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Keywords:

  • modifier gene;
  • Van der Woude;
  • popliteal pterygium;
  • cleft;
  • lip pit

ABSTRACT

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES
  9. Supporting Information

Van der Woude syndrome is the most common form of syndromic orofacial clefting, accounting for 1–2% of all patients with cleft lip and/or cleft palate. Van der Woude and popliteal pterygium syndromes are caused by mutations in IRF6, but phenotypic variability within and among families with either syndrome suggests that other genetic factors contribute to the phenotypes. The aim of this study was to identify common variants acting as genetic modifiers of IRF6 as well as genotype–phenotype correlations based on mutation type and location. We identified an association between mutations in the DNA-binding domain of IRF6 and limb defects (including pterygia). Although we did not detect formally significant associations with the genes tested, borderline associations suggest several genes that could modify the VWS phenotype, including FOXE1, TGFB3, and TFAP2A. Some of these genes are hypothesized to be part of the IRF6 gene regulatory network and may suggest additional genes for future study when larger sample sizes are also available. We also show that families with the Van de Woude phenotype but in whom no mutations have been identified have a lower frequency of cleft lip, suggesting there may be locus and/or mutation class differences in Van de Woude syndrome. © 2013 Wiley Periodicals, Inc.


INTRODUCTION

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES
  9. Supporting Information

Van der Woude syndrome (VWS; OMIM #119300) is the most common form of syndromic clefting, accounting for approximately 2% of all patients with cleft lip and/or palate, with a prevalence of 1/34,000 live births [Burdick, 1986]. The VWS is highly penetrant, with penetrance estimated to range from 89% to 99%, but with remarkably variable expressivity [Burdick et al., 1985]. Individuals with VWS can present with a range of clefting phenotypes, including cleft lip (CL), cleft lip and palate (CLP), and cleft palate only (CP). The primary feature distinguishing VWS from nonsyndromic clefting is the presence of paramedian lower lip pits or mounds [Van Der Woude, 1954]. Lip pits are observed in 83% of individuals diagnosed with VWS and are the only phenotypic feature seen in 44% of affected individuals [Burdick et al., 1985]. Lip pits and orofacial clefts are also observed in popliteal pterygium syndrome (PPS; OMIM #119500), but unlike VWS, PPS is further characterized by skin folds, genital anomalies, syndactyly, oral adhesions, and ankyloblepharon [Gorlin et al., 1968]. Even within families, there can be a broad range of severity in each of these two disorders, suggesting that other genetic or environmental factors play roles in determining the phenotypic features of VWS and PPS.

VWS and PPS are allelic, autosomal dominant disorders caused by mutations in interferon regulatory factor 6 (IRF6) [Kondo et al., 2002]. IRF6 belongs to a family of nine transcription factors with a highly conserved DNA-binding domain and a less conserved protein-binding domain [Tamura et al., 2008]. Mutations in patients with VWS are divided between missense and truncation mutations (including whole gene deletions), suggesting that haploinsufficiency is a likely mechanism underlying VWS [de Lima et al., 2009]. On the other hand, there is a strong correlation between PPS and missense mutations at residues of the DNA-binding domain predicted to contact the DNA, suggesting that PPS-causing mutations have a different effect on IRF6 function than do VWS-causing mutations. Despite these general genotype–phenotype differences between PPS and VWS, notably, mutations causing VWS are enriched in the DNA-binding domain and can also occur at residues contacting DNA [de Lima et al., 2009; Little et al., 2009]. Therefore, the presence and severity of the additional PPS-associated phenotypic features may in part be due to additional genetic or environmental factors.

We hypothesized that genetic variation in or around genes contributing to other forms of clefting contribute to the variable phenotype of individuals affected with VWS and PPS. Although hundreds of genes have been implicated in clefting through genetic and expression studies in humans and mice [Dixon et al., 2011; Marazita, 2012], here we perform a candidate gene association study of VWS and PPS to identify common genetic variation associated with these phenotypes using genes identified in genome-wide association or human mutation searches as high priority candidates. We also examined phenotypic correlations with the type and location of IRF6 mutations in these families.

