Weight loss-induced cellular stress in subcutaneous adipose tissue and the risk for weight regain in overweight and obese adults.

BACKGROUND/OBJECTIVE: Weight loss is often followed by weight regain after the dietary intervention (DI). Cellular stress is increased in adipose tissue of obese individuals. However, the relation between cellular stress and weight regain is unclear. Previously, we observed increased adipose tissue cellular stress of participants regaining weight compared with participants maintaining weight loss. In the current study, we further investigated the relation between weight regain and changes in the expression of stress-related genes and stress protein levels to determine possible predictors of weight regain. PARTICIPANTS/METHODS: In this randomized controlled trial, sixty-one healthy overweight/obese participants followed a DI of either a 5-week very-low-calorie diet (500 kcal per day) or a 12-week low-calorie diet (1250 kcal per day; WL period) with a subsequent 4-week weight stable diet (WS period), and a 9-month follow-up. The WL and WS period taken together was named the DI. Abdominal subcutaneous adipose tissue biopsies were collected in 53 participants for microarray and liquid chromatography-mass spectrometry analysis. RNA and protein levels for a broad set of stress-related genes were correlated to the weight regain percentage. RESULTS: Different gene sets correlated to weight regain percentage during WS and DI. Bioinformatics clustering suggests that during the WS phase-defined genes for actin filament dynamics, glucose handling and nutrient sensing are related to weight regain. HIF-1 (hypoxia-inducible factor-1) is indicated as an important regulator. With regard to DI, clustering of correlated genes indicate that LGALS1, ENO1 and ATF2 are important nodes for conferring risk for weight regain. CONCLUSIONS: Our present findings indicate that the risk for weight regain is related to expression changes of distinct sets of stress-related genes during the first 4 weeks after returning to energy balance, and during the DI. Further research is required to investigate the mechanistic significance of these findings and find targets for preventing weight regain.

Adipose tissue gene expression is differentially regulated with different rates of weight loss in overweight and obese humans.

BACKGROUND/OBJECTIVES: Moderate weight loss (WL) can ameliorate adverse health effects associated with obesity, reflected by an improved adipose tissue (AT) gene expression profile. However, the effect of rate of WL on the AT transcriptome is unknown. We investigated the global AT gene expression profile before and after two different rates of WL that resulted in similar total WL, and after a subsequent weight stabilization period. SUBJECTS/METHODS: In this randomized controlled trial, 25 male and 28 female individuals (body mass index (BMI): 28-35 kg m-2) followed either a low-calorie diet (LCD; 1250 kcal day-1) for 12 weeks or a very-low-calorie diet (VLCD; 500 kcal day-1) for 5 weeks (WL period) and a subsequent weight stable (WS) period of 4 weeks. The WL period and WS period together is termed dietary intervention (DI) period. Abdominal subcutaneous AT biopsies were collected for microarray analysis and gene expression changes were calculated for all three periods in the LCD group, VLCD group and between diets (ΔVLCD-ΔLCD). RESULTS: WL was similar between groups during the WL period (LCD: -8.1±0.5 kg, VLCD: -8.9±0.4 kg, difference P=0.25). Overall, more genes were significantly regulated and changes in gene expression appeared more pronounced in the VLCD group compared with the LCD group. Gene sets related to mitochondrial function, adipogenesis and immunity/inflammation were more strongly upregulated on a VLCD compared with a LCD during the DI period (positive ΔVLCD-ΔLCD). Neuronal and olfactory-related gene sets were decreased during the WL period and DI period in the VLCD group. CONCLUSIONS: The rate of WL (LCD vs VLCD), with similar total WL, strongly regulates AT gene expression. Increased mitochondrial function, angiogenesis and adipogenesis on a VLCD compared with a LCD reflect potential beneficial diet-induced changes in AT, whereas differential neuronal and olfactory regulation suggest functions of these genes beyond the current paradigm.

Diet-induced weight loss decreases adipose tissue oxygen tension with parallel changes in adipose tissue phenotype and insulin sensitivity in overweight humans.

