Vol 1, No 1 (2013)  ISSN 2167‐8677 (online)  DOI 10.5195/d3000.2013.9     http://dentistry3000.pitt.edu This work is licensed under a Creative Commons Attribution 3.0 United States License.  This site is published by the University Library System, University of Pittsburgh as part of its D‐Scribe Digital Publishing Program and is cosponsored  by the University of Pittsburgh Press.  Lack of association between IRF6 polymorphisms and nonsyndromic oral clefts in South Indian population Venkatesh Babu Gurramkonda1, Jyotsna Murthy2, Altaf Hussain Syed2, and Bhaskar VKS Lakkakula1   1 Department of Biomedical Sciences, Sri Ramachandra University, Chennai, India.  2 Department of Plastic Surgery, Sri Ramachandra University, Chennai, India.  Abstract Objec ve:  This present  study  is  aimed  to  inves gate  the  associa on between  interferon  regulatory factor 6 (IRF6), single nucleo de polymorphisms (SNPs), and nonsyndromic cle   lip  without  without  cle   palate  (NSCLP)  in  the  South  Indian  popula on.   Subject and Methods: For this study, 190 unrelated NSCLP pa ents and 189 controls with‐ out cle s were genotyped with rs2235371 (V2741) and rs642961 SNPs using PCR‐RFLP. The  associa ons between NSCLP groups and  IRF6 gene polymorphisms, as well as haplotypes,  were analyzed using chi‐squared test and 95% confidence interval (95%CI) of the odds ra ‐ os were calculated with the control groups as reference.    Results: For controls,  the minor allele  frequencies of both variants, V2741 and  rs642961,  were 7.1%  and 21.1%,  respec vely. Genotype data  for both  variants  in  control  and  cle   groups follow the Hardy Weinberg Equilibrium. Between cases with NSCLP and controls, the  two SNPs  showed no differences  in  frequencies of  the genotypes or alleles. The pairwise  linkage  disequilibrium  (LD)  values  (D’=1  and  r2=0.027)  between V2741  and  rs642961  re‐ vealed that these two SNPs are not  in strong LD. Haplotype G‐T showed a significantly re‐ duced  risk  for  oral  cle s  (p<0.001)  and  haplotype  A‐T  increased  the  risk  for  oral  cle s  (p=0.043). Gene‐gene  interac on showed that the higher risk group contains more GG‐CC  combina on of cases that the controls, but this model was not significantly associated with  cle  status (p=0.136)  Conclusion: In conclusion, while IRF6 is strongly associated in other popula ons, this study  demonstrated that variants  in IRF6 may play a role  in NSCLP  in a South Indian popula on,  but other genes are expected to play a role in this popula on as well.   Cita on: Gurramkonda VB, Murthy J, Syed AH, and  Lakkakula BVKS (2013) Evidence of associa on between  IRF6 polymorphisms and nonsyndromic oral cle s in  South Indian popula on. Den stry 3000 1:a001  doi:10.5195/d3000.2013.9  Received: March 25, 2013  Accepted: June 20, 2013  Published: August 1, 2013  Copyright: ©2013 Gurramkonda et al. This is an open‐ access ar cle licensed under a Crea ve Commons  A ribu on 3.0 United States License.  Email: lvksbhaskar@gmail.com Introduc on Cleft lip with or without cleft palate is an extremely complex orofacial birth defect and is found to be more common in Asian and Asian‐American populations and less common in Africans and African‐Americans. Roughly 70% of CLP cases are nonsyndrom‐ ic, occurring as an isolated condition, while the remaining 30% of CLP cases are present in association with syndromes [1]. Converg‐ ing lines of evidence suggest that the non‐ syndromic cleft lip with or without cleft palate (NSCLP) involves interplay of both genetic and environmental factors. NSCLP most often occurs as an isolated de‐ fect in families with no history of clefts. NSCLP gene identification is difficult be‐ cause of varying levels of penetrance, sex differences, and environmental overlays that increase etiological heterogeneity [2]. Despite having a substantial genet‐ ic component, only a fraction of all predis‐ posing genes have been convincingly con‐ firmed as playing role in NSCLP. Many genes associated with syndromic cases of CLP have been identified to contribute to the incidence of NSCLP [3]. This approach led to the identification of several genes that con‐ tribute to the