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Otosclerosis and vitamin D receptor gene polymorphism☆

Yavuz Selim Yıldırım, MDa, b,⁎, Tayfun Apuhan, MDc,Selma Düzenli, MDd, Ali Osman Arslan, MDd

a Department of Otorhinolaryngology and Head and Neck Surgery, Bezmialem Vakif University, Faculty of Medicine, Istanbulb Department of Otorhinolaryngology and Head and Neck Surgery, Haseki Training and Research Hospital, Istanbulc Department of Ear Nose Throat Clinics, Abant Izzet Baysal University, Medical Faculty, Bolu, Turkeyd Department of Genetics, Abant Izzet Baysal University, Medical Faculty, Bolu, Turkey

A R T I C L E I N F O

☆ Conflict of interest: none; financial disclo⁎ Corresponding author. Department of Oto

Medicine, Adnan Menderes Bulvarı Vatan CaE-mail address: dryavuzselim@yahoo.com

0196-0709/$ – see front matter © 2013 Elsevhttp://dx.doi.org/10.1016/j.amjoto.2013.03.01

A B S T R A C T

Article history:Received 11 March 2013

Objective: The possible genetic relationship between otosclerosis and Vitamin D Receptor(VDR) gene polymorphism is uncertain. The aim of this study is to assess associationbetween 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 60controls to investigate the association of the VDR gene polymorphisms FokI, BsmI, ApaI, andTaq Iwith otosclerosis. The patient and control DNAwas genotyped for; VDR Bsm I (rs1544410),VDR Apa I (rs7975232), VDR Taq I (rs731236) and VDR Fok I (rs2228570) gene. Primer, simpleprobe sequences was genotyped by RT-PCR restriction fragment length polymorphism.Results: There was a statistically significant association between VDR gene and otosclerosisin polymorphism Taq I, Apa I and Bsm I. There was no significant association between VDRgene and otosclerosis in polymorphism Foq I.Conclusion: Three polymorphisms (Taq I, Apa I and Bsm I) in the VDR gene appear to beassociated to susceptibility to otosclerosis disorder with otosclerosis patients.

© 2013 Elsevier Inc. All rights reserved.

1. Introduction

Otosclerosis is a common, often hereditary disorder of thebony labyrinth of the inner ear characterized by a disorderedbone remodeling in the otic capsule, most commonly anteriorto the stapedio-vestibular joint (oval window) in the area ofthe fissula ante fenestram [1,2]. Despite many research bynumerous investigators during the last few decades, theetiopathogenesis of otosclerosis is still not fully understood.Different theories have been postulated to explain the etiologyof otosclerosis, including endocrine, hormonal, autoimmune,environmental and viral factors. However, it is certain that

sures: none; sponsor: nonrhinolaryngology and Hddesi 34093 Fatih/İstanbu(Y.S. Yıldırım).

ier Inc. All rights reserve6

genetic factors play a significant role in the disease [3,4]. Inotosclerosis, compared to other bones, the otic capsuleundergoes very little bone remodeling and abnormal resorp-tion and redeposition of bone are present. This abnormal boneremodeling causes fixation of the stapes due to invasion ofotosclerotic foci at the stapediovestibular joint [2].

The vitamin D endocrine system is involved in a widevariety of biological processes including bone metabolism,regulation of cell proliferation and differentiation and mod-ulation of the immune response [5]. The role of Vitamin D andvitamin D receptor (VDR) in skeletal metabolism is wellknown. VDR gene plays an important role in the Vitamin D

e.ead and Neck Surgery, Bezmialem Vakif University, Faculty ofl, Turkey. Tel.: +90 212 453 17 10; fax: +90 212 621 75 80.

d.

