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. Author manuscript; available in PMC: 2013 May 1.
Published in final edited form as: Am J Med Genet A. 2013 Mar 5;161(5):965–972. doi: 10.1002/ajmg.a.35749

Replication of Genome Wide Association Identified Candidate Genes Confirm the Role of Common and Rare Variants in PAX7 and VAX1 in the Etiology of Non-syndromic CL(P)

Azeez Butali 1,*, Satoshi Suzuki 1,2,6,*, Margaret E Cooper 3, Adela M Mansilla 1, Karen Cuenco 3, Elizabeth J Leslie 1, Yasushi Suzuki 2, Teruyuki Niimi 2, Masahiko Yamamoto 6, Gongorjav Ayanga 9, Tudevdorj Erkhembaatar 9, Hiroo Furukawa 2,6, Kumiko Fujiwawa 2, Hideto Imura 2, Aline L Petrin 1, Nagato Natsume 2, Terri H Beaty 4, Mary L Marazita 3,5, Jeffery C Murray 1,7,8
PMCID: PMC3634899  NIHMSID: NIHMS420947  PMID: 23463464

Abstract

Following recent genome wide association studies (GWAS), significant genetic associations have been identified for several genes with non-syndromic cleft lip with or without cleft palate (CL(P). To replicate two of these GWAS signals, we investigated the role of common and rare variants in the PAX7 and VAX1 genes. TaqMan genotyping was carried out for SNPs in VAX1 and PAX7 and Transmission Disequilibrium Test (TDT) was performed to test for linkage and association in each population. Direct sequencing in and around the PAX7 and VAX1 genes in 1,326 individuals of European and Asian ancestry was done. TDT analysis showed strong associations with markers in VAX1 (rs7078160, p=2.7E-06 and rs475202, p=0.0002) in a combined sample of Mongolian and Japanese CL (P) case-parent triads. Analyses using parent-of-origin effects showed significant excess transmission of the minor allele from both parents with the effect in the mothers (p=6.5E-05, OR (transmission) =1.91) more striking than in the fathers (p=0.004, OR (transmission) =1.67) for VAX1 marker rs7078160 in the combined Mongolian and Japanese samples when all cleft types were combined. The rs6659735 trinucleotide marker in PAX7 was significantly associated with all the US cleft groups combined (p=0.007 in all clefts and p=0.02 in CL(P)). Eight rare missense mutations found in PAX7 and two rare missense mutations in VAX1. Our study replicated previous GWAS findings for markers in VAX1 in the Asian population, and identified rare variants in PAX7 and VAX1 that may contribute to the etiology of CL(P). Determining the role of rare variants clearly warrants further investigation.

Keywords: GWAS, PAX7, VAX1, CL(P)

INTRODUCTION

Clefts of the lip and cleft palate are the most common craniofacial birth defects with a worldwide birth prevalence of approximately 1/700 [Mossey and Little, 2002]. These complex traits are associated with an increased infant mortality and significant morbidity through adult life and require multidisciplinary treatment for their management [Jugessur et al., 2009]. The development of the craniofacial structures is a complex process that involves the coordinated growth of multiple independently derived primordia. Genetic and environmental factors or their interactions may influence the growth of these primordia and lead to abnormal development of facial structures which then results in clefting of the lip, the primary or secondary palate, or a combination of these sites [Jugessur et al., 2009].

Although the etiology of orofacial clefts is complex, several genetic and environmental risk factors have been identified [Dixon et al., 2011]. Linkage and association studies have provided significant statistical evidence for candidate genes such as interferon regulatory factor six (IRF6) [Rahimov et al., 2008] and forkhead box E1 (FOXE1) [Marazita et al., 2004; Moreno et al., 2009]. Other candidate genes suggested to play a role include bone morphogenetic protein four (BMP4) [Suzuki et al., 2009], tumour protein 63 (TP63) [Scapoli et al., 2008], the jagged2 gene (JAG2) [Vieira et al., 2005], fibroblast growth factor and their receptors (FGFs, FGFRs)[Riley et al., 2007], poliovirus receptor-like 1 (PVRL1) [Turhani et al.,2005; Avila et al., 2006], cysteine-rich secretory protein LCCL domain containing 2 (CRISPLD2) [Chiquet et al., 2007] and muscle segment homeobox one (MSX1) [Jezewski et al., 2003].