MATERIALS AND METHODS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES
  9. Supporting Information

Candidate Genes

Twenty-eight SNPs from 12 candidate genes or loci (IRF6, 8q24, MAFB, ABCA4, VAX1, FOXE1, TGFA, TFAP2A, FGFR2, BMP4, TGFB3, and RIPK4) were chosen for analysis. SNPs for IRF6, 8q24, MAFB, ABCA4, and VAX1 were selected from SNPs with the lowest P-values from previous genome-wide association studies (GWAS) [Birnbaum et al., 2009; Beaty et al., 2010; Mangold et al., 2010; Ludwig et al., 2012]. Several SNPs near TFAP2A and RIPK4 were selected because mutations in these genes cause clefting syndromes with phenotypes that overlap VWS or PPS [Milunsky et al., 2008; Kalay et al., 2012; Mitchell et al., 2012]. FOXE1, TGFA, FGFR2, BMP4, and TGFB3 were selected because of compelling statistical or supporting biological evidence [Dixon et al., 2011; Marazita, 2012]. Supplemental eTable I (see Supporting Information Online) summarizes the genes selected and types of supporting data available in the literature. The SNPs for TFAP2A, RIPK4, FOXE1, TGFA, FGFR2, BMP4, and TGFB3 were chosen to interrogate the haplotype block structure of the relevant gene while also considering practicality and cost.

Samples

A total of 1,057 DNA samples from 225 families were used, with a total of 567 affected individuals. The families are from the United States, Philippines, Brazil, Colombia, and other European countries (Supplemental eTable II in supporting information online). Approval for all protocols was obtained from the Institutional Review Boards at the University of Iowa. All subjects were examined by a clinical geneticist or genetic counselor, and diagnoses were made as described [Kondo et al., 2002]. Affected subjects were given a phenotypic classification of cleft lip (CL), cleft lip with cleft palate (CLP), cleft palate (CP), lip pits only (PO), CL with lip pits (PCL), CLP with lip pits (PCLP), or CP with lip pits (PCP). Additional phenotypic classifications included the presence of dental anomalies (hypodontia, dental aplasia, or malocclusion), limb anomalies (cutaneous syndactyly, polydactyly, or contractures), or popliteal pterygia (Table I). Finally, family level phenotypes were assigned for each of these classifications based on the presence or absence of each phenotype in the affected family members (including family members without a DNA sample, there were 816 affected individuals).

Table I. Phenotypic Classification of Affected Individuals
Main phenotypes# Individuals
Note
  1. There were individuals with unknown laterality (14 PCL, 73 PCLP, 28 CLP, 19 CL).

Cleft lip and lip pits (PCL)55
Bilateral CL20
Unilateral CL21
Cleft lip and palate and lip pits (PCLP)182
Bilateral CL77
Unilateral CL32
Cleft palate and lip pits (PCP)128
Lip pits only (PO)184
Cleft palate (CP)87
Cleft lip and palate (CLP)54
Bilateral CL19
Unilateral CL7
Cleft lip (CL)24
Bilateral CL4
Unilateral CL1
Total main phenotypes714
Other phenotypes
Dental anomalies64
Limb anomalies57
Pterygia15
Total with other phenotypes136

Genotyping, Sequencing, Mutation Classification

Mutation analysis for IRF6 was performed as described [de Lima et al., 2009]. Mutations for affected individuals in this study are available in Supplemental Table III. Genotype analysis was performed as described [Rahimov et al., 2008]. IRF6 mutations were classified as truncating (large deletions, frameshift, nonsense, splicing, and those predicted to alter the translation start site) or nontruncating (missense mutations and small in-frame deletions or insertions). Mutations were also classified by domain [de Lima et al., 2009]. Within the DNA-binding domain, mutations were divided into those that contact DNA (P12, W13, L14, W40, A43, R45, H46, W60, K66, E69, A77, K80, Q82, R84, C85, N88, K89, K101, and E102) and those that do not contact DNA.