BACKGROUND/OBJECTIVES: Although adipose tissue (AT) hypoxia is present in rodent models of obesity, evidence for this in humans is limited. Here, we investigated the effects of diet-induced weight loss (WL) on abdominal subcutaneous AT oxygen tension (pO2), AT blood flow (ATBF), AT capillary density, AT morphology and transcriptome, systemic inflammatory markers and insulin sensitivity in humans. SUBJECTS/METHODS: Fifteen overweight and obese individuals underwent a dietary intervention (DI), consisting of a 5-week very-low-calorie diet (VLCD, 500 kcal day-1; WL), and a subsequent 4-week weight stable diet (WS). Body composition, AT pO2 (optochemical monitoring), ATBF (133Xe washout), and whole-body insulin sensitivity were determined, and AT biopsies were collected at baseline, end of WL (week 5) and end of WS (week 9). RESULTS: Body weight, body fat percentage and adipocyte size decreased significantly during the DI period. The DI markedly decreased AT pO2 and improved insulin sensitivity, but did not alter ATBF. Finally, the DI increased AT gene expression of pathways related to mitochondrial biogenesis and non-mitochondrial oxygen consumption. CONCLUSIONS: VLCD-induced WL markedly decreases abdominal subcutaneous AT pO2, which is paralleled by a reduction in adipocyte size, increased AT gene expression of mitochondrial biogenesis markers and non-mitochondrial oxygen consumption pathways, and improved whole-body insulin sensitivity in humans.

The effects of 30 days resveratrol supplementation on adipose tissue morphology and gene expression patterns in obese men.

Polyphenolic compounds, such as resveratrol, have recently received widespread interest because of their ability to mimic effects of calorie restriction. The objective of the present study was to gain more insight into the effects of 30 days resveratrol supplementation on adipose tissue morphology and underlying processes. Eleven healthy obese men were supplemented with placebo and resveratrol for 30 days (150 mg per day), separated by a 4-week washout period in a double-blind randomized crossover design. A postprandial abdominal subcutaneous adipose tissue biopsy was collected to assess adipose tissue morphology and gene expression using microarray analysis. Resveratrol significantly decreased adipocyte size, with a shift toward a reduction in the proportion of large and very-large adipocytes and an increase in small adipocytes. Microarray analysis revealed downregulation of Wnt and Notch signaling pathways and upregulation of pathways involved in cell cycle regulation after resveratrol supplementation, suggesting enhanced adipogenesis. Furthermore, lysosomal/phagosomal pathway and transcription factor EB were upregulated reflecting an alternative pathway of lipid breakdown by autophagy. Further research is necessary to investigate whether resveratrol improves adipose tissue function.

Promoter haplotype combinations of the platelet-derived growth factor alpha-receptor gene predispose to human neural tube defects.

Neural tube defects (NTDs), including anencephaly and spina bifida, are multifactorial diseases that occur with an incidence of 1 in 300 births in the United Kingdom. Mouse models have indicated that deregulated expression of the gene encoding the platelet-derived growth factor alpha-receptor (Pdgfra) causes congenital NTDs (refs. 2-4), whereas mutant forms of Pax-1 that have been associated with NTDs cause deregulated activation of the human PDGFRA promoter. There is an increasing awareness that genetic polymorphisms may have an important role in the susceptibility for NTDs (ref. 6). Here we identify five different haplotypes in the human PDGFRA promoter, of which the two most abundant ones, designated H1 and H2 alpha, differ in at least six polymorphic sites. In a transient transfection assay in human bone cells, the five haplotypes differ strongly in their ability to enhance reporter gene activity. In a group of patients with sporadic spina bifida, haplotypes with low transcriptional activity, including H1, were under-represented, whereas those with high transcriptional activity, including H2 alpha, were over-represented. When testing for haplotype combinations, H1 homozygotes were fully absent from the group of sporadic patients, whereas H1/H2 alpha heterozygotes were over-represented in the groups of both sporadic and familial spina bifida patients, but strongly under-represented in unrelated controls. Our data indicate that specific combinations of naturally occurring PDGFRA promoter haplotypes strongly affect NTD genesis.

The insulin-like growth factor type-2 receptor gene is imprinted in the mouse but not in humans.

In mouse, four genes have been found to undergo genomic imprinting resulting in differential expression of maternally and paternally inherited alleles. To determine whether the cognate genes are also subject to imprinting in humans, we have studied allele-specific expression patterns of insulin-like growth factor 2, IGF2-receptor and H19 in human fetal and adult tissues. In keeping with previous findings in mice, our results indicate that in human fetal tissues the paternal H19 alleles is inactive. IGF2 is monoallelically expressed in various tissues but surprisingly not in adult human liver. The human IGF2R gene, another classic example of imprinting in mice, was found to be expressed from both alleles. We provide the first direct evidence for differential imprinting in the human and murine genome.

