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Background Neural tube defects (NTDs), including spina bifida and anencephaly, are the second most common birth defect with an incidence of 1/1000. Genetic factors are believed to contribute to NTD risk and family-based studies can be useful for identifying such risk factors. Methods We ascertained 1066 NTD families (1467 affected patients), including 307 multiplex NTD families. We performed pedigree analysis to describe the inheritance patterns, pregnancy outcomes, and recurrence risks to relatives of various types. Results Myelomeningocele or spina bifida (66.9%) and cranial defects (17.7%) were the most common NTD subtypes observed. The overall male:female ratio for affected individuals was 0.82, and there were even fewer males among individuals with an upper level NTD (0.62). Among twins, 2 of the 5 monozygotic twins and only 3 of 35 dizygotic twins were concordant, while 27% of the same sex twins were concordant, but none of the different sex twins. The estimated 6.3% recurrence risk to siblings (CI 0.04-0.08) is consistent with previous reports. Families with two or more affected individuals show a higher proportion of female transmitters (p = 0.0002). Additionally, the number of affected relatives in maternal compared to paternal lineages was more than double (p = 0.006). There were significantly more miscarriages, infant deaths, and stillborn pregnancies of the maternal aunts and uncles (p Conclusions Our data provide several lines of evidence consistent with a maternal effect, as well as a sex-influenced effect, in the etiology of NTDs.

BACKGROUND: Neural tube defects (NTDs) are considered complex, with both genetic and environmental factors implicated. To date, no major causative genes have been identified in humans despite several investigations. The first genomewide screen in NTDs demonstrated evidence of linkage to chromosomes 7 and 10. This screen included 44 multiplex families and consisted of 402 microsatellite markers spaced ∼10 cM apart. Further investigation of the genomic screen data identified a single large multiplex family, pedigree 8776, as primarily driving the linkage results on chromosome 7. METHODS: To investigate this family more thoroughly, a high-density single nucleotide polymorphism (SNP) screen was performed. Two-point and multipoint linkage analyses were performed using both parametric and nonparametric methods. RESULTS: For both the microsatellite and SNP markers, linkage analysis suggested the involvement of a locus or loci proximal to the telomeric regions of chromosomes 2q and 7p, with both regions generating a LOD* score of ∼3.0 using a nonparametric identity by descent relative sharing method. CONCLUSIONS: The regions with the strongest evidence for linkage map proximal to the telomeres on these two chromosomes. In addition to mutations and/or variants in a major gene, these loci may harbor a microdeletion and/or translocation; potentially, polygenic factors may also be involved. This single family may be promising for narrowing the search for NTD susceptibility genes. Birth Defects Research (Part A) 76:499-505, 2006.

BACKGROUND Vitamin A (retinol), in the form of retinoic acid (RA), is essential for normal development of the human embryo. Studies in the mouse and zebrafish have shown that retinol is metabolized in the developing spinal cord and must be maintained in a precise balance along the anteroposterior axis. Both excess and deficiency of RA can affect morphogenesis, including failures of neural tube closure. METHODS We chose to investigate 5 genes involved in the metabolism or synthesis of RA, ALDH1A2, CYP26A1, CYP26B1, CRABP1, and CRABP2, for their role in the development of human neural tube defects, such as spina bifida. RESULTS An association analysis using both allelic and genotypic single-locus tests revealed a significant association between the risk for spina bifida and 3 polymorphisms in the gene ALDH1A2; however, we found no evidence of a significant multilocus association. CONCLUSIONS These results may suggest that polymorphisms in ALDH1A2 may influence the risk for lumbosacral myelomeningocele in humans. Birth Defects Research (Part A), 2005. © 2005 Wiley-Liss, Inc.

Folate supplementation appears to reduce the risk for neural tube defects (NTDs). Methylenetetrahydrofolate reductase (MTHFR) is a candidate gene in the folate metabolism pathway that has been extensively studied in different human populations. We examined the risk associated with having the thermolabile variant (TT) of MTHFR in a study of 175 American Caucasians with NTDs and their families. We found a significant association in patients compared with 195 unrelated controls [odds ratio (OR) = 2.13, 95% confidence interval (95% CI) = 1.11-4.09)], but not in mothers (OR = 1.29, 95% CI = 0.622-2.67) or in fathers (OR = 1.45, 95% CI = 0.681-3.09). We found no evidence for unequal transmission from parents to an affected child (p > 0.10). We failed to find a previously reported association for a combined haplotype for MTHFR and cystathionine beta-synthase, except in subjects with NTDs compared with 559 pooled controls (OR = 2.87, 95% CI = 1.03-8.03). We found no evidence for an association for a novel CA-repeat polymorphism identified in a gene closely linked to MTHFR (p > 0.10). Our studies continue to suggest that additional candidate genes other than MTHFR may be responsible for an increased risk to NTD in some American Caucasian families.