isolated clefting [4‐9]. Van Der Woude syndrome (VWS; OMIM 119300) is the most common autosomal dominant clefting syndrome and is distinguished by the presence of highly characteristic pitting of lower lip mucosa and CLP [10]. The VWS locus was initially mapped to hu‐ man chromosome 1q32–q41 region that harbors interferon regulatory factor 6 (IRF6) gene [11]. Numerous mutations in the IRF6 were reported to cause VWS [11‐ 14]. The IRF6 gene encodes a transcription fac‐ tor characterized by a highly conserved DNA‐binding domain in addition to a less well‐conserved protein interaction domain [15]. Several polymorphisms in the IRF6 gene have been studied to check their asso‐ ciation with cleft lip and palate, but the re‐ sults are inconclusive. Genome‐wide and candidate gene studies and a subse‐ quent meta‐analysis have identified IRF6 as a plausible gene contributing to cleft lip and palate in different ethnicities [16‐18]. The present study is aimed to investigate the association between IRF6 single nucleotide polymorphisms and NSCLP in a South Indi‐ an population. We chose two markers:   Lack of associa on between IRF6 polymorphisms and nonsyndromic oral cle s in South Indian popula on  Vol 1, No 1 (2013)    DOI 10.5195/d3000.2013.9  http://dentistry3000.pitt.edu 2 rs2235371 (V274I), a non‐synonymous SNP, and rs642961, located in the newly identified IRF6 enhancer region. In addition to this, HapMap data on these polymor‐ phisms in GIH samples (Gujarati ‐ a West Indian population) showed that these SNPs are polymorphic and located in two differ‐ ent linkage disequilibrium (LD) blocks. Since the present study population is near‐ est to GIH, it is likely that these two poly‐ morphisms are the best fit for the study undertaken. Materials and Methods Subjects: This study included a total of 379 individu‐ als of South Indian origin. One hundred ninety unrelated NSCLP patients who were admitted for palatoplasty and lip repair were ascertained at Sri Ramachandra cleft and craniofacial centre, Sri Ramachandra University, Chennai, India. To determine their individual phenotype status, all of the cases were examined by two clinicians. Oral clefts with other congenital malfor‐ mations or major developmental disorders were excluded from the study. All of the cases are isolated, nonsyndromic oral clefts and were classified into two groups: cleft lip with or without cleft palate (CLP) and cleft palate only (CPO). All patients with oral clefts were screened for history, consan‐ guinity, affected members in family and relatives, gestational history, drug intake, smoking, alcohol consumption, etc. We re‐ cruited 189 age‐ and sex‐matched normal children without family history of birth de‐ fects and considered this as the control group. Power and sample size calculation software (version 2.1.31) was used to calcu‐ late the sample sizes in the study. We used an uncorrected chi‐squared statistic to evaluate this null hypothesis. Based on power analysis, a study with 190 case pa‐ tients and 190 controls was large enough to detect a significant odds ratio (OR) of 0.5 with a power of 85.8% and an alpha of 5% and a minor allele frequency difference of 0.15 between cases and controls. The study was approved by the Institutional Ethics Committee of Sri Ramachandra University, Chennai, India. As many of the subjects are under 15 years of age, consent was request‐ ed from their parents. Three milliliters of blood sample was collected from all the participants after obtaining the informed consent. Genotyping: Genomic DNA was extracted from the sam‐ ples by phenol chloroform extraction and ethanol precipitation protocol [19]. The V274I and rs642961 SNPs of the IRF6 gene were amplified by polymerase chain‐ reaction (PCR) with primers published elsewhere [20]. Genotyping for the two SNPs was carried out by restriction diges‐ tion of the PCR products with Mbol and BstNI restriction enzymes, respectively. For the V274I polymorphism, Mbol digests the G allele in five fragments [322, 177, 80, 35, and 33 base pairs (bp)], whereas the A allele adds another restriction site, allowing the 322 bp fragment to be digested into two smaller pieces of 235 and 87 