Interaction between IRF6 and TGFA Genes Contribute to the Risk of Nonsyndromic Cleft Lip/Palate

Citation: ( Interaction between IRF6 and TGFA Genes Contribute to the Risk of Nonsyndromic Cleft Lip/Palate Ariadne Letra Walid Fakhouri Renata F. Fonseca Renato Menezes Inga Kempa Joanne L. Prasad Toby G. McHenry Andrew C. Lidral Lina Moreno Jeffrey C. Murray Sandra Daack-Hirsch Mary L. Marazita Eduardo E. Castilla Baiba Lace Ieda M. Orioli Jose M. Granjeiro Brian C. Schutte Alexandre R. Vieira Dale J. Hedges, University of Miami, United States of America Previous evidence from tooth agenesis studies suggested IRF6 and TGFA interact. Since tooth agenesis is commonly found in individuals with cleft lip/palate (CL/P), we used four large cohorts to evaluate if IRF6 and TGFA interaction contributes to CL/P. Markers within and flanking IRF6 and TGFA genes were tested using Taqman or SYBR green chemistries for casecontrol analyses in 1,000 Brazilian individuals. We looked for evidence of gene-gene interaction between IRF6 and TGFA by testing if markers associated with CL/P were overtransmitted together in the case-control Brazilian dataset and in the additional family datasets. Genotypes for an additional 142 case-parent trios from South America drawn from the Latin American Collaborative Study of Congenital Malformations (ECLAMC), 154 cases from Latvia, and 8,717 individuals from several cohorts were available for replication of tests for interaction. Tgfa and Irf6 expression at critical stages during palatogenesis was analyzed in wild type and Irf6 knockout mice. Markers in and near IRF6 and TGFA were associated with CL/P in the Brazilian cohort (p,1026). IRF6 was also associated with cleft palate (CP) with impaction of permanent teeth (p,1026). Statistical evidence of interaction between IRF6 and TGFA was found in all data sets (p = 0.013 for Brazilians; p = 0.046 for ECLAMC; p = 1026 for Latvians, and p = 0.003 for the 8,717 individuals). Tgfa was not expressed in the palatal tissues of Irf6 knockout mice. IRF6 and TGFA contribute to subsets of CL/P with specific dental anomalies. Moreover, this potential IRF6-TGFA interaction may account for as much as 1% to 10% of CL/P cases. The Irf6-knockout model further supports the evidence of IRF6-TGFA interaction found in humans. - Competing Interests: The authors have declared that no competing interests exist. Introduction Oral-facial clefts are common birth defects with an incidence of 12 in 1000 live births, thus comprising almost one-half of all craniofacial anomalies. They impose adverse health, social, and economic implications for the affected individuals and their families [1]. Although the mortality and morbidity of an infant born with a cleft lip and or a cleft palate has improved greatly in the last century, it is still elevated for infants born with multiple additional anomalies. Among the consequences of being born with clefts are shorter life span and increased risk for all major causes of death when compared to individuals without clefts [2]. Cleft lip with or without cleft palate (herein called cleft lip/ palate) can be classified as nonsyndromic or syndromic based on the presence of other associated congenital defects. Approximately 2050% of all cleft cases are associated with one of more than 400 syndromes [3]. Syndromic forms usually present Mendelian inheritance patterns, which allow identification of causal genes. Nonsyndromic cleft lip/palate however, is considered a genetically complex trait with no clearly recognizable inheritance pattern [4]. Identifying the key genes responsible for the genesis of cleft lip/ palate is fundamental for elucidating the pathogenetic mechanisms and developing measures for its management and prevention. Studies have estimated that 314 genes interacting multiplicatively may be involved in the etiology of cleft lip/palate [5], and a variety of genes have been associated and suggested to play a role in the genetic susceptibility to cleft lip/palate [4]. To date, the most consistent finding for the genetic etiology of nonsyndromic cleft lip/palate has been the association of the interferon regulatory factor 6 (IRF6) gene at 1q32 [6], previously identified as etiologic for Van der Woude syndrome which includes cleft lip/palate as part of the clinical spectrum [7]. A particularly strong overtransmission of the ancestral allele V at a V274I polymorphism (rs2235371) was detected in individuals of Asian and South American ancestry from 8,003 individuals representing ten distinct populations. Attributable risk calculations suggested IRF6 could contribute to as much as 12% of all cleft cases [6]. Intriguingly, additional studies with different populations have consistently shown positive association between markers in IRF6 and cleft lip/palate [822]. The frequency of the V274I risk allele is over 97% in European and African populations making it an unlikely candidate for the etiological mutation. The association of the transforming growth factor alpha (TGFA) gene at 2p13 with cleft lip/palate has also rendered intriguing results. TGFA was the first gene associated to nonsyndromic cleft lip/palate in a case-control study [23]. Several studies followed with rather discrepant results; in turn, comparison among studies with TGFA has been somewhat difficult due to unaccounted variations in study design, markers tested and percentages of patients with positive family history [24]. Meta-analytic approaches [25,26] concluded that TGFA plays a small but significant role in cleft lip/palate with odds ratios indicating a modest effect size. Instead of an effector gene, TGFA has been regarded as a]modifier to the clefting phenotype [24]. Evidence from tooth agenesis studies suggested that IRF6 and TGFA genes may interact [27,28]. Tooth agenesis is a common congenital anomaly where one or more permanent teeth are absent and is a frequent observation in individuals with cleft lip/ palate. Therefore interaction between IRF6 and TGFA in tooth agenesis may also be relevant to cleft lip/palate. Since tooth agenesis is commonly found in individuals with cleft lip/palate, we used three large samples of cleft cases to test for interaction between IRF6 and TGFA in the etiology of the cleft phenotype. Results of Case-control Comparisons Tables 1 and 2 summarize the studied Brazilian samples and genetic markers. There were no evidences of deviation from Hardy-Weinberg equilibrium for any of the markers in cases and controls (data not shown). Table 3 summarizes the linkage disequilibrium relationships of the markers studied. Table 4 summarizes the results of the association analysis obtained for Brazilian Caucasian cases (N = 406) and controls (N = 285) for each marker studied, according to each cleft subphenotype. When comparing Brazilian cleft cases with controls, we observed an association between the intronic marker rs2902345 with cleft lip/palate (P,0.001). For IRF6, we found significant association between the V274I polymorphism (rs2235371) with complete left cleft lip/palate (P,0.001). An (...truncated)