Cystathionine Β-synthase Gene Research Articles

Hydrogen sulphide-related thiol metabolism and nutrigenetics in relation to hypertension in an elderly population

Hydrogen sulphide (H(2)S) is a gaseous signalling molecule that regulates blood flow and pressure. It is synthesised from cysteine via cystathionine β-synthase and cystathionine γ-lyase. We examined whether thiol precursors of H(2)S, transsulphuration pathway gene variants (CBS-844ins68 and CTH-G1364T) and key B-vitamin cofactors might be critical determinants of hypertension in an elderly Australian population. An elderly Australian retirement village population (n=228; age 65-96years, 91 males and 137 females) was assessed for the prevalence of two transsulphuration pathway-related variant genes associated with cysteine synthesis and hence H(2)S production. Thiols were determined by HPLC, genotypes by PCR and dietary intake by food frequency questionnaire. Homocysteine levels were statistically higher in the hypertensive phenotype (p=0.0399), but there was no difference for cysteine or glutathione. Using nominal logistic regression, cysteine, CTH-G1364T genotype, dietary synthetic folate and vitamin B(6) predicted clinical phenotype (determined as above/below 140/90mm Hg) and then only in female subjects (p=0.0239, 0.0178, 0.0249 and 0.0371, respectively). Least-squares regression supports cysteine being highly inversely predictive of diastolic blood pressure: p and r (2) values <0.0001 and 0.082; 0.0409 and 0.046; and <0.0001 and 0.113 for all subjects, males and females, respectively. Additionally, CTH-G1364T genotype predicts diastolic blood pressure in males (p=0.0217; r (2)=0.083), but contrasts with observations for females. Overall, analyses, including stepwise regression, suggest cysteine, dietary natural and synthetic folate, vitamins B(6) and B(12), and both genetic variants (CTH-C1364T and CBS-844ins68) are all aetiologically relevant in the regulation of blood pressure. Hydrogen sulphide is a vasorelaxant gasotransmitter with characteristics similar to nitric oxide. Cysteine and the G1364T and 844ins68 variants of the cystathionine γ-lyase and cystathionine β-synthase genes, respectively, are the biological determinants of H(2)S synthesis, and all three are shown here to influence the hypertensive phenotype. Additionally, B-vitamin cofactors for these three enzymes may also be important determinants of blood pressure.

Genetic polymorphisms modulate the folate metabolism of Brazilian individuals with Down syndrome

Individuals with Down syndrome (DS) carry three copies of the Cystathionine β-synthase (CβS) gene. The increase in the dosage of this gene results in an altered profile of metabolites involved in the folate pathway, including reduced homocysteine (Hcy), methionine, S-adenosylhomocysteine (SAH) and S-adenosylmethionine (SAM). Furthermore, previous studies in individuals with DS have shown that genetic variants in genes involved in the folate pathway influence the concentrations of this metabolism's products. The purpose of this study is to investigate whether polymorphisms in genes involved in folate metabolism affect the plasma concentrations of Hcy and methylmalonic acid (MMA) along with the concentration of serum folate in individuals with DS. Twelve genetic polymorphisms were investigated in 90 individuals with DS (median age 1.29 years, range 0.07-30.35 years; 49 male and 41 female). Genotyping for the polymorphisms was performed either by polymerase chain reaction (PCR) based techniques or by direct sequencing. Plasma concentrations of Hcy and MMA were measured by liquid chromatography-tandem mass spectrometry as previously described, and serum folate was quantified using a competitive immunoassay. Our results indicate that the MTHFR C677T, MTR A2756G, TC2 C776G and BHMT G742A polymorphisms along with MMA concentration are predictors of Hcy concentration. They also show that age and Hcy concentration are predictors of MMA concentration. These findings could help to understand how genetic variation impacts folate metabolism and what metabolic consequences these variants have in individuals with trisomy 21.

Brain phenotype of transgenic mice overexpressing cystathionine β-synthase.

