All PhD Theses

I.P.C. Krapels

The etiology of orofacial clefts


A scientific essay in Medical Sciences

DOCTORAL THESIS defended in public on 10th of November 2005


Nonsyndromic orofacial clefts (OFC) are frequently encountered birth defects in man, affecting approximately 1-2 per 1,000 newborns worldwide. The medical and psychosocial implications of OFC for the individual and his/her family are severe. The majority of patients with an OFC require multidisciplinary treatment up until adulthood. Therefore, there is a strong imperative towards the understanding of the causes in order to develop measures for the primary prevention of OFC in the future. As for most congenital malformations the exact pathogenesis of nonsyndromic OFC is not fully unraveled due to the complexity of the biological processes involved and the genetic and phenotypic heterogeneity. Increasing evidence indicates that nonsyndromic OFC should be considered as a complex trait, in which genetic and environmental determinants interact and express the aberrant phenotype. As the genetic components cannot be modified yet, the main aim of this thesis was to provide more insight in the role of amendable factors as nutrition, smoking and other lifestyle factors in the etiology of nonsyndromic OFC. This has been described in the chapters 3 to 7 of this thesis. In part I and II of the thesis the results of a case-control triad study are described. This was an unique study in the Netherlands, in which the ten largest cleft lip and palate team and the parents and patients organization for clefts (VSOP/BOSK) contributed to the recruitment of children with a nonsyndromic OFC and both of their parents. The controls comprised families of a healthy child without OFC or other major congenital malformations. They were recruited via the case parents, nurseries and public health centers. All participants were Dutch Caucasians. Part I focuses on the role of nutrition in the etiology of nonsyndromic OFC. The maternal nutritional status was investigated both at the level of dietary intake as well as at a biochemical level via the determination of the concentrations of myo-inositol, glucose and zinc. In part II we describe the associations between parental characteristics, health and lifestyle factors in the periconceptional period, polymorphisms in genes involved in biotransformation pathways and the risk of having offspring affected with a nonsyndromic OFC.

In chapter 3 and chapter 4 we investigated the role of maternal dietary intake before conception on the risk for offspring affected with an OFC. As a proxy for the preconceptional nutritional intake, we collected maternal dietary intake data, covering a period of 4 weeks before the study moment, being approximately 24 months after conception of the index child, through a validated food frequency questionnaire. With the exception of a relative lack of fiber and iron and an excess of saturated fat, comparisons to the Recommended Daily Allowances and the National Food Consumption Survey demonstrated that the overall nutrient intake was adequate in both case mothers and controls. The 182 mothers of a nonsyndromic child with an OFC showed lower intakes of all macronutrients, vitamins and minerals compared to the 173 controls. Of great interest is our finding that increased dietary intakes of thiamine, pyridoxine, vegetable protein, fiber, ascorbic acid, magnesium, and iron decreased OFC risk. These nutrients are mainly found in vegetables, fruits grain products and bread. This effect was most prominent in women who daily took folic acid supplements 4 weeks before until 8 weeks after conception. Although, there is no clear explanation for this unexpected interaction, this finding may suggest that these women are more health conscious and emphasizes that both adequate nutrition as well as the use of folic acid supplements in the periconceptional period is important in the prevention of nonsyndromic OFC in the offspring.

At the moment of investigation, 24 months after conception of the index child, we also obtained a venous blood samples from mothers and children for the determination of the concentrations of myo-inositol and glucose in serum and zinc in red blood cells. The results of 76 children with a nonsyndromic cleft lip with or without cleft palate (CLP), their mothers (n=66), 77 control children and their mothers (n=81) are described in chapter 5. Children with a CLP and their mothers had lower concentrations of zinc compared to the controls. Maternal myo-inositol concentrations below or equal to 13.5 µmol/L and zinc concentrations below or equal to 189 µmol/L, 2-3 fold increased CLP risk and children with low myo-inositol (≤ 21.5 µmol/L) and low zinc concentrations (≤ 118 µmol/L) were 3-times more likely to have a CLP. Glucose concentrations were not associated with CLP risk. Of special interest was the correlation between maternal and child myo-inositol and zinc concentrations in cases, possibly implicating a common (genetic) factor.

In chapter 6, we studied multiple parental characteristics, lifestyle and health factors in the periconceptional period as potential risk factors for OFC in the offspring. Twenty-four months after conception of the index child, both parents of a child with an OFC and control parents filled out a questionnaire on demographic factors and periconceptional and first trimester exposures. Data of 319 parents of a child with a nonsyndromic CLP, 68 parents of a child with a cleft palate only (CPO) and 223 control parents were analyzed. A low parental education, a family history for CLP, maternal medication use for illnesses, first trimester common cold and paternal smoking were associated with increased CLP risk. Pregnancy planning and folic acid supplement use from 4 weeks before to 8 weeks after conception reduced CLP risk. Mostly comparable results were obtained for the CPO group. These data confirm the multifactorial etiology of OFC and delineate amendable risk factors and possibilities for OFC prevention.

The interaction between parental smoking and polymorphisms in biotransformation enzymes was investigated in chapter 7. Both the EPHX and the GSTP1 enzyme are involved in the biotransformation of compounds present in cigarette smoke. Polymorphisms in the genes encoding for these enzymes may result in an altered enzymatic activity and thus in an altered detoxification of toxic compounds. The EPHX exon 3 113Tyr>His polymorphism, in which tyrosine is replaced by histidine, and EPHX exon 4 139His>Arg polymorphism, in which histidine is replaced by arginine, decrease and increase EPHX enzyme activity, respectively. For GSTP1, the mutation in exon 5 105 Iso>Val, in which isoleucine is replaced by valine, results in a decreased enzyme activity. The TDT-test data revealed that polymorphisms in EPHX exon 3 and exon 4 were associated with CLP. Homozygosity for EPHX exon 3 polymorphisms in mothers significantly decreased CLP risk by 70 percent. Fathers carrying at least one polymorphic EPHXexon 3 allele showed nearly 2-fold increased risk. Mothers and children with at least one polymorphic EPHX exon 4 allele tended to have a 30% to 40% reduced CLP risk. Homozygosity for GSTP1 exon 5 polymorphisms in mothers and children was associated with an approximately 3-fold increased risk. With the exception of paternal smoking and polymorphisms in EPHX exon 4 that 2.5-fold increased CLP risk, no significant gene-environment interactions could be demonstrated

In the general discussion (chapter 8), we discuss these findings in the context of methodological issues such as the ascertainment of the cases and the controls, measurement errors, confounding and statistical power. We also place our findings in the light of the existing literature, in order to contribute to a better picture of the etiology of OFC. In addition, we make suggestions for future research. Our research on associations between nutrition and birth defects is rather new and the findings emphasize the importance of further investigation in this field. The continued search for new environmental and genetic factors, both of the mother and the father, will be essential to unravel the complex etiology of OFC in order to contribute to their prevention in the future. These goals can be met by large scale case control triad studies, in which special attention should be paid to subtypes of clefts as well. This thesis emphasizes that both maternal and paternal exposures in the periconceptional period may affect palatogenesis in their offspring. A maternal diet low in vegetables, fruits, grain products and bread, low paternal education, positive family history for CLP, maternal medication use or illnesses, refraining from periconceptional folic acid supplement use, first trimester common cold and paternal smoking may contribute to OFC risk. Most of these factors are modifiable and should be targets for preventive actions. Health authorities, health care workers, nutritional companies and health educators should be aware of their position to optimize the nutritional status of women in reproductive age. Because it is very difficult to achieve intakes of folate according to the recommended daily allowances, periconceptional nutrition should be completed with a supplement containing folic.