Does Smoking Lead to Baby's With Cleft Lips?
Epidemiology. Author manuscript; bachelor in PMC 2017 Aug 17.
Published in final edited form equally:
PMCID: PMC5560035
NIHMSID: NIHMS87880
Maternal smoking and oral clefts
The role of detoxification pathway genes
Rolv T. Lie
iSection for Epidemiology and Medical Statistics, Department of Public Health and Primary Health Intendance, University of Bergen, Norway and Medical Nascence Registry of Norway, Norwegian Constitute of Public Health, Bergen, Norway
Allen J Wilcox
2Epidemiology Branch, National Institute of Environmental Wellness Sciences, Durham, NC, USA
Jack Taylor
2Epidemiology Branch, National Institute of Environmental Health Sciences, Durham, NC, USA
HÃ¥kon Chiliad. Gjessing
threeNorwegian Institute of Public Health, Oslo, Kingdom of norway and Section for Epidemiology and Medical Statistics, Department of Public Health and Primary Health Care, University of Bergen, Norway
Ola Didrik Saugstad
fourDepartment of Pediatrics, The National Hospital, Oslo, Norway
Frank Aabyholm
5Department of Plastic Surgery, The National Hospital, Oslo, Norway
Halvard Vindenes
half-dozenDepartment of Plastic Surgery, Haukeland Infirmary, Bergen, Norway
Abstract
Background
There is evidence for an effect of cigarette smoking on risk of oral clefts. There are as well hypothetical pathways for a biological effect involving toxic chemicals in cigarette fume.
Methods
We performed a combined example-command and family unit-triad written report of babies born in Kingdom of norway with oral clefts in the catamenia 1996 to 2001, with 88% participation amongst cases (northward=573) and 76% participation among controls (north=763). Mothers completed a questionnaire 3 months after birth of the baby. DNA was nerveless from parents and children, and assayed for genes related to detoxification of compounds of cigarette smoke (NAT1, NAT2, CYP1A1, GSTP1, GSTT1 and GSTM1).
Results
For isolated cleft lip (with or without scissure palate) there was a dose-response result of smoking in the first trimester. The odds ratio rose from 1.6 (95% CI: 1.0 - 2.5) for passive smoking to 1.9 (95% CI: 0.ix - 4.0) for mothers who smoked more than x cigarettes per twenty-four hours. There was little bear witness of an association with cleft palate. Genetic analyses used both instance-control and family-triad data. In instance-triads we found an association between a NAT2 haplotype and isolated cleft lip (RR of 1.6 in single dose and 2.5 in double dose), just with fiddling evidence of interaction with smoking. Other genes did not testify associations, and previously described interactions with smoking were not confirmed.
Conclusion
Start-trimester smoking was clearly associated with risk of scissure lip. This effect was not modified past variants of genes related to detoxification of compounds of cigarette smoke.
Oral clefts are amongst the most common nascency defects.1 The defects result from nonclosure of specific facial structures in calendar week 5 through 9 of pregnancy, and require extensive surgical and complementary treatment. For unexplained reasons, Norway has i of the highest recorded prevalences of these defects, particularly of cleft lip (ane.5 per k births).two , three
Maternal smoking is an established risk cistron for oral clefts. A contempo meta-analysis of 24 studies estimated that mothers who smoked during pregnancy had a 1.3 fold increased take a chance of having a baby with cleft lip with or without cleft palate, and a 1.2-fold risk of cleft palate alone.4 The biological mechanisms that might underlie this association are unknown. Tobacco smoke contains a large number of toxic chemicals.5 , 6 Studies of smoking-related cancers have establish variations in cancer chance associated with variants of genes that regulate detoxification pathways.7 , 8 The same detoxification genes may also impact the chance of oral clefts,9 - 12 modifying the smoking event on clefting to create interaction between smoking and allelic variants. Relevant detoxification during early pregnancy may occur both in the child and in the mother. The genes of both should therefore be considered in studies of birth outcomes.13
Several candidate genes are related to detoxification of components of cigarette smoke. The arylamine N-acetyltransferases (NAT1 and NAT2) are xenobiotic-metabolizing enzymes that play an important role in the metabolic activation of carcinogenic amines present in cigarette smoke.10 , eleven , 14 Cytochrome P450 (CYP1A1) is related to the bioactivation of chemicals such as dioxin in cigarette fume.fifteen , 16 The glutathione S-transferase (GST) enzymes affect the detoxification and secretion of compounds of cigarette smoke.9 , 11 , 12 , 17
Nosotros explored the consequence of maternal smoking on clefting risk, and the modifying effects of candidate detoxification genes through a population-based case-control, family triad written report in Norway.
