Clin Oral Invest DOI 10.1007/s00784-014-1305-3

ORIGINAL ARTICLE

Family-based association analysis between nonsyndromic cleft lip with or without cleft palate and IRF6 polymorphism in an Iranian population Nayereh Nouri & Mehrdad Memarzadeh & Francesco Carinci & Francesca Cura & Luca Scapoli & Narges Nouri & Fariba Jafary & Maryam Sedghi & Leyli Sadri & Mansoor Salehi

Received: 28 December 2013 / Accepted: 15 August 2014 # Springer-Verlag Berlin Heidelberg 2014

Abstract Objectives Nonsyndromic cleft lip with or without cleft palate (NSCL/P) is a common birth defect which is strongly associated with genetic factors. Previous studies in several populations showed a significant correlation between IRF6 rs642961 polymorphism and NSCL/P. The aim of this study is to indicate the correlation of IRF6 rs642961 polymorphism and NSCL/P in Iranian NSCL/P families. N. Nouri : M. Sedghi : M. Salehi Medical Genetics Laboratory, Alzahra University Hospital, Isfahan University of Medical Sciences, Isfahan, Iran N. Nouri : M. Memarzadeh : N. Nouri Cleft Lip and Palate Clinic, Isfahan University of Medical Sciences, Isfahan, Iran M. Memarzadeh Department of Pediatric Surgery, Emam Hossein Hospital, Isfahan University of Medical Sciences, Isfahan, Iran F. Carinci Department of Morphology, Surgery and Experimental Medicine, University of Ferrara, Ferrara, Italy F. Cura : L. Scapoli Department of Specialist Diagnostic and Experimental Medicine, University of Bologna, Bologna, Italy F. Jafary Young Researchers and Elite Club, Falavarjan Branch, Islamic Azad University, Isfahan, Iran L. Sadri Students’ Research Center, School of Dentistry, Isfahan University of Medical Sciences, Isfahan, Iran M. Salehi (*) Department of Genetics and Molecular Biology, Medical School, Isfahan University of Medical Sciences, Isfahan, Iran e-mail: [email protected]

Material and methods In this study, we analyzed IRF6 rs642961 genotype in 352 individuals from 102 Iranian nuclear families affected by NSCL/P using iPlex assay on a Sequenom MassARRAY platform. Hardy–Weinberg equilibrium and Mendelian error checking were performed by Haploview 4.2. Allelic association analysis was conducted with family-based association tests implemented in FBAT program v2.03. Results The family-based association analysis revealed no significant association between IRF6 rs642961 genotypes and an increased NSCL/P risk. Conclusions In contrast to other Asian populations, our study indicates that the IRF6 rs642961 polymorphism cannot be a risk factor for NSCL/P in an Iranian population. Clinical relevance Genetic factors have an important role in NSCL/P, among which interferon regulatory factor 6 (IRF6) has been reported as a risk factor for NSCL/P in several populations; however, our data indicated no significant association between IRF6 polymorphism and NSCL/P in an Iranian population. Keywords Cleft lip . Cleft palate . Polymorphism . IRF6

Introduction Nonsyndromic cleft lip with or without cleft palate (NSCL/P) is the most common craniofacial abnormality characterized by an incomplete separation between nasal and oral cavities without any other associated anomaly [1–3]. Prevalence of NSCL/P that depends on geographical origin is approximately 1–2 per 1,000 live births worldwide; the highest rates of NSCL/P were reported in Asian and American populations and the lowest in African populations [3–5]. NSCL/P is a multifactorial disease associated with genetic and environmental risk factors [6–8]. Genetic factors play an important role in increased risk of NSCL/P, and a number of candidate genes have been reported

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to be associated with NSCL/P, among which interferon regulatory factor 6 (IRF6) is an important one [5, 6, 9–11]. IRF6 gene located on chromosome 1q32.3–q41 encodes a protein that plays an important role in early development. The product of this gene is considered to contribute in connective tissue formation [12, 13]. Significant association between various common polymorphisms and NSCL/P has been reported in different populations [5, 6, 9]. The rs642961 is a functional SNP in the IRF6 promoter region disrupting the transcription factor AP-2a binding site. This SNP is able to directly influence the risk of NSCL/P and is accounted for 18 % attributable risk for isolated cleft lip [14]. In this study, we investigated correlation between IRF6 rs642961 polymorphism and the risk of nonsyndromic CL/P in a subset of Iranian patients.

Materials and methods Patients affected by nonsyndromic cleft lip with or without cleft palate (NSCL/P) were recruited at the cleft lip and palate clinic of the Isfahan University of Medical Science. Pedigree analysis and medical history were taken to assess the nonsyndromic CL/ P status and to exclude known teratogenic influences. The patients and their relatives were asked about the presence of other disorders in the family and environmental exposures such as smoking, alcohol, and use of medication such as phenytoin and warfarin during the periconceptional period. Peripheral blood samples were taken from the patients and their firstdegree relatives after obtaining signed informed consent, and genomic DNAwas extracted by Qiagen DNA Mini Kit (cat No: 51304). Sample study was composed of 102 nuclear families, mostly proband–parents triads, but additional family members were also occasionally included (Table 1). Genotyping of rs642961 was performed on 352 individuals from 102 nuclear families by iPlex assay on a Sequenom MassARRAY platform. Hardy–Weinberg equilibrium and Mendelian error checking were performed by Haploview 4.2 [15]. Allelic association analysis was conducted with family-based association tests implemented in FBAT program v2.03 [16]. The FBAT statistic reduces to the more familiar transmission/ disequilibrium test (TDT) statistic when the dataset consists of Table 1 The number of individuals genotyped in each family

