Computational protocol: Joint Testing of Genotypic and Gene-Environment Interaction Identified Novel Association for BMP4 with Non-Syndromic CL/P in an Asian Population Using Data from an International Cleft Consortium

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Protocol publication

[…] DNA for cases and their biological parents were obtained from a variety of types of biological specimens: whole blood, buccal brush/swab, saliva, mouthwash or dried blood spots.122 single nucleotide variants (SNVs) in and around BMP4 were genotyped using the Illumina Human610-Quad v.1_B Bead Chip at the Center for Inherited Disease Research at the Johns Hopkins University of the US . Based on the genomic structure of NM_130850.2 or NM_ 001202.3 for BMP4 ( , these SNVs are located 3′ (n = 114), introns (intron_1 for rs762642 and intron_3 for rs2071047) and 5′ (n = 6) of the gene, respectively.In addition, 25 imputed SNVs located between the two genotyped SNPs (rs7152946 and rs1001161) which are adjacent to rs7156227 were also used in the current study. Imputation was carried out based on the 1000 genomes reference population using IMPUTE2 by the GENEVA coordinating center . [...] The 122 genotyped and 25 imputed SNVs were all screened in Asian and European trios separately by Haploview (v4.2, using parents' data . SNPs that passed the quality control (QC) criteria among all genotyped and imputed SNVs in and around the gene were valid for association and linkage assessment. The QC flags for each SNP are: Mendelian errors >5%, missing genotyping call rate>5%, minor allele frequency (MAF) <1%, and deviates from Hardy-Weinberg Equilibrium (HWE, P>0.05/122 = 0.00041 for genotyped and P>0.05/25 = 0.002 for imputed SNVs, respectively).For Asian trios, the numbers of SNPs used in analyses were 117 (110 genotyped plus 7 imputed for those informative for ETS or both ETS and VIT), and 118 (111 genotyped plus 7 imputed for those informative for VIT). 120 (113 genotyped plus 7 imputed) SNPs were used in all analyses for European trios. Specifically, 10 and 3 genotyped SNVs were dropped due to MAF<1% in Asian and European parents, respectively; other genotyped SNVs were dropped due to linkage disequilibrium (LD, as measured by r2 = 1) with another SNP. All 18 imputed SNVs were dropped due to MAF<1%. The lowest genotyping call rates for Asian and European trios were 99.2% and 98.7%, respectively (data not shown). [...] To capture evidence of linkage and association between BMP4 and NSCL/P that may be missed by the allelic or genotypic TDT, the additive conditional logistic regression model for gTDT was extended to include additional GxE terms as Logit[P(case)] = βgG+∑[β(g*e_i)G*Ei] to jointly consider the effects of a genetic marker and interaction(s) with maternal environmental exposure(s). In this model, Ei represents a dummy variable denoting status for selected maternal environmental exposure , . Linkage and association signals can be captured by the likelihood ratio test (LRT) comparing a full model containing both G and GxE terms to a null model without containing any terms. The degrees of freedom for the LRT can be computed by the number of parameters in the larger model minus that in the smaller, nested model. When Ei is coded as 0 and 1 for the unexposed and exposed respectively, exp(βg) represents the OR for being a case carrying one copy of the minor allele (compared to non-carriers) for unexposed trios to any environmental factors considered in the GxE interaction terms. While exp[βg+∑β(g*e_i)] estimates the OR of being a case carrying one copy of the minor allele for trios with any or any combination of the maternal environmental exposures considered in the GxE interaction terms. A 1df Wald test can be used to test for statistical significance for each term in the model. Relevant ORs for carrying two copies of the minor allele can be estimated as exp(2βg) and exp[2(βg+∑β(g*e_i))], respectively. The corresponding 95% confidence intervals (CIs) for these ORs can be estimated considering the associated standard errors.Because maternal exposure status is the same between the observed case and all three pseudo-controls, tests for the independent effects of any maternal environmental exposure are not possible. Risk for exposed carriers can only be assessed by comparison to exposed non-carriers, while estimation of OR for exposed carriers by comparison to either unexposed carriers or unexposed non-carriers is not possible under a case-parent trio design.In the Asian trios, interactions with maternal ETS and VIT (in addition to G) were first individually tested and then simultaneously considered using additive conditional logistic regression models. Two and 3 df LRTs were used to identify significant linkage and associations accordingly. ORs for being a case carrying one copy of the minor allele were estimated for trios with various exposure statuses for ETS and VIT (exposed to both ETS and VIT, ETS only, VIT only, neither ETS nor VIT). To avoid the potential influence of ALCOHOL and SMK exposures on these tests, analyses simultaneously considering interactions with ETS and VIT were repeated for the most significant SNP (rs7156227) using trios with no exposure to either ALCOHOL or SMK. These repeated analysis were performed among all Asian combined NSCL/P trios (n = 704), Chinese only NSCL/P trios (n = 609), Asian and Chinese non-syndromic cleft lip only (NSCLO) and NSCLP trios, as well as NSCL/P trios from the two largest groups (Taiwan and Shandong). For European trios, interactions with each of the four investigated maternal environmental exposures were all considered individually and simultaneously (a 5df LRT). ORs were estimated for the two largest groups of European trios: trios without exposure to any of the four environmental factors (n = 64) and trios had exposure to VIT only (n = 98).Statistical analyses considering G and one GxE terms were performed using TRIO Package in R (version 3.0.0) , available at Analyses considering G and more than one GxE terms were carried out using SPSS software package version 20.0 (IBM SPSS Statistics).Statistical power for relevant tests was estimated using QUANTO software with Bonferroni corrected significance level (α = 0.05/117 (for trios informative for ETS and combined ETS&VIT) and 0.05/118 (for trios informative for VIT) for Asian and α = 0.05/120 for European trios) ( Other parameters required for power estimation were obtained either from the parents' genotypic data (number of trios, MAF and maternal environmental exposure rates) or from the fitted conditional logistic regression models (ORs for corresponding G and GxE terms). […]

Pipeline specifications

Software tools IMPUTE, Haploview, SPSS
Applications Miscellaneous, GWAS
Organisms Homo sapiens, Nicotiana tabacum
Diseases Cleft Lip, Cleft Palate