Reference
Complete the bibliographic reference for the article according to AJE format.

van Rooij IA , Wegerif MJ, Roelofs HM, Peters WH, Kuijpers-Jagtman AM, Zielhuis GA , Merkus HM, Steegers-Theunissen RP. Smoking, genetic polymorphisms in biotransformation enzymes, and nonsyndromic oral clefting: a gene-environment interaction. Epidemiology 2001;12(5):502-7

Category of HuGE information
Specify the types of information (from the list below) available in the article:

1. Prevalence of gene variant
2. Gene-disease association
3. Gene-environment interaction
4. Gene-gene interaction
5. Genetic test evaluation/monitoring

1. Gene prevalence
2. Gene-disease associationI
3. Gene-environment association

Study hypotheses or purpose
The authors study hypotheses or main purpose for conducting the study.

Hypothesis:  Epidemiologic evidence suggests that smoking is weakly associated with risk for (OFC). Several enzyme systems metabolize (activate and deactivate) the substances present in tobacco smoke, including aromatic hydrocarbons. The study tests the hypothesis that two alleles that increase the activation (CYP1A1) or decrease the detoxification of aromatic hydrocarbons (null GSTT1) interact with maternal smoking in increasing the risk for OFC in the fetus.

Gene(s)
Identification of the following:

1. Gene name
2. Chromosome location
3. Gene product/function
4. Alleles
5. OMIM #

1. Gene: Glutathione S-Transferase, Theta-1 (GSTT1)
2. Chromosome location:  22q11.2
3. Gene product/function: In humans, the GSTT1 enzyme is involved in the biotransformation of low-molecular-weight halogenated compounds and reactive epoxides; thus, it has been suggested that the enzyme may act as a detoxification sink.
4. Alleles:  A proportion of the population, varying from 12%-20% in Europeans to 65% in Asians carries a null polymorphism (deletion). Such people do not express the gene and therefore do not have any GSST1 enzyme activity.
5. OMIM #: 600436

1. Gene: Cytochrome P450, subfamily I, polypeptide 1; CYP1A1
2. Chromosome location:  15q22-q24
3. Gene product/function: CYP1A1 encodes a phase 1 cytochrome P450 enzyme that metabolizes polycyclic aromatic hydrocarbons (PAHs) such as benzo[a]pyrene that are found in cigarette smoke and in the products of fossil fuel combustion. Such metabolism can lead to the bioactivation of such compounds, and their product can damage cellular components if not rapidly conjugated in a phase 2 reaction.
4. Alleles:  A polymorphism has been reported in exon 7, resulting in a replacement of an isoleucine with a valine in position 462.  Homozygotes and heterozygotes for this variant have higher enzymatic activity than do Common-type carriers, which could lead to an increased production of toxicants.
5. OMIM #: 108330

Environmental factor(s)
Identification of the major environmental factors studied (infectious, chemical, physical, nutritional, and behavioral)

Health outcome(s)
Identification of the major health outcome(s) studied

1. Cleft lip with or without cleft palate, cleft palate

1. Case-control
2. Cohort 
3. Cross-sectional
4. Descriptive or case series
5. Clinical trial
6. Population screening

Case definition
For study designs 2, 3, and 6, the following are defined, where available:

1. Case selection criteria
2. Exclusion criteria
3. Gender
4. Race/ethnicity
5. Age
6. Time period
7. Geographic location
8. Number of participants

1. Disease case definition: Oral-facial clefts (OFC), defined as cleft lip with or without cleft palate, or cleft palate. Diagnoses were assessed by individual centers contributing cases.
2. Exclusion criteria: Not mentioned if certain cases were excluded (e.g., those with other congenital anomalies). See also general inclusion criteria below.
3. Gender: Both males and females included
4. Race/ethnicity: Caucasians only
5. Age: Cases had to be between 9 months and 3 years of age at time of recruitment.
6. Time period: Not mentioned
7. Geographic location: Cases were recruited from nine cleft teams in the Netherlands .
8. Number of participants: 113 infants and children with OFC and their mothers

Control definition  
For study design 1, the following are defined, if available. 

