Transcobalamin II receptor polymorphisms are associated with increased risk for neural tube defects.

OBJECTIVE: Women who have low cobalamin (vitamin B(12)) levels are at increased risk for having children with neural tube defects (NTDs). The transcobalamin II receptor (TCblR) mediates uptake of cobalamin into cells. Inherited variants in the TCblR gene as NTD risk factors were evaluated. METHODS: Case-control and family-based tests of association were used to screen common variation in TCblR as genetic risk factors for NTDs in a large Irish group. A confirmatory group of NTD triads was used to test positive findings. RESULTS: 2 tightly linked variants associated with NTDs in a recessive model were found: TCblR rs2336573 (G220R; p(corr)=0.0080, corrected for multiple hypothesis testing) and TCblR rs9426 (p(corr)=0.0279). These variants were also associated with NTDs in a family-based test before multiple test correction (log-linear analysis of a recessive model: rs2336573 (G220R; RR=6.59, p=0.0037) and rs9426 (RR=6.71, p=0.0035)). A copy number variant distal to TCblR and two previously unreported exonic insertion-deletion polymorphisms were described. CONCLUSIONS: TCblR rs2336573 (G220R) and TCblR rs9426 represent a significant risk factor in NTD cases in the Irish population. The homozygous risk genotype was not detected in nearly 1000 controls, indicating that this NTD risk factor may be of low frequency and high penetrance. 9 other variants are in perfect linkage disequilibrium with the associated single nucleotide polymorphisms. Additional work is required to identify the disease-causing variant. Our data suggest that variation in TCblR plays a role in NTD risk and that these variants may modulate cobalamin metabolism.

Low-dose folic acid lowers plasma homocysteine levels in women of child-bearing age.

BACKGROUND: Ongoing clinical trials are investigating whether lowering plasma homocysteine reduces the risk of vascular disease. If so, food fortification with folic acid will be the likely result, and sub-optimal amounts are likely to be preferred, for safety reasons. Dose-finding studies are needed before the outcomes of these trials, to establish the benefits and risks of folic acid consumption over the widest intake range likely to be encountered. AIM: To find the lowest dose of folic acid that effectively reduces plasma homocysteine in premenopausal women. DESIGN: Double-blind, randomized placebo-controlled trial. METHODS: Women of child-bearing age (n=95) were randomly allocated to 0, 100, 200, or 400 microg/day of folic acid. Red-cell folate and plasma homocysteine were measured at baseline and after 10 weeks supplementation. RESULTS: Median red cell folate levels increased significantly in the 200 microg(p=0.0001) and 400 microg(p=0.0001) groups; but not in the placebo (0 microg) (p=0.25) or the 100 microg (p=0.5) groups. Only the 200 microg and the 400 microg groups had significant decreases in plasma homocysteine, (p=0.04 and p=0.0008, respectively). However, when subjects whose initial plasma homocysteine was <8 micromol/l (already optimally low) were removed from the analysis, there were significant plasma homocysteine decreases in all three treatment groups, but not the placebo group. DISCUSSION: In this sub-population, low doses of folic acid significantly lower plasma homocysteine. This could be achieved safely by fortification.

Outcome of hip fracture in older Irish women: a 2-year follow-up of subjects in a case-control study.

To assess outcome after hip fracture in older Irish women, 106 consecutive females aged over 50 years admitted to a general hospital with a hip fracture were compared to 89 age- and gender-matched controls from the same catchment area. Interview-based data were collected on socio-demographic factors, mobility and activities of daily living before recruitment and 2 years later. Information was also collected on residence, further falls and fractures and use of health and community support services during the 2-year period. Mortality at 2 years was higher in cases (23.6%) compared to controls (10.1%; P = 0.01). Cases were significantly less mobile and more dependent in the activities of daily living. Of the cases who were community dwellers at baseline, 26.6% were institutionalised at 2 years compared with 9.2% of controls (P = 0.01). During the 2 years cases were significantly more likely to have multiple falls and a further hip or pelvic fracture. Hospital and nursing home admissions and use of physiotherapy, day centre and home help services were also significantly greater among cases. The marked adverse impact of hip fracture reported in this study underlines the importance of public health strategies to prevent these injuries in older people.

