Lucina Suarez, PhD Kate Hendricks, MD Texas Department of Health Neural Tube Defects Among Mexican Americans Living on the Texas-Mexico Border: Effects of Folic Acid and Dietary Folate Lucina Suarez, PhD Kate Hendricks, MD Texas Department of Health Lucina Suarez, PhD. is a Senior Scientist and head of the Epidemiology Research and Evaluation Section of the Texas Department of Health. She was a co-investigator on the Texas Neural Tube Defect Project Kate Hendricks, MD is head of the Infectious Disease Epidemiology and Surveillance Division at the Texas Department of Health. She was the primary investigator for the Texas Neural Tube Defect Project Please send comments and questions to Zunera Gilani at zunera.gilani@tdh.state.tx.us Prepared by Zunera Gilani, 2002

Suggested Readings Suarez L, Hendricks KA, Cooper SP, et al. Neural tube defects among Mexican Americans living on the US-Mexico border : Effects of folic acid and dietary folate. Am J Epidemiol 2000; 152: 1017-23. Shaw GM, Schaffer D, Velie EM,et al. Periconceptional vitamin use, dietary folate, and the occurrence of neural tube defects. Epidemiology 1995; 6: 219-26. Harris JA, Shaw GM. Neural tube defects-why are rates high among populations of Mexican descent? Environ Health Perspect 1995; 103 (suppl 6): 163-4. Hendricks KA, JS Simpson, Larsen RD. Neural tube defects along the Texas-Mexico border, 1993-1995. Am J Epidemiol 1999;149 : 1119-27.

Neural Tube Defects NTDs are malformations of the developing brain and spinal cord NTD closure occurs during the fourth week of embryosis The most common NTDs are anencephaly and spina bifida Numerous studies have shown a 50% to 100% reduction in NTDs by taking .4 mg of folic acid daily NTD prevalence is higher among Mexican Americans (9-16 per 10,000) than non-Hispanic whites (6 per 10,000) and African Americans (5 per 10,000)

Background Cameron County, on the Texas-Mexico border, had the highest prevalence in the US since the 1970s--29 per 10,000 in 1991 In 1991 an unusually large cluster of anencephalic births occurred in Brownsville, Texas (Cameron County) In one hospital six affected deliveries occurred in six weeks A Texas Department of Health-CDC joint investigation of this cluster revealed that NTDs were endemic among the entire Texas-Mexico border population This recognition of an NTD problem along the border prompted long-term active surveillance of NTDs, a case-control study, and a primary prevention program Texas Mexico Cameron County Brownsville

US Prevalence per 10,000 Births Hawaii 1988-94 7.2 Iowa 1985-90 9.0 California 1989-91 9.3 Atlanta 1990-91 9.9 Arkansas 1985-89 10.3 South Carolina 1992-94 16.0 Contrast the Cameron County prevalence (29 per 10,000) with those across the U.S.

Prevalence in Mexican Americans (per 10,000 births) Cameron County, Texas 1990-92 21.2 Texas-Mexico border 1993-95 14.9 Mexico-born 15.1 US-born 9.5 California 1989-91 Mexico-born 16.0 US-born 6.8 The high NTD prevalence seen in Cameron County in the early 1990s dropped somewhat in the subsequent years, but levels remain high along the border Mexican American women migrating to the US have NTD risks that are intermediate between those of women living in Mexico (36 per 10,000) and US born Mexican American women (7-10 per 10,000) The progressive shift in risk toward the lower risk among US non-Hispanic whites suggests a shift toward more folate-rich diets However, Mexican American women of childbearing age consume more dietary folate than other North American women, though they use multivitamins less often

Folic Acid Supplements Estimated Risk Reduction from Clinical Trials Author Location NTD Type Study Size Risk Reduction Laurence 1981 Wales Recurrence 111 57% Smithells 1983 England 1052 88% Vergel 1990 Cuba 214 100% MRC 1991 Europe 1195 72% Kirke 1992 Ireland 261 Czeizel 1992 Hungary Occurrence 4156 Milunsky 1989 USA Occurrence cohort 13870 73% All clinical trials of folic acid and NTD risk have been carried out among populations outside the US The Milunsky study, conducted in Massachusetts among women undergoing maternal serum alpha fetoprotein screening at 16 weeks gestation, was actually an observational cohort study The Vergel study showed a strong protective effect among Cuban women

Folic Acid Supplements Estimated Risk Reduction from Case-Control Studies Author Location Case:Control Risk Reduction Winship, 1984 England 764:764 86% Mulinare, 1988 Atlanta 181:1480 60% Mills, 1989 Illinois-California 565:567 11% Martinez-Frias, 1992 Spain 16736:16574 30% Bower, 1992 Australia 75:150 89% Werler, 1993 Boston-Philadelphia 232:1558 Shaw, 1995 California 295:247 35% The study by Shaw, et al is the only U.S. study that included a population of Mexican descent The Martinez-Frias study, a hospital-based case-control study in a Spanish population, reported a 30% risk reduction for postconception folic acid use Cuban and Spanish population are distinctly different from Mexican Americans in terms of Amerindian genetic admixture and NTD-related risk factors like diabetes and obesity