BackgroundThe cystathionine β-synthase (CBS) gene, located on human chromosome 21q22.3, is a good candidate for playing a role in the Down Syndrome (DS) cognitive profile: it is overexpressed in the brain of individuals with DS, and it encodes a key enzyme of sulfur-containing amino acid (SAA) metabolism, a pathway important for several brain physiological processes.Methodology/Principal FindingsHere, we have studied the neural consequences of CBS overexpression in a transgenic mouse line (60.4P102D1) expressing the human CBS gene under the control of its endogenous regulatory regions. These mice displayed a ∼2-fold increase in total CBS proteins in different brain areas and a ∼1.3-fold increase in CBS activity in the cerebellum and the hippocampus. No major disturbance of SAA metabolism was observed, and the transgenic mice showed normal behavior in the rotarod and passive avoidance tests. However, we found that hippocampal synaptic plasticity is facilitated in the 60.4P102D1 line.Conclusion/SignificanceWe demonstrate that CBS overexpression has functional consequences on hippocampal neuronal networks. These results shed new light on the function of the CBS gene, and raise the interesting possibility that CBS overexpression might have an advantageous effect on some cognitive functions in DS.

Transcobalamin 2 variant associated with poststroke homocysteine modifies recurrent stroke risk

The Vitamin Intervention for Stroke Prevention trial found an association between baseline poststroke homocysteine (Hcy) and recurrent stroke. We investigated genes for enzymes and cofactors in the Hcy metabolic pathway for association with Hcy and determined whether associated single nucleotide polymorphisms (SNPs) influenced recurrent stroke risk. Eighty-six SNPs in 9 candidate genes (BHMT1, BHMT2, CBS, CTH, MTHFR, MTR, MTRR, TCN1, and TCN2) were genotyped in 2,206 subjects (83% European American). Associations with Hcy measures were assessed using linear regression models assuming an additive genetic model, adjusting for age, sex, and race and additionally for baseline Hcy when postmethionine load change was assessed. Associations with recurrent stroke were evaluated using survival analyses. Five SNPs in the transcobalamin 2 (TCN2) gene were associated with baseline Hcy (false discovery rate [FDR]-adjusted p = 0.049). TCN2 SNP rs731991 was associated with recurrent stroke risk in the low-dose arm of the trial under a recessive model (log-rank test p = 0.009, hazard ratio 0.34). Associations with change in postmethionine load Hcy levels were found with 5 SNPs in the cystathionine β-synthase (CBS) gene (FDR-adjusted p < 0.031). TCN2 variants contribute to poststroke Hcy levels, whereas variants in the CBS gene influence Hcy metabolism. Variation in the TCN2 gene also affects recurrent stroke risk in response to cofactor therapy.

Association between cystathionine β-synthase gene tagging single nucleotide polymorphisms and essential hypertension in the Kazakh population in Xijiang

近年研究发现血浆HCY水平升高与EH有着密切的联系.CBS和MTHFR是HCY代谢过程中的2个关键酶,除营养因素外,CBS和MTHFR基因缺陷对血浆总HCY水平的影响[1]可间接导致EH,这已成为研究热点;而CBS作为HCY代谢途径中的关键酶之一,当其缺陷时可导致高同型半胱氨酸血症.目前,许多研究表明,CBS基因多态性与心脑血管疾病发生有关[2].目前,有关SNP与疾病的关联研究,除在SNP位点选择上注意了功能区(启动子和外显子)外,由于不同连锁标记间还存在LD.为增加基因分型的有效性,还需进行tSNP与疾病的关联性研究。

Homocysteine homeostasis in the rat is maintained by compensatory changes in cystathionine β-synthase, betaine-homocysteine methyltransferase, and phosphatidylethanolamine N-methyltransferase gene transcription occurring in response to maternal protein and folic acid intake during pregnancy and fat intake after weaning

The reactions of the methionine/homocysteine pathway are mediated by several enzymes, including phosphatidylethanolamine N-methyltransferase, cystathionine β-synthase, and betaine-homocysteine methyltransferase. Homocysteine homeostasis is regulated by these enzymes. We hypothesized here that the protein and folic acid content in the maternal diet affects methionine/homocysteine metabolism in the progeny. To test this hypothesis, pregnant rats were fed a diet with normal protein and normal folic acid levels (a modified casein-based AIN-93G diet), a protein-restricted and normal folic acid diet, a protein-restricted and folic acid-supplemented diet, or a normal protein and folic acid-supplemented diet. The progeny were fed either the modified AIN-93G diet or a high-fat lard-based diet. Progeny were analyzed for expression of the phosphatidylethanolamine N-methyltransferase, cystathionine β-synthase, and betaine-homocysteine methyltransferase genes in the liver and for serum homocysteine concentration. Interactions between prenatal and postnatal nutrition were also determined. The progeny of the dams fed the diets supplemented with folic acid showed decreased expression of all 3 genes (P < .001). An interaction effect between the protein and folic acid content in the maternal diet contributed to this down-regulation (P < .001), and the postweaning diet modified these effects. Serum homocysteine concentrations were approximately 15% higher in the male rats (P < .01), but neither prenatal nutrition nor the postweaning diet affected it significantly. We conclude that maternal diet during gestation has an important effect on the transcription level of these 3 genes, but changes in gene expression were not associated with significant changes in progeny homocysteine concentrations.

