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Published in final edited form as: Genet Epidemiol. 2008 Sep;32(6):505–512. doi: 10.1002/gepi.20323

Differential Parental Transmission of Markers in RUNX2 Among Cleft Case-Parent Trios From Four Populations

Jae Woong Sull 1, Kung-Yee Liang 1, Jacqueline B Hetmanski 1, Margaret Daniele Fallin 1, Roxann G Ingersoll 3, Jiwan Park 3, Yah-Huei Wu-Chou 4, Philip K Chen 4, Samuel S Chong 5, Felicia Cheah 5, Vincent Yeow 6, Beyoung Yun Park 6, Sun Ha Jee 1,7, Ethylin Wang Jabs 2,8, Richard Redett 2, Euiju Jung 1, Ingo Ruczinski 2, Alan F Scott 2, Terri H Beaty 1,*
PMCID: PMC2841966  NIHMSID: NIHMS149449  PMID: 18357615

Abstract

Isolated cleft lip with or without cleft palate (CL/P) is among the most common human birth defects, with a prevalence around 1 in 700 live births. The Runt-related transcription factor 2 (RUNX2) gene has been suggested as a candidate gene for CL/P based largely on mouse models; however, no human studies have focused on RUNX2 as a risk factor for CL/P. This study examines the association between markers in RUNX2 and isolated, nonsyndromic CL/P using a case-parent trio design, while considering parent-of-origin effects. Case-parent trios from four populations (77 from Maryland, 146 from Taiwan, 35 from Singapore, and 40 from Korea) were genotyped for 24 single nucleotide polymorphisms (SNPs) in the RUNX2 gene. We performed the transmission disequilibrium test on individual SNPs. Parent-of-origin effects were assessed using the transmission asymmetry test and the parent-of-origin likelihood ratio test (PO-LRT). When all trios were combined, the transmission asymmetry test revealed a block of 11 SNPs showing excess maternal transmission significant at the P < 0.01 level, plus one SNP (rs1934328) showing excess paternal transmission (P = 0.002). For the 11 SNPs showing excess maternal transmission, odds ratios of being transmitted to the case from the mother ranged between 3.00 and 4.00. The parent-of-origin likelihood ratio tests for equality of maternal and paternal transmission were significant for three individual SNPs (rs910586, rs2819861, and rs1934328). Thus, RUNX2 appears to influence risk of CL/P through a parent-of-origin effect with excess maternal transmission.

Keywords: RUNX2, oral cleft, maternal transmission effects, parent-of-origin

INTRODUCTION

Oral clefts are one of the most common birth defects in humans and represent a significant public health problem both in terms of the medical and economic burden for affected individuals and their families. Nonsyndromic cleft lip with or without palate (CL/P) is “complex” or “multifactorial” in its etiology, in that both genes and environmental risk factors control risk [Wyszynski et al., 1997; Cobourne, 2004]. Although several candidate genes have been extensively studied in different populations (transforming growth factor α (TGFA), IRF6, BCL3, RARA, etc.), only a few genes have been shown to contain mutations that appear causal (MSX1, PVRL1, etc.), and these are rare and often show incomplete penetrance [van den Boogaard et al., 2000; Zucchero et al., 2004; Carinci et al., 2007].

The Runt-related transcription factor 2 (RUNX2 [MIM 600211]) gene is located on chromosome 6p, where studies of multiplex families have yielded evidence for linkage [Eiberg et al., 1987; Marazita et al., 2004]. RUNX2 (CBFA1) is a critical transcription regulator involved in both bone and tooth formation [Aberg et al., 2004]. Mutations in this gene have been associated with cleidocranial dysplasia [CCD; Mundlos et al., 1997; Otto et al., 1997; Cooper et al., 2001]. Several studies have reported that RUNX2 may lead to cleft palate in mice [Ducy et al., 1999; Aberg et al., 2004]. However, to date no study has focused on whether the RUNX2 gene is a risk factor for CL/P in humans.

It is important to consider parent-of-origin effects when studying birth defects because maternal genotype controls the in utero environment of the developing fetus, and separating maternal genotypic effects from imprinting effects remains an important question [Weinberg and Umbach, 2005; Wilkins and Haig, 2003]. Maternal parent-of-origin effects have been suggested for several genes associated with non-syndromic CL/P [Mossey et al., 1998; Martinelli et al., 2001; van Rooij et al., 2003; Jugessur et al., 2003; Rubini et al., 2005]. In this paper, we tested for association between markers in RUNX2 and risk of CL/P in 298 case-parent trios specifically considering parent-of-origin effects.