Genome wide association studies (GWAS) have also identified new candidate genes and regions that are associated with non-syndromic cleft lip with or without cleft palate (CL(P)). The first GWAS for CL(P) identified a strong association of non-syndromic CL(P) to markers in a gene desert on chromosome 8q24.21[Birnbuam et al., 2009] in a German case-control sample, which was replicated in another study of European Americans [Grant et al., 2009]. In an extension of the German study, Mangold et al. [2010] reported two new loci associated with non syndromic CL(P) at chromosome 17q.22 and chromosome 10q25.3. A GWAS carried out on CL(P) case-parent trios from an international consortium [Beaty et al., 2010]showed evidence of linkage and association at a genome-wide level of significance for chromosome 8q24 (consistent with previous studies) and for the IRF6 gene. In the same case-parent trio study, two other loci with evidence for association at genome-wide significance were identified in or near two novel candidate genes: ABCA4 on chromosome 1p22.1 and MAFB on chromosome 20q.12. They also reported three potential candidate genes where markers attained or approached genome wide significance: PAX7 on chromosome 1p.36, VAX1 on chromosome 10q25.3 and NTN1 on chromosome 17p.13.

A replication study carried out using Mesoamerican populations revealed a significant association between CL(P) and genetic risk factors in IRF6, chr.8q24 and 10q25 loci using single SNP association analysis [Rojas-Martinez et al.,2010]. Similarly, another replication study in Estonia reported a significant association with a single nucleotide polymorphism (SNP) on chr.10q25, confirming its association with CL(P) in this Baltic population [Nikopensius et al., 2010].

The present study tested for the effects of common and rare variants in PAX7 and VAX1 genes as a follow up to the GWAS signals reported by Beaty et al. [2010] and recently confirmed in a meta-analysis by Ludwig et al [2012]. The associated SNPs in chromosome 1p36 previously reported in GWAS results [Beaty et al., 2010] are approximately 2 kb and 20 kb upstream of the PAX7 gene itself and over 100 kb centromeric to the KLHDC7A gene. The KLHCD7A gene does not have any known biological function in the craniofacial region. Therefore we choose to examine the PAX7 gene because of its proximity to the signal and the biological role it plays in neural crest development [Mansouri et al., 1996]. The most strongly associated SNP, rs7078160 on chr10q25 reported in the GWAS by Beaty et al. [2010] (p=1.07x10−7) and Mangold et al. [2010] (p=1.1x10−7) lies 50 kb 3′ of VAX1 and about 50 kb 5′ of KIAA1598. The KIAA1598 gene product is predicted to be involved in signals for neuronal polarization. It is possible the causative SNPs are in either of these genes or in other nearby genes. However, we choose VAX1 as a strong candidate because the SNP rs7078160 is in strong linkage disequilibrium (LD) with SNPs in and around VAX1 and also based on the biological role it plays in the craniofacial region. Replication of the GWAS signals using independent samples complemented by direct sequencing of these two genes to identify novel sequence variations in and around each gene in cases and controls was carried out. PAX7 is a member of the paired box containing gene family which includes nine different genes. These PAX genes are regulatory transcriptional proteins encoding a family of highly conserved DNA-binding transcription factors [Underhill, 2000]. The VAX1 gene is a homeodomain transcription factor which encodes the ventral anterior homeobox 1 protein [Hallonet et al., 1998].

MATERIALS AND METHODS

Study design

The patients were examined to identify the presence of other congenital anomalies and other major structural anomalies and excluded if these were present [Murray et al., 1997]. Parents were interviewed to obtain information on family history of orofacial clefts. The University of Iowa Institutional Review Board (IRB) gave approval for sample collection (approval number 199804080) in conjunction with local approval in the Philippines (approval number 199804081) after approval by the IRB’s of all local sites. The Ethics Committee of the School of Dentistry, Aichi-Gakuin University, approved sample collection in Mongolia and Japan (approval number 11) in conjunction with local IRB approval.

A total of 5,421 individuals (including triads from Japan, Iowa, Mongolia and family members from Philippines multiplex families) were genotyped and all analyses were performed over sub-groups by cleft type and by family history(Table I). Often families had both CLO and CLP in their family history and they formed a separate subgroup (CLO and CLP). An overall cleft lip with or without cleft palate (CL ± CP) subgroup was formed (CL(P)) from the three distinct groups of families mentioned above. The CL(P) group was distinct from those families with a history of cleft palate only (CPO). The CPO group included families with a history of CPO as well as those with a history of cleft palate as well as CL ± CP. Some analyses were conducted on the overall all cleft group formed from the CL(P) and the CPO group. Analyses were also carried out for the overall Asian group by pooling the data from the Philippines, Japanese, and Mongolian studies. Finally, an overall test was conducted by pooling Asian and Iowan samples. In the Beaty et al. [2010] GWAS some genes were shown to be population specific while others played a role in multiple populations suggesting that ancestral background is a factor that should be considered in cleft etiology. For example, in Beaty et al. [2010] study, a GWAS significant SNP in the MAFB gene was observed only in the Asian population, while the GWAS significant SNP in the chromosome 8q.24 locus was observed only in the European population.