A gene for autosomal dominant sacral agenesis maps to the holoprosencephaly region at 7q36.

Sacral agenesis is a rare disorder of uncertain incidence that has been reported in diverse populations. Although usually sporadic and most commonly associated with maternal diabetes, there is a hereditary form which may occur in isolation or with a presacral mass (anterior meningocele and/or presacral teratoma) and anorectal abnormalities, which constitute the Currarino triad (MIM 176450). The radiological hallmark of hereditary sacral agenesis is a hemi-sacrum (sickle-shaped sacrum) with intact first sacral vertebra. Bowel obstruction is the usual neonatal presentation, but, unlike other neural tube defects, adult presentation is not uncommon. The major pathology is confined to the pelvic cavity and may present as a space-occupying lesion or meningitis due to ascending infection. All recurrences in families have been compatible with autosomal dominant inheritance except for those associated with the isomerism gene at Xq24-q27.1 (ref. 3). Several associated cytogenetic defects have been reported, including 7q deletions. Previous studies failed to detect linkage to HLA markers, but we now present evidence for a location on 7q36. The same region also contains a gene for holoprosencephaly, an early malformation of the extreme rostral end of the neural tube.

Male pseudohermaphroditism due to a homozygous missense mutation of the luteinizing hormone receptor gene.

Leydig cell hypoplasia is a rare autosomal recessive condition that interferes with normal development of male external genitalia in 46,XY individuals. We have studied two Leydig cell hypoplasia patients (siblings born to consanguineous parents), and found them to be homozygous for a missense mutation (Ala593Pro) in the sixth transmembrane domain of the luteinizing hormone (LH) receptor gene. In vitro expression studies showed that this mutated receptor binds human choriogonadotropin with a normal KD, but the ligand binding does not result in increased production of cAMP. We conclude that a homozygous LH receptor gene mutation underlies the syndrome of autosomal recessive congenital Leydig cell hypoplasia in this family. These results have implications for the understanding of the development of the male genitalia.

Mapping a gene for Noonan syndrome to the long arm of chromosome 12.

Noonan syndrome is characterized by typical facies, short stature and congenital cardiac defects. Approximately half of all cases are sporadic, but autosomal dominant inheritance with variable expression is well established. We have performed a genome-wide linkage analysis in a large Dutch kindred with autosomal dominant Noonan syndrome, and localized the Noonan syndrome gene to chromosome 12 (Zmax = 4.04 at 0 = 0.0). Linkage analysis using chromosome 12 markers in 20 smaller, two-generation families gave Zmax = 2.89 at 0 = 0.07, but haplotype analysis showed non-linkage in one family. These data imply that a gene for Noonan syndrome is located on chromosome 12q, between D12S84 and D12S366.

Localization of the gene for Cowden disease to chromosome 10q22-23.

Cowden disease (CD) (MIM 158350), or multiple hamartoma syndrome, is a rare autosomal dominant familial cancer syndrome with a high risk of breast cancer. Its clinical features include a wide array of abnormalities but the main characteristics are hamartomas of the skin, breast, thyroid, oral mucosa and intestinal epithelium. The pathognomonic hamartomatous features of CD include multiple smooth facial papules, acral keratosis and multiple oral papillomas. The pathological hallmark of the facial papules are multiple trichilemmomas. Expression of the disease is variable and penetrance of the dermatological lesions is assumed to be virtually complete by the age of twenty. Central nervous system manifestations of CD were emphasized only recently and include megalencephaly, epilepsy and dysplastic gangliocytomas of the cerebellum (Lhermitte-Duclos disease, LDD). Early diagnosis is important since female patients with CD are at risk of developing breast cancer. Other lesions include benign and malignant disease of the thyroid, intestinal polyps and genitourinary abnormalities. To localize the gene for CD, an autosomal genome scan was performed. A total of 12 families were examined, resulting in a maximum lod score of 8.92 at theta = 0.02 with the marker D10S573 located on chromosome 10q22-23.

Circulating ACE is a predictor of weight loss maintenance not only in overweight and obese women, but also in men.