Predictors of first hip fracture and mortality post fracture in older women.

BACKGROUND: Hip fracture causes significant morbidity and mortality in older women. AIM: To document factors contributing to the risk of hip fracture in older women and to assess the effect of hip fracture on subsequent mortality. METHODS: Case-control study of 89 women with hip fracture and 89 controls, with two-year follow-up. Singh index and bone mineral density were calculated. RESULTS: Osteoporotic indices did not differ significantly between cases and controls. Significant predictors of risk were sleeping tablets, perception of health as fair/poor and a lower mental status score. Patients were 3.57 times more likely to die in the first year after fracture, with no difference between the groups in year two. After adjustment, hip fracture did not remain significantly associated with mortality. Inability to walk 100 yards alone prior to fracture and lower social class were significantly associated with mortality at 12 months. Age alone was significantly associated at 12-24 months. CONCLUSIONS: Factors related to falls and fracture may be more discriminatory predictors of hip fracture risk than osteoporosis in older females. Medications for sleep should be prescribed with caution. Hip fracture may have an independent effect on one year mortality, this effect is not seen in the second year.

Association between historically high frequencies of neural tube defects and the human T homologue of mouse T (Brachyury).

The human T developmental gene has been implicated in the etiology of neural tube defects (NTDs) on the basis both of mouse studies of its homologue, T (Brachyury), and of allelic association in a Caucasian population. We have investigated the frequency of the T allelic variant TIVS7-2 in 218 Irish NTD case-parent triads. This population showed the same trend as previously reported, with an excess of the TIVS7-2 allele among cases. Log-linear modeling of case and maternal genotypic effects within families indicated that TIVS7-2 was elevated in cases (relative risk, RR = 1.36) but not in mothers (RR = 0.91). The TIVS7-2 allele is markedly associated with cases born before 1980 (RR = 2.09; CI = 1.23-3.55; corrected p = 0.030), but not with more recent cases (RR = 0.92). Cases carrying a TIVS7-2 allele did not show any increased tendency to be homozygous for the thermolabile variant of the folate-dependent enzyme 5,10-methylene tetrahydrofolate reductase, which is an established genetic risk factor for NTDs. Since the incidence of NTDs has declined markedly in Ireland over the last few decades, we suggest that the T-associated risk is potentiated by nutritional or environmental risk factor(s), the impact of which have been diminishing over time.

Folate status and neural tube defects.

Periconceptional folic acid supplementation prevents approximately 70% of neural tube defects (NTDs). While most women carrying affected fetuses do not have deficient blood folate levels, the risk of having an NTD affected child is inversely correlated with pregnancy red cell folate levels. Current research is focused on the discovery of genetic abnormalities in folate related enzymes which might explain the role of folate in NTD prevention. The first candidate gene to emerge was the C677T variant of 5,10-methylenetetrahydrofolate reductase. Normal subjects who are homozygous for the mutation (TT) have red cell folate status some 20% lower than expected. It is now established that the prevalence of the TT genotype is significantly higher among spina bifida cases and their parents. Nevertheless, our studies show that the variant does not account for the reduced blood folate levels in many NTD affected mothers. We conclude that low maternal folate status may in itself be the most important risk factor for NTDs and that food fortification may be the only population strategy of benefit in the effort to eliminate NTDs.

The influence of 5,10 methylenetetrahydrofolate reductase genotypes on enzyme activity in placental tissue.

OBJECTIVE: To study the effects of heterozygosity and homozygosity for the C677T mutation of the 5,10 methylenetetrahydrofolate reductase (MTHFR) gene on the activity of this enzyme in placental tissue from pregnancies not affected by neural tube defect. DESIGN: Placental tissue was genotyped for the C677T variants of MTHFR. Total enzyme activity and residual activity after heating to 46 degrees C for 5 minutes was then measured. SETTING: A teaching hospital. SAMPLE: Placental samples (n = 200), one from each of 200 uncomplicated term deliveries. MAIN OUTCOME MEASURES: Total and residual enzyme activity for MTHFR. RESULTS: Placentae heterozygous for the C677T mutation of the MTHFR gene had significantly lower total enzyme activity than those without the mutation; the lowest activity occurred in homozygotes for the mutation. The same pattern was seen in relation to enzyme activity after heating. CONCLUSION: This study demonstrates that reduced enzyme activity is associated with the C677T variant of MTHFR in placental tissue. This is an important metabolic step in folic acid metabolism and pro-

Methylenetetrahydrofolate reductase thermolabile variant and oral clefts.