MATERIALS AND METHODS
Recruitment
An overview of the design and recruitment of the report is given in Figure 1. All cases born in Kingdom of norway 1996-2001 and referred for clefts surgery were ascertained through the two surgical departments responsible for all facial clefts repairs in Norway. Affected babies are routinely referred for surgery shortly after birth, at which fourth dimension the family was invited to participate in the study.
Description of recruitment of case- and command families and completeness of different components of the study.
During the same period of time, controls were selected randomly from all alive births with a probability of 4/1000 (with small-scale adjustments during the study) using an automated procedure in the Medical Birth Registry of Norway. Controls were invited by mail service through the delivering physician. More details of the design of the study have been published elsewhere.18
Biological samples
Case mothers and fathers were asked to donate both claret and cheek swabs. We also asked permission to draw a blood sample from the child during surgery, and to retrieve from the centralized screening lab in Norway the left-over portion of the kid's sample collected for PKU testing.
In control families, the mother and child provided cheek swab samples, and fathers provided swabs for babies built-in after November 1998 (half-way through the study). We besides retrieved the PKU samples from control babies.
Questionnaires
All participating mothers were asked to complete two questionnaires, i on a broad spectrum of atmospheric condition and exposures and one on nutrition. Copies of these translated to English may exist constitute at our web site (http://dir.niehs.nih.gov/direb/studies/ncl/question.htm.).
Closure of the lip occurs around week five of embryonic life (before many women are enlightened of their pregnancy) and is followed by closure of the palate around week 9. Nosotros explicitly directed our questions nearly smoking to the three first months of pregnancy. The first questionnaire contained detailed questions on smoking both before pregnancy and during the first three months of the pregnancy. Mothers reported average number of cigarettes smoked per mean solar day (or per month, if less than one per day).
We also asked the mother the average number of hours per twenty-four hour period she was inside two meters of a person who was smoking. Passive smoking exposure was divers equally the exposure of a non-smoker to a smoker (inside two meters) for at least two hours a day. A categorical dose-response variable was created for smoking, with passive smoking as the everyman level,one-5 cigarettes a day every bit the 2nd category, 6-ten cigarettes a day every bit the tertiary, and eleven or more cigarettes a solar day every bit the fourth and highest level of smoking.
The smoking information was tested for reliability against prospectively collected data on smoking from the female parent's starting time prenatal visit (typically around calendar week 10 of pregnancy). Some mothers who smoked earlier in pregnancy might accept stopped smoking earlier the prenatal visit. More important, differential recall between cases and controls should be evident in a comparison of the prenatal report and the post-commitment questionnaire. Evidence of differential recall would indicate response bias, and advise that prospective smoking information is a more reliable data-source in our analyses.
Case information
We obtained surgical records for cases from the hospitals. These records contained information on details of the oral fissure too as diagnoses of syndromes or other accompanying defects. We besides retrieved the Medical Birth Registry record, which contained additional information on birth defects diagnoses, and we asked the mother about diagnoses of the child. A case with any accompanying nativity defect or diagnosis of a syndrome reported from whatever source was categorized as a "not-isolated" case. All other cases were categorized as "isolated".
Genetic assays
Deoxyribonucleic acid was extracted from blood of parents and child in the case grouping and from cheek swabs from parents and child in the control group. For NAT1 nosotros assayed two SNPs with labels T1088A (rs1057126) and C1095A (rs15661). For NAT2 nosotros assayed the SNPs C481T (rs1799929), G590A (rs1799930) and G857A (rs1799931). Ii SNPs of CYP1A1 were assayed: M1-T400C (rs4646903) and T1101C (rs1048943) and two SNPs of GSTP1: A114V (rs1799811) and A1517G (rs947894). SNP assays were based on a Masscode™ organisation. The SNPs selected for this study within each gene are believed to have functional effects on enzyme activity and are candidates for biological furnishings on risk. Nosotros likewise identified individuals who were homozygous for nil-variants of GSTM1 and GSTT1.
Statistical analyses
Analyses of maternal smoking
Nosotros tested for a chief effect of the exposure (maternal smoking) using a traditional instance-command analysis of the 2 example categories cleft lip with or without cleft palate and cleft palate simply. We repeated the analysis for isolated cases but (excluding cases with additional defects). Unconditional logistic regression models in STATA five.nine were used to clarify the case-command data for effects of smoking. Odds ratios (ORs) from these analyses are good estimates of relative risks (RRs) since facial clefts are rare weather. We estimate both rough ORs and ORs adjusted for female parent's education, work status, booze intake, folate supplementation and dietary folate, multi-vitamin supplementation, father'southward income, and calendar year of baby's birth.
