Progressive cerebral edema associated with high methionine levels and betaine therapy in a patient with cystathionine β‐synthase (CBS) deficiency

Cystathionine β‐synthase (CBS) deficiency, the most common form of homocystinuria, is an autosomal recessive inborn error of homocysteine metabolism. Treatment of B6‐nonresponsive patients centers on lowering homocysteine and its disulfide derivatives (tHcy) by adherence to a methionine‐restricted diet. However, lifelong dietary control is difficult. Betaine supplementation is used extensively in CBS‐deficient patients to lower plasma tHcy. With betaine therapy, methionine levels increase over baseline, but usually remain below 1,500 μmol/L, and these levels have not been associated with adverse affects. We report a child with B6‐nonresponsive CBS deficiency and dietary noncompliance whose methionine levels reached 3,000 μmol/L on betaine, and who subsequently developed massive cerebral edema without evidence of thrombosis. We investigated the etiology by determining methionine and betaine metabolites in our patient, and several possible mechanisms for her unusual response to betaine are discussed. We conclude that the cerebral edema was most likely precipitated by the betaine therapy, although the exact mechanism is uncertain. This case cautions physicians to monitor methionine levels in CBS‐deficient patients on betaine and to consider betaine as an adjunct, not an alternative, to dietary control. © 2002 Wiley‐Liss, Inc.     

The use of betaine in the treatment of elevated homocysteine

Elevation of homocysteine is implicated in multiple medical conditions, including classical homocystinuria, a variety of remethylation disorders, and most recently in coronary artery disease. Betaine is a methyl donor agent that is beneficial in lowering homocysteine through the remethylation of methionine. Betaine therapy alone has been shown to prevent vascular events in homocystinuria and may have clinical benefits in other hyperhomocysteinemic disorders when used as adjunctive therapy. Betaine does raise the methionine level and cerebral edema has occurred when plasma methionine exceeds 1000 micromol/L. Thus the plasma methionine as well as homocysteine must be monitored in patients receiving betaine.     

Gene-gene interaction between the cystathionine beta-synthase 31 base pair variable number of tandem repeats and the methylenetetrahydrofolate reductase 677C > T polymorphism on homocysteine levels and risk for neural tube defects

INTRODUCTION: Most studies showed that mothers of children with NTD have elevated homocysteine levels pointing to a disturbed homocysteine metabolism as a risk factor for NTD. Folate lowers homocysteine levels by remethylation of homocysteine to methionine. Homocysteine can be irreversibly converted to cystathionine by the vitamin B6-dependent enzyme CBS. Recently, our group showed that a 31 bp VNTR in the CBS gene was associated with decreased CBS activity and increased tHcy levels after methionine loading in a CVD population. AIM: The aim of our study was to investigate whether this VNTR influences tHcy levels and risk for NTD. In addition, we assessed the role of vitamin B6 as an effect modifier in this possible interaction. We examined possible gene-gene interaction with the MTHFR 677C > T polymorphism. We screened genomic DNA of 88 NBD patients, 100 mothers, 88 fathers, and 505 controls for this CBS 31 bp VNTR. RESULTS: In this study population five different alleles with 16,17, 18, 19, and 21 times the 31 bp repeat were observed that constituted 10 different genotypes. The most common 18/18 VNTR genotype was associated with higher tHcy levels compared with the 17/18 and 18/19 VNTR genotypes. Vitamin B6 levels did not influence this association. In addition, no association with risk for NTD was found. Combination of the CBS VNTR with the MTHFR 677C > T polymorphism revealed an additional increase in homocysteine levels in 18-18 individuals compared with 17-18 peers within subjects homozygous mutant for the MTHFR 677C > T polymorphism. CONCLUSIONS: The present study indicates that the number of 31 bp repeat elements in the CBS gene influences tHcy levels. This VNTR seems not to be associated with an increased risk for NTD.     