Genetic polymorphisms of the enzymes involved in DNA methylation and synthesis in elite athletes

Physical exercise induces adaptive changes leading to a muscle phenotype with enhanced performance. We first investigated whether genetic polymorphisms altering enzymes involved in DNA methylation, probably responsible of DNA methylation deficiency, are present in athletes' DNA. We determined the polymorphic variants C667T/A1298C of 5,10-methylenetetrahydrofolate reductase (MTHFR), A2756G of methionine synthase (MTR), A66G of methionine synthase reductase (MTRR), G742A of betaine:homocysteine methyltransferase (BHMT), and 68-bp ins of cystathionine β-synthase (CBS) genes in 77 athletes and 54 control subjects. The frequency of MTHFR (AC), MTR (AG), and MTRR (AG) heterozygous genotypes was found statistically different in the athletes compared with the control group (P=0.0001, P=0.018, and P=0.0001), suggesting a reduced DNA methylating capacity. We therefore assessed whether DNA hypomethylation might increase the expression of myogenic proteins expressed during early (Myf-5 and MyoD), intermediate (Myf-6), and late-phase (MHC) of myogenesis in a cellular model of hypomethylated or unhypomethylated C2C12 myoblasts. Myogenic proteins are largely induced in hypomethylated cells [fold change (FC)=Myf-5: 1.21, 1.35; MyoD: 0.9, 1.47; Myf-6: 1.39, 1.66; MHC: 1.35, 3.10 in GMA, DMA, respectively] compared with the control groups (FC=Myf-5: 1.0, 1.38; MyoD: 1.0, 1.14; Myf-6: 1.0, 1.44; MHC: 1.0, 2.20 in GM, DM, respectively). Diameters and length of hypomethylated myotubes were greater then their respective controls. Our findings suggest that DNA hypomethylation due to lesser efficiency of polymorphic MTHFR, MS, and MSR enzymes induces the activation of factors determining proliferation and differentiation of myoblasts promoting muscle growth and increase of muscle mass.

Novel alleles of 31-bp VNTR polymorphism in the human cystathionine β-synthase (CBS) gene were detected in healthy Asians

A 31-bp variable number of tandem repeats (VNTR) polymorphism of the cystathionine β-synthase (CBS) gene was earlier reported in Caucasians of predominantly European descent and Indo-Caucasoid populations. We report here for the first time, the detection of allele 20, which was absent in Caucasian and Indo-Caucasoid populations, as a common allele present in Singaporean Chinese (6.25%), Indians (11.7%), and Malays (11.5%). Hence, allele 20 might be a specific allele for Asian populations. A relatively common allele 19 found in the Caucasian and Indo-Caucasoid populations (10.4%-10.6%) was absent in the Asian samples of this study. Therefore, allele 19 might be a specific allele for the Caucasian populations. A novel and rare allele 13, which was not reported before in the Caucasian and Indo-Caucasoid populations, was found in 0.5% of Singaporean Chinese as genotype 13/17 heterozygotes. The presence of alleles 13 and 20 were verified by DNA sequencing. There were five new genotypes (13/17, 16/20, 17/20, 18/20 and 20/20) not reported before in the Caucasian and Indo-Caucasoid populations, detected in this study. Nine genotypes (15/18, 16/18, 16/21, 17/19, 18/19, 18/21, 19/19, 19/21 and 21/21) which were present in the Caucasian and/or Indo-Caucasoid populations were absent in this study. Our results showed that CBS 31-bp VNTR polymorphism has a distinct genetic difference in allele and genotype frequencies between the European Caucasians, Indo-Caucasoid and Asian populations.

