The effect of folic acid fortification on plasma folate and total homocysteine concentrations.

BACKGROUND: In 1996, the Food and Drug Administration issued a regulation requiring all enriched grain products to be fortified with folic acid to reduce the risk of neural-tube defects in newborns. Fortification (140 microg per 100 g) began in 1996, and the process was essentially complete by mid-1997. METHODS: To assess the effect of folic acid fortification on folate status, we measured plasma folate and total homocysteine concentrations (a sensitive marker of folate status) using blood samples from the fifth examination (January 1991 to December 1994) of the Framingham Offspring Study cohort for baseline values and the sixth examination (January 1995 to August 1998) for follow-up values. We divided the cohort into two groups on the basis of the date of their follow-up examination: the study group consisted of 350 subjects who were seen after fortification (September 1997 to March 1998), and the control group consisted of 756 subjects who were seen before fortification (January 1995 to September 1996). RESULTS: Among the subjects in the study group who did not use vitamin supplements, the mean folate concentrations increased from 4.6 to 10.0 ng per milliliter (11 to 23 nmol per liter) (P<0.001) from the baseline visit to the follow-up visit, and the prevalence of low folate concentrations (<3 ng per milliliter [7 nmol per liter]) decreased from 22.0 to 1.7 percent (P< 0.001). The mean total homocysteine concentration decreased from 10.1 to 9.4 micromol per liter during this period (P<0.001), and the prevalence of high homocysteine concentrations (>13 micromol per liter) decreased from 18.7 to 9.8 percent (P<0.001). In the control group, there were no statistically significant changes in concentrations of folate or homocysteine. CONCLUSIONS: The fortification of enriched grain products with folic acid was associated with a substantial improvement in folate status in a population of middle-aged and older adults.     

Correlation between cystathionine beta synthase gene polymorphisms,plasma homocysteine and idiopathic mental retardation in Indian individuals from Kolkata

Deficiency in cystathionine beta synthase (CBS) enzyme sometimes leads to hyperhomocysteinemia/homocystinuria, conditions often associated with mental retardation (MR). In this investigation, association of idiopathic MR (IMR) with six CBS gene polymorphisms and fasting total plasma homocysteine (plHcy) was explored. Nuclear families with IMR probands (N = 180) and control subjects (N = 106) were recruited. Genomic DNA was subjected to PCR amplification and RFLP analysis. plHcy was measured by enzyme immunoassay. Data obtained was subjected to statistical analyses. Linkage disequilibrium between polymorphic sites was computed. T833C/844ins68 polymorphism revealed significant maternal transmission in IMR cases. The 31 bpVNTR 21 repeat allele was significantly higher in male IMR cases as compared to sex-matched controls (P = 0.004). A significant difference was also noticed in genotype frequencies of male IMR cases (P = 0.005). Four other sites, G919A, C1105T, G1316A and G1330A, were not polymorphic in the studied population. While no significant contribution of any particular genotype was observed, plHcy level was significantly higher in male IMR cases as compared to sex-matched controls (P = 0.0001). The data presented here is probably indicative of a higher risk of IMR in male subjects in association with two CBS polymorphisms and mild elevation in plHcy concentration.     

胸苷酸合成酶对结肠癌5-FU治疗耐药的影响

目的 探讨结肠癌对5-FU产生耐药的机制，揭示不同胸苷酸合成酶(TS)蛋白表达水平的结肠癌患者对5-Fu治疗的敏感性。方法 采用免疫组化S-P法检测60例结肠癌组织及正常对照组中TS蛋白的表达情况。结果 正常结肠黏膜组织中的TS表达水平高于结肠癌组织，结肠癌组织中TS表达水平与5-Fu化疗患者的预后呈负相关，与患者的性别、有无淋巴结转移及癌组织的分化程度无关。结论 结肠癌组织中TS的表达水平可以作为患者对5-Fu为主的化疗疗效及预后的判断指标。     

