Anorectal atresia is a serious birth defect of largely unknown etiology but candidate genes have been identified in animal studies and human syndromes. Because alterations in the activity of these genes might lead to anorectal atresia, we selected 71 common variants predicted to be in transcription factor binding sites, CpG windows, splice sites, and miRNA target sites of 25 candidate genes, and tested for their association with anorectal atresia. The study population comprised 150 anorectal atresia cases and 623 control infants without major malformations. Variants predicted to affect transcription factor binding, splicing, and DNA methylation in WNT3A, PCSK5, TCF4, MKKS, GLI2, HOXD12, and BMP4 were associated with anorectal atresia based on a nominal P value <0.05. The GLI2 and BMP4 variants are reported to be moderately associated with gene expression changes (Spearman’s rank correlation coefficients between −0.260 and 0.226). We did not find evidence for interaction between maternal pre-pregnancy obesity and variants in MKKS, a gene previously associated with obesity, on the risk of anorectal atresia. Our results for MKKS support previously suggested associations with anorectal malformations. Our findings suggest that more research is needed to determine whether altered GLI2 and BMP4 expression is important in anorectal atresia in humans.
anorectal malformations; imperforate anus; hindgut; congenital abnormalities
Association between the C677T polymorphism of the methylene tetrahydrofolate reductase (MTHFR) gene and congenital heart disease (CHD) is contentious.
Methods and Results
We compared genotypes between CHD cases and controls, and between mothers of CHD cases and controls. We placed our results in context by conducting metaanalyses of previously published studies. Among 5,814 cases with primary genotype data and 10,056 controls, there was no evidence of association between MTHFR C677T genotype and CHD risk (OR 0.96 [95% CI 0.87-1.07]). A random-effects meta-analysis of all studies (involving 7,697 cases and 13,125 controls) suggested the presence of association (OR 1.25 [95% CI 1.03-1.51]; p=0.022), but with substantial heterogeneity among contributing studies (I2=64.4%), and evidence of publication bias. Meta-analysis of large studies only (defined by a variance of the log OR less than 0.05), which together contributed 83% of all cases, yielded no evidence of association (OR 0.97 [95% CI 0.91-1.03]), without significant heterogeneity (I2=0). Moreover, meta-analysis of 1,781 mothers of CHD cases (829 of whom were genotyped in this study) and 19,861 controls revealed no evidence of association between maternal C677T genotype and risk of CHD in offspring (OR 1.13 [95% CI 0.87-1.47]). There was no significant association between MTHFR genotype and CHD risk in large studies from regions with different levels of dietary folate.
The MTHFR C677T polymorphism, which directly influences plasma folate levels, is not associated with CHD risk. Publication biases appear to substantially contaminate the literature with regard to this genetic association.
congenital heart disease; MTHFR; genetic association; folate; Mendelian randomization
We conducted a population-based case-control study of single nucleotide polymorphisms (SNPs) in selected genes to find common variants that play a role in the etiology of limb deficiencies (LD)s. Included in the study were 389 infants with LDs of unknown cause and 980 unaffected controls selected from all births in New York State (NYS) for the years 1998 to 2005. We used cases identified from the NYS Department of Health (DOH) Congenital Malformations Registry. Genotypes were obtained for 132 SNPs in genes involved in limb development (SHH, WNT7A, FGF4, FGF8, FGF10, TBX3, TBX5, SALL4, GREM1, GDF5, CTNNB1, EN1, CYP26A1, CYP26B1), angiogenesis (VEGFA, HIF1A, NOS3), and coagulation (F2, F5, MTHFR). Genotype call rates were >97% and SNPs were tested for departure from Hardy-Weinberg expectations by race/ethnic subgroups. For each SNP, odds ratios (OR)s and confidence intervals (CI)s were estimated and corrected for multiple comparisons for all LDs combined and for LD subtypes. Among non-Hispanic white infants, associations between FGF10 SNPs rs10805683 and rs13170645 and all LDs combined were statistically significant following correction for multiple testing (OR=1.99; 95% CI=1.43-2.77; uncorrected p=0.000043 for rs10805683 heterozygous genotype, and OR=2.37; 95% CI=1.48-3.78; uncorrected p=0.00032 for rs13170645 homozygous minor genotype). We also observed suggestive evidence for associations with SNPs in other genes including CYP26B1 and WNT7A. Animal studies have shown that FGF10 induces formation of the apical ectodermal ridge and is necessary for limb development. Our data suggest that common variants in FGF10 increase the risk for a wide range of non-syndromic limb deficiencies.