bp. Digestion of the rs642961 PCR products with BstNI results in three fragments (213, 33, and 30 bp) for the C allele and two fragments (246 and 30 bp) for the T allele [20]. Statistical analysis: Allele frequencies were estimated based on the gene count method. The genotypic fre‐ quencies for V274I and rs642961 were evaluated for Hardy‐Weinberg equilibrium by using a Monte Carlo permutation test implemented in the HWSIM program [21]. All frequencies were in agreement with Hardy‐Weinberg equilibrium. The associa‐ tions between NSCLP groups and IRF6 gene polymorphisms, as well as haplotypes, were analyzed using chi‐squared test, and 95% confidence interval (95% CI) for the odds ratios were calculated with the control group as reference. Linkage disequilibrium values of D′ and r2 were estimated using HaploView 3.12 [22]. Haplotypes were con‐ structed using ARLEQUIN program [23]. Multifactor Dimensionality Reduction (MDR) 2.0.beta.8.4 software was used to detect the gene‐gene interactions[24]. Results Genotyping by RFLP and electrophoresis on the DNA samples of all cleft and control individuals was performed. Hardy‐ Weinberg expectations were fulfilled in controls for both V274I (p=0.964) and rs642961 (p=0.260) SNPs. For V274I, the distribution of mutant allele and genotypes was not significantly different between NSCLP groups and controls (Table 1). Mu‐ tant allele (Ile) was less in controls (7.1%) than the cleft groups (CLP 9.9% and CPO 11.7%) and the difference is not statistically significant for CLP and CPO with ORs of 1.41 (CI, 0.80‐2.50; p=0.208) and 1.73 (CI, 0.60‐ 4.33; p=0.219), respectively (Table 2). Comparison of individual genotype fre‐ quencies between NSCLP groups and con‐ trols did not reveal association between the V274I genotype and type of cleft (Table 1). Although, in the present study the mutant genotypes (GA+AA) increased the risk in both NSCLP groups, the increase in risk is not statistically significant (Table 1). Simi‐ larly, the enhancer polymorphism (rs642961) also failed to demonstrate sig‐ nificant differences in genotype frequencies between controls and NSCLP groups (Table 1). The pairwise LD values (D′=1 and r2=0.027) between V274I and rs642961 also revealed that these two SNPs are not in strong LD. The haplotypes, constructed by using two polymorphic SNPs, are provided in Table 3. The G‐T haplotype was the sec‐ ond major haplotype in both cases (10.0%) and controls (20.0%) and showed a signifi‐ cantly reduced risk for oral clefts (p<0.001). Whereas, the rare haplotype A‐T, formed of two minor alleles of V274I and rs642961 polymorphisms, increased the risk for oral clefts (p=0.043). Using the MDR analysis, the best MDR models for the studied SNP SNP. ID  Group  GG (%)  GA (%)  AA (%)  p value  OR (95% CI) GG vs. GA  OR (95% CI)  GG vs. AA  OR (95% CI) GG vs. (GA+AA)  V274I  Control  164(86.32)  25(13.16)  1(0.53) CLP  129(81.24)  29(8.24)  1(0.63) 0.419 1.47(0.82‐2.64) 1.27(0.07‐20.52)  1.47(0.82‐2.60) CPO  23(76.67)  7(23.33)  0(0.00) 0.318 1.99(0.77‐5.13) ‐  1.73(0.60‐4.33) Total  152(80.42)  36(19.5)  1(0.53) 0.295 1.55(0.86‐2.81) 1.08(0.01‐85.2)  1.4(0.86‐3.75)   Group  CC (%)  CT (%)  TT (%)  p value  OR (95% CI) CC vs. CT  OR (95% CI)  CC vs. TT  OR (95% CI) CC vs. (CT+TT)  rs642961  Control  121(63.68)  58(30.53)  11(5.79) CLP  90(56.60)  55(34.59)  14(8.81) 0.323 1.27(0.80‐2.01) 1.7(0.74‐3.94)  1.34(0.87‐2.06) CPO  22(73.33)  8(26.57)  0(0.00) 0.326 0.76(0.31‐1.80) ‐  0.64(0.26‐1.50) Total  112(59.26)  63(34.59)  14(8.81) 0.633 1.17(0.74‐1.87) 1.38(0.56‐3.41)  1.21(0.78‐1.86) Table 1: Results of association tests with IRF6 gene polymorphisms in case and control groups.   