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. Author manuscript; available in PMC: 2020 Nov 1.
Published in final edited form as: Birth Defects Res. 2019 Dec 11;112(3):234–244. doi: 10.1002/bdr2.1630

PBX-WNT-P63-IRF6 pathway in nonsyndromic cleft lip and palate

Lorena Maili 1, Ariadne Letra 2,3, Renato Silva 3,4, Edward P Buchanan 5, JB Mulliken 6, MR Greives 7, JF Teichgraeber 7, SJ Blackwell 8, Rohit Ummer 3, Ryan Weber 3, Brett Chiquet 3,9, Susan H Blanton 10, Jacqueline T Hecht 1,3
PMCID: PMC7325739  NIHMSID: NIHMS1586609  PMID: 31825181

Abstract

Nonsyndromic cleft lip and palate (NSCLP) is one of the most common craniofacial anomalies in humans, affecting more than 135,000 newborns worldwide. NSCLP has a multifactorial etiology with more than 50 genes postulated to play an etiologic role. The genetic pathway comprised of Pbx-Wnt-p63-Irf6 genes was shown to control facial morphogenesis in mice and proposed as a regulatory pathway for NSCLP. Based on these findings, we investigated whether variation in PBX1, PBX2, and TP63, and their proposed interactions were associated with NSCLP. Fourteen single nucleotide variants (SNVs) in/nearby PBX1, PBX2, and TP63 were genotyped in 780 NSCLP families of nonHispanic white (NHW) and Hispanic ethnicities. Family-based association tests were performed for individual SNVs stratified by ethnicity and family history of NSCLP. Gene-gene interactions were also tested. A significant association was found for PBX2 rs3131300 and NSCLP in combined Hispanic families (p= 0.003) while nominal association was found for TP63 rs9332461 in multiplex Hispanic families (p=0.005). Significant haplotype associations were observed for PBX2 in NHW (p= 0.0002) and Hispanic families (p=0.003), and for TP63 in multiplex Hispanic families (0.003). An independent case-control group was used to validate findings, and significant associations were found with PBX1 rs6426870 (p=0.007) and TP63 rs9332461 (p=0.03). Gene-gene interactions were detected between PBX1/PBX2/TP63 with IRF6 in NHW families, and between PBX1 with WNT9B in both NHW and Hispanic families (p<0.0018). This study provides the first evidence for a role of PBX1 and PBX2, additional evidence for the role of TP63, and support for the proposed PBX-WNT-TP63-IRF6 regulatory pathway in the etiology of NSCLP.

INTRODUCTION

Craniofacial development is complex and involves multiple biological processes with precise timing of cell growth, movement and fusion. During embryogenesis, neural crest cells give rise to five facial primordia: the frontonasal prominence, the paired maxillary (MXP), and paired mandibular processes (Dixon, Marazita, Beaty, & Murray, 2011; Jiang, Bush, & Lidral, 2006). The edges of the frontonasal process then begin to invaginate and the nasal placodes appear, defining the medial (MNP) and lateral (LNP) nasal processes (Jiang et al., 2006; Marazita & Mooney, 2004; Senders, Peterson, Hendrickx, & Cukierski, 2003; Som & Naidich, 2013). As these tissues proliferate, the MXP displaces the LNP superiorly, allowing for contact with the MNP and then fuses with the MNP to form a complete upper lip and primary palate (Jiang et al., 2006; Senders et al., 2003; Som & Naidich, 2013). Defects in cellular migration, differentiation, proliferation or apoptosis during these developmental stages can lead to incomplete fusion in the primary or secondary palate and manifest as an orofacial cleft (Dixon et al., 2011). While orofacial clefting is associated with more than 400 syndromes, the majority of the cases are isolated, or nonsyndromic, where affected individuals do not have additional structural abnormalities (Mossey, Little, Munger, Dixon, & Shaw, 2009; Online Mendelian Inheritance in Man, 1995; Stuppia et al., 2011; Wyszynski, 2002).