Disposition of homocysteine in subjects heterozygous for homocystinuria due to cystathionine beta-synthase deficiency: relationship between genotype and phenotype

We have investigated 31 subjects from five unrelated families with one or more members with cystathionine beta-synthase (CBS) deficiency. On the basis of their CBS genotype, the subjects were grouped as normal (n = 11) or heterozygotes (n = 20). Based on pyridoxine effect in the probands, the heterozygotes were further classified as pyridoxine-responsive (n = 9) or non-responsive (n = 11). Heterozygous subjects had normal fasting total plasma homocysteine (tHcy), but median urinary tHcy excretion rate was significantly elevated compared to healthy controls (0.39 micromol/h vs 0.24 micromol/h, P < 0.05). An abnormal tHcy response after methionine loading identified 73% of the pyridoxine non-responsive heterozygotes, but only 33% of the pyridoxine responsive participants. The increase in cystathionine or the change in tHcy relative to cystathionine did not improve diagnostic accuracy of the methionine loading test. After Hcy loading, the maximal increase in tHcy was significantly elevated, whereas t(1/2) was normal in heterozygotes. In conclusion, a single biochemical test cannot discriminate CBS heterozygotes from controls. Abnormal tHcy response after methionine loading was the most sensitive test. Our data suggest that the urinary tHcy excretion rate is a simple, non-invasive approach for studying mild disturbances in Hcy metabolism.     

Prevention of brain disease from severe 5,10-methylenetetrahydrofolate reductase deficiency

Over a four-year period, we collected clinical and biochemical data from five Amish children who were homozygous for missense mutations in 5,10-methylenetetrahydrofolate reductase (MTHFR c.1129C>T). The four oldest patients had irreversible brain damage prior to diagnosis. The youngest child, diagnosed and started on betaine therapy as a newborn, is healthy at her present age of three years. We compared biochemical data among four groups: 16 control subjects, eight heterozygous parents, and five affected children (for the latter group, both before and during treatment with betaine anhydrous). Plasma amino acid concentrations were used to estimate changes in cerebral methionine uptake resulting from betaine therapy. In all affected children, treatment with betaine (534+/-222 mg/kg/day) increased plasma S-adenosylmethionine, improved markers of tissue methyltransferase activity, and resulted in a threefold increase of calculated brain methionine uptake. Betaine therapy did not normalize plasma total homocysteine, nor did it correct cerebral 5-methyltetrahydrofolate deficiency. We conclude that when the 5-methyltetrahydrofolate content of brain tissue is low, dietary betaine sufficient to increase brain methionine uptake may compensate for impaired cerebral methionine recycling. To effectively support the metabolic requirements of rapid brain growth, a large dose of betaine should be started early in life.     

Expression of mutant human cystathionine beta-synthase rescues neonatal lethality but not homocystinuria in a mouse model

Cystathionine beta-synthase (CBS) deficiency is a recessive genetic disorder in humans characterized by elevated levels of total plasma homocysteine (tHcy) and frequent thrombosis in humans. The I278T mutation is the most common mutation found in human CBS-deficient patients. The T424N mutation was identified as a mutation in human CBS that could restore function to I278T in Saccharomyces cerevisiae. In this report, we have engineered mice that express human I278T and I278T/T424N proteins from a metallotheinein-driven transgene. These transgene-containing mice were then bred to CBS knockout animals (Cbs-) to generate mice that express only human I278T or I278T/T424N protein. Both the I278T and the I278T/T424N transgenes are able to entirely rescue the previously described neonatal mortality phenotype despite the animals having a mean tHcy of 250 microm. The transgenic Cbs-/- animals exhibit facial alopecia, have moderate liver steatosis and are slightly smaller than heterozygous littermates. In contrast to human CBS deficiency, these mice do not exhibit extreme methioninemia. The mutant proteins are stable in the liver, kidney and colon, and liver extracts have only 2-3% of the CBS enzyme activity found in wild-type mice. Surprisingly, the I278T/T424N enzyme had exactly the same activity as the I278T enzyme indicating that T424N is unable to suppress I278T in mice. Our results show that elevated tHcy per se is not responsible for the neonatal lethality observed in Cbs-/- animals and suggests that CBS protein may have a function in addition to its role in homocysteine catabolism. These transgenic animals should be useful in the study of homocysteine related human disease.     

