Trajectories of Occupational Cognitive Demands and Risk of Mild Cognitive Impairment and Dementia in Later Life: The HUNT4 70+ Study.

BACKGROUND AND OBJECTIVES: The cognitive reserve hypothesis posits that cognitively stimulating work delays the onset of mild cognitive impairment (MCI) and dementia. However, the effect of occupational cognitive demands across midlife on the risk of these conditions is unclear. METHODS: Using a cohort study design, we evaluated the association between registry-based trajectories of occupational cognitive demands from ages 30-65 years and clinically diagnosed MCI and dementia in participants in the HUNT4 70+ Study (2017-19). Group-based trajectory modeling identified trajectories of occupational cognitive demands, measured by the routine task intensity (RTI) index (lower RTI indicates more cognitively demanding occupation) from the Occupational Information Network. Multinomial regression was implemented to estimate the relative risk ratios (RRRs) of MCI and dementia, after adjusting for age, sex, education, income, baseline hypertension, obesity, diabetes, psychiatric impairment, hearing impairment, loneliness, smoking status, and physical inactivity assessed at HUNT1-2 in 1984-1986 and 1995-1997. To handle missing data, we used inverse probability weighting to account for nonparticipation in cognitive testing and multiple imputation. RESULTS: Based on longitudinal RTI scores for 305 unique occupations, 4 RTI trajectory groups were identified (n = 7,003, 49.8% women, age range 69-104 years): low RTI (n = 1,431, 20.4%), intermediate-low RTI (n = 1,578, 22.5%), intermediate-high RTI (n = 2,601, 37.1%), and high RTI (n = 1,393, 19.9%). Participants in the high RTI group had a higher risk of MCI (RRR 1.74, 95% CI 1.41-2.14) and dementia (RRR 1.37, 95% CI 1.01-1.86), after adjusting for age, sex, and education compared with participants in the low RTI group. In a sensitivity analysis, controlling for income and baseline health-related factors, the point estimates were not appreciably changed (RRR 1.66, 95% CI 1.35-2.06 for MCI, and RRR 1.31, 95% CI 0.96-1.78 for dementia). DISCUSSION: People with a history of cognitively stimulating occupations during their 30s, 40s, 50s, and 60s had a lower risk of MCI and dementia older than 70 years, highlighting the importance of occupational cognitive stimulation during midlife for maintaining cognitive function in old age. Further research is required to pinpoint the specific occupational cognitive demands that are most advantageous for maintaining later-life cognitive function.

Marital Histories and Associations With Later-Life Dementia and Mild Cognitive Impairment Risk in the HUNT4 70+ Study in Norway.

Objectives: Earlier studies suggest that being married in later life protects against dementia, and that being single in old age increases the risk of dementia. In this study, we examine midlife marital status trajectories and their association with dementia and mild cognitive impairment (MCI) at ages 70 plus using a large population based sample from Norway. Methods: Based on a general population sample linked to population registries (N = 8706), we used multinomial logistic regression to examine the associations between six types of marital trajectories (unmarried, continuously divorced, intermittently divorced, widowed, continuously married, intermittently married) between age 44 and 68 years from national registries and a clinical dementia or a MCI diagnosis after age 70. We estimated relative risk ratios (RRR) and used mediation analyses adjusting for education, number of children, smoking, hypertension, obesity, physical inactivity, diabetes, mental distress, and having no close friends in midlife. Inverse probability weighting and multiple imputations were applied. The population attributable fraction was estimated to assess the potential reduction in dementia cases due to marital histories. Results: Overall, 11.6% of the participants were diagnosed with dementia and 35.3% with MCI. Dementia prevalence was lowest among the continuously married (11.2%). Adjusting for confounders, the risk of dementia was higher for the unmarried (RRR = 1.73; 95% CI: 1.24, 2.40), continuously divorced (RRR = 1.66; 95% CI: 1.14, 2.43), and intermittently divorced (RRR = 1.50; 95% CI: 1.09, 2.06) compared to the continuously married. In general, marital trajectory was less associated with MCI than with dementia. In the counterfactual scenario, where all participants had the same risk of receiving a dementia diagnosis as the continuously married group, there would be 6.0% fewer dementia cases. Discussion: Our data confirm that staying married in midlife is associated with a lower risk of dementia and that divorced people account for a substantial share of dementia cases.

Associations between epigenetic age acceleration and infertility.