Genetic analyses
Family triads were the master basis for genetic assay. The triad blueprint had the advantage of being immune to effects of population stratification. It also provides more information for haplotype reconstruction, which may compensate for a slightly lower statistical ability in single marker analyses.19
All SNPs were outset assessed for Hardy-Weinberg equilibrium (and whatever signs of deviance from Mendelian manual of alleles) in the control triads after November 1998, when we began to include collection of father's DNA. The absence of data from control fathers in the first half of the report is unrelated to genetic characteristics of the fathers, and thus has no result on genetic results, other than reducing power.
We restricted our analysis of genetic effects to case types for which smoking had a clear consequence. This genetic analysis was based on the case triads.xiii , twenty If the assumptions of Hardy-Weinberg equilibrium holds, we used analyses with a program called Haplin, both for effects of single SNPs and for analyses of haplotypes reconstructed from the SNP markers.twenty Haplin is implemented as a package in R (world wide web.uib.no/smis/gjessing/genetics/software/haplin/). The methods used past Haplin are generalizations of other family-based methods such as the TDT and log-linear models.xiii , 21 Estimation was done both for haplotypes carried by the child and for haplotypes carried past the mother. Haplin uses maximum likelihood methods and the EM algorithm. This analytic method allows triads with missing genotype data or missing family members (usually fathers) to exist included in the estimation. This assumes that genetic data are missing randomly. Haplin estimates the relative risk for a single dose of a haplotype (heterozygotes combining with any other haplotype) and also for a double dose (homozygotes). The reference category for each estimated haplotype upshot is the grouping not carrying that detail haplotype. When only 2 haplotypes enter the interpretation, the reference category is the single category of homozygotes for the common haplotype.
For SNPs or haplotypes associated with clefts in the family-based analyses, nosotros starting time estimated gene-surround interaction using case-triads. Haplin was used to compare the gene effects between case triads of smoking mothers and non-smoking mothers. A likelihood-based examination for departure in gene furnishings was calculated.
We also supplemented these analyses with case-control analyses, incorporating smoking and the relevant genetic variants. Although family triads provide some information about haplotypes, unique identification of haplotypes is non possible for every individual. When haplotypes were ambiguous, we estimated probability weights for the culling haplotypes for the mother and the child using the EM algorithm and maximum-likelihood interpretation in Haplin. These probability weights were generated separately for instance triads and control triads. Example-control analyses were used with these haplotypes (for both mother and child) to verify the master furnishings of haplotypes and to estimate interaction with smoking. Case command analyses were performed by logistic regression with probability weights in STATA 5.9.
We had to use the case-control arroyo to analyze these genotypes, since standard family-based association analyses require more than explicit identification of all genotypes. In a set of supplementary analyses nosotros besides tested previously-reported associations with GSTT1, GSTM1 and NAT1.nine - 12
RESULTS
Table 1 provides descriptive information for mothers and fathers of cases and controls. Based on the retrospective questionnaire, 42% of example mothers and 32% of control mothers reported smoking during the get-go trimester. This information could be subject to recall bias. To explore this possibility, we compared our smoking information with prospectively-collected information on smoking from the mother's first prenatal visit (at an average of 10 weeks of gestation). Prospective data was missing for iii cases and forty controls.
Table 1
Demographic and other characteristics of mothers, fathers and children,cases and controls, Norway 1996-2001.