limb deficiencies; polymorphisms; FGF10
Triad families are routinely used to test association between genetic variants and complex diseases. Triad studies are important and popular since they are robust in terms of being less prone to false positives due to population structure. In practice, one may collect not only complete triads, but also incomplete families such as dyads (affected child with one parent) and singleton monads (affected child without parents). Since there is a lack of convenient algorithms and software to analyze the incomplete data, dyads and monads are usually discarded. This may lead to loss of power and insufficient utilization of genetic information in a study.
We develop likelihood-based statistical models and likelihood ratio tests to test for association between complex diseases and genetic markers by using combinations of full triads, parent-child dyads, and affected singleton monads for a unified analysis. A likelihood is calculated directly to facilitate the data analysis without imputation and to avoid computational complexity. This makes it easy to implement the models and to explain the results.
By simulation studies, we show that the proposed models and tests are very robust in terms of accurately controlling type I error evaluations, and are powerful by empirical power evaluations. The methods are applied to test for association between transforming growth factor alpha (TGFA) gene and cleft palate in an Irish study.
Association mapping of complex diseases; Likelihood ratio tests; Transmission disequilibrium tests
We previously suggested links between specific XPD mutations in the fetal genome and the risk of placental maldevelopment and preeclampsia, possibly due to impairment of Transcription Factor (TF)IIH-mediated functions in placenta. To identify the underlying mechanisms, we conducted the current integrative analysis of several relevant transcriptome data sources. Our meta-analysis revealed downregulation of TFIIH subunits in preeclamptic placentas. Our overall integrative analysis suggested that, in the presence of hypoxia and oxidative stress, EGFR signaling deficiency, which can be caused by TFIIH impairment as well as by other mechanisms, results in ATF3 upregulation, inducing mediators of clinical symptoms of preeclampsia such as FLT1 and ENG. EGFR- and ATF3-dependent pathways play prominent roles in cancer development. We propose that dysregulation of these canonical cancer molecular pathways occurs in preeclampsia and delineate the relevance of TFIIH, providing etiologic clues which could eventually translate into a therapeutic approach.
Hirschsprung’s disease (HSCR) results from failed colonization of the embryonic gut by enteric neural crest cells (ENCCs); colonization requires RET proto-oncogene (RET) signaling. We sequenced RET to identify coding and splice-site variants in a population-based case group and we tested for associations between HSCR and common variants in RET and candidate genes (ASCL1, HOXB5, L1CAM, PHOX2B, PROK1, PROKR1) chosen because they are involved in ENCC proliferation, migration, and differentiation in animal models. We conducted a nested case-control study of 304 HSCR cases and 1 215 controls. Among 38 (12.5%) cases with 34 RET coding and splice-site variants, 18 variants were previously unreported. We confirmed associations with common variants in HOXB5 and PHOX2B but the associations with variants in ASCL1, L1CAM, and PROK1 were not significant after multiple comparisons adjustment. RET variants were strongly associated with HSCR (P values between 10−3 and 10−31) but this differed by race/ethnicity: associations were absent in African-Americans. Our population-based study not only identified novel RET variants in HSCR cases, it showed that common RET variants may not contribute to HSCR in all race/ethnic groups. The findings for HOXB5 and PHOX2B provide supportive evidence that genes regulating ENCC proliferation, migration, and differentiation could be risk factors for HSCR.
congenital abnormalities; enteric nervous system; Hirschsprung disease; RET
Growing evidence of cell-to-cell transmission of neurodegenerative disease (ND)–associated proteins (NDAPs) (ie, tau, Aβ, and α-synuclein) suggests possible similarities in the infectious prion protein (PrPsc) in spongiform encephalopathies. There are limited data on the potential human-to-human transmission of NDAPs associated with Alzheimer disease (AD) and other non-PrPsc ND.
To examine evidence for human-to-human transmission of AD, Parkinson disease (PD), and related NDAPs in cadaveric human growth hormone (c-hGH) recipients.
We conducted a detailed immunohistochemical analysis of pathological NDAPs other than PrPsc in human pituitary glands. We also searched for ND in recipients of pituitary-derived c-hGH by reviewing the National Hormone and Pituitary Program (NHPP) cohort database and medical literature.