同型半胱氨酸代谢酶基因突变多态性与妊娠高血压综合征遗传易感性研究

目的探讨同型半胱氨酸代谢酶—甲基四氢叶酸还原酶(MTHFR)、蛋氨酸合成酶(MS)和胱硫醚-β合成酶(CBβS)基因多态性在妊娠高血压综合征(妊高征)发病中的作用地位。方法荧光偏振免疫分析法测定血浆总Hcy浓度;聚合酶链反应-限制性片段长度多态性技术(PCR-RFLP)检测MTHFRC677T、MSA2756G、MTRRA66G和CBβS844ins68基因多态性。结果病例组MTHFRC677TC/T基因型频率显著高于正常对照组,总的突变T等位基因频率显著高于对照组(P<0.05);病例组MS野生型A等位基因频率明显高于对照组,而突变型G等位基因频率显著低于对照组。结论MTHFRC677T基因突变是妊高征发生的遗传风险因素;MSA2756多态性改变是妊高征的保护因子。二者均可作为妊高征预后的检测指标。     

Influence of the cystathionine beta-synthase 844ins68 and methylenetetrahydrofolate reductase 677C>T polymorphisms on folate and homocysteine concentrations

A high homocysteine, low folate phenotype is a feature of many diseases. The effect of the cystathionine beta-synthase (CBS) 844ins68 polymorphism on homocysteine and folate concentrations was examined alone and in the context of the 5,10-methylenetetrahydrofolate reductase (MTHFR) 677C>T polymorphism in a Northwestern European male population. The MTHFR 677TT genotype is known to be associated with increased homocysteine and decreased folate relative to CT heterozygotes and CC homozygotes in this and other populations. MTHFR 677TT homozygotes who were also CBS 844ins68 carriers had homocysteine and folate concentrations similar to those of individuals with the MTHFR 677CT and CC genotypes. Homocysteine levels in MTHFR 677TT subjects carrying the CBS 844ins68 allele were 24.1% lower than in non-carriers (6.66 vs 8.77 micromol/l, P=0.045), and serum folate levels were 27.7% higher (11.16 vs 8.74 nmol/l, P=0.034). These findings suggest that the CBS 844ins68 allele 'normalizes' homocysteine and folate levels in MTHFR 677TT individuals.     

Effect of genetic variation in the human S-adenosylhomocysteine hydrolase gene on total homocysteine concentrations and risk of recurrent venous thrombosis

Hyperhomocysteinemia is an independent and graded risk factor for arterial vascular disease and venous thrombosis. It is still debated via which mechanism homocysteine (Hcy) causes vascular disease. S-adenosylhomocysteine hydrolase (AHCY) catalyses the reversible hydrolysis of S-adenosylhomocysteine (AdoHcy) to Hcy. As an increase in AdoHcy, a strong inhibitor of many methyltransferases, is observed in hyperhomocysteinemic individuals, AdoHcy may play a role in the development of cardiovascular diseases by inhibiting transmethylation reactions. We sequenced the entire coding region and parts of the untranslated regions (UTRs) of the AHCY gene of 20 patients with recurrent venous thrombosis in order to identify genetic variation within this gene. We identified three sequence variants in the AHCY gene: a C > T transition in the 5' UTR (-34 bp C > T), a missense mutation in exon 2, which mandates an amino-acid conversion at codon 38 (112 C > T; Arg38Trp) and a silent mutation in exon 4 (390 C > T; Asp130Asp). We studied the effect of the first two variants on total plasma Hcy and venous thrombosis risk in a case-control study on recurrent venous thrombosis. The two polymorphisms under study seem to have no evident effect on tHcy. The adjusted relative risk of venous thrombosis associated with the 112CT genotype compared with 112CC individuals was 1.27 (95% CI 0.55-2.94), whereas the -34CT genotype confers a risk of 1.25 (95% CI 0.44-3.52) compared with the wild-type genotype at this locus. However, the wide confidence intervals do not allow firm conclusions to be drawn.     