STUDY QUESTION: Is the use of ART, a proxy for infertility, associated with epigenetic age acceleration? SUMMARY ANSWER: The epigenetic age acceleration measured by Dunedin Pace of Aging methylation (DunedinPoAm) differed significantly between non-ART and ART mothers. WHAT IS KNOWN ALREADY: Among mothers who used ART, epigenetic age acceleration may be associated with low oocyte yield and poor ovarian response. However, the difference in epigenetic age acceleration between non-ART and ART mothers (or even fathers) has not been examined. STUDY DESIGN, SIZE, DURATION: The Norwegian Mother, Father and Child Cohort Study (MoBa) recruited pregnant women and their partners across Norway at around 18 gestational weeks between 1999 and 2008. Approximately 95 000 mothers, 75 000 fathers and 114 000 children were included. Peripheral blood samples were taken from mothers and fathers at ultrasound appointments or from mothers at childbirth, and umbilical cord blood samples were collected from the newborns at birth. PARTICIPANTS/MATERIALS, SETTING, METHODS: Among the MoBa participants, we selected 1000 couples who conceived by coitus and 894 couples who conceived by IVF (n = 525) or ICSI (n = 369). We measured their DNA methylation (DNAm) levels using the Illumina MethylationEPIC array and calculated epigenetic age acceleration. A linear mixed model was used to examine the differences in five different epigenetic age accelerations between non-ART and ART parents. MAIN RESULTS AND THE ROLE OF CHANCE: We found a significant difference in the epigenetic age acceleration calculated by DunedinPoAm between IVF and non-ART mothers (0.021 years, P-value = 2.89E-06) after adjustment for potential confounders. Further, we detected elevated DunedinPoAm in mothers with tubal factor infertility (0.030 years, P-value = 1.34E-05), ovulation factor (0.023 years, P-value = 0.0018) and unexplained infertility (0.023 years, P-value = 1.39E-04) compared with non-ART mothers. No differences in epigenetic age accelerations between non-ART and ICSI fathers were found. DunedinPoAm also showed stronger associations with smoking, education and parity than the other four epigenetic age accelerations. LIMITATIONS, REASONS FOR CAUTION: We were not able to determine the directionality of the causal pathway between the epigenetic age accelerations and infertility. Since parents' peripheral blood samples were collected after conception, we cannot rule out the possibility that the epigenetic profile of ART mothers was influenced by the ART treatment. Hence, the results should be interpreted with caution, and our results might not be generalizable to non-pregnant women. WIDER IMPLICATIONS OF THE FINDINGS: A plausible biological mechanism behind the reported association is that IVF mothers could be closer to menopause than non-ART mothers. The pace of decline of the ovarian reserve that eventually leads to menopause varies between females yet, in general, accelerates after the age of 30, and some studies show an increased risk of infertility in females with low ovarian reserve. STUDY FUNDING/COMPETING INTEREST(S): This study was partly funded by the Research Council of Norway (Women's fertility, project no. 320656) and through its Centres of Excellence Funding Scheme (project no. 262700). M.C.M. has received funding from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme (grant agreement number 947684). The authors declare no conflict of interest. TRIAL REGISTRATION NUMBER: N/A.

Detecting differentially methylated regions using a fast wavelet-based approach to functional association analysis.

BACKGROUND: We present here a computational shortcut to improve a powerful wavelet-based method by Shim and Stephens (Ann Appl Stat 9(2):665-686, 2015. https://doi.org/10.1214/14-AOAS776 ) called WaveQTL that was originally designed to identify DNase I hypersensitivity quantitative trait loci (dsQTL). RESULTS: WaveQTL relies on permutations to evaluate the significance of an association. We applied a recent method by Zhou and Guan (J Am Stat Assoc 113(523):1362-1371, 2017. https://doi.org/10.1080/01621459.2017.1328361 ) to boost computational speed, which involves calculating the distribution of Bayes factors and estimating the significance of an association by simulations rather than permutations. We called this simulation-based approach "fast functional wavelet" (FFW), and tested it on a publicly available DNA methylation (DNAm) dataset on colorectal cancer. The simulations confirmed a substantial gain in computational speed compared to the permutation-based approach in WaveQTL. Furthermore, we show that FFW controls the type I error satisfactorily and has good power for detecting differentially methylated regions. CONCLUSIONS: Our approach has broad utility and can be applied to detect associations between different types of functions and phenotypes. As more and more DNAm datasets are being made available through public repositories, an attractive application of FFW would be to re-analyze these data and identify associations that might have been missed by previous efforts. The full R package for FFW is freely available at GitHub https://github.com/william-denault/ffw .