University-based academic center and agencies of the US Department of Health and Human Services.
Thirty-four routine autopsy subjects (10 non-ND controls and 24 patients with ND) and a US cohort of c-hGH recipients in the NHPP.
Main Outcome Measures
Detectable NDAPs in human pituitary sections and death certificate reports of non-PrPsc ND in the NHPP database.
We found mild amounts of pathological tau, Aβ, and α-synuclein deposits in the adeno/neurohypophysis of patients with ND and control patients. No cases of AD or PD were identified, and 3 deaths attributed to amyotrophic lateral sclerosis (ALS) were found among US NHPP c-hGH recipients, including 2 of the 796 decedents in the originally confirmed NHPP c-hGH cohort database.
Conclusions and Relevance
Despite the likely frequent exposure of c-hGH recipients to NDAPs, and their markedly elevated risk of PrPsc-related disease, this population of NHPP c-hGH recipients does not appear to be at increased risk of AD or PD. We discovered 3 ALS cases of unclear significance among US c-hGH recipients despite the absence of pathological deposits of ALS-associated proteins (TDP-43, FUS, and ubiquilin) in human pituitary glands. In this unique in vivo model of human-to-human transmission, we found no evidence to support concerns that NDAPs underlying AD and PD transmit disease in humans despite evidence of their cell-to-cell transmission in model systems of these disorders. Further monitoring is required to confirm these conclusions.
Mutations in XPD (ERCC2), XPB (ERCC3), and TTD-A (GTF2H5), genes involved in nucleotide excision repair and transcription, can cause several disorders including trichothiodystrophy (TTD) and xeroderma pigmentosum (XP). In this study, we tested the hypothesis that mutations in the XPD gene affect placental development in a phenotype-specific manner. To test our hypothesis and decipher potential biologic mechanisms, we compared all XPD-associated TTD (n=43) and XP (n=37) cases reported in the literature with respect to frequencies of gestational complications. Our genetic epidemiologic investigations of TTD and XP revealed that the exact genetic abnormality was relevant to the mechanism leading to gestational complications such as preeclampsia. Through structural mapping, we localized the preeclampsia-associated mutations to a C-terminal motif and the helicase surfaces of XPD, most likely affecting XPD's binding to cdk-activating kinase (CAK) and p44 subunits of transcription factor (TF) IIH. Our results suggested a link between TTD- but not XP-associated XPD mutations, placental maldevelopment and risk of pregnancy complications, possibly due to impairment of TFIIH-mediated functions in placenta. Our findings highlight the importance of the fetal genotype in development of gestational complications, such as preeclampsia. Therefore, future studies of genetic associations of preeclampsia and other placental vascular complications may benefit from focusing on genetic variants within the fetal DNA.
trichothiodystrophy; xeroderma pigmentosum; XPD; TFIIH; human fetal development
Many neural tube defects can be prevented if women take folic acid around the time of conception. However, the majority of women do not take folic acid at the critical time, so the US government required that food be fortified with folic acid effective January 1, 1998. Whether the amount being added was sufficient to prevent all folate-related neural tube defects has been hotly debated. Mosley et al. (Am J Epidemiol. 2008;169(1):9–17) found no evidence that folic acid supplement use or dietary folate intake was related to neural tube defects, indicating that fortified food is probably providing sufficient folic acid to prevent folate-related defects. Because data on the effectiveness of fortification in the United States are scarce, this is an important contribution. There is great interest in the other effects of fortification. Folic acid reduces homocysteine levels, and homocysteine has been linked to cardiovascular disease and cancer. On the basis of current evidence, however, it seems unlikely that fortification will reduce cardiovascular disease rates. Its effect on cancer remains unclear. Folic acid may be useful in primary prevention but may also stimulate the growth of existing malignancies or premalignant lesions. Although these issues remain unresolved, Mosley et al. have provided important data to address the primary question: Does fortification prevent folate-related neural tube defects?