Large-scale population-based metabolic phenotyping of thirteen genetic polymorphisms related to one-carbon metabolism

Several polymorphisms of genes involved in one-carbon metabolism have been identified. The reported metabolic phenotypes are often based on small studies providing inconsistent results. This large-scale study of 10,601 population-based samples was carried out to investigate the association between a panel of biochemical parameters and genetics variants related to one-carbon metabolism. Concentrations of total homocysteine (tHcy), folate, vitamin B(12) (cobalamin), methylmalonic acid (MMA), vitamin B(2) (riboflavin), vitamin B(6) (PLP), choline, betaine, dimethylglycine (DMG), cystathionine, cysteine, methionine, and creatinine were determined in serum/plasma. All subjects were genotyped for 13 common polymorphisms: methylenetetrahydrofolate reductase (MTHFR) c.665C>T (known as 677C>T; p.Ala222Val) and c.1286A>C (known as 1298A>C; p.Glu429Ala); methionine synthase (MTR) c.2756A>G (p.Asp919Gly); methionine synthase reductase (MTRR) c.66A>G (p.Ile22Met); methylenetetrahydrofolate dehydrogenase (MTHFD1) c.1958G>A (p.Arg653Gln); betaine homocysteine methyltransferase (BHMT) c.716G>A (known as 742G>A; p.Arg239Gln); cystathionine beta-synthase (CBS) c.844_845ins68 and c.699C>T (p.Tyr233Tyr); transcobalamin-II (TCN2) c.67A>G (p.Ile23Val) and c.776C>G (p.Pro259Arg); reduced folate carrier-1 (SLC19A1) c.80G>A (p.Arg27His); and paraoxonase-1 (PON1) c.163T>A (p.Leu55Met) and c.575A>G (p.Gln192Arg). The metabolic profile in terms of the measured vitamins and metabolites were investigated for these 13 polymorphisms. We confirmed the strong associations of MTHFR c.665C>T with tHcy and folate, but also observed significant (P<0.01) changes in metabolite concentrations according to other gene polymorphisms. These include MTHFR c.1286A>C (associations with tHcy, folate and betaine), MTR c.2756A>G (tHcy), BHMT c.716G>A (DMG), CBS c.844_845ins68 (tHcy, betaine), CBS c.699C>T (tHcy, betaine, cystathionine) and TCN2 c.776C>G (MMA). No associations were observed for the other polymorphisms investigated.     

维生素B6与人类基因组健康研究进展

维生素B6作为叶酸代谢途径中丝氨酸羟甲基转移酶(serine hydroxymethyltransferase,SHMT)、胱硫醚β-合成酶(cystathionineβ-synthase,CBS)、甜菜碱羟甲基转移酶(betaine hydroxymethyltransferase,BHMT)等酶的辅酶,一方面涉及丝氨酸、四氢叶酸到甘氨酸、5,10-甲基四氢叶酸的可逆转化,另一方面协助完成同型半胱氨酸到甲硫氨酸和胱硫醚的代谢.因此维生素B6对于维持基因组、表基因组稳定性以及促进同型半胱氨酸代谢可能具有一定作用,从而在肿瘤、癌症、心脑血管疾病的防范上可能起到积极作用.本文综述了维生素B6代谢功能研究进展、维生素B6缺乏临床表征及引发的并发症和营养缺陷症,为人类基因组健康、公共卫生健康和饮食供给提供参考.