Nonsyndromic cleft lip and palate (NSCLP) is among the most common birth defects in humans, with a birth prevalence of 1 in 700 – 2500, depending on population/ethnicity, and affecting over 4000 infants in the US each year (Centers for Disease & Prevention, 2004; Hashmi, Waller, Langlois, Canfield, & Hecht, 2005; Parker et al., 2010). NSCLP has multifactorial etiology in which numerous genes and environmental factors individually or interactively may contribute to the phenotype (Carter, 1976; Dixon et al., 2011; Mossey et al., 2009). Over the years, numerous studies have successfully identified many genes/loci contributing to NSCLP (Beaty, Marazita, & Leslie, 2016; Dixon et al., 2011; Kousa & Schutte, 2016). Previous linkage, candidate gene and genome-wide association studies using family-based or case-control populations, and studies in animal models have provided evidence for the role of MSX1, IRF6, TFAP2A, CRISPLD2, 8q24 locus, TP63, and WNT genes, among other genes/loci in NSCLP (Barrow et al., 2002; Beaty et al., 2016; Dixon et al., 2011; Leoyklang, Siriwan, & Shotelersuk, 2006). More recently, whole exome sequencing (WES) studies have confirmed the role of variants in known NSCLP genes (IRF6, CDH1, CRISPLD2, FGFR2 and PAX7) and identified novel candidate genes (CTNND1, PLEKHA7, PLEKHA5 and ESRP2) (Bureau et al., 2014; Cox et al., 2018; Pengelly et al., 2015). Furthermore, there is increasing evidence supporting an additive role for gene-gene interactions in NSCLP, with modifier phenotypic effects (Carlson et al., 2017; Chiquet et al., 2018; Li et al., 2015; D. Liu et al., 2018; Velazquez-Aragon et al., 2016; Zhou et al., 2019).

In this context, a novel genetic pathway comprised of Pbx-Wnt-p63-Irf6 genes was shown to control murine facial morphogenesis and was proposed to be an important regulatory pathway for NSCLP (Ferretti et al., 2011). Pbx genes (Pbx1, −2, −3) and their respective encoded proteins are considered Hox factors, which increase Hox DNA-binding specificity and are important players during skeletal development (Capellini et al., 2006; Capellini et al., 2008; Tumpel, Wiedemann, & Krumlauf, 2009). Compound Pbx mutant mice presented with craniofacial abnormalities and fully penetrant bilateral cleft lip and palate, which was attributed to altered Wnt signaling at the midfacial region (Ferretti et al., 2011). Expression of Wnt9b, Wnt3, and p63 was not detected in the midface of compound Pbx mutant mice in comparison to wild type littermates; meanwhile Fgf8 and Irf6 expression was dramatically reduced (Ferretti et al., 2011). This led to the conclusion that loss of Pbx genes in the mouse midfacial region disrupts this Wnt-p63-Irf6 regulatory pathway, which in turn causes facial morphogenesis defects resulting in cleft lip/palate (Ferretti et al., 2011). Based on these findings, we asked whether variation in genes in the proposed PBX-WNT-P63-IRF6 pathway and their potential interactions might contribute to NSCLP.

MATERIALS AND METHODS

This study was approved by the University of Texas Health Science Center Committee for Protection of Human Subjects (HSC-MS-03–090 and HSC-DB-11–0492). An overview of the methodology workflow is represented in Supplemental Figure 1.

Family-based study

The NSCLP family-based group consisted of 2,233 individuals belonging to 780 families, 243 multiplex families (145 nonHispanic White (NHW); 87 Hispanic) and 564 simplex parent-child trios (335 NHW and 213 Hispanic) (Table 1) (Chiquet et al., 2008; Chiquet et al., 2011; Letra et al., 2014). Each family was ascertained through a NSCLP proband from one of four craniofacial centers: Boston Children’s Hospital, Texas Children’s Hospital, Shriners Hospital for Children and the McGovern Medical School Craniofacial Center. Probands and relatives were examined to exclude syndromic forms of orofacial clefting. Information about race/ethnicity was obtained by self-report. After informed consent, blood and/or saliva samples were collected and genomic DNA was extracted using established protocols (Chiquet et al., 2008).

Table 1.