anencephaly; folic acid; food, fortified; neural tube defects; spinal dysraphism
Individual studies of the genetics of neural tube defects (NTDs) contain results on a small number of genes in each report. To identify genetic risk factors for NTDs, we evaluated potentially functional single nucleotide polymorphisms (SNPs) that are biologically plausible risk factors for NTDs but that have never been investigated for an association with NTDs, examined SNPs that previously showed no association with NTDs in published studies, and tried to confirm previously reported associations in folate-related and non-folate-related genes. We investigated 64 SNPs in 34 genes for association with spina bifida in up to 558 case-families (520 cases, 507 mothers, 457 fathers) and 994 controls in Ireland. Case-control and mother-control comparisons of genotype frequencies, tests of transmission disequilibrium, and log-linear regression models were used to calculate effect estimates. Spina bifida was associated with over-transmission of the LEPR (leptin receptor) rs1805134 minor C allele (genotype relative risk (GRR): 1.5; 95% confidence interval (CI): 1.0, 2.1; P = 0.0264) and the COMT (catechol-O-methyltransferase) rs737865 major T allele (GRR: 1.4; 95% CI: 1.1, 2.0; P = 0.0206). After correcting for multiple comparisons, these individual test P-values exceeded 0.05. Consistent with previous reports, spina bifida was associated with MTHFR 677C>T, T (Brachyury) rs3127334, LEPR K109R, and PDGFRA promoter haplotype combinations. The associations between LEPR SNPs and spina bifida suggest a possible mechanism for the finding that obesity is a NTD risk factor. The association between a variant in COMT and spina bifida implicates methylation and epigenetics in NTDs.
congenital abnormalities; folic acid; neural tube defects; single nucleotide polymorphism; spina bifida
Suggestive, but not conclusive, studies implicate many genetic variants in oral cleft etiology. We used a large, ethnically homogenous study population to test whether reported associations between nonsyndromic oral clefts and 12 genes (CLPTM1, CRISPLD2, FGFR2, GABRB3, GLI2, IRF6, PTCH1, RARA, RYK, SATB2, SUMO1, TGFA) could be confirmed.
Thirty-one single nucleotide polymorphisms (SNPs) in exons, splice sites, and conserved non-coding regions were studied in 509 patients with cleft lip with or without cleft palate (CLP), 383 with cleft palate only (CP), 838 mothers and 719 fathers of patients with oral clefts, and 902 controls from Ireland. Case-control and family-based statistical tests were performed using isolated oral clefts for the main analyses.
In case-control comparisons, the minor allele of PTCH1 A562A (rs2066836) was associated with reduced odds of CLP (OR: 0.29, 95% CI: 0.13–0.64 for homozygotes) whereas the minor allele of PTCH1 L1315P (rs357564) was associated with increased odds of CLP (OR: 1.36, 95% CI: 1.07–1.74 for heterozygotes and OR: 1.56, 95% CI: 1.09–2.24 for homozygotes). The minor allele of one SUMO1 SNP, rs3769817 located in intron 2, was associated with increased odds of CP (OR: 1.45, 95% CI: 1.06–1.99 for heterozygotes). Transmission disequilibrium was observed for the minor allele of TGFA V159V (rs2166975) which was over-transmitted to CP cases (P=0.041).
For 10 of the 12 genes, this is the largest candidate gene study of nonsyndromic oral clefts to date. The findings provide further evidence that PTCH1, SUMO1, and TGFA contribute to nonsyndromic oral clefts.
cleft lip; cleft palate; congenital abnormalities
Neural tube defects (NTDs) are common birth defects (~1 in 1000 pregnancies in the US and Europe) that have complex origins, including environmental and genetic factors. A low level of maternal folate is one well-established risk factor, with maternal periconceptional folic acid supplementation reducing the occurrence of NTD pregnancies by 50-70%. Gene variants in the folate metabolic pathway (e.g., MTHFR rs1801133 (677 C > T) and MTHFD1 rs2236225 (R653Q)) have been found to increase NTD risk. We hypothesized that variants in additional folate/B12 pathway genes contribute to NTD risk.
A tagSNP approach was used to screen common variation in 82 candidate genes selected from the folate/B12 pathway and NTD mouse models. We initially genotyped polymorphisms in 320 Irish triads (NTD cases and their parents), including 301 cases and 341 Irish controls to perform case–control and family based association tests. Significantly associated polymorphisms were genotyped in a secondary set of 250 families that included 229 cases and 658 controls. The combined results for 1441 SNPs were used in a joint analysis to test for case and maternal effects.