Association of Methylenetetrahydrofolate Reductase C677T and Cystathionine β-Synthase Polymorphisms in Cardiovascular Disease in the Algerian Population

Polymorphisms in the methylenetetrahydrofolate reductase (MTHFR) and cystathionine β-synthase (CBS) genes, involved in the intracellular metabolism of homcysteine, can result in hyperhomocysteinemia. The objective of this study was to evaluate prevalence estimates of MTHFR C677T and the CBS insertion of 68-bp (844ins68) polymorphisms among individuals with cardiovascular disease (CVD). In total, 131 patients (61 men and 70 women) were hospitalized in the Cardiology Department in CHU of Sétif, Algeria. The control group included 147 apparently healthy adults (82 women and 65 men). The genetic analysis of the MTHFR C677T polymorphism was performed by real-time polymerase chain reaction on a Light Cycler; the CBS genotype was analyzed by polymerase chain reaction in a thermal cycler. The frequency of the TT genotype was 16.1% in the patient group and 14.3% in the control group. The CT genotype constituted 43.5% and 40.1% in the patient group and the control group, respectively. There was no significant difference in the occurrence of the TT genotype between the studied groups. The frequency of C677T/MTHFR in male and female patients was 16.4% and 15.7% for the TT genotype, respectively. There was no significant difference in T allele frequencies between sexes. However, the frequency of C677T homozygotes in the patients was higher in men with CVD than that in corresponding control subjects (40.2% vs. 29.2%), but the difference was not statistically significant. The coexistence of the MTHFR 677TT genotype and the common CBS 844ins68 variant was lower among patients. The MTHFR C677T and CBS 844ins68 variants tested in this study, individually or combined, are not associated with CVD in the Algerian population.

Frequency of Prothrombotic Risk Factors in Patients with Deep Venous Thrombosis and Controls: Their Implications for Thrombophilia Screening in Chilean Subjects

In this work, we evaluated the frequency of prothrombotic defects associated with deep venous thrombosis (DVT) in southern Chilean subjects. A total of 261 individuals, 87 patients with DVT confirmed by Doppler ultrasonography and 174 controls, were included in this study. Factor V and factor VIII levels, activated protein C (APC) resistance, and lupus anticoagulant detection were assayed by clotting methods. Basal homocysteine was quantified by immunoassay, and the polymorphisms in factor V (F5), methylenetetrahydrofolate reductase (MTHFR), and cystathionine β-synthase (CBS) genes were genotyped by molecular methods. The most frequent defects were APC resistance, hyperhomocysteinemia, and increased levels of factor VIII. We observed a complete absence of the F5 G1691A variant in the studied population, and the frequency of MTHFR C677T polymorphism was significantly different between patients and controls (odds ratio = 3.2; 95% confidence interval, 1.513-6.735; p = 0.016). In addition, subjects carrying the homozygous MTHFR 677TT genotype exhibited higher levels of plasma homocysteine. Our data suggest that the APC resistance is the most important defect in Chilean patients with DVT. However, this phenotype is not associated with the presence of the F5 G1691A variant. In addition, only MTHFR C677T polymorphism constituted a molecular biomarker of DVT in Chilean population.

G runs in Cystathionine β-Synthase c.833C/c.844_845ins68 mRNA are splicing silencers of pathogenic 3′ splice sites

The c.844_845ins68 is an evolutionary conserved polymorphism of the Cystathionine β-Synthase gene that segregates with the pathogenic c.833C mutation and consists of a 68nt insertion duplicating the 3′ splice site between intron 7 and exon 8. The gene rearrangement brought two GGGG runs close to each other and generated a splicing control element that allows the constitutive selection of the more distal 3′ splice site in the c.844_854ins68 carriers. In this study, we have characterized functionally the two G4 runs within the duplication and have found that they work as silencers of the upstream potentially pathogenic 3′ splice sites has been functionally characterized. This selection allows skipping of both the 68nt-insertion and the c.833C mutation, and is essential to preserve the wild-type ORF. Knocking down hnRNP H and F expression modulated the rescue of the proximal 3′ splice site more than hnRNP H alone. These observations suggest that hnRNP H/F contribute jointly to prevention of CBS deficiency in c.844_854ins68 carriers by silencing the potentially pathogenic upstream acceptor site.