We investigated the T locus as a candidate gene in a series of patients and families with lumbosacral myelomeningocele. Single-strand conformation polymorphism (SSCP) analysis was used to identify sequence variation in all 8 exons and in intron 7 of this locus. We found evidence of substantial polymorphism within this locus, as previously reported [Papapetrou et al., 1999, J Med Genet 36:208-213], and moderately significant evidence of linkage disequilibrium with the CacI polymorphism of exon 8. However, when the locus was considered as a whole, with all single nucleotide polymorphisms (SNPs) integrated into a haplotype, there was no evidence for linkage disequilibrium. In addition, we did not identify any new sequence variants. Thus, we conclude that the T locus is not a major locus for human NTDs in this sample.

BACKGROUND Neural tube defects (NTDs) are the second most common birth defects, after congenital heart defects. Telomerase, the reverse transcriptase that maintains telomere DNA, has been shown to be important for neural tube development and bilateral symmetry in the brain. In knockout mice null for the telomerase RNA component (TERC), telomere loss results in the failure of neural tube closure, primarily at the forebrain and midbrain. METHODS We investigated TERC for variants that may predispose to human NTDs in 477 NTD cases with a variety of phenotypic presentations. RESULTS Two novel single nucleotide polymorphisms were identified in the human TERC sequence but showed no association with the NTD phenotype. CONCLUSIONS Variants in TERC are unlikely to be a major risk factor for the most common form of human NTDs, lumbosacral myelomeningocele. Birth Defects Research (Part A), 2004. © 2004 Wiley-Liss, Inc.

BACKGROUND: NTDs are considered complex disorders that arise from an interaction between genetic and environmental factors. NTD family 8776 is a large multigenerational Caucasian family that provides a unique resource for the genetic analysis of NTDs. Previous linkage analysis using a genome-wide SNP screen in family 8776 with multipoint nonparametric mapping methods identified maximum LOD* scores of ∼3.0 mapping to 2q33.1–q35 and 7p21.1–pter. METHODS: We ascertained an additional nuclear branch of 8776 and conducted additional linkage analysis, fine mapping, and haplotyping. Expression data from lymphoblast cell lines were used to prioritize candidate genes within the minimum candidate intervals. Genomic copy number changes were evaluated using BAC tiling arrays and subtelomeric fluorescent in situ hybridization probes. RESULTS: Increased evidence for linkage was observed with LOD* scores of ∼3.3 for both regions. Haplotype analyses narrowed the minimum candidate intervals to a 20.3 Mb region in 2q33.1–q35 between markers rs1050347 and D2S434, and an 8.3 Mb region in 7p21.1–21.3 between a novel marker 7M0547 and rs28177. Within these candidate regions, 16 genes were screened for mutations; however, no obvious causative NTD mutation was identified. Evaluation of chromosomal aberrations using comparative genomic hybridization arrays, subtelomeric fluorescent in situ hybridization, and copy number variant detection techniques within the 2q and 7p regions did not detect any chromosomal abnormalities. CONCLUSIONS: This large NTD family has identified two genomic regions that may harbor NTD susceptibility genes. Ascertainment of another branch of family 8776 and additional fine mapping permitted a 9.1 Mb reduction of the NTD candidate interval on chromosome 7 and 37.3 Mb on chromosome 2 from previously published data. Identification of one or more NTD susceptibility genes in this family could provide insight into genes that may affect other NTD families. Birth Defects Research (Part A), 2008. © 2008 Wiley-Liss, Inc.

