Abstract

Non-syndromic cleft lip with or without cleft palate (NSCLP) is a common birth defect. Genetic and environmental factors have been causally implicated and studies have begun to delineate genetic contributions. The Wnt genes are involved in regulating mid-face development and upper lip fusion and are therefore strong candidates for an etiological role in NSCLP. Furthermore, the clf1 region in A/WyN clefting susceptible mice contains the Wnt3 and Wnt9B genes. To assess the role of the Wnt family of genes in NSCLP, we interrogated seven Wnt genes (Wnt3, Wnt3A, Wnt5A, Wnt7A, Wnt8A, Wnt9B and Wnt11) in our well-defined NSCLP dataset. Thirty-eight single nucleotide polymorphisms were genotyped in 132 multiplex NSCLP families and 354 simplex parent–child trios. In the entire dataset, single-nucleotide polymorphisms (SNPs) in three genes, Wnt3A (P = 0.006), Wnt 5A (P = 0.002) and Wnt11 (P = 0.0001) were significantly associated with NSCLP after correction for multiple testing. When stratified by ethnicity, the strongest associations were found for SNPs in Wnt3A (P = 0.0007), Wnt11 (P = 0.0012) and Wnt8A (P = 0.0013). Multiple haplotypes in Wnt genes were associated with NSCLP, and gene–gene interactions were observed between Wnt3A and both Wnt3 and Wnt5A (P = 0.004 and P = 0.039, respectively). This data suggests that alteration in Wnt gene function may perturb formation and/or fusion of the facial processes and predispose to NSCLP.

INTRODUCTION

Non-syndromic cleft lip with or without cleft palate (NSCLP) is a common birth defect with a prevalence of 1/700 live births (1–3). NSCLP is a complex malformation caused by the interaction of multiple genes and environmental factors (4). Genetic causality has been slow to be identified, but variations in a small number of genes have been shown to contribute to a non-syndromic orofacial clefting phenotype, including interferon regulatory 6 (IRF6), Msh homeobox homolog 1 (MSX1), transforming growth factor alpha (TGFα), and cysteine-rich secretory protein Limulus factor C-related region (LCCL) domain containing 2 (CRISPLD2) (4–12). Approximately 12–25% of the genetic variation causing NSCLP is estimated to have been identified (4,10,13,14).

In an effort to identify additional NSCLP genetic loci, we interrogated the Wnt gene family members. These genes were chosen because Wnt3A, Wnt5A, Wnt8A and Wnt11 play important roles in murine craniofacial embryogenesis; Wnt expression is observed in the upper lip and primary and secondary palates and Wnts are involved in regional specification of the vertebrate face (15–22). Further evidence supporting Wnt genes as possible clefting loci comes from the inbred A/WySn mouse strain where ∼5–30% are born with a cleft lip and palate (CL/P) (23). Two cleft loci, clf1 and clf2, were mapped in these mice (23). Wnt3 and Wnt9B genes are in the clf1 region; however, no causative variation within these two genes has been identified. An inserted transposable element, unique to the A/WySn strain, was found 6.6 kb downstream of the Wnt9B gene. This insertion is thought to inhibit normal Wnt9B expression and contribute to the A/WySn clefting phenotype (24). Additionally, mice heterozygous for the clf1 locus and Wnt9B-null alleles exhibit penetrance of up to 67% for cleft lip (25). Wnt expression and A/WySn murine studies indicate that the Wnt family of genes plays a critical role in the development of the lip and palate. This study was undertaken to interrogate seven Wnt genes (Wnt3, Wnt3A, Wnt5A, Wnt7A, Wnt8A, Wnt9B and Wnt11) to determine if variation in members of this gene family is associated with NSCLP.

RESULTS

All single-nucleotide polymorphisms (SNPs) were in Hardy–Weinberg equilibrium. Comparisons of SNP allele frequencies, after Bonferroni correction, showed significant differences between European Americans and Hispanics (P < 0.001) in three of the seven genes (Table 1). Therefore, in addition to an overall analysis of the complete dataset, the data were stratified by ethnicity and examined separately. There was no evidence for linkage under any of the tested parametric models or with non-parametric methods for any of the regions (data not shown).

Table 1.