Validation of an immunoassay for measurement of plasma total homocysteine

Hyperhomocysteinemia is an evolving cardiovascular risk factor. It is imperative that a simple, precise, and accurate assay be available in the clinical laboratory. The aim of this study was to evaluate an automated fluorescence polarization immunoassay for homocysteine. The assay had excellent precision at normal and high levels (intra-assay and interassay coefficients of variation < 5%). The method was linear from 0.24 to 50 mumol/L and displayed good correlation with a high-performance liquid chromatography (HPLC) method. There was no significant interference detectable in icteric and hyperlipidemic samples, but hemolysis resulted in a significant negative bias. While homocysteine levels were not increased in smokers, patients with renal failure had significantly higher levels compared with control subjects. This automated assay requires no sample preparation, displays excellent precision, shows good correlation with HPLC, and, thus, is favored over HPLC for use in the clinical laboratory. The main indications for measuring plasma homocysteine levels will be in the early diagnosis of cobalamin deficiency, patients with cardiovascular disease and few or no established risk factors, and patients with unexplained venous thromboembolic disease.     

Lack of consistent association between endothelial nitric oxide synthase gene polymorphisms, homocysteine levels and recurrent pregnancy loss in tunisian women

PROBLEM: Polymorphisms of the endothelial nitric oxide synthase (eNOS) gene have been associated with reduced vascular NO production or increased level of homocysteine, and evaluated as risk factors for recurrent pregnancy loss (RPL). Therefore, in this case-control study, we aimed to determine the effects of some eNOS functional polymorphisms: the 27-bp intron 4 repeat, the 894G/T of exon 7, and the promoter substitution -786T/C, in women with RPL. METHOD OF STUDY: We genotyped 350 patients with RPL and 200 healthy women by polymerase chain reaction (PCR) and restriction fragment length polymorphism-PCR (RFLP-PCR). The homocysteine total plasma concentrations (tHcy) were determined by enzyme-linked immunosorbent assay (ELISA). RESULTS: None of the eNOS polymorphisms-related alleles, genotypes, and haplotypes were associated with RPL. The tHcy were similar between patients and controls; no significant association between tHcy levels and eNOS genotypes could be evidenced. CONCLUSION: The present study identified a lack of association between eNOS gene polymorphisms, the risk of RPL and tHcy levels.     

Influence of pepsinogen gene polymorphisms on serum pepsinogen

We identified pepsinogen C (PGC) gene polymorphisms by means of PCR, which amplified DNA in the region within the intron between exons 7 and 8, and by 6% polyacrylamide gel electrophoresis. Six alleles were found in a Japanese population. The frequencies of these alleles in 408 unrelated Japanese individuals were 0·074, 0·026, 0·335, 0·237, 0·016 and 0·314, respectively. The serum pepsinogen II level significantly decreased in the order of the allele 6 homozygote, the allele 6 heterozygote and the other genotypes (χ²=7·850, d.f. =2, p =0·020). These findings indicated that the genetic background of serum pepsinogen should be considered when screening for stomach cancer by this procedure.     

Multicenter analytical evaluation of an automated immunoassay for total plasma homocysteine

A fully automated immunoassay for total plasma homocysteine assay was evaluated at four centers. To measure total homocysteine, oxidized forms of homocysteine in serum and plasma were reduced by dithiothreitol and assayed by a competitive fluorescence polarization technique. The assay had within-run precision from 0.9 to 3.0% and total precision from 2.8 to 4.1% for control materials with homocysteine concentrations of approximately 7, 12.5, and 25 micromol/L, a sensitivity of 0.35 micromol/L, good parallelism upon dilution, and analytical recovery ranging from 97.4 to 103.8%. The immunoassay correlated with four different HPLC assays for homocysteine, yielding a slope of 0.98, an intercept of -0.19 micromol/L, and a correlation coefficient of 0.966 for 440 paired samples. The reference range, determined with plasma samples from 609 males and 600 females, yielded a mean of 9.17+/-2.86 micromol/L, with a central 95% range of 4.78-15.43 micromol/L. The immunoassay is a suitable alternative to HPLC and may be useful in screening persons with high risk of coronary artery disease.     