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pathway, and belongs to the steroid hormone family ofnuclear receptors which are responsible for the transcription-al regulation of a number of hormone responsive genes.Polymorphismswithin the genemay potentially influence thevitamin D expression and stability of mRNA. Recent studieshave well-characterized four VDR polymorphisms Fok1, Bsm1,Apa1 and Taq1 [5,6]. The VDR gene is located on the long armof chromosome 12 (12q12-14), consisting of at least fivepromoter regions [7].

The aim of this study is to assess association betweenotosclerosis and VDR gene polymorphisms VDR Bsm I(rs1544410), VDR Apa I (rs7975232), VDR Taq I (rs731236) andVDR Fok I (rs2228570).

Table 1 – Demographic characteristics of study andcontrol groups.

Age-Mean(Min–Max)

Sex(Female–male)

N (Case)

Study 45, 56 (29–66) 16 F, 9 M 25Control 35, 40 (27–69) 38 F, 22 M 60

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 clinicsof Haseki Training and Research Hospital, Istanbul and Abantİzzet Baysal University Bolu, Turkey and age matchedcontrols. The study was conducted with the approval of theEthics Committee of the Medical Faculty of Abant Izzet BaysalUniversity and it was conducted in accordance with theDeclaration of Helsinki.

We performed the study between June 2010 and March2012 in our two tertiary referral center. Clinical diagnosis ofstapes fixation was based on otoscopic, audiometric, tympa-nometric and surgical findings. Audiological testing wasperformed 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 rememberedmeasles virus infection in childhood and all the patientshave anti-measles vaccination. All the patients were operatedon under general anesthesia. Teflon prostheses with adiameter of 0.6–0.4 mm were used in stapedotomy, aftersurgical otosclerotic stapes fixation confirmed 25 patientsincluded in the study.

Hearing histories were taken from patients and controlsand a physical examination was performed. Those withexternal auditory canal pathology, chronic otitis media,ossicular chain discontinuity, previous ear surgery, tympano-sclerosis, sensorineural hearing loss, profound hearing lossdue to otosclerosis (over the 85 dB HL), bone and kidneydisease, and genetic disease were excluded from the study.During the study period one patient due to persistentstapedial artery and one patient due to ossicular chainabnormality were excluded from the study.

All the control group patients have normal hearing andnormal otoscopic examination. None of them have familyhistory.

2.1. Genetic analysis

All subjects gave informed consent for participation. Bloodsamples were taken from patients and controls. Serumsamples were stored at −20 °C until studied. Biochemicalparameters and genotype analyses were performed in Abantİzzet Baysal University School of Medicine in the genetic

research laboratory and biochemistry laboratory. Blood sam-ples were collected after overnight fasting in EDTA-coatedtubes for genotype. DNA was extracted from peripheral bloodsamples using high pure PCR template preparation kit (RocheDiagnostics Mannheim Germany). A high-throughput assayfor VDR locus genotyping was used for all four SNPs. For eachSNP a single hybridization-basedmethodwas established on ahigh-resolution platform, the 96-well Roche LightCycler® 480(LC480) instrument.

PCR mix contained 14.4 μl PCR grade water (H2O), 1.0 μlReagent mix, 2.0 μl FastStart DNA master Hybprobe (RocheDiagnostics Mannheim Germany), 1.6 μl MgCI2 (25 mM) and5.0 μl DNA. Themixturewas distributed to themicroplatewells,15 μl for each reaction. The plate was placed into the cycler,after centrifugation. The patient and control DNA was geno-typed for VDR Bsm I (rs1544410), VDR Apa I (rs7975232), Taq I(rs731236), VDR Fok I (rs2228570) in theVDR gene. Primer, simpleprobe sequences and RT-PCR (LightCycler 480) were used.

2.2. Statistical analysis

Unconditional logistic regression was used to estimaterelative risk (odds ratio) of otosclerosis in relation to VDRgenotypes. Heterogeneity was considered significant when anominal p value of 0.05 was found. Analyses were performedwith EpiInfo version 3.5.3.