METHODS

SAMPLE DESCRIPTION

As part of an international study of oral clefts, we collected data on case-parent trios recruited through treatment centers in Maryland (MD, Johns Hopkins and University of Maryland), the Chang Gung Memorial Hospital in Taiwan (TW), KK Women’s and Children’s Hospital in Singapore (SP), and Yonsei Medical Center in South Korea (KR). Research protocols were reviewed and approved by institutional review boards at each institution. Table I lists the gender of all CL/P probands. All parents of probands were unaffected in the SP, TW, and KR trios, but four parents among the 77 MD trios also had an oral cleft. All probands underwent clinical genetics evaluations (including assessing other congenital anomalies or major developmental delays) and were classified as having an isolated, nonsyndromic CL/P.

TABLE I.

Gender among 298 nonsyndromic CL/P cases from four populations

CL/P cases
Population Total (n) Male (n) Female (n)
Taiwan 146 95 51
Singapore 35 24 11
Korea 40 22 18
Maryland 77 44 33
Total 298 185 113
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CL/P, cleft lip with or without cleft palate.

SNP SELECTION, DNA, AND GENOTYPING

Single nucleotide polymorphisms (SNPs) were selected in a region surrounding RUNX2 on chromosome 6p21, with a goal of identifying one SNP per 5 kb of physical distance. Variants with “SNP scores” (an assessment of design quality of the Illumina assay (San Diego, CA) based on a proprietary algorithm) above 0.6, high validation levels in dbSNP (this included validation levels where the submitter had validated the SNP on multiple platforms), and high heterozygosity levels (particularly in multiple populations) were given higher priority during the selection process. From 26 selected SNPs, 24 were polymorphic in all four populations (Table II).

TABLE II.

SNP minor allele frequencies among parents of 298 CL/P cases from four populations

Minor allele frequency
No. SNP Name Physical locationa Minor allele Taiwan Singapore Korea Maryland
1 rs7771980 45,497,267 C 0.041 0.068 0.086 0.069
2 rs2677104 45,503,851 C 0.273 0.322 0.233 0.150
3 rs2819855 45,508,372 A 0.446 0.457 0.480 0.395
4 rs2819854 45,512,506 A 0.403 0.440 0.395 0.458
5 rs910586 45,518,290 T 0.039 0.042 0.053 0.283
6 rs2819853 45,520,635 G 0.036 0.042 0.039 0.283
7 rs765724 45,525,096 T 0.039 0.042 0.053 0.289
8 rs1343799 45,529,608 G 0.036 0.042 0.039 0.283
9 rs2819861 45,532,918 A 0.036 0.042 0.039 0.293
10 rs2790103 45,540,243 T 0.040 0.042 0.053 0.276
11 rs2790093 45,545,462 G 0.036 0.042 0.039 0.279
12 rs2790098 45,550,428 C 0.036 0.042 0.053 0.279
13 rs4714854 45,555,912 T 0.036 0.042 0.053 0.279
14 rs9472494 45,559,814 T 0.041 0.042 0.086 0.300
15 rs2396442 45,564,959 C 0.049 0.042 0.086 0.274
16 rs1934328 45,573,731 T 0.295 0.212 0.250 0.454
17 rs7773875 45,580,276 C 0.253 0.186 0.178 0.189
18 rs7771889 45,594,185 G 0.293 0.254 0.243 0.276
19 rs10485422 45,597,170 A 0.031 0.017 0.072 0.226
20 rs6904353 45,601,061 T 0.034 0.025 0.086 0.416
21 rs13207392 45,604,434 T 0.034 0.025 0.087 0.264
22 rs7748231 45,609,530 G 0.034 0.025 0.086 0.417
23 rs10948237 45,615,272 A 0.034 0.025 0.086 0.417
24 rs1928533 45,617,802 C 0.029 0.025 0.072 0.400
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SNP, single nucleotide polymorphism; CL/P, cleft lip with or without cleft palate.

a

Based on NCBI Human Genome build 35.1.

