Analysis of Parent-of-Origin Effects on the X Chromosome in Asian and European Orofacial Cleft Triads Identifies Associations with DMD, FGF13, EGFL6, and Additional Loci at Xp22.2

Abstract

Background: Although both the mother's and father's alleles are present in the offspring, they may not operate at the same level. These parent-of-origin (PoO) effects have not yet been explored on the X chromosome, which motivated us to develop new methods for detecting such effects. Orofacial clefts (OFCs) exhibit sex-specific differences in prevalence and are examples of traits where a search for various types of effects on the X chromosome might be relevant. Materials and Methods: We upgraded our R-package Haplin to enable genome-wide analyses of PoO effects, as well as power simulations for different statistical models. 14,486 X-chromosome SNPs in 1,291 Asian and 1,118 European case-parent triads of isolated OFCs were available from a previous GWAS. For each ethnicity, cleft lip with or without cleft palate (CL/P) and cleft palate only (CPO) were analyzed separately using two X-inactivation models and a sliding-window approach to haplotype analysis. In addition, we performed analyses restricted to female offspring. Results: Associations were identified in "Dystrophin" (DMD, Xp21.2-p21.1), "Fibroblast growth factor 13" (FGF13, Xq26.3-q27.1) and "EGF-like domain multiple 6" (EGFL6, Xp22.2), with biologically plausible links to OFCs. Unlike EGFL6, the other associations on chromosomal region Xp22.2 had no apparent connections to OFCs. However, the Xp22.2 region itself is of potential interest because it contains genes for clefting syndromes [for example, "Oral-facial-digital syndrome 1" (OFD1) and "Midline 1" (MID1)]. Overall, the identified associations were highly specific for ethnicity, cleft subtype and X-inactivation model, except for DMD in which associations were identified in both CPO and CL/P, in the model with X-inactivation and in Europeans only. Discussion/Conclusion: The specificity of the associations for ethnicity, cleft subtype and X-inactivation model underscores the utility of conducting subanalyses, despite the ensuing need to adjust for additional multiple testing. Further investigations are needed to confirm the associations with DMD, EGF16, and FGF13. Furthermore, chromosomal region Xp22.2 appears to be a hotspot for genes implicated in clefting syndromes and thus constitutes an exciting direction to pursue in future OFCs research. More generally, the new methods presented here are readily adaptable to the study of X-linked PoO effects in other outcomes that use a family-based design.

Keywords: GWAS; Haplin; X chromosome; birth defects; case-parent triads; genetic epidemiology; orofacial clefts; parent-of-origin.

Figure 1

An illustration of the Haplin model for parent-of-origin (PoO) effects on the X chromosome. The red arrows show the relative risks associated with girls inheriting the risk allele “a” from the mother (RR_m) or from the father (RR_f). Under the multiplicative risk model illustrated here, the relative risk increase for “aa” girls, i.e., girls inheriting allele “a” from both the mother and the father, is RRaa = RR_m^*RR_f. The ratio RR_m/RR_f = 1.4 is a measure of the PoO effect (blue arrow). The risk increase for boys when inheriting the “a” allele is RR_B = 2.0. Under the assumption of X-inactivation (A), the risk increase for girls inheriting “aa” is the same as that for a single “a” in boys, i.e., RRaa = RR_B. Under the assumption of no X-inactivation (B), the risk increase for girls inheriting “a” from the mother is the same as that for boys, i.e., RR_m = RR_B. In the model without X-inactivation, when a girl inherits “a” from the father as well, this may lead to a higher total risk increase for girls than for boys inheriting the one “a” from the mother. In this illustration, RRaa = RR_m^*RR_f = 2.86 > RR_B. The model allows different baseline risks for girls and for boys, here 0.4 and 0.5%, respectively.

Figure 2

Single-marker and haplotype analyses in the Asian and European samples without stratification by child's sex. The Manhattan plots show the single-marker and haplotype analyses based on the model without and with X-inactivation in females, respectively. For convenience, we have added a vertical line corresponding to a Bonferroni-corrected p-value cutoff of 10⁻⁴.

Figure 3

Single-marker and haplotype analyses in the Asian and European girls only. The Manhattan plots show the single-marker and haplotype analyses of the girls only. For convenience, we have added a vertical line corresponding to a Bonferroni-corrected p-value cutoff of 10⁻⁴.

Figure 4

Statistical power for a single SNP. (A) Shows the simulated power for the model restricted to girls only, (B) shows the simulated power for the model without X-inactivation, and (C) shows the simulated power for the model with X-inactivation. The figures on the left depict the power with increasing relative risk ratios (RRRs) (increasing values of RR_m; RR_f = 1) and differing sample sizes of case-parent triads, using a fixed minor allele frequency (MAF) of 0.2. The figures on the right-hand side show the power with varying RRRs and MAFs, assuming a total of 300 case-parent triads for the girls-only analyses in (A) and 600 case-parent triads in (B,C) (assuming an equal distribution of boys and girls). We used a nominal significance level of 0.05 throughout.

Figure 5

Genes located in chromosomal region Xp22.2. This collage of screenshots of chromosomal region Xp22.2 was generated using the Ensembl genome browser (http://www.ensembl.org/) (Kersey et al., ; Yates et al., 2016). The three genes identified in this study are shown in boxes, in addition to the “Oral-facial-digital syndrome 1” (OFD1) gene, in which we had identified associations in our previous study on candidate genes on the X chromosome (Jugessur et al., 2012a), and the “Midline 1” (MID1) gene.