Description of family-based and case-control NSCLP datasets

Ethnicity NSCLP Datasets
Family-Based Case-Control2
Families Individuals Individuals
Simplex Multiplex Total Unaffected Affected Total Cases Controls Total
NHW1 335 145 480 934 553 1487 504 441 945
Hispanic 213 87 300 531 315 846 -- -- --
Total 548 232 780 1465 868 2333 504 441 945
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1

NHW = nonHispanic white

2

Unrelated individuals of European Caucasian ancestry

Genotyping

Fourteen single nucleotide variants (SNVs) in/nearby PBX1, PBX2, and TP63 genes were selected based on heterozygosity (minor allele frequency > 0.15%), location in gene, and linkage disequilibrium blocks surrounding each gene as previously described (Chiquet et al., 2008) (Table 2). HaploView was used to test for linkage disequilibrium and to identify tagging SNVs (Barrett, Fry, Maller, & Daly, 2005). Genotyping was performed using Taqman genotyping assays and genotypes were detected on a ViiA7 Automatic Sequence Detection System (Life Technologies, Foster City, CA, USA). Control individuals with known genotypes (positive controls) as well as non-template negative control samples were included on all genotyping reactions.

Table 2.

SNV alleles and frequencies by ethnicity

Gene Chr Base positiona dbSNP ID Allelesb SNV Locationc NHW MAFd Hispanic MAFe Controls MAF
PBX1 1q23 164523953 rs6426870 CT 5’ (– 867) 0.27 0.47 0.34
164607346 rs1618566 AG Intron 2 0.38 0.26 0.34
164658057 rs10800043 CT Intron 2 0.21 0.27 0.25
164723835 rs7543038 GT Intron 2 0.39 0.272 0.34
164782006 rs3767367 AG Intron 6 0.18 0.14 0.17
PBX2 6p21.3 32158319 rs176095 AG 5’ (−340) 0.20 0.15 0.20
32155581 rs204993 AG Intron 4 0.26 0.18 0.22
32151934 rs3131300 AG 3’ (+576) 0.17 0.11 0.12
TP63 3q28 189341790 rs9332461 AG 5’ (−7388) 0.38 0.26 0.33
189421319 rs4575879 AG Intron 1 0.37 0.31 0.37
189451290 rs4607088 CT Intron 1 0.39 0.49 0.43
189497616 rs4686529 AG Intron 3 0.46 0.38 0.50
189596855 rs1515490 AG Intron 10 0.24 0.22 0.24
189641053 rs11706540 TC 3’ (+25988) 0.29 0.22 0.29
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Chr= chromosomal location

a

Ensembl GRCh37 reference assembly position

b

Most common allele listed first.

c

Distance from transcription start site in base pairs for upstream and downstream SNVs

d

Minor allele frequency

e

corresponding frequency in Hispanic of nonHispanic White minor allele

Data Analysis

Family-based single SNV association analysis was performed using the Family-Based Association Test (FBAT) (Laird, Horvath, & Xu, 2000). The “-e” extension was applied to correct for complex pedigree structures (Laird & Lange, 2006). Association in the Presence of Linkage (APL) test was used for the individual and pairwise-association analyses (Chung, Schmidt, & Martin, 2011). Analyses were stratified by ethnicity and presence/absence of family history of NSCLP. For all analyses, the Bonferroni method for multiple testing was used with a corrected p-value ≤0.0036 (0.05/14 SNVs) considered significant.

Transmission of all possible intragenic 2, 3 and 4-SNV haplotypes was examined using the Haplotype Based Association Test (HBAT) function in FBAT (Laird et al., 2000; Laird & Lange, 2006). APL was used to detect gene-gene interactions between the studied SNVs with 14 variants in additional known NSCLP genes (WNT3, WNT9B and IRF6) for which genotype data was available (Supplemental Table 1) (Blanton et al., 2010; Chiquet et al., 2008). The significance threshold was based on the number of possible gene-gene SNV combinations and was set at p-value≤0.0019 (0.05/27 total SNVs).

Bioinformatic prediction of SNV function

Bioinformatic prediction of variant function was assessed using Genomic Evolutionary Rate Profiling (GERP) score to estimate the evolutionary constraint rates for individual nucleotide positions (Cooper et al., 2005; Davydov et al., 2010). GERP scores, which range from −12 to +6, indicate constraint when positive (Cooper et al., 2005; Davydov et al., 2010). Ensembl Variant Effect Predictor (VEP) was used to identify the effect of each variant on the gene transcript. Genome Wide Annotation of Variants (GWAVA) was used to annotate variants in noncoding regions for potential functionality; this tool calculates a score for each SNV, with scores higher than 0.5 indicating functionality (Ritchie, Dunham, Zeggini, & Flicek, 2014; Yourshaw, Taylor, Rao, Martin, & Nelson, 2015). HaploReg V4.1, which employs Roadmap Epigenomics and ENCODE data, sequence conservation across mammals (SiPhy), and eQTL data, was used to further assess functionality annotations (Ward & Kellis, 2012, 2016). HaploReg also annotates promoter histone marks, enhancer histone marks, DNAse hypersensitivity, proteins bound by ChIP-seq experiments, binding motifs, and expression quantitative trait loci (eQTLs) on various cell lines and tissues. Lastly, the Genotype-Tissue Expression (GTEx) portal was utilized to gain insights into the predicted effects of associated SNVs in different human tissues (Consortium et al., 2017).

