Simulating autosomal genotypes with realistic linkage disequilibrium and a spiked-in genetic effect.

BACKGROUND: To evaluate statistical methods for genome-wide genetic analyses, one needs to be able to simulate realistic genotypes. We here describe a method, applicable to a broad range of association study designs, that can simulate autosome-wide single-nucleotide polymorphism data with realistic linkage disequilibrium and with spiked in, user-specified, single or multi-SNP causal effects. RESULTS: Our construction uses existing genome-wide association data from unrelated case-parent triads, augmented by including a hypothetical complement triad for each triad (same parents but with a hypothetical offspring who carries the non-transmitted parental alleles). We assign offspring qualitative or quantitative traits probabilistically through a specified risk model and show that our approach destroys the risk signals from the original data. Our method can simulate genetically homogeneous or stratified populations and can simulate case-parents studies, case-control studies, case-only studies, or studies of quantitative traits. We show that allele frequencies and linkage disequilibrium structure in the original genome-wide association sample are preserved in the simulated data. We have implemented our method in an R package (TriadSim) which is freely available at the comprehensive R archive network. CONCLUSION: We have proposed a method for simulating genome-wide SNP data with realistic linkage disequilibrium. Our method will be useful for developing statistical methods for studying genetic associations, including higher order effects like epistasis and gene by environment interactions.

Gene-methylation interactions: discovering region-wise DNA methylation levels that modify SNP-associated disease risk.

BACKGROUND: Current technology allows rapid assessment of DNA sequences and methylation levels at a single-site resolution for hundreds of thousands of sites in the human genome, in thousands of individuals simultaneously. This has led to an increase in epigenome-wide association studies (EWAS) of complex traits, particularly those that are poorly explained by previous genome-wide association studies (GWAS). However, the genome and epigenome are intertwined, e.g., DNA methylation is known to affect gene expression through, for example, genomic imprinting. There is thus a need to go beyond single-omics data analyses and develop interaction models that allow a meaningful combination of information from EWAS and GWAS. RESULTS: We present two new methods for genetic association analyses that treat offspring DNA methylation levels as environmental exposure. Our approach searches for statistical interactions between SNP alleles and DNA methylation (G ×Me) and between parent-of-origin effects and DNA methylation (PoO ×Me), using case-parent triads or dyads. We use summarized methylation levels over nearby genomic region to ease biological interpretation. The methods were tested on a dataset of parent-offspring dyads, with EWAS data on the offspring. Our results showed that methylation levels around a SNP can significantly alter the estimated relative risk. Moreover, we show how a control dataset can identify false positives. CONCLUSIONS: The new methods, G ×Me and PoO ×Me, integrate DNA methylation in the assessment of genetic relative risks and thus enable a more comprehensive biological interpretation of genome-wide scans. Moreover, our strategy of condensing DNA methylation levels within regions helps overcome specific disadvantages of using sparse chip-based measurements. The methods are implemented in the freely available R package Haplin ( https://cran.r-project.org/package=Haplin ), enabling fast scans of multi-omics datasets.

Genetic variants in GRHL3 and risk for neural tube defects: A case-control and case-parent triad/control study.

BACKGROUND: Neural tube defects (NTDs) are the most common severe birth defects with complex etiologies. Previous studies conducted on animals have suggested that the Grhl3 gene is essential for closure of the spinal neural tube, but little evidence from human studies on the variants of GRHL3 gene has been provided, especially the common genetic variants. METHODS: To investigate the relationship between common genetic variants of GRHL3 and the risk for NTDs, we performed a case-control study and a case-parent triad/control study. Fast-target enrichment sequencing was performed to screen exon regions from 503 NTD cases, and three tag SNPs (single nucleotide polymorphisms, including rs12030057, rs2486668, and rs545809) were selected according to the sequencing results. Then, Sequenom MassARRAY genotyping was performed in 757 case parents and 519 controls to obtain genotype information of the target variant sites among all NTD triads and controls. RESULTS: The genotype distributions of all SNPs were in accordance with Hardy-Weinberg Equilibrium (HWE) in the control population. In the case-control study, significant associations were found between C27G genetic variants on rs2486668 and risk for spina bifida and encephalocele, respectively, under different genetic models. Consistently, in the case-parent triad/control study, GG genotype on rs2486668 was associated with increased risk for spina bifida, with a RR of 2.15 (95% CI: 1.20-3.83). However, no parent-of-origin effect was found for any tag SNPs. CONCLUSION: The GRHL3 C67G missense variant may increase the risk for spina bifida and encephalocele phenotypes.

