American Journal of Medical Genetics Part C (Seminars in Medical Genetics) 154C:62 72 (2010)

A R T I C L E

Risk Factors for Non-Syndromic Holoprosencephaly in the National Birth Defects Prevention Study
AS, ERIC A. MILLER,* SONJA A. RASMUSSEN, ANNA MARIA SIEGA-RIZ, JAIME L. FRI MARGARET A. HONEIN AND THE NATIONAL BIRTH DEFECTS PREVENTION STUDY
Holoprosencephaly (HPE) is a complex structural brain anomaly that results from incomplete cleavage of the forebrain. The prevalence of HPE at birth is low, and risk factors have been difcult to identify. Using data from a large multi-state population-based case-control study, we examined risk factors for non-syndromic HPE. Data from maternal telephone interviews were available for 74 infants with HPE and 5871 controls born between 1997 and 2004. Several characteristics and exposures were examined, including pregnancy history, medical history, maternal diet and use of nutritional supplements, medications, tobacco, alcohol, and illegal substances. We used w2-tests and logistic regression (excluding women with pre-existing diabetes) to examine associations with HPE. Except for diet (year before pregnancy) and sexually transmitted infections (STIs) (throughout pregnancy), most exposures were examined for the time period from the month before to the third month of pregnancy. HPE was found to be associated with pre-existing diabetes (w2 6.0; P 0.01), aspirin use [adjusted odds ratio (aOR) 3.4; 95% condence interval (CI) 1.66.9], lower education level (aOR 2.5; 95%CI 1.15.6), and use of assisted reproductive technologies (ART) (crude OR 4.2; 95%CI 1.313.7). Consistent maternal folic acid use appeared to be protective (aOR 0.4; 95%CI 0.21.0), but the association was of borderline statistical signicance. While some of these ndings support previous observations, other potential risk factors identied warrant further study. Published 2010 Wiley-Liss, Inc.{ KEY WORDS: holoprosencephaly; forebrain defect; epidemiology

as JL, Honein MA, the National How to cite this article: Miller EA, Rasmussen SA, Siega-Riz AM, Fr Birth Defects Prevention Study. 2010. Risk factors for non-syndromic holoprosencephaly in the National Birth Defects Prevention Study. Am J Med Genet Part C Semin Med Genet 154C:6272.

The ndings and conclusions in this report are those of the authors and do not necessarily represent the ofcial position of the Centers for Disease Control and Prevention. Eric A. Miller, PhD, is the Epidemiology Manager for the Texas Cancer Registry and former epidemiologist for the National Center on Birth Defects and Developmental Disabilities. His research interests include etiology of and risk factors for birth defects, preterm birth, and cancer. Sonja A. Rasmussen, MD, MS, is a Pediatrician and Clinical Geneticist and currently serves as Senior Scientist at the National Center on Birth Defects and Developmental Disabilities at the Centers for Disease Control and Prevention in Atlanta. Her research interests include the identication of risk factors for birth defects, morbidity and mortality associated with birth defects and genetic conditions, and the impact of infections on the pregnant woman and her embryo or fetus. Dr. Siega-Riz is a Professor of Epidemiology and Nutrition. She serves as the Associate Chair and Program Leader of the Reproductive, Perinatal, and Pediatric program area in the Department of Epidemiology. Her research interests include maternal nutritional status and its relationship with birth outcomes, gestational weight gain, and obesity development. as, MD, is a Professor Emeritus of Pediatrics at the University of South Florida in Jaime L. Fr Tampa, and currently a Visiting Scientist at the National Center on Birth Defects and Developmental Disabilities at the Centers for Disease Control and Prevention in Atlanta. He is a Clinical Geneticist whose research has focused in the denition of patterns of malformation and the epidemiology of birth defects. Margaret A. Honein, Ph.D., MPH is an Epidemiologist and Chief of the Birth Defects Surveillance and Epidemiology Branch in the National Center on Birth Defects and Developmental Disabilities, Centers for Disease Control and Prevention, Atlanta, GA. Grant sponsor: Centers for Disease Control and Prevention; Grant number: U50/CCU613232. *Correspondence to: Eric A. Miller, Cancer Epidemiology and Surveillance Branch - MC 1928, Texas Department of State Health Services, PO Box 149347, Austin, TX 78714-9347, USA. E-mail: eric.miller@dshs.state.tx.us DOI 10.1002/ajmg.c.30244 Published online 26 January 2010 in Wiley InterScience (www.interscience.wiley.com)

INTRODUCTION
Holoprosencephaly (HPE) is a clinically and causally heterogeneous structural brain defect that has been found to be associated with chromosome abnormalities, single gene disorders, and certain teratogenic agents [Rasmussen et al., 1996; Wallis and Muenke, 2000; Muenke and Gropman, 2008; Bendavid et al., 2010; Solomon et al., 2010a, 2010b]. The defect, which is due to failed or insufcient cleavage of the forebrain [DeMyer et al., 1964], results in a spectrum of craniofacial anomalies that varies from cyclopia to a single central incisor [DeMyer et al., 1964]. Although HPE is the most common human forebrain defect with an estimated prevalence of 1:250 conceptuses [Edison and Muenke, 2003], its prevalence at birth is low, ranging between 0.9

Published 2010 Wiley-Liss, Inc. { This article is a US Government work and, as such, is in the public domain in the United States of America.

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and 1.2 per 10 000 live births [Croen et al., 1996; Rasmussen et al., 1996]. This low birth prevalence has hindered the conduct of populationbased analyses to examine risk factors. To date, only two case-control studies assessing maternal exposures that are not reported as part of vital statistics or other administrative processes have been conducted. The rst study analyzed data from the California Birth Defects Monitoring Program and identied several possible risk factors, including foreign maternal birthplace; early age at menarche; higher gravidity; history of diabetes or anemia; periconceptional exposure to alcohol or tobacco; respiratory illness, including exposures to medication for respiratory illnesses; and exposure to salicylate-containing medications [Croen et al., 2000]. The second study analyzed data from the Latin American Collaborative Study of Congenital Malformations, a hospital-based registry of births in South American countries. These authors identied maternal diabetes, u, vaginal bleeding, and use of sex hormones during the rst trimester as possible HPE risk factors [Orioli and Castilla, 2007]. Using data from the National Birth Defects Prevention Study (NBDPS), we examined several risk factors for non-syndromic HPE and did an extensive assessment of diet and nutrient intake, which has not been previously reported.