Folic acid can prevent neural tube defects; in some cases the mechanism is probably a correction of a metabolic defect caused by thermolabile methylenetetrahydrofolate reductase (MTHFR) found in increased frequency in cases. It is less clear whether folic acid can prevent oral clefts, in part because it is not known whether thermolabile MTHFR is more common in those with oral clefts. This study examined the prevalence of the mutation (677 C-->T) that causes thermolabile MTHFR in subjects with oral clefts from a national Irish support group, and an anonymous control group randomly selected from a neonatal screening program covering all births in Ireland. Eighty-three of 848 control subjects were homozygous (TT) thermolabile MTHFR (9.8%). This defect was almost three times as common in the 27 subjects (25.9%) with isolated cleft palate (odds ratio 3.23, 95% confidence interval 1.32 -7.86, P = 0. 02) and somewhat more common in the 66 subjects with cleft lip with or without cleft palate (15.2%, odds ratio 1.65, 95% confidence interval 0.81-3.35, P = 0.20). When the two groups with different etiologies were combined, the overall odds ratio was 2.06 (95% confidence interval 1.16-3.66, P = 0.02). In the Irish population homozygosity for the common folate-related polymorphism associated with thermolabile MTHFR is significantly more frequent in those with isolated cleft palate, and could be etiologically important. Am. J. Med. Genet. 86:71-74, 1999. Published 1999 Wiley-Liss, Inc.

Methionine synthase: high-resolution mapping of the human gene and evaluation as a candidate locus for neural tube defects.

Periconceptual folate supplementation has been found to prevent the occurrence of many neural tube defects (NTDs). Consequently, genetic variation in folate metabolism genes is expected to contribute to the risk for neural tube defects. Methionine synthase catalyzes the vitamin B(12)-dependent conversion of homocysteine and 5-methyltetrahydrofolate to methionine and tetrahydrofolate. The observation that homocysteine and vitamin B(12) levels are independent predictors of NTD risk suggested that methionine synthase could be a candidate gene for NTDs. To assess the role of the MS gene in NTDs, we performed high-resolution physical mapping of the MS locus, isolated highly polymorphic markers linked to the MS gene, and tested for an association between specific MS alleles and NTDs. We mapped the MS gene to a position between 909 and 913 cR(10000) on chromosome 1 by radiation hybrid mapping. Polymorphic markers D1S1567 and D1S1568 map to locations no more than 900 and 194 kb from the MS gene, respectively. The segregation of these polymorphic markers was measured in 85 Irish NTD families. No allele of either marker showed a significant association with NTDs using the transmission disequilibrium test. A lack of association was also observed for the D1919G missense mutation within the gene. Our results suggest that inherited variation in the MS gene does not contribute to NTD risk in this population.

The "thermolabile" variant of methylenetetrahydrofolate reductase and neural tube defects: An evaluation of genetic risk and the relative importance of the genotypes of the embryo and the mother.

Recent reports have implicated the "thermolabile" (T) variant of methylenetetrahydrofolate reductase (MTHFR) in the causation of folate-dependent neural tube defects (NTDs). We report herein the largest genetic study of NTD cases (n=271) and families (n=218) to date, establishing that, in Ireland, the "TT" genotype is found in 18.8% of cases versus 8.3% of controls (odds ratio 2.57; confidence interval [CI] 1.48-4.45; P=.0005). The maternal and paternal TT genotypes have intermediate frequencies of 13.8% and 11.9%, respectively, indicating that the predominant MTHFR-related genetic effect acts via the TT genotype of the developing embryo. Analysis of the 218 family triads of mother, father, and affected child with log-linear models supports this interpretation, providing significant evidence that the case TT genotype is associated with NTDs (P=.02) but no evidence of a maternal TT genotypic effect (P=. 83). The log-linear model predicted that the risk of NTDs conferred by the case TT genotype is 1.61 (CI 1.06-2.46), consistent with the paramount importance of the case TT genotype in determining risk. There is no compelling evidence for more than a modest additional risk conferred by a maternal TT genotype. These results favor a biological model of MTHFR-related NTD pathogenesis in which suboptimal maternal folate status imposes biochemical stress on the developing embryo, a stress it is ill-equipped to tolerate if it has a TT genotype.

