1. 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
Prepared by Zunera Gilani, 2002

2. 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:
Shaw GM, Schaffer D, Velie EM,et al. Periconceptional vitamin use, dietary folate, and the occurrence of neural tube defects. Epidemiology 1995; 6:
Harris JA, Shaw GM. Neural tube defects-why are rates high among populations of Mexican descent? Environ Health Perspect 1995; 103 (suppl 6):
Hendricks KA, JS Simpson, Larsen RD. Neural tube defects along the Texas-Mexico border,
Am J Epidemiol 1999;149 :

3. 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)

4. 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

5. US Prevalence per 10,000 Births
Hawaii
Iowa
California
Atlanta
Arkansas
South Carolina
Contrast the Cameron County prevalence (29 per 10,000) with those across the U.S.

6. Prevalence in Mexican Americans (per 10,000 births)
Cameron County, Texas
Texas-Mexico border
Mexico-born
US-born
California
Mexico-born
US-born
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

7. 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

8. 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

9. Shaw 1995 : Results on Folic Acid Supplements by Race/Ethnicity
OR 95% CI
Mexican American
Non-Hispanic White
African American
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

10. Shaw 1995: Results on Combined Folate Intake from Vitamins and Diet
Folate µg Mexican American Non-Hispanic
< referent referent
( ) ( )
( ) ( )
≥ ( ) ( )
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

11. 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

12. 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

13. 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

14. 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 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

15. 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

16. 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

17. Study Participation Rates
Case women Control women
Identified for study
Completed interview
Refused interview
Moved
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

18. 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

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

20. Crude ORs for NTD Risk Factors
95% CI
Age >25 years
0.9
Mexico-born
1.1
Education 7+ years
0.6
Cigarette smoking
1.8
Alcohol use
1.3
Diabetes
1.6
Obesity
Prior pregnancy loss
1.9
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

21. 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

22. 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

23. 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

24. 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,
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

25. Effect of Preconceptional Use of Folic Acid-Containing Vitamins on NTD Risk
OR 95% CI
Crude
Adjusted
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

26. Effect of Dietary Folate on NTD Risk
Daily Folate Intake (mg/day) OR
95% CI
Adj OR†
1.0‡
referent 
1.0
0.7
0.8
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

27. Effect of Combined Dietary and Supplemental Folate
Folate Intake
(mg/day) OR
95% CI
Adj OR†
95% CI
<.400
1.0‡
referent
0.8
0.9
≥1.000
0.7
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 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

28. 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

29. 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

30. 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

31. 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

32. 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

33. 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

34. 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