BACKGROUND: Circulating angiotensin-converting enzyme (ACE) was identified as a predictor of weight loss maintenance in overweight/obese women of the Diogenes project. OBJECTIVE: To investigate whether ACE acted also as a predictor in men of the Diogenes study and to compare it with that in women. DESIGN: Subjects, who lost ≥ 8% of body weight induced by low-caloric diet in an 8-week weight loss period, were assigned to weight loss maintenance with dietary intervention for 6 months. SUBJECTS: 125 overweight/obese healthy men from eight European countries who completed whole intervention. MEASUREMENTS: Concentrations and activity of serum ACE at baseline and after the 8-week weight loss, in addition to anthropometric and physiological parameters. RESULTS: Serum ACE concentration decreased by 11.3 ± 10.6% during the weight loss period in men. A greater reduction is associated with less body weight regain during the maintenance period (r=0.227, P=0.012). ACE change was able to predict a weight regain ≤ 20% after 6 months, with an odds ratio of 1.59 (95% confidence interval (CI): 1.09-2.33, P=0.016) for every 10% reduction, which was independent of body mass index and weight loss. The prediction power was weaker in men than in women, but without a significant sex difference (P=0.137). In pooled subjects (N=218), the odds ratio was 1.96 (95% CI: 1.46-2.64, P<0.001). CONCLUSIONS: A greater reduction of ACE during weight loss is favorable for weight maintenance in both men and women. This can offer useful information for personalized advice to improve weight loss maintenance. It also confirms the role of ACE in the metabolic pathways of weight regulation.

A comparison between the abdominal and femoral adipose tissue proteome of overweight and obese women.

Body fat distribution is an important determinant of cardiometabolic health. Lower-body adipose tissue (AT) has protective characteristics as compared to upper-body fat, but the underlying depot-differences remain to be elucidated. Here, we compared the proteome and morphology of abdominal and femoral AT. Paired biopsies from abdominal and femoral subcutaneous AT were taken from eight overweight/obese (BMI ≥ 28 kg/m2) women with impaired glucose metabolism after an overnight fast. Proteins were isolated and quantified using liquid chromatography-mass spectrometry, and protein expression in abdominal and femoral subcutaneous AT was compared. Moreover, correlations between fat cell size and the proteome of both AT depots were determined. In total, 651 proteins were identified, of which 22 proteins tended to be differentially expressed between abdominal and femoral AT after removal of blood protein signals (p < 0.05). Proteins involved in cell structure organization and energy metabolism were differently expressed between AT depots. Fat cell size, which was higher in femoral AT, was significantly correlated with ADH1B, POSTN and LCP1. These findings suggest that there are only slight differences in protein expression between abdominal and femoral subcutaneous AT. It remains to be determined whether these differences, as well as differences in protein activity, contribute to functional and/or morphological differences between these fat depots.

SNP analyses of postprandial responses in (an)orexigenic hormones and feelings of hunger reveal long-term physiological adaptations to facilitate homeostasis.

BACKGROUND: The postprandial responses in (an)orexigenic hormones and feelings of hunger are characterized by large inter-individual differences. Food intake regulation was shown earlier to be partly under genetic control. OBJECTIVE: This study aimed to determine whether the postprandial responses in (an)orexigenic hormones and parameters of food intake regulation are associated with single nucleotide polymorphisms (SNPs) in genes encoding for satiety hormones and their receptors. DESIGN: Peptide YY (PYY), glucagon-like peptide 1 and ghrelin levels, as well as feelings of hunger and satiety, were determined pre- and postprandially in 62 women and 41 men (age 31+/-14 years; body mass index 25.0+/-3.1 kg/m(2)). Dietary restraint, disinhibition and perceived hunger were determined using the three-factor eating questionnaire. SNPs were determined in the GHRL, GHSR, LEP, LEPR, PYY, NPY, NPY2R and CART genes. RESULTS: The postprandial response in plasma ghrelin levels was associated with SNPs in PYY (215G>C, P<0.01) and LEPR (326A>G and 688A>G, P<0.01), and in plasma PYY levels with SNPs in GHRL (-501A>C, P<0.05) and GHSR (477G>A, P<0.05). The postprandial response in feelings of hunger was characterized by an SNP-SNP interaction involving SNPs in LEPR and NPY2R (668A>G and 585T>C, P<0.05). Dietary restraint and disinhibition were associated with an SNP in GHSR (477G>A, P<0.05), and perceived hunger with SNPs in GHSR and NPY (477G>A and 204T>C, P<0.05). CONCLUSIONS: Part of the inter-individual variability in postprandial responses in (an)orexigenic hormones can be explained by genetic variation. These postprandial responses represent either long-term physiological adaptations to facilitate homeostasis or reinforce direct genetic effects.