| CASES | CONTROLS | ||
|---|---|---|---|
| Cleft lip1 Northward = 377 | Cleft palate only Northward = 196 | North = 763 | |
| N (%) | North (%) | N (%) | |
| Mother | |||
| Current marital status | |||
| Married | 182 (48) | 91 (46) | 405 (53) |
| Alive-in | 177 (47) | 96 (49) | 329 (43) |
| Singletwo | 18 (five) | 85 (4) | 28 (3) |
| Missing | 0 (0) | 1 (i) | 1 (ane) |
| Maternal age (yrs) | |||
| < xx | eight (2) | six (three) | 12 (one) |
| xx – 29 | 199 (53) | 105 (54) | 408 (54) |
| 30 – 39 | 165 (44) | 81 (41) | 328 (43) |
| 40+ | 5 (1) | 4 (2) | 15 (2) |
| Parity | |||
| 1 | 151 (40) | 88 (45) | 292 (38) |
| 2 | 138 (37) | 63 (32) | 290 (38) |
| 3 | 63 (17) | 36 (xviii) | 132 (17) |
| 4+ | 25 (half dozen) | 9 (5) | 49 (seven) |
| Pedagogy | |||
| < Highschool | 70 (19) | 23 (12) | 87 (11) |
| Loftier school | 94 (25) | 48 (25) | 211 28) |
| Technical college | 69 (18) | 41 (21) | 153 (xx) |
| ii - 4 year college | 124 (33) | 72 (36) | 265 (34) |
| Academy | 19 (5) | eleven (half dozen) | 46 (vi) |
| Other | 1 (<ane) | 1 (<1) | 1 (<1) |
| Employment in early pregnancy | |||
| Yes | 297 (79) | 158 (81) | 646 (85) |
| No | 80 (21) | 38 (19) | 117 (xv) |
| Country of birth | |||
| Kingdom of norway | 354 (94) | 177 (90) | 720 (94) |
| Other | 23 (6) | nineteen (10) | 43 (6) |
| Cigarette smoking | |||
| No exposure | 152 (40) | 92 (47) | 414 (54) |
| Passive only | 58 (15) | 32 (xvi) | 106 (xiv) |
| Agile, 1 – 53 | 93 (25) | 36 (18) | 142 (19) |
| Agile, 6 – ten3 | 49 (13) | 31 (xvi) | 73 (10) |
| Active, 11+3 | 25 (vii) | 5 (3) | 28 (4) |
| Folic acid supplementiv | |||
| No supplement | 240 (64) | 119 (61) | 453 (59) |
| ane – 399 μg | 86 (23) | 46 (23) | 165 (22) |
| 400+ μg | 51 (14) | 31 (xvi) | 145 (nineteen) |
| Dietary folate, μg (quartiles) | |||
| 0 – 171 | |||
| 172 – 214 | 111 (31) | 62 (33) | 176 (25) |
| 215 – 264 | 87 (25) | 44 (23) | 173 (25) |
| 265+ | 79 (22) | 35 (19) | 178 (25) |
| 77 (22) | 47 (25) | 17 seven (25) | |
| Multivitaminsiv | |||
| Yes | 123 (33) | 71 (36) | 279 (37) |
| No | 254 (67) | 325 (64) | 484 (63) |
| Alcoholic beverages5 | |||
| 0 | 230 (61) | 120 (61) | 527 (69) |
| 1-3 | 70 (xix) | 37 (nineteen) | 123 (16) |
| four-6 | 26 (7) | 17 (9) | twoscore (5) |
| 7+ | 45 (12) | 22 (11) | 68 (9) |
| missing | 6 (2) | 0 | v (i) |
| Mother with facial cleft | |||
| Yeah | ten (3) | 6 (3) | ii (0.2) |
| No | 367 (97) | 190 (97) | 761 (99+) |
| Father | |||
| Age (yrs) | |||
| < xx | three (1) | 0 (0) | three (ane) |
| 20 – 39 | 341 (91) | 179 (92) | 696 (91) |
| 40+ | 30 (viii) | 16 (viii) | 63 (8) |
| Yearly income (Kr) | |||
| No income | vii (2) | i (1) | 15 (ii) |
| < 150,000 | 20 (five) | 11 (5) | 44 (6) |
| 151 – 200,000 | 63 (17) | 31 (16) | 96 (12) |
| 201 – 250,000 | 94 (25) | 59 (thirty) | 186 (24) |
| 251,000+ | 166 (44) | 85 (43) | 380 (50) |
| Missing | 27 (7) | 9 (five) | 42 (6) |
| Country of nativity | |||
| Norway | 348 (92) | 182 (93) | 709 (93) |
| Other | 29 (8) | 14 seven) | 54 (vii) |
| Father with facial cleft | |||
| Yes | 14 (4) | 6 (3) | ii (0.two) |
| No | 363 (96) | 190 (97) | 761 (99+) |
| Infant | |||
| Cases with other birth defects | |||
| Yeah | 63 (17) | 78 (40) | -- |
| No | 314 | 118 (60) | |
| (83) | |||
| Controls with any nascence defect | |||
| Yes | -- | -- | 38 (v) |
| No | 725 (95) | ||
Overall, fewer mothers were recorded as smokers in the prospective information, suggesting either that they had stopped smoking earlier the prenatal visit or that they underreported smoking at their dr. visit. All mothers except one (a control mother) who had been identified at the prenatal visit as smokers were also identified as smokers past the post-nativity questionnaire (Table 2). Amidst case mothers who reported their smoking in the retrospective questionnaire, 48% reported this only in the questionnaire. This proportion was about the aforementioned for control mothers (49%). The biggest difference betwixt the two data sources was for mothers smoking 1 to 5 cigarettes per twenty-four hour period. Notwithstanding, the proportion of smokers in this category added by the questionnaire was very similar for mothers of cases and controls (64% vs. 62%).