Abstract：

Vitamin B6 is a cofactor for the serine hydroxymethyltransferase ( SHMT ), cystathionine β-synthase (CBS) and betaine hydroxymethyltransferase (BHMT) in folate metabolism. SHMT catalyzes the reversible interconversion of serine and tetrahydrofolate ( THF ) to glycine and 5,10- methylene THF. The metabolism of homocysteine to methionine and cysteine is initiated by the enzymes CBS and BHMT. So the evidence indicates that vitamin B6 plays an important role in the maintenance of genome,epigenetics stability and homocysteine metabolism, suggesting vitamin B6' s critical role in the prevention of tumour, cancer and cardiovascular disease. This article will review and discuss the metabolic functions of vitamin B6, some associated diseases due to vitamin B6 deficiency, and new insights from recently published studies regarding prevention and treatment potentials.     

Hyperkeratosis in cystathionine beta synthase-deficient mice: an animal model of hyperhomocysteinemia

Cystathionine beta synthase (CBS) is a crucial regulator of plasma concentrations of homocysteine. Severe hyperhomocysteinemia due to CBS deficiency confers diverse clinical manifestations. Patients with severe hyperhomocysteinemia have fine hair and thin skin, but it is unclear whether these changes are related to CBS deficiency or are coincidental. To investigate these aspects of hyperhomocysteinemia, we characterized skin abnormalities of CBS-deficient mice, a murine model of severe hyperhomocysteinemia. Histological and histomorphometric analyses revealed that CBS-deficient mice have wrinkled skin with hyperkeratinosis of the epidermis and thinning of the dermis.     

Association of a 31 bp VNTR in the CBS gene with postload homocysteine concentrations in the Framingham Offspring Study

Elevated total plasma homocysteine concentrations (tHcy), both fasting and post-methionine load, have been established as risk factors for vascular disease. Recently, we described the association of a 31 bp variable number of tandem repeats (VNTR) in the cystathionine beta-synthase (CBS) gene with both CBS enzyme activity and tHcy concentrations. In the present study, we determined the 31 bp VNTR genotypes in 2598 individuals of the Framingham Offspring Study and studied the association between this genotype and fasting, 2-h post-methionine load and delta (ie increase upon methionine loading) tHcy concentrations in 1416 subjects. We observed a positive association between the number of repeat units of the CBS 31 bp VNTR and both postload and delta tHcy concentrations. Adjustment for possible effect modifying factors like age, sex and vitamin (B6, B12 and folate) status did not change this observation. We hereby confirm the results of our earlier study, in which we found that this 31 bp VNTR is a genetic determinant of post-methionine load tHcy concentrations. Since also post-methionine load tHcy concentrations are found to be associated with an increased risk for cardiovascular disease (CVD), this 31 bp VNTR may be considered a risk factor for CVD.     

Polygenic association with total homocysteine in the post-folic acid fortification era: The CARDIA study

Elevated plasma concentration of total homocysteine (tHcy) has been linked with many diseases. tHcy is associated with a variety of factors, including polymorphisms in genes involved in homocysteine metabolism. It is not clear whether US-mandated fortification of grain products with folic acid has affected the association of genetic variants with tHcy levels. We determined tHcy concentrations in sera from 997 Caucasians and 692 African Americans participants in the Coronary Artery Risk Development in Young Adults (CARDIA) study before and after folic acid fortification. DNA was genotyped for variants present in four genes involved in homocysteine metabolism: cystathionine β-synthase (CBS) 844ins68, methionine synthase (MS) 2756A>G; methionine synthase reductase (MTRR) 66A>G and methylenetetrahydrofolate reductase (MTHFR) 677C>T and 1298A>C. A greater number of African Americans were homozygous for the MS 2756GG, MTRR 66GG and CBS 844ins68 genotypes compared to Caucasians, while prevalence of MTHFR 677TT and 1298CC genotypes was substantially lower in African Americans compared to Caucasians. The overall variance in tHcy levels at y 0, 7 and 15 that can be explained by the combined presence of all five variants increased slightly over time in Caucasians (17%, y 0; 21%, y 7; and 26%, y 15) and in African Americans (13%, y 0; 17% y 7; and 18% y 15) largely due to decrease in tHcy variance.     