Shaw 1995 : Results on Folic Acid Supplements by Race/Ethnicity OR 95% CI Mexican American 1.0 0.4-2.1 Non-Hispanic White 0.6 0.4-1.1 African American 0.5 0.1-3.2 Shaw’s study looked at whether periconceptual intake of supplemental or dietary folate reduced the risk of having an NTD-affected pregnancy The data for the population based case-control study came from the California Birth Defects Monitoring Program Compare the 40% risk reduction observed in non-Hispanic whites to the lack of an effect among Hispanics Shaw concluded that folic acid supplements may not protect populations of Mexican American descent

Shaw 1995: Results on Combined Folate Intake from Vitamins and Diet Folate µg Mexican American Non-Hispanic <228 referent referent 228-399 1.0 (0.4-2.4) 0.6 (0.2-1.7) 400-999 0.8 (0.3-1.9) 0.6 (0.2-1.5) ≥1000 0.6 (0.2-2.0) 0.3 (0.1-0.8) The protective effect from daily folate intake (>1mg) in non-Hispanic whites was double that in Hispanic women It is not clear why the NTD risk in Mexican Americans would not be amenable to folic acid The migrant NTD risk pattern of Mexican women would seem to implicate an environmental cause such as folate deficient diets rather than an underlying genetic defect Nonetheless, a genetic heterogeneity in metabolizing folic acid may exist Other complicating factors include higher prevalences of obesity and diabetes (NTD risk factors) in Mexican Americans which may modify effects of folic acid

Specific Aims of Texas Border Study To determine the extent that preconception intake of: supplemental folic acid dietary folate alone folic acid and folate from combined sources reduces the risk of NTDs in Mexican American women We wanted to determine if folic acid was effective in reducing NTD risk in Mexican American women along the Texas-Mexico border We placed special interest in whether our findings would be consonant or dissonant with Shaw’s study in California

Case Definition Cases were ascertained through active surveillance of the 14 counties along the Texas-Mexico border (Jan 1995-May 2000) Cases were identified from multiple sources including hospitals, birthing centers, midwives, genetic clinics, abortion clinics Data for this study came from the Texas Department of Health’s Neural Tube Defect Project

Population-based Case-Control Study Cases included all clinically apparent NTDs at all gestational ages prenatally diagnosed, induced or spontaneously aborted still or live born Controls were randomly selected from normal live births, frequency matched by hospital and year Cases were defined as infants or fetuses who had anencephaly, spina bifida, or encephalocele identified at birth or prenatally between January 1995 and May 2000 We identified control women from residents of the study area who had normal births during the same time period Subjects were approached about study enrollment either prenatally or in the hospital at the time of delivery or pregnancy termination

Measurement Instruments In-person interviews, English or Spanish, one month postpartum Extensive mother questionnaire health and reproductive history, demographics, nutritional supplements, drug use, environmental exposures 98-item food frequency questionnaire Exposure in the periconceptional period, 3 months before to 3 months after conception Mother Questionnaire The interview instrument was modeled after the Centers for Disease Control and Prevention’s 1993 mother questionnaire for birth defects risk factor surveillance Food Frequency Questionnaire The Food frequency questionnaire was developed by TDH for this study population Questions were based on 100 24-hour dietary recalls among new enrollees in the federal Women, Infants, and Children (WIC) nutrition program in Cameron, El Paso, Hidalgo, and Webb counties The enrollees in WIC, poor Mexican American women of childbearing age, typified the high risk border population

Main Exposure Variables Folic acid supplements prenatal vitamins, multivitamins or single-ingredient folic acid tablets Dietary folate based on food frequency questionnaire Combined supplemental folic acid and dietary folate Folic Acid Supplements Women were asked whether they had taken prenatal vitamins, multi-vitamins, or single-ingredient folic acid tablets during the periconceptual period 3 months before and after conception For each month during the 6 month period, interviewers recorded the type of folic acid supplement used, number of pills taken daily/weekly, and number of days/weeks in which vitamins were taken Prenatal vitamins were assumed to contain 1 mg of folic acid, multivitamins or single folic acid tablets were assumed to contain .4 mg For analysis, exposure to folic acid was defined as use of folic-acid containing vitamins during the 3 months before conception Reported use of vitamins during this time period is thought to closely reflect true intake during neural tube closure (~1 month postconception) This exposure definition is the same as the one used by Shaw Dietary Folate Dietary folate intake measured with the 98-item food frequency questionnaire For each food item, women estimated their usual frequency of intake (average number of times per month/week/day) over the 6 month periconceptual period Exposure categories for dietary folate intake were based on quartiles derived from the distribution of the controls Combined Folate Exposure We combined average daily dose of folic acid supplements and average daily dietary folate intake