SNP alleles and frequencies by ethnicity

Gene SNP Chr Base pair Order in gene Allelesa European American MAFb Hispanic corresponding frequencyc P-value 
Wnt3 rs142167 17 42150418 A/G 0.258 0.523 <0.0001 
Wnt3 rs7216231 17 42170907 A/G 0.055 0.409 <0.0001 
Wnt3 rs199525 17 42203002 G/T 0.22 0.159 0.07 
Wnt3 rs70602 17 42214876 C/T 0.219 0.149 0.041 
Wnt3 rs199501 17 42217772 A/G 0.256 0.538 <0.0001 
Wnt3 rs199498 17 42220763 C/T 0.241 0.489 <0.0001 
Wnt3 rs111769 17 42227151 C/T 0.416 0.258 <0.0001 
Wnt3 rs3851781 17 42246300 C/T 0.474 0.657 <0.0001 
Wnt3 hCV1139279 17 42248220 C/G 0.198 0.11 0.005 
Wnt3 rs9890413 17 42256448 10 A/G 0.363 0.215 0.0002 
Wnt3A rs708111 226257988 C/T 0.483 0.524 0.290 
Wnt3A rs708114 226264119 C/T 0.469 0.557 0.034 
Wnt3A rs3094912 226276438 A/T 0.454 0.5 0.228 
Wnt3A rs3121310 226291447 A/G 0.308 0.382 0.055 
Wnt3A rs752107 226313974 C/T 0.308 0.356 0.175 
Wnt3A rs1745420 226318355 C/G 0.144 0.178 0.193 
Wnt5A rs566926 55487719 A/C 0.261 0.333 0.55 
Wnt7A rs1124480 13832970 C/T 0.459 0.418 0.274 
Wnt7A rs9840696 13840076 A/G 0.375 0.392 0.651 
Wnt7A rs6778046 13846680 C/T 0.369 0.415 0.212 
Wnt7A rs9863149 13858361 C/T 0.315 0.216 0.004 
Wnt7A rs934450 13862730 A/G 0.329 0.257 0.037 
Wnt7A rs1433354 13872246 A/G 0.305 0.453 <0.0001 
Wnt7A rs6442414 13881839 A/G 0.312 0.221 0.008 
Wnt7A rs11128663 13887851 C/G 0.4 0.297 0.005 
Wnt7A rs4685048 13897736 A/C 0.487 0.412 0.046 
Wnt8A rs4835761 137445768 A/G 0.469 0.489 0.619 
Wnt8A rs2040862 137447888 C/T 0.172 0.108 0.030 
Wnt8A rs2306110 137455986 A/C 0.477 0.489 0.765 
Wnt9B rs2165846 17 42296365 A/G 0.421 0.720 <0.0001 
Wnt9B rs1530364 17 42306776 A/G 0.261 0.317 0.128 
Wnt9B rs197915 17 42345521 A/G 0.434 0.432 0.972 
Wnt11 rs663746 11 75571777 C/T 0.498 0.474 0.524 
Wnt11 rs1533763 11 75578175 A/T 0.209 0.172 0.272 
Wnt11 rs1533767 11 75583448 A/G 0.267 0.279 0.718 
Wnt11 rs689095 11 75591735 A/G 0.307 0.324 0.667 
Wnt11 rs596339 11 75594155 A/G 0.338 0.412 0.06 
Wnt11 rs1568507 11 75596967 A/T 0.315 0.324 0.82 
Gene SNP Chr Base pair Order in gene Allelesa European American MAFb Hispanic corresponding frequencyc P-value 
Wnt3 rs142167 17 42150418 A/G 0.258 0.523 <0.0001 
Wnt3 rs7216231 17 42170907 A/G 0.055 0.409 <0.0001 
Wnt3 rs199525 17 42203002 G/T 0.22 0.159 0.07 
Wnt3 rs70602 17 42214876 C/T 0.219 0.149 0.041 
Wnt3 rs199501 17 42217772 A/G 0.256 0.538 <0.0001 
Wnt3 rs199498 17 42220763 C/T 0.241 0.489 <0.0001 
Wnt3 rs111769 17 42227151 C/T 0.416 0.258 <0.0001 
Wnt3 rs3851781 17 42246300 C/T 0.474 0.657 <0.0001 
Wnt3 hCV1139279 17 42248220 C/G 0.198 0.11 0.005 
Wnt3 rs9890413 17 42256448 10 A/G 0.363 0.215 0.0002 
Wnt3A rs708111 226257988 C/T 0.483 0.524 0.290 
Wnt3A rs708114 226264119 C/T 0.469 0.557 0.034 
Wnt3A rs3094912 226276438 A/T 0.454 0.5 0.228 
Wnt3A rs3121310 226291447 A/G 0.308 0.382 0.055 
Wnt3A rs752107 226313974 C/T 0.308 0.356 0.175 
Wnt3A rs1745420 226318355 C/G 0.144 0.178 0.193 
Wnt5A rs566926 55487719 A/C 0.261 0.333 0.55 
Wnt7A rs1124480 13832970 C/T 0.459 0.418 0.274 
Wnt7A rs9840696 13840076 A/G 0.375 0.392 0.651 
Wnt7A rs6778046 13846680 C/T 0.369 0.415 0.212 
Wnt7A rs9863149 13858361 C/T 0.315 0.216 0.004 
Wnt7A rs934450 13862730 A/G 0.329 0.257 0.037 
Wnt7A rs1433354 13872246 A/G 0.305 0.453 <0.0001 
Wnt7A rs6442414 13881839 A/G 0.312 0.221 0.008 
Wnt7A rs11128663 13887851 C/G 0.4 0.297 0.005 
Wnt7A rs4685048 13897736 A/C 0.487 0.412 0.046 
Wnt8A rs4835761 137445768 A/G 0.469 0.489 0.619 
Wnt8A rs2040862 137447888 C/T 0.172 0.108 0.030 
Wnt8A rs2306110 137455986 A/C 0.477 0.489 0.765 
Wnt9B rs2165846 17 42296365 A/G 0.421 0.720 <0.0001 
Wnt9B rs1530364 17 42306776 A/G 0.261 0.317 0.128 
Wnt9B rs197915 17 42345521 A/G 0.434 0.432 0.972 
Wnt11 rs663746 11 75571777 C/T 0.498 0.474 0.524 
Wnt11 rs1533763 11 75578175 A/T 0.209 0.172 0.272 
Wnt11 rs1533767 11 75583448 A/G 0.267 0.279 0.718 
Wnt11 rs689095 11 75591735 A/G 0.307 0.324 0.667 
Wnt11 rs596339 11 75594155 A/G 0.338 0.412 0.06 
Wnt11 rs1568507 11 75596967 A/T 0.315 0.324 0.82 

aMost common allele are listed first.

bMinor allele frequency.

cFrequency in Hispanic of European American minor allele.

Pedigree disequilibrium test (PDT) and Geno-PDT results for the entire dataset are presented in Table 2. Out of the seven genes interrogated, five have SNPs that meet a nominal threshold of 0.05. If we correct the significance level for the number of genes tested, then SNPs in three genes (Wnt3A, Wnt5A and Wnt11) still yield significant results (P < 0.007). Table 3 and Supplementary Material, Tables S1 and S2 contain the results of PDT, Geno-PDT a]nd association in the presence of linkage (APL) analyses when the families were grouped by ethnicity and also subdivided according to the presence or absence of family history (FH). SNPs meeting a nominal threshold (P-value < 0.05) were found in each gene, generally by multiple methods. In the overall European American sample, and when stratified by FH, associations were identified with multiple SNPs in Wnt3, Wnt5A and Wnt11 (Table 3). In addition, SNPs in Wnt7A were significant in the total European American sample and in the positive FH subgroup, while an SNP in Wnt9B was significant only in the positive FH subgroup. Multiple SNPs in Wnt3A were significant in the total European American sample and in the negative FH subgroup, and an SNP in Wnt8A was significant in the negative FH subgroup.

Table 2.

