Methods

A prospective and paired case-control study (1:1) was conducted from October 2008 to April 2010 by inviting 360 parents of a child with isolated CHD and 360 parents of a child without any congenital malformations to participate. The study subjects were the parents of pediatric patients who had been admitted to our pediatric cardiac center and the parents of a healthy child who had been recruited in collaboration with the maternity and pediatric units. The matching criteria for the controls were the absence of a congenital malformation and age compatible with the observed age range of the patients. The exclusion criteria were mothers who reported inconsistent use of B vitamin and folate supplements in the periconception period; and an inability to obtain complete information about the occupational, demographic, and lifestyle data for both parents. The present study was conducted with informed consent of every participant, and the local ethical research committee approved it.

Both case and control parents completed a structured questionnaire on the demographic, preconceptional, and lifestyle exposures. We derived environmental and occupational exposure data from specific questions on potential teratogens/mutagens that have been linked to human reproductive impairment, including ionizing radiation, solvents, pesticides, asbestos, and heavy metals.

The paternal questionnaire carefully evaluated the 3-month period before conception, according to the duration of spermatogenesis (about 70 days). The maternal questionnaire covered the period before conception to the end of the first trimester of pregnancy. The first trimester of pregnancy was chosen because that it is most critical period for embryogenesis associated with increased sensitivity to environmental factors. The maternal questionnaire gathered information on personal characteristics and lifestyle habits, health, pregnancy aspects, medication during pregnancy, occupational, and other potential harmful exposures (e.g., diagnostic radiation exposure). The paternal questionnaire collected data on personal characteristics, health, occupational history, and other potential harmful exposures (e.g., diagnostic radiation exposure). Parental illness data included diseases of the thyroid, kidney, liver, gut, stomach, pancreas, gastrointestinal, respiratory, and urinary disorders.

Using a case-only design, DNA was obtained from 180 patients with CHD (104 males, age 4.9 ± 5.8 years). Genotyping analysis was performed by our laboratory staff, who were unaware of the clinical data. Genomic DNA was extracted from peripheral blood leukocytes. The GSTM1 and GSTT1 genotypes were determined using a co-amplification polymerase chain reaction approach with the GSTM4 gene, which is never deleted, as the internal control to distinguish the null genotypes from aborted polymerase chain reaction. Primer sequences, annealing temperatures, and digest conditions were performed according to previously published protocols. The internal standard fragment amplified from the GSTM4 gene was 157 bp. A 230-bp fragment was amplified for the GSTM1 gene, and a 480-bp fragment was obtained for the GSTT1 gene. The absence of amplified products was consistent with the null genotypes.

For statistical analysis, all determinants were dichotomized, except for the age variables. We considered as smokers those who smoked ≥3 cigarettes/day at conception; exsmokers were those who had stopped smoking ≥6 months before study inclusion; and nonsmokers were those who had never smoked. The smokers were also divided into groups according to smoking level: medium smokers (3 to 14 cigarettes/day) and heavy smokers (≥15cigarettes/day). The consumption frequency of alcoholic beverages (beer, wine, liquor) was classified in drinkers who drank ≥3 alcoholic drinks/day (beer, wine, liquor) and nondrinkers, who drank <3 drinks/day.

Given the relatively small number of exposed parents in any particular environmental and occupational category, statistical analysis for toxicant exposure was performed, including both environmental and occupational exposure to x-rays, chemicals, anesthetics, industrial cleaning agents and solvents, exhaust and welding fumes, paint/varnish/thinner, asbestos, heavy metals, and pesticides.

Statistical analyses of the data were conducted using the StatView statistical package, version 5.0.1 (Abacus Concepts, Berkeley, California). Data are expressed as the mean ± SD. Differences between the mean values of 2 continuous variables were evaluated using the unpaired Student t test. Differences in noncontinuous variables and genotype distribution were tested using chi-square analysis. Unconditional logistic regression analysis was used to estimate the odds ratios (ORs) and 95% confidence intervals (CIs) for the association between CHD and parental exposure. The ORs were also adjusted for potential confounding factors. A 2-tailed p value <0.05 was chosen as the level of significance. In the genetic study, we used a case-only study design because it is considered the most powerful method to study gene–gene and gene–environment interaction in disease etiology, achieving greater statistical power than a case-control study of the same size. With independence assumed between the exposure and the genotype, case-only ORs for the relevant interactions and 95% CIs were estimated by using the presence or absence of the GST variant as the dependent dichotomous variable in the logistic regression models that tested for an association with parental exposure. The study was powered to allow an interaction of magnitude of 2.3 with ≥80% power and an error rate of 5% under a sample size of 170 only-cases.