Using data from the National Birth Defects Prevention Study (NBDPS), we examined several risk factors for non-syndromic HPE and did an extensive assessment of diet and nutrient intake, which has not been previously reported.
MATERIALS AND METHODS
The NBDPS is a large, ongoing, population-based case-control study examining risk factors for over 30 major birth

defects. Because the focus of the NBDPS is on identifying risk factors for birth defects of unknown etiology, infants known or strongly suspected to have a condition of known etiology (chromosome abnormality or single-gene condition) are excluded from the study. Information on case infants is reviewed by a clinical geneticist at each site before inclusion to ensure that case denitions for the study are met [Rasmussen et al., 2003]. Additional information on the methods of this study has been published previously [Yoon et al., 2001]. This analysis included infants from 10 states (Arkansas, California, Georgia, Iowa, Massachusetts, New Jersey, New York, North Carolina, Texas, and Utah) with dates of birth on or after October 1, 1997 through December 31, 2004. Infants with varying degrees of HPE that had been conrmed postnatally, either by imaging studies or postmortem examination, were included in this analysis. In addition to the review at each site, clinical information on these infants was reviewed by a single clinical geneticist (JLF) to ensure that all met the case inclusion criteria and to classify the infant as having isolated HPE (no additional unrelated major defect) or multiple defects (one or more unrelated major defects in addition to the HPE) [Rasmussen et al., 2003]. Controls were a random sample of liveborn infants without birth defects selected from the same geographic regions (based on maternal residence at delivery) and time period. The NBDPS has been approved by the Institutional Review Boards at CDC and all participating sites. Telephone interviews with mothers were conducted by trained interviewers using a standardized questionnaire between 6 weeks and 24 months after the estimated date of delivery (median months to interview: cases 10, controls 7). The participation rates were 59% for mothers of infants with HPE and 69% among control mothers. The extensive interview included questions about demographic characteristics, pregnancy history, pregnancy intention, use of fertility procedures, medical history (including diabetes, hypertension, infections, or fevers), maternal diet

and use of nutritional supplements, medications, tobacco, alcohol, and illicit substances (including paternal use). In this analysis, most exposures were examined from the month before pregnancy to the third month of pregnancy (periconceptional period). Because some sexually transmitted infections (STIs) can be latent during pregnancy and women might not be tested during the periconceptional period, STIs were examined from the month before pregnancy through delivery. Use of folic acid and folic acid-containing multivitamins was examined as any use during the periconceptional period and as consistent use, dened as use in each month during the periconceptional period. Information on pregnancy intention was based on responses to a question on use of contraception and a question asking if at the time of pregnancy, the mother wanted to become pregnant then, wait until later, or not become pregnant at all [Dott et al., 2009]. Use of any fertility treatment during pregnancy was based on a gateway question: Did you or the father take any medications or have any procedures to help you become pregnant? We also examined use of assisted reproductive technologies (ART), which was dened as any procedure where sperm and egg were medically handled, and included in vitro fertilization, gamete intrafallopian transfer, zygote intrafallopian transfer, tubal embryo transfer, and intracytoplasmic sperm injection. Analysis of use of sex hormones during the periconceptional period was limited to progesterone because it was the only hormone use reported by mothers of infants with HPE. Dietary Exposure Dietary habits were collected for the year before pregnancy using a modied 58question Willett food frequency questionnaire [Willett et al., 1987]. Calculation of micronutrients from foods consumed was based on the USDA National Nutrient Database Standard Reference 16-1 (http://www.nal.usda. gov/fnic/foodcomp/Data/SR16-1/sr161.html). We rst examined the association with HPE for 58 foods ascertained

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by the questionnaire by frequency of consumption, (none, below median, above median) and by grams consumed per food item (none, below median, above median). Foods were also grouped into categories (e.g., vegetables, fruits, etc.) using grams consumed per food item and examined using quartiles. The associations between quartiles of specic macro- and micronutrients and HPE were also examined. Specic nutrients examined included protein, fat, ber, cholesterol, folate (total folate, dietary folate, and folic acid from foods), calcium, iron, selenium, sodium, potassium, caffeine, and vitamins A, B2, C, D, and E. Medians and quartiles were determined based on consumption among control mothers. All models of nutrients controlled for total energy intake using a nutrient density method [Willett et al., 1997] and nal adjusted models for nutrients and foods included all the variables selected for the other exposure models. Because of the large number of foods and nutrients examined, only selected results are presented. Full results are available by request. Statistical Analysis The analysis examined HPE separately for isolated defects and for all cases, which includes HPE classied as having multiple defects. The clinical characteristics of infants (e.g., birthweight) with HPE were examined descriptively and compared with controls when possible, using w2-tests to determine statistical differences. Birthweight and preterm delivery were only examined among liveborn singleton births. Demographic characteristics, pregnancy characteristics, and various exposures during pregnancy were compared between mothers of infants with HPE and control mothers by crude (cOR) and adjusted odds ratios (aOR) with corresponding 95% condence intervals (CI). We examined the association with preexisting diabetes as a characteristic of pregnancy, but because diabetes has been consistently associated with birth defects, women with pre-existing diabetes (cases n 2; controls n 32) were

excluded from all other exposure risk factor analyses. We used multivariable logistic regression to examine associations with HPE for exposures that had 5 exposed cases. Confounding was assessed separately for each exposure variable and separately for all HPE cases combined and isolated HPE using a 10% change in estimate criterion. Any variable found

their own associations with HPE in multivariable models, therefore, only the results for the non-imputed data are presented.

RESULTS
Interview data were available from mothers of 74 infants with HPE and 5871 controls for this analysis. Most infants were classied as having isolated HPE (n 50, or 68%) (Table I). Ninetysix percent of infants with HPE in this analysis were liveborn; however, only 61% were alive at the time of interview. Among liveborn singleton births, infants with HPE were more likely than controls to be born preterm or very preterm (P < 0.001) or with low or very low birthweight (P < 0.001). For all HPE, a higher percentage of infants were female or from a multiple gestation pregnancy (twins or higher) compared to controls, but the associations did not reach statistical signicance. There was also an association with diabetes before pregnancy and having an infant with HPE (P 0.01). A higher percentage of mothers of infants with HPE were found to be aged 19 years, of lower education (<high school), lower family income (<$20K), Hispanic ethnicity, and were interviewed in Spanish, compared to controls (Table II). However, only the association with lower education level among all cases of HPE remained statistically signicant in the adjusted models. Results did not meaningfully differ for isolated HPE compared to all HPE cases. Among pregnancy characteristics (Table III), there was a strong crude association with use of ART (cOR 4.2; 95%CI 1.3 13.7). However, there were only three infants with HPE who were reported to be conceived through ART and all were by in vitro fertilization. Other pregnancy characteristics with evidence of associations with HPE were having an unwanted pregnancy and having an STI or urinary tract infection (UTI). However, these associations were not statistically signicant or of borderline statistical signicance in the adjusted models. Among infants with isolated

