Association of TFAP2A gene polymorphism with susceptibility to non-syndromic cleft lip with or without palate risk in south Indian population.

The aetiology of non-syndromic cleft lip with or without cleft palate (NSCL/P) is complex involving multiple interacting genes and environmental factors. The primary objective of the present study was to investigate the role of TFAP2A gene single nucleotide polymorphisms (SNPs) in the pathogenesis of NSCL/P. In this study, 173 unrelated NSCL/P patients and 176 controls without clefts were genotyped with TFAP2A rs1675414 (Exon 1), rs3798691 (Intron 1), and rs303050 (Intron 4) variants by allele-specific amplification using the KASPar SNP genotyping system. The method of multifactor dimensionality reduction (MDR) was used to analyze gene-gene interactions. TFAP2A polymorphisms are not found to be associated with non-syndromic cleft lip with or without cleft palate (NSCL/P) at either the genotype or allele levels. No linkage disequilibrium (LD) was found between TFAP2A variants. MDR analysis did not show a significant effect of the TFAP2A gene polymorphisms on susceptibility to NSCL/P (p > 0.05). These results suggest that the analyzed variations in TFAP2A gene might not be associated with NSCL/P pathogenesis in south Indian population.

Introduction

Orofacial clefts are among the commonest birth defects with a worldwide frequency of 1 in 750 live births. About 70% of clefts are non-syndromic that occur in isolated conditions while the remaining 30% are syndromic clefts that are associated with other cognitive or structural anomalies. Based on different clinical manifestations, non-syndromic cleft lip and palate (NSCL/P) is categorized into two groups - cleft lip with or without cleft palate (CL/P) and cleft palate only (CPO) (Mossey et al., 2009). CPO is less common than CL/P. The incidence of non-syndromic clefts shows variations depending upon economic, geographical and genetic backgrounds. Asians and Native Americans exhibited the highest prevalence rates, followed by Caucasians and Africans (Murthy and Bhaskar, 2009).

Over the last decade, numerous linkage and association studies have been performed to identify the genes that predispose to NSCL/P. The development of lip and palate is known to be dependent on a spectrum of signaling molecules, transcription factors and growth factors. However, the relative contribution of individual susceptibility genes varies across different populations, thereby accentuating the need for replication of association studies in different populations. Transcription factors are DNA-binding proteins capable of activating or repressing gene regulation. The transcription factor AP-2 alpha, encoded by the TFAP2A gene, is another important functional candidate for the non-syndromic oral clefts. The gene coding for AP-2 alpha transcription factor (TFAP2A) is located on 6p24-p22.3 and composed of seven exons with the last six exons encoding the majority of the protein. AP-2 alpha protein acts as a sequence specific DNA-binding transcription factor, recognizing and binding to the specific GC-rich regions that are present in the cis-regulatory regions of several cellular genes. Thus it regulates gene expression during the embryogenesis of eye, ear, face, body wall and limbs (Ahituv et al., 2004).

The first evidence to prove that TFAP2A gene is a risk factor for cleft lip/palate is derived from the breakpoints within 375 to 930 kb of the 5′-end of the TFAP2A gene in 3 patients that showed balanced translocations [46,XX,t(6;9)(p23;q22.3)] involving 6p (Davies et al., 1995, Davies et al., 2004). Several genome-wide linkage scans have provided evidence for linkage between 6p23 region that harbor TFAP2A gene and NSCL/P in different populations (Carinci et al., 1995, Prescott et al., 2000, Moreno et al., 2004, Schultz et al., 2004). The TFAP2A has been identified as a causative gene for Branchio-Oculo-Facial Syndrome (BOFS) that commonly presents with facial clefting (Li et al., 2013). The present study aims to investigate the role of TFAP2A gene polymorphisms in the pathogenesis of NSCL/P in south Indian population.

Materials & methods

Subjects

The study included a total of 349 individuals of both sexes, consisting of 176 NSCL/P cases and 173 controls. All the subjects were recruited at Sri Ramachandra Cleft and Craniofacial Centre, Sri Ramachandra University, Chennai, India. The case groups were examined by two surgeons to assess the exact cleft phenotype. Cases with possible specific malformations and those with intellectual disability or other anomalies were excluded from the study. Subjects without family history of clefting were recruited as controls. The study was approved by Institution Ethics Committee of Sri Ramachandra University (Ref:IEC-NI/II/OCT/25/60 dated 12.01.2012) and informed consent was obtained from all the participants. Consent was collected from parent or legal guardian when the subject was a minor.