Low blood folates in NTD pregnancies are only partly explained by thermolabile 5,10-methylenetetrahydrofolate reductase: low folate status alone may be the critical factor.

Thermolabile 5,10-methylenetetrahydrofolate reductase (MTHFR) is the first folate-related variant to be associated with an increased risk of neural tube defects (NTDs). The variant causes high plasma homocysteine levels and reduced red cell folate (RCF) levels, both of which have also been linked to an increased risk of NTDs. We examined the relationship between folate status and presence of the common mutation MTHFR C677T in 82 NTD-affected and 260 control mothers. Homozygosity for the TT genotype was associated with very low folate status among both the cases (n = 13) and the controls (n = 21). However, after exclusion of TT homozygotes, only 10% of the remaining 240 controls had RCF levels less than 200 microg/L compared with 29% of the 69 cases (odds ratio, 3.67; 95% confidence interval, 1.88-7.18; P < 0.001), and those with RCF less than 150 microg/L had eight times higher risk of NTD than subjects with levels over 400 microg/L. Plasma homocysteine levels of non-TT cases were also higher than those of controls (P = 0.047). This study shows that homozygosity for the C677T MTHFR variant cannot account for reduced blood folate levels in many NTD-affected mothers. Thus, a strategy of genetic screening of all childbearing women for this variant would be ineffective as a method of primary prevention of NTDs. The data suggest that low maternal folate status is itself the major determinant of NTD risk, or else that other folate-dependent genetic variants confer risk through the reduction of folate levels. These results emphasize the importance of a food-fortification program as a population strategy for reducing the occurrence of NTDs.

Analysis of the human Sonic Hedgehog coding and promoter regions in sacral agenesis, triphalangeal thumb, and mirror polydactyly.

The human Sonic Hedgehog gene (SHH) is one of the vertebrate homologs related to the Drosophila segment polarity gene hedgehog. The entire coding and promoter region of the SHH gene, including 2 kb 5' of the transcriptional start site has been screened for mutations in families with autosomal dominant sacral agenesis and autosomal dominant triphalangeal thumb, two conditions previously known to be linked to 7q36. We have also studied the SHH gene in five families with mirror polydactyly associated with tibial hemimelia and in 51 unrelated patients with neural tube defects. Except for two sequence variants in exon 3, no mutations were found in these disease categories. OFF

Minimum effective dose of folic acid for food fortification to prevent neural-tube defects.

BACKGROUND: Although a daily supplement of 400 micrograms folic acid has been shown to prevent neural-tube defects (NTD), most women do not take the recommended supplement. Thus, food fortification is to be introduced in the USA and is being considered in the UK. Because of safety concerns, the USA has chosen a level of fortification that will increase the average woman's intake by only 100 micrograms. Such an increase, although safe, may be ineffective; but a trial to assess its efficacy would be unethical. Because women with red-cell folate concentrations above 400 micrograms/L have a very low risk of NTD, we undertook a randomised trial of several folic acid doses to find out how much is needed to reach this protective concentration. METHODS: We screened 323 women. 172 with red-cell folate between 150 micrograms/L and 400 micrograms/L were invited to take part in the trial. 121 women were randomly assigned placebo or 100 micrograms, 200 micrograms, or 400 micrograms daily of additional folic acid. Compliance was monitored by having the women sign a dated sheet when taking the tablet. 95 women completed the 6-month study. FINDINGS: There were significant increases in red-cell folate in all folic acid groups. The placebo group showed no significant change. The median incremental changes and median post-treatment concentrations were 67 micrograms/L (95% CI 43-120) and 375 micrograms/L (354-444) in the 100 micrograms/day group, 130 micrograms/L (108-184) and 475 micrograms/L (432-503) in the 200 micrograms/day group, and 200 micrograms/L (125-312) and 571 micrograms/L (481-654) in the 400 micrograms/day group. INTERPRETATION: A fortification programme that delivered 400 micrograms folic acid daily to women would protect against NTD, but at the expense of unnecessarily high exposure for many people. Delivery of 200 micrograms daily is also effective against NTD and safer for the general population. Based on projections from the positive folate balance in the group that received 100 micrograms daily, this dose taken continually, as it will be in fortified food, will also produce an important decrease in NTD.