Table I.

Family based association analysis for Iowa case triads for all cleft sub-types using markers for PAX7 and VAX1.

Gene SNP CLP (Na=65) CLO (N=46) CL(P)b (N=114) CPO (N=40) Totalc (N=157)
VAX1 rs7078160 0.10 0.39 0.08 0.81 0.10
rs4752028 0.13 1.00 0.26 0.32 0.10
PAX7 rs4920520 0.56 0.75 0.92 0.27 0.66
rs766325 0.31 0.34 0.68 0.72 0.94
rs6659735 0.05 0.34 0.02 0.37 0.007
Family based association analysis for all Asian populations combined for all cleft sub-types using markers for PAX7 and VAX1.
Gene SNP CLP (Na=270) CLO (N=103) CL(P)b (N=440) CPO (N=54) Totalc (N=494)
VAX1 rs7078160 6.00E-07 0.02 4.6E-07 0.008 2.3E-08
rs4752028 0.0003 0.01 0.00002 0.78 0.00004
PAX7 rs4920520 0.91 0.47 0.70 0.78 0.75
rs766325 0.91 0.37 0.42 0.70 0.48
Family based association analysis for all four populations combined for all cleft sub-types using markers for PAX7 and VAX1.
Gene Location CLP (Na=335) CLO (N=149) CL(P)b (N=554) CPO (N=94) Totalc (N=651)
VAX1 rs7078160 1.5E-07 0.01 1.1E-07 0.02 7.4E-09
rs4752028 0.0001 0.02 0.00001 0.34 9.8E-06
PAX7 rs4920520 0.87 0.45 0.70 0.54 0.61
rs766325 0.60 0.84 0.61 0.70 0.58
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CLO is cleft lip only, CPO is cleft palate only, CLP is cleft lip and palate and CL(P) is cleft lip with or without cleft palate.

a

Number of informative families

b

CL(P) includes families with both CL and CLP in their cleft history

c

Total includes cleft families where cleft type is unknown.

We sequenced a total of 1,326 individuals with clefts in all four populations (Tables II and III). Missense mutations were compared to missense mutations present in the 1000 genome database [http://www.1000genomes.org/] and exome variant sequence database [http://snp.gs.washington.edu/EVS/].

Table II.

Newly described variants in PAX7 and nucleotide changes following sequence analysis of Iowan, Philippine, Mongolian and Japanese individuals.

GRCh37/hg19 Amino Acid Change Iowa Filipinos Polyphen
Cases Controls Cases Controls
c.189,614G>C G411R 0/81 0/81 1/92 0/87 Possibly damaging
c.189,617G>A G412S 1/81 0/81 0/92 0/87 Benign
c.062,366G>A G466S 0/81 0/81 1/810 0/87 Benign
c.088,401C>T 0/81 0/81 1/92 0/87
c.018,438C>A 0/81 0/81 1/92 0/87
c.957,713C>T 2/81 2/82 11/86 4/89
c.958,083C>A 3/81 0/82 0/86 0/89
c.957,933C>T 0/81 0/82 1/86 0/89
c.072014C>A 2/83 0/82 0/86 0/86
c.072317G>A 1/83 0/82 0/86 0/86
c.189576C>T P397L 0/81 1/81 0/92 0/87 Probably damaging
GRCh37/hg19 Mongolian Japanese
Cases Controls Cases Controls
c.958,104G>A A3T 1/87 NDb 0/88 NDb Benign
c.958,140G>A A15T 0/87 ND 1/87 ND Benign
c.958,168G>A G24E 0/87 ND 2/88 ND Possibly damaging
c.961,014T>C 1/87 ND 0/88 ND
c.961,088G>T 2/85 ND 0/88 ND
c.961,515C>- 1/87 ND 0/88 ND
c.029895A>T 8/87 ND 0/88 ND
c.062,129A>C M387L 1/87 ND 0/88 ND Benign
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b

Not determined

Table III.