Endothelial nitric oxide synthase gene polymorphisms in Fabry's disease

The gene encoding endothelial nitric oxide synthase (eNOS) is involved in abnormalities in nitric oxide (NO) synthesis that mediates functional damage of vascular cells, especially of endothelial cells (ECs), a common characteristic in cardiovascular diseases. In Fabry's disease, the characteristic mutation in the alpha-galactosidase A (alpha-gal A) gene induces large deposits of glycosphingolipids, particularly concentrated in ECs, a process associated with endothelial dysfunction. To determine whether in addition to alpha-gal A gene mutations, eNOS genetic variations are implicated in this process, we examined the genotypes of the missense Glu298Asp (G894T) variant in exon 7 and 27-bp tandem repeats in intron 4 (4b/a) in 19 patients with Fabry's disease, and 39 normal volunteers. The results showed that both varials have a significant association with Fabry's disease. The frequencies of mutant Glu/Asp + Asp/Asp genotypes and Asp allele are significantly higher in Fabry's disease (68.4%, p = 0.044, and 47.4%, p = 0.022, respectively) than in controls (46.7% and 25%, respectively). The frequencies of eNOS 4b/a polymorphisms are also significantly different in Fabry's disease when compared to controls. The mutant 4b/a + 4a/a genotype frequencies are 55.5% (p = 0.032) and 4a allele 27.8% (p = 0.05) compared with controls (23.1% and 12.8%, respectively). These results indicate that more than half of the patients with Fabry's disease carry the Glu298Asp variant ( approximately 68%) and/or the 4b/a polymorphism ( approximately 55%). To the best of our knowledge, this is the first report showing an influence of eNOS gene polymorphisms in patients with Fabry's disease.     

Thymidylate synthase gene polymorphisms and markers of DNA methylation capacity

BackgroundDNA methylation plays a critical role in gene regulation and has been implicated in the etiology of chronic disease including atherosclerosis, neural degeneration and cancer. One-carbon metabolism serves two critically important functions: one concerning the production of purines and thymidine for DNA synthesis and the other related to the provision of methyl groups through the metabolism of methionine. Critical intermediates of methionine metabolism relevant to DNA methylation include S-adenosylmethionine (SAM), a universal methyl donor, and S-adenosylhomocysteine (SAH), a potent inhibitor of most methylation reactions. Thymidine synthesis, catalyzed by the crucial enzyme thymidylate synthase (TS), competes with methionine metabolism for a common substrate. Three functional polymorphisms in the TS gene have been identified including: (i) the thymidylate synthase enhancer region (TSER) tandem repeat polymorphism and (ii) the G to C single nucleotide polymorphism (G/C SNP) both of which occur in the 5′untranslated region (UTR) of the TS gene; and (iii) the 6-bp deletion at base pair 1494 (TS1494del6) located in the 3′UTR.PurposeThe purpose of this research was to investigate the relationship between TS polymorphisms and concentrations of SAM and SAH, markers of DNA methylation capacity.MethodsThe study population consisted of 395 healthy male and female volunteers from Kingston, Ontario and Halifax, Nova Scotia, Canada between 2006 and 2008. The effect of each TS polymorphism on SAM and SAH concentrations was investigated, and further analyses were conducted on categorization of polymorphisms based on 5′ or 3′UTR. The combined effect of TS polymorphisms on SAM and SAH concentrations was also investigated, in addition to interactions between polymorphisms in TS and MTHFR 677C>T and interactions between TS polymorphisms and serum folate and vitamin B₁₂ status.ResultsNo associations were observed between TS polymorphisms and concentrations of SAM and SAH. Analysis of interaction between TS and MTHFR polymorphisms on SAH levels revealed a significant interaction with TS 3′polymorphism and MTHFR C677T (p = 0.03). As well, interactions between TS 3′polymorphism and serum folate (p = 0.03) and the combined effect of TS polymorphisms and serum folate on SAH levels (p = 0.04) were found.ConclusionsThe findings of this research provide evidence that SAH, a marker of methylation capacity, is influenced by genetic and environmental factors and their interactions.     

Plasma homocysteine measurement: a study of pre-analytical variation factors for conditions for total plasma homocysteine concentration