Analysis

All SNPs were tested for departures from Hardy–Weinberg equilibrium (HWE) using a threshold of P = 0.001 but recognizing that the mixed ancestry of our populations make HWE testing a poor metric for data quality (Table II). The Family Based Association Test (FBAT) [Horvath et al., 2001] was used to test for association of both single markers and haplotypes with VWS phenotypes. We utilized the -e option in FBAT, which allows for the adjustment for multiple affecteds or multiple nuclear families within a pedigree by adjusting the variance used in the Z-score calculation. Table I summarizes the phenotypes of affected individuals. Larger groups for analysis were made by combining all families; all individuals with a cleft lip (CL/P: CL or CLP); all individuals with a cleft palate (CP/L: CLP or CP); all individuals with lip pits (LP: PO, PCL, PCLP, and PCP); and those with clefts but no lip pits. The threshold P-value for modifier analysis was determined using a Bonferroni correction (P = 0.05/196 (12 loci × 14 phenotypes) = 0.0003).

Table II. Alleles and Frequencies of SNPs Studied
ChrSNPbp (Hg19)GeneMinor alleleMajor alleleMAFNHWE
  1. MAF, minor allele frequency.

  2. N = number of founders genotyped.

1rs56042694553438ABCA4GA0.4333390.023
1rs48193194570016ABCA4AC0.3892910.122
1rs414781194575056ABCA4AG0.4082890.112
1rs2235371209964080IRF6AG0.053390.117
1rs642961209989270IRF6AG0.1873290.162
2rs290234570716599TGFATC0.453190.031
2rs45430570736219TGFAGA0.3163070.038
6rs30305010403183TFAP2ACT0.1763220.383
6rs167541410412188TFAP2ACT0.4082980.082
8rs9875251299461548q24AC0.2033080.006
9rs3758249100614140FOXE1AG0.3123160.034
9rs4460498100620412FOXE1TC0.2852790.002
10rs7078160118827560VAX1AG0.1862690.003
10rs3750817123332577FGFR2TC0.3983240.027
10rs2981582123352317FGFR2TC0.3922970.580
14rs1756354417522BMP4TC0.4863220.044
14rs226862576439634TGFB3CT0.203300.101
14rs228479276443579TGFB3CT0.2993080.049
14rs1048387376508770TGFB3AG0.3433120.163
20rs1782094339268516MAFBTC0.3853120.683
20rs1304124739269074MAFBCT0.3853210.592
20rs1169625739270816MAFBTC0.3993010.585
21rs317153243160015RIPK4AG0.4092850.272
21rs727810343163288RIPK4AC0.1332900.359
21rs658623543174436RIPK4CA0.4932830.594
21rs385070543180208RIPK4CT0.2602751.000
21rs230910743184597RIPK4AG0.2412760.452
21rs491994443185765RIPK4AC0.3102950.294

We performed genotype–phenotype correlation analyses between IRF6 mutations and family level phenotypes with Fisher's exact test using STATA. Exclusion criteria for this analysis included all individuals diagnosed with VWS but without a known IRF6 mutation, individuals without specific descriptions of the craniofacial features (i.e., cleft type or presence of lip pits), and individuals with a cleft phenotype but where the familial IRF6 mutation was not identified by direct sequencing (i.e., potential phenocopies). We then assumed that all affected individuals within the same family shared the same mutation. A broader genotype–phenotype correlation analysis compared family-level phenotypes between IRF6 mutation-positive families and IRF6 mutation-negative families. The threshold P-value for these genotype–phenotype correlation analyses was also calculated using a Bonferroni correction (P = 0.05/11 phenotypes = 0.005).