Nearly 70 SNPs in 30 genes were found to be associated with NTDs at the p < 0.01 level. The ten strongest association signals (p-value range: 0.0003–0.0023) were found in nine genes (MFTC, CDKN2A, ADA, PEMT, CUBN, GART, DNMT3A, MTHFD1 and T (Brachyury)) and included the known NTD risk factor MTHFD1 R653Q (rs2236225). The single strongest signal was observed in a new candidate, MFTC rs17803441 (OR = 1.61 [1.23-2.08], p = 0.0003 for the minor allele). Though nominally significant, these associations did not remain significant after correction for multiple hypothesis testing.
To our knowledge, with respect to sample size and scope of evaluation of candidate polymorphisms, this is the largest NTD genetic association study reported to date. The scale of the study and the stringency of correction are likely to have contributed to real associations failing to survive correction. We have produced a ranked list of variants with the strongest association signals. Variants in the highest rank of associations are likely to include true associations and should be high priority candidates for further study of NTD risk.
Neural tube defects; Spina bifida; Folic acid; One-carbon metabolism; Candidate gene
Both taking folic acid-containing vitamins around conception and consuming food fortified with folic acid have been reported to reduce omphalocele rates. Genetic factors are etiologically important in omphalocele as well; our pilot study showed a relationship with the folate metabolic enzyme gene methylenetetrahydrofolate reductase (MTHFR). We studied 169 non-aneuploid omphalocele cases and 761 unaffected, matched controls from all New York State births occurring between 1998 and 2005 to look for associations with single nucleotide polymorphisms (SNPs) known to be important in folate, vitamin B12, or choline metabolism. In the total study population, variants in the transcobalamin receptor gene (TCblR), rs2232775 (Q8R), and the MTHFR gene, rs1801131 (1298A>C), were significantly associated with omphalocele. In African-Americans significant associations were found with SNPs in genes for the vitamin B12 transporter (TCN2) and the vitamin B12 receptor (TCblR). A SNP in the homocysteine-related gene, betaine-homocysteine S-methyltransferase (BHMT), rs3733890 (R239Q), was significantly associated with omphalocele in both African-Americans and Asians. Only the TCblR association in the total population remained statistically significant if Bonferroni correction was applied. The finding that transcobalamin receptor (TCblR) and transporter (TCN2) SNPs and a BHMT SNP were associated with omphalocele suggests that disruption of methylation reactions, in which folate, vitamin B12, and homocysteine play critical parts, may be a risk factor for omphalocele. Our data, if confirmed, suggest that supplements containing both folic acid and vitamin B12 may be beneficial in preventing omphaloceles.
omphalocele; folate; vitamin B12; homocysteine; transcobalamin; transcobalamin receptor
Although methylenetetrahydrofolate reductase, a folate enzyme gene, has been associated with idiopathic male infertility, few studies have examined other folate-related metabolites and genes. We investigated whether idiopathic male infertility is associated with variants in folate, vitamin B12 (B12) and total homocysteine (tHcy)-related genes and measured these metabolites in blood. We conducted a case–control study that included 153 men with idiopathic infertility and 184 fertile male controls recruited at the Fertility Center and Antenatal Care Center, University Hospital, Malmö and Lund, Sweden. Serum folate, red cell folate (RCF), serum B12, plasma tHcy and semen quality were measured. Subjects were genotyped for 20 common variants in 12 genes related to folate/B12/homocysteine metabolism. Metabolite concentrations and genotype distributions were compared between cases and controls using linear and logistic regression with adjustment for covariates. The phosphatidylethanolamine N-methyltransferase (PEMT) M175V and TCblR rs173665 polymorphisms were significantly associated with infertility (P = 0.01 and P = 0.009, respectively), but not with semen quality. Among non-users of supplements, infertile men had lower serum folate concentrations than fertile men (12.89 vs. 14.73 nmol l−1; P = 0.02), but there were no significant differences in RCF, B12 or tHcy. Folate, B12 and tHcy concentrations were not correlated with any semen parameters. This study provides little support for low folate or B12 status in the pathogenesis of idiopathic male infertility. Although additional data are needed to confirm these initial findings, our results suggest that PEMT and TCblR, genes involved in choline and B12 metabolism, merit further investigation in idiopathic male infertility.
folate; idiopathic male infertility; semen quality; vitamin B12
Periconceptional use of folic acid prevents most neural tube defects (NTDs). Whether folic acid and/or multivitamins can prevent other congenital anomalies is not clear. This study tested whether maternal blood levels of folate and vitamin B12 in pregnancies affected by congenital malformations excluding NTDs are lower when compared to non-affected pregnancies.