parent-affected child trios, and like the TDT, FBAT is immune to confounding by admixture. FBAT allows valid testing of association with different sampling structure and missing marker allele information. When parental genotypes are missing, this method computes a test statistic by conditioning on genotypes of any observed parents and offspring, adjusting for admixture. FBAT decomposes pedigrees into individual nuclear families and treats them as independent or with the –e option computes an empirical test statistic to account for correlation. The additive and dominant genetic models were applied for each allelic association test, which examines the transmission of the SNP alleles from parents to the affected offspring. In order to evaluate the power of the study, i.e., the smallest detectable effect size of IRF6 susceptibility allele, a genetic power calculation was performed using the on-line tool at web site http://pngu.mgh.harvard.edu/~purcell/gpc/[17]. We found that a genotype relative risk of 2.4, for both heterozygotes and homozygotes, ensure a power higher than 80 %, in case of a type I error rate of 0.05, disease prevalence of 0.001, and allele frequency of 0.2.

Results The genotyping step was successful with a call rate 0.97 %. No Mendelian error or deviation from Hardy–Weinberg expectations was detected (P value=1). The variant allele frequency was 0.21, a value that was similar to those observed in other populations. The family-based association analysis revealed no evidence of association between the variant allele and NSCL/P under the additive as well as dominant and recessive models (Table 2). Positive values of Z score indicated that the variant allele A was slightly over transmitted to affected individuals.

Discussion This family-based association study was performed to test whether the functional SNP rs642961 in IRF6 promoter was associated with NSCL/P in an Iranian population. SNP

Number of families

Number of members genotyped in each family

Table 2 Family-based association tests of the allele A at rs642961 using FBAT statistics under different models. The Z score, that has an approximate χ2 distribution, was used to test the null hypothesis of no association in the presence of linkage

60

3

Model

Familya

Z score

P value

34 4 2 1 1

4 5 6 7 8

Additive Dominant Recessive

56 52 17

0.31 0.44 −0.12

0.76 0.66 0.90

a

Number of families that are actually informative for the test. It mainly depends on the heterozygosity of the SNP

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rs642961 was firstly reported by Rahimov et al. to be strongly associated with risk of NSCL/P especially with cleft lip only (NSCLO) but not cleft palate only (NSCPO) [14]. Other studies on Polish, Northern Chinese, Chinese Han, and Central European population indicated a strong association between rs642961 polymorphism and NSCL/P [18–20, 6]. In the meta-analysis of 20 published case–control studies by Wang et al., a significant association between rs642961 A allele and an increased risk of NSCL/P was indicated. In addition, another polymorphism in the IRF6 gene (rs2235371) was also reported to be in association with NSCL/P risk [21]. In another study by Wan Park et al. in four populations including European American, Taiwanese, Singaporean, and Korean, a significant association between genetic variants in the IRF6 gene and an increased risk of NSCL/P was identified. Authors in this study highlighted that rs2235371 (V274I) is not the causative mutation in NSCL/P; however, it is in linkage disequilibrium with other causal mutation in IRF6. Another interesting finding of this study was that SNPs in association with NSCL/P differed between populations under study. This study highlighted the importance of the IRF6 gene polymorphisms in increased risk of NSCL/P [9]. However, there are studies which underscore the importance of some of the IRF6 gene polymorphisms in NSCL/P. Paranaíba et al. identified no significant correlation between rs2235371 and rs642961 and NSCL/P in the Brazilian population [22]. In a Swedish population, no association between rs642961 and NSCL/P was reported [23], and in a Honduran population no association between rs642961and NSCL/P was identified; however, rs2235371 was found to be associated with NSCL/P [24]. In another study by Blanton et al., no significant association between NSCL/P and rs642961 was indicated in Hispanic and nonHispanic white NSCL/P families [25]. While there are several studies worldwide investigating the role of IRF6 polymorphisms and mutations in NSCL/P, there is no study in an Iranian population. In the present study, no significant association between IRF6 rs642961 polymorphism and NSCL/P was indicated in an Iranian population subset. This evidence could indicate that the functional rs642961, supposed able to alter the IRF6 expression level, is not a susceptibility factor in an Iranian population. However, we cannot exclude that the investigated sample did not fully represent the Iranian population. Possible reasons for failure to detect association between rs642961 and NSCL/P are possible effects of different environmental factors and different ethnicity background of participants. Several linkage studies have demonstrated that the IRF6 gene is associated with NSCL/P; however, the variants responsible for this association are not fully elucidated. Variants reported to be the underlying cause of this association differs in different populations. This suggests that different polymorphisms influence the increased risk of NSCL/P in different populations. All together, it is highly recommended to perform association analysis of rs642961 and additional polymorphisms in isolated ethnic populations in Iran to verify results obtained in this study.

Acknowledgment This work was funded by grant number 189,087 from deputy for research, Isfahan University of Medical Sciences, Isfahan, Iran. The authors wish to sincerely thank the patients and their family that assisted us to carry out this research. Conflict of Interest The authors declare that they have no conflict of interest.

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