Table two
Data on maternal smoking in first trimester collected by retrospective questionnaire compared with prospective data on smoking from first prenatal visit.
| Prospective information1 | |||||
|---|---|---|---|---|---|
| Retrospective questionnaire informationtwo | Smoker | Non- smoker | Total | % smokers added past questionnaire3 | |
| All cases | Non smoker | 1 | 331 | 332 | |
| (n=570) | 1- v cigarettesiv | 47 | 82 | 129 | 64% |
| half-dozen-x cigarettes4 | 58 | 22 | 80 | 28% | |
| 11+ cigarettesfour | 19 | 10 | 29 | 35% | |
| All smokers | 124 | 114 | 238 | 48% | |
| | |||||
| All controls | Non smoker | 0 | 494 | 494 | |
| (n=723) | ane- 5 cigarettesiv | fifty | 83 | 133 | 62% |
| 6-10 cigarettes4 | 48 | 21 | 69 | xxx% | |
| 11+ cigarettesiv | 19 | 8 | 27 | xxx% | |
| All smokers | 117 | 112 | 229 | 49% | |
Thus, there was no evidence of differential recall of smoking by cases and controls in our questionnaire. The fact that just one-half of the women who reported retrospectively that they smoked in the commencement trimester besides reported current smoking at their prenatal visit suggests that the prospective information did not adequately capture first-trimester smoking. Nosotros have therefore used the questionnaire information on smoking in our primary analyses.
Effects of maternal smoking
There was little show of an effect of smoking on the hazard of cleft palate only. When we restricted to isolated cleft palate (Table 3), the test-for-trend p-value was 0.74. Use of the prospective information on smoking did not alter this (p=0.37).
Table 3
Odds ratio (OR) for isolated cleft palate (without cleft lip) with categories of maternal smoking in start trimester
| Rough assay | Adjusted analysis1 | |||||
|---|---|---|---|---|---|---|
| Level of smoking | Cases (due north=118) | Controls (n=763) | OR | 95% CI | OR | 95% CI |
| No smoking | 59 | 414 | 1.00 | Ref. | 1.00 | Ref. |
| Only passivetwo | 17 | 106 | 1.xiii | 0.63 – 2.01 | 1.05 | 0.55 – 2.00 |
| one- v cigarettes3 | 20 | 142 | 0.99 | 0.57 – 1.70 | 0.81 | 0.45 – 1.44 |
| 6-ten cigarettes3 | 21 | 73 | 2.02 | 1.16 – 3.52 | 1.82 | 0.98 – 3.39 |
| 11+ cigarettes3 | one | 28 | 0.25 | 0.03 – one.88 | 0.29 | 0.04 – 2.26 |
In contrast, there was a strong and consequent dose-response effect of smoking for cleft lip (with or without fissure palate), including a modest increased chance with passive smoking. Restricting to 314 isolated cases of fissure lip, the hazard ranged from i.6-fold for passive smoking (adjusted OR=one.59, 95% CI: 1.02-2.47) to virtually two-fold when mothers smoked more than 10 cigarettes per day (adjusted OR=1.92, 95% CI: 0.92-iv.01) (Table iv, test-for-trend p-value=0.001). The estimated associations were similar when nosotros used smoking recorded at get-go prenatal visit (exam-for-tendency p-value=0.04). Amidst the fissure lip cases without other defects ("not-isolated") there was lilliputian evidence of an effect of smoking (overall p-value=0.54; data not shown). Extrapolating from these population based data, we estimate that 19% of isolated cleft lip cases in Norway may be attributable to maternal smoking in the first trimester.
Table four
Odds ratio (OR) for isolated cleft lip (with or without crevice palate) with categories of maternal smoking in first trimester
| Crude analysis | Adjusted analysis1 | |||||
|---|---|---|---|---|---|---|
| Level of smoking | Cases (north=314) | Controls (n=763) | OR | 95% CI | OR | 95% CI |
| No smoking | 118 | 414 | one.0 | Ref. | ane.0 | Ref. |
| Just passive2 | 48 | 106 | 1.59 | 1.07 - 2.36 | 1.59 | ane.02 - 2.47 |
| 1- 5 cigarettesiii | 82 | 142 | two.03 | 1.44 - 2.85 | one.62 | 1.12 - 2.36 |
| half-dozen-10 cigarettes3 | 46 | 73 | 2.21 | 1.45 - 3.37 | one.87 | ane.15 - 3.04 |
| 11+ cigarettes3 | 20 | 28 | ii.51 | 1.36 - 4.61 | 1.92 | 0.92 - four.01 |
Family unit triad analyses of smoking detoxification genes
SNPs
The call charge per unit of our SNP-assays varied between 88 and 96% (Table 5). None of the SNPs in our report had meaning deviance from Hardy-Weinberg equilibrium or mendelian transmission of alleles among control triads. In that location was strong linkage disequilibrium amid SNPs within each factor in our data.