A genome-wide scan of cleft lip triads identifies parent-of-origin interaction effects between ANK3 and maternal smoking, and between ARHGEF10 and alcohol consumption.

Background: Although both genetic and environmental factors have been reported to influence the risk of isolated cleft lip with or without cleft palate (CL/P), the exact mechanisms behind CL/P are still largely unaccounted for. We recently developed new methods to identify parent-of-origin (PoO) interactions with environmental exposures (PoOxE) and applied them to families with children born with isolated cleft palate only. Here, we used the same genome-wide association study (GWAS) dataset and methodology to screen for PoOxE effects in the larger sample of CL/P triads. Methods: Genotypes from 1594 complete triads and 314 dyads (1908 nuclear families in total) with CL/P were available for the current analyses. Of these families, 1024 were Asian, 825 were European and 59 had other ancestries. After quality control, 341,191 SNPs remained from the original 569,244. The exposures were maternal cigarette smoking, use of alcohol, and use of vitamin supplements in the periconceptional period. The methodology applied in the analyses is implemented in the R-package Haplin. Results: Among Europeans, there was evidence of a PoOxSmoke effect for ANK3 with three SNPs (rs3793861, q=0.20, p=2.6e-6; rs7087489, q=0.20, p=3.1e-6; rs4310561, q=0.67, p=4.0e-5) and a PoOxAlcohol effect for ARHGEF10 with two SNPs (rs2294035, q=0.32, p=2.9e-6; rs4876274, q=0.76, p=1.3e-5). Conclusion: Our results indicate that the detected PoOxE effects have a plausible biological basis, and thus warrant replication in other independent cleft samples. Our demonstration of the feasibility of identifying complex interactions between relevant environmental exposures and PoO effects offers new avenues for future research aimed at unravelling  the complex etiology of cleft lip defects.

A Genome-Wide Search for Gene-Environment Effects in Isolated Cleft Lip with or without Cleft Palate Triads Points to an Interaction between Maternal Periconceptional Vitamin Use and Variants in ESRRG.

Background: It is widely accepted that cleft lip with or without cleft palate (CL/P) results from the complex interplay between multiple genetic and environmental factors. However, a robust investigation of these gene-environment (GxE) interactions at a genome-wide level is still lacking for isolated CL/P. Materials and Methods: We used our R-package Haplin to perform a genome-wide search for GxE effects in isolated CL/P. From a previously published GWAS, genotypes and information on maternal periconceptional cigarette smoking, alcohol intake, and vitamin use were available on 1908 isolated CL/P triads of predominantly European or Asian ancestry. A GxE effect is present if the relative risk estimates for gene-effects in the offspring are different across exposure strata. We tested this using the relative risk ratio (RRR). Besides analyzing all ethnicities combined ("pooled analysis"), separate analyses were conducted on Europeans and Asians to investigate ethnicity-specific effects. To control for multiple testing, q-values were calculated from the p-values. Results: We identified significant GxVitamin interactions with three SNPs in "Estrogen-related receptor gamma" (ESRRG) in the pooled analysis. The RRRs (95% confidence intervals) were 0.56 (0.45-0.69) with rs1339221 (q = 0.011), 0.57 (0.46-0.70) with rs11117745 (q = 0.011), and 0.62 (0.50-0.76) with rs2099557 (q = 0.037). The associations were stronger when these SNPs were analyzed as haplotypes composed of two-SNP and three-SNP combinations. The strongest effect was with the "t-t-t" haplotype of the rs1339221-rs11117745-rs2099557 combination [RRR = 0.50 (0.40-0.64)], suggesting that the effects observed with the other SNP combinations, including those in the single-SNP analyses, were mainly driven by this haplotype. Although there were potential GxVitamin effects with rs17734557 and rs1316471 and GxAlcohol effects with rs9653456 and rs921876 in the European sample, respectively, none of the SNPs was located in or near genes with strong links to orofacial clefts. GxAlcohol and GxSmoke effects were not assessed in the Asian sample because of a lack of observations for these exposures. Discussion/Conclusion: We identified significant interactions between vitamin use and variants in ESRRG in the pooled analysis. These GxE effects are novel and warrant further investigations to elucidate their roles in orofacial clefting. If validated, they could provide prospects for exploring the impact of estrogens and vitamins on clefting, with potential translational applications.