Validation study

The case-control NSCLP group was used to validate the family-based results. It was comprised of 945 unrelated individuals, 504 individuals with NSCLP and 441 control individuals without NSCLP or family history of NSCLP. Subjects were recruited under local IRB-approved protocols and written informed consent at the Hospital of Rehabilitation and Craniofacial Anomalies, Bauru Dental School, University of Sao Paulo, and at the Center for Treatment of Craniofacial Anomalies, Rio de Janeiro, Brazil. Only Caucasian individuals (from the Southeastern region of Brazil and of predominantly European ancestry) were included. Ethnicity was self-reported for up to 2 generations. All SNVs were genotyped as described above. Chi square and Fisher’s exact tests, as implemented in PLINK v.1.07 (Purcell et al., 2007), were used to detect differences in genotype and allele frequencies for each SNV between cases and controls, with p-values ≤ 0.05 considered significant.

RESULTS

All SNVs were in Hardy-Weinberg equilibrium. In the family-based analysis, SNVs in all three genes interrogated met the nominal association threshold of p≤0.05 (Table 3). After Bonferroni correction, the most significant individual SNV associations were between PBX2 rs3131300 (p=0.003) with NSCLP in Hispanic families (Table 3). Analyses of 2-, 3-, and 4-window haplotypes revealed altered transmission of PBX2 alleles involving rs3131300 in both Hispanic and NHW families. Interestingly, the alternate allele G in rs3131300 was consistently over transmitted together with the ancestral alleles of rs176095 (A) and rs204993 (A) in NHW, whereas in Hispanic families over transmission of both alternate alleles in rs3131300 and rs204993 (G-G) was detected (p<0.003; Table 4). Additional TP63 variant haplotypes were also significantly associated with NSCLP in Hispanics; in these families the transmission of the alternate alleles in rs4607088 and rs4686529 (T-G) were detected in combination with the ancestral alleles in rs9332461 (A), rs4575879 (A) and rs1515490 (A) (p<0.003; Table 4).

Table 3.

Association results by ethnicity and pedigree type

Gene SNV Population
NHW# Hispanic NHW
All Multiplex All Multiplex Simplex Case -Control
FBAT FBAT-e APL FBAT FBAT-e APL FBAT FBAT-e APL FBAT FBAT-e APL FBAT FBAT-e APL PLINK
PBX1 rs6426870 -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- 0.006
rs3767367 0.04 0.05 -- -- -- -- -- -- -- -- -- -- -- -- -- --
rs1618566 -- -- -- -- -- -- <0.05 -- -- -- -- -- -- -- -- --
PBX2 rs204993 -- -- -- -- -- -- <0.05 <0.05 -- -- -- -- 0.05 -- 0.02 --
rs176095 -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- --
rs3131300 -- -- -- 0.02 0.02 0.04 0.003 0.003 0.03 -- -- -- 0.01 0.014 0.02 --
TP63 rs9332461 -- -- -- -- -- -- -- -- -- 0.005 0.01 -- -- -- -- 0.03
rs4686529 -- -- 0.02 -- -- -- -- -- -- -- -- -- -- -- -- --
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p-values ≤ 0.05 are shown

p-values ≤ 0.0036 are significant after Bonferroni correction (in bold)

FBAT = Family Based Association Test, FBAT -e = option for extended pedigrees

APL = Association in the Presence of Linkage

#

Results for NHW Simplex families are not shown because they were not significant

Table 4.