Lack of associa on between IRF6 polymorphisms and nonsyndromic oral cle s in South Indian popula on  Vol 1, No 1 (2013)    DOI 10.5195/d3000.2013.9  http://dentistry3000.pitt.edu 3 combination is shown in Figure 1. The two‐ SNP model containing the IRF6 V274I and rs642961 markers had a testing accuracy (TA) of 0.541 and cross‐validation con‐ sistency (CVC) of 10/10. However, this model was not significantly associated with cleft status (p=0.136); the higher risk group contains more GG‐CC combination of cases than the controls (Figure 1). Discussion Interferon regulatory factor 6 (IRF6) is a member of the IRF family of transcription factors that share a highly conserved helix– turn–helix DNA‐binding domain and a less conserved protein‐binding domain. The function of IRF6 is still unknown because it is not linked to the regulatory pathways or functions associated with other IRF family members. However, amino acid sequence alignment analysis demonstrated that the IRF6 is exhibiting 89% similarity with IRF5, which plays a major role in interferon acti‐ vation and tumor suppression [25]. Although a role for IRF6 during embryonic development has been identified, its func‐ tion and regulation remain unknown [26, 27].However, recent studies show that IRF6 is regulated primarily through the enhancer where rs642961 resides by p63 and AP2‐ alpha [28]. Under normal conditions, the vertical pala‐ tal shelves elevate above the tongue, grow horizontally towards each other, and come in contact at the medial edge epithelium (MEE) region along the facial midline. Sub‐ sequently, the thin medial edge epithelial lining is eliminated and the surrounding mesenchyme migrates inward and fuses the palatal cleft. During this process, IRF6 ex‐ pression in the MEE rapidly increases, lead‐ ing to dramatic morphological and cell spec‐ ification changes, allowing palatal fusion [12]. A recent study demonstrated that IRF6 knockout mice failed to express TGFA in palatal tissues [29]. Furthermore, IRF6 ex‐ pression in MME is mediated by TGFβ sig‐ naling [30]. The functional polymorphism rs2235371(820G>A) replaces a valine with an isoleucine at amino acid position 274 (V274I) of the SMIR‐binding domain of IRF6. Another variant, rs642961 (G>A), is located 10 kilobases upstream of the tran‐ scription start site of IRF6. This disrupts the binding site of the transcription factor AP‐ 2α, which plays role in craniofacial devel‐ opment [31]. Analysis of two important IRF6 gene poly‐ morphisms in 190 NSCLP patients and 189 controls of South Indian origin has not sup‐ ported the association at neither genotype nor allele level with NSCLP. Haplotype anal‐ ysis, however, provided indication that IRF6 contributes to NSCLP in the studied popula‐ tion. Over‐transmission of 274V allele in CLP subjects has been identified as a risk factor in Asians and South Americans, but it is not as strong in European populations [16]. A replication study that was conducted using four IRF6 SNPs that have high hetero zygosity showed evidence for altered transmission of three of the four SNPs in Italians with CLP [32]. This positive associa‐ tion between NSCLP and IRF6 was con‐ firmed in American populations [33], and Belgian populations [34]. Comparison of CLP family members and controls revealed that the GG genotype increased the risk of CLP in Thai populations [35]. The V274I polymorphism was significantly associated with NSCLP in different populations, such as South America [36], Chile [37], West China [38], Honduras [39], Norway [40] and Span‐ ish Honduras [39]. Conversely, positive association was not reported in a German population where the frequency of 274V allele is 99.4% [41]. Although TDT haplo‐ type analysis showed significant association between NSCLP and IRF6 haplotypes, V274I is not associated with NSCLP in Chileans and Chinese, where the V274 allele frequen‐ cy is 74% and 75%, respectively [37, 42]. In an Indian population, the V274I alone con‐ tributed to minor risk, but the risk is in‐ creased when the V274 allele is present in homozygous condition in combination with MTHFR 677CT [43]. Based on the published sources, the V274I polymorphism showed wide variations in the minor allele frequencies (274I allele); Africans 0%, Europeans 0% to 10% [16], Hispanic and non‐Hispanic populations 7% and 22%, respectively [44]. However, East Asians and Southeast Asian populations reported the highest frequencies: 34% and 42%, respectively [16]. HapMap data also showed wide variations in the V274I minor allele frequency in world populations with highest frequency in CHD (42.1 %), CHB (41.1 %), JPT (40.6 %) and MEX (16.7 %) populations, whereas lowest frequency was observed in YRI (0.7 %), LWK (0.5 %), ASW (1.8 %), TSI (0.5 %) and CEU (3.2 %) popu‐ lations. Gujarati Indian population (GIH; 8.6 %) exhibited fairly lesser frequencies than the East Asian populations. This high fre‐ quency of the 274V allele in Indian and Eu‐ ropean populations yielded a nonsignificant trend of positive association with cleft lip and palate [16]. The rs642961 SNP showed slight variations in European populations, ranging from 24% to 25% [31], and in Hispanic and non‐       V274I        Group  G (%)  A (%)  p value  OR (95% CI) Control  353(92.8)  27(7.10)  Reference CLP  287(90.25)  31(9.74)  0.208  1.41(0.80‐2.50) CPO  53(88.3)  7(11.66)  0.219  1.73(0.60‐4.33) Total  340(89.94)  38(10.05)  0.17  1.46(0.85‐2.52)       rs642961        C (%)  T (%)  p value  OR (95% CI) Control  300(78.94)  80(21.05)  Reference CLP  235(73.89)  83(30.81)  0.116  1.32(0.92‐1.91) CPO  52(86.66)  8(0.13)  0.164  1.58(0.24‐1.32) Total  287(75.92)  91(25.07)  0.319  1.190(0.83‐1.70) Haplotype Control (%) Oral clefts (%)  OR(95%CI) p‐value G‐C 277 (72.9) 296 (78.3)  Reference G‐T 76 (20.0) 38 (10.0)  0.47(0.31 ‐ 0.71) <0.001 A‐C 23 (6.1) 31 (8.3)  1.26(0.72 ‐2.22) 0.419 A‐T 4 (1.1) 13 (3.3)  3.04(0.98 ‐ 9.44) 0.043 Table 2: Results of allelic association tests  for  IRF6 gene polymorphisms  in case  and control groups.  Figure 1: Graphical representation of inter‐ action analysis between V274I and rs642961 in NSCLP by MDR. Value within each cell is combined genotypes and color‐coding represents degree of risk. Dark grey is high‐risk, light grey is low risk, empty cell is not a possible combination. Table 3: Associa on between IRF6 haplotypes and NSCLP.    Lack of associa on between IRF6 polymorphisms and nonsyndromic oral cle s in South Indian popula on  Vol 1, No 1 (2013)    DOI 10.5195/d3000.2013.9  http://dentistry3000.pitt.edu 4 Hispanic populations 22% and 25%, respec‐ tively [44]. However, in Han Chinese popu‐ lations, it was reported as low as 15% [45]. The IRF6 enhancer polymorphism (rs642961) exhibited a dose‐dependent effect of A allele with cleft lip alone, but not cleft palate alone [31, 46]. A highly signifi‐ cant association between rs642961 and NSCLP was observed in Central Europe [47, 48], Poland [49], and China populations [42, 50, 51]. In contrast to this, negative associa‐ tion was found in Brazilian [20, 52], Spanish Honduras [39], Swedish, and Finnish NSCLP families [53]. In conclusion, while IRF6 is strongly associ‐ ated in other populations, this study demonstrated that variants in IRF6 may play a role in NSCLP in a south Indian popu‐ lation, but it is expected that other genes may play a role in this population as well. Conflict of interest: There are no conflicts of interests. Acknowledgements: The authors acknowledge funding from the Indian Coun‐ cil of Medical Research (ICMR), Government of India (Project Ref. No. 56/15/2007‐BMS). References 1. Current  concepts  in  genetics  of  nonsyndromic  clefts; Murthy  J, Bhaskar L;  Indian  J Plast Surg.  2009;42(1):68‐81.  epub  date:  2009/11/03.  PIMD:19881024.  2. Face  facts:  genes,  environment,  and  clefts;  Murray  JC;  Am  J  Hum  Genet.  1995;57(2):227‐ 32.epub date:1995/08/01. PIMD:7668246.  3. Genetics  of  cleft  lip  and  palate:  syndromic  genes  contribute  to  the  incidence  of  non‐ syndromic  clefts;  Stanier  P,  Moore  GE;  Hum  Mol  Genet.  2004;13  Spec  No  1:R73‐81.epub  date:2004/01/15.PIMD:14722155.  4. Splitting p63; van Bokhoven H, Brunner HG; Am  J  Hum  Genet.  2002;71(1):1‐13.epub  date:2002/05/31.PIMD:12037717.  5. Craniofacial  expression  of  human  and murine  TBX22 correlates with the cleft palate and anky‐ loglossia  phenotype  observed  in  CPX  patients;  Braybrook C, Lisgo S, Doudney K, Henderson D,  Marcano AC, Strachan T, et al.; Hum Mol Genet.  2002;11(22):2793‐804.epub  date:2002/10/11.PIMD:12374769.  6. TBX22 mutations are a  frequent  cause of  cleft  palate; Marcano AC, Doudney K, Braybrook C,  Squires  R,  Pa�on MA,  Lees MM,  et  al.;  J Med  Genet.  2004;41(1):68‐74.epub  date:2004/01/20.PIMD:14729838.  7. Mutations in FOXC2 (MFH‐1), a forkhead family  transcription factor, are responsible for the he‐ reditary  lymphedema‐distichiasis  syndrome;  Fang J, Dagenais SL, Erickson RP, Arlt MF, Glynn  MW,  Gorski  JL,  et  al.;  Am  J  Hum  Genet.  