Continuous combined hormone replacement therapy with oral 17beta-estradiol and norethisterone acetate improves homocysteine metabolism in postmenopausal women.

OBJECTIVE: To evaluate the effect of a continuous combined oral hormone replacement therapy (HRT) on basal and post-methionine load homocysteine levels in postmenopausal women. DESIGN: Twenty-two postmenopausal women (PMW) were randomly allocated to receive either continuous combined oral HRT (2 mg of estradiol plus 1 mg of norethisterone acetate; n = 11) or no treatment (controls, n = 11) for 6 months. A methionine oral load (0.1 g/kg body weight) was performed in each subject at time 0 and after 6 months. Serum homocysteine levels were measured by high-performance liquid chromatography in samples collected at time 0 and at 4, 8, and 24 h after the methionine load, while levels of vitamin B6 (by high-performance liquid chromatography) and B12 and folate (both by ELISA) were assayed in samples collected at time 0. RESULTS: Serum levels of glucose and body mass index increased in treated PMW, whereas folate decreased in controls. In treated PMW, basal homocysteine tended to decrease (10.6 +/- 3.3 micromol/L vs. 9.62 +/- 2.8 micromol/L, p = 0.062), whereas in controls it significantly increased (10.7 +/- 2.65 micromol/L vs. 12.17 +/- 3.89 micromol/L, p < 0.05). This increase was not significant after correction for vitamin status (p = 0.072). Homocysteine values 4 h (31.9 +/- 13.53 micromol/L vs. 39.83 +/- 22.53 micromol/L, p < 0.05) and 8 h (35.1 +/- 13.13 vs. 43.34 +/- 22.15 micromol/L) after methionine, and integrated homocysteine response to methionine (392.5 +/- 133.8 micromol/24 h vs. 458.8 +/- 104.8 micromol/24 h; p < 0.05), were significantly reduced in HRT-treated, but not in untreated, PMW. CONCLUSIONS: Continuous combined oral HRT with17beta-estradiol plus norethisterone acetate reduces homocysteine levels, mainly after a methionine load. This effect seems to be independent of vitamin status and may have positive implications for the prevention of cardiovascular diseases in PMW.     

Infantile hypermethioninemia and hyperhomocysteinemia due to high methionine intake: a diagnostic trap

Studies were carried out to identify the cause of combined severe hypermethioninemia and moderate hyperhomocysteinemia in a cluster of 10 infants ascertained between 1999 and early 2001. Although several were thought initially to have cystathionine beta-synthase (CBS) deficiency and treated accordingly, CBS deficiency and other known genetic causes of hypermethioninemia were ruled out by assay of CBS activity in fibroblasts of four patients and by assays of plasma cystathionine and S-adenosylmethionine. Retrospective data on dietary methionine intakes and plasma concentrations of methionine and related metabolites established that the hypermethioninemia in nine of the 10 babies was related to ingestion of an infant protein hydrolysate formula, the methionine content of which had been increased from May 1998 to February 2001. The formula in question has now been reformulated and is no longer available. The 10th infant manifested similar metabolic abnormalities while receiving TPN containing excessive methionine. Brain MRI abnormalities indicative of cerebral edema, most marked in the cerebral cortex and posterior brainstem, occurred in two patients near times of extreme hypermethioninemia. Metabolic and MRI abnormalities resolved when the methionine intake decreased. A third infant had a normal MRI 1 day after the formula was changed. The possible relationship between extreme hypermethioninemia and cerebral edema is discussed and a working hypothesis offered to explain the relative sensitivity of the inferior colliculi, based upon the facts that this is the region most active in glucose utilization and that Na(+),K(+)-ATPase is inhibited by methionine and related metabolites.     

Relation between plasma homocysteine concentration, the 844ins68 variant of the cystathionine beta-synthase gene, and pyridoxal-5'-phosphate concentration.