Genomic DNA samples were prepared from peripheral blood by the protein precipitation method described previously [Bellus et al., 1995]. DNA concentration was determined using the PicoGreen® dsDNA Quantitation Kit (Molecular Probes, Inc., Eugene, OR) and all DNA samples were stored at −20°C. A 4 μg aliquot of each genomic DNA sample was dispensed into a bar-coded 96-well microtiter plate at a concentration of 100 ng/μl and genotyped for SNP markers using the Illumina Golden-Gate™ chemistry with Sentrix® Array Matrices from the manufacturer [Oliphant et al., 2002] at the SNP Center of the Genetic Resources Core Facility, a part of the McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins School of Medicine. Two duplicates and four Centre d’Etude du Polymorphisme Humain (CEPH) controls were included on each plate to evaluate genotyping consistency within and between plates and to insure correct orientation. Genotypes were generated on a BeadLab 1000 system (San Diego, CA) [Fan et al., 2003].

STATISTICAL ANALYSIS

Within each population, the minor allele frequency (MAF) was computed among parents. Pairwise linkage disequilibrium (LD) was computed as r2 for all SNPs using the Haploview program [Barrett et al., 2005]. LD blocks were identified from the pairwise LD in each population separately (Fig. 1). Four blocks of LD were identified, consisting of 4, 11, 3, and 6 SNPs, respectively. As an initial screening, single-SNP and two to five-SNP haplotypes were analyzed using the family-based association test (FBAT) program, where empirical P-values were calculated for each test [Laird et al., 2000; Rabinowitz and Laird, 2000]. For this FBAT analysis, each population was analyzed separately for individual SNPs and haplotypes. Clayton’s extension of the transmission disequilibrium test (TDT) incorporated into STATA 8.2 (College Station, TX) [Spielman et al., 1993; Cordell et al., 2004] was used on individual SNPs to test for evidence of linkage and LD in the combined sample of 298 trios.

Fig. 1.

Fig. 1

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Linkage disequilibrium as measured by r2 in RUNX2 among parents of CL/P children from four populations. White: r2 = 0. Shades of gray: 0<r2<1. Black: r2 = 1. RUNX2, Runt-related transcription factor 2; CL/P, cleft lip with or without cleft palate.

Parent-of-origin analyses were conducted in the combined sample in several ways. Firstly, parent-of-origin effects were examined using the transmission asymmetry test (TAT) suggested by Weinberg et al. [1998], which is similar to the TDT but excludes matings between two heterozygotes (where transmission can be ambiguous). The TAT was stratified into separate allelic tests for fathers and mothers. Next, we used the likelihood-based approach proposed by Weinberg [1999] to test for parent-of-origin effects. This log-linear model considers the three mating types in which the mother and the father carry different numbers of variant alleles, with further stratification by the number of alleles inherited by the child. Like Weinberg, we will call this latter method the “parent-of-origin likelihood ratio test” (PO-LRT). This model considers maternally mediated in utero effects (maternal genotypic effects on the phenotype of the fetus), which could otherwise confound assessment of parent-of-origin effects, along with a separate term for imprinting [Weinberg, 1999]. Here, imprinting reflects a differential transmission of alleles to the affected child from mothers vs. fathers. This PO-LRT was executed using the LEM software (Tilburg, The Netherlands) [van Den Oord and Vermunt, 2000].

To assess the overall significance of these TAT findings for maternal and paternal transmission given the observed pattern of LD among SNPs in RUNX2 while considering multiple tests (see Fig. 1), we conducted a permutation test for each of the inferred LD blocks in this gene (consisting of k = 4, 11, 3, and 6 SNPs, respectively). Therefore, the block became the unit of analysis. A Bonferroni correction for each individual SNP P-value would be unacceptably conservative, in light of the high dependence between SNPs within a block. Generating P-values for each LD block permitted correcting for multiple comparisons with fewer and largely independent P-values (i.e. in RUNX2 there were four LD blocks to be tested, compared to 24 SNPs). We generated 10,000 data sets by randomly selecting the transmitted/nontransmitted status of the minor allele across all SNPs within a block for each trio. In this manner, LD structure was maintained, but any potential information regarding the transmission status was destroyed, as would occur under a true null hypothesis. Thus, this permutation test gives a clear assessment of the likelihood of the observed data under the null hypothesis of no preferential transmission of alleles. Many different test statistics can be devised to generate a block-specific P-value. In our analysis, the final inference was based on the arithmetic average of all of the −log10(P-values) within a block (i.e. a function of the geometric mean of these 10,000 P-values on simulated replicates). Histograms of these mean −log10(P-values) are plotted separately for the maternal and paternal transmissions from the 10,000 permutation tests along with the observed data.