We measured pregnancy red cell folate (RCF), vitamin B12, and homocysteine (tHcy) concentrations in blood samples taken at the first antenatal clinic in Dublin maternity hospitals in 1986–1990 when vitamin supplementation was rare. The cases were mothers who delivered a baby with a congenital malformation other than NTD identified by the Dublin EUROCAT Registry; controls were a systematic sample of mothers of offspring without congenital malformations from the same hospitals in the same time period.
The median maternal levels of RCF and tHcy did not differ significantly between cases and controls for any of the congenital malformation groups examined (RCF: all malformations 275.9 ug/L v controls 271.2; p=0.77; tHcy: all malformations 7.5 umol/L v controls 7.6; p=0.57). In an unadjusted analysis vitamin B12 was significantly higher in case-mothers whose babies had cleft palate only (p=0.006), musculoskeletal malformations (p=0.034) and midline defects (p=0.039) but not after adjustment for multiple testing.
Our data suggest that low maternal folate and B12 levels or high tHcy levels in early pregnancy are not associated with all congenital malformations excluding NTDs. Fortification with folic acid or B12 may not have a beneficial effect in the prevention of these anomalies.
folic acid; folate; NHANES; folic acid fortification
New technology for large-scale genotyping has created new challenges for statistical analysis. Correcting for multiple comparison without discarding true positive results and extending methods to triad studies are among the important problems facing statisticians. We present a one-sample permutation test for testing transmission disequilibrium hypotheses in triad studies, and show how this test can be used for multiple single nucleotide polymorphism (SNP) testing. The resulting multiple comparison procedure is shown in the case of the transmission disequilibrium test to control the familywise error. Furthermore, this procedure can handle multiple possible modes of risk inheritance per SNP. The resulting permutational procedure is shown through simulation of SNP data to be more powerful than the Bonferroni procedure when the SNPs are in linkage disequilibrium. Moreover, permutations implicitly avoid any multiple comparison correction penalties when the SNP has a rare allele. The method is illustrated by analyzing a large candidate gene study of neural tube defects and an independent study of oral clefts, where the smallest adjusted p-values using the permutation procedure are approximately half those of the Bonferroni procedure. We conclude that permutation tests are more powerful for identifying disease-associated SNPs in candidate gene studies and are useful for analysis of triad studies.
Exchangeable; familywise error rate; linkage disequilibrium; power
To study the effect of in-utero alcohol exposure on the insulin-like growth factor axis (IGF) and leptin during infancy and childhood, considering that exposed children may exhibit pre- and postnatal growth retardation.
We prospectively identified heavily drinking pregnant women who consumed on average 4 or more drinks of ethanol per day (≥48 g/day) and assessed growth in 69 of their offspring and an unexposed control group of 83 children, measuring serum IGF-I (radioimmunoassay), IGF-II (immunoradiometric assay, IRMA), insulin-like growth factor-binding protein 3 (IGFBP-3) (IRMA) and leptin (IRMA) at 1 month and 1, 2, 3, 4, and 5 years of age.
IGF-II levels increased with age in both groups, but the rate of increase was significantly higher in exposed children, and levels were significantly higher in ethanol-exposed children at 3, 4, and 5 years of age. In exposed children, IGF-I levels were higher at 3 and 4 years and leptin levels were significantly lower at 1 and 2 years. Exposed subjects showed a much lower correlation between IGF-I and growth parameters than unexposed subjects.
Exposure to ethanol during pregnancy increases IGF-I and IGF-II and decreases leptin during early childhood. The increase in serum IGF-II concentrations in ethanol-exposed children suggests that this hormone should be explored as a potential marker for prenatal alcohol exposure.