Newly described variants in VAX1 and nucleotide changes following sequence analysis of Iowan, Philippine, Mongolian and Japanese individuals.

GRCh37/hg19 Amino acid change Iowans Philippines Polyphen
Cases Controls Cases Controls
c.898,982>C 0/92 0/81 1/91 1/88
rs148741605C>T 0/92 0/78 13/270 2/270
c.840,949G>A 6/91 6/72 0/90 0/82
c.840,439C>A 0/91 2/78 7/86 6/85
GRCh37/hg19 Mongolian Japanese
Cases Controls Cases Controls
c.897,839C>T 8/171 NDb 0/94 NDb
c.896,171G>T 1/171 ND 0/94 ND
c.896,062T>G M117R 1/171 0/92 0/94 ND Probably damaging
c.893,947C>T 0/169 0/92 1/72 ND
c.893,882G>T 1/169 0/92 1/72 ND
c.893,824G>T A233S 1/169 0/92 0/72 ND Benign
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b

Not determined

Genotyping analysis

We included 206 non-syndromic case-parent triads from Mongolia, 98 case-parent triads from Japan, 157 case-parent triads from Iowa, and 190 extended pedigrees from the Philippines multiplex families in this study. We performed TaqMan genotyping (Applied biosystems) using four SNPs in potential candidate genes from previous GWAS (VAX1 rs7078160, rs4752028 and PAX7 rs4920520, rs766325) reported to show association (Beaty et al., 2010; Mangold et al., 2010). TaqMan genotyping of a variant rs14874160>T in the 5′ UTR found through sequencing of the VAX1 gene was also performed on 644 Philippine cases and 112 Philippine controls.

Sequencing Analyses

Direct sequencing was used to search for sequence variations in coding regions and conserved non-coding regions within 1Kb of the PAX7 (NM_001135254.1 8 exons) and the VAX1 genes (NM_001112704.1 4 exons) in 180 CL(P) patients (90 from Iowa and 90 from Philippines) and 180 controls (90 from Iowa and 90 from Philippines). An additional 630 cases from the Philippines were sequenced for the PAX7 gene. We also sequenced 265 patients (171 Mongolians and 94 Japanese) and 92 Mongolian controls. Primers were designed using Primer 3 and optimized to the optimal annealing temperature with details available on request through the Murray lab website (see web resources). PCR products were sent for sequencing using an ABI 3730XL (Functional Biosciences, Inc., Madison, WI). Chromatograms were transferred to a Unix workstation, base-called with PHRED (v.0.961028), assembled with PHRAP (v. 0.960731), scanned by POLYPHRED (v. 0.970312), and viewed with the CONSED program (v. 4.0).

Statistical Analyses

Transmission disequilibrium was tested using the Family Based Association Test (FBAT) under the additive model for the Philippine multiplex families. PLINK software was used for the TDT test in the Iowa, Japanese and Mongolian trios. The PLINK software [Purcell et al., 2007] was also used to estimate transmitted and un-transmitted allele counts, calculate odds ratios (affected transmitted allele count divided by unaffected transmitted allele count), and parental effects (separate maternal/paternal transmission counts).

We performed gene-gene (GxG) interaction analyses on the family data. We specifically targeted the PAX7 and VAX1 genes and their possible interaction with SNPs in the other cleft associated genes/regions: ABCA4, IRF6, 8q region, FOXE1, and MAFB. Our primary gene-gene interaction evaluation was conducted using conditional logistic regression. A model containing one term for each SNP as a main effect and one interaction term was compared to another model without the interaction term. Assessment was based on a one degree of freedom Wald Chi-Square statistic contrasting these two models. Comparison of these models was implemented using R(trio) “full” option [Schwender et al., 2011; Schwender et al., 2012)] In addition, we considered the Cordell epistatic model [Cordell et al., 2002; Cordell et al., 2004] (with four interaction terms). However, there were convergence failures as a result of the sparseness in the data for specific SNP combinations. Therefore, the only the results from the one degree of freedom method are reported. The SNPs’ linkage disequilibrium (LD) and minor allele frequencies were not problematic in the regression modeling.

RESULTS

Replication of markers in PAX7 and VAX1 genes in four populations

Mendelian errors were removed from the data by PLINK in executing the allelic TDT. Iowa patient-parent triads and Filipino multiplex families were genotyped for the PAX7 SNP rs6659735. The threshold for significance was set as p< 0.05 and when we corrected for multiple testing, p<0.001 was significant.

