Genome-wide analysis of parent-of-origin effects in non-syndromic orofacial clefts

Abstract

Parent-of-origin (PofO) effects, such as imprinting are a phenomenon where the effect of variants depends on parental origin. Conventional association studies assume that phenotypic effects are independent of parental origin, and are thus severely underpowered to detect such non-Mendelian effects. Risk of orofacial clefts is influenced by genetic and environmental effects, the latter including maternal-specific factors such as perinatal smoking and folate intake. To identify variants showing PofO effects in orofacial clefts we have used a modification of the family-based transmission disequilibrium test to screen for biased transmission from mothers and fathers to affected offspring, biased ratios of maternal versus paternal transmission, and biased frequencies of reciprocal classes of heterozygotes among offspring. We applied these methods to analyze published genome-wide single-nucleotide polymorphism (SNP) data from ∼2500 trios mainly of European and Asian ethnicity with non-syndromic orofacial clefts, followed by analysis of 64 candidate SNPs in a replication cohort of ∼1200 trios of European origin. In our combined analysis, we did not identify any SNPs achieving conventional genome-wide significance (P<5 × 10(-8)). However, we observed an overall excess of loci showing maternal versus paternal transmission bias (P=0.013), and identified two loci that showed nominally significant effects in the same direction in both the discovery and replication cohorts, raising the potential for PofO effects. These include a possible maternal-specific transmission bias associated with rs12543318 at 8q21.3, a locus identified in a recent meta-analysis of non-syndromic cleft (maternal-specific P=1.5 × 10(-7), paternal-specific P=0.17). Overall, we conclude from this analysis that there are subtle hints of PofO effects in orofacial clefting.

Figure 1

Summary of the method used to screen for parent-of-origin effects in orofacial clefts.

Figure 2

Manhattan plot showing the results of four tests for 22 autosomes performed using all ethnic groups (Europeans and Asians) and disease subtypes (CL/P and CPO) combined. The y axis shows the −log₁₀ P-value for each test and the red and blue lines represent thresholds of P<1 × 10⁻⁵ (suggestive association) and 0.05 (nominal significance), respectively. Green dots represent loci showing suggestive associations identified by the four tests (see Methods). Each locus is labeled based on the test showing P<1 × 10⁻⁵: P# for PAT, M# for MAT, PofO# for PofO test and H# for HET. The P-values generated by four tests for each locus are shown in all four panels using the same label. For example, all SNPs at M11 loci have P<10⁻⁵ in MAT (above red line), while the same SNPs show P>0.05 in PAT (below blue line). These SNPs show nominal significance in PofO and HET (between blue and red line).

Figure 3

A global bias for maternal genetic effects in the etiology of OFCs. In every comparison using different subtypes of orofacial clefts and ethnicities (All: all ethnic groups, Eur: Europeans and Asn: Asians), we observed that the number of maternal-specific signals equals or exceeds the number of paternal-specific signals. A significant excess (P<0.05) of maternal-specific signals is observed in three categories, as indicated. Bar plots show the number of independent loci (LD blocks) with P<1 × 10⁻⁴ identified using the MAT (gray) and PAT (black) tests. A similar excess of maternal associations was also observed using a more stringent significance threshold of P<10⁻⁵, although due to the smaller number of loci, these differences did not reach statistical significance (data not shown).

Figure 4

Loci on 2q35 and 8q21.3 show suggestive evidence of PofO effects in NSCL/P in both discovery and replication cohorts. (a) A paternal-specific signal at SNP rs719325 on chromosome 2q35 in combined European and Asian analysis (P_GWAS: P_PAT=8.07 × 10⁻⁶, P_MAT=0.69, P_comb: P_PAT=5.43 × 10⁻⁸, P_MAT=0.12). (b) A maternal-specific signal at SNP rs12543318 on chromosome 8q21.3 in European-only analysis (P_GWAS: P_PAT=0.31, P_MAT=2.89 × 10⁻⁶, P_comb: P_PAT=0.27, P_MAT=7.85 × 10⁻⁷). The squares and diamond represent P-values from the discovery GWAS and combined analysis, respectively.