We used multivariable logistic regression to examine associations with HPE for exposures that had 5 exposed cases. Confounding was assessed separately for each exposure variable and separately for all HPE cases combined and isolated HPE using a 10% change in estimate criterion.

to be a confounder in a model was included in all multivariable models for consistency. The variables included in all nal models were maternal age (19, 20 34, 35), maternal education (<high school, high school graduate, >high school), maternal race/ethnicity (Non-Hispanic white/other, Non-Hispanic black, Hispanic), previous pregnancy (0, 1, 1), periconceptional aspirin use (yes, no), and periconceptional folic acid/multivitamin use (yes, no). Because of the large number of missing values for income [cases n 9 (12.5%); controls n 940 (10.5%)] and prepregnancy BMI [cases n 7 (9.7%); controls n 233 (4.0%)], we used multiple imputation, based on maternal age, race/ethnicity, and maternal and paternal education level and job status (working, not working, student) to determine the inuence the missing data might have had on associations. Imputed prepregnancy BMI and income did not appreciably change other associations or

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TABLE I. Clinical Features and Comparisons of Infants with Non-syndromic Holoprosencephaly (Cases; n 74) and Controls (n 5871) All cases Controls n (%) Birth defect classication Isolated NA Multiple NA Birthweighta Very low (<1500) 32 (0.6) Low (1500 2499) 231 (4.1) Normal (2500 3999) 4798 (84.7) Macro (4000) 603 (10.7) Sex Female 2914 (49.6) Male 2952 (50.3) Gestational agea Very preterm 59 (1.0) (<32 weeks) Preterm (32 402 (7.0) 36 weeks) Term (37 weeks) 5226 (91.9) Multiple pregnancy Singleton 5691 (97.0) Twins or greater 174 (3.0) Pre-existing diabetes Yes 32 (0.6) No 5839 (99.5) Birth outcome Live birth 5871b (100) Fetal death 0 (20 weeks) Pregnancy termination 0 Live birth survival at interview Yes 5859 (99.9) No 8 (0.01)
a b

Isolated cases w P-value NA

2

n (%) 50 (67.6) 24 (32.4) 4 (6.1) 9 (13.6) 49 (74.2) 4 (6.0) 40 (54.1) 33 (44.6) 5 (7.7) 11 (16.7) 50 (75.8) 69 (93.2) 5 (6.8) 2 (2.7) 72 (97.3) 71 (96.0) 2 (2.7) 1 (1.4) 43 (60.6) 28 (39.4)

n (%) 50 0 3 (6.3) 5 (10.4) 37 (77.1) 3 (6.3) 25 (50.0) 25 (50.0) 3 (6.3) 7 (14.6) 38 (79.2)

w2 P-value NA

<0.01

<0.01

0.38

0.96

<0.01

<0.01

0.06

48 (96.0) 2 (4.0) 0 50 (100) 50 (100) 0 0

0.67

0.01

0.60

NA

NA

<0.01

35 (70.0) 15 (30.0)

<0.01

Among liveborn singleton births. Controls were selected from live births only.

HPE, the association with unwanted pregnancy in the adjusted model was attenuated toward the null. HPE was inversely associated with consistent use of a folic acid supplement or a folic acid-containing multivitamin during the periconceptional period (Table IV). In the crude and adjusted models, consistent use of a folic acid supplement or a multivitamin containing folic acid was associated with a 60% reduced odds of all HPE and a 50% reduced odds of isolated HPE. The association was of borderline statistical

signicance in the adjusted model for all HPE and non-signicant for isolated HPE. In contrast to ibuprofen and acetaminophen, there was a strong positive association with aspirin use (Table IV). Mothers of infants with HPE were more than three times more likely to have taken aspirin during the periconceptional period compared to controls (aOR 3.4; 95%CI 1.6 6.9). Among infants with isolated HPE, the association was somewhat attenuated (aOR 2.0; 95%CI 0.7 5.5). There was an increased odds of HPE among

women who used progesterone during the periconceptional period (cOR 2.5; 95% 0.7 7.3) but there were only three mothers of infants with HPE who reported any use. There was little evidence of an association between HPE and maternal periconceptional use of alcohol, tobacco, and illicit drugs in this analysis, but the odds of HPE associated with paternal drug use were elevated, and this nding was stronger with isolated HPE. When looking at individual foods consumed, after adjustment for other

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TABLE II. Associations Between Maternal Demographic Characteristics and Having an Infant with Non-syndromic Holoprosencephaly (Cases n 72; Controls n 5839), Excluding Women With Pre-existing Diabetes All cases Controls n (%) Maternal age 19 years 632 (10.8) 20 34 4392 (75.2) 35 815 (14.0) Maternal race/ethnicity Hispanic 1312 (22.6) Non-hispanic black 665 (11.4) Non-hispanic white 3507 (60.3) Other 334 (5.7) Language of interview English 5439 (93.2) Spanish/translated 400 (6.8) Maternal country of origin U.S. 4710 (80.8) Other 1122 (19.2) Maternal education <High school 982 (16.9) High school graduate 2839 (48.7) Post high school 2008 (34.5) degree Income <$20K 1667 (31.9) $20K 3557 (68.1) Maternal state of residence Arkansas 711 (12.2) California 787 (13.5) Georgia 620 (10.6) Iowa 657 (11.3) Massachusetts 742 (12.7) New Jersey 575 (9.9) New York 519 (8.9) North Carolinac 287 (4.9) Texas 683 (11.7) Utahc 258 (4.4)
a b

Isolated cases aOR (95%CI) 1.2 (0.6 2.6) 1.0 (Ref) 1.1 (0.5 2.3) 1.4 (0.8 2.5) 1.0 (0.5 2.2) 1.0 (Ref) 1.0 (Ref) 1.1 (0.5 2.5) 1.0 (Ref) 0.7 (0.3 1.3) 2.5 (1.1 5.6) 1.5 (0.7 2.8) 1.0 (Ref)
b

n (%) 13 (18.1) 50 (69.4) 9 (12.5) 26 (36.1) 9 (12.5) 35 (48.6) 2 (2.8) 63 (87.5) 9 (12.5) 57 (79.2) 15 (20.8) 24 (33.8) 33 (46.5) 14 (19.7)

cOR (95%CI) 1.8 (1.0 3.3) 1.0 (Ref) 1.0 (0.5 2.0) 2.1 (1.2 3.4) 1.4 (0.7 2.9) 1.0 (Ref) 1.0 (Ref) 1.9 (1.0 3.9) 1.0 (Ref) 1.1 (0.6 2.0) 3.5 (1.8 6.8) 1.7 (0.9 3.1) 1.0 (Ref)

n (%) 8 (16.0) 34 (68.0) 8 (16.0) 17 (34.0) 8 (16.0) 25 (50.0) 0 43 (86.0) 7 (14.0) 39 (78.0) 11 (22.0) 15 (30.0) 23 (46.0) 12 (24.0)

cORa (95%CI) 1.6 (0.8 3.5) 1.0 (Ref) 1.3 (0.6 2.7) 2.0 (1.1 3.7) 1.8 (0.8 4.1) 1.0 (Ref)

aORb (95%CI) 1.2 (0.5 3.1) 1.0 (Ref) 1.4 (0.6 3.2) 1.5 (0.7 3.1) 1.6 (0.7 3.7) 1.0 (Ref) 1.0 (Ref) 1.5 (0.6 4.0) 1.0 (Ref) 0.8 (0.3 1.7) 1.9 (0.7 4.8) 1.2 (0.6 2.6) 1.0 (Ref)