RESULTS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES
  9. Supporting Information

Genetic Modifiers of the VWS Phenotype

To test for genetic modifiers of the VWS phenotype, we classified the VWS phenotype into 11 phenotypic groups and genotyped 225 families for 28 SNPs (Table II). The results of the TDT analyses are depicted in Figure 1, and the most significant results are detailed in Table III (all results are available in Supplemental eTable IV in Supporting Information Online). None of the results reached formal levels of significance; however, several results were interesting in that they had P values (uncorrected) of less than 0.05. For example, rs3758249 (FOXE1) was associated with CL/P (P = 0.03) as was the G-C haplotype of FOXE1 (P = 0.007). Similarly, rs2235371 (IRF6) and rs7278103 (RIPK4) were also associated with CL/P (P = 0.02 for both SNPs).

image

Figure 1. Results of TDT for phenotypic subgroups. A: TDT for main clefting phenotypes: VWS, Van der Woude syndrome; CL, cleft lip; CLP, cleft lip and palate; CP, cleft palate. B: TDT for cleft lip phenotypes: CL ± P, cleft lip with or without cleft palate; BL CL, bilateral cleft lip; UL CL, unilateral cleft lip. C: TDT for lip pit phenotypes: no lip pits, lip pits only, and lip pits with or without a cleft.

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Table III. Most Significant Results From Single Marker and Haplotype Analyses
GeneSNPAssociated alleleVWSCL/PCP/LBilateral CLPOLip pits
  • —, global P-value > 0.05.

  • CL/P, cleft lip with or without cleft palate; CP/L, cleft palate with or without cleft lip; CL, cleft lip; PO: lip pits only.

  • *

    Global P-values < 0.05.

Single marker analysis
IRF6rs2235371A0.050.020.040.010.390.02
TGFArs2902345C0.0950.630.330.510.140.03
FOXE1rs3758249G0.250.030.210.010.480.41
FGFR2rs2981582T0.680.080.720.040.560.32
TGFB3rs2284792T0.020.400.340.430.030.02
RIPK4rs7278103T0.3220.020.260.040.030.66
Haplotype analysis*
IRF6rs2235371–rs642961A-G0.01
TGFArs2902345–rs454305C-A0.05
TFAP2Ars303050–rs1675414T-T0.01
FOXE1rs3758249–rs4460498G-C0.007
RIPK4rs3171532–rs7278103A-A0.05

Lip pits are a distinguishing feature of VWS but are not always present, so we looked for associations with the presence or absence of lip pits (1C). The SNPs rs2235371 (IRF6), rs2902345 (TGFA), and rs2268625 (TGFB3) were associated with lip pits, without regard for cleft phenotype (P = 0.03) while rs2268625 (TGFB3) and rs7278103 (RIPK4) were associated with PO (P = 0.03 for both SNPs). Although neither TFAP2A SNP was significant independently, the T-T haplotype was associated with PO (P = 0.01). We did not observe any associations with clefts without lip pits.

Another indicator of genetic modifiers of the VWS phenotype could be an association between ancestry and these phenotypes. Genetic markers can be used to distinguish continental population groups on the basis of differing minor allele frequencies between populations [Rosenberg 2002]. Therefore, variants with differing allele frequencies between populations could act as modifiers of the VWS phenotype in a population-specific manner. To determine if VWS phenotypic heterogeneity was associated with ancestry/population, we analyzed the phenotypes of VWS families with IRF6 mutations by population, when known. While there was variation in the frequency of these phenotypes between populations, these differences were not significantly different.

Mutations in the DNA-Binding Domain Are Associated With Limb Defects

We next performed association analyses in 124 families to determine if either the type or location of IRF6 mutations was correlated with any VWS or PPS phenotypes (Table IV). Neither missense mutations nor truncation mutations were associated with any of the clefting phenotype categories. In 5 of the 124 families, the only phenotype present was lip pits; there was no association between the mutation type or domain and the risk of having a cleft.

Table IV. Association Analysis of VWS Phenotypes With Types and Locations of IRF6 Mutations in 124 Families
PhenotypeMutation typeMutations contacting DNAIRF6 domains
P-valueAssociated mutation typeP-valueP-valueAssociated domain
  1. DBD, DNA-binding domain; PBD, protein-binding domain.

  2. a

    Includes individuals with or without lip pits.