RESULTS

Among these 24 SNPs, there was considerable variation in allele frequency among parents from MD and the three Asian populations (Table II). From the allele frequencies given in Table II, it is clear that some markers showed sharp distinctions between MD and Asian samples, while others did not. Measures of genetic distance based on either individual SNPs or haplotypes also varied considerably (data not shown). The three Asian populations (TW, SP, and KR) had very low MAFs for SNPs 5–15 and 19–24 compared to the MD trios, while SNPs 2–4 and 16–18 had similar MAFs in all four populations. For genes in the former group, obviously there will be fewer informative matings (e.g. matings involving heterozygotes) among Asian populations, but trios from all populations can still provide some information. Patterns of LD across the entire gene were calculated within each population, and Figure 1 shows that LD patterns among the 24 polymorphic SNPs were similar across all populations.

In conventional FBAT analysis of individual SNPs and haplotypes (ignoring parent-of-origin), only SNP 16 showed empirical significance at the 1% level among KR trios, and haplotypes including this marker were also significant. However, none of the other populations yielded evidence of linkage and association for SNPs in RUNX2 when parent-of-origin was not considered (data not shown). When all markers were screened in the combined data set of all four populations using the TDT without considering parent-of-origin, the odds ratio of transmission for the minor allele, OR(transmission), was significant for SNP 10 (OR = 1.62, P = 0.039), SNP 16 (OR = 1.40, P = 0.014), and SNP 18 (OR = 1.35, P = 0.032) (Table III).

TABLE III.

Number of transmitted (T) or nontransmitted (NT) minor alleles in 298 CL/P cases (all populations combined) from TDT and estimated odds ratios of transmission OR(transmission) ignoring parent-of-origin

TDT
No. SNP name T NT P-value ORa
1 rs7771980 28 29 0.895 0.97
2 rs2677104 91 92 0.941 0.99
3 rs2819855 135 115 0.206 1.17
4 rs2819854 137 122 0.351 1.12
5 rs910586 47 32 0.091 1.47
6 rs2819853 44 31 0.133 1.42
7 rs765724 47 33 0.112 1.42
8 rs1343799 44 31 0.133 1.42
9 rs2819861 44 29 0.079 1.52
10 rs2790103 47 29 0.039 1.62
11 rs2790093 44 30 0.104 1.47
12 rs2790098 45 31 0.108 1.45
13 rs4714854 45 31 0.108 1.45
14 rs9472494 50 36 0.131 1.39
15 rs2396442 53 36 0.072 1.47
16 rs1934328 125 89 0.014 1.40
17 rs7773875 97 75 0.093 1.29
18 rs7771889 120 89 0.032 1.35
19 rs10485422 37 28 0.264 1.32
20 rs6904353 43 37 0.502 1.16
21 rs13207392 41 32 0.292 1.28
22 rs7748231 44 37 0.437 1.19
23 rs10948237 46 36 0.269 1.28
24 rs1928533 41 36 0.569 1.14
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Shading in the table indicates inferred LD blocks.

T, transmitted; NT, not transmitted; OR, odds ratio; CL/P, cleft lip with or without cleft palate; TDT, transmission disequilibrium test; LD, linkage disequilibrium; SNP, single nucleotide polymorphism.

a

OR(transmission): odds ratio of transmission for the minor allele.

Parent-of-origin effects were first investigated by stratifying informative transmissions and nontransmissions by parental source (paternal and maternal) for all SNPs in the combined data set (Table IV). TAT revealed 11 contiguous SNPs (SNPs 5–15) with excess maternal transmission significant at the P < 0.01 level, and SNP 16 that showed excess paternal transmission (P = 0.002). For SNPs 5–15, the estimated OR(transmission) from the mother was statistically significant (ranging from 3.00 to 4.00) in TAT analysis. However, the PO-LRTs were only significant for SNP 5 (P = 0.036, OR(transmission) = 3.59), SNP 9 (P = 0.036, OR(transmission) = 3.73), and SNP 16 (P = 0.029, OR (transmission) = 0.44; Table IV, last column). PO-LRTs gave estimated risk ratios for an imprinting effect ranging between 2.51 and 3.73 for the 11 contiguous SNPs (SNPs 5–15), suggesting excess maternal transmission of this 46.7 kb region.