Results of association testing in complete dataseta

Gene SNP PDT Geno-PDT 
Wnt3 rs142167 0.147 0.278 
Wnt3 rs7216231 0.359 0.584 
Wnt3 rs199525 0.353 0.636 
Wnt3 rs70602 0.132 0.302 
Wnt3 rs199501 0.190 0.249 
Wnt3 rs199498 0.269 0.220 
Wnt3 rs111769 0.498 0.331 
Wnt3 rs3851781 0.914 0.219 
Wnt3 hCV1139279 0.299 0.519 
Wnt3 rs9890413 0.853 0.741 
Wnt3A rs708111 0.127 0.310 
Wnt3A rs708114 0.378 0.570 
Wnt3A rs3094912 0.334 0.352 
Wnt3A rs3121310 0.034 0.088 
Wnt3A rs752107 0.021 0.022 
Wnt3A rs1745420 0.006 0.009 
Wnt7A rs1124480 0.290 0.314 
Wnt5A D3S3719 0.320 0.340 
Wnt5A rs566926 0.002 0.008 
Wnt5A D3S2408 0.757 0.358 
Wnt7A rs9840696 0.272 0.483 
Wnt7A rs6778046 0.172 0.049 
Wnt7A rs9863149 0.183 0.415 
Wnt7A rs934450 0.209 0.454 
Wnt7A rs1433354 0.228 0.437 
Wnt7A rs6442414 0.665 0.091 
Wnt7A rs11128663 0.229 0.332 
Wnt7A rs4685048 0.010 0.049 
Wnt8A rs4835761 0.490 0.067 
Wnt8A rs2040862 0.306 0.045 
Wnt8A rs2306110 0.535 0.164 
Wnt9B rs2165846 0.962 0.971 
Wnt9B rs1530364 0.869 0.939 
Wnt9B rs197915 0.469 0.149 
Wnt11 rs663746 0.083 <0.001 
Wnt11 rs1533763 0.199 0.378 
Wnt11 rs1533767 0.099 0.009 
Wnt11 rs689095 0.386 0.151 
Wnt11 rs596339 0.747 0.747 
Wnt11 rs1568507 0.124 0.178 
Gene SNP PDT Geno-PDT 
Wnt3 rs142167 0.147 0.278 
Wnt3 rs7216231 0.359 0.584 
Wnt3 rs199525 0.353 0.636 
Wnt3 rs70602 0.132 0.302 
Wnt3 rs199501 0.190 0.249 
Wnt3 rs199498 0.269 0.220 
Wnt3 rs111769 0.498 0.331 
Wnt3 rs3851781 0.914 0.219 
Wnt3 hCV1139279 0.299 0.519 
Wnt3 rs9890413 0.853 0.741 
Wnt3A rs708111 0.127 0.310 
Wnt3A rs708114 0.378 0.570 
Wnt3A rs3094912 0.334 0.352 
Wnt3A rs3121310 0.034 0.088 
Wnt3A rs752107 0.021 0.022 
Wnt3A rs1745420 0.006 0.009 
Wnt7A rs1124480 0.290 0.314 
Wnt5A D3S3719 0.320 0.340 
Wnt5A rs566926 0.002 0.008 
Wnt5A D3S2408 0.757 0.358 
Wnt7A rs9840696 0.272 0.483 
Wnt7A rs6778046 0.172 0.049 
Wnt7A rs9863149 0.183 0.415 
Wnt7A rs934450 0.209 0.454 
Wnt7A rs1433354 0.228 0.437 
Wnt7A rs6442414 0.665 0.091 
Wnt7A rs11128663 0.229 0.332 
Wnt7A rs4685048 0.010 0.049 
Wnt8A rs4835761 0.490 0.067 
Wnt8A rs2040862 0.306 0.045 
Wnt8A rs2306110 0.535 0.164 
Wnt9B rs2165846 0.962 0.971 
Wnt9B rs1530364 0.869 0.939 
Wnt9B rs197915 0.469 0.149 
Wnt11 rs663746 0.083 <0.001 
Wnt11 rs1533763 0.199 0.378 
Wnt11 rs1533767 0.099 0.009 
Wnt11 rs689095 0.386 0.151 
Wnt11 rs596339 0.747 0.747 
Wnt11 rs1568507 0.124 0.178 

aAll SNPs with P < 0.01 are shown and P ≤ 0.007 are bolded.

Table 3.

Results of association testing by ethnicity and FHa

Gene SNP All Positive FH Negative FH 
  PDT Geno-PDT APL PDT Geno-PDT APL PDT Geno-PDT APL 
European Americans 
 Wnt3 rs70602 0.154 0.308 0.041 0.309 0.585 0.009 0.299 0.426 0.365 
 Wnt3 rs199498 0.162 0.239 0.161 0.087 0.139 0.006 1.000 0.941 0.947 
 Wnt3 rs9890413 0.948 0.698 0.593 0.932 0.216 0.504 0.840 0.005 0.835 
 Wnt3A rs752107 0.023 0.027 0.003 0.31 0.441 0.125 0.0178 0.027 0.009 
 Wnt5A rs566926 0.005 0.016 0.004 0.043 0.107 0.049 0.0462 0.144 0.02 
 Wnt8A rs2040862 0.352 0.055 0.061 0.581 0.372 0.085 0.009 0.016 0.001 
 Wnt9B rs197915 0.160 0.051 0.573 0.072 0.180 0.003 0.926 0.060 0.310 
 Wnt11 rs663746 0.067 0.001 0.449 0.104 0.047 0.278 0.361 0.014 0.849 
 Wnt11 rs1533767 0.114 0.012 0.001 0.744 0.010 0.100 0.006 0.016 0.003 
Hispanics 
 Wnt3A rs708111 NS NS NS 0.034 0.135 0.007 0.297 0.426 0.005 
 Wnt3A rs3094912 0.343 0.572 0.031 NS NS NS 0.162 0.240 <0.001 
 Wnt3A rs1745420 0.083 0.157 <0.001 0.096 0.264 0.009 0.346 0.376 0.008 
Gene SNP All Positive FH Negative FH 
  PDT Geno-PDT APL PDT Geno-PDT APL PDT Geno-PDT APL 
European Americans 
 Wnt3 rs70602 0.154 0.308 0.041 0.309 0.585 0.009 0.299 0.426 0.365 
 Wnt3 rs199498 0.162 0.239 0.161 0.087 0.139 0.006 1.000 0.941 0.947 
 Wnt3 rs9890413 0.948 0.698 0.593 0.932 0.216 0.504 0.840 0.005 0.835 
 Wnt3A rs752107 0.023 0.027 0.003 0.31 0.441 0.125 0.0178 0.027 0.009 
 Wnt5A rs566926 0.005 0.016 0.004 0.043 0.107 0.049 0.0462 0.144 0.02 
 Wnt8A rs2040862 0.352 0.055 0.061 0.581 0.372 0.085 0.009 0.016 0.001 
 Wnt9B rs197915 0.160 0.051 0.573 0.072 0.180 0.003 0.926 0.060 0.310 
 Wnt11 rs663746 0.067 0.001 0.449 0.104 0.047 0.278 0.361 0.014 0.849 
 Wnt11 rs1533767 0.114 0.012 0.001 0.744 0.010 0.100 0.006 0.016 0.003 
Hispanics 
 Wnt3A rs708111 NS NS NS 0.034 0.135 0.007 0.297 0.426 0.005 
 Wnt3A rs3094912 0.343 0.572 0.031 NS NS NS 0.162 0.240 <0.001 
 Wnt3A rs1745420 0.083 0.157 <0.001 0.096 0.264 0.009 0.346 0.376 0.008 

aAll SNPs with P < 0.01 are shown and P ≤ 0.007 are bolded.