1.0 (Ref) 2.2 (1.0 5.0) 1.0 (Ref) 1.2 (0.6 2.3) 2.6 (1.2 5.5) 1.4 (0.7 2.7) 1.0 (Ref)

32 (50.8) 31 (49.2) 8 (11.1) 13 (18.1) 3 (4.2) 10 (13.9) 7 (9.7) 7 (9.7) 7 (9.7) 2 (2.8) 15 (20.8) 1 (1.4)

2.2 (1.3 3.6) 1.0 (Ref) 0.7 (0.3 1.7) 1.0 (Ref) 0.3 (0.1 1.0) 0.6 (0.2 1.5) 0.8 (0.4 1.9) 0.7 (0.3 1.9) 0.8 (0.3 2.1) 0.4 (0.1 1.9) 1.3 (0.6 2.8) 0.2 (0.1 1.8)

1.5 (0.8 2.8) 1.0 (Ref) 0.9 (0.3 2.3) 1.0 (Ref) 0.4 (0.1 1.3) 0.9 (0.3 2.5) 1.4 (0.5 3.4) 0.9 (0.3 2.4) 1.1 (0.4 3.1) 0.6 (0.1 2.7) 1.4 (0.6 2.9) 0.4 (0.1 2.9)

20 (45.5) 24 (54.6) 5 (10.0) 8 (16.0) 3 (6.0) 5 (10.0) 8 (16.0) 5 (10.0) 5 (10.0) 1 (2.0) 9 (18.0) 1 (2.0)

1.8 (1.0 3.2) 1.0 (Ref) 0.7 (0.2 2.1) 1.0 (Ref) 0.5 (0.1 1.8) 0.7 (0.2 2.3) 1.1 (0.4 2.8) 0.9 (0.3 2.6) 0.9 (0.3 2.9) 0.3 (0.1 2.8) 1.3 (0.5 3.4) 0.4 (0.1 3.1)

1.4 (0.6 2.9) 1.0 (Ref) 0.8 (0.2 2.9) 1.0 (Ref) 0.5 (0.1 2.0) 1.2 (0.4 4.2) 1.7 (0.6 5.0) 1.1 (0.3 3.5) 1.3 (0.4 4.2) 0.4 (0.1 3.6) 1.3 (0.5 3.4) 0.6 (0.1 5.3)

Crude odds ratio. Adjusted odds ratio controlling for maternal age, race/ethnicity, education, previous pregnancies, folic acid/multivitamin use, aspirin use. c North Carolina and Utah joined the NBDPS in 2003 and therefore contributed fewer cases and controls.

variables, we found statistically signicant and borderline statistically signicant inverse associations with the highest levels of intake compared to no intake for skim/lowfat milk (aOR 0.5; 95%CI 0.2 0.9), yogurt (aOR 0.5; 95%CI 0.3 0.9), butter (aOR 0.5; 95%CI 0.3 0.9), string beans

(aOR 0.6; 95%CI 0.3 1.0), broccoli (aOR 0.6; 95%CI 0.3 1.0), chicken (aOR 0.6; 95%CI 0.4 1.0), and sh (aOR 0.5; 95%CI 0.3 1.0) (Table V). Although the point estimates were similar for isolated HPE, only the association with broccoli remained statistically signicant in the adjusted mod-

els. A signicant positive association was found for hot dog consumption for all HPE (aOR 1.9; 95%CI 1.1 3.2) and isolated HPE (aOR 2.3; 95%CI 1.2 4.3). When combining foods into groups (all vegetables, green vegetables, fruits, fruits and vegetables, all meats excluding sh, and all meats excluding

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TABLE III. Associations Between Pregnancy Characteristics and Having an Infant with Non-syndromic Holoprosencephaly, Excluding Women with Pre-existing Diabetes
All cases cORa (95% CI) 1.0 (Ref) 1.1 (0.6 2.1) 1.3 (0.8 2.4) 1.2 (0.7 2.0) 1.0 (Ref) 0.9 (0.3 2.9) 1.0 (Ref) 4.2 (1.3 13.7) 1.0 (Ref) 1.0 (Ref) 1.1 (0.6 1.9) 2.9 (1.6 5.2) 1.6 (0.7 3.9) 1.0 (Ref) 1.0 (0.5 1.8) 0.9 (0.4 1.9) 0.9 (0.5 1.9) 1.0 (Ref) 1.6 (0.7 3.6) 1.0 (Ref) 0.8 (0.5 1.4) 0.6 (0.3 1.1) 1.5 (0.8 2.9) 2.7 (1.2 6.3) 1.7 (0.8 3.6) 0.8 (0.4 1.8) 1.0 (Ref) 1.3 (0.5 3.6) 1.0 (Ref) 1.3 (0.7 2.4) 1.0 (Ref) aORb (95% CI) 1.0 (Ref) 1.2 (0.6 2.3) 1.5 (0.8 2.9) 1.1 (0.6 2.1) 1.0 (Ref) 1.0 (Ref) 0.9 (0.5 1.6) 1.9 (1.0 3.6) 1.5 (0.6 3.5) 1.0 (Ref) 0.9 (0.5 1.6) 0.8 (0.4 1.6) 1.0 (0.5 2.1) 1.0 (Ref) 1.5 (0.7 3.3) 1.0 (Ref) 0.8 (0.5 1.4) 0.7 (0.4 1.2) 1.7 (0.9 3.3) 2.1 (0.9 5.1) 1.7 (0.8 3.6) 0.9 (0.4 1.9) 1.0 (Ref) 1.3 (0.7 2.5) 1.0 (Ref) Isolated cases cORa (95% CI) 1.0 (Ref) 1.0 (0.5 2.1) 1.3 (0.7 2.6) 1.1 (0.6 2.1) 1.0 (Ref) 0.9 (0.2 3.6) 1.0 (Ref) 4.0 (1.0 16.9) 1.0 (Ref) 1.0 (Ref) 1.0 (0.5 1.9) 1.8 (0.8 3.9) 1.5 (0.5 4.3) 1.0 (Ref) 0.9 (0.4 1.9) 1.0 (0.4 2.2) 1.3 (0.6 2.7) 1.0 (Ref) 1.7 (0.7 4.3) 1.0 (Ref) 0.8 (0.4 1.5) 0.5 (0.2 1.1) 1.7 (0.8 3.5) 2.6 (0.9 7.2) 1.9 (0.8 4.4) 0.7 (0.2 1.8) 1.0 (Ref) 1.4 (0.4 4.5) 1.0 (Ref) 1.4 (0.7 2.8) 1.0 (Ref) aORb (95% CI) 1.0 (Ref) 1.1 (0.5 2.4) 1.3 (0.6 2.9)