Leptin-adiposity relationship changes, plus behavioral and parental factors, are involved in the development of body weight in a Dutch children cohort.

BACKGROUND: The development of body weight is determined by different factors, namely genetic, behavioral, parental and physiological. OBJECTIVE: To investigate whether genetic, behavioral, parental and physiological factors are involved and the extent of involvement in the development of body weight at ages 12 and 13 y in a Dutch children cohort. METHODS: In a Dutch cohort of 94 children at ages 12 and 13 y, we determined anthropometric measurements, body composition, leptin concentrations, TFEQ scores, physical activity, as well as 3 polymorphisms, and in the parents we determined anthropometric measurements and TFEQ scores. RESULTS: 11% of the children in the cohort were classified as overweight. The genotype frequency distributions of the PPARy2, GRL and CNTF genes at ages 12 and 13 y were not significantly different for the overweight children compared to the lean children. Overweight children showed higher dietary restraint and disinhibition scores. Overweight children's parents had a higher BMI, dietary restraint and disinhibition scores, compared to lean children's parents. A peak in leptin concentrations between 7 and 13 y was shown at 12 y. In lean boys, the decrease in leptin concentrations between 12 and 13 y was related to an increase in fat free mass. At the age of 12 y predominantly the physiological factors were predictors for body weight, and at the age of 13 y both the physiological and behavioral factors were predictors for body weight. CONCLUSION: We conclude from this longitudinal study, that leptin appeared to play an important role in the development of body weight during puberty, in addition to behavioral and parental factors.

Biallelic expression of the H19 and IGF2 genes in human testicular germ cell tumors.

BACKGROUND: Genomic imprinting, resulting in the nonequivalence of expression of homologue genes depending on their parental origin, is an important determinant of the developmental potential of embryonic cells. The expression of two genes, one termed H19 and the other IGF2, has been found to be necessary for proper embryonal development in humans. Both the murine and human H19 and IGF2 genes are normally characterized by monoallelic expression. PURPOSE: Because testicular germ cell tumors of adults originate from an early germ cell and, to a certain extent, mimic normal embryonal development, we investigated the patterns of allelic expression of the H19 and IGF2 genes in these tumors to determine if genomic imprinting, or a disturbance of it, is involved in their pathogenesis. METHODS: Specimens of normal tissue and tumor tissue were obtained from 20 patients with testicular germ cell tumors; 10 of the patients had seminomas and 10 had nonseminomatous germ cell tumors. To determine if there was heterozygosity of the Alu I and Apa I restriction site polymorphism in the H19 and IGF2 genes, respectively, DNA was isolated from cells of the peripheral blood of these patients, then subjected to polymerase chain reaction (PCR) amplification, restriction enzyme digestion, and electrophoresis in agarose gels. If heterozygosity was determined, a similar analysis was performed on complementary DNA (cDNA) obtained from matched tumor RNA by reverse transcription and subsequent PCR amplification (RT-PCR). If monoallelic expression was found, matched tumor DNA was studied for possible deletions. RESULTS: Tumor samples from 14 of 20 and 11 of 20 patients were informative for allelic expression patterns of the H19 and IGF2 genes, respectively. Analysis of the products of RT-PCR showed biallelic expression of the H19 gene in 12 testicular germ cell tumors (patients numbered 6, 8-13, 15, 16, and 18-20) and of the IGF2 gene in 10 testicular germ cell tumors (patients numbered 1, 3, 6, 8-13, and 15-20). The three remaining tumors (patients numbered 2, 4, and 5) had lost the nonexpressed allele. CONCLUSIONS: In contrast to normally developing embryos, testicular germ cell tumors show a consistent expression of both parental alleles of the H19 and IGF2 genes. IMPLICATIONS: Testicular germ cell tumors of adults may develop from precursor cells in which the imprinting has been either erased or subjected to a consistent relaxation of its effect.