When examining the entire Hispanic sample or when stratifying by FH, associations were identified for SNPs in Wnt3A and Wnt9B. Two SNPs were significant in the total sample; one of these was significant in the negative FH subgroup. One SNP in Wnt11 was significant in the positive FH subgroup and when the sample was considered as a whole. Lastly, an SNP in Wnt3 was significant in the positive FH subgroup. Overall, across ethnicity, the same SNPs were significant in four genes: Wnt3A (two SNPs), Wnt7A (four SNPs), Wnt11 (one SNP) and Wnt9B (one SNP) and both ethnicities had significant SNPs in Wnt3, although there was no overlap.

The results of haplotype analysis are shown in Table 4. Because of the small sample size, data were not stratified by FH. In both European American and Hispanic samples, four haplotypes in Wnt3A and one in Wnt8A were significantly associated (Table 4). Wnt11 haplotypes were only significant in the European American sample (Table 4). While some of the SNPs within genes were in significant linkage disequilibrium (LD), most of the SNPs in the significantly overtransmitted haplotypes had r2 < 0.3 (Supplementary Material, Table S3).

Table 4.

Overtransmitted haplotypes by ethnicity

Gene SNP1 SNP ordera SNP2 SNP order Overtransmitted haplotype P-valueb 
European Americans 
 Wnt3 rs142167 rs199525 2.1 0.049 
 Wnt3 rs7216231 rs199525 2.1 0.033 
 Wnt3 rs199525 rs70602 1.2 0.05 
 Wnt3A rs708111 rs752107 2.1 0.010 
 Wnt3A rs708114 rs752107 1.1 0.014 
 Wnt3A rs3094912 rs752107 1.1 0.037 
 Wnt3A rs3121310 rs752107 2.1 0.017 
 Wnt3A rs752107 rs1745420 1.2 0.001 
 Wnt8A rs4835761 rs2306110 1.1 0.05 
 Wnt11 rs663746 rs1533767 2.2 0.047 
 Wnt11 rs1533763 rs596339 2.2 0.012 
 Wnt11 rs1533767 rs1568507 2.2 0.013 
Hispanics 
 Wnt3 rs199498 rs9890413 10 2.2 0.047 
 Wnt3A rs708111 rs3121310 1.1 0.022 
 Wnt3A rs708111 rs752107 1.1 0.008 
 Wnt3A rs708111 rs1745420 1.2 0.007 
Wnt3A rs708114 rs3094912 2.1 0.004 
 Wnt3A rs708114 rs1745420 2.2 <0.001 
 Wnt3A rs3094912 rs3121310 1.1 <0.001 
 Wnt3A rs3094912 rs752107 1.1 <0.001 
 Wnt3A rs3094912 rs1745420 1.2 0.003 
 Wnt3A rs3121310 rs752107 1.1 0.027 
 Wnt3A rs3121310 rs1745420 1.2 <0.001 
 Wnt3A rs752107 rs1745420 1.2 <0.001 
 Wnt8A rs4835761 rs2306110 2.2 0.049 
Gene SNP1 SNP ordera SNP2 SNP order Overtransmitted haplotype P-valueb 
European Americans 
 Wnt3 rs142167 rs199525 2.1 0.049 
 Wnt3 rs7216231 rs199525 2.1 0.033 
 Wnt3 rs199525 rs70602 1.2 0.05 
 Wnt3A rs708111 rs752107 2.1 0.010 
 Wnt3A rs708114 rs752107 1.1 0.014 
 Wnt3A rs3094912 rs752107 1.1 0.037 
 Wnt3A rs3121310 rs752107 2.1 0.017 
 Wnt3A rs752107 rs1745420 1.2 0.001 
 Wnt8A rs4835761 rs2306110 1.1 0.05 
 Wnt11 rs663746 rs1533767 2.2 0.047 
 Wnt11 rs1533763 rs596339 2.2 0.012 
 Wnt11 rs1533767 rs1568507 2.2 0.013 
Hispanics 
 Wnt3 rs199498 rs9890413 10 2.2 0.047 
 Wnt3A rs708111 rs3121310 1.1 0.022 
 Wnt3A rs708111 rs752107 1.1 0.008 
 Wnt3A rs708111 rs1745420 1.2 0.007 
Wnt3A rs708114 rs3094912 2.1 0.004 
 Wnt3A rs708114 rs1745420 2.2 <0.001 
 Wnt3A rs3094912 rs3121310 1.1 <0.001 
 Wnt3A rs3094912 rs752107 1.1 <0.001 
 Wnt3A rs3094912 rs1745420 1.2 0.003 
 Wnt3A rs3121310 rs752107 1.1 0.027 
 Wnt3A rs3121310 rs1745420 1.2 <0.001 
 Wnt3A rs752107 rs1745420 1.2 <0.001 
 Wnt8A rs4835761 rs2306110 2.2 0.049 

aSNP order: order of SNP within the gene.

bAll SNPs with P < 0.05 are shown and P ≤ 0.007 are bolded.

Generalized estimating equations (GEE) analysis identified significant gene–gene interactions for SNPs in Wnt3A and Wnt3 (P = 0.0037) in the European American sample, and SNPs in Wnt3A and Wnt5A in the Hispanic sample (P = 0.0388) (Table 5). Models for interaction considering dominant and recessive main effects were tested in the European American sample. The best model for increased susceptibility was when at least one copy of the rarer allele was present at each locus (P = 0.03, data not shown). Interestingly, when there was homozygosity for the common allele in Wnt3, the rare allele in Wnt3A was protective (P = 0.007, data not shown). The Hispanic sample was too small to evaluate the main effects.

Table 5.

Results of gene–gene interaction analysisa

Ethnicity Gene1/SNP Gene2/SNP GEE P-value 
European American Wnt3A/rs752107 Wnt3/rs70602 0.004 
Hispanic Wnt3A/rs1745420 Wnt5A/rs566926 0.039 
Ethnicity Gene1/SNP Gene2/SNP GEE P-value 
European American Wnt3A/rs752107 Wnt3/rs70602 0.004 
Hispanic Wnt3A/rs1745420 Wnt5A/rs566926 0.039 

aSNPs with P < 0.05 are shown and P < 0.007 is bolded.