2000;67(6):1382‐8.epub  date:2000/11/15.PIMD:11078474.  8. FOXC2  truncating  mutation  in  distichiasis,  lymphedema, and cleft palate; Bahuau M, Hou‐ dayer  C,  Tredano  M,  Soupre  V,  Couderc  R,  Vazquez MP; Clin Genet. 2002;62(6):470‐3.epub  date:2002/12/18.PIMD:12485195.  9. A  novel  loss‐of‐function  mutation  in  TTF‐2  is  associated  with  congenital  hypothyroidism,  thyroid  agenesis  and  cleft palate; Castanet M,  Park SM, Smith A, Bost M, Leger J, Lyonnet S, et  al.; Hum Mol Genet. 2002;11  (17):2051‐9.epub  date:2002/08/08.PIMD:12165566.  10. Fistula  labii  inferioris congenita and  its associa‐ tion with cleft lip and palate; Van Der Woude A;  Am J Hum Genet. 1954;6(2):244‐56.epub   11. A preliminary gene map for the Van der Woude  syndrome critical region derived from 900 kb of  genomic  sequence  at  1q32‐q41;  Schutte  BC,  Bjork  BC,  Coppage  KB, Malik MI,  Gregory  SG,  Scott  DJ,  et  al.;  Genome  Res.  2000;10(1):81‐ 94.epub date:2000/01/25.PIMD:10645953.  12. Mutations  in  IRF6  cause  Van  der Woude  and  popliteal  pterygium  syndromes;  Kondo  S,  Schutte BC, Richardson RJ, Bjork BC, Knight AS,  Watanabe  Y,  et  al.; Nat Genet.  002;32(2):285‐ 9.epub date:2002/09/10.PIMD:12219090.  13. A combined targeted mutation analysis of  IRF6  gene would be useful  in  the  first  screening of  oral  facial  clefts; Wu‐Chou YH,  Lo  LJ, Chen KT,  Chang  CS,  Chen  YR;  BMC  Med  Genet.  2013;14:37.  epub  date:2013/03/21.  PIMD:23510002.  14. Comparative analysis of IRF6 variants in families  with  Van  der Woude  syndrome  and  popliteal  pterygium syndrome using public whole‐exome  databases; Leslie EJ, Standley J, Compton J, Bale  S,  Schutte  BC,  Murray  JC;  Genet  Med.  2012.epub date:2012/11/17.PIMD:23154523.  15. IRF family of transcription factors as regulators  of host defense; Taniguchi T, Ogasawara K, Ta‐ kaoka  A,  Tanaka  N;  Annu  Rev  Immunol.  001;19:623‐55.  epub  date:2001/03/13.  PIMD:11244049.  16. Interferon  regulatory  factor 6  (IRF6) gene vari‐ ants and  the  risk of  isolated cleft  lip or palate;  Zucchero  TM,  Cooper  ME,  Maher  BS,  Daack‐ Hirsch  S,  Nepomuceno  B,  Ribeiro  L,  et  al.;  N  Engl  J  Med.  2004;351(8):769‐80.  epub  date:2004/08/20. PIMD:15317890.  17. Breakthroughs in the genetics of orofacial cleft‐ ing; Mangold E, Ludwig KU, Nothen MM; Trends  Mol  Med.  2011;17(12):725‐33.epub  date:2011/09/03.PIMD:21885341.  18. Genome‐wide meta‐analyses  of  nonsyndromic  cleft lip with or without cleft palate identify six  new  risk  loci; Ludwig KU, Mangold E, Herms S,  Nowak S, Reutter H, Paul A, et al.; Nat Genet.  2012;44(9):968‐71.epub  date:2012/08/07.PIMD:22863734.  19. Molecular Cloning: A Laboratory Manual., Cold  Spring  Harbor  Laboratory  Press,  Cold  Spring  Harbor, NY.; Sambrook J, Fritsch EF, Maniatis T.  1989. epub   20. Lack  of  association  between  IRF6  polymor‐ phisms  (rs2235371  and  rs642961)  and  non‐ syndromic  cleft  lip and/or palate  in a Brazilian  population; Paranaiba LM, Bufalino A, Martelli‐ Junior H, de Barros  LM, Graner  E,  Coletta RD;  Oral  Dis.  2010;16(2):193‐7.epub  date:2009/09/29. PIMD:19780991.  21. Population  genetics  of  a  functional  variant  of  the  dopamine  betahydroxylase  gene  (DBH);  Cubells  JF,  Kobayashi  K,  Nagatsu  T,  Kidd  KK,  Kidd  JR,  Calafell  F,  et  al.;  Am  J  Med  Genet.  1997;74(4):374‐9.epub  date:1997/07/25.  PIMD:9259372.  22. Haploview: analysis and visualization of LD and  haplotype maps; Barrett JC, Fry B, Maller J, Daly  MJ;  Bioinformatics.  2005;21(2):263‐5.epub  date:2004/08/07. PIMD:15297300.  23. Arlequin suite ver 3.5: a new series of programs  to perform population genetics analyses under  Linux and Windows; Excoffier L, Lischer HE; Mol  Ecol  Resour.  2010;10(3):564‐7.epub   date:2011/05/14. PIMD:21565059.  24. Multifactor  dimensionality  reduction  software  for detecting gene‐gene and gene‐environment  interactions; Hahn  LW, Ritchie MD, Moore  JH;  Bioinformatics.  2003;19(3):376‐82.epub  date:2003/02/14. PIMD:12584123.  