Fetal alcohol syndrome; Pregnancy; Alcohol abuse; Insulin-like growth factor I; Insulin-like growth factor II
Although methylenetetrahydrofolate reductase, a folate enzyme gene, has been associated with idiopathic male infertility, few studies have examined other folate-related metabolites and genes. We investigated whether idiopathic male infertility is associated with variants in folate, vitamin B12 (B12) and total homocysteine (tHcy)-related genes and measured these metabolites in blood. We conducted a case–control study that included 153 men with idiopathic infertility and 184 fertile male controls recruited at the Fertility Center and Antenatal Care Center, University Hospital, Malmö and Lund, Sweden. Serum folate, red cell folate (RCF), serum B12, plasma tHcy and semen quality were measured. Subjects were genotyped for 20 common variants in 12 genes related to folate/B12/homocysteine metabolism. Metabolite concentrations and genotype distributions were compared between cases and controls using linear and logistic regression with adjustment for covariates. The phosphatidylethanolamine N-methyltransferase (PEMT) M175V and TCblR rs173665 polymorphisms were significantly associated with infertility (P=0.01 and P=0.009, respectively), but not with semen quality. Among non-users of supplements, infertile men had lower serum folate concentrations than fertile men (12.89 vs. 14.73 nmol l−1; P=0.02), but there were no significant differences in RCF, B12 or tHcy. Folate, B12 and tHcy concentrations were not correlated with any semen parameters. This study provides little support for low folate or B12 status in the pathogenesis of idiopathic male infertility. Although additional data are needed to confirm these initial findings, our results suggest that PEMT and TCblR, genes involved in choline and B12 metabolism, merit further investigation in idiopathic male infertility.
folate; idiopathic male infertility; semen quality; vitamin B12
Children with autism spectrum disorder (ASD) are reported to have decreased bone cortical thickness (BCT). Vitamin D plays an important physiological role in bone growth and development, so deficiency of vitamin D could contribute to decreased BCT. The goal of this study was to compare plasma 25(OH)D concentration in three groups of Caucasian males age 4 to 8 years old: (1) ASD and an unrestricted diet (n=40), (2) ASD and a casein-free diet (n=9), and (3) unaffected controls (n=40). No significant group differences were observed (p=0.4). However, a total of 54 (61%) of the children in the entire cohort had a plasma 25(OH)D concentration of less than 20ng/mL, similar to findings of low 25(OH)D concentrations in population-based studies. Children with ASD should be monitored for vitamin D deficiency.
Emerging hypotheses suggest a causal role for prenatal androgen exposure in some cases of autism spectrum disorders (ASD). The ratios of the lengths of the bones of the 2nd to the 4th digit (2D:4D) are purported to be markers for prenatal androgen exposure and to be established early in gestation. Elongation of the 4th digit in response to testosterone is said to reduce 2D:4D in males versus females. We examined the ratios of bones from the left hand radiographs of 75 boys and 6 girls 4–8 years of age, diagnosed with ASD, to evaluate digit ratio as a marker for gestational androgen exposure. Contrary to our expectations, girls had reduced 2D:4D compared to boys but the difference was not significant (Cohen’s D 0.51–0.66, P>0.05). The limited sample size for this study and the absence of a referent group precluded providing robust estimates for girls and identifying possible statistical differences between the sexes. Tanner-Whitehouse 3 (TW3) rating of finger bone growth suggested relative immaturity of the 4th relative to the 2nd digits. Positive correlations were detected for 2D:4D ratios, body mass index (r=0.23, P=0.039), chronologic age (r=0.35, P=0.001), and skeletal age (r=0.42, P<0.0001). The TW3 ratings and associations between 2D:4D ratios and indicators of growth suggest that digits develop at different rates. This asynchronous development may produce differences in 2D:4D over time which could lead to erroneous interpretation of androgen exposure in utero among young ASD children.
Autism spectrum disorder; digit ratio; hyperandrogenic hypothesis; measurement error
Polymorphisms in folate-related genes have emerged as important risk factors in a range of diseases including neural tube defects (NTDs), cancer and coronary artery disease (CAD). Having previously identified a polymorphism within the cytoplasmic folate enzyme, MTHFD1, as a maternal risk factor for NTDs; we considered the more recently identified mitochondrial paralogue, MTHFD1L as a candidate gene for NTD association. We identified a common deletion/insertion polymorphism, rs3832406, c.781-6823ATT(7-9), that influences splicing efficiency and is strongly associated with NTD risk. Three alleles of rs3832406 were detected in the Irish population with varying number of ATT repeats; Allele 1 consists of ATT7, while Alleles 2 and 3 consist of ATT8 and ATT9 respectively. Allele 2 of this triallelic polymorphism showed a decreased case risk as demonstrated by case-control logistic regression (P= 0.002) and by transmission disequilibrium test (TDT) (P= 0.001); while Allele 1 showed an increased case risk. Allele 3 showed no influence on NTD risk and represents the lowest frequency allele (0.15). Additional SNP genotyping in the same genomic region provides additional supportive evidence of an association. We demonstrate that two of the three alleles of rs3832406 are functionally different and influence the splicing efficiency of the alternate MTHFD1L mRNA transcripts.