Genotyping

3 ml peripheral blood samples were collected from all the subjects. DNA was isolated from the leukocytes using a standard procedure. Three SNPs reported in a previous study (Shi et al., 2011), [rs1675414 (Exon 1), rs3798691 (Intron 1), and rs303050 (Intron 4)] were selected for analysis. Genotyping was performed using KASPar chemistry (KBioscience Ltd., Hoddesdon, UK), which is a competitive allele-specific PCR-SNP genotyping system that uses FRET quencher cassette oligos (Didenko, 2001, Cuenca et al., 2013). On the basis of the fluorescence obtained, the allele call data were viewed graphically as a scatter plot for each marker assayed using the SNPViewer (http://www.lgcgenomics.com).

Statistical analysis

Hardy-Weinberg equilibrium (HWE) was assessed for all polymorphisms in both cases and controls groups by using chi-square test. Allele frequencies were estimated by the gene counting method (Ceppellini et al., 1955). Comparison of genotype and allele frequencies among cases and the control groups were analyzed by the chi-square test. Odds ratio (OR) and 95% confidence intervals (CI) were calculated using wild type genotypes or allele as reference group. Pairwise linkage disequilibrium (LD) was computed as both of D′ and r2 for TFAP2A gene by using of the Haploview (Barrett et al., 2005) Genotype-genotype interaction was calculated by using Multifactor Dimensionality Reduction (MDR) 3.0.2 software (Hahn et al., 2003).

Results

The genotype frequencies of three TFAP2A variants were distributed according to the Hardy-Weinberg equilibrium among the controls. There were no significant differences in genotypic distribution between cases and controls of the three variants (Table 1). Odds ratio analysis showed that the variant rs3798691 was associated with decreasing risk trend of oral clefts while the other two variants, namely rs1675414 and rs303050 were associated with an increased risk of oral clefts. However, none of the models mentioned above reached statistical significance (Table 1). Genotype and allele frequency distribution of TFAP2A variants by cleft phenotype was depicted in Fig. S1. In subgroup analysis by cleft phenotype, no significant associations were found for all TFAP2A polymorphisms (Supplementary Table S1). Measures of pairwise LD presented in Fig. 1 revealed that the TFAP2A polymorphisms were in LD as evidenced by lower r2 values. Haplotype analysis was not informative.

Table 1

Results of association tests with TFAP2A gene polymorphisms in NSCL/P and control groups.

SNP	Genotype	Control n (%)	Clefts n (%)	OR (95%CI)	p value
rs1675414	GG	56 (31.8)	46 (26.6)	Reference	0.297⁎
GA	83 (47.2)	96 (55.5)	1.41 (0.86–2.29)
AA	37 (21.0)	31 (17.9)	1.02 (0.55–1.88)
GA + AA	120 (68.2)	127 (73.3)	1.29 (0.81–2.04)	0.282
G	195 (55.4)	188 (54.3)
A	157 (44.6)	158 (45.7)	1.04 (0.77–1.40)	0.77
rs3798691	GG	168 (95.5)	167 (96.5)	Reference	0.608⁎
GC	8 (4.5)	6 (3.5)	0.75 (0.26–2.22)
CC	0 (0)	0 (0)	−
GC + CC	8 (4.5)	6 (3.5)	0.75 (0.26–2.22)	0.608
G	344 (97.7)	340 (98.3)
C	8 (2.3)	6 (1.7)	0.76 (0.26–2.21)	0.611
rs303050	TT	155 (88.1)	147 (85.0)	Reference	0.194⁎
TC	19 (10.8)	26 (15.0)	1.44 (0.77–2.72)
CC	2 (1.1)	0 (0)	−
TC + CC	21 (11.9)	26 (15.0)	0.85 (0.46–1.58)	0.61
T	329 (93.5)	268 (92.5)
C	23 (6.5)	26 (7.5)	1.38 (0.77–2.49)	0.269

p value by ᵡ2 test (df = 2), p value by ᵡ2 test (df = 1).