Controls (n) Previous pregnancies 0 1 2 Previous miscarriage Yes No Any fertility procedure Yes No Assisted reproductive technologies Yes No Pregnancy intention Intended Mistimed/ambivalent Unwanted Prepregnancy body mass index (BMI) Underweight (BMI < 18.5) Normal (BMI 18.5 24.9) Overweight (BMI 25 29.9) Obese (BMI  30) High blood pressure Yes No Gestational diabetes Yes No Infectionsc Any Respiratory Any non-respiratory Sexually transmitted Urinary tract Any fever Yes No X-ray Yes No Any scand Yes No
a b

n 18 20 34 18 54 3 69 3 69 36 19 17 6 36 14 9 9 63 7 65 22 13 12 6 8 7 65 4 67 12 59

n 13 13 24 12 38 2 48 2 48 28 14 8 4 26 9 7 8 42 5 45 15 8 9 4 6 4 46 3 47 9 41

1699 1740 2397 1294 4542 264 5572 58 5572 3521 1728 574 319 3156 1246 885 768 5062 362 5477 2044 1571 675 191 396 679 5160 254 5519 804 5023

1.0 (Ref) 1.0 (Ref) 0.8 (0.4 1.7) 1.2 (0.5 2.8) 1.5 (0.5 4.3) 1.0 (Ref) 0.8 (0.4 1.7) 0.8 (0.4 2.0) 1.2 (0.6 2.6) 1.0 (Ref) 1.5 (0.6 3.9) 1.0 (Ref) 0.9 (0.5 1.6) 0.6 (0.3 1.2) 1.7 (0.8 3.6) 1.9 (0.8 4.5) 1.4 (0.7 3.0) 1.0 (Ref)

Crude odds ratio. Odds ratios adjusted for maternal age, race/ethnicity, education, previous pregnancies, folic acid/multivitamin use, and aspirin use. Multivariable models run only for variables with 5 exposed infants with HPE. c The reference group for each infection is lack of the specic infection from the month before pregnancy through the third month, except for sexually transmitted infections which were examined from the month before pregnancy through the end of pregnancy. d Includes X-rays, CT/CAT scans, MRI, and radionucleotides.

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TABLE IV. Associations Between Non-dietary Exposures from the Month Before Pregnancy Through the Third Month of Pregnancy and Having an Infant with Non-syndromic Holoprosencephaly, Excluding Women with Pre-existing Diabetes All Cases Controls (n) Folic acid/multivitamins No folic acid/multivitamin use Any folic acid/multivitamin use Folic acid/multivitamin use each month Aspirin No aspirin use Any aspirin use Acetaminophen No acetaminophen use Any acetaminophen use Ibuprofen No ibuprofen use Any ibuprofen use Progesterone No progesterone use Any progesterone use Exposure to tobacco Non-smoker Any smoking <1/2 pack/day 1/2 pack/day Environmental tobacco smoke Exposure to alcohol No drinking Any alcohol use <1.5 drinks per day 1.5 drinks per day Binge drinking (4 drinks) Illicit drug use No maternal use Maternal use No paternal use Paternal use
a b

Isolated Cases aOR (95%CI) 1.0 (Ref) 0.8 (0.4 1.4) 0.4 (0.2 1.0)
b

n 17 55 13

cOR (95%CI) 1.0 (Ref) 0.6 (0.3 1.0) 0.4 (0.2 0.8)

n 12 38 11

cORa (95%CI) 1.0 (Ref) 0.5 (0.3 1.0) 0.5 (0.2 1.1)

aORb (95%CI) 1.0 (Ref) 0.7 (0.4 1.5) 0.5 (0.2 1.5)

858 4981 1685

5579 260 2513 3326 4596 1243 5729 110 3888 1111 319 790 840 3660 2150 915 1246 770 5582 248 5196 543

63 9 27 45 55 17 69 3 45 14 3 11 13 47 25 8 17 9 69 3 58 11

1.0 (Ref) 3.1 (1.5 6.2) 1.0 (Ref) 1.3 (0.8 2.0) 1.0 (Ref) 1.1 (0.7 2.0) 1.0 (Ref) 2.3 (0.7 7.3) 1.0 (0Ref) 0.9 (0.5 1.7) 0.8 (0.3 2.6) 1.2 (0.6 2.3) 1.3 (0.7 2.5) 1.0 (0Ref) 0.9 (0.6 1.5) 0.7 (0.3 1.4) 1.0 (0.6 1.8) 1.0 (0.5 2.1) 1.0 (0Ref) 1.0 (0.3 3.1) 1.0 (0Ref) 1.8 (0.9 3.5)

1.0 (Ref) 3.4 (1.6 6.9) 1.0 (Ref) 1.5 (0.9 2.5) 1.0 (Ref) 1.3 (0.7 2.3) 1.0 (Ref) 0.9 (0.5 1.8) 0.6 (0.2 2.1) 1.0 (0.5 2.2) 1.1 (0.6 2.1) 1.0 (Ref) 0.9 (0.5 1.5) 0.9 (0.4 2.0) 1.2 (0.6 2.2) 0.9 (0.5 1.6) 1.0 (Ref) 0.7 (0.2 2.4) 1.0 (Ref) 1.5 (0.8 2.9)

46 4 19 31 37 13 48 2 33 9 0 9 8 35 15 5 10 5 48 2 40 9

1.0 (Ref) 1.9 (0.7 5.2) 1.0 (Ref) 1.2 (0.7 2.2) 1.0 (Ref) 1.3 (0.7 2.5) 1.0 (Ref) 2.2 (0.5 9.0) 1.0 (Ref) 1.0 (0.5 2.0) 1.3 (0.6 2.8) 1.1 (0.5 2.4) 1.0 (Ref) 0.7 (0.4 1.3) 0.6 (0.2 1.5) 0.8 (0.4 1.7) 0.7 (0.3 1.9) 1.0 (Ref) 0.9 (0.2 3.9) 1.0 (Ref) 2.2 (1.0 4.5)