Haplotype results from NSCLP families

Gene Population SNVs Alleles◊ P-value*
PBX2 NHW All rs3131300 rs204993 G A 0.0002
rs3131300 rs176095 G A 0.0005
rs3131300 rs204993 rs176095 G A A 0.0006
NHW Simplex rs3131300 rs204993 G A 0.0002
rs3131300 rs176095 G A 0.002
rs3131300 rs204993 rs176095 G A A 0.0003
Hispanic All rs3131300 rs204993 G G 0.003
TP63 Hispanic Multiplex rs9332461 rs4575879 A A 0.003
rs9332461 rs4607088 rs4686529 A T G 0.003
rs9332461 rs4607088 rs1515490 A T A 0.003
rs9332461 rs4607088 rs4686529 rs1515490 A T G A 0.003
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◊

Minor alleles shown in bold

*

APL test, significant if p-values ≤ 0.0036 (in bold)

In the case-control group, significant associations were found for PBX1 rs6426870 (p=0.007) and TP63 rs9332461 (p=0.03); no association was found for PBX2 (Table 3).

Gene-gene interaction analyses suggested numerous potential biological interactions, with the majority observed in the NHW families. In the NHW families, considering multiplex and all families, interactions were found between PBX1/PBX2/TP63 with IRF6, followed by PBX1 with WNT9B (p≤0.0018). In Hispanics, evidence of interaction was also found between PBX1 and WNT9B (p=0.0007). No significant interactions were observed between TP63 and either PBX or WNT genes (Table 5).

Table 5.

Summary of gene-gene interaction calculations in NSCLP families

Gene 1 SNV Gene 2 SNV NHW Hispanic
All multiplex simplex All multiplex simplex
PBX1 rs10800043 WNT3 rs199525 0.0026
rs6426870 WNT9B rs1530364 0.0007
rs3767367 rs199498 0.0010
rs10800043 IRF6 rs2235371 0.0017 0.0026
rs1618566 0.0018 0.0008
rs3767367 0.0003
rs6426870 0.0004 0.0002
rs7543038 0.0013 0.0026
rs6426870 rs642961 0.0023
PBX2 rs176095 WNT9B rs1530364 0.0023
PBX2 rs176095 IRF6 rs2235371 0.0003 0.0007
rs204993 0.0001 0.0001
rs3131300 0.0007 0.0022
TP63 rs1515490 IRF6 rs2235371 0.0009
rs4575879 0.0001 0.0001
rs4607088 0.0031 0.0011
rs4686529 0.0004
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*

interactions with p-values ≤ 0.0036 are shown; significant if p ≤ 0.0018 (in bold)

Bioinformatic predictions of the potential functional effects of associated SNVs on gene expression are listed in Supplemental Table 2. The downstream associated PBX2 variant rs3131300 showed evidence of evolutionary conservation, with a GERP score of 2.9. This variant was classified as functional by GWAVA and as a modifier of the PBX2 gene transcript by Ensembl VEP. The base pair change at this SNV affected 13 protein binding motifs, including BDP1, KLF4, KLF7, PLAG1 and p300. Enhancer histone marks were present at the SNV location, further implicating functionality. PBX2 rs3131300 was also a significant expression quantitative trait locus (eQTL) in whole blood samples, where individuals with an A allele show higher PBX2 expression (Consortium et al., 2017) (Supplemental Figure 2). The 5’ PBX1 rs6426870 variant had a neutral GERP score but was classified as functional by GWAVA and harbored 6 altered binding motifs, including ones for FOXD1, FOXK1, FOXL1 and YY1. Additionally, this variant is also a significant eQTL in whole blood, and individuals with a C allele show higher PBX1 expression (Supplemental Figure 2). The upstream TP63 variant rs9332461 was predicted to affect the protein-binding motif for NKX2–1 and presented enhancer histone marks in blood and muscle tissues. This variant was also a significant eQTL in lung tissue, with the A allele leading to higher TP63 expression (Supplemental Figure 2).

DISCUSSION

In this study, we investigated the role of the PBX-WNT-TP63-IRF6 pathway in NSCLP because this gene module resulted in clefting in Pbx-deficient mice (Ferretti et al., 2011). The contribution of IRF6, TP63 and individual WNT genes has been extensively studied in NSCLP and data strongly supports them as cleft susceptibility genes in humans (Dixon et al., 2011). However, evidence regarding the role of PBX genes in NSCLP, individually and/or interactively with additional genes is still lacking. We tested whether variants in PBX and TP63 were associated with NSCLP phenotypes in a large family-based group and in an independent case-control validation group. We also performed gene-gene interaction calculations considering the studied genes and additional known cleft genes. Overall, our results with two independent datasets provide additional support for the contribution of the proposed PBX-WNT-TP63-IRF6 regulatory pathway to NSCLP risk. A significant association with PBX2 was found in our NSCLP families and additional significant associations were found for PBX1 and TP63 in the case-control group. Additionally, we observed evidence of altered allele transmission and significant gene-gene interactions between PBX2-IRF6, PBX1-WNT9B-IRF6, and TP63-IRF6, that further support the biological mechanisms previously proposed (Ferretti et al., 2011).