Thermolabile variant of 5,10-methylenetetrahydrofolate reductase associated with low red-cell folates: implications for folate intake recommendations.

BACKGROUND: The dietary reference values for folate, as for other nutrients, are targeted to the general and supposedly normal population, not people with special needs, such as those with genetic or metabolic abnormalities or diseases. However, 5-15% of general populations are homozygous for a thermolabile variant of 5,10-methylenetetrahydrofolate reductase (C677T) which causes mild hyperhomocysteinaemia and is positively associated with the development of vascular disease and the risk of neural-tube defects. If tissue-folate status is compromised in large sectors of the population by this or other genetic variants, the present dietary reference values may need to be changed. METHODS: We identified the C677T genotype and measured red-cell folate concentrations in two groups of healthy women (pregnant, 242, not pregnant, 318). We then analysed the effect of genotype on red-cell folates, which are a reliable marker for tissue folate stores. FINDINGS: In the pregnant group there were 20 TT homozygotes, 114 wild-type CC homozygotes, and 108 CT heterozygotes. In the non-pregnant group, the numbers were 41, 148, and 129. In both pregnant and non-pregnant groups, red-cell folate was significantly lower among TT homozygous than CC homozygous women (mean 252 [95% CI 202-317] vs 347 [321-372] micrograms/L, p = 0.002 for pregnant women; 284 [250-327] vs 347 [342-372] micrograms/L, p = 0.01 for non-pregnant women). Plasma folate was also significantly lower in TT homozygous than in CC homozygous women in the pregnant group (p = 0.009) but not in the non-pregnant group. INTERPRETATION: These results suggest that a substantial minority of people in general populations may have increased folate needs. Future studies may show the presence of other common genetic variants that interact with particular nutrients and place doubts on the validity of assuming "normality" for nutrient requirements in any general population.

Are common mutations of cystathionine beta-synthase involved in the aetiology of neural tube defects?

Mildly elevated maternal plasma homocysteine (Hcy) levels (hyperhomocysteinemia) have recently been observed in some neural tube defect (NTD) pregnancies. Plasma levels of Hcy are governed by both genetic and nutritional factors and the aetiology of NTDs is also known to have both genetic and nutritional components. We therefore examined the frequency of relatively common mutations in the enzyme cystathionine beta-synthase (CBS), which is one of the main enzymes that controls Hcy levels, in the NTD population. Neither the severely dysfunctional G307S CBS allele nor the recently reported 68 bp insertion/I278T CBS allele was observed at increased frequency in the cases relative to controls. We therefore conclude that loss of function CBS alleles do not account for a significant proportion of NTDs in Ireland.

Evaluation of quantitative CT vertebral bone mineral density measurement and the Singh index in elderly females with hip fractures--a case control study.

This study aims to evaluate the ability of quantitative computed tomography (QCT) bone mineral density (BMD) measurement of vertebral bodies to predict risk of hip fracture. We also examine the predictive value of the radiographic Singh index and its relationship to the vertebral BMD. The vertebral BMD (using a QCT protocol) and radiographic Singh index were evaluated in 86 white females who had sustained a hip fracture after minor trauma. 86 age-matched female controls were also studied. All patients were post-menopausal, the age range was 52-95 years. BMD values were found to be low in both the study group and controls; there was no statistically significant difference between the groups. A low Singh index did not correlate with hip fracture, nor did it correlate with low vertebral BMD measurement. We conclude that vertebral BMD and radiographic Singh index are not reliable predictors of hip fracture in the elderly female.