Cleftinga
Cleft lip0.05Truncation1.000.27 
Cleft lip and palate0.70 0.160.71 
Cleft palate0.05Missense1.000.71 
Lip pits
Cleft only0.36 1.000.62 
Lip pits only0.86 0.260.01PBD
Lip pits0.69 0.100.30 
Other features
Limb defects0.03Missense0.0020.002DBD
Pterygia0.30 0.020.04DBD
Dental anomalies0.46 1.000.54 

Limb defects were associated with missense mutations (P = 0.03). Families with missense mutations were 3.1 times more likely to have limb defects than those with truncation mutations (95% confidence interval (CI) 1.05–9.56). We found that mutations in the DNA-binding domain, and more specifically in residues contacting DNA, were significantly associated with limb defects (P = 0.002 and P = 0.001, respectively). Families with mutations at these residues were 13 times more likely to have limb defects than those with mutations elsewhere in the DNA-binding domain (CI: 2.6–65.2). Interestingly, these associations were not driven by the IRF6 p.Arg84Cys or p.Arg84His mutations, which commonly cause PPS, as families with missense mutations and limb defects were evenly split between p.Arg84Cys or p.Arg84His and other mutations. Although PPS is highly associated with mutations in the DNA-binding domain, we did not detect strong associations with the pterygia phenotype. This result was likely due to insufficient power as there were only eight families with pterygia.

We also detected a nominally significant association between mutations in the protein-binding domain and PO (P = 0.01). Families with mutations in this domain were 2.9 times more likely to have family members with PO than families with mutations in the rest of IRF6 (P = 0.008, OR = 2.9; CI: 1.3–6.8). Within the families that had PO and mutations in this domain, the mutation type was evenly divided between truncation and missense; no single mutation predominated.

Mutation Negative Families Are Less Likely to Have Cleft Lip

Of the 225 families included in this study, 56 (25%) did not have detectable mutations in IRF6. We compared the phenotypes of these mutation-negative families with the 124 families with IRF6 mutations (Table V, 2). Mutations in IRF6 were significantly associated with all cleft lip phenotypes (P = 2.1 × 10−4 for CL/P, OR = 3.5; CI: 1.8–6.9). In addition, while 95.2% of families with IRF6 mutations had lip pits, only 89.3% of mutation-negative families had lip pits (P = 0.14). While this difference was not statistically significant, the differences in the lip pit phenotype led to the question of whether these mutation-negative families would exhibit linkage between their phenotypes and the IRF6 locus. The average number of individuals per family was small (5.2), with small numbers of affected individuals (average = 2.5), so there was insufficient power to perform linkage analyses. We used TDT to look for association between syndrome occurrence in these 56 families and the 12 candidate genes/loci. This analysis showed a nonsignificant association with rs10483873 (TGFB3, P = 0.02), but the number of informative families was too low to draw conclusions (N informative families < 18).

Table V. Comparison of Mutation-Positive and Mutation-Negative VWS Families
Phenotype% Mutation-positive families (N = 124)% Mutation-negative families (N = 56)P-valueOR [95% CI]
  1. a

    Includes individuals with or without lip pits.

Cleftinga
Cleft lip30.78.90.0014.51 [1.7–12.2]
Cleft lip and palate66.944.60.0062.51 [1.3–4.8]
Cleft palate54.048.20.47 
Lip pits
Cleft only39.542.90.74 
Lip pits only44.441.10.68 
Lip pits95.289.30.20 
Other features
Limb defects20.214.30.41 
Pterygia6.53.60.73 
Dental anomalies16.110.70.49 
image

Figure 2. Comparison of phenotypes between mutation-positive and mutation-negative VWS families. Fifty-six mutation-negative families (white) and 124 families with IRF6 mutations (black) were compared for 11 phenotypes. CL, cleft lip; CLP, cleft lip and palate; CL/P, cleft lip with or without cleft palate; CPO, cleft palate only; CP/L, cleft palate with or without cleft lip. **P < 0.01, ***P < 0.0001.