25. Virus‐induced heterodimer  formation between  IRF‐5 and IRF‐7 modulates assembly of the IFNA  enhanceosome in vivo and transcriptional activ‐ ity  of  IFNA  genes;  Barnes  BJ,  Field  AE,  Pitha‐ Rowe  PM;  J  Biol  Chem.  2003;278  (19):16630‐ 41.epub date:2003/02/26. PIMD:12600985.26.   26. Abnormal  skin,  limb  and  craniofacial morpho‐ genesis  in mice deficient  for  interferon  regula‐ tory  factor  6  (Irf6);  Ingraham  CR,  Kinoshita  A,  Kondo  S, Yang B,  Sajan  S, Trout KJ, et al.; Nat  Genet.  2006;38(11):1335‐40.  epub  date:2006/10/17. PIMD:17041601.  27. Irf6  is  a  key  determinant  of  the  keratinocyte  proliferation‐differentiation  switch;  Richardson  RJ, Dixon  J, Malhotra S, Hardman MJ, Knowles  L,  Boot‐Handford  RP,  et  al.;  Nat  Genet.  2006;38(11):1329‐34.  epub  date:2006/10/17.  PIMD:17041603.  28. Genome‐wide  analysis  of  p63  binding  sites  identifies  AP‐2  factors  as  coregulators  of  epi‐ dermal  differentiation; McDade  SS,  Henry  AE,  Pivato GP, Kozarewa  I, Mitsopoulos C, Fenwick  K,  et  al.; Nucleic Acids  Res.  2012;40(15):7190‐ 206. epub date:2012/05/11. PIMD:22573176.  29. Interaction  between  IRF6  and  TGFA  genes  contribute  to  the  risk  of  nonsyndromic  cleft  lip/palate;  Letra  A,  Fakhouri  W,  Fonseca  RF,  Menezes  R,  Kempa  I,  Prasad  JL,  et  al.;  PLoS  ONE. 2012;7(9):e45441. Epub date:2012/10/03.  PIMD:23029012.  30. Smad4‐Irf6  genetic  interaction  and  TGF  beta‐ mediated  IRF6 signaling cascade are crucial  for  palatal fusion in mice; Iwata J, Suzuki A, Pelikan  RC, Ho TV, Sanchez‐Lara PA, Urata M, et al.; De‐ velopment.  2013;140(6):1220‐30.  epub  date:2013/02/15. PIMD:23406900.  31. Disruption  of  an  AP‐2alpha  binding  site  in  an  IRF6  enhancer  is  associated  with  cleft  lip;  Rahimov  F, Marazita ML,  Visel  A,  Cooper ME,  Hitchler  MJ,  Rubini  M,  et  al.;  Nat  Genet.  2008;40(11):1341‐7.epub  date:2008/10/07.  PIMD:18836445.   32. Strong  evidence  of  linkage  disequilibrium  be‐ tween  polymorphisms  at  the  IRF6  locus  and  nonsyndromic  cleft  lip  with  or  without  cleft  palate, in an Italian population; Scapoli L, Palm‐ ieri A, Martinelli M, Pezzetti F, Carinci P, Togn‐ on M, et al.; Am J Hum Genet. 2005;76(1):180‐ 3.epub date:2004/11/24. PIMD:15558496.    Lack of associa on between IRF6 polymorphisms and nonsyndromic oral cle s in South Indian popula on  Vol 1, No 1 (2013)    DOI 10.5195/d3000.2013.9  http://dentistry3000.pitt.edu 5 33. Variation  in  IRF6  contributes  to  nonsyndromic  cleft lip and palate; Blanton SH, Cortez A, Stal S,  Mulliken  JB,  Finnell  RH,  Hecht  JT;  Am  J Med  Genet  A.  2005;137A(3):259‐62.epub  date:2005/08/13. PIMD:16096995.  34. Interferon  regulatory  factor‐6:  a  gene  predis‐ posing to isolated cleft lip with or without cleft  palate  in  the  Belgian  population; Ghassibe M,  Bayet B, Revencu N, Verellen‐Dumoulin C, Gille‐ rot  Y,  Vanwijck  R,  et  al.;  Eur  J  Hum  Genet.  2005;13(11):1239‐42.epub  date:2005/09/01.  PIMD:16132054.  35. Significant  association  between  IRF6  820G‐>A  and  non‐syndromic  cleft  lip  with  or  without  cleft  palate  in  the  Thai  population;  Srichomthong  C,  Siriwan  P,  Shotelersuk  V;  J  Med  Genet.  2005;42(7):e46.  Epub  date:2005/07/05. PIMD:15994871.  36. 844ins68  in  the  cystathionine  beta‐synthase  gene  in  Israel  and  review of  its distribution  in  the world; Zoossmann‐Diskin A, Gazit E, Peleg L,  Shohat  M,  Turner  D;  Anthropol  Anz.  2004;62(2):147‐55.  Epub  date:2004/07/02.  PIMD:15228193.  37. Linkage  disequilibrium  between  IRF6  variants  and nonsyndromic cleft  lip/palate  in  the Chile‐ an population; Suazo J, Santos JL, Jara L, Blanco  R;  Am  J  Med  Genet  A.  2008;146A(20):2706‐ 8.epub date: 2008/09/18. PIMD:18798331.  38. Association between  IRF6  SNPs and oral  clefts  in West China; Huang Y, Wu J, Ma J, Beaty TH,  Sull  JW,  Zhu  L,  et  al.;  J  Dent  Res.  2009;88(8):715‐8.epub  date:2009/09/08.  PIMD:19734457.  39. Association of common variants, not rare muta‐ tions,  in  IRF6  with  nonsyndromic  clefts  in  a  Honduran  population;  Larrabee  YC,  Birkeland  AC,  Kent  DT,  Flores  C,  Su  GH,  Lee  JH,  et  al.;  Laryngoscope.  