MTHFD1L; NTD; Splicing; Polymorphism; Association; Folate; Mitochondria
Genetic variants in MTHFD1 (5,10-methylenetetrahydrofolate dehydrogenase/ 5,10-methenyltetrahydrofolate cyclohydrolase/ 10-formyltetrahydrofolate synthetase), an important folate metabolic enzyme, are associated with a number of common diseases, including neural tube defects (NTDs). This study investigates the promoter of the human MTHFD1 gene in a bid to understand how this gene is controlled and regulated. Following a combination of in silico and molecular approaches, we report that MTHFD1 expression is controlled by a TATA-less, Initiator-less promoter and transcription is initiated at multiple start sites over a 126bp region. We confirmed the presence of three database polymorphisms (dbSNP) by direct sequencing of the upstream region (rs1076991 C>T, rs8010584 G>A, rs4243628 G>T), with a fourth (dbSNP rs746488 A>T) not found to be polymorphic in our population and no novel polymorphisms identified. We demonstrate that a common SNP rs1076991 C>T within the window of transcriptional initiation exerts a significant effect on promoter activity in vitro. We investigated this SNP as a potential risk factor for NTDs in a large homogenous Irish population and determined that it is not an independent risk factor, but, it does increase both case (χ2 = 11.06, P = 0.001) and maternal (χ2 = 6.68, P = 0.01) risk when allele frequencies were analysed in combination with the previously identified disease-associated p.R653Q (c.1958 G>A; dbSNP rs2236225) polymorphism. These results provide the first insight into how MTHFD1 is regulated and further emphasise its importance during embryonic development.
MTHFD1; NTD; Functional; SNP; R653Q; Promoter
Genetic and environmental factors contribute to the etiology of neural tube defects (NTDs). While periconceptional folic acid supplementation is known to significantly reduce the risk of NTDs, folate metabolic pathway related factors do not account for all NTDs. Evidence from mouse models indicates that the tumor protein p53 (TP53) is involved in implantation and normal neural tube development. To determine whether genetic variation in the TP53 might contribute to NTD risk in humans, we constructed a high resolution linkage disequilibrium (LD) map of the TP53 genomic region based on genotyping 21 markers in an Irish population. We found that nine of these variants can be used to capture the majority of common variation in the TP53 genomic region. In contrast, the 3-marker haplotype commonly reported in the TP53 literature offers limited coverage of the variation in the gene. We used the expanded set of polymorphisms to measure the influence of TP53 on NTDs using both case-control and family-based tests of association. We also assayed a functional variant in the p53 regulator MDM2 (rs2279744). Alleles of three noncoding TP53 markers were associated with NTD risk. A case effect was seen with the GG genotype of rs1625895 in intron 6 (OR = 1.37 [1.04-1.79], p=0.02). A maternal effect was seen with the 135/135 genotype of the intron 1 VNTR (OR = 1.86 [1.16-2.96], p=0.01) and the TT genotype of rs1614984 (RR = 0.58 [0.37-0.91], p=0.02). As multiple comparisons were made, these cannot be considered definitive positive findings and additional investigation is required.
neural tube defects; spina bifida; p53; TP53; MDM2; linkage disequilibrium
Wang and Sheffield (2005) showed that it is preferable to use a robust model that incorporated constraints on the genotype relative risk rather than rely on a model that assumes the disease operates in a recessive or dominant fashion. Wang and Sheffield’s method is applicable to case-control studies, but not to family based studies of case children along with their parents (triads). We show here how to implement analogous constraints while analyzing triad data. The likelihood, conditional on the parents genotype, is maximized over the appropriately constrained parameter space. The asymptotic distribution for the maximized likelihood ratio statistic is found and used to estimate the null distribution of the test statistics. The properties of several methods of testing for association are compared by simulation. The constrained method provides higher power across a wide range of genetic models with little cost when compared to methods that restrict to a dominant, recessive, or multiplicative model, or make no modeling restriction. The methods are applied to two SNPs on the methylenetetrahy-drofolate reductase (MTHFR) gene with neural tube defect (NTD) triads.
conditional distribution; genetic risk model; likelihood ratio test; power