Table 5
Genetic SNP information and analyses. Relative risks of isolated cleft lip are risks associated with the rare allele in single or double dose
| Control triads (n=417)one | Isolated cleft lip (with or without scissure palate) triads (n=314) | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Call rate (%) | Frequency rare allele (%) | Non H-Due west equilibrium (p-value) | Non-mendelian transmission (p-value) | Result of rare allele | Fetal alleles | Maternal alleles | ||||
| RR | 95% CI | RR | 95% CI | |||||||
| NAT1 | T1088A/ rs1057126 | 93.7 | 19.3 | 0.88 | 0.70 | Single dose Double dose | 1.0 1.0 | 0.7-1.4 0.5-2.0 | ane.1 0.4 | 0.8-1.four 0.two-1.1 |
| C1095A/ rs15661 | 94.8 | 24.eight | 0.85 | 0.92 | Single dose Double dose | 1.1 i.3 | 0.8-one.4 0.7-2.3 | i.1 0.seven | 0.viii-one.5 0.3-i.iv | |
| | ||||||||||
| NAT2 | C481T/ rs1799929 | 88.4 | 43.two | 0.60 | 0.33 | Single dose Double dose | 0.9 ane.1 | 0.6-ane.ii 0.vii-1.7 | 1.ii ane.one | 0.eight-one.6 0.7-1.8 |
| G590A/ rs1799930 | 92.0 | 28.1 | 0.28 | 0.09 | Single dose Double dose | 1.three 2.0 | 0.9-1.8 1.two-3.5 | 1.0 one.0 | 0.8-1.4 0.5-1.8 | |
| G857A/ rs1799931 | 96.4 | 2.seven | 0.35 | 0.59 | Unmarried dose Double dose | 0.half-dozen - | 0.three-1.1 - | 0.ix - | 0.v-1.7 - | |
| | ||||||||||
| CYP1A1 | M1-T400C/ rs4646903 | 92.2 | 9.7 | 0.87 | 0.98 | Single dose Double dose | 0.eight 0.7 | 0.6-1.2 0.ii-2.vi | 1.1 2.ii | 0.8-1.7 0.8-5.nine |
| T1101C/ rs1048943 | 95.ii | ii.9 | 0.78 | 0.32 | Single dose Double dose | one.9 27 | 0.eight-iv.five 3.7-202 | 0.9 14 | 0.iv-two.0 two.4-82 | |
| | ||||||||||
| GSTP1 | A114V/ rs1799811 | 95.0 | 8.two | 0.41 | 0.07 | Single dose Double dose | 0.9 - | 0.vi-1.4 - | 0.vii 0.4 | 0.5-1.ane 0.one-3.i |
| A1517G/ rs947894 | ninety.3 | 33.8 | 0.ten | 0.98 | Single dose Double dose | 1.one 0.ix | 0.8-one.5 0.5-1.4 | ane.0 0.7 | 0.vii-i.3 0.4-one.ane | |
Since our analysis of smoking showed a articulate association only for isolated crevice lip, we limited our analyses of smoking detoxification genes to this case group. There was some bear witness of an effect of the NAT2 SNP G590A/rs1799930 (Tabular array v). Children heterozygous for the rare variant appeared to have a ii-fold risk (RR=2.0, 95% conviction interval (CI): 1.ii-3.5). The rare variant CYP1A1 SNP T1101C/rs1048943 (iii% allele frequency) was estimated with high risks for homozygotes, although only ii cases were homozygous for this variant. No other SNPs — whether carried by the mother or the infant — appeared to be associated with cleft lip.
Haplotypes
The numbers of haplotypes that could be reconstructed from the SNPs (and that were prevalent enough to be included in the estimation) ranged from two for NAT1 and CYP1A1 to three for GSTP1 and four for NAT2 (Fig. 2).
Estimated relative risks for haplotypes of NAT1, NAT2, CYP1 and GSTP1 from triads of isolated cleft lip cases. Relative risks are estimated separately for the kid's and the mother's haplotypes. Estimation is done with the estimator plan HAPLIN accounting for missing information and unknown stage by the EM-algorithm. For NAT1 and CYP1 in that location were only two haplotypes involved in estimation, and ane of the homozygotes is used as reference category. For NAT2 and GSTP1 the reference is reciprocal (all other haplotypes) and estimates are shown for unmarried dose (left signal) and double dose (right signal) for all haplotypes.