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DISCUSSION

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES
  9. Supporting Information

VWS and PPS have considerable variability in phenotypes within and between families, prompting us to evaluate genetic variation in genes other than IRF6 (that are known to be involved in craniofacial development) as modifiers of the VWS phenotype. Orofacial clefts are a heterogeneous group of disorders, and epidemiological [Grosen 2010] and biological [Rahimov 2008] data suggest that CL and CLP may have separate genetic etiologies. However, except for several interesting but not formally significant associations with IRF6, we did not detect associations between the candidate genes and the major clefting phenotypes (CL, CLP, CP). The rs2235371 SNP (IRF6) was associated using uncorrected values for significance with several phenotypes, but the minor allele frequency of this SNP was low (5%). Furthermore, the observed association was with the minor allele (A), which is contrary to the previous associations with nonsyndromic clefting [Zucchero et al., 2004; Rahimov et al., 2008]. This result may reflect linkage of VWS to this SNP, rather than suggesting a role for this SNP as a modifier.

Clefts involving the lip are also heterogeneous, and may be either unilateral or bilateral; to date, no SNPs have been specifically associated with unilateral or bilateral CL. We found nonsignificant associations of FOXE1 and RIPK4 with CL/P and bilateral CL. FOXE1 was identified as a cleft candidate gene by a genome-wide linkage scan and subsequent fine-mapping [Marazita et al., 2004; Moreno et al., 2009]. Expression of Foxe1 has been described in the epithelium during fusion of the medial nasal and maxillary processes, consistent with a role in CL.

The feature distinguishing VWS from nonsyndromic clefting is the presence of paramedian lower lip pits. The association of TFGB3 with lip pits regardless of the presence of a cleft suggests a role for TGFB3 in the development of lip pits, although the role of TGFB3 in lip development has not been specifically examined. The TGFB3 gene has been associated with clefting through animal models, in which Tgfb3−/− mice have a cleft palate [Kaartinen 1995; Proetzel et al., 1995]. We found evidence for TGFA as a modifier of the lip pit phenotype in VWS; recently IRF6 and TGFA were shown to interact to increase risk for nonsyndromic CL/P [Letra 2012]. A role for TFAP2A in lip pits is not surprising as mutations in TFAP2A cause branchiooculofacial syndrome, which has overlapping features with VWS, including cleft lip, cleft palate, and upper lip pits [Milunsky 2008]. In addition, TFAP2A binds to an enhancer element upstream of IRF6 [Rahimov et al., 2008; Fakhouri et al., 2012]. In contrast, a role for RIPK4 in lip pits is unclear because a feature distinguishing Bartsocas–Papas syndrome (BPS) from PPS is the absence of lower lip pits in BPS. However, Ripk4 is a transcriptional target of p63, which also regulates Irf6, so it is possible that variability in this pathway modifies the VWS or PPS phenotype, including the presence of lip pits.

We did not identify associations with SNPs near 8q24, MAFB, ABCA4, or VAX1, all of which have been associated with nonsyndromic clefting by GWAS [Birnbaum et al., 2009; Beaty et al., 2010; Mangold et al., 2010], or with candidate genes FGFR2 or BMP4. While these results could indicate that common genetic variation at these loci does not contribute to VWS phenotypic variation, they may also reflect a lack of power. One limitation of this study was the sample size. Although we included a large number of families with VWS, the number of informative families for the various analyses (ranging from 0 to 76) depended on both the frequency of each phenotype in these families and on the minor allele frequencies of the SNPs. Another limitation was the ethnic heterogeneity of the samples, which included families from Brazil, Colombia, the Philippines, and the United States, but 33% of the families were from unspecified populations (Supplemental eTable II in Supporting Information Online).

We also examined the types and locations of IRF6 mutations to determine if these contributed to specific VWS phenotypes. We would have predicted that truncation mutations, which are most obviously damaging to the protein, would be associated with more severe phenotypes such as CLP. We did not find such an association, suggesting that both missense and truncation mutations result in haploinsufficiency of IRF6 and result in similar phenotypes. The exception to this is the strong association between missense mutations in the DNA-binding domain (and more specifically, residues that contact DNA) with limb defects. This result was expected and was consistent with previous data showing a strong association between mutations at residues contacting DNA and PPS [de Lima 2009]. In addition, we found a nonsignificant association between mutations in the protein-binding domain and lip pits only. There was no association when we grouped all individuals with lip pits together, suggesting that mutations in the protein-binding domain may not be related to the development of lip pits but rather may protect against the development of clefts.

