Characteristics of case and control mothers are reported elsewhere (Schmidt et al., 2009
). As reported previously, of the 768 NTDs and 4143 controls with interview data, 761 (99.1%) cases and 4108 (99.2%) controls had data on total caffeine intake (Schmidt et al., 2009
). The majority of mothers (87.3% case and 84.5% control) reported caffeine intake in the year prior to pregnancy. Additionally, 537 (70.7%) case and 2850 (69.8%) control mothers consumed caffeinated beverages during pregnancy. Caffeine consumption in the year prior to pregnancy was associated with slightly elevated risk for all NTDs combined (OR: 1.3, 95% CI: 1.0–1.6), that was strongest for spina bifida and encephalocele, and did not increase with higher levels of intake (Schmidt et al., 2009
Of the families with interview data, a total of 328 (42.7%) case and 1320 (32.1%) control families returned buccal samples. Of these families, 306 (93.3%) case and 1271 (96.3%) control families met eligibility criteria and 669 control families were selected for genotyping. For both cases and controls, mothers in families who returned buccal samples were more likely to be non-Hispanic white and native born than mothers in families who did not return samples (). Mothers in case families who returned samples were more likely to be college-educated, to have household annual incomes above $50,000, to report drinking alcohol during pregnancy, and to be of higher parity, and were less likely to be obese. Most maternal characteristics of families who were genotyped did not differ from those who returned samples. The frequency of case and control mothers reporting caffeine intake was similar among those with and those without samples returned and genotyped (), and the adjusted OR for any caffeine consumption among genotyped families was similar to that for all interviewed participants (OR: 1.42, 95% CI: 0.95–2.13).
Characteristics of Case and Control Mothers by Availability of Biologic Samples and Selection for Genotyping
Genotyping success rates for mother, father, and infant samples are presented with genotype and phenotype frequencies for each gene variant in . The observed genotype proportions for CYP1A2*1F, NAT2 481C>T and 590G>A in controls were not significantly different from Hardy-Weinberg expectations (χ2 = 2.3, 3.5, and 1.0, respectively).
Genotypes and Phenotypes for Genotyped Case and Control Family Members
Maternal and infant CYP1A2*1F
risk estimates near 1.0 for the heterozygous C/A genotypes supported grouping heterozygotes with homozygous C/C genotypes. An association was found between maternal CYP1A2*1F
fast oxidation status and increased risk for NTDs () which remained significant after a Bonferroni correction for multiple comparisons (p
=0.01). No association was found for infant CYP1A2*1F
fast oxidation status (). Findings for maternal and infant CYP1A2*1F
in the case-control logistic regression and case-parent triad log-linear analyses were similar to those presented for the hybrid log-linear design (Supplemental Digital Content 2, Table S1
). When stratified by reported race/ethnicity, the association between maternal CYP1A2*1F
and NTDs was stronger for non-Hispanic white mothers (OR: 1.77, 95% CI: 1.20–2.61) than for other mothers (OR: 1.17, 95% CI: 0.69–2.00). The association for maternal CYP1A2*1F
was also somewhat stronger when the infant was also a fast oxidizer (OR: 1.90, 95% CI: 0.87–4.17) compared to when the infant was a slow oxidizer (OR: 1.26, 95% CI: 0.64–2.50).
Odds Ratios (OR) for Associations between CYP1A2 *1F Gene Variants and NTDs Using a Hybrid Log-Linear Method Combining Case-control and Case-Parent Trio Study Designs
Both tests for population structure bias indicated that for NAT2 acetylator status the case-control component was vulnerable to bias due to population stratification and thus only the case-parent triad log-linear findings were reliable. The family-based log-linear approach, which controlled for this bias by stratifying on parental mating type, demonstrated an association between infant slow NAT2 acetylation status and NTDs with adjustment for maternal NAT2 acetylation status (RR: 2.00, 95% CI: 1.10–3.64). This association was also found using the ETDT (χ2 = 13.0, p < 0.01). There was a suggestion of an inverse association between slow maternal NAT2 acetylation status and NTDs when compared to rapid acetylators (RR adjusted for infant NAT2 acetylation status: 0.52, 95% CI: 0.27–1.03). This association was strongest for mothers who reported non-Hispanic white race/ethnicity (RR: 0.34, 95% CI: 0.14–0.81).
Caffeine by gene effect modification
Maternal gene variants
A slightly increased OR for the association between any maternal caffeine intake and NTDs was observed for children of mothers who were fast CYP1A2*1F
oxidizers (A/A) (OR: 1.61, 95% CI: 0.87–2.98) compared to children of mothers who were slow oxidizers (OR: 1.31, 95% CI: 0.65–2.62). Slow maternal NAT2
acetylation status was also associated with an increased OR between maternal caffeine intake and NTDs (OR: 1.85, 95% CI: 0.82–4.18) compared to those with rapid NAT2
acetylator status (OR: 1.31, 95% CI: 0.72–2.40). When stratified across both maternal CYP1A2*1F
oxidation and NAT2
acetylation status, the risk estimate for the association between NTDs and caffeine intake in the year prior to pregnancy was most elevated for the offspring of mothers who were both fast oxidizers and slow acetylators (). For women with planned pregnancies, we similarly observed the highest risk estimate for maternal intake of caffeinated beverages during pregnancy for those who were fast CYP1A2 oxidizers and slow NAT2 acetylators (Supplemental Digital Content 3, Table S2
Association between Maternal Caffeine Intake and NTD-affected Pregnancies Stratified by Maternal and Infant CYP1A2*1F Oxidizer and NAT2 Acetylator Status
Infant gene variants
Stratified case-control results revealed significantly greater estimated risk of NTDs associated with any maternal caffeine intake in the year prior to pregnancy for infants with fast oxidation status compared to those with slow oxidation status (; p
for interaction = 0.03). This effect remained after adjusting for maternal CYP1A2*1F
oxidation status (data not shown). A similar, though non-significant, effect was observed for caffeinated beverage intake during pregnancy for mothers with planned pregnancies (Table S2
). The QPL analyses also demonstrated an interaction effect between infant CYP1A2*1F
oxidation status and maternal caffeine intake as a continuous variable among case families. The interaction estimate for the association between each additional 100 mg caffeine and fast infant CYP1A2*1F oxidation status was 2.40 (95% CI: 1.10–5.21) using complete triads. The OR for maternal caffeine intake was slightly greater for rapid NAT2
acetylator infants than for slow acetylator infants in both the case-control () and family-based QPL analyses (data not shown).