Results

The demographic characteristics of the study population are listed in Table 1 . The mothers of the patients with CHD had a significantly lower age than the controls (p = 0.01). No significant differences between the 2 groups were found for paternal characteristics. The CHD cases had a significantly lower birth weight (p = 0.01) compared to the control children. The children with CHD were also more often conceived with artificial fertilization (p = 0.05).

The parental exposure to environmental factors and CHD risk is listed in Table 2 . Maternal smoking was not significantly associated with an increased risk of CHD (OR 1.2, 95% CI 0.7 to 1.8). Fathers who were smokers showed an increased risk of CHD in their offspring (OR 1.7, 95% CI 1.1 to 2.6, p = 0.02), especially fathers who were heavy smokers (≥15 cigarettes/day) had a high risk of having children with CHD ( Figure 1 ) . Parental alcohol use did not influence the risk of CHD, and neither did the existence of a chronic disease or disease in the first trimester of pregnancy. Exposure to diagnostic radiographs was more frequently seen among the case-parents. Both maternal (OR 2.6, 95% CI 1.6 to 4.2, p <0.0001) and paternal (OR 2.5, 95% CI 1.6 to 3.8, p <0.0001) occupational/environmental exposure to toxicants increased the risk of CHD. The specific occupational and professional risk factors for CHD are listed in Table 3 . In addition, the exposure of both parents to toxicants showed a significant additive risk (OR 4.5, 95% CI 2.5 to 8.3, p <0.0001) of CHD in the offspring ( Figure 2 ) . On multivariate analysis, a father’s heavy smoking (OR 1.9, 95% CI 1.1 to 3.3) and maternal exposure (OR 2.1, 95% CI 1.2 to 3.6) and paternal exposure (OR 1.8, 95% CI 1.1 to 3.0) to toxicants were the main determinants of CHD risk. In the case-only study, we considered the association between GST polymorphisms in children and parental exposure. The frequency of GSTM1 null genotype was 57.7%, and the frequency of the GSTT1 null genotype was 16.7%. The genotype distribution was within the range previously reported for GSTM1 and GSTT1 null genotypes in other white populations. We did not detect an interactive effect of GSTM1 or GSTT1 null genotypes and active maternal and paternal smoking ( Table 4 ). The risk of CHD tended to be greater in children carrying the GSTM1 and GSTT1 null genotypes who had parents exposed to toxicants. A significant interaction was found for the combined null GSTs and both maternal (OR 3.6, 95% CI 1.1 to 11.2, p = 0.03) and paternal (OR 3.3, 95% CI 1.0 to 10.8, p = 0.03) exposure to toxicants ( Figure 3 ) . Finally, children with the combined null GST genotypes had a greater risk (OR 6.5; 95% CI 1.5 to 28.0) than children carrying wild-type GST genes when both parents were exposed ( Figure 3 ).

Parental smoking and CHD risk.

Interactive effect between maternal and paternal exposures on CHD risk.

Table 4

Gene–environment interactions for combination of glutathione S-transferase (GST) genes and parental exposure

Positive Parental Exposure	GSTM1		OR (CI 95%)	p Value
	Present	Absent
Mother
Active smoking				0.3
Negative	54	61	1.0
Positive	19	31	1.4 (0.7–2.9)
Diagnostic x-ray exposure				0.1
Negative	61	70	1.0
Positive	17	32	1.6 (0.8–3.2)
Other toxicant exposures				0.2
Negative	52	58	1.0
Positive	26	44	1.5 (0.8–2.9)
Father
Active smoking				0.8
Negative	34	45	1.0
Positive	36	45	1.0 (0.5–1.8)
Diagnostic x-ray exposure				0.6
Negative	60	75	1.0
Positive	18	27	1.2 (0.6–2.4)
Other toxicant exposures				0.1
Negative	45	47	1.0
Positive	33	55	1.6 (0.9–2.9)
	GSTT1
Mother
Active smoking				0.1
Negative	93	22	1.0
Positive	45	5	0.5 (0.2–1.3)
Diagnostic x-ray exposure				0.9
Negative	109	22	1.0
Positive	41	8	1.0 (0.4–2.3)
Other toxicant exposures				0.08
Negative	96	14	1.0
Positive	54	16	2.0 (0.9–4.5)
Father
Active smoking				0.4
Negative	63	16	1.0
Positive	69	12	0.7 (0.3–1.4)
Diagnostic x-ray exposure				0.1
Negative	109	26	1.0
Positive	26	4	0.4 (0.1–1.2)
Other toxicant exposures				0.09
Negative	81	11	1.0
Positive	69	19	2.0 (0.9–4.5)

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