Genetic determinants of facial clefting: analysis of 357 candidate genes using two national cleft studies from Scandinavia

Abstract

Background: Facial clefts are common birth defects with a strong genetic component. To identify fetal genetic risk factors for clefting, 1536 SNPs in 357 candidate genes were genotyped in two population-based samples from Scandinavia (Norway: 562 case-parent and 592 control-parent triads; Denmark: 235 case-parent triads).

Methodology/principal findings: We used two complementary statistical methods, TRIMM and HAPLIN, to look for associations across these two national samples. TRIMM tests for association in each gene by using multi-SNP genotypes from case-parent triads directly without the need to infer haplotypes. HAPLIN on the other hand estimates the full haplotype distribution over a set of SNPs and estimates relative risks associated with each haplotype. For isolated cleft lip with or without cleft palate (I-CL/P), TRIMM and HAPLIN both identified significant associations with IRF6 and ADH1C in both populations, but only HAPLIN found an association with FGF12. For isolated cleft palate (I-CP), TRIMM found associations with ALX3, MKX, and PDGFC in both populations, but only the association with PDGFC was identified by HAPLIN. In addition, HAPLIN identified an association with ETV5 that was not detected by TRIMM.

Conclusion/significance: Strong associations with seven genes were replicated in the Scandinavian samples and our approach effectively replicated the strongest previously known association in clefting--with IRF6. Based on two national cleft cohorts of similar ancestry, two robust statistical methods and a large panel of SNPs in the most promising cleft candidate genes to date, this study identified a previously unknown association with clefting for ADH1C and provides additional candidates and analytic approaches to advance the field.

Figure 1. TRIMM analyses of the Norwegian and Danish samples.

Schweder-Spjøtvoll plot of p-values for (A) isolated cleft lip with or without cleft palate (I-CL/P) and (B) isolated cleft palate (I-CP). All genes with p-values ≤0.1 are shown on the X-axis and ordered according to observed p-values (Y-axis). Genes with p-values ≤0.05 are highlighted in red. The sloping line represents the expected uniform distribution under the null (of no effect). Genes with p-values ≤0.1 in both the Norwegian and Danish samples are indicated by vertical lines connecting the upper (Norway) and lower (Denmark) plots.

Figure 2. HAPLIN analyses of the Norwegian and Danish samples.

Schweder-Spjøtvoll plot of p-values for (A) isolated cleft lip with or without cleft palate (I-CL/P) and (B) isolated cleft palate (I-CP). All genes with p-values ≤0.1 are shown on the X-axis and ordered according to observed p-values (Y-axis). Genes with p-values ≤0.05 are highlighted in red. The sloping line represents the expected uniform distribution under the null (of no effect). Genes that had p-values ≤0.1 in both the Norwegian and Danish samples are indicated by vertical lines connecting the upper (Norway) and lower (Denmark) plots.

Figure 3. Quantile-quantile (QQ) plots of p-values.

The QQ plot compares the distribution of the observed Fisher-combined p-values (−log scale) for both populations with an expected uniform distribution under the null (sloping line). The plots for I-CL/P and I-CP are provided separately for TRIMM in (A) and (B), respectively, and the corresponding plots for HAPLIN are shown in (C) and (D). Gene labels for the top six most significant genes are displayed in each plot.

Figure 4. Genomic location of the seven genes identified in both populations by TRIMM and HAPLIN.

Fisher-combined p-values are shown for all genes with p-values ≤0.05 in both the Norwegian and Danish populations for either analysis. The distance between genes on the same chromosome is indicated in megabases (Mb). Gene structure is shown in red for coding regions and blue for untranslated regions, with exon boxes connected by intron lines. The seven identified genes are on the minus strand compared to the reference sequence and are scaled relative to one another based on the length shown in kilobases (kb). Each SNP is represented by a symbol for its functional status as indicated at the bottom of the figure. Two SNPs represented by a square with a star were considered in the region of IRF6, but actually fell within the neighboring gene TRAF3IP3.