Polymorphisms of 5,10-Methylenetetrahydrofolate Reductase and Cystathionine β-Synthase Genes as a Risk Factor for Neural Tube Defects in Sétif, Algeria

Background: Neural tube defects (NTD) are severe congenital malformations due to a failure in neural tube formation at the beginning of pregnancy. The etiology of NTD is multifactorial, with environmental and genetic determinants. We suggest a study of gene-gene interactions regarding the possible association of NTD with specific mutations of 5,10-methylenetetrahydrofolate reductase (MTHFR) and cystathionine β-synthase (CBS) genes. Patients and Methods: The genetic analysis of the MTHFR C677T polymorphism was performed by real-time polymerase chain reaction (PCR) on a Light Cycler, the CBS genotype was analyzed by PCR in a thermal cycler. Ninety-two mothers who had conceived NTD children and 48 fathers were investigated. A group of 147 adults, including 82 apparently healthy women, was used as control. Results: Among control mothers, 35 (43%) were heterozygous for the C677T variant and 14 (17%) were TT homozygous. Among the cases, 25 (52%) out of 48 mothers and 22 (46%) out of 48 fathers carried the T allele; 9 mothers (19%) and 5 fathers (10%) had the TT genotype. A homozygous C677T mutation was not an NTD risk factor in this preliminary study in an Algerian population; a possible gene-gene interaction between the MTHFR C677T polymorphism and the CBS 844ins68 has also been examined in relation to NTD, but no such association has been shown. There was a statistically significant difference between the heterozygosity genotype frequency of CBS polymorphisms in mothers with a previous child with NTD compared with the mother controls (odds ratio: 3.72; 95% CI: 1.59–8.73). Conclusion: Our results with Algerian NTD mothers did not show a significant association for any group, suggesting that the thermolabile variant C677T in the MTHFR gene is not a risk factor for a mother to have NTD offspring; rather, folic acid supplementation or fortification should become mandatory for all women of reproductive age in Algeria.

A Novel Mutation of Cystathionine β-synthase Gene in a Thai Boy With Homocystinuria

The authors reported a 14-year-old boy who presented with multiple organs thrombosis and subluxation of lens. His diagnosis of homocystinuria was delayed owing to the unrecognition of the disease resulting in significant morbidity. The mutation analysis showed one novel mutation that can explain the high level of plasma homocysteine.

Cystathionine beta-synthase p.S466L mutation causes hyperhomocysteinemia in mice.

Missense mutations in the cystathionine β-synthase (CBS) gene are the most common cause of clinical homocystinuria in humans. The p.S466L mutation was identified in a homocystinuric patient, but enzymatic studies with recombinant protein show this mutant to be highly active. To understand how this mutation causes disease in vivo, we have created mice lacking endogenous mouse CBS and expressing either wild-type (Tg-hCBS) or p.S466L (Tg-S466L) human CBS under control of zinc inducible metallothionein promoter. In the presence of zinc, we found that the mean serum total homocysteine (tHcy) of Tg-S466L mice was 142 ± 55 µM compared to 16 ± 13 µM for hCBS mice. Tg-S466L mice also had significantly higher levels of total free homocysteine and S-adenosylhomocysteine in liver and kidney. Only 48% of Tg-S466L mice had detectable CBS protein in the liver, whereas all the Tg-hCBS animals had detectable protein. Surprisingly, CBS mRNA was significantly elevated in Tg-S466L animals compared to Tg-hCBS, implying that the reduction in p.S466L protein was occurring due to posttranscriptional mechanisms. In Tg-S466L animals with detectable liver CBS, the enzyme formed tetramers and was active, but lacked inducibility by S-adenosylmethionine (AdoMet). However, even in Tg-S466L animals that had in vitro liver CBS activity equivalent to Tg-hCBS animals there was significant elevation of serum tHcy. Our results show that p.S466L causes homocystinuria by affecting both the steady state level of CBS protein and by reducing the efficiency of the enzyme in vivo. Hum Mutat 29(8), 1048–1054, 2008. © 2008 Wiley-Liss, Inc.