The only haplotype associated with isolated crack lip was the C-A-Yard haplotype of NAT2 (referring to the variants of the SNPs rs1799929, rs1799930 and rs1799931 respectively) when this haplotype was carried by the child. This haplotype carries the rare A-allele of G590A/rs1799930 and the common alleles of rs1799929 and rs1799931. A unmarried copy of this haplotype increased the risk of isolated cleft lip 1.half-dozen-fold (RR=ane.60, 95% CI:i.10-ii.xl) and a double dose of the haplotype increased the risk 2.v-fold (RR=2.50, 95%CI:i.40-4.60). The outcome of haplotypes carrying the very rare variant of CYP1A1 T1101C/rs1048943 could not be estimated.
Using haplotype data derived from the family triads, we then used a instance-control approach to estimate haplotype effects for the C-A-G haplotype of NAT2. This did not confirm our family unit-based analysis. For the child's haplotypes, there was no increased risk for the C-A-One thousand haplotype in single dose (OR=1.0, 95% CI: 0.8-1.4) and merely a 1.2-fold gamble in double dose (OR=one.2, 95% CI: 0.8-1.9).
Gene-environment interaction analyses
We explored interaction by creating a dichotomous variable of maternal smoking exposure. In case-triad analyses with Haplin, the association of the C-A-M haplotype of NAT2 was not substantially different among children with smoking mothers (RR= 1.5 in single dose and two.7 in double dose) and children with non-smoking mothers (RR=1.8 in single dose and 2.iii in double dose) (overall p-value of difference p=0.20). Similarly, there was no evidence of effect modification by smoking for the unmarried SNPs NAT2 rs1799930 and CYP1A1 rs1048943 (overall p-values of 0.93 and 0.81 respectively).
There was no indication of interaction between the child'due south NAT2 C-A-Yard haplotype and maternal smoking (p of interaction = 0.72). For the single SNP NAT2 rs1799930 we found no prove of association in example-control analyses (p=0.37) and no prove of interaction with smoking (p=0.15). The variant of CYP1A1 rs1048943 was besides rare to be studied in case-control analyses. Results were once again similar when the prospective information on smoking was used.
Supplementary analyses
We establish no evidence of association of the nil variants of GSTT1 or GSTM1 with isolated cleft lip (Table vi) or of interaction between GSTT1 zilch in the child and maternal smoking (p of interaction was 0.lx). The risk was not changed when both mother and child had the GSTT1 nix variant (OR=0.9, 95% CI: 0.4-ii.2). When both mother and child had the GSTM1 null variant, still, the risk was ii-fold (OR=two.2, 95% CI: i.ane-four.4). When restricting to mothers exposed to tobacco fume, risks were not elevated for children with the NAT1 1088 AA genotype compared to children with TT (OR=ane.3, 95% CI:0.5-3.ii) of for children with the NAT1 1095 AA genotype compared to CC (OR=1.3, 95% CI: 0.half-dozen-2.seven). There was no increased gamble of scissure lip for children with zip variants of both GSTT1 and GSTM1 when the female parent smoked (OR=1.0, 95% CI: 0.3-3.2).
Table 6
Odds ratio (OR) for isolated cleft lip with or without cleft palate for the zip variants of GSTT1 and GSTM1
| Number of homozygotes | ||||
|---|---|---|---|---|
| Genotype | Cases (%1) | Controls (%i) | ORii | 95% CIthree |
| Kid homozygous for GSTT1 aught | 33 (13) | 65 (15) | .78 | .49 - 1.25 |
| Mother homozygous for GSTT1 nix | 46 (19) | 66 (16) | one.32 | .86- 2.03 |
| Kid homozygous for GSTM1 null | 145 (59) | 232 (54) | ane.17 | .83 - i.65 |
| Mother homozygous for GSTM1 null | 138 (56) | 227 (53) | 1.05 | .74 - 1.47 |
Word
We found persuasive evidence of an association between mothers' smoking and the risk of cleft lip in her offspring, only no evidence that genetic variation in several detoxification enzymes afflicted or modified this risk.
This effect of maternal smoking on crack lip is consistent with several reports in the literature.3 , 4 , 22 - 26 For cleft palate, past bear witness is less consistent. Some studies have found increased risk for cleft palate with smoking,23 , 26 , 27 merely not all.24 , 25 While we cannot exclude the possibility of an effect of maternal smoking on fissure palate only in our data, the evidence is much weaker than for scissure lip.