Confounding Variables maternal age education annual income country of birth cigarette smoking alcohol use diabetes obesity gravidity previous pregnancy loss prenatal care oral contraceptive use Potentially confounding variables were identified from a review of all previous NTD case-control studies Only maternal age, education, obesity, and previous still birth or miscarriage proved to be important risk factors or confounders in preliminary analysis These were later included in the adjustment of estimates

Study Participation Rates Case women Control women Identified for study 225 378 Completed interview 184 225 Refused interview 26 101 Moved 15 52 We had a higher participation rate among case women (82%) than among control women (60%) About twice as many control women refused to be interviewed or had moved out of the study area

Distribution of Cases NTD type Pregnancy outcome 83 anencephaly 84 spina bifida 17 encephalocele Gestation 41 <20 weeks 70 >35 weeks Pregnancy outcome 94 live births 27 still births 4 miscarriages 59 elective terminations The NTD types were distributed as expected: 45% anencephaly, 46% spina bifida, 9% encephalocele Active surveillance identified NTDs from a variety of pregnancy outcomes Because controls were normal live births, gestational differences exist between cases and controls This affected the length of the recall period at the time of the interview, since interviews were held roughly one month postpartum for both case and control women

Demographic and Risk Factor Characteristics The study population is poor, of low education and equally divided into U.S. or Mexico-born

Crude ORs for NTD Risk Factors 95% CI Age >25 years 0.9 0.6-1.3 Mexico-born 1.1 0.8-1.7 Education 7+ years 0.6 0.3-1.0 Cigarette smoking 1.8 1.0-3.0 Alcohol use 1.3 0.8-1.9 Diabetes 1.6 0.7-3.3 Obesity 1.1-2.8 Prior pregnancy loss 1.9 1.1-3.3 As in other populations, prior unproductive pregnancy, defined as prior miscarriage or stillbirth, and obesity were strong risk factors among Mexican Americans The risk of NTD was comparable between U.S.-born and Mexico-born women

Daily Vitamin Use Among Case and Control Women A substantial proportion of both case and control women did not consume vitamins at all, prenatal or otherwise, during the 6-month periconceptual period Consumption of folic acid vitamins before conception was extremely low among case and control women Roughly half of case and control women did not consume folic acid containing vitamins at all during pregnancy

Use of Folic Acid-Containing Vitamins More than half of case and control women had started taking vitamins only after conception Vitamin use, predominantly in the form of prenatal vitamins, occurred well after the time of neural tube closure in the second and third month of pregnancy In this poor population, use of multivitamins was rare

Frequency Distribution of Average Daily Dietary Folate Intake The frequency distribution of dietary folate consumption for control women is shifted slightly to the right of case women, that is towards higher intakes Note that folate from dietary intake is in the polyglutamated form, which is only about 50% bioavailable Folic acid in the form of supplements is almost 100% bioavailable

Periconceptional Vitamin Use Case Women Control Women No. % None (6 month interval) 102 44.7 126 5.3 Preconception Any use 1-3 months before conception 11 6.0 10 4.4 Daily use every month before conception 5 2.7 9 4.0 Postconception (use 1-3 months after) 55.4 56.0 Periconceptual vitamin use among Mexican American case women with neural tube defect-affected pregnancies and control women in 14 Texas-Mexico border counties, 1995-1999 Vitamin use was defined as maternal use of multivitamins, prenatal vitamins, or single ingredient folic acid tablets from 3 months before conception to 3 months after conception Only 11 case and 10 control women reported daily use of folic acid-containing vitamins throughout the entire preconception period

Effect of Preconceptional Use of Folic Acid-Containing Vitamins on NTD Risk OR 95% CI Crude 0.7 0.2-2.1 Adjusted 0.8 0.2-2.6 Odds ratios are compared to non-users alone Adjusted for education, age, previous pregnancy loss, and obesity We observed a modest 20% reduction in risk due to folic acid This estimate had a confidence interval which overlapped with the null; this is compatible with no effect

Effect of Dietary Folate on NTD Risk Daily Folate Intake (mg/day) OR 95% CI Adj OR† .925-.431 1.0‡ referent  .432-.613 1.0 0.6-1.7 0.6-1.8 .614-.786 0.7 0.4-1.1 0.4-1.2 .787-2.10 0.4-1.3 0.8 0.4-1.4 Odds ratios for quartiles of average daily dietary folate intake during the periconceptual period showed a reduced risk for both the third and fourth quartiles (30%), when compared with the lowest quartile 21 women who took folic acid preconceptually, 7 women without food frequency data, and five additional women without obesity data were excluded † Adjusted for maternal age, education, obesity, and previous still birth or miscarriage ‡Referent