3. Results

We identified 25 eligible patient and 60 controls to investigatethe association of the VDR polymorphisms FokI, BsmI, ApaI,and TaqI with otosclerosis. Demographic characteristics ofstudy and control groups are shown in Table 1 and meta-analysis of the relation between the Apa1, Bsm I, Fok I, Taq Ipolymorphism in the vitamin D receptor gene with otoscle-rosis is shown in Table 2.

The frequencies of Taq I genotypes determined fromotosclerosis patients were 32 % for CC, 32% for TC, and 36%for TT. We observed statistically significant increased preva-lence of the CC genotype in otosclerosis patients comparedwith normal healthy groups (p = 0.005).

The frequencies of FokI genotypes determined fromotosclerosis patients were 52% for CC, 48% for CT, and 0% forTT. We observed no significant association between VDR geneand otosclerosis patients in polymorphism Foq I comparedwith normal healthy groups (p = 0.382).

The frequencies of Apa I genotypes determined fromotosclerosis patients were 8% for CC, 44% for CA, and 48% forAA. We observed statistically significant increased prevalence

Table 2 – The characteristics of case–control studies included in ameta-analysis of the relation between the Apa1, Bsm I, FokI, Taq I polymorphism in the vitamin D receptor gene with otosclerosis.

Studiedpolymorphism

Genotypedistribution

Patients(n = 25)

Control(n = 60)

OR(odds ratio)

PMantel-Haenszel

χ2

Taq I TT 9 (%36) 29 (%48,3) 0.60<0.21<OR<1.73 0.297 1.09TC 8 (%32) 26 (%43,3) 0.62<0.20<OR<1.82 0.331 0.94CC 8 (%32) 5 (%8,3) 5,18<1.30<OR<21.51 0.005 7.63

25 60Fok I CC 13 (%52) 25 (%41,67) 1.52<0.54<OR<4.30 0.382 0.76

CT 12 (%48) 29 (%48,3) 0.99<0.35<OR<2.78 0.977 0.00TT 0 6 (%10) 0.00<0.000<OR<2.22 0.100 2.69

25 60Apa I CC 2 (%8) 13 (%21,67) 0.31<0.04<OR<1.67 0.132 2.27

CA 11 (%44) 32 (%53,3) 0.69<0.24<OR<1.94 0.432 0.61AA 12 (%48) 15 (%25) 2.77<0.94<OR<8.28 0.037 4.31

25 60Bsm I AA 8 (%32) 5 (%8,3) 5.18<1.30<OR<21.51 0.005 7.63

AG 10 (%40) 29 (%48,3) 0.71<0.25<OR<2.03 0.482 0.49GG 7 (%28) 26 (%43,3) 0.51<0.16<OR<1.55 0.186 1.75

25 60

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of the AA genotype in otosclerosis patients compared withnormal healthy groups (p = 0.037).

The frequencies of Bsm I genotypes determined fromotosclerosis patients were 32% for AA, 40% for AG, and 28%for GG. We observed statistically significant increased preva-lence of the AA genotype in otosclerosis patients comparedwith normal healthy groups (p = 0.005).

4. Discussion

Vitamin D is initially metabolized to the intermediatecompound 25-hydroxyvitamin D in the liver and thensubsequently binds to intracellular receptors, which regulategene expression. The vitaminD receptor (VDR) is an importantregulator of the vitamin D pathway, which involves theconversion of serum 25-hydroxyvitamin D into the activehormone, 1,25-dihydroxyvitamin D. The vitamin D receptor(VDR) is required for actions of vitamin D [6].