1.0 (Ref) 2.0 (0.7 5.5) 1.0 (Ref) 1.5 (0.8 2.7) 1.0 (Ref) 1.5 (0.8 2.8) 1.0 (Ref) 0.9 (0.4 2.1) 1.4 (0.6 3.3) 1.0 (0.5 2.3) 1.0 (Ref) 0.9 (0.5 1.6) 0.7 (0.3 1.8) 1.0 (0.5 2.0) 0.9 (0.4 1.9) 1.0 (Ref) 1.9 (0.9 4.0)

Crude odds ratio. Odds ratios adjusted for maternal age, race/ethnicity, education, previous pregnancies, folic acid/multivitamin use, and aspirin use. Multivariable models run only for variables with 5 exposed infants with HPE.

sh and poultry) by grams consumed, there was some evidence of inverse associations with green vegetables, fruits, and fruits and vegetables in adjusted models but none of the associations were statistically signicant. We found no strong evidence of any associations between HPE and specic nutrients.

DISCUSSION
This population-based study examining characteristics and exposures associated with non-syndromic HPE found a number of associations consistent with previous literature, but also identied some new potential risk factors. Infant characteristics associated with HPE

included being born preterm or very preterm and of low or very low birthweight, factors that are inter-related and that have been associated with numerous birth defects [Mili et al., 1991; Rasmussen et al., 2001]. Among maternal demographic characteristics and exposures examined, the strongest evidence for associations with HPE was found for

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TABLE V. Associations Between Infants with Non-syndromic Holoprosencephaly and Quartiles (Q) of Selected Nutrients and Food Groups, Excluding Women with Pre-existing Diabetes All cases Controls (n) Selected individual foods Skim/lowfat milk No intake Below median intake Above median intake Yogurt No intake Below median intake Above median intake Butter No intake Below median intake Above median intake String beans No intake Below median intake Above median intake Broccoli No intake Below median intake Above median intake Chicken No intake/below median intakec Above median intake Fish No intake Below median intake Above median intake Hot dogs No intake Below median intake Above median intake Food groups (g) Vegetables Quartile (Q) 1 (45.5) Q2 (45.6 78.8) Q3 (78.9 126.7) Q4 (>126.7) Green vegetables Q1 (12.3) Q2 (12.4 29.1) Q3 (29.2 52.0) Q4 (>52.0) Fruit Q1 (172.8) Q2 (172.9 305.6) n ORa (95%CI) aOR (95%CI)
b

Isolated cases n ORa (95%CI) aORb (95%CI)

2553 1064 2197 2434 1460 1920 2569 1140 2105 1783 1081 2950 1463 1445 2906 1618 4196 1794 1987 2033 2643 984 2187

44 15 13 40 18 14 40 15 17 38 3 31 28 15 29 30 42 31 23 18 24 9 39

1.0 (Ref) 0.8 (0.5 1.5) 0.3 (0.2 0.6) 1.0 (Ref) 0.8 (0.4 1.3) 0.4 (0.2 0.8) 1.0 (Ref) 0.8 (0.5 1.5) 0.5 (0.3 0.9) 1.0 (Ref) 0.1 (0.04 0.4) 0.5 (0.3 0.8) 1.0 (Ref) 0.5 (0.3 1.0) 0.5 (0.3 0.9) 1.0 (Ref) 0.5 (0.3 0.9) 1.0 (Ref) 0.7 (0.4 1.2) 0.5 (0.3 0.9) 1.0 (Ref) 1.0 (0.5 2.2) 2.0 (1.2 3.3)

1.0 (Ref) 1.1 (0.6 2.0) 0.5 (0.2 0.9) 1.0 (Ref) 0.8 (0.5 1.5) 0.5 (0.3 0.9) 1.0 (Ref) 0.9 (0.5 1.6) 0.5 (0.3 0.9) 1.0 (Ref) 0.2 (0.1 0.5) 0.6 (0.3 1.0) 1.0 (Ref) 0.6 (0.3 1.1) 0.6 (0.3 1.0) 1.0 (Ref) 0.7 (0.4 1.1) 1.0 (Ref) 0.7 (0.4 1.2) 0.5 (0.3 1.0) 1.0 (Ref) 1.1 (0.5 2.4) 1.9 (1.1 3.2)

27 13 10 25 13 12 27 9 14 24 2 24 22 10 18 18 32 23 17 10 15 5 30

1.0 (Ref) 1.2 (0.6 2.2) 0.4 (0.2 0.9) 1.0 (Ref) 0.9 (0.4 1.7) 0.6 (0.3 1.2) 1.0 (Ref) 0.8 (0.4 1.6) 0.6 (0.3 1.2) 1.0 (Ref) 0.2 (0.03 0.6) 0.6 (0.3 1.1) 1.0 (Ref) 0.5 (0.2 1.0) 0.4 (0.2 0.8) 1.0 (Ref) 0.7 (0.4 1.2) 1.0 (Ref) 0.7 (0.4 1.3) 0.4 (0.2 0.8) 1.0 (Ref) 0.9 (0.3 2.5) 2.4 (1.3 4.5)

1.0 (Ref) 1.4 (0.7 2.9) 0.6 (0.3 1.3) 1.0 (Ref) 1.0 (0.5 1.9) 0.7 (0.3 1.4) 1.0 (Ref) 0.7 (0.3 1.6) 0.6 (0.3 1.2) 1.0 (Ref) 0.2 (0.04 0.7) 0.7 (0.4 1.2) 1.0 (Ref) 0.5 (0.2 1.1) 0.4 (0.2 0.8) 1.0 (Ref) 0.8 (0.4 1.4) 1.0 (Ref) 0.6 (0.3 1.3) 0.3 (0.2 0.7) 1.0 (Ref) 0.9 (0.3 2.5) 2.3 (1.2 4.3)

1458 1459 1459 1459 1444 1455 1475 1461 1457 1460

18 21 17 16 27 19 10 16 20 16

1.0 (Ref) 1.2 (0.6 2.2) 0.9 (0.5 1.8) 0.9 (0.5 1.7) 1.0 (Ref) 0.7 (0.4 1.3) 0.4 (0.2 0.8) 0.6 (0.3 1.1) 1.0 (Ref) 0.8 (0.4 1.5)

1.0 (Ref) 1.3 (0.7 2.5) 1.1 (0.5 2.1) 0.8 (0.4 1.7) 1.0 (Ref) 0.8 (0.4 1.4) 0.4 (0.2 0.9) 0.7 (0.3 1.3) 1.0 (Ref) 0.8 (0.4 1.6)