Effect of Combined Dietary and Supplemental Folate Folate Intake (mg/day) OR 95% CI Adj OR† 95% CI <.400 1.0‡ referent .400-.999 0.8 0.5-1.3 0.9 0.5-1.4 ≥1.000 0.7 0.3-1.3 0.3-1.4 Combining the estimated daily folate intake from diet with the estimated daily dose from vitamins showed modest risk reductions of 20% and 30% for daily intakes of 0.40-0.99 mg and ≥1.0 mg respectively These risk reductions were further diminished when data were adjusted All estimates had 95% confidence intervals that overlapped with the null 8 women without food frequency data and five women without obesity values were excluded †Adjusted for maternal age, education, obesity, and previous still birth or child birth ‡Referent

Main Findings Mild to no benefit in using folic acid containing vitamins (20% reduction) 30% risk reduction for combined intakes of ≥1.0 mg per day, adjusted for covariates The fact that so few Mexican American women in this study consumed multivitamins during the critical time period severely limited what could be determined about supplemental folic acid and NTD risk in this population Combining the estimated folic intakes from supplements and dietary sources did not reveal any strong effects Our findings are consonant with Shaw 1995 who also showed modest effects for combined sources (20% - 40%) The risk reductions for Hispanics contrast sharply with the much greater reductions seen among non-Hispanic White women at the same levels (in the Shaw study) The modest effects of combined intakes are probably due to the intake ratios of dietary folate to supplemental folic acid Hispanic women receive a smaller proportion of the bioavailable form (folic acid) than other women

Underlying Population Factors Economically disadvantaged and medically underserved Distinct gene admixture with Amerindian population Traditional yet atypical dietary pattern Environmental contaminants pesticides or fumonisins Higher frequencies of specific folate pathway gene polymorphisms The modest or weak effect of folate among Mexican Americans suggests that levels are insufficient for achieving reductions seen in other populations or insufficient for overcoming other underlying risk factors Host susceptibility factors could center on a genetic or acquired variability in the intake, uptake, or metabolism of folic acid There could be interference from environmental toxicants such as pesticides from farm work or other occupational exposures to organic solvents Fumonisin, a naturally occurring mycotoxin in corn (a very common food in this population), has recently been shown to affect cellular folate uptake

Study Limitations Low power Low prevalence of exposure Homogeneous dietary exposure Given the study size and the 3% exposure to folic acid, the power to detect a 50% reduction in risk was dismally low (21 percent) At such low levels of exposure to folic acid containing vitamins, the study would require 6 times the existing number of cases and controls (~1200) (α=.05; β=.80) Diets in this population were relatively homogeneous compared to other U.S. populations According to Geoffrey Rose 1985, this would have decreased the probability of identifying folate as a component cause of NTDs

Potential Biases Food Frequency Questionnaire FFQs less valid among low-educated populations FFQs overestimate absolute levels of folate intake Referenced a single period from 3 months before to 3 months after conception (pregnancy influenced recall) An overestimate of true folate intake among Mexican American women would have made effects appear weaker than they were The FFQ doesn’t distinguish postconception time from preconception time Many women adhere to a healthier diet during pregnancy These women may have selectively recalled healthy (folate-rich) foods consumed after the neural tube closed (~1 month postconception) rather than their more relevant preconception intake If case and control women misclassified their exposures to the same extent, estimated odds ratios would have been biased towards the null

Potential Biases Differential recall period between cases and controls (ie, gestational age) Differential participation rates (60% vs 82%) Controls demographically similar to source population Given the earlier termination of NTD-affected pregnancies, at the time of interview, control women were recalling exposures further in the past than case women Although the case-control difference in recall time was only about 1 month, the shorter recall time for case women may have caused them to remember exposures somewhat more accurately than control women Combined with the possibility that women with affected pregnancies would recall exposures more carefully, this differential exposure misclassification also would have attenuated observed protective folate effects If selection bias produced the weak or null folate effect, then control women with higher socioeconomic status (better diets and more vitamin use) must have refused participation more often However, we know from vital statistics data that control women in the study were remarkably similar to the entire border population

Inconclusive results because of methodological problems Almost no exposure to folic acid Homogeneous dietary exposures Sources of bias would push estimated effects toward the null The question of whether folic acid reduces NTD risk in Mexican Americans to the extent observed in other populations remains unresolved because of methodological problems

Recommendations Test this hypothesis in a Mexican American population more highly exposed to folic acid Pursue all recommended prevention strategies Motivate Mexican American women of childbearing age to take folic acid routinely Fortify foods consumed by this population