The PAX7 trinucleotide SNP (rs6659735) showed significant association (p= 0.007) in all the cleft groups combined in the Iowa population (Table I). There was strong evidence of linkage and association with markers in VAX1 for CL(P) (rs7078160, p=2.7E-06 and rs4752028 p=0.0002 respectively) in both Mongolian and Japanese case-parent triads. The results in the CPO group were not significant (p=0.14 and p=0.87, respectively). For all clefts combined, the results were p=8.9E-06 and p=0.0006, respectively for Mongolian and Japanese case-parent triads) (Table I). A significant association with the CL(P) group was also observed among the Filipino multiplex families for rs70781860 (p= 0.001). No statistically significant findings were observed for VAX1 in the Iowa sample (p=0.10 for all clefts combined and p=0.08 for the CL(P) group). There was strong evidence of association with SNPs in VAX1 (rs7078160 and rs4752028) in the combined Asian samples (p=2.3E-08 and p=0.00004 respectively) (Table I). When data were analyzed from all four populations and all clefts combined, a significant association was also observed for markers in VAX1 (rs7078160, p=7.4E-09 and rs4752028, p=9.8E-06). The CL(P) cleft group contributed the most to the association (p=1E-07 and p=0.00002). Interestingly, the CPO group also contributed to significant association findings in Asians, but for only rs7078160 (p=0.008) (Table I). The VAX1 c.80130G>T marker (identified through sequencing) analyses suggested that the non-ancestral rare allele is not over-transmitted to patients (p=0.72 for all clefts combined and p-values ranging between 0.48 and 1 for the other cleft subgroups) (Supplementary table I).

Analyses using parent-of-origin effects showed significant excess transmission of the minor allele from both parents with the effect in the mothers (p=6.5E-05, OR (transmission) =1.91) more striking than in the fathers (p=0.004, OR (transmission) =1.67) for VAX1 marker rs7078160 in the combined Mongolian and Japanese samples when all cleft types were combined. Affected males were mostly responsible for the rs7078160 SNP association with all clefts in the combined Japanese and Mongolian case-parent trios (male cases: p=6.4E-06 and female cases: p=0.02). In males with clefts, parental transmission effects were also different: (rs7078160; maternal (p=3.4E-05), OR=2.65 for and paternal (p=0.03), OR=1.69). No statistically significant parental transmission effects were seen in the affected female analysis (paternal (p=0.08) and maternal (p=0.14)).

Sequencing

Identification of variants in PAX7

We found 19 new variants, eight of which were missense mutations and three were synonymous mutations (Table II). The other newly identified variants were in introns. For cases with the missense mutations, we sequenced the samples for the available parent (in this case the mothers) but did not find the variants in them. Since we did not have samples from the fathers in these families, we cannot make any conclusions concerning the mode of inheritance (i.e., whether the missense variant segregated in the family or it was a de novo variant).

Identification of variants in VAX1

We found 12 new variants in VAX1 (Table III). One of the SNPs rs14874160G>T showed significant association (p= 0.007) with CL(P) using the Fisher’s exact test) when analyzed in 270 cases and 270 controls in the Philippines samples (Supplementary Table II).

Two of the new VAX1 variants were missense mutations, (pMet117Arg) c. 96026T>G and (p.Ala233Ser) c.93824G>T, found only in Mongolian cases. A canonical splice acceptor variant c.96171G>T at position-1 was found in a Mongolian case. Two more variants were synonymous, found in a single Japanese case and in a Mongolian case) in coding regions (Table III). A Proline in-frame insertion (p.A233_P234insP) c.698_699insCCC was found in a single Filipino case and both parents.

Gene-gene interactions (GxG)

None of the GxG results reached the threshold for significance under the multiple testing done in this project (i.e., p-values ≤ 0.001), however there were a few GxG results that were nominally significant and warrant future investigations. For example, in the Asian group the MAFB rs17820943 SNP interacted with the PAX7 rs4920520 SNP (p=0.03) and with VAX1 rs7078160 (p=0.05) and VAX1 rs4752028 (p=0.03). Also in Asians, VAX1 rs7078160 interacts with FOXE1 rs3758249 (p=0.01). In the combined sample across all populations, there was evidence for interaction of VAX1 rs4752028 with 8q rs987525 (p=0.03) and with MAFBrs17820943 (p=0.05).