DISCUSSION

In this study, we interrogated Wnt3, Wnt3A, Wnt5A, Wnt7A, Wnt8A, Wnt9B and Wnt11 to determine if any of them are implicated in the etiology of NSCLP. In the overall dataset (Table 2), SNPs in five of the genes (Wnt3A, Wnt7A, Wnt5A, Wnt8A and Wnt11) had nominal P-values of 0.05 or less. Three of these, Wnt3A, Wnt5A and Wnt11 remained significant after correction for multiple testing. Among the subsets of data, the strongest evidence for an association was found in the European American sample for SNPs primarily in Wnt3, Wnt3A, Wnt7A, Wnt8A, Wnt9B and Wnt11 (Table 3), although only SNPs in Wnt3, Wnt3A, Wnt5A, Wnt9 and Wnt11, meet the more restrictive P-value of 0.007. In the Hispanic sample, the strongest associations were found with SNPs in Wnt3A, although some evidence for association was also observed in Wnt3, Wnt7A, Wnt9B and Wnt11 (Table 3). SNPs in five genes (Wnt3, Wnt3A, Wnt7A, Wnt9B and Wnt11) had at least suggestive evidence for association in both ethnicities; in four of these genes (excluding Wnt3), eight of the associated SNPs were identical. Moreover, with the exception of Wnt7A, multiple haplotypes in these same genes were also associated with NSCLP (Table 4). This pattern is quite striking, although the analyses performed are not independent and the haplotype results were not corrected for multiple testing.

The issue of correcting for multiple tests is complicated when multiple methodologies are performed, and neither the tests nor all the SNPs are independent. Moreover, which correction factor to use is further complicated when the SNPs are in genes considered viable candidates from other scientific evidences. Therefore, we elected to report results meeting a nominal P-value of 0.05. In addition, for the association studies of the individual SNPs, we have employed a correction factor of 7 (0.05/7 = 0.007) to take into consideration the number of genes evaluated. This represents a generally acceptable correction strategy; one not as stringent as a strict Bonferroni, yet still taking the number of totally independent analyses into account. This correction is not employed in the haplotype or gene–gene interaction analyses, as these are truly exploratory in nature and are restricted by sample size. Clearly, replication of these studies is critical, as well as examining gene sequences to identify etiologic susceptibility mutations.

Interestingly, three of these genes, Wnt3A, Wnt11 and Wnt8A, are involved in neural crest cell (NCC) differentiation and migration. NCCs are derived in the neural tube and migrate into the pharyngeal/branchial arches and differentiate into connective tissue and bone of the head and neck (26,27). Several mouse studies have demonstrated that genes inactivated in NCCs (i.e. Tgfbr2, Tcof1 and Ap-2alpha) cause orofacial clefting, as well as other malformations (28–30).

Wnt3A, in mice, has been shown to control the fate of both mesenchymal and NCCs in the craniofacial processes and to regulate palatal fusion (22). Embryonic expression of Wnt3A is upregulated by fibroblast growth factor 8, which is highly expressed in the facial primordia and is also an important regulator of craniofacial development (31,32). Additionally, Wnt3A signaling is also decreased when fibroblast growth factor receptor 1, which regulates the epithelial-mesenchymal transformation necessary for normal palatal development, is inactivated (31). We found that variation in Wnt3A and Wnt3A haplotypes are associated with NSCLP (Tables 2–4). Additionally, we showed that variation in Wnt3A interacts with other Wnt genes expressed in NCCs in NSCLP (Table 5). Of the six SNPs genotyped in Wnt3A, five were found to be associated with NSCLP. rs752107 is in the 3′-UTR region of the gene and the other SNPs were either intronic or flanked the gene. Therefore, while none of these SNPs code for the Wnt3A protein, the results suggest that Wnt3A is involved in the etiology of NSCLP.

Embryogenic studies have shown that Wnt11 is necessary for directing migrating NCCs, which will later form ectomesenchyme of the developing face (17,26). Also, bone morphogenic protein 4 (BMP4), which functions in the same pathway as Msx homeobox genes, is necessary for upper labial fusion in mice (32,33), and downregulates Wnt11 during normal urogenital development (34). Inactivation of BMP4 causes cleft lip in mice; therefore, the relationship between BMP4 and Wnt11 suggests that variation in Wnt11 could play an etiological role in NSCLP (33). Of the four SNPs in Wnt11 that were associated with our European American sample, irrespective of FH, the most significant association was with rs1533767, a synonymous SNP in exon 3 (P = 0.001; Table 3). The other three SNPs were in non-coding regions. While synonymous SNPs do not alter protein sequence, these SNPs may be in linkage disequilibrium with a functional mutation or they might alter amino acid translation time. Either scenario could be detrimental to gene function (35). Haplotypes of Wnt11 SNPs were also associated with clefting in the European American sample (Table 4), giving further support that Wnt11 may play an etiological role in the development of NSCLP in that population.

We previously identified a region on chromosome 3p21–14 between two short-tandem repeat (STR) markers that may harbor a clefting gene (36). Wnt5A is a candidate gene in this region because in situ hybridization studies have shown that Wnt5A is expressed in the frontonasal prominence and maxillary process, which fuse to form the primary palate (21). Wnt5A intronic SNP rs566926 (P = 0.001) is associated with NSCLP in the overall European American sample. While this SNP does not alter protein sequence, recent studies have shown associations with intronic SNP variants in other complex disorders (5,7,10,11,37–39). Gene–gene interaction between Wnt5A and Wnt3A was observed in the Hispanic sample (P = 0.039; Table 5), suggesting that variation in genes that regulate neural crest cells (NCCs) from cell fate determination (Wnt3A) to differentiation (Wnt5A) may predispose to an orofacial cleft.

While there is clearly overlap in the associated SNPs between ethnicities and across FH, there are some differences. These differences may be the result of a number of factors. The sample sizes in some stratifications are not extensive, so that genes with a smaller effect may not be detected. It is also likely that there are different genes acting in different populations. Our negative FH subgroups are likely to be composed of two different susceptible populations. One group has a higher genetic risk, and by chance, there is only a single affected individual and another group that has a lower genetic risk. The genes may be different between the two groups. Additionally, this model suggests that variation in individual Wnt genes and/or multiple genes acting to direct craniofacial morphogenesis, but on different genetic backgrounds, may ultimately be used to define those at high and low risk for NSCLP. This would explain the multiplex families in which NSCLP is present in several relatives but not in a Mendelian pattern. It would also explain those families that appear to have a lower liability.

Given the extensive role that the Wnt signaling pathway genes plays in craniofacial development, and the number of orofacial clefting syndromes caused by mutations in Wnt genes, it is not surprising that we found association with SNPs in the Wnt genes in our NSCLP sample (15,16,18,19,40–42). Of importance is the finding that variation within Wnt genes specifically involved in NCC development and migration (Wnt3A, Wnt5A and Wnt11) were the most strongly associated with NSCLP. Wnt genes clearly play a critical role during cephalogenesis and our results suggest that variation in the Wnt family of genes plays an etiological role in NSCLP.