Of 1,000 births worldwide, in one embryo the neural tube will fail to close properly 28 days after conception, resulting in some form of neural tube defect (NTD). Failed closure at the cranial end, known as anencephaly, is a lethal condition, whereas failed closure at the caudal end usually results in a myelomeningocele. NTDs are the most common debilitating birth defect. Familial studies indicate a significant genetic component to NTDs, with a 40-fold increase in risk in first-degree relatives (Elwood et al. 1992). Myriad environmental exposures have been implicated in the development of NTDs; most notably, a significant decrease in risk can be achieved by maternal folic acid supplementation before conception. The mechanism by which dietary folate supplementation prevents NTDs is poorly understood (MRC Vitamin Study Research Group 1991). Folic acid derivatives are essential for the synthesis of DNA, cell division, tissue growth, and DNA methylation (Morrison et al. 1998). Methylation enables proper gene expression and chromosome structure maintenance, both of which are critical in the developing embryo (Razin and Kantor 2005). The folate and methionine cycles are linked by the conversion of homocysteine to methionine (Figure 1). In the absence of food frequency data, maternal vitamin supplementation can also serve as a proxy for overall health because of the positive correlation between supplement intake, diet, and a healthy lifestyle (Slesinski et al. 1996). Vitamin supplementation is an important cofactor to consider when studying nutritionally related genes. Figure 1 The folate and methionine cycles highlighting the 11 genes included in this study. Substrates are shown in rectangular boxes; enzymes are shown in ellipses. Adapted from Nijhout et al. (2004) and Reed et al. (2004). Substrate abbreviations: AdoHcy, S ... Animal models demonstrate that periconceptional folate supplementation protects against congenital defects in the face, neural tube, and conotruncal region of the heart. Low folate could directly limit its availability to cells or indirectly disrupt methionine metabolism, thereby increasing homocysteine in the maternal serum (Rosenquist and Finnell 2001). Either mechanism implicates folate receptor and methionine–homocysteine regulatory genes. Folate enters cells by folate receptor 1 [FOLR1; GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"NM_016725","term_id":"262331571","term_text":"NM_016725"}}NM_016725 (http://www.ncbi.nih.gov/GenBank)] and folate receptor 2 (FOLR2; GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"NM_000803","term_id":"166064049","term_text":"NM_000803"}}NM_000803) or carrier-mediated internalization by solute carrier family 19 member 1(SLC19A1; GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"U15939","term_id":"1222522","term_text":"U15939"}}U15939), also known as reduced folate carrier protein 1. Transcobalamin II (TCN2; GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"NM_000355","term_id":"296080702","term_text":"NM_000355"}}NM_000355) imports vitamin B12, cobalamin, a cofactor for another folate enzyme, 5-methyltetrahydrofolate-homocys-teine methyltransferase (MTR; GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"NM_000254","term_id":"169790922","term_text":"NM_000254"}}NM_000254).The reactions within the folate metabolism cycle can be very complex, with methylenetetrahydrofolate dehydrogenase 1 (MTHFD1; GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"J04031","term_id":"187464","term_text":"J04031"}}J04031), serine hydroxymethyl-tranferase 1 (SHMT1; GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"NM_004169","term_id":"528881072","term_text":"NM_004169"}}NM_004169), and 5,10-methylenetetrahy-drofolate reductase (MTHFR ; GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"NM_005957","term_id":"260898771","term_text":"NM_005957"}}NM_005957) being widely studied in the NTD literature. MTHFR rs1801133 is the most frequently investigated polymorphism in NTDs with conflicting results in different populations: Dutch and Irish populations associate the TT allele with risk (Shields et al. 1999; van der Put et al. 1995), whereas a protective effect is seen in Italians (De Marco et al. 2002) and other populations have no evidence of association (Gonzalez-Herrera et al. 2002; Revilla et al. 2003; Stegmann et al. 1999). This polymorphism also has a confirmed role heart disease (Frosst et al. 1995). Homocysteine can accumulate from low dietary folate, cobalamin, and/or genetic factors (Morrison et al. 1998; Ramsbottom et al. 1997) and is elevated in some NTD mothers (Mills et al. 1995; Steegers-Theunissen et al. 1994). Homocysteine itself may be teratogenic (Rosenquist et al. 1996) or impair substrates for methylation reactions (Essien and Wannberg 1993). Enzymes that degrade homocysteine regulate homocysteine levels; for example, MTR converts homocysteine to methionine and folate to tetrahydrofolate (Trembath et al. 1999). 5-Methyltetrahydrofolate-homocysteine methyltransferase reductase (MTRR; GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"AF025794","term_id":"2981302","term_text":"AF025794"}}AF025794) maintains MTR in its active state. Betaine-homocysteine methyltransferase (BHMT; GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"BC012616","term_id":"15214971","term_text":"BC012616"}}BC012616) remethylates homocysteine to methionine with a betaine cofactor (Morin et al. 2003). Cystathionine-betasynthase (CBS; GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"NM_000071","term_id":"209862802","term_text":"NM_000071"}}NM_000071) controls homocysteine levels by degrading homocysteine into cystathionine (Morrison et al. 1998). Detecting moderate effects of multiple folate genes will be particularly difficult if they are interactive or additive with environmental impacts (Morrison et al. 1998). This complex pathway has several known metabolic interactions, such as MTRR maintaining MTR in an active state. Previous studies found an association of MTHFR and MTRR (Gueant-Rodriguez et al. 2003; Wilson et al. 1999) plus CBS and the MTHFR thermolabile variant with NTDs (Afman et al. 2003; Ramsbottom et al. 1997; Speer et al. 1999). Thus, genes involved in folate metabolism are compelling candidates for NTDs, from both a genetic and an environmental perspective.