Otosclerosis is a multi-factor, often hereditary disease ofbone remodeling disorder of the human otic capsule.Although, the etiology is not fully understood, it is a complexand multifactorial disease caused by a combination ofdifferent genetic and environmental factors and their in-teractions [2]. Besides the latter, viral infection, inflammatorymechanisms, autoimmunity, as well as hormonal and manymetabolic and environmental factors have been involved inthe development of otosclerosis. But a great part of theetiopathogenesis of the disease is still unknown. Autosomaland sex-linked models, dominant and recessive and combi-nations of these have all been proposed. The epidemiologicalstudies on otosclerosis suggest an autosomal dominant modeof inheritancewith reduced penetrance of approximately 40%.Genetic linkage studies have shown the presence of six locilocated on chromosomes 15q, 7q, 6p, 16q, 3q and 6qrespectively. But no causative genes have been identifiedand we have little information of the molecular processinvolved in this disease [8].

Osteogenesis imperfecta is an autosomal dominant geneticdisease of decreased or absent type I collagen synthesis. It wasproposed that otosclerosis was related to mild osteogenesisimperfecta with genetic defects in type I collagen. A signifi-cant association was found between otosclerosis and the typeI collagen (COL1A1) gene using three different polymorphicmarkers within the gene [9,10]. McKenna et al. [11] alsosuggested a possible link between otosclerosis and osteopo-rosis. Chen et al. [12] identified that susceptibility andprotective haplotypes were significantly associated withotosclerosis in the Caucasian population. At least in someotosclerotic patients, COL1A1 gene polymorphisms wereassociated with the disease [10].

A prospective study with otosclerosis was undertaken toinvestigate the possible etiologic role of vitamin D undernutrition. Vitamin D deficiency is probably a factor in theetiology of some cases of otosclerosis since the deafnessresulting from cochlear involvement may be reversible [13].The VDR is an intracellular hormone receptor that specificallybinds the biologically active form of calcitriol or vitamin D,1,25-dihydroxyvitamin D and interacts with specific nucleo-tide sequences of target genes to produce a variety of biologiceffects [14].

VDR gene plays an important role in the Vitamin Dpathway. VDR protein is known to display polymorphicvariation and belongs to the steroid hormone family ofnuclear receptors which are responsible for the transcription-al regulation of a number of hormone responsive genes.Studies showed that VDR is expressed in normal mammarytissue, andmore than 80% of breast tumor specimens are VDRpositive [7,15]. The VDR harbors several known functionalpolymorphisms. Several of these polymorphisms in VDR genehave been commonly investigated [5]. But to our knowledge,there is no study undertaken in otosclerosis patients, whichshows the distribution of the VDR polymorphisms.

We focused on the four well-characterized polymor-phisms: Bsm I (rs 1544410), (SNPs) Fok I (rs 10735810), Apa1(rs7975232) and Taq1 (rs731236). The Fok1 restriction enzymeidentifies a polymorphic site in exon 2 at the 5’ end of the VDR

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gene. Three other polymorphisms have been identified bytheir restriction endonuclease cleavage sites (Bsm1, Apa1 andTaq1) [7]. These four SNPswere analyzed for their effect on theetiology of otosclerosis.

According to our results, BsmI, ApaI and TaqI polymor-phisms showed statistical significance for increased risk ofotosclerosis, although, our test population was small. Thesethree polymorphisms are promising SNPs of the etiology forotosclerosis. We found the BsmI AA, ApaI AA and TaqI CCpolymorphisms, which are defined as the mutant SNPs, asstatistically significant for our patient group, when comparedwith the control group.

Knowledge of the molecular and functional consequencesof the VDR polymorphisms is crucial to fully appreciate theirsignificance and understand their potential clinical implica-tions. Since there are almost no studies on otosclerosis, whereVDR polymorphisms were analyzed; we have no point ofcomparison. But we can postulate that our findings areinteresting and could be of value, when more similar studiesare conducted in the future.

In conclusion, this is one of the earliest studies to searchfor an association between VDR gene polymorphisms andotosclerosis patients. Additional data are needed to clarifywhether these SNPs are related to otosclerosis risk.

Acknowledgments

This research was supported by the Abant Izzet BaysalUniversity Scientific Research Committee.

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