Confirming genes influencing risk to cleft lip with/without cleft palate in a case-parent trio study

Abstract

A collection of 1,108 case-parent trios ascertained through an isolated, nonsyndromic cleft lip with or without cleft palate (CL/P) was used to replicate the findings from a genome-wide association study (GWAS) conducted by Beaty et al. (Nat Genet 42:525-529, 2010), where four different genes/regions were identified as influencing risk to CL/P. Tagging SNPs for 33 different genes were genotyped (1,269 SNPs). All four of the genes originally identified as showing genome-wide significance (IRF6, ABCA4 and MAF, plus the 8q24 region) were confirmed in this independent sample of trios (who were primarily of European and Southeast Asian ancestry). In addition, eight genes classified as 'second tier' hits in the original study (PAX7, THADA, COL8A1/FILIP1L, DCAF4L2, GADD45G, NTN1, RBFOX3 and FOXE1) showed evidence of linkage and association in this replication sample. Meta-analysis between the original GWAS trios and these replication trios showed PAX7, COL8A1/FILIP1L and NTN1 achieved genome-wide significance. Tests for gene-environment interaction between these 33 genes and maternal smoking found evidence for interaction with two additional genes: GRID2 and ELAVL2 among European mothers (who had a higher rate of smoking than Asian mothers). Formal tests for gene-gene interaction (epistasis) failed to show evidence of statistical interaction in any simple fashion. This study confirms that many different genes influence risk to CL/P.

Figure 1

Evidence for linkage & association from genotypic TDT in 1,108 CL/P case-parent trios for four genes/regions identified as genome-wide significant by Beaty et al. (2010). (note differences in scale of Y-axis for chr. 8q24).

Figure 2

Significance of meta-analyses of p-values from genotypic TDT on CL/P case-parent trios from the replication study and the original GWAS for three genes identified as second tier hits: NTN1, PAX7 and COL8A1/FILIP1L1. Each of these genes achieved genome-wide significance in this meta-analysis based on called genotypes from imputed genotypic probabilities in the GWAS trios and the observed genotypes in the replication trios (n.b. the scale of the Y axis varies).

Figure 3

QQ plots for tests of GxG interaction using 647 SNPs in 12 genes showing some marginal effect of genotype

Figure 4

Significance of genotypic TDT model including a term for GxSmoking interaction in two genes among 654 European CL/P case-parent trios (where the exposure rate for maternal smoking was 18%). Blocks of SNPs showing evidence of GxSmoking interaction are highlighted in black.

Figure 5

Predicted OR(CL/P|unexposed carrier) and OR(CL/P|exposed carrier) in two genes showing evidence of GxSmoking interaction among 645 European CL/P case-parent trios. Open circles represent exposed infants with one risk allele (i.e. those whose mothers smoked) and black dots represent unexposed carrier infants. P-values from 1 df test for GxSmoking interaction are shown along the x-axis. A) 25 SNPs in GRID2, B) 26 SNPs in ELAVL2.