Parent-of-origin-environment interactions in case-parent triads with or without independent controls.

With case-parent triad data, one can frequently deduce parent of origin of the child's alleles. This allows a parent-of-origin (PoO) effect to be estimated as the ratio of relative risks associated with the alleles inherited from the mother and the father, respectively. A possible cause of PoO effects is DNA methylation, leading to genomic imprinting. Because environmental exposures may influence methylation patterns, gene-environment interaction studies should be extended to allow for interactions between PoO effects and environmental exposures (i.e., PoOxE). One should thus search for loci where the environmental exposure modifies the PoO effect. We have developed an extensive framework to analyze PoOxE effects in genome-wide association studies (GWAS), based on complete or incomplete case-parent triads with or without independent control triads. The interaction approach is based on analyzing triads in each exposure stratum using maximum likelihood estimation in a log-linear model. Interactions are then tested applying a Wald-based posttest of parameters across strata. Our framework includes a complete setup for power calculations. We have implemented the models in the R software package Haplin. To illustrate our PoOxE test, we applied the new methodology to top hits from our previous GWAS, assessing whether smoking during the periconceptional period modifies PoO effects on cleft palate only.

Genome-wide analysis of parent-of-origin interaction effects with environmental exposure (PoOxE): An application to European and Asian cleft palate trios.

Cleft palate only is a common birth defect with high heritability. Only a small fraction of this heritability is explained by the genetic variants identified so far, underscoring the need to investigate other disease mechanisms, such as gene-environment (GxE) interactions and parent-of-origin (PoO) effects. Furthermore, PoO effects may vary across exposure levels (PoOxE effects). Such variation is the focus of this study. We upgraded the R-package Haplin to enable direct tests of PoOxE effects at the genome-wide level. From a previous GWAS, we had genotypes for 550 case-parent trios, of mainly European and Asian ancestry, and data on three maternal exposures (smoking, alcohol, and vitamins). Data were analyzed for Europeans and Asians separately, and also for all ethnicities combined. To account for multiple testing, a false discovery rate method was used, where q-values were generated from the p-values. In the Europeans-only analyses, interactions with maternal smoking yielded the lowest q-values. Two SNPs in the 'Interactor of little elongation complex ELL subunit 1' (ICE1) gene had a q-value of 0.14, and five of the 20 most significant SNPs were in the 'N-acetylated alpha-linked acidic dipeptidase-like 2' (NAALADL2) gene. No evidence of PoOxE effects was found in the other analyses. The connections to ICE1 and NAALADL2 are novel and warrant further investigation. More generally, the new methodology presented here is easily applicable to other traits and exposures in which a family-based study design has been implemented.

Folic acid supplementation use and the MTHFR C677T polymorphism in orofacial clefts etiology: An individual participant data pooled-analysis.

BACKGROUND: This study examines gene-environment interaction between the MTHFR C667T polymorphism and folic acid in the etiology of orofacial clefts (OFC). We used a pooled-analytical approach on four studies that used similar methods. METHODS: We used logistic regression to analyze the pooled sample of 1149 isolated cases and 1161 controls. Fetal and maternal MTHFR C677T genotypes, and maternal periconceptional exposure to smoking, alcohol, vitamin containing folic acid and folic acid supplements were contrasted between the cleft types [non-syndromic clefts lip or without cleft palate (CL(P)) and non-syndromic cleft palate (CP)] and control groups. RESULTS: There was a reduced risk of CL(P) with maternal folic acid use (p = 0.008; OR = 0.70, 95% CI: 0.65-0.94) and with supplements containing folic acid (p = 0.028, OR = 0.80, 95% CI: 0.65-0.94). Maternal smoking increased the risk of both CL(P) (p < 10 e-3; OR = 1.62, 95% CI: 1.35-1.95) and CP (p = 0.028; OR = 1.38, 95% CI: 1.04-1.83). No significant risk was observed with either maternal or fetal MTHFR C677T genotypes. CONCLUSION: This individual participant data (IPD) meta-analysis affords greater statistical power and can help alleviate the problems associated with aggregate-level data-sharing. The result of this IPD meta-analysis is consistent with previous reports suggesting that folic acid and smoking influence OFC outcomes.

Application of a novel hybrid study design to explore gene-environment interactions in orofacial clefts.