Origin and evolution of the c.844_845ins68/c.833T>C mutations within the cystathionine β-synthase gene in great apes

The c.[833C; 844_845ins68] is a common haplotype of the human cystathionine β-synthase gene among healthy individuals. This polymorphism (5–40% allelic frequency in different populations) consists of the c.844_845ins68 insertion that segregates in cis with the pathogenic c.833T>C substitution (p.I278T). Through genotyping of primates, we have found that gorillas, chimpanzees and bonobos are homozygous for the 68 bp insertion, c.844_845ins68. In gorillas and bonobos, the c.844_845ins68 lesion segregates in cis with the wild-type c.833T variant, whilst chimpanzees present the human haplotype. These genetic evidences suggest that the origin of the 68 bp insertion might be dated back to 6–8 million years ago, and that the c.833T>C substitution occurred within the allele carrying the insertion. The evolutionary conservation of this peculiar haplotype supports the hypothesis of its protective effects against cardiovascular diseases.

Enhanced susceptibility to arterial thrombosis in a murine model of hyperhomocysteinemia

AbstractHyperhomocysteinemia is a risk factor for thrombosis, but the mechanisms are not well defined. We tested the hypothesis that hyperhomocysteinemia accelerates arterial thrombosis in mice. Mice heterozygous for a targeted disruption of the cystathionine β-synthase gene (Cbs+/–) and wild-type littermates (Cbs+/+) were fed either a control diet or a high methionine/low folate (HM/LF) diet for 6 to 8 months to produce graded hyperhomocysteinemia. The time to occlusion of the carotid artery after photochemical injury was shortened by more than 50% in Cbs+/+ or Cbs+/– mice fed the HM/LF diet (P < .001 versus control diet). Carotid artery thrombosis was not accelerated in mice deficient in endothelial nitric oxide synthase (Nos3), which suggests that decreased endothelium-derived nitric oxide is not a sufficient mechanism for enhancement of thrombosis. Cbs+/+ and Cbs+/– mice fed the HM/LF diet had elevated levels of reactive oxygen species in the carotid artery, increased aortic expression of the NADPH oxidase catalytic subunit, Nox4, and decreased activation of anticoagulant protein C in the aorta (P < .05 versus control diet). We conclude that hyperhomocysteinemia enhances susceptibility to arterial thrombosis through a mechanism that is not caused by loss of endothelium-derived nitric oxide but may involve oxidative stress and impairment of the protein C anticoagulant pathway.

Detection and allele-frequencies of the 833T>C, 844ins68 and a novel mutation in the cystathionine β-synthase gene

Background The most common 833T>C/844ins68 in cis double mutation in the cystathionine beta synthase (CBS) gene probably is non-pathogenic because the 68-bp insertion eliminates the 833T>C mutation due to alternative splicing. However, allele frequency and effects of the isolated 833T>C mutation are unclear. Method DNA was isolated from 500 volunteers and used directly for PCR-RFLP of CBS gene exon-8. A new primer design was developed to create annealing sites upstream and downstream of exon-8 for forward and reverse primers, respectively. The design was made to exclude sequence homology of the forward primer with the insert fragment and to introduce an internal Bsr1 digestion site. Results A new 9276G>A mutation was found in intron 8. Because of this mutation, an extra Bsr1 digestion site is created in intron 8. In Caucasian volunteers, the following allele frequencies were found: 833T>C=0.2%, 833T>C/844ins68=10.2%, and 9276G>A=0.2%. Conclusion The developed PCR-RFLP method is able to detect the 833T>C mutation, the 833T>C/844ins68 polymorphism as well as a new 9276G>A mutation in intron 8. Further study should explore the effect of the isolated 9276G>A mutation.

Hyperhomocysteinemic Mice Have Increased Susceptibility to Carotid Artery Thrombosis.