Although statistically we only detected associations that met nonstringent (P < 0.05) significance, it is notable that they tended to be with genes related to the IRF6 gene regulatory network, including TFAP2A [Rahimov 2008], FOXE1 [Venza 2011], and TGFB3 [Knight 2006]. Recently, the IRF6 gene-regulatory network has been expanded to include Pbx-1, 2, and 3 regulating p63 through Wnt9b and Wnt3 [Ferretti 2011]. We propose that these genes and TP63, which regulates IRF6 through the enhancer MCS9.7 [Rahimov et al., 2008; Moretti et al., 2010; Thomason et al., 2010; Fakhouri et al., 2012], be investigated in future modifier studies.

The MCS9.7 enhancer was not associated in this study (through rs642961); however, its expression pattern does not completely recapitulate the expression of IRF6 [Fakhouri 2012], suggesting that IRF6 expression is controlled by additional regulatory elements. It is possible that variation in these yet to be discovered regulators and regulatory elements contribute to the variable expressivity of VWS and PPS. We also showed that the mutation-negative families are less likely to have individuals with cleft lip. The causal mutations in the IRF6-mutation-negative families may lie in these undiscovered regulatory elements. A second locus for VWS has been reported on 1p34 by linkage [Koillinen 2001], and a mutation has been described in WDR65, although not in the linkage family [Rorick 2011]. Locus heterogeneity could explain the phenotypic differences in the mutation-negative families. Although the association with TGFB3 was not formally significant, this gene may contribute directly or indirectly (as a modifier) to the development of VWS in the families in which a mutation has not been identified.

Although VWS is highly penetrant, there are individuals who are obligate carriers with no phenotypic expression, and VWS family members with only a cleft or only lip pits may have additional subtle phenotypes. There is increasing evidence that subclinical phenotypes are part of the expression of nonsyndromic clefting risk genes in family members with no overt cleft. For example, sub-epithelial discontinuities in the orbicularis oris muscle (OOM) [Neiswanger et al., 2007; Rogers 2008] and lip print whorls [Neiswanger 2009] are significantly increased in relatives of individuals with clefts versus controls with no family history of craniofacial anomalies. Notably, we found that the unaffected father of an individual with VWS had OOM discontinuities and carried the same mutation as his child (unpublished results, Schutte, Marazita). Also, lip print whorls are seen in the same paramedian region where lip pits are found, even when no pitting or mounding is evident [Neiswanger 2009]. Therefore, additional assessment of such subclinical phenotypes could also be instrumental in the genetic dissection of VWS phenotypic expression.

Here, we hypothesized that common variation contributes to VWS and PPS phenotypic expression, but it is equally likely that rare variants play a role. As sequencing costs have decreased, exome and whole genome sequencing have been used to uncover the causes of dozens of Mendelian disorders [Bamshad 2011]. We still do not have a good understanding of the variable expressivity seen in many of these disorders, including VWS. Eventually, we will need to revisit the families in which a mutation has already been identified using deep phenotyping and exome sequencing, targeted sequencing, or whole genome sequencing, to fully understand the genetic contributions to these Mendelian phenotypes.

ACKNOWLEDGMENTS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES
  9. Supporting Information

We sincerely thank all of the families who participated in this study and all administrative, clinical, and laboratory staff who helped make this work possible, especially M. Adela Mansilla and Aline Petrin for their support and technical assistance. This research was supported by National Institutes of Health grants R37-DE008559 (JCM), R01-DE013513 (BCS), R01-DE016148 (MLM), and the FaceBase consortium (U01-DE020057).

REFERENCES

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES
  9. Supporting Information

Additional supporting information may be found in the online version of this article at the publisher's web-site.

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ajmga36133-sm-0001-SuppData-S1.docx38K

Table SI. Summary of Data Supporting Candidate Genes

Table SII. Origin of VWS/PPS Families

Table SIII. IRF6 Mutations in VWS and PPS in this study

Table SIV. TDT Results for All SNPs and Phenotypes

Table SV. Comparison of Phenotypes in VWS Families With IRF6 Mutations by Population

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.