A model for the excision of introns 1 and 2 from adenoviral major late pre-messenger RNAs.

Total steady-state RNA was extracted from nuclei of HeLa cells late after infection with adenovirus serotype 2. Most of the nuclear RNA is transcribed from the major late transcription unit (16.2 to 100.0 map units). To study the cleavage reactions involved in the splicing of leaders 1 and 2, we have used the S1 nuclease mapping technique with restriction fragments located in the region of intron 1 as DNA probes. The S1 mapping data showed that in total nuclear RNA, RNA species accumulate from which the 5' part of intron 1 has been excised, but which still contain the 3' part of the intron. This indicates that intron 1 can be removed in a stepwise fashion following the 5' to 3' direction. We have compared the nucleotide sequences from the ends of the putative processing intermediates. The internal cleavage sites do not resemble the consensus 5' or 3' splice site sequences. However, they show considerable homology to the sequence 5' A-T-G-A-T-G-G-C-A-T 3', which may act as a signal for internal cleavage. The intermediates are present in both the poly(A)+ and poly(A)- RNA fractions, although with different relative intensities. Primer extension experiments have been performed in which a primer, located with its left end in leader 2, is extended into intron 1. The results show that there may be a cleavage site as short as 35 nucleotides before the 3' splice site. Cleavage at the 3' splice site seems to be rapidly followed by ligation of leader 1 to leader 2. A model for RNA splicing based on these findings and data from the literature is presented.

Alternative splicing pathways exist in the formation of adenoviral late messenger RNAs.

Total rapidly labeled RNA was extracted from nuclei of HeLa cells late after infection with adenovirus type 2. Most of this nuclear RNA is transcribed from the major late transcription unit (16.2 to 100.0 map units (m.u.)). To study the cleavage reactions that are involved in RNA splicing, we used the S1 nuclease mapping technique with HindIII B (16.8 to 31.5 m.u.) and XhoI F (15.5 to 22.4 m.u.) restriction fragments as DNA probes. The S1 mapping data showed that both intron 1 (16.3 to 19.1 m.u.) and intron 2 (19.4 to 26.2 m.u.) can be excised in more than one step. Kinetic labeling and pulse--chase experiments demonstrated that certain cleavage sites in the RNA are used within three minutes after the start of transcription, while other cleavages occur only after a significant time-lag. The use of 5.6-dichloro-1-beta-D-ribofuranosylbenzimidazole enabled us to label the nuclear RNA exclusively from the 5' end. Such an approach showed that the first cleavages are introduced in the nascent RNA before the RNA polymerase has passed more than 2000 nucleotides beyond the cleavage site. The chronology of the appearance of processing intermediates that results from cleavage of the RNA indicates that preferentially intron 1 is removed before intron 2. This is an agreement with the finding that leader 1 is ligated to leader 2 before ligation of leader 2 to leader 3 takes place. However, we have found that alternative splicing pathways exist in the excision of introns 1 and 2, demonstrating that cleavage in intron 2 may occur before intron 1 is attacked by the splicing enzymes.

Decreased methylene tetrahydrofolate reductase activity due to the 677C-->T mutation in families with spina bifida offspring.

Periconceptional folate intake reduces both the occurrence and recurrence risk of neural tube defects. Plasma homocysteine levels can be elevated in mothers of a child with a neural tube defect, suggesting a dysfunctional folate metabolism. Very recently we showed that a common 677C-->T mutation in the 5,10-methylene tetrahydrofolate reductase gene, causing thermolability of the enzyme, is a risk factor for spina bifida offspring. Restriction enzyme analysis of the genomic 5,10-methylene tetrahydrofolate reductase polymerase chain reaction fragment revealed a significantly higher prevalence of a +/+ genotype among spina bifida patients and their mothers. The risk for spina bifida offspring is the strongest if both the mother and her child have the mutation in the homozygous state. Enzymatic analysis showed that homozygosity for the 677C-->T mutation causes a decreased 5,10-methylene tetrahydrofolate reductase activity, resulting in elevated plasma homocysteine and red blood cell folate levels and lowered plasma folate and cysteine values. This extended study demonstrates that a nucleotide substitution in the coding region of 5,10-methylene tetrahydrofolate reductase, resulting in reduced activity and an impaired homocysteine and folate metabolism, is a genetic risk factor for spina bifida.