Orofacial clefts are common birth defects with strong evidence for both genetic and environmental causal factors. Candidate gene studies combined with exposures known to influence the outcome provide a highly targeted approach to detecting GxE interactions. We developed a new statistical approach that combines the case-control and offspring-parent triad designs into a "hybrid design" to search for GxE interactions among 334 autosomal cleft candidate genes and maternal first-trimester exposure to smoking, alcohol, coffee, folic acid supplements, dietary folate and vitamin A. The study population comprised 425 case-parent triads of isolated clefts and 562 control-parent triads derived from a nationwide study of orofacial clefts in Norway (1996-2001). A full maximum-likelihood model was used in combination with a Wald test statistic to screen for statistically significant GxE interaction between strata of exposed and unexposed mothers. In addition, we performed pathway-based analyses on 28 detoxification genes and 21 genes involved in folic acid metabolism. With the possible exception of the T-box 4 gene (TBX4) and dietary folate interaction in isolated CPO, there was little evidence overall of GxE interaction in our data. This study is the largest to date aimed at detecting interactions between orofacial clefts candidate genes and well-established risk exposures.

Assessing the impact of nicotine dependence genes on the risk of facial clefts: An example of the use of national registry and biobank data.

BACKGROUND: Maternal smoking during pregnancy has been associated with risk of facial clefts in offspring, but causation has not yet been established. It is possible that the effect of maternal smoking on facial clefts is mediated through genes that are involved in nicotine dependence. Gamma-aminobutyric acid B receptor 2 (GABBR2), dopa decarboxylase (DDC), and cholinergic receptor nicotinic alpha 4 (CHRNA4) are three examples of genes that have previously shown strong associations with nicotine dependence. METHODS: We used a population-based sample of 377 case-parent trios of cleft lip with or without cleft palate (CL/P) and 762 control-parent trios from Norway (1996-2001) to investigate whether variants in GABBR2, DDC and CHRNA4 are associated with maternal first-trimester smoking and with clefting risk. We used HAPLIN (Gjessing et al. 2006), a statistical software tailored for family-based association tests, to perform haplotype-based analyses on 12 SNPs in these genes (rs10985765, rs1435252, rs3780422, rs2779562, and rs3750344 in GABBR2; rs2060762, rs3757472, rs1451371, rs3735273, and rs921451 in DDC; rs4522666 and rs1044393 in CHRNA4). RESULTS: When analyzed one at a time, there was little evidence of association between any of the 12 SNPs and maternal first-trimester smoking. In haplotype analyses, however, one copy of the maternal G-G-c-G-c haplotype in DDC was linked with smoking prevalence (odds ratio: 1.5; 95% confidence interval: 1.0-2.1). This same haplotype also increased the risk of isolated CL/P in offspring by 1.5-fold with one copy and 2.4-fold with two copies (Ptrend = 0.06). No statistically significant associations were detected with GABBR2 and CHRNA4. CONCLUSIONS: Despite strong associations previously reported between nicotine dependence and variants in GABBR2, DDC and CHRNA4, these genes were poor predictors of maternal first-trimester smoking in our data. The direct association of the DDC haplotype with CL/P suggests that this haplotype may either have direct effects on clefts or it may influence clefting risks through other yet unexplored risk behavior(s).

GENES AS INSTRUMENTS FOR STUDYING RISK BEHAVIOR EFFECTS: AN APPLICATION TO MATERNAL SMOKING AND OROFACIAL CLEFTS.

This study uses instrumental variable (IV) models with genetic instruments to assess the effects of maternal smoking on the child's risk of orofacial clefts (OFC), a common birth defect. The study uses genotypic variants in neurotransmitter and detoxification genes relateded to smoking as instruments for cigarette smoking before and during pregnancy. Conditional maximum likelihood and two-stage IV probit models are used to estimate the IV model. The data are from a population-level sample of affected and unaffected children in Norway. The selected genetic instruments generally fit the IV assumptions but may be considered "weak" in predicting cigarette smoking. We find that smoking before and during pregnancy increases OFC risk substantially under the IV model (by about 4-5 times at the sample average smoking rate). This effect is greater than that found with classical analytic models. This may be because the usual models are not able to consider self-selection into smoking based on unobserved confounders, or it may to some degree reflect limitations of the instruments. Inference based on weak-instrument robust confidence bounds is consistent with standard inference. Genetic instruments may provide a valuable approach to estimate the "causal" effects of risk behaviors with genetic-predisposing factors (such as smoking) on health and socioeconomic outcomes.

Complete mutation screening and haplotype characterization of the BRCA1 gene in 61 familial breast cancer patients from Norway.