In the family-based analysis, PBX2 rs3131300 was significantly associated with NSCLP in Hispanics and nominally associated in NHW families. Interestingly, PBX2 haplotypes including this variant were significantly associated in both ethnic groups. In multiplex Hispanic families, a marginal association was observed for TP63 rs9332461, and four haplotypes containing this variant were significantly associated. Haplotype-based associations are thought to be powerful approaches in addition to single variant analysis for complex diseases because the inclusion of flanking variants has the potential to capture cis-interactions (N. Liu, Zhang, & Zhao, 2008). In the case-control analysis, the most significant associations were seen with PBX1 rs6426870 and TP63 rs9332461, whereas no association was found for PBX2.

Family-based and case-control studies have different strengths in the types of associations detected. Family studies have the advantage that family members share a common genetic background and are also more likely to share environmental factors; whereas, case-control studies can be well-powered when of adequate sample size and controlled for population admixture effects (Evangelou, Trikalinos, Salanti, & Ioannidis, 2006; Laird & Lange, 2008). In this context, it is not unexpected to find that different variant associations from the family-based and case-control groups. The aim of validation studies is to obtain similar findings under modified influencing factors such as ethnic background, phenotype, or sampling scheme. As a consequence, results from validation studies can be different from those obtained with the discovery analysis because of both random and systematic variation (Igl, Konig, & Ziegler, 2009).

The associated variants in this study are all located in noncoding regions, downstream (rs3131300) and upstream (rs6426870 and rs9332461) of their respective genes, and therefore of potential functional significance. Although determining the biological effects of these variants will require functional studies, their location and predicted functions suggest biological relevance to craniofacial development and/or NSCLP. PBX2 rs3131300 is located in a conserved region associated with enhancer histone marks in skin, muscle and nerve tissues, and predicted to alter 13 transcription factor binding motifs, including Klf4 and p300. Interestingly, missense variants in Klf4, an important regulator of periderm differentiation, have also been identified in NSCLP cases (H. Liu et al., 2016). P300 is a multifunctional coactivator protein that exhibits protein/histone acetyltransferase activity and is essential for normal embryonic development and adult tissue homeostasis (Bhattacherjee et al., 2009; Warner, Smith, Smolenkova, Pisano, & Greene, 2016). Loss of p300 function in humans and in mice leads to craniofacial defects, possibly due to altered WNT and TGF-β signaling (Warner et al., 2016).

Craniofacial enhancer activity has recently been implicated in controlling facial morphogenesis during development and influencing the incidence of cleft phenotypes in mice (Attanasio et al., 2013; Uslu et al., 2014). PBX2 rs3131300 is noted as a significant eQTL in the GTEx database with allele-specific differences in gene expression, the A allele being associated with higher expression of PBX2 in whole blood. In turn, the alternate allele G, associated with NSCLP in our family-based analysis, may be predicted to decrease PBX2 expression. Although speculative, the association of this decreased function variant in humans would be in agreement with the observed effects of the reported murine Pbx regulatory module (Ferretti et al., 2011). Bioinformatic analysis of PBX1 rs6426870 also suggests potential functional effects with predicted alterations in 6 binding motifs, among them FOXD1, FOXF2, FOXK1, and FOXL1. Fox proteins have been shown to regulate palate, facial cartilage and tooth development (Leslie et al., 2017; Lidral et al., 2015; J. Xu et al., 2016; P. Xu et al., 2018). Among these, ample evidence exists suggesting that FOXE1 is associated with NSCLP risk, therefore additional studies addressing the potential relationship between PBX1 and FOX genes are warranted (Carlson et al., 2017). Additionally, in this study, NSCLP individuals had a higher frequency of the PBX1 rs6426870 T allele, which was shown to be an eQTL associated with lower PBX1 expression in whole blood. This suggests that this variant might contribute to lower PBX1 expression, corroborating the findings in the mouse model (Ferretti et al., 2011). Lastly, TP63 rs9332461 alternate allele G was predicted to have preferential binding to NKX2–1, a transcription factor in brain, lung and thyroid development (Manoli & Driever, 2014; Minocha et al., 2017). It was also located in enhancer mark-rich regions in bone and muscle tissues. Of note, these predictions are only suggestive as they obtained from gene expression data in whole blood, as there is currently a lack of a publicly available embryonic craniofacial cell/tissue database.

