Search

Abstract Background Spina bifida is a malformation of the neural tube and is the most common of neural tube defects (NTDs). The etiology of spina bifida is largely unknown, although it is thought to be multi-factorial, involving multiple interacting genes and environmental factors. Mutations in transcriptional co-activator genes-Cited2, p300, Cbp, Tfap2α, Carm1 and Cart1 result in NTDs in murine models, thus prompt us to investigate whether homologues of these genes are associated with NTDs in humans. Methods Data and biological samples from 297 spina bifida cases and 300 controls were derived from a population-based case-control study conducted in California. 37 SNPs within CITED2, EP300, CREBBP, TFAP2A, CARM1 and ALX1 were genotyped using an ABI SNPlex assay. Odds ratios and 95% confidence intervals were calculated for alleles, genotypes and haplotypes to evaluate the risk for spina bifida. Results Several SNPs showed increased or decreased risk, including CITED2 rs1131431 (OR = 5.32, 1.04~27.30), EP300 rs4820428 (OR = 1.30, 1.01~1.67), EP300 rs4820429 (OR = 0.50, 0.26~0.50, in whites, OR = 0.7, 0.49~0.99 in all subjects), EP300 rs17002284 (OR = 0.43, 0.22~0.84), TFAP2A rs3798691 (OR = 1.78, 1.13~2.87 in Hispanics), CREBBP rs129986 (OR = 0.27, 0.11~0.69), CARM1 rs17616105 (OR = 0.41, 0.22~0.72 in whites). In addition, one haplotype block in EP300 and one in TFAP2A appeared to be associated with increased risk. Conclusions Modest associations were observed in CITED2, EP300, CREBBP, TFAP2A and CARM1 but not ALX1. However, these modest associations were not statistically significant after correction for multiple comparisons. Searching for potential functional variants and rare causal mutations is warranted in these genes.

Abstract Background Neonatal blood, obtained from a heel stick and stored dry on paper cards, has been the standard for birth defects screening for 50 years. Such dried blood samples are used, primarily, for analysis of small-molecule analytes. More recently, the DNA complement of such dried blood cards has been used for targeted genetic testing, such as for single nucleotide polymorphism in cystic fibrosis. Expansion of such testing to include polygenic traits, and perhaps whole genome scanning, has been discussed as a formal possibility. However, until now the amount of DNA that might be obtained from such dried blood cards has been limiting, due to inefficient DNA recovery technology. Results A new technology is employed for efficient DNA release from a standard neonatal blood card. Using standard Guthrie cards, stored an average of ten years post-collection, about 1/40th of the air-dried neonatal blood specimen (two 3 mm punches) was processed to obtain DNA that was sufficient in mass and quality for direct use in microarray-based whole genome scanning. Using that same DNA release technology, it is also shown that approximately 1/250th of the original purified DNA (about 1 ng) could be subjected to whole genome amplification, thus yielding an additional microgram of amplified DNA product. That amplified DNA product was then used in microarray analysis and yielded statistical concordance of 99% or greater to the primary, unamplified DNA sample. Conclusion Together, these data suggest that DNA obtained from less than 10% of a standard neonatal blood specimen, stored dry for several years on a Guthrie card, can support a program of genome-wide neonatal genetic testing.

Abstract Background Folic acid taken in early pregnancy reduces risks for delivering offspring with several congenital anomalies. The mechanism by which folic acid reduces risk is unknown. Investigations into genetic variation that influences transport and metabolism of folate will help fill this data gap. We focused on 118 SNPs involved in folate transport and metabolism. Methods Using data from a California population-based registry, we investigated whether risks of spina bifida or conotruncal heart defects were influenced by 118 single nucleotide polymorphisms (SNPs) associated with the complex folate pathway. This case-control study included 259 infants with spina bifida and a random sample of 359 nonmalformed control infants born during 1983–86 or 1994–95. It also included 214 infants with conotruncal heart defects born during 1983–86. Infant genotyping was performed blinded to case or control status using a designed SNPlex assay. We examined single SNP effects for each of the 118 SNPs, as well as haplotypes, for each of the two outcomes. Results Few odds ratios (ORs) revealed sizable departures from 1.0. With respect to spina bifida, we observed ORs with 95% confidence intervals that did not include 1.0 for the following SNPs (heterozygous or homozygous) relative to the reference genotype: BHMT (rs3733890) OR = 1.8 (1.1–3.1), CBS (rs2851391) OR = 2.0 (1.2–3.1); CBS (rs234713) OR = 2.9 (1.3–6.7); MTHFD1 (rs2236224) OR = 1.7 (1.1–2.7); MTHFD1 (hcv11462908) OR = 0.2 (0–0.9); MTHFD2 (rs702465) OR = 0.6 (0.4–0.9); MTHFD2 (rs7571842) OR = 0.6 (0.4–0.9); MTHFR (rs1801133) OR = 2.0 (1.2–3.1); MTRR (rs162036) OR = 3.0 (1.5–5.9); MTRR (rs10380) OR = 3.4 (1.6–7.1); MTRR (rs1801394) OR = 0.7 (0.5–0.9); MTRR (rs9332) OR = 2.7 (1.3–5.3); TYMS (rs2847149) OR = 2.2 (1.4–3.5); TYMS (rs1001761) OR = 2.4 (1.5–3.8); and TYMS (rs502396) OR = 2.1 (1.3–3.3). However, multiple SNPs observed for a given gene showed evidence of linkage disequilibrium indicating that the observed SNPs were not individually contributing to risk. We did not observe any ORs with confidence intervals that did not include 1.0 for any of the studied SNPs with conotruncal heart defects. Haplotype reconstruction showed statistical evidence of nonrandom associations with TYMS, MTHFR, BHMT and MTR for spina bifida. Conclusion Our observations do not implicate a particular folate transport or metabolism gene to be strongly associated with risks for spina bifida or conotruncal defects.