2011;121(8):1756‐9.  epub  date:2011/07/28. PIMD:21792966.  40. Genetic  variants  in  IRF6  and  the  risk  of  facial  clefts: single‐marker and haplotype‐based anal‐ yses  in  a  population‐based  case‐control  study  offacial clefts in Norway; Jugessur A, Rahimov F,  Lie RT, Wilcox AJ, Gjessing HK, Nilsen RM, et al.;  Genet  Epidemiol.  2008;32(5):413‐24.  epub  date:2008/02/19. PIMD:18278815.  41. The  IRF6  p.274V  polymorphism  is  not  a  risk  factor  for  isolated  cleft  lip;  Hering  R,  Grund‐ mann K; Genet Med. 2005;7(3):209; author  re‐ ply  ‐10.  Epub  date:2005/03/19.  PIMD:15775759.  42. Association  Between  Interferon  Regulatory  Factor  6  Gene  Polymorphisms  and  Nonsyn‐ dromic Cleft Lip With or Without Cleft Palate in  a Chinese Population; Zhou Q, Li M, Zhu W, Guo  J, Wang Y, Li Y, et al.; Cleft Palate Craniofac  J.  2013. epub date:2013/03/21. PIMD:23509905.  43. MTHFR 677TT alone and  IRF6 820GG  together  with MTHFR  677CT,  but  not MTHFR  A1298C,  are  risks  for  nonsyndromic  cleft  lip  with  or  without cleft palate in an Indian population; Ali  A,  Singh  SK,  Raman  R;  Genet  Test  Mol  Bi‐ omarkers.  2009;13(3):355‐60.epub  date:2009/05/08. PIMD:19419265.  44. Ethnic Heterogeneity of IRF6 AP‐2a Binding Site  Promoter SNP Association With Nonsyndromic  Cleft Lip and Palate; Blanton SH, Burt A, Garcia  E, Mulliken JB, Stal S, Hecht JT; Cleft Palate Cra‐ niofac  J.  2010;47(6):574‐7.  epub  date:2010/11/03. PIMD:21039277.  45. Low erythrocyte  folate status and polymorphic  variation  in folate‐related genes are associated  with risk of neural tube defect pregnancy; Rel‐ ton  CL, Wilding  CS,  Laffling AJ,  Jonas  PA,  Bur‐ gess  T,  Binks  K,  et  al.;  Mol  Genet  Metab.  2004;81(4):273‐81.epub  date:2004/04/03.  PIMD:15059614.  46. Genome  scan,  fine‐mapping,  and  candidate  gene analysis of nonsyndromic cleft  lip with or  without cleft palate  reveals phenotype‐specific  differences  in  linkage  and  association  results;  Marazita  ML,  Lidral  AC,  Murray  JC,  Field  LL,  Maher  BS,  Goldstein McHenry  T,  et  al.;  Hum  Hered.  2009;68(3):151‐70.epub  date:2009/06/13. PIMD:19521098.  47. Polymorphisms  in  the  folate‐metabolizing  genes MTR, MTRR, and CBS and breast cancer  risk; Weiner  AS,  Boyarskikh  UA,  Voronina  EN,  Selezneva  IA,  Sinkina  TV,  Lazarev  AF,  et  al.;  Cancer  Epidemiol.  2012;36(2):e95‐e100.  epub  date:2012/01/13. PIMD:22236648.  48. IRF6 gene variants in Central European patients  with  non‐syndromic  cleft  lip  with  or  without  cleft palate; Birnbaum S, Ludwig KU, Reutter H,  Herms S, de Assis NA, Diaz‐Lacava A, et al.; Eur J  Oral  Sci.  2009;117(6):766‐9.  epu‐ date:2010/02/04. PIMD:20121942.  49. Association between genetic variants of report‐ ed candidate genes or  regions and  risk of cleft  lip  with  or  without  cleft  palate  in  the  polish  population; Mostowska A, Hozyasz KK, Wojcicki  P,  Biedziak  B,  Paradowska  P,  Jagodzinski  PP;  Birth  Defects  Res  A  Clin  Mol  Teratol.  2010;88(7):538‐45.  epub  date:2010/06/15.  PIMD:20544801.  50. Single‐nucleotide polymorphisms  (SNPs) of  the  IRF6 and TFAP2A in nonsyndromic cleft lip with  or without  cleft  palate  (NSCLP)  in  a  northern  Chinese population; Shi J, Song T, Jiao X, Qin C,  Zhou  J;  Biochem  Biophys  Res  Commun.  2011;410(4):732‐6.epub  date:2011/06/21.  PIMD:21683068.  51. IRF6  polymorphisms  are  associated with  non‐ syndromic  orofacial  clefts  in  a  Chinese  Han  population; Pan Y, Ma J, Zhang W, Du Y, Niu Y,  Wang  M,  et  al.;  Am  J  Med  Genet  A.  2010;152A(10):2505‐11. epubdate:2010/08/28.  PIMD:20799332.  52. IRF6 is a risk factor for nonsyndromic cleft lip in  the  Brazilian  population;  Brito  LA,  Bassi  CF,  Masotti C, Malcher C, Rocha KM, Schlesinger D,  et al.; Am J Med Genet A. 2012;158A(9):2170‐5.  epub date:2012/08/14. PIMD:22887868.  53. Association and Mutation Analyses of  the  IRF6  Gene  in Families With Nonsyndromic and Syn‐ dromic  Cleft  Lip  and/or  Cleft  Palate;  Pegelow  M, Koillinen H, Magnusson M,  Fransson  I, Un‐ neerg P, Kere  J,  et al.; CleftPalate Craniofac  J.  2013. epub date:2013/02/12. PIMD:23394314.