We found an effect of passive smoking on cleft lip among non-smoking mothers defining passive smoking every bit having a smoking person closer than 2 meters for at least ii hours per twenty-four hours. Passive smoking — defined in a variety of ways — has been studied previously. Shaw et al. defined passive smoking past closeness to a smoker, and constitute increased run a risk among offspring of exposed women.23 No associations were institute in studies defining exposure based on duration of exposure and degree of smokiness in the air,26 or as whatsoever exposure to smoke for a non-smoking mother.28
Our case-control study of smoking has several strengths. It is population based, with virtually 100% ascertainment of clinically verified cases during a divers time period. Data drove was done in the first months after birth, which should have reduced recall problems for smoking and other exposures. Participation was reasonably skillful both for cases (88%) and controls (76%). Since the difference betwixt prospective and retrospective report of smoking was nearly identical for cases and controls, we may assume that reporting bias of smoking was minimal. Our data advise that but one-half the mothers who smoked in the starting time trimester reported this at their beginning antenatal visit, either because they had stopped smoking when they became aware of their pregnancy or considering they didn't want to tell their doctor. We as well had all-encompassing information on relevant confounders for statistical aligning of our estimates.
Genetic analyses
The metabolism genes studied here accept been of interest for cancer and interactions with smoking because of their activating/deactivating activities for carcinogens. Since mutation acquired past the same carcinogens is a possible machinery for oral clefts, these genes are also relevant candidate genes to explicate an effect of smoking on oral clefts.
Since the consequence of smoking in our data seemed to be restricted to isolated cleft lip, we limited our genetic analyses for the detoxification genes to this case-grouping. Nosotros found some testify of an effect of NAT2 in the case-family unit-based analysis, although this was non confirmed in the case-control analysis. The NAT2 haplotype associated with crevice lip carries the A-allele of the G590A-SNP (rs1799930). This variant is known to reduce expression and stability of NAT2 immunoreactive poly peptide and reduce acetylation activity,29 , 30 and has previously been found to exist associated with cleft lip.11 There was, all the same, no prove in our information that smoking modified an event of the NAT2-haplotype on risk of crevice lip. NAT2 may still have a existent event on scissure lip, but apparently not by interaction with smoking.
We also institute an indication in our case-triad analyses of an effect of CYP1A1 T1101C/rs1048943, apparent as a strong dominant effect of the rare allele. The association was created by only a few instance families and could not be verified in instance-command analyses. Larger studies would be needed to study an involvement of this variant with the smoking outcome. Other associations previously described for children with variants of NAT1, GSTT1 and GSTM1 for cleft lip were not replicated in our data.9 - 12
Combining two study designs
The genetic aspects of our study were strengthened by being able to combine a family triad design with a case-control design.31 In studies with genetic information for both case-parent triads and control-parent triads, the integrated use of the two analytic approaches has several advantages. Commencement, the ii structures of genetic analysis recoup for each other's weakness. Genetic case-control analyses can be vulnerable to admixture problems in the population, even in relatively homogeneous populations such as the Norwegian.32 , 33 Instance-triad analyses overcome this difficulty, but in turn may be vulnerable to deviance from Mendelian transmission of alleles. Control-parent triads permit a cheque for Mendelian transmission. 2nd, the integrated arroyo provides a check for consistency between the furnishings estimated by case-triad and example-control analyses. A failure to detect consistency betwixt the two approaches (as occurred with our NAT2 analysis) raises doubts about associations that otherwise might exist regarded as strong in a single approach.
Limitations
Although our overall participation was expert, participation was lower for controls than for cases, which creates the possibility of differential participation and bias. The credible specificity of an event of smoking to one example-group may signal that such bias did non bear upon our results, just the possibility cannot be ruled out.
While we validated our smoking data against prospectively collected information, we cannot dominion out incompleteness in our information on smoking. If some smoking mothers in our study were categorized as exposed only to passive smoking, this could bias the passive smoking finding.
Nosotros did non take Dna for genetic analyses for control fathers recruited before November 1998. This created a substantial number of missing fathers in our data. Furthermore, our study used DNA from blood cells for case-triads and buccal cells for control triads. If these ii sources of DNA gave different genotyping results, our example-control analyses of genetic markers could exist biased. There is however little evidence for such differences betwixt buccal cell and blood cell DNA.34 , 35 To assess the probability of genotyping errors more than generally, we performed a blinded second genotyping of a random 10 per cent of our samples, which showed an over-all concordance rate of 99.4%. Concordance was not different for cases and controls. Virtually of the errors were for the null-variant assays.
In sum, we found strong show that maternal smoking in the first trimester causes cleft lip. There was some evidence that a functional variant of NAT2 is associated with cleft lip, independent of smoking, although nosotros could not confirm this in our case-control assay. In our exploration of genes involved with metabolism of cigarette fume toxicants, we were not able to demonstrate interactions of smoking with NAT1, NAT2, CYP1A1, GSTP1, GSTT1 or GSTM1. Previously reported associations were not confirmed.
Acknowledgments
This research was supported by the Intramural Research Program of the NIH, National Institute of Environmental Health Sciences, The Norwegian Research Council, Helse Vest and NIH (2RO1 DE-11948-04).
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Source: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5560035/
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