DISCUSSION

Significant genetic associations have been identified for 11 genes or loci with non-syndromic cleft lip with or without cleft palate (CL(P)) in five GWAS reports including a recent meta-analysis [Ludwig et al., 2012]. To replicate two of these GWAS signals, we investigated the role of common and rare variants in the PAX7 and VAX1 genes.

Pax7 is important in neural crest development and required for the expression of the neural crest markers Slug, Sox9, Sox10 and HNK-1 in vivo [Basch et al., 2006]. Evidence from animal models suggests Vax1 has biological functions in the craniofacial region [Hallonet et al., 1998]. Since the older SNP arrays were less comprehensive for non-Caucasian ethnicities and do not include rare variants [Pritchard, 2001; McCarthy et al., 2008], we sequenced PAX7 and VAX1 in individuals from Iowa, Japan, Mongolia and Philippines and identified eight rare missense variants in PAX7 and two in VAX1. None of the PAX7 rare variants in the cases were found in over 6500 individuals in the 1000 genome and whole exome variant sequence databases [Leslie and Murray, 2012]. However, the rare variant (p.Pro937Leu) c.89576C>T found in a control individual from Iowa, was also found in five out of 2188 chromosomes in the 1000 Genomes database. In VAX1, one of the missense mutations (p.Met117Arg) c.96062T>G is predicted to be probably damaging by Polyphen. These variants may affect protein structure and function. None of these missense variants were found in the 1000 genome and whole exome sequence databases. A recent study reported a missense mutation in VAX1 in an individual with bilateral microphthalmia, cleft lip and palate, small optic nerves, hippocampal malformations, corpus callosum agenesis, and absence of the pineal gland [Slavotinek et al., 2011].

Rare coding variants have been reported in re-sequencing of genes implicated by GWAS [Johansen et al., 2010] and significant GWAS SNPs may actually be reflecting signals from multiple associated functional rare variants that have large effects on risks [Dickson et al., 2010]. Therefore, it is possible that these rare variants that have profound effects on the disease risks are responsible for the missing heritability even if there is no evidence of Mendelian segregation [Keinan and Clark, 2012; Tennessen et al., 2012].

In the present study, we found statistically significant association with all clefts types and VAX1 GWAS SNPs in the three Asian populations combined (rs7078160p=2.3E-08 and rs4752028 p=0.00004) and all four populations combined (rs7078160 p=7.4E-09 and rs4752028 p=9.8E-06).

Significant association signals were observed using the same reported VAX1 GWAS markers in the Asian case-parent trios. The findings of this study support previous GWAS [Beaty et al., 2010, Mangold et al., 2010] and is consistent with the replication studies reporting significant association with VAX1 [Nikopensius et al., 2010, Rojas-Martinez et al., 2010; Ludwig et al., 2012]. Our findings suggest the GWAS signals are in LD with the unobservable functional variants associated with NSCL(P). This assumption is supported by the evidence which suggest that GWAS signals can extend across multiple LD blocks [Dickson et al., 2010].

In conclusion, our study has replicated previous findings from GWAS for markers in VAX1 across three independent Asian populations, and identified rare variants in PAX7 that may contribute to the etiology of CL(P). Determining the specific role of these rare variants in the etiology of CL(P) will require further investigations. We also found some evidence of interactions between SNPs in different genes suggesting they may act in the same pathway. These findings provide solid support for PAX7 and VAX1 as genes playing a role in human CLP.

Supplementary Material

Supplementary Table

Acknowledgments

We are particularly grateful to the many patients and their families who participated in this study. We were greatly assisted by many people including those in the Division of Research and Treatment for Oral and Maxillofacial Congenital Anomalies, Aichi-Gakuin University, Japanese Cleft Palate Foundation (Aichi-Gakuin University, Nagoya, Japan), a medical organization that provides clinical care for underserved populations in Japan, Vietnam, Mongolia, and Cambodia, and physicians and volunteers in Mongolia. We sincerely appreciate the outstanding support in Iowa of Sandy Daack Hirsh, Susie McConnell, Nancy Davin, Erin Brothers Smith, Raul Petrin, and Tamara Busch. The project was supported by the National Institute for Dental and Craniofacial Research (NIH) grants K99-DE022378-01, R37-DE08559 and R01-DE016148, and U01-DE-018993 to the consortium for GWAS genotyping and analysis. KAKENHI-Grant-in-Aid for Young Scientist (B) No. 20791560 (Aichi-Gakuin), Face Base grant U01 DE-20057.

Footnotes

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