Hyperhomocysteinemia is a risk factor for cardiovascular disease, stroke, and venous thrombosis. In animal models, hyperhomocysteinemia produces endothelial dysfunction due to decreased bioavailability of endothelium-derived nitric oxide (NO). Because NO has antithrombotic properties, we tested the hypothesis that hyperhomocysteinemia accelerates photochemically-induced carotid artery thrombosis induced in mice. Mice heterozygous for a targeted disruption of the cystathionine β-synthase (Cbs) gene (Cbs+/−) and wild type littermates (Cbs+/+) were fed either a control diet or a high methionine/low folate (HM/LF) diet for 8 months. Plasma total homocysteine was elevated by the HM/LF diet compared with the control diet in both Cbs+/+ (11.6±1.2 vs. 3.7±0.5 μmol/L; p<0.05) and Cbs+/− mice (26.4±3.8 vs. 6.2±0.6 μmol/L; p<0.001). The time to stable occlusion after photochemical injury (rose bengal and green laser) of the carotid artery was 50% shorter in mice fed the HM/HF diet (17.5±2.3 and 18.2±4.0 minutes for Cbs+/+ and Cbs+/− mice, respectively) compared with mice fed the control diet (44.0±9.1 and 31.4±4.7 minutes for Cbs+/+ and Cbs+/− mice, respectively; p<0.001). Using the oxidative dye, dihydroethidium (DHE), increased production of superoxide was detected in Cbs+/+ and Cbs+/− mice fed the HF/HM diet compared with the control diet. To determine whether lack of endothelium-derived NO may accelerate carotid artery thrombosis, mice deficient in endothelial nitric oxide synthase (Nos3−/ −) were also studied. Time to occlusion did not differ between Nos3−/ − mice and wild type mice (55.0±10.2 vs. 42.1±7.1 minutes, p=0.31). We conclude that hyperhomcysteinemia increases susceptibility to experimental thrombosis through a mechanism that is independent of endothelium-derived NO, but may involve oxidative stress.

Cerebral vascular dysfunction mediated by superoxide in hyperhomocysteinemic mice.

Hyperhomocysteinemia is an emerging risk factor for stroke, but little is known about effects of hyperhomocysteinemia on cerebral vascular function. We tested the hypothesis that chronic hyperhomocysteinemia in mice causes endothelial dysfunction in cerebral arterioles through a mechanism that involves superoxide. Mice heterozygous for a targeted disruption of the cystathionine beta-synthase gene (Cbs+/-) and their wild type littermates (Cbs+/+) were fed either a control diet or a high-methionine diet for 10 to 12 months. Plasma total homocysteine was elevated with the high-methionine diet compared with the control diet for both Cbs+/+ (7.9+/-1.0 versus 5.0+/-0.5 micromol/L; P<0.05) and Cbs+/- (20.5+/-3.1 versus 8.2+/-0.9 micromol/L; P<0.001) mice. Dilatation of cerebral arterioles ( approximately 30 microm baseline diameter) was measured in vivo in response to the endothelium-dependent dilator acetylcholine or the endothelium-independent dilator nitroprusside. Vasodilatation to acetylcholine was impaired with the high-methionine diet compared with the control diet for both Cbs+/+ and Cbs+/- mice (P<0.01). Dilatation of arterioles to acetylcholine was restored toward normal by the superoxide scavenger tiron (P<0.05). Vasodilatation to nitroprusside was not influenced by Cbs genotype or diet. Dihydroethidium (DHE) staining for vascular superoxide was elevated in Cbs+/- mice fed the high-methionine diet and was inhibited by apocynin or Nomega-nitro-l-arginine methyl ester, implicating NAD(P)H oxidase and nitric oxide synthase as potential sources of superoxide. Superoxide is a key mediator of endothelial dysfunction in the cerebral circulation during diet-induced hyperhomocysteinemia.

Characterization of cystathionine β-synthase gene mutations in homocystinuric Venezuelan patients: identification of one novel mutation in exon 6

This study describes for the first time the cystathionine β-synthase (CBS) gene mutations in Venezuelan patients. A total of five disease-causing mutations were identified in 9 out of 10 independent chromosomes. Four of the mutations have been previously described (G85R, T191M, D234N, and D444N) and a novel mutation was found (Q243X). Two common polymorphisms (699C/T and 1080C/T) were found in the CBS gene. Mutation analysis was performed using a combined screening approach for CBS mutations: restriction analysis, single-strand conformational polymorphism (SSCP) scanning, and sequencing. All the mutations were detected in homozygous state, except for Q243X, detected in three heterozygous siblings. Each one of the patients studied presented a different mutation. All mutations and polymorphisms detected involved hypermutable CpG sites, except for the novel mutation Q243X. The most common mutations I278T and G307S were not found in any of the patients. The CBS mutations present in each country differ from each other depending on the demographic profile; therefore, specific mutations scanning must be performed in each population for diagnosis and prognosis purposes.