18 21 17 16 27 19 10 16 20 16

1.0 (Ref) 1.3 (0.6 2.8) 1.2 (0.5 2.7) 1.1 (0.5 2.5) 1.0 (Ref) 0.4 (0.2 0.9) 0.3 (0.1 0.6) 0.5 (0.2 1.0) 1.0 (Ref) 0.8 (0.4 1.7)

1.0 (Ref) 1.3 (0.6 2.9) 1.2 (0.5 2.8) 0.9 (0.4 2.2) 1.0 (Ref) 0.5 (0.2 1.0) 0.3 (0.1 0.7) 0.5 (0.2 1.0) 1.0 (Ref) 0.8 (0.4 1.6)

(Continued )

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TABLE V. (Continued) All cases Controls (n) Q3 (305.7 483.4) Q4 (483.4) Fruits and vegetables Q1 (242.0) Q2 (242.1 394.4) Q3 (394.5 599.2) Q4 (599.2) All meat, not including sh Q1 (66.0) Q2 (66.1 96.4) Q3 (96.5 135.7) Q4 (135.7) Meat, not including chicken or sh Q1 (35.3) Q2 (35.4 60.0) Q3 (60.1 96.3) Q4 (96.3) Nutrients (g) Cholesterol Q1 (115.9) Q2 (116.0 146.3) Q3 (146.5 183.3) Q4 (>183.3) Total fat intake Q1 (27.6) Q2 (27.7 32.3) Q3 (32.4 36.7) Q4 (>36.7) Total folate intake Q1 (197.1) Q2 (197.2 250.8) Q3 (250.9 314.7) Q4 (>314.7) Total energy intake Q1 (1125.5) Q2 (1125.6 1458.8) Q3 (1458.9 1928.5) Q4 (>1928.5) Total protein intake Q1 (38.5) Q2 (38.6 44.6) Q3 (44.7 50.5) Q4 (>50.5)
a b

Isolated cases aORb (95%CI) 0.9 (0.4 1.7) 0.7 (0.4 1.4) 1.0 (Ref) 1.4 (0.7 2.7) 0.8 (0.4 1.7) 0.8 (0.4 1.7) 1.0 (Ref) 0.9 (0.4 1.8) 0.7 (0.3 1.5) 1.3 (0.7 2.5) 1.0 (Ref) 0.9 (0.4 1.8) 0.7 (0.3 1.5) 1.3 (0.7 2.5) n 18 18 17 23 14 18 10 14 8 18 17 15 13 27 OR (95%CI) 0.6 (0.2 1.3) 0.8 (0.4 1.6) 1.0 (Ref) 1.2 (0.6 2.6) 0.8 (0.3 1.8) 0.8 (0.4 1.9) 1.0 (Ref) 1.4 (0.6 3.2) 0.8 (0.3 2.0) 1.8 (0.8 3.9) 1.0 (Ref) 1.4 (0.6 3.2) 0.8 (0.3 2.0) 1.8 (0.8 3.9)
a

n 18 18 17 23 14 118 17 15 13 27 17 15 13 27

OR (95%CI) 0.9 (0.5 1.7) 0.9 (0.5 1.7) 1.0 (Ref) 1.4 (0.7 2.5) 0.8 (0.4 1.7) 1.1 (0.5 2.1) 1.0 (Ref) 0.9 (0.4 1.8) 0.8 (0.4 1.6) 1.6 (0.9 2.9) 1.0 (Ref) 0.9 (0.4 1.8) 0.8 (0.4 1.6) 1.6 (0.9 2.9)

aORb (95%CI) 0.5 (0.2 1.2) 0.5 (0.2 1.2) 1.0 (Ref) 1.2 (0.6 2.5) 0.7 (0.3 1.6) 0.6 (0.3 1.4) 1.0 (Ref) 1.4 (0.6 3.1) 0.8 (0.3 1.9) 1.4 (0.6 3.2) 1.0 (Ref) 1.4 (0.6 3.1) 0.8 (0.3 1.9) 1.4 (0.6 3.2)

1459 1459 1458 1459 1459 1459 1458 1459 1459 1459 1458 1459 1459 1459

1458 1458 1461 1460 1467 1464 1427 1479 1511 1474 1470 1471 1459 1459 1459 1460 1456 1462 1467 1452

19 15 18 20 19 13 19 21 20 20 15 17 16 18 15 23 23 18 21 10

1.0 (Ref) 0.8 (0.4 1.6) 1.0 (0.5 1.9) 1.1 (0.6 2.1) 1.0 (Ref) 0.7 (0.3 1.4) 1.1 (0.6 2.0) 1.1 (0.6 2.1) 1.0 (Ref) 1.0 (0.5 1.9) 0.8 (0.4 1.5) 0.9 (0.5 1.7) 1.0 (Ref) 1.1 (0.6 2.2) 0.9 (0.5 1.9) 1.4 (0.8 2.7) 1.0 (Ref) 0.8 (0.4 1.5) 1.0 (0.5 1.8) 0.5 (0.2 1.0)

1.0 (Ref) 0.8 (0.4 1.6) 1.0 (0.5 2.0) 1.1 (0.6 2.1) 1.0 (Ref) 0.7 (0.4 1.5) 1.1 (0.6 2.1) 1.3 (0.7 2.5) 1.0 (Ref) 1.1 (0.6 2.0) 0.8 (0.4 1.5) 0.9 (0.4 1.7) 1.0 (Ref) 1.0 (0.5 2.1) 0.7 (0.3 1.6) 0.7 (0.3 1.8) 1.0 (Ref) 1.0 (0.5 1.8) 1.2 (0.6 2.2) 0.6 (0.3 1.4)

14 8 10 18 14 9 12 15 15 15 9 11 11 12 12 15 14 14 14 8

1.0 (Ref) 0.6 (0.2 1.4) 0.7 (0.3 1.7) 1.3 (0.7 2.7) 1.0 (Ref) 0.7 (0.3 1.5) 0.9 (0.4 2.0) 1.1 (0.5 2.3) 1.0 (Ref) 1.0 (0.5 2.1) 0.6 (0.3 1.4) 0.8 (0.4 1.7) 1.0 (Ref) 1.1 (0.5 2.5) 1.1 (0.5 2.5) 1.4 (0.6 3.0) 1.0 (Ref) 1.0 (0.5 2.2) 1.1 (0.5 2.3) 0.6 (0.3 1.5)

1.0 (Ref) 0.6 (0.2 1.4) 0.7 (0.3 1.7) 1.3 (0.6 2.6) 1.0 (Ref) 0.7 (0.3 1.6) 1.0 (0.4 2.1) 1.2 (0.6 2.6) 1.0 (Ref) 1.0 (0.5 2.1) 0.6 (0.3 1.4) 0.7 (0.3 1.6) 1.0 (Ref) 1.0 (0.4 2.3) 0.8 (0.3 2.0) 0.7 (0.2 2.52) 1.0 (Ref) 1.2 (0.5 2.5) 1.2 (0.6 2.7) 0.8 (0.3 1.9)

Nutrients adjusted for total energy intake using the nutrient density method. Odds ratios adjusted for maternal age, race/ethnicity, education, previous pregnancies, folic acid/multivitamin use, and aspirin use. Multivariable models run only for variables with 5 exposed infants with HPE. c There were only two mothers of infants with HPE with no chicken intake. Therefore, odds ratios were calculated comparing above median to below median intake.