A genome-wide scan of cleft lip triads identifies parent-of-origin interaction effects between ANK3 and maternal smoking, and between ARHGEF10 and alcohol consumption.

Background: Although both genetic and environmental factors have been reported to influence the risk of isolated cleft lip with or without cleft palate (CL/P), the exact mechanisms behind CL/P are still largely unaccounted for. We recently developed new methods to identify parent-of-origin (PoO) interactions with environmental exposures (PoOxE) and applied them to families with children born with isolated cleft palate only. Here, we used the same genome-wide association study (GWAS) dataset and methodology to screen for PoOxE effects in the larger sample of CL/P triads. Methods: Genotypes from 1594 complete triads and 314 dyads (1908 nuclear families in total) with CL/P were available for the current analyses. Of these families, 1024 were Asian, 825 were European and 59 had other ancestries. After quality control, 341,191 SNPs remained from the original 569,244. The exposures were maternal cigarette smoking, use of alcohol, and use of vitamin supplements in the periconceptional period. The methodology applied in the analyses is implemented in the R-package Haplin. Results: Among Europeans, there was evidence of a PoOxSmoke effect for ANK3 with three SNPs (rs3793861, q=0.20, p=2.6e-6; rs7087489, q=0.20, p=3.1e-6; rs4310561, q=0.67, p=4.0e-5) and a PoOxAlcohol effect for ARHGEF10 with two SNPs (rs2294035, q=0.32, p=2.9e-6; rs4876274, q=0.76, p=1.3e-5). Conclusion: Our results indicate that the detected PoOxE effects have a plausible biological basis, and thus warrant replication in other independent cleft samples. Our demonstration of the feasibility of identifying complex interactions between relevant environmental exposures and PoO effects offers new avenues for future research aimed at unravelling  the complex etiology of cleft lip defects.

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.

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.

Child apolipoprotein E gene variants and risk of cerebral palsy: estimation from case-parent triads.

OBJECTIVE: To use case-parent triad data to investigate if cerebral palsy (CP) is associated with variants of the APOE gene, the rs59007384 SNP of the TOMM40 gene or combined haplotypes of the two genes. STUDY DESIGN: DNA was analyzed in buccal swabs from 235 children with CP, their parents and a sibling. The relative risks (RR) with 95% confidence intervals (CI) that the children would have a distribution of APOE genotypes, rs59007384 variants or combined haplotypes deviating from Mendelian inheritance were estimated. RESULTS: Children with CP were more likely than expected to carry the APOEε3 allele (RR 7.5; CI: 0.99-53.7 for heterozygotes and 10.3; CI: 1.4-79.6 for homozygotes), and to have the haplotype of APOEε3 and rs59007384 G (RR 2.4; CI: 1-5.7 for heterozygotes, RR 3.7; CI: 1.4-9.5 for homozygotes) whereas the distribution was as expected for rs59007384 alone. In the subgroup analyses the findings were confined to children born preterm. Among siblings the distribution of these genes was as expected according to Mendelian inheritance. CONCLUSION: We speculate that children with APOEε2/APOEε4 alleles are more likely to die following cerebral injury in utero, resulting in a higher than expected proportion of children with CP carrying the APOEε3 allele.