Mutations in the Breast-Cancer-1 (BRCA1) gene are the major cause of familial breast/ovarian cancer. Among familial breast cancer only, 15-20% have been suggested to have a deleterious mutation in BRCA1. A highly sensitive method (REF-SSCP) was applied to screen the open reading frame and the 5'UTRs of BRCA1 for mutations. The patient cohort comprised 61 unrelated moderate to high risk breast cancer patients from Western-Norway. Only one known deleterious BRCA1 mutation (c.816-817delGT) was found in two of the 61 patients (3.3%). Four haplotypes were established based on nine known single nucleotide polymorphisms. Two patients had a novel deletion (c.-33_-29delAAAAA) in the 5'UTR, and a novel amino acid substitution (L523W) was found in one patient. Size variations analysis in the 5'UTR was repeated in a cohort of 159 unrelated familial breast/ovarian cancer patients and 94 healthy blood donors. Two patients were identified with 5'UTR (c.-30 to -60) variations (CAAAA)5 and (CAAAA)7, instead of the (CAAAA)6-repeat. All of the identified 5'UTR size variations were localized between the start codon and the most stable secondary structures previously proposed for the exon 1b transcript. No such alterations were found among the healthy blood donors but association studies of the 5'UTR variations within the respective families were not conclusive.

Cleft palate, transforming growth factor alpha gene variants, and maternal exposures: assessing gene-environment interactions in case-parent triads.

We have previously reported a threefold risk of cleft palate only (CPO) among children homozygous for the less common allele A2 at the TaqI marker site of the transforming growth factor alpha gene (TGFA) (Jugessur et al. [2003a] Genet. Epidemiol. 24:230-239). Here we assess possible interaction between the child's TGFA TaqI A2A2 genotype and maternal cigarette smoking, alcohol consumption, use of multivitamins and folic acid. This was done by comparing the strength of genetic associations between strata of exposed and unexposed case-parent triads. We also looked for possible gene-gene interaction with the polymorphic variant C677T of the folic acid-metabolizing gene MTHFR. We analyzed a total of 88 complete CPO triads selected from a population-based study of orofacial clefts in Norway (May 1996-1998). No evidence of interaction was observed with either smoking or alcohol use. The risk associated with two copies of the A2 allele at TGFA TaqI was strong among children whose mothers did not use folic acid (relative risk=4.5, 95% confidence interval=1.3-15.7), and was only marginal among children whose mothers reported using folic acid (RR=1.4, 95% CI=0.2-12.7). Although the interaction between the child's genotypes at TGFA TaqI and MTHFR-C677T was not statistically significant, the effect of the TGFA TaqI A2A2 genotype appeared to be stronger among children with either one or two copies of the T-allele at C677T (RR=4.0, 95% CI=1.1-13.9) compared to children homozygous for the C-allele (RR=1.7, 95% CI=0.2-15.7). In conclusion, we find little evidence of interaction between the child's genotypes at TGFA TaqI and various exposures for cleft palate, with the possible exception of folic acid intake.

Exploring the effects of methylenetetrahydrofolate reductase gene variants C677T and A1298C on the risk of orofacial clefts in 261 Norwegian case-parent triads.

Folic acid and the methylenetetrahydrofolate reductase (MTHFR) gene have both been implicated in the etiology of orofacial clefts. The authors selected 261 case-parent triads (173 cases with cleft lip with or without cleft palate (CL/P) and 88 cases with cleft palate only (CPO)) from a Norwegian population-based study of orofacial clefts (May 1996-1998). A case-parent triad design was used to examine whether MTHFR variants C677T and A1298C, and their haplotypes, are risk factors for orofacial clefts. Among CL/P cases, the child's genotype at C677T or A1298C did not influence the risk. However, children of mothers carrying the C677T variant allele had a lower risk of CL/P. For CPO, children carrying the C677T variant allele had about a twofold increased risk, whereas the mother's genotypes did not contribute to the risk. The haplotype-based transmission/disequilibrium test showed that except for 677T/1298A (p = 0.06), none of the other haplotypes showed evidence of excess transmission to the offspring. The authors also explored interaction of C677T with maternal use of folic acid among children with CPO. Surprisingly, the risk associated with the child's carrying either CT or TT was higher (fourfold) when the mother used folic acid. These findings suggest a possible role of MTHFR and folic acid in the causation of orofacial clefts, but the strength and direction of these effects remain to be clarified.