significant association between VDR gene and otosclerosis in polymorphism Taq I , Apa I and Bsm I . There was no significant association between VDR gene and otosclerosis in polymorphism Foq I .
Conclusion
Three polymorphisms ( Taq I , Apa I and Bsm I ) in the VDR gene appear to be associated to susceptibility to otosclerosis disorder with otosclerosis patients.
1
Introduction
Otosclerosis is a common, often hereditary disorder of the bony labyrinth of the inner ear characterized by a disordered bone remodeling in the otic capsule, most commonly anterior to the stapedio-vestibular joint (oval window) in the area of the fissula ante fenestram . Despite many research by numerous investigators during the last few decades, the etiopathogenesis of otosclerosis is still not fully understood. Different theories have been postulated to explain the etiology of otosclerosis, including endocrine, hormonal, autoimmune, environmental and viral factors. However, it is certain that genetic factors play a significant role in the disease . In otosclerosis, compared to other bones, the otic capsule undergoes very little bone remodeling and abnormal resorption and redeposition of bone are present. This abnormal bone remodeling causes fixation of the stapes due to invasion of otosclerotic foci at the stapediovestibular joint .
The vitamin D endocrine system is involved in a wide variety of biological processes including bone metabolism, regulation of cell proliferation and differentiation and modulation of the immune response . The role of Vitamin D and vitamin D receptor (VDR) in skeletal metabolism is well known. VDR gene plays an important role in the Vitamin D pathway, and belongs to the steroid hormone family of nuclear receptors which are responsible for the transcriptional regulation of a number of hormone responsive genes. Polymorphisms within the gene may potentially influence the vitamin D expression and stability of mRNA. Recent studies have well-characterized four VDR polymorphisms Fok1 , Bsm1 , Apa1 and Taq1 . The VDR gene is located on the long arm of chromosome 12 (12q12-14), consisting of at least five promoter regions .
The aim of this study is to assess association between otosclerosis and VDR gene polymorphisms VDR Bsm I (rs1544410), VDR Apa I (rs7975232), VDR Taq I (rs731236) and VDR Fok I (rs2228570).
2
Material and methods
This case–control study was conducted on patients 18–70 years of age who had applied to ear nose and throat clinics of Haseki Training and Research Hospital, Istanbul and Abant İzzet Baysal University Bolu, Turkey and age matched controls. The study was conducted with the approval of the Ethics Committee of the Medical Faculty of Abant Izzet Baysal University and it was conducted in accordance with the Declaration of Helsinki.
We performed the study between June 2010 and March 2012 in our two tertiary referral center. Clinical diagnosis of stapes fixation was based on otoscopic, audiometric, tympanometric and surgical findings. Audiological testing was performed on all subjects (air conduction at 250, 500, 1000, 2000, 4000, and 8000 Hz; bone conduction at 500, 1000, 2000, and 4000 Hz). Air–bone gap at 1000 Hz was at least 30 dB HL, lower than 85 dB HL. None of the patients remembered measles virus infection in childhood and all the patients have anti-measles vaccination. All the patients were operated on under general anesthesia. Teflon prostheses with a diameter of 0.6–0.4 mm were used in stapedotomy, after surgical otosclerotic stapes fixation confirmed 25 patients included in the study.
Hearing histories were taken from patients and controls and a physical examination was performed. Those with external auditory canal pathology, chronic otitis media, ossicular chain discontinuity, previous ear surgery, tympanosclerosis, sensorineural hearing loss, profound hearing loss due to otosclerosis (over the 85 dB HL), bone and kidney disease, and genetic disease were excluded from the study. During the study period one patient due to persistent stapedial artery and one patient due to ossicular chain abnormality were excluded from the study.
All the control group patients have normal hearing and normal otoscopic examination. None of them have family history.
2.1
Genetic analysis
All subjects gave informed consent for participation. Blood samples were taken from patients and controls. Serum samples were stored at − 20 °C until studied. Biochemical parameters and genotype analyses were performed in Abant İzzet Baysal University School of Medicine in the genetic research laboratory and biochemistry laboratory. Blood samples were collected after overnight fasting in EDTA-coated tubes for genotype. DNA was extracted from peripheral blood samples using high pure PCR template preparation kit (Roche Diagnostics Mannheim Germany). A high-throughput assay for VDR locus genotyping was used for all four SNPs. For each SNP a single hybridization-based method was established on a high-resolution platform, the 96-well Roche LightCycler® 480 (LC480) instrument.