Abstract Background The reduced folate carrier (RFC1) is an integral membrane protein and facilitative anion exchanger that mediates delivery of 5-methyltetrahydrofolate into mammalian cells. Adequate maternal-fetal transport of folate is necessary for normal embryogenesis. Targeted inactivation of the murine RFC1 gene results in post-implantation embryolethality, but daily folic acid supplementation of pregnant dams prolongs survival of homozygous embryos until mid-gestation. At E10.5 RFC1-/- embryos are developmentally delayed relative to wildtype littermates, have multiple malformations, including neural tube defects, and die due to failure of chorioallantoic fusion. The mesoderm is sparse and disorganized, and there is a marked absence of erythrocytes in yolk sac blood islands. The identification of alterations in gene expression and signaling pathways involved in the observed dysmorphology following inactivation of RFC1-mediated folate transport are the focus of this investigation. Results Affymetrix microarray analysis of the relative gene expression profiles in whole E9.5 RFC1-/- vs. RFC1+/+ embryos identified 200 known genes that were differentially expressed. Major ontology groups included transcription factors (13.04%), and genes involved in transport functions (ion, lipid, carbohydrate) (11.37%). Genes that code for receptors, ligands and interacting proteins in the cubilin-megalin multiligand endocytic receptor complex accounted for 9.36% of the total, followed closely by several genes involved in hematopoiesis (8.03%). The most highly significant gene network identified by Ingenuity™ Pathway analysis included 12 genes in the cubilin-megalin multiligand endocytic receptor complex. Altered expression of these genes was validated by quantitative RT-PCR, and immunohistochemical analysis demonstrated that megalin protein expression disappeared from the visceral yolk sac of RFC1-/- embryos, while cubilin protein was widely misexpressed. Conclusion Inactivation of RFC1 impacts the expression of several ligands and interacting proteins in the cubilin-amnionless-megalin complex that are involved in the maternal-fetal transport of folate and other nutrients, lipids and morphogens such as sonic hedgehog (Shh) and retinoids that play critical roles in normal embryogenesis.

Abstract Background CFL1 encodes human non-muscle cofilin (n-cofilin), which is an actin-depolymerizing factor and is essential in cytokinesis, endocytosis, and in the development of all embryonic tissues. Cfl1 knockout mice exhibit failure of neural tube closure at E10.5 and die in utero. We hypothesized that genetic variation within the human CFL1 gene may alter the protein's function and result in defective actin depolymerizing and cellular activity during neural tube closure. Such alterations may be associated with an increased risk for neural tube defects (NTDs). Methods Having re-sequenced the human CFL1 gene and identified five common single nucleotide polymorphisms (SNPs) in our target population, we investigated whether there existed a possible association between the genetic variations of the CFL1 gene and risk of spina bifida. Samples were obtained from a large population-based case-control study in California. Allele association, genotype association and haplotype association were evaluated in two different ethnicity groups, non-Hispanic white and Hispanic white. Results Homozygosity for the minor alleles of the SNPs studied (rs652021, rs665306, rs667555, rs4621 and rs11227332) appeared to produce an increased risk for spina bifida. Subjects with the haplotype composed of all minor alleles (CCGGT) appeared to have increased spina bifida risk (OR = 1.6, 95% CI: 0.9~2.9), however, this finding is not statistically significant likely due to limited sample size. Conclusion The sequence variation of human CFL1 gene is a genetic modifier for spina bifida risk in this California population.

Abstract Background Neural tube defects (NTDs) are among the most common of all human congenital defects. Over the last two decades, accumulating evidence has made it clear that periconceptional intake of folic acid can significantly reduce the risk of NTD affected pregnancies. This beneficial effect may be related to the ability of folates to donate methyl groups for critical physiological reactions. Choline is an essential nutrient and it is also a methyl donor critical for the maintenance of cell membrane integrity and methyl metabolism. Perturbations in choline metabolism in vitro have been shown to induce NTDs in mouse embryos. Methods This study investigated whether single nucleotide polymorphisms (SNPs) in human choline kinase A (CHKA) gene and CTP:phosphocholine cytidylytransferase (PCYT1A) gene were risk factors for spina bifida. Fluorescence-based allelic discrimination analysis was performed for the two CHKA intronic SNPs hCV1562388 (rs7928739) and hCV1562393, and PCYT1A SNP rs939883 and rs3772109. The study population consisted of 103 infants with spina bifida and 338 non-malformed control infants who were born in selected California counties in the period 1989–1991. Results The CHKA SNP hCV1562388 genotypes with at least one C allele were associated with a reduced risk of spina bifida (odds ratio = 0.60, 95%CI = 0.38–0.94). The PCYT1A SNP rs939883 genotype AA was associated with a twofold increased risk of spina bifida (odds ratio = 1.89, 95% CI = 0.97–3.67). These gene-only effects were not substantially modified by analytic consideration to maternal periconceptional choline intake. Conclusion Our analyses showed genotype effects of CHKA and PCYT1A genes on spina bifida risk, but did not show evidence of gene-nutrient interactions. The underlying mechanisms are yet to be resolved.