MATERIALS AND METHODS

Study population and sample preparation

The study sample was composed of 132 multiplex NSCLP families and 235 simplex parent–child trios and 199 duos which have been described previously (36,43,44). Each family was ascertained through a proband affected with NSCLP from one of three craniofacial centers: Children's Hospital, Boston, Texas Children's Hospital, Houston, and the University of Texas Craniofacial Clinic, Houston. Probands and relatives were examined to exclude syndromes known to be associated with any type of orofacial clefting. Ethnicity (Hispanic versus European American) was determined by self-report. After obtaining informed consent, blood or saliva samples were collected and DNA was extracted using either the Roche DNA Isolation Kit for Mammalian Blood (Roche, Switzerland) or the Oragene Purifier for saliva (DNA Genotek, Inc., Ontario, Canada) following the manufacturer's protocol.

Genotyping

Thirty-eight SNPs in seven candidate genes [Wnt3 (three flanking and seven intragenic), Wnt3A (two flanking and four intragenic), Wnt5A (one intragenic), Wnt7A (two flanking and seven intragenic), Wnt8A (two flanking and one intragenic), Wnt9B (one flanking and two intragenic) and Wnt11 (2 flanking and 4 intragenic)] and two STRs flanking Wnt5A were evaluated in this study (Table 1). SNPs were selected based on heterozygosity, tagging ability and position in or around the gene as previously described (5). SNPs were genotyped using ABI TaqMan Genotyping Assays following manufacturer's protocol and were detected on an ABI 7900HT Sequence Detection System (Foster City, CA, USA). Two microsatellite markers flanking Wnt5A were genotyped, as previously described (UCSC database – genome.ucsc.edu) (36).

Statistical methods

In the initial association analysis, the entire data set was considered. In addition, probands were stratified by ethnicity alone or by ethnicity and FH. Allele frequency differences and Hardy–Weinberg equilibrium were calculated using SAS (v9.1). Pairwise linkage disequilibrium values (D′ and r2) were calculated using GOLD (graphical overview of linkage disequilibrium) (45). To extract the maximum amount of information from the dataset, multiple approaches for assessing linkage and/or association were used. Parametric and non-parametric linkage analyses were conducted using multipoint engine for rapid likelihood inferences (MERLIN) (46) and linkage parameters were defined, as previously reported (43). The PDT, Geno-PDT and APL test were used to evaluate evidence for association (47–49). This panel of analytic tools was chosen because each has different strengths and assumptions. PDT is an extension of the transmission disequilibrium test and allows for the incorporation of extended pedigrees in the analysis of allelic association. The Geno-PDT examines association between marker genotypes and disease while APL allows for missing parental genotypes. Each program may be more powerful for certain genetic models and for different pedigree structures. In addition, APL can be used to examine multi-marker haplotypes; it was employed to look for overtransmission of two-marker haplotypes within genes. However, since APL makes genotype assignments based on allele frequencies, it is not appropriate for analyzing multi-ethnic datasets where the allele frequencies can vary greatly between the specific groups. Lastly, gene–gene interactions of SNPs found to be significant with association studies were examined by GEE as implemented in SAS (50). Only two-way interactions were tested with GEE because of the limited sample size.

SUPPLEMENTARY MATERIAL

Supplementary Material is available at HMG Online.

FUNDING

This work was funded by NIH grant 5 R01-DE011931 and Shriners Hospital for Children grant to J.T.H. B.T.C. is a trainee under NIH grant 5T32DE015355-05.

ACKNOWLEDGEMENTS

We thank Maria Elena Serna and Dr. Syed Shahrukh Hashmi for recruiting patients and managing our databases. Dr William Scott provided thoughtful discussion and advice on GEE analyses. This study was approved by the IRB of the University of Texas Health Science Center at Houston (HSC-MS-03-090).

Conflict of Interest statement. None declared.

REFERENCES

1
Gorlin
R.J.
Cohen
J.M.M.
Levin
L.S.
Syndromes of the Head and Neck
 , 
1990
3rd edn.
New York
Oxford University Press
2
Hashmi
S.S.
Waller
D.K.
Langlois
P.
Canfield
M.
Hecht
J.T.
Prevalence of nonsyndromic oral clefts in Texas: 1995–1999
Am. J. Med. Genet. A
 , 
2005
, vol. 
134
 (pg. 
368
-
372
)
3
Gorlin
R.J.
Cohen
M.M.
Hennekam
R.C.M.
Syndromes of the Head and Neck
 , 
2001
4th edn.
New York
Oxford University Press
4
Lidral
A.C.
Moreno
L.M.
Progress toward discerning the genetics of cleft lip
Curr. Opin. Pediatr
 , 
2005
, vol. 
17
 (pg. 
731
-
739
)
5
Chiquet
B.T.
Lidral
A.C.
Stal
S.
Mulliken
J.B.
Moreno
L.M.
Arco-Burgos
M.
Valencia-Ramirez
C.
Blanton
S.H.
Hecht
J.T.
CRISPLD2: A novel NSCLP candidate gene
Hum. Mol. Genet
 , 
2007
, vol. 
16
 (pg. 
2241
-
2248
)
6
Ghassibe
M.
Bayet
B.
Revencu
N.
Verellen-Dumoulin
C.
Gillerot
Y.
Vanwijck
R.
Vikkula
M.
Interferon regulatory factor-6: a gene predisposing to isolated cleft lip with or without cleft palate in the Belgian population
Eur. J. Hum. Genet
 , 
2005
, vol. 
13
 (pg. 
1239
-
1242
)
7
Scapoli
L.
Palmieri
A.
Martinelli
M.
Pezzetti
F.
Carinci
P.
Tognon
M.
Carinci
F.
Strong evidence of linkage disequilibrium between polymorphisms at the IRF6 locus and nonsyndromic cleft lip with or without cleft palate, in an Italian population
Am. J. Hum. Genet
 , 
2005
, vol. 
76
 (pg. 
180
-
183
)
8
Srichomthong
C.
Siriwan
P.
Shotelersuk
V.
Significant association between IRF6 820G->A and non-syndromic cleft lip with or without cleft palate in the Thai population
J. Med. Genet
 , 
2005
, vol. 
42
 pg. 
e46
 