TABLE IV.

Number of transmitted (T) or non-transmitted (NT) minor alleles to 298 CL/P cases (all populations combined)a from TAT and estimated odds ratios, plus parent-of-origin likelihood ratio test results about inequality of maternal vs. paternal transmission

Paternal
 Maternal
TAT
 TAT
 PO-LRTb
No. SNP name T NT P-value ORc T NT P-value ORc ORd P-value
1 rs7771980 9 8 0.808 1.13 16 18 0.732 0.89 0.79 0.692
2 rs2677104 25 30 0.500 0.83 22 24 0.768 0.92 1.10 0.811
3 rs2819855 36 34 0.811 1.06 37 25 0.128 1.48 1.40 0.342
4 rs2819854 35 36 0.906 0.97 37 29 0.325 1.28 1.32 0.417
5 rs910586 15 13 0.705 1.15 20 5 0.003 4.00 3.59 0.036
6 rs2819853 14 12 0.695 1.17 18 5 0.007 3.60 3.19 0.063
7 rs765724 15 13 0.705 1.15 20 6 0.006 3.33 2.97 0.065
8 rs1343799 14 12 0.695 1.17 18 5 0.007 3.60 3.19 0.063
9 rs2819861 13 12 0.841 1.08 19 5 0.004 3.80 3.73 0.036
10 rs2790103 16 11 0.336 1.45 20 5 0.003 4.00 2.86 0.092
11 rs2790093 15 12 0.564 1.25 18 5 0.007 3.60 2.99 0.079
12 rs2790098 15 12 0.564 1.25 19 6 0.009 3.17 2.60 0.110
13 rs4714854 15 12 0.564 1.25 19 6 0.009 3.17 2.60 0.110
14 rs9472494 15 14 0.853 1.07 22 7 0.005 3.14 2.99 0.051
15 rs2396442 17 14 0.590 1.21 24 8 0.005 3.00 2.51 0.086
16 rs1934328 41 17 0.002 2.41 35 33 0.808 1.06 0.44 0.029
17 rs7773875 33 21 0.102 1.57 32 32 1.000 1.00 0.65 0.245
18 rs7771889 36 18 0.014 2.00 40 31 0.285 1.29 0.64 0.238
19 rs10485422 15 13 0.705 1.15 17 6 0.022 2.83 2.42 0.135
20 rs6904353 13 14 0.847 0.93 18 11 0.194 1.64 1.78 0.294
21 rs13207392 16 15 0.857 1.07 19 7 0.019 2.71 2.50 0.102
22 rs7748231 13 13 1.000 1.00 18 11 0.194 1.64 1.64 0.373
23 rs10948237 13 14 0.847 0.93 18 11 0.194 1.64 1.78 0.294
24 rs1928533 12 13 0.841 0.92 15 13 0.705 1.15 1.27 0.671
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Shading in the table indicates inferred LD blocks.

T, transmitted; NT, not transmitted; OR, odds ratio; CL/P, cleft lip with or without cleft palate; TAT, transmission asymmetry test; LD, linkage disequilibrium; SNP, single nucleotide polymorphism.

a

TAT analysis was used on trios with matings between two heterozygous parents deleted.

b

Parent-of-origin likelihood ratio test (PO-LRT) includes separate terms for imprinting.

c

OR(transmission): odds ratio of transmission of the minor allele.

d

OR: odds ratio for imprinting effect (i.e. differential transmission from mothers and from fathers).

The overall significance of the findings for maternal and paternal transmissions in the TAT was assessed by permutation tests to minimize the problem of multiple comparisons while preserving all information about LD. Excess transmission from the mother to the affected child was present for almost all of the SNPs in the 11-SNP block containing SNPs 5–15, while no obvious pattern of excess paternal transmission existed relative to the permuted values generated under the H0. Using the arithmetic mean of the −log10(P-values) over all 11 SNPs in this block, only 37 out of the 10,000 permuted replicates exceeded the observed test statistic for maternal transmission (Fig. 2, left side), and thus, this result is statistically significant at a 5% level after correcting for the number of tests (four tests, one for each block). On the other hand, there was no evidence of deviation from independence among paternal transmissions (P-value = 0.645, without correction).