PCR mix contained 14.4 μl PCR grade water (H2O), 1.0 μl Reagent mix, 2.0 μl FastStart DNA master Hybprobe (Roche Diagnostics Mannheim Germany), 1.6 μl MgCI2 (25 mM) and 5.0 μl DNA. The mixture was distributed to the microplate wells, 15 μl for each reaction. The plate was placed into the cycler, after centrifugation. The patient and control DNA was genotyped for VDR Bsm I (rs1544410), VDR Apa I (rs7975232), Taq I (rs731236), VDR Fok I (rs2228570) in the VDR gene. Primer, simple probe sequences and RT-PCR (LightCycler 480) were used.
2.2
Statistical analysis
Unconditional logistic regression was used to estimate relative risk (odds ratio) of otosclerosis in relation to VDR genotypes. Heterogeneity was considered significant when a nominal p value of 0.05 was found. Analyses were performed with EpiInfo version 3.5.3.
2
Material and methods
This case–control study was conducted on patients 18–70 years of age who had applied to ear nose and throat clinics of Haseki Training and Research Hospital, Istanbul and Abant İzzet Baysal University Bolu, Turkey and age matched controls. The study was conducted with the approval of the Ethics Committee of the Medical Faculty of Abant Izzet Baysal University and it was conducted in accordance with the Declaration of Helsinki.
We performed the study between June 2010 and March 2012 in our two tertiary referral center. Clinical diagnosis of stapes fixation was based on otoscopic, audiometric, tympanometric and surgical findings. Audiological testing was performed on all subjects (air conduction at 250, 500, 1000, 2000, 4000, and 8000 Hz; bone conduction at 500, 1000, 2000, and 4000 Hz). Air–bone gap at 1000 Hz was at least 30 dB HL, lower than 85 dB HL. None of the patients remembered measles virus infection in childhood and all the patients have anti-measles vaccination. All the patients were operated on under general anesthesia. Teflon prostheses with a diameter of 0.6–0.4 mm were used in stapedotomy, after surgical otosclerotic stapes fixation confirmed 25 patients included in the study.
Hearing histories were taken from patients and controls and a physical examination was performed. Those with external auditory canal pathology, chronic otitis media, ossicular chain discontinuity, previous ear surgery, tympanosclerosis, sensorineural hearing loss, profound hearing loss due to otosclerosis (over the 85 dB HL), bone and kidney disease, and genetic disease were excluded from the study. During the study period one patient due to persistent stapedial artery and one patient due to ossicular chain abnormality were excluded from the study.
All the control group patients have normal hearing and normal otoscopic examination. None of them have family history.
2.1
Genetic analysis
All subjects gave informed consent for participation. Blood samples were taken from patients and controls. Serum samples were stored at − 20 °C until studied. Biochemical parameters and genotype analyses were performed in Abant İzzet Baysal University School of Medicine in the genetic research laboratory and biochemistry laboratory. Blood samples were collected after overnight fasting in EDTA-coated tubes for genotype. DNA was extracted from peripheral blood samples using high pure PCR template preparation kit (Roche Diagnostics Mannheim Germany). A high-throughput assay for VDR locus genotyping was used for all four SNPs. For each SNP a single hybridization-based method was established on a high-resolution platform, the 96-well Roche LightCycler® 480 (LC480) instrument.
PCR mix contained 14.4 μl PCR grade water (H2O), 1.0 μl Reagent mix, 2.0 μl FastStart DNA master Hybprobe (Roche Diagnostics Mannheim Germany), 1.6 μl MgCI2 (25 mM) and 5.0 μl DNA. The mixture was distributed to the microplate wells, 15 μl for each reaction. The plate was placed into the cycler, after centrifugation. The patient and control DNA was genotyped for VDR Bsm I (rs1544410), VDR Apa I (rs7975232), Taq I (rs731236), VDR Fok I (rs2228570) in the VDR gene. Primer, simple probe sequences and RT-PCR (LightCycler 480) were used.
2.2
Statistical analysis
Unconditional logistic regression was used to estimate relative risk (odds ratio) of otosclerosis in relation to VDR genotypes. Heterogeneity was considered significant when a nominal p value of 0.05 was found. Analyses were performed with EpiInfo version 3.5.3.
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