An increase in homocysteine, a sulphur amino acid, is nowdays considered as a risk factor for cardiovascular diseases, and is independent of other risk factors. Reference range for total plasma homocysteine level in adults is usually 5-15 mmol/l. Hyperhomocysteinemia is defined as a fasting total plasma homocysteine level > 15 mmol/l. There may be also graded increased risks for subjects with homocysteinemia from 10 to 15 mmol/l. However, no threshold has been defined, partly because of the lack of standardization in pre-analytical and analytical steps. The aim of the present work was to evaluate three pre-analytical parameters on plasma homocysteine levels: i) the influence of three anticoagulants (EDTA, sodium citrate and lithium heparin); ii) the delay period of blood sample on ice before centrifugation; and iii) the advantages of strong acidic citrate at room temperature. The mean concentrations of total plasma homocysteine were different in function of the anticoagulant. These differences (EDTA minus lithium heparin or EDTA minus sodium citrate) were less than 10% however the used methods and could explain the good correlation between the results. However we recommend to keep the anticoagulant constant in the same study. When EDTA blood samples were immediately put on crushed ice, the maximum delay period before centrifugation could reach 4 hours. If ice is unavailable, strong acidic citrate at room temperature is a good alternative until for 4 hours.     

Structure and regulation of cysD, the homocysteine synthase gene of Aspergillus nidulans

The A. nidulans cysD gene encoding homocysteine synthase (O-acetyl-L-homoserine sulphydrylase) has been isolated by functional complementation of a cysD11 mutation. The gene contains five short introns and codes for a protein of 437 amino acids. The protein shows homology with bacterial and yeast O-acetyl- and O-succinyl-homoserine sulphydrylases, particularly from Schizosaccharomyces pombe, Saccharomyces cerevisiae and Kluyveromyces lactis. The cysD cDNA is able to complement a S. cerevisiae mutation impairing homocysteine synthase. Synthesis of the cysD mRNA is down-regulated by a high concentration of methionine in growth medium without sulphate and up-regulated under sulphur limitation. A comparison of cysD genomic and cDNA copies, derived from different A. nidulans strains, revealed a marked DNA-sequence polymorphism manifested mostly by silent point mutations. There was, however, much less polymorphism in the protein sequence. Received: 18 July / 27 October 1997     

Subunit complementation of thymidylate synthase in Plasmodium falciparum bifunctional dihydrofolate reductase-thymidylate synthase

Thymidylate synthase of Plasmodium falciparum dihydrofolate reductase-thymidylate synthase (PfDHFR-TS) functions as a dimeric enzyme with extensive contact between the two TS domains. Structural data of PfDHFR-TS shows that the formation of the two TS active sites involves contribution of the amino acid residues from both TS domains. Arg-470 donated from the adjoining domain is shown to hydrogen-bond to dUMP, while Cys-490 is a key nucleophile for TS catalysis by attacking C-6 of dUMP. However, mutants of the two series could complement one another, giving rise to active enzyme. By means of subunit complementation assay using Arg-470 and Cys-490 mutants, it is shown that co-transformants of both TS-inactive Arg-470 and Cys-490 mutants can complement the growth of thymidine auxotroph chi2913RecA(DE3) by formation of a functional TS heterodimer contributing from both Arg-470 and Cys-490 mutant subunits. 6-[3H]-FdUMP thymidylate synthase activity assay further elaborate the essence of restoration of TS activity. The TS k(cat) value of the R470D+C490A heterodimer is decreased by half from that of the wild-type PfDHFR-TS. However, the Km values for dUMP and CH2H4folate of the R470D+C490A heterodimer are similar to those of wild-type enzyme, indicating that the catalytic efficiency of the functional TS from the R470D+C490A heterodimer is similar to the wild-type TS enzyme in P. falciparum DHFR-TS.     

Assignment of human gene encoding thymidylate synthase to chromosome 18 using interspecific cell hybrids between thymidylate synthase-Negative mouse mutant cells and human diploid fibroblasts

We have constructed interspecific somatic cell hybrids between a thymidineauxotrophic mutant cell line of mouse FM3A cells that lacks thymidylate synthase and human diploid fibroblasts derived from a male patient with fragile X-linked mental retardation. Twenty primary hybrid clones were isolated independently, all of which exhibited the thymidine-prototrophic phenotype. Segregation of the hybrid cells in nonselective culture conditions gave rise to thymidine-auxotrophic hybrid clones. Both electrophoretic assay of thymidylate synthase activity and karyotype analysis of the segregants revealed a strong correlation between the expression of the human form of the enzyme and the presence of human chromosome 18. Thus, it is concluded that the functional gene for human thymidylate synthase, designated TS,is located on this chromosome.