Elsevier

Annals of Epidemiology

Volume 11, Issue 6, August 2001, Pages 434-442
Annals of Epidemiology

Original report
A Case-Control Study of Nonsyndromic Oral Clefts in Maryland

https://doi.org/10.1016/S1047-2797(01)00222-8Get rights and content

Abstract

PURPOSE: Isolated, nonsyndromic oral clefts cases (n = 171) and unaffected controls (n = 182) were used to identify both genetic and environmental risk factors.

METHODS: Infants born in Maryland between 1992 to 1998 with an isolated, nonsyndromic oral cleft [cleft lip (CL), cleft lip and palate (CLP), or cleft palate (CP)] were recruited and exposure plus family history data were collected. Controls were unaffected infants. DNA was collected from all cases and their parents, plus controls.

RESULTS: No statistically significant association was found between any of the following: maternal smoking, vitamin use, urinary tract infection, or recreational drug use in either univariate analysis or after adjusting for maternal age and education. More control mothers reported alcohol use during the critical time period of pregnancy (one month before conception through the first trimester) as compared to case mothers. There was a 10-fold increase in risk to siblings of cases as compared to siblings of controls. Markers at four candidate genes were examined: transforming growth factor α (TGFα), transforming growth factor β3 (TGFβ3), MSX1, and BCL3. Only MSX1 showed significant differences in allele frequencies between CP cases and controls. MSX1 also showed significant evidence of linkage disequilibrium with a susceptibility gene controlling risk for CP.

CONCLUSION: Most environmental risk factors examined here gave little evidence of association with risk to isolated, nonsyndromic oral clefts, although any alcohol consumption seemed protective. MSX1 showed evidence of linkage disequilibrium in both case-control and case-parent trio analysis.

Introduction

Oral clefts include cleft lip (CL), cleft lip and palate (CLP), and cleft palate (CP), and collectively they represent a common, nonlethal birth defect of complex and heterogeneous etiology (1). Because CL and CLP share similarities in both epidemiologic characteristics (e.g., sex ratio) and embryologic timing (the secondary palate forms later than the lip and primary palate), CL and CLP are typically grouped together into cleft lip with/without cleft palate (CL/P). Although the proportion of all oral cleft cases representing a recognized malformation syndrome varies, more infants born with CP have a recognizable syndrome as compared to infants with CL/P (2). In a recent California study, the highest proportion of nonisolated clefts was seen among CP infants (53%), and 14 and 32% of CL and CLP cases, respectively, had another malformation (3). When conducting population-based studies of oral clefts, it is a major challenge to distinguish isolated, nonsyndromic from syndromic or nonisolated forms of oral clefts, because both family history information and direct physical examination of affected individual(s) are needed (4).

A number of environmental factors have been examined as risk factors for oral clefts, including maternal smoking, exposure to antiepileptic drugs, antiemetic agents, vitamin use during the periconceptual period, maternal metabolic factors, alcohol consumption and exposure to agricultural chemicals (5). Maternal smoking, the best studied environmental risk factor, is associated with a slight increase in risk for both CL/P and CP (6).

Other environmental risk factors may also be important. In a population-based case-control study from Iowa, Manger et al. (7) showed a higher risk of isolated CL/P associated with increasing maternal alcohol consumption. Romitti et al. (8) showed similar results using an extended dataset from this same population. Christensen et al. (9); however, found that fewer case mothers reported drinking alcohol than did control mothers (59% vs. 65%) before the pregnancy. Shaw et al. (10) found that maternal prenatal vitamin supplements decreased the first occurrence of CL/P. Czeizel et al. (11) also showed a protective effect of folic acid in a Hungarian study and later suggested this effect may depend on dose (12). Clinical trial data to confirm this protective effect of vitamin supplementation are lacking, however.

There is also a strong genetic component to oral clefts. The ratio of risk in first degree relatives of cases as compared to the general population is as high as 30 (13). Different models of inheritance have been proposed, including single gene models with reduced penetrance, multifactorial models, and various combinations of both 14, 15. Multilocus or oligogenic models have also been suggested 16, 17, 18, but no one model can yet fully explain observed familial aggregation of oral clefts.

Ardinger et al. (19) was the first to use a case-control design to test candidate genes. They found a significant statistical association between case status and two of 12 markers in five genes, with an intronic Taq1 marker in the transforming growth factor alpha (TGFα) gene showing the strongest association. Similar studies, primarily in Caucasians, have shown differences in allele frequencies between cleft cases and controls, but not all were statistically significant. Mitchell (20) argued that heterogeneity in the proportion of bilateral clefts and cases with a positive family history across studies could explain some of this inconsistency. Lidral et al. (21) recently found a higher allele frequency in a population-based series of controls in Iowa, which reduced the association between oral clefts and this TGFα marker.

Other candidate genes also show association with oral clefts, including retinoic acid receptor A (RARA) 22, 23 and an anonymous marker on chromosome 4q (24). The Msh homeobox homolog 1 gene, MSX1, was reported to be associated with CP in cases from Iowa 21, 8, but no such association was seen in a Philippine population (25). In the Iowa case-control study, Lidral et al. (21) also found an association with TGFβ3 and risk of CL/P.

Two case-control studies have raised the possibility that gene-environment interaction may determine risk to oral clefts 26, 27. In particular, maternal smoking was shown to be a strong risk factor for CP in the presence of the rarer allele at the TaqI marker in TGFα, but not in its absence 26, 27. Other studies have failed to confirm this, however 28, 8, 9. Romitti et al. (8) did show evidence for similar gene-environment interaction when testing markers in TGFβ3 and MSX1 and exposure to maternal smoking on risk of CP, as well as apparent interactions between exposure to maternal alcohol consumption and these two loci on risk of CL/P.

In the current study, the case-control design was used to examine both environmental and genetic risk factors for oral clefts, and to test for possible interactions. Individual effects of risk factors were compared among CP and CL/P in univariate analyses, and after adjustment for confounders. Where DNA was available on both parents, the case-parent trio design was also used to test these genetic markers for evidence of linkage disequilibrium.

Section snippets

Case Ascertainment

As described in (28), cases of oral clefts born between June, 1992 and December, 1998 were identified through treatment centers in Maryland, plus the Craniofacial Clinic of the Children's National Medical Center in Washington, DC (which treats many Maryland residents). Records of the Maryland Birth Defects Reporting and Information System (BDRIS) were also reviewed as a population-based ascertainment source, because oral clefts are included among the 12 sentinel defects reported to this

Smoking

Patterns and amount of maternal smoking around the time of conception and during the first trimester of pregnancy were examined as potential risk factors. Smoking exposure changed during pregnancy and varied slightly among groups. For example, 22% of mothers of CL/P cases reported smoking during the 3 months before pregnancy and 19% during the first trimester, but only 12% of these mothers reported continuous smoking during the second and third trimesters. In addition to ceasing, smoking

Discussion

In the current study, we compared infants with confirmed isolated, nonsyndromic oral clefts to unaffected controls to test a number of genetic and environmental risk factors using a case-control design. Although some debate remains about the use of unaffected infants versus malformed infants as controls in retrospective studies of birth defects 38, 39, 40, recall bias seems unlikely to lead to erroneous inferences except in extreme circumstances (41). Here we tested for effects of maternal

Selected Abbreviations and Acronyms

CL = cleft lip

CLP = cleft lip and palate

CP = cleft palate

CL/P = cleft lip with/without cleft palate

TGFα = transforming growth factor α

TGFβ3 = transforming growth factor β3

RARA = retinoic acid receptor A

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