Evidence of multiple gene-gene interactions was detected between the studied variants in PBX1, PBX2, and TP63 with variants in other known cleft genes for which genotype data was available in our NSCLP families (Blanton et al., 2010; Blanton et al., 2005). The majority of the interactions identified in the present study were found in the NHW families, and included multiple markers in PBX1/PBX2/TP63 with IRF6 rs2235371. Interactions between PBX1 and WNT9B were also found in both NHW and Hispanic families. The IRF6 rs2235371 variant results in a valine to isoleucine substitution at position 274 (V274I) of the protein and has been consistently reported in association with NSCLP in many populations (Birnbaum et al., 2009; Blanton et al., 2010; Blanton et al., 2005; Jugessur et al., 2008; Park et al., 2007; T.M. Zucchero et al., 2003; T. M. Zucchero et al., 2004). Moreover, previous studies have demonstrated the important role of IRF6, TP63 and WNT pathway genes and their interactions in NSCLP and in craniofacial development (Brugmann et al., 2007; Gritli-Linde, 2010; Jiang et al., 2006; Leslie et al., 2017; D. Liu et al., 2018; Mani, Jarrell, Myers, & Atit, 2010; Mazemondet et al., 2011; Reynolds et al., 2019; Song et al., 2009; Wang, Song, & Zhou, 2011). The results of this study reflect statistical probabilities of gene-gene interactions, and yet revealed population-specific variant combinations in our NHW and Hispanic populations that further highlight the heterogeneous nature of NSCLP with many genes with etiologic and/or modifier roles contributing to the condition (Carter, 1976; Dixon et al., 2011; Mossey et al., 2009). In the study by Ferretti et al., a Pbx-Wnt-Tp63-Irf6 regulatory module was proposed based on the observations that mice lacking Pbx genes in the cephalic ectoderm exhibited fully penetrant cleft lip/palate and disruption of the Wnt-p63-Irf6 regulatory network caused by suppression of midfacial apoptosis (Ferretti et al., 2011). In our study, this regulatory model in humans was mainly reflected on the observed interactions between PBX1 with WNT9B and IRF6, and between PBX2 with IRF6 (Figure 1). Identification of potential biological interactions with purely statistical methods is easily overinterpreted, and utilizing a family-based approach limits the methods available (Ionita-Laza, Lee, Makarov, Buxbaum, & Lin, 2013; Laird & Lange, 2008). The observed statistical gene-gene interactions in the present study do not claim biological interactions; rather, they support the already existing biological evidence for the genes investigated (Ferretti et al., 2011). Additional biological studies addressing the relevance of the interactions identified in this study should further our knowledge of the complex genetic architecture of NSCLP.

Figure 1.

Figure 1

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PBX genes and their interactions contribute to clefting in mice and humans. In the pathway proposed by Ferretti et al (29), murine Pbx proteins bind to an intergenic region between Wnt3 and Wnt9b and regulate p63 expression, which in turn affects Irf6 . In humans, evidence from the present and previously published studies support the role of individual genes in the pathway in nonsyndromic cleft lip and palate (NSCLP). The results of our statistical gene-gene interaction analyses further support the observations in animal models and identified novel interactions contributing to NSCLP

The results of this study provide the first evidence for a role of PBX1/PBX2, additional evidence for the role of TP63, and support for the proposed PBX-WNT-TP63-IRF6 regulatory pathway in the etiology of human NSCLP. Studies focusing on identifying genes and regulatory networks that when disrupted lead to NSCLP have the potential to advance knowledge of the condition and directives for early diagnosis and prevention.

Supplementary Material

Supplementary material

ACKNOWLEDGMENTS

We thank the NSCLP families for participating in this study, Maria Elena Serna, Rosa Martinez, and Dr. Syed Hashmi for recruiting patients and families and for database management. This study was supported by NIH grants R01-DE11931 (to JTH and SHB) and R00-DE018954 (to AL). The authors have no conflict of interest to declare.

Footnotes

DATA AVAILABILITY

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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