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pre-existing diabetes, consistent folic acid/multivitamin use, aspirin use, lower education level, and use of ART.

Among maternal demographic characteristics and exposures examined, the strongest evidence for associations with HPE was found for preexisting diabetes, consistent folic acid/multivitamin use, aspirin use, lower education level, and use of ART.
Modest evidence for association with HPE was found for unwanted pregnancies, having an STI or UTI, use of progesterone, and certain foods consumed as part of the mothers diet. The associations found with diabetes, aspirin, Hispanic ethnicity, and lower educational level have been previously reported [Croen et al., 2000; Orioli and Castilla, 2007]. And consistent with the earlier studies, a higher percentage of infants with HPE (all HPE) were female compared to controls, although the difference was not statistically signicant in our study. We did not nd associations with foreign maternal birthplace, gravidity, tobacco use, or alcohol intake, which had also been previously reported. We were unable to assess age at menarche, history of anemia, or vaginal bleeding as possible risk factors because these data are not collected in the NBDPS. It is unclear why aspirin was found to be associated with HPE. Although no association was found by Orioli and Castilla [2007], Croen et al. [2000] found a non-statistically signicant odds ratio of 2.5 with aspirin and salicylatecontaining medicines. Additionally, salicylates have been implicated in anecdotal evidence from case reports [Khudr and Olding, 1973; Benawra et al., 1980]. In this study, there was no evidence of a dose response relationship based on frequency of use (data not shown) and the timing of aspirin exposure likely has

more misclassication than other variables because most exposed women reported that they took aspirin as needed. There are still questions regarding the effectiveness of using low-dose aspirin to prevent recurrence of miscarriage and to improve in vitro fertilization success [James et al., 2008; Schisterman et al., 2009]. Therefore, it is important to understand if its use increases the risk for HPE. In this analysis, one of the three women with an infant with HPE who reported in vitro fertilization also reported periconceptional aspirin exposure. Given that over 1% of births in the U.S. in 2005 were conceived through ART [Wright et al., 2008], it is important to follow up on the association between ART and HPE observed in this study, even though it is based on only three exposed mothers of infants with HPE. This nding is consistent with other studies showing an increased risk of congenital malformations with ART and IVF [Olson et al., 2005; Reefhuis et al., 2008], although we are unaware of any studies specically examining HPE. Whether the ndings in these studies are related to ART or to the underlying infertility is unknown and difcult to disentangle. We found no association between HPE and low cholesterol intake, despite the fact that mutations in several genes that are dependent on cholesterol for cell signaling, including Sonic Hedgehog (SHH), SIX3, ZIC2, GLI2, TGIF, and PTCH, have been identied in association with non-syndromic HPE [Cohen and Shiota, 2002; Muenke and Gropman, 2008]. In addition, defects in the synthesis of cholesterol, which is required for the expression of the SHH protein [Wolf, 1999], have been found in some cases of syndromic HPE [Wever et al., 2010]. Because of these observations, it has been hypothesized that low levels of dietary cholesterol intake and prenatal consumption of drugs that interfere with cholesterol synthesis might contribute to the risk for the defect as well [Edison and Muenke, 2003; Haas and Muenke, 2010]. In this analysis, no mothers of infants with HPE reported taking cholesterol-lowering medications.

Although we found evidence of an association with folic acid supplementation, there was no apparent association with dietary folate consumption. This may be reective of the greater bioavailability of folic acid compared to dietary folate and the observation that blood folate levels have been found to increase more in response to folic acid supplementation than increases in dietary folate intake [Neuhouser et al., 1998; Elkin and Higham, 2000; Matsuzaki et al., 2008]. Alternatively, it may be that consistent use of a supplement that contains folic acid is a behavior more easily remembered than recalling foods consumed in the year prior to pregnancy or that residual confounding exists. It is possible that some unmeasured factor, which may not be related to folic acid supplementation, was associated with consistent use of taking a supplement and accounts for the association. There was no evidence of associations between other nutrient intakes and HPE identied. However, based on the associations found for specic foods (i.e., higher intakes of skim milk, yogurt, vegetables, chicken/turkey, and sh being associated with a lower risk and higher intakes of hot dogs being associated with a higher risk of HPE), there was some suggestion that diet quality might be a risk factor. To our knowledge, no population-based study has reported on the association of HPE with maternal food group intake. More work is warranted to understand if this is truly related to this birth defect and not merely a proxy for other healthy behaviors or socioeconomic status. A number of limitations must be acknowledged in this study. We examined a wide variety of exposures in this analysis. Because so little is known about risk factors for HPE, this was intended to be a hypothesis-generating study and we did not adjust for multiple testing. In addition, this analysis is based on selfreported exposures. Women who had an infant with HPE might be more or less likely to report unhealthy and socially undesirable behaviors, such as alcohol intake, smoking, and drug use compared to mothers of healthy infants. Furthermore, differences in the characteristics of

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the women who did not participate in the study compared to those who did participate could potentially bias the results. However, controls participating in the NBDPS were recently found to be generally representative of the underlying population from which they were selected [Cogswell et al., 2009]. Another possible limitation is that infants with mild HPE might not have been identied in time for the mother to be interviewed before the child was 24 months of age. Conversely, severe forms of HPE might not be identied if a termination occurs early in pregnancy. This study also has a number of important strengths. Even with only 74 cases, this is one of the largest studies of risk factors for HPE to date. The NBDPS conducts an extensive interview of participants allowing us to examine a wide range of possible risk factors and control for other exposures in our analysis. Another strength is that clinical information that has been abstracted from medical records was reviewed by a clinical geneticist, to ensure that included cases met the criteria for inclusion. This investigation of HPE suggested some additional risk factors that should be further investigated. Unfortunately, because HPE is rare among live births, additional large population-based studies might not be feasible. More data from in vitro and animal models might be necessary to identify the most biologically plausible risk factors.

ACKNOWLEDGMENTS
The authors thank Dr. Tunu Loponi for her assistance with this analysis. We also thank the participating families and the staff and scientists at all National Birth Defects Prevention Study sites. The work was supported by the Centers for Disease Control and Prevention (U50/ CCU613232).

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