Fig. 2.

Fig. 2

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Permutation tests under H0 for LD block 2 for maternal and paternal transmission under TAT on 298 CL/P trios. Results of permutation tests for maternal and paternal transmissions in the largest inferred LD block (11 SNPs) in RUNX2. A total of 10,000 data sets were generated by randomly selecting transmitted/nontransmitted status of the minor allele for all SNPs within the LD block for each case. Using the arithmetic mean of the 11 −log10 P-values (calculated for each of the 10,000 data sets) as test statistic, we find that only 37 out of the 10,000 permuted data sets exceeded the test statistic for maternal transmission in the observed data (left), while 6,450 out of the 10,000 permutation-based data sets exceeded the test statistic for paternal transmission (right). LD, linkage disequilibrium; TAT, transmission asymmetry test; CL/P, cleft lip with or without cleft palate; RUNX2, Runt-related transcription factor 2; SNP, single nucleotide polymorphism.

Similar examination of the other LD blocks in RUNX2 showed no evidence of excess transmission, either maternal or paternal for blocks 1 and 4 (data not shown). However, in the third LD block there was evidence of excess paternal transmission yielding a statistically significant empirical P-value (0.011 corrected for multiple tests). SNPs within this block had higher heterozygosity values and showed relatively little variation in allele frequencies among populations (see Table II). Furthermore, two of the three SNPs in this block (rs1934328 and rs7771889) showed evidence of linkage and LD when parent-of-origin was ignored (see Table III). SNP rs1934328 also yielded a nominally significant value from the PO-LRT with an estimated OR(imprinting) = 0.44 (P-value = 0.029), rejecting the null hypothesis of equal transmission from mothers and fathers (see Table IV).

DISCUSSION

Our study of case-parent trios from different populations (comprising a total of 298 case-parent trios) showed consistent evidence of linkage in the presence of association (LD) for multiple SNPs in RUNX2 only when parent-of-origin effects were considered. In a phenotypic study of RUNX2 mutants in mice, Aberg et al. [2004] reported cleft palate and early opening of the eyelids among mutant mice embryos. Yamachika et al. [2001] also reported a heterozygous C⃗ T transition in exon 3 of the RUNX2 gene in a patient with CCD and a cleft lip. In a cohort study of CCD patients, Cooper et al. [2001] found a significantly increased prevalence of submucosal cleft palate. Recently, Khan et al. [2006] suggested that CBFA2, a gene related to RUNX2, may be associated with enlarged cranial sutures with or without cleft palate.

The RUNX2 gene encodes a nuclear protein with a Runt DNA-binding domain and is a member of the RUNX2 family of transcription factors. RUNX2 contains nine alternatively spliced exons. Exons 1 and 2 contain alternatively utilized promoter regions and an ATG translational start codon. There are three alternative versions of exon 5 (exons 5, 5.1, and 5.2) and two alternative versions of exon 6 [exons 6 and 6.1; [Terry et al., 2004]. A sporadic case of CCD was found to be caused by heterozygosity at a G⃗ A transition at codon 283 in exon 5 of RUNX2 [Mundlos et al., 1997].

In our study, we analyzed 24 SNPs spanning 120.5 kb of the RUNX2 gene. We screened for parent-of-origin effects and 11 adjacent SNPs in RUNX2 showed evidence of excess maternal transmission. This region with excess maternal transmission falls within the same LD block in all four populations (Fig. 1), located between exons 4 and 5. The PO-LRT for inequality between maternal and paternal transmissions seldom exceeded marginal levels of significance for these 11 SNPs, likely due to the limited number of informative matings. The total number of informative fathers and mothers is modest, but after deleting heterozygous × heterozygous matings there were equal numbers of informative mothers and fathers (29 of each) for SNP rs910586, which showed the most significant result. Complete confirmation, of course, still requires larger samples of trios, larger numbers of informative heterozygotes, and more complete coverage with additional polymorphic SNPs.