9
Suzuki
Y.
Jezewski
P.A.
Machida
J.
Watanabe
Y.
Shi
M.
Cooper
M.E.
Viet le
T.
Nguyen
T.D.
Hai
H.
Natsume
N.
, et al.  . 
In a Vietnamese population, MSX1 variants contribute to cleft lip and palate
Genet. Med
 , 
2004
, vol. 
6
 (pg. 
117
-
125
)
10
Zucchero
T.M.
Cooper
M.E.
Maher
B.S.
Daack-Hirsch
S.
Nepomuceno
B.
Ribeiro
L.
Caprau
D.
Christensen
K.
Suzuki
Y.
Machida
J.
, et al.  . 
Interferon regulatory factor 6 (IRF6) gene variants and the risk of isolated cleft lip or palate
N. Engl. J. Med
 , 
2004
, vol. 
351
 (pg. 
769
-
780
)
11
Blanton
S.H.
Cortez
A.
Stal
S.
Mulliken
J.B.
Finnell
R.H.
Hecht
J.T.
Variation in IRF6 contributes to nonsyndromic cleft lip and palate
Am. J. Med. Genet. A
 , 
2005
, vol. 
137
 (pg. 
259
-
262
)
12
Vieira
A.R.
Association between the transforming growth factor alpha gene and nonsyndromic oral clefts: a HuGE review
Am. J. Epidemiol
 , 
2006
, vol. 
163
 (pg. 
790
-
810
)
13
Jezewski
P.A.
Vieira
A.R.
Nishimura
C.
Ludwig
B.
Johnson
M.
O'Brien
S.E.
Daack-Hirsch
S.
Schultz
R.E.
Weber
A.
Nepomucena
B.
, et al.  . 
Complete sequencing shows a role for MSX1 in non-syndromic cleft lip and palate
J. Med. Genet
 , 
2003
, vol. 
40
 (pg. 
399
-
407
)
14
Riley
B.M.
Mansilla
M.A.
Ma
J.
Daack-Hirsch
S.
Maher
B.S.
Raffensperger
L.M.
Russo
E.T.
Vieira
A.R.
Dode
C.
Mohammadi
M.
, et al.  . 
Impaired FGF signaling contributes to cleft lip and palate
Proc. Natl Acad. Sci. USA
 , 
2007
, vol. 
104
 (pg. 
4512
-
4517
)
15
Brugmann
S.A.
Goodnough
L.H.
Gregorieff
A.
Leucht
P.
ten Berge
D.
Fuerer
C.
Clevers
H.
Nusse
R.
Helms
J.A.
Wnt signaling mediates regional specification in the vertebrate face
Development
 , 
2007
, vol. 
134
 (pg. 
3283
-
3295
)
16
Cadigan
K.M.
Nusse
R.
Wnt signaling: a common theme in animal development
Genes Dev
 , 
1997
, vol. 
11
 (pg. 
3286
-
3305
)
17
De Calisto
J.
Araya
C.
Marchant
L.
Riaz
C.F.
Mayor
R.
Essential role of non-canonical Wnt signalling in neural crest migration
Development
 , 
2005
, vol. 
132
 (pg. 
2587
-
2597
)
18
Girotti
M.
Zingg
H.H.
Gene expression profiling of rat uterus at different stages of parturition
Endocrinology
 , 
2003
, vol. 
144
 (pg. 
2254
-
2265
)
19
Kemp
C.
Willems
E.
Abdo
S.
Lambiv
L.
Leyns
L.
Expression of all Wnt genes and their secreted antagonists during mouse blastocyst and postimplantation development
Dev. Dyn
 , 
2005
, vol. 
233
 (pg. 
1064
-
1075
)
20
Lewis
J.L.
Bonner
J.
Modrell
M.
Ragland
J.W.
Moon
R.T.
Dorsky
R.I.
Raible
D.W.
Reiterated Wnt signaling during zebrafish neural crest development
Development
 , 
2004
, vol. 
131
 (pg. 
1299
-
1308
)
21
Yamaguchi
T.P.
Bradley
A.
McMahon
A.P.
Jones
S.
A Wnt5a pathway underlies outgrowth of multiple structures in the vertebrate embryo
Development
 , 
1999
, vol. 
126
 (pg. 
1211
-
1223
)
22
Yamaguchi
T.P.
Takada
S.
Yoshikawa
Y.
Wu
N.
McMahon
A.P.
T (Brachyury) is a direct target of Wnt3a during paraxial mesoderm specification
Genes Dev
 , 
1999
, vol. 
13
 (pg. 
3185
-
3190
)
23
Juriloff
D.M.
Harris
M.J.
Dewell
S.L.
A digenic cause of cleft lip in A-strain mice and definition of candidate genes for the two loci
Birth Defects Res. A Clin. Mol. Teratol
 , 
2004
, vol. 
70
 (pg. 
509
-
518
)
24
Juriloff
D.M.
Harris
M.J.
Dewell
S.L.
Brown
C.J.
Mager
D.L.
Gagnier
L.
Mah
D.G.
Investigations of the genomic region that contains the clf1 mutation, a causal gene in multifactorial cleft lip and palate in mice
Birth Defects Res. A Clin. Mol. Teratol
 , 
2005
, vol. 
73
 (pg. 
103
-
113
)
25
Juriloff
D.M.
Harris
M.J.
McMahon
A.P.
Carroll
T.J.
Lidral
A.C.
Wnt9b is the mutated gene involved in multifactorial nonsyndromic cleft lip with or without cleft palate in A/WySn mice, as confirmed by a genetic complementation test
Birth Defects Res. A Clin. Mol. Teratol
 , 
2006
, vol. 
76
 (pg. 
574
-
579
)
26
Nanci
A.
Ten Cate's Oral Histology - Development, Structure, and Function
 , 
2003
6th edn.
Los Angelos
Mosby
27
Chai
Y.
Maxson
R.E.
Jr
Recent advances in craniofacial morphogenesis
Dev. Dyn
 , 
2006
, vol. 
235
 (pg. 
2353
-
2375
)
28
Brewer
S.
Feng
W.
Huang
J.
Sullivan
S.
Williams
T.
Wnt1-Cre-mediated deletion of AP-2alpha causes multiple neural crest-related defects
Dev. Biol
 , 
2004
, vol. 
267
 (pg. 
135
-
152
)
29
Dixon
J.
Jones
N.C.
Sandell
L.L.
Jayasinghe
S.M.
Crane
J.
Rey
J.P.
Dixon
M.J.
Trainor
P.A.
Tcof1/Treacle is required for neural crest cell formation and proliferation deficiencies that cause craniofacial abnormalities
Proc. Natl Acad. Sci. USA
 , 
2006
, vol. 
103
 (pg. 
13403
-
13408
)
30
Ito
Y.
Yeo
J.Y.
Chytil
A.
Han
J.
Bringas
P.
Jr
Nakajima
A.
Shuler
C.F.
Moses
H.L.
Chai
Y.
Conditional inactivation of Tgfbr2 in cranial neural crest causes cleft palate and calvaria defects
Development
 , 
2003
, vol. 
130
 (pg. 
5269
-
5280
)
31
Ciruna
B.
Rossant
J.
FGF signaling regulates mesoderm cell fate specification and morphogenetic movement at the primitive streak
Dev. Cell
 , 
2001
, vol. 
1
 (pg. 
37
-
49
)
32
Jiang
R.
Bush
J.O.
Lidral
A.C.
Development of the upper lip: Morphogenetic and molecular mechanisms
Dev. Dyn
 , 
2006
, vol. 
235
  