1. Control selection criteria
2. Matching variables
3. Exclusion criteria 
4. Gender
5. Race/ethnicity
6. Age
7. Time period
8. Geographic location
9. Number of participants

1. Control selection criteria: Variable, but essentially this is a heterogeneous, convenient sample. Approximately half of control mothers and children were enrolled by case mothers among their friends and neighbors. The other half were recruited through unknown procedures, in nurseries and “infant welfare centers” in and around the city of Nijmegen . Age and race of the child appeared to be the only other selection criteria (see below). Control children had to be unrelated to the case children and free of congenital malformations.
2. Matching variables: Though matching was not explicitly mentioned, selection criteria ensured that control children were of the same age range and race (White) than the case children.
3. Gender: Both males and females included
4. Race/ethnicity: Caucasians only
5. Age: control children had to be between 9 months and 3 years of age at time of recruitment.
6. Time period: not mentioned
7. Geographic location: Netherlands with 47% of control children and mothers being recruited from in and around the city of Nijmegen whereas the remaining were recruited by the case mothers (presumably from a wider area in the Netherlands)
8. Number of participants: 104 infants and children and their mothers

1. Environmental factor
2. Exposure assessment
3. Exposure definition
4. Number of participants with exposure data (%
   of total eligible)

1. Environmental factor: Cigarette Smoking
2. Exposure assessment: Self reported, by written questionnaire sent to home. Missing or unclear answers were followed up by telephone.
3. Exposure definition: Information was gathered on average number of cigarettes smoked per day, whether and in which week after conception the women had ceased or reduced smoking, and how many cigarettes the women smoked before stopping. The exposure window investigated was from three months prior to conception through 3 months after conception.
4. Number of Participants with exposure data: 113 case mothers and 104 control mothers (unclear what percentage these subjects represent of total eligible subjects)

1. Gene
2. DNA source
3. Methodology
4. Number of participants genotyped (% of total eligible) 

1. Gene: GSTT1 and CYP1A1
2. DNA source: Cytology brushes
3. Methodology: Cells were extracted from the brushes and buccal cell DNA was prepared (references). GSTT1 polymorphisms were detected by polymerase chain reaction (PCR) using one primer located in the deleted area followed by gel electrophoresis to identify the presence or absence of the 480-bp fragment indicative of the GSTT1 gene.
   CYPA1A polymorphisms were determined by PCR and restriction enzyme digestion. The primer was designed so that the rare (valine) allele results in the loss of a Nco1 restriction site and is visible at gel electrophoresis as a single large 195-bp fragment in place of the two expected two smaller (163 and 32-bp) fragments.
4. Number of Participants genotyped: Not stated. It appears from Table 3 that between 80% and 86% of infants and mothers were genotyped for at least one of the two loci.

1. Prevalence of gene variant
2. Gene-disease association
3. Gene-environment interaction
4. Gene-gene interaction
5. Genetic test evaluation/monitoring

Prevalence of gene variants are summarized in Table 1. Note that controls are a convenient sample (friends of cases plus other children),so that the prevalence among controls is not necessarily representative of the general population.

Table 1. Prevalence of Genotypes

Gene-disease associations are summarized in Table 2. Overall, the gene-disease associations are unremarkable, with ORs being close to 1 (except for an OR of 1.9, based on two exposed cases; the others were 1.3 or less).

Table 2. Gene-Disease Associations-Infants

Table 2. Gene-Disease Associations-Mother

Gene-environment interactions are summarized in Tables 3 and 4. The major finding of the study related to a possible interaction between smoking and the GSTT1 null genotype (Table 3). The risk of having a baby with OFC among smokers with the null genotype was increased threefold compared to nonsmokers with the Common-type genotype. If the child also carried the null genotype, the relative risk was increased to 4.9. Interestingly, in this sample, smoking alone (i.e., in the presence of a Common-type genotype) did not appear to be a risk factor for OFC (Ors  between 0.7 and 0.9, depending on the case group considered).

Table 3. Gene-Environment Interaction: Maternal Smoking, GSTT1 Genotypes, and the Risk for Oral Clefting

Table 4. Gene-Environment Interaction: CYP1A1, Maternal Smoking, and the Risk for Oral Clefting

Attributable fraction: The authors estimate that from their data, approximately 7% of OFC cases in Netherlands can be attributed to the combination of smoking and GSTT1 genotype.

The authors conclude that maternal smoking is not an independent risk factor for OFC, but is one when combined with certain genotypes. The authors acknowledge that these findings are supported by some previous studies but are contradicted by several others. The authors also point out that their findings are biologically plausible and that their findings on possible gene-environment interactions are unlikely to be due to bias or confounding.

Comments
Provide additional insight, including methodologic issues and/or concerns about the study

The main finding of a possible interaction between smoking and GSTT1 null genotype is interesting, yet it awaits replication. The lack of marginal effects of smoking is also somewhat surprising. Ideally, this study should be independently replicated using a population based sample of cases and controls. The authors define their study as population-based, but this statement is difficult to justify, particularly given the use of a heterogeneous and convenient control group. From a public health perspective, the finding that the null genotype is not a risk factor for OFC in the absence of smoking and that smoking has a wide range of other adverse effects suggests that screening for GSTT1 genotype is not a useful public health measure when attempting to reduce the incidence of OFC.