None of the SNPs in TNNC2 were in HWE in the NHW discovery dataset and were removed from the association analyses; all remaining SNPs in the NHW were in HWE. All TNNC2 SNPs were in HWE in the Hispanic dataset and were therefore included in the association analyses. Only rs2074877 in MYH13 was out of HWE in the Hispanic discovery dataset and was removed from analyses. Allele frequencies differed significantly between the NHW and Hispanic groups for SNPs in fourteen of the fifteen examined genes (). Therefore, the data were stratified by ethnicity. Parametric and nonparametric linkage analysis found no evidence for linkage (data not shown).
Overall, nominal evidence for association was found for SNPs in twelve of fifteen genes in the discovery datasets (p<0.05) (). For the NHW dataset, evidence for association was seen for SNPs in six genes: MYBPH, TPM2, TNNT3, TPM1, MYH13 and MYH3. Three SNPs in MYH3 had altered transmission primarily in the NHW multiplex subset. All other associations involved a single SNP in each of the five other genes. In the Hispanic dataset, there was evidence for altered transmission in eleven genes (). Five of these genes, MYBPH, TPM2, TNNT3, TPM1 and MYH13, also had SNPs with altered transmission in the NHW dataset; only one SNP was common to both datasets (MYH13/rs17690195). In addition, several genes had multiple SNPs with altered transmission (MYL1 (3), TNNT3 (3), MYH8 (4), MYH4 (3), MYH1 (2) and MYH2 (2)).
Single SNP association by ethnicity*,†
When 2-SNP haplotypes were considered, altered transmission was found for five genes in the NHW group (p<0.01) (). Two of these genes, ACTA1 and MYH8, did not have individually altered transmitted SNPs. Three different MYH13 haplotypes had altered transmission; none of the haplotypes included the individual SNPs with altered transmission (). The two TPM2 haplotypes both contained rs1998303, which had altered transmission in the single SNP analyses. In the Hispanic discovery dataset, three MYH13 haplotypes had altered transmission (); only one contained rs17690195, which had altered transmission in the single SNP analysis (). There was no overlap between the NHW MYH13 haplotypes and the Hispanic MYH13 haplotypes, and only one SNP (MYH13/rs2240579) was common to both ethnicities.
2-SNP haplotype transmission – discovery population*,†
Numerous potential gene interactions were identified in both the NHW and Hispanic discovery datasets (p<0.01) (). The only gene interaction present in both datasets was TPM1 and MYH13, although the same SNPs were not involved in the two datasets. SNPs in ACTA1, MYH1, MYH13, MYH2, MYH4, MYH3, MYH8, MYL1, TNNT3, TPM1 and TPM2 were involved in interactions in both ethnic groups.
Gene interactions between SNPs in different muscle contraction genes*,†
Three genes (TNNI2, MYBPC2 and TNNC2) did not have any SNPs meeting our criteria for follow-up in the validation datasets. In the family-based validation dataset, only two SNPs in the single SNP analyses demonstrated any evidence for altered transmission, TNNT3/rs2734495 (p=0.04) and TPM1/rs1972041 (p=0.000074)(data not shown). The TPM1 result is supported by the 2-SNP analyses in the validation dataset where only TPM1 haplotypes had altered transmission (). All four of the significant haplotypes contained rs1972041. In the case-control dataset, only nominal evidence for association was seen with rs1248828 in TPM1 (p=0.04) in the Hispanic subset; there were no associations in the NHW subset (data not shown).
2-SNP haplotype transmission - validation population*,†
Further examination of the NHW maternal, paternal and proband TNNC2 genotype frequencies revealed that only the maternal genotypes deviated from HWE, suggesting the presence of a maternal genetic effect. summarizes the results of log-linear models assessing maternal and inherited genotypic effects. For rs383112, significant associations were observed with both the maternal and inherited genotypes (p=0.02 and 0.03, respectively). The maternal genotype for rs383112 was associated with a 1.38-fold increased risk (CT versus CC; 95% CI: 1.13–1.72) of clubfoot in offspring, while a protective inherited genotypic effect was conferred with a relative risk of 0.77 (CT versus CC; 95% CI: 0.50–0.99). In addition, a significant protective inherited genotypic effect (p=0.02), with a relative risk of 0.74 (TG versus TT; 95% CI: 0.48–0.97), was found for rs4629.
Results of log-linear modeling for TNNC2 in the NHW case-parent triads*,+