Excess maternal transmission could reflect genomic imprinting or maternal genotype effects. Maternal genotypic effects for nonsyndromic CL/P have also been reported for several other candidate genes. van Rooij et al. [2003] reported that mothers carrying the TT genotype at the C677T variant in MTHFR showed an increased risk of having a child with CL/P. Martinelli et al. [2001] also observed an increase in TT homozygotes among mothers of CL/P subjects, with a risk ratio of 2.51 (1.00–6.14). However, Jugessur et al. [2003] found that children of mothers carrying the T variant allele had a slightly lower risk of CL/P. Interplay between maternal and fetal genotypes has been suggested for the gene coding for the TGFA [Mossey et al., 1998]. However, in a study of 134 Italian CL/P trios, Rubini et al. [2005] reported an association between the c844ins68 variant in the cystathionine β-synthase gene and nonsyndromic CL/P, and showed an 18.7-fold increase in risk for CL/P when the child received the c844ins68 allele from the mother rather than the father.

In the present study, log-linear models suggested by Weinberg [1999] to discriminate between maternal genotype and child genotype revealed a possible maternal imprinting effect for multiple SNPs in RUNX2. Maternal genotype effects were not individually significant for all 11 of these contiguous SNPs. Recently, Young et al. [2007] reported that RUNX2 transcription factors reinforce cell fate through an epigenetic mechanism that retains phenotypic gene expression patterns after cell division. RUNX2 also regulates expression of GNAS, a maternally imprinted gene [Bertaux et al., 2006; Weinstein et al., 2004]. In an analysis by real-time polymerase chain reaction of zebrafish RUNX2 genes, Flores et al. [2004] reported that RUNX2b was expressed only as a maternal transcript. They also detected maternal expression of RUNX2b during early embryogenesis in zebrafish.

Even though this candidate gene study involved a modest number of SNPs, addressing the issue of multiple comparisons is imperative before an overall statement about the significance of findings can be made. Since SNPs in strong LD typically have highly correlated P-values, adjusting significance levels via the overly conservative Bonferroni correction is particularly undesirable. Therefore, we conducted permutation tests for each of the four LD blocks and adjusted empirical P-values for the number of blocks, resulting in largely independent P-values. In the second LD block with 11 SNPs, we found strong evidence against the null hypothesis only for maternal transmission (the empirical P-value of 0.0037 would still be significant after correcting for multiple comparisons); but no such evidence for excess paternal transmission was seen. Thus, this 47 kb region of the gene seems to show excess maternal transmission to offspring with CL/P.

Interestingly, the next LD block of three SNPs with higher allele frequencies (and more informative) showed evidence of excess paternal transmission in these data. One SNP (rs1934328) in this block yielded significant evidence of imprinting when analyzed alone with the PO-LRT, giving an estimated OR(-imprinting) = 0.44, which was significantly less than what would be expected under the null hypothesis (H0:OR = 1). Since this model included separate terms for maternal genotype effects, this raises the possibility of imprinting in RUNX2.

The case-parent trio design offers the advantage of testing directly for maternal vs. paternal effects, and allows separating these from effects of the fetal genotype vs. parental origin in a robust manner [Cordell et al., 2004; Starr et al., 2005; Sinsheimer et al., 2003]. Another advantage of this design is that it minimizes issues of confounding that plague traditional case-control designs. This feature permitted pooling trios from four diverse populations into a combined test of allelic effects on the OR(transmission) to the affected child, while testing for parent-of-origin effects. The present study suggests maternal transmission effects for markers in RUNX2 and risk of nonsyndromic CL/P, although further work is still needed to confirm its ultimate impact on risk.

WEB RESOURCES

HAPLOVIEW: http://www.broad.mit.edu/mpg/haploview/index.php/

Online Mendelian Inheritance in Man (OMIM): http://www.ncbi.nlm.nih.gov/Omim/

Acknowledgments

Contract grant sponsor: National Institute of Dental and Craniofacial Research; Contract grant numbers: R21-DE-013707, R01-DE-014581; Contract grant sponsor: NIH; Contract grant numbers: R01 HL 090577; Contract grant sponsor: Korean Research Foundation; Contract grant numbers: 2004-041-E00104.

This research was supported by R21-DE-013707 and R01-DE-014581 from the National Institute of Dental & Craniofacial Research, NIH R01 HL 090577, and Korean Research Foundation (2004-041-E00104 and 2005-214-E00042). We thank all participants who donated samples for this multicenter study of oral clefts, as well as the staff at each participating site and institution. We also thank Gerald Raymond for his assistance in screening patients at Hopkins, and Katherine Zarfas for her technical help in the analysis.

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