Fig. S1

Genotype and allele frequency distribution of the TFAP2A polymorphisms with respect to cleft phenotypes.

A: genotype frequencies and B: Allele frequencies.

Fig. 1

Paired linkage disequilibrium statistics of TFAP2A polymorphisms.

Color coding represents the D′/LOD values and the values in cells are r2 multiplied by 100. Values presented at the bottom of the figure are D′ and r2, above and below diagonal, respectively.

Multi-dimensional reductionality scaling approach was employed to evaluate the epistasis and to characterize the potential interactions of TFAP2A gene polymorphisms on NSCL/P risk (Table 2). The best model predicted for 1, 2 and 3 locus interactions were not significant. Both two locus (rs1675414 and rs303050) and three locus (rs1675414, rs3798691 and rs303050) interaction models were selected 10 out of 10 times by MDR based on a cross-validation consistency (CVC) score of 100% and predictive accuracy of 52.8% (Table 2). Furthermore, we observed a lesser mutual information for the combined effect of any two polymorphisms, in contrast to rs303050 alone (I = 0.84%) or rs1675414 alone (I = 0.50%), as depicted in entropy-based interaction dendrograms (Fig. 2).

Table 2

Interaction models by MDR analysis.

Factors	CVC	TA	p value
rs1675414	10/10	0.542	0.119
rs1675414, rs303050	10/10	0.528	0.091
rs1675414, rs3798691, rs303050	10/10	0.528	0.075

CVC: Cross validation consistency; TA: Testing accuracy.

Fig. 2

Entropy graph for gene–environment interaction and NSCL/P.

The blue color indicating highest level of redundancy, and gold represents the independence and a midway point between synergy and redundancy.

Discussion

Analysis of three SNPs within the TFAP2A gene did not show any significant association with NSCL/P. No significant linkage disequilibrium was detected between markers. MDR analysis did not show a significant effect of the TFAP2A gene polymorphisms on susceptibility to NSCL/P. Similar findings were also observed in the Northern Chinese population (Shi et al., 2011). In contrast to the above mentioned results, both single marker and haplotypes were associated with NSCL/P in a family based association analysis of Italian NSCL/P triads (Martinelli et al., 2011). Earlier studies observed that loss of AP-2 alpha caused severe craniofacial, limb and skin defects (Schorle et al., 1996, Zhang et al., 1996). Further, AP-2 alpha-null chimeras exhibit midline craniofacial clefting, acrania, and a failure of sternal fusion (Nottoli et al., 1998). Furthermore, tissue specific deletion of AP-2 alpha gene revealed that palatal development is independent of neural tube closure, but neural crest-specific expression of AP-2 alpha is essential for particular aspects of craniofacial development (Brewer et al., 2004). Numerous patients with Branchio-Oculo-Facial Syndrome have mutations in the DNA binding domain of TFAP2A, suggesting importance of downstream transcriptional targets (Tekin et al., 2009, Milunsky et al., 2011). While some previous studies demonstrated the binding of TFAP2A to the IRF6 enhancer (MCS-9.7) and regulate IRF6 expression (McDade et al., 2012), other experiments suggested that the IRF6 binds to upstream of TFAP2C, but not TFAP2A (Botti et al., 2011). A recent dual-fluorescence transgenic analysis of MCS-9.7, unambiguously demonstrated the in vivo effect of rs642961 SNP on enhancer activity (Bhatia et al., 2015). Although the TFAP2A is involved in orofacial development in mice, very few studies have investigated the role of TFAP2A in the pathogenesis NSCL/P. There is a definite need for more studies to confirm the role of TFAP2A variants in the causation of NSCL/P.

The following are the supplementary data related to this article.

Table S1

Results of association test with TFAP2A gene polymorphisms in sub-phenotypic groups of NSCL/P.

Conflict of interest

There are no conflicts of interests.

Authors contribution

LVKSB, SAH and JM defined the research theme. LVKSB and GVB designed methods and experiments, carried out the laboratory experiments. LVKSB and GVB analyzed the data, interpreted the results and wrote the paper. All authors have contributed to, seen and approved the manuscript.