spc1
33
Liu
W.
Sun
X.
Braut
A.
Mishina
Y.
Behringer
R.R.
Mina
M.
Martin
J.F.
Distinct functions for Bmp signaling in lip and palate fusion in mice
Development
 , 
2005
, vol. 
132
 (pg. 
1453
-
1461
)
34
Miyazaki
Y.
Oshima
K.
Fogo
A.
Hogan
B.L.
Ichikawa
I.
Bone morphogenetic protein 4 regulates the budding site and elongation of the mouse ureter
J. Clin. Invest
 , 
2000
, vol. 
105
 (pg. 
863
-
873
)
35
Kimchi-Sarfaty
C.
Oh
J.M.
Kim
I.W.
Sauna
Z.E.
Calcagno
A.M.
Ambudkar
S.V.
Gottesman
M.M.
A ‘silent’ polymorphism in the MDR1 gene changes substrate specificity
Science
 , 
2007
, vol. 
315
 (pg. 
525
-
528
)
36
Blanton
S.H.
Bertin
T.
Serna
M.E.
Stal
S.
Mulliken
J.B.
Hecht
J.T.
Association of chromosomal regions 3p21.2, 10p13, and 16p13.3 with nonsyndromic cleft lip and palate
Am. J. Med. Genet. A
 , 
2004
, vol. 
125
 (pg. 
23
-
27
)
37
Li
M.
Atmaca-Sonmez
P.
Othman
M.
Branham
K.E.
Khanna
R.
Wade
M.S.
Li
Y.
Liang
L.
Zareparsi
S.
Swaroop
A.
, et al.  . 
CFH haplotypes without the Y402H coding variant show strong association with susceptibility to age-related macular degeneration
Nat. Genet
 , 
2006
, vol. 
38
 (pg. 
1049
-
1054
)
38
Maller
J.
George
S.
Purcell
S.
Fagerness
J.
Altshuler
D.
Daly
M.J.
Seddon
J.M.
Common variation in three genes, including a noncoding variant in CFH, strongly influences risk of age-related macular degeneration
Nat. Genet
 , 
2006
, vol. 
38
 (pg. 
1055
-
1059
)
39
McWhinney
S.R.
Boru
G.
Binkley
P.K.
Peczkowska
M.
Januszewicz
A.A.
Neumann
H.P.
Eng
C.
Intronic single nucleotide polymorphisms in the RET protooncogene are associated with a subset of apparently sporadic pheochromocytoma and may modulate age of onset
J. Clin. Endocrinol. Metab
 , 
2003
, vol. 
88
 (pg. 
4911
-
4916
)
40
Niemann
S.
Zhao
C.
Pascu
F.
Stahl
U.
Aulepp
U.
Niswander
L.
Weber
J.L.
Muller
U.
Homozygous WNT3 mutation causes tetra-amelia in a large consanguineous family
Am. J. Hum. Genet
 , 
2004
, vol. 
74
 (pg. 
558
-
563
)
41
Nusse
R.
Wnt signaling in disease and in development
Cell Res
 , 
2005
, vol. 
15
 (pg. 
28
-
32
)
42
Pfeiffer
R.A.
Stoss
H.
Voight
H.J.
Wundisch
G.F.
Absence of fibula and ulna with oligodactyly, contractures, right-angle bowing of femora, abnormal facial morphology, cleft lip/palate and brain malformation in two sibs: a possibly new lethal syndrome
Am. J. Med. Genet
 , 
1988
, vol. 
29
 (pg. 
901
-
908
)
43
Blanton
S.H.
Bertin
T.
Patel
S.
Stal
S.
Mulliken
J.B.
Hecht
J.T.
Nonsyndromic cleft lip and palate: four chromosomal regions of interest
Am. J. Med. Genet. A
 , 
2004
, vol. 
125
 (pg. 
28
-
37
)
44
Stein
J.
Mulliken
J.B.
Stal
S.
Gasser
D.L.
Malcolm
S.
Winter
R.
Blanton
S.H.
Amos
C.
Seemanova
E.
Hecht
J.T.
Nonsyndromic cleft lip with or without cleft palate: evidence of linkage to BCL3 in 17 multigenerational families
Am. J. Hum. Genet
 , 
1995
, vol. 
57
 (pg. 
257
-
272
)
45
Abecasis
G.R.
Cookson
W.O.
GOLD–graphical overview of linkage disequilibrium
Bioinformatics
 , 
2000
, vol. 
16
 (pg. 
182
-
183
)
46
Abecasis
G.R.
Cherny
S.S.
Cookson
W.O.
Cardon
L.R.
Merlin–rapid analysis of dense genetic maps using sparse gene flow trees [see comment]
Nat. Genet
 , 
2002
, vol. 
30
 (pg. 
97
-
101
)
47
Martin
E.R.
Bass
M.P.
Gilbert
J.R.
Pericak-Vance
M.A.
Hauser
E.R.
Genotype-based association test for general pedigrees: the genotype-PDT
Genet. Epidemiol
 , 
2003
, vol. 
25
 (pg. 
203
-
213
)
48
Martin
E.R.
Monks
S.A.
Warren
L.L.
Kaplan
N.L.
A test for linkage and association in general pedigrees: the pedigree disequilibrium test
Am. J. Hum. Genet
 , 
2000
, vol. 
67
 (pg. 
146
-
154
)
49
Chung
R.H.
Hauser
E.R.
Martin
E.R.
The APL test: extension to general nuclear families and haplotypes and examination of its robustness
Hum. Hered
 , 
2006
, vol. 
61
 (pg. 
189
-
199
)
50
Hancock
D.B.
Martin
E.R.
Li
Y.J.
Scott
W.K.
Methods for interaction analyses using family-based case-control data: conditional logistic regression versus generalized estimating equations
Genet. Epidemiol
 , 
2007
, vol. 
8
 (pg. 
883
-
893
)