A moderately elevated plasma total homocysteine (tHcy), whether measured during fasting or post-methionine load (PML), is recognized as a risk factor for coronary artery diseases (CAD). Cystathionine beta-synthase (CBS), a key enzyme in the transsulfuration pathway, is important for the metabolism of homocysteine. In recent years, a relatively prevalent mutation, the 844ins68 (68-bp insertion), was found to be carried by about 12% of the general population. In the current investigation, we studied 741 individuals with respect to the effect of the 68-bp insertion of the CBS gene on fasting and PML tHcy, and also determined the level of pyridoxal-5'-phosphate (vitamin B(6)), a cofactor of the CBS enzyme. Our results showed that the mean fasting and PML increase in tHcy levels were lower in individuals carrying the 844ins68 variant compared to those without the insertion; although only the difference in PML increase in tHcy reached statistical significance (P = 0.02). When these individuals were divided into two groups based on vitamin B(6) concentration, the PML increase in tHcy was significantly lower in individuals heterozygous for the insertion compared to those without the insertion only in the group of individuals whose vitamin B(6) concentrations were below the sample median (38.0 nmol/L). We speculate that the 68-bp insertion is associated with somewhat higher levels of CBS enzyme activity, and that the effect of this becomes more pronounced in the presence of relatively low concentrations of pyridoxal-5'-phosphate, a cofactor of the CBS enzyme.     

A 31 bp VNTR in the cystathionine beta-synthase (CBS) gene is associated with reduced CBS activity and elevated post-load homocysteine levels

Molecular defects in genes encoding enzymes involved in homocysteine metabolism may account for mild hyperhomocysteinaemia, an independent and graded risk factor for cardiovascular disease (CVD). Although heterozygosity for cystathionine beta-synthase (CBS) deficiency has been excluded as a major genetic cause of mild hyperhomocysteinaemia in vascular disease, mutations in (non-)coding DNA sequences may lead to a mildly decreased CBS expression and, consequently, to elevated plasma homocysteine levels. We assessed the association between a 31 bp VNTR, that spans the exon 13-intron 13 boundary of the CBS gene, and fasting, post-methionine load and increase upon methionine load plasma homocysteine levels in 190 patients with arterial occlusive disease, and in 381 controls. The 31 bp VNTR consists of 16, 17, 18, 19 or 21 repeat units and shows a significant increase in plasma homocysteine concentrations with an increasing number of repeat elements, in particular after methionine loading. In 26 vascular disease patients the relationship between this 31 bp VNTR and CBS enzyme activity in cultured fibroblasts was studied. The CBS enzyme activity decreased with increasing number of repeat units of the 31 bp VNTR. RT-PCR experiments showed evidence of alternative splicing at the exon 13-intron 13 splice junction site. The 31 bp VNTR in the CBS gene is associated with post-methionine load hyperhomocysteinaemia that may predispose individuals to an increased risk of cardiovascular diseases.     

Are heterocygotes for classical homocystinuria at risk of vitamin B12 and folic acid deficiency?

OBJECTIVES/DESIGN: Comparative cross-sectional study to assess homocysteine and vitamin status in carriers of CBS gene mutations. METHOD: Subjects included 34 parents (13 males, 21 females, age 27-59 years) of 30 patients with classical homocystinuria due to homozygous cystathionine beta-synthase deficiency. Control subjects were matched for gender and age (13 males, 21 females, age 25-59 years). All subjects were of Qatari origin, had normal liver and renal function tests and had not taken drugs or vitamin supplements prior to the study. The concentrations of homocysteine, folic acid and vitamins B6 and B12 in blood were determined after an overnight fast. RESULTS: Heterozygous carriers had significantly increased fasting levels of homocysteine compared to controls (9.1 vs. 8.1 micromol/l, P=0.012). Both folic acid (328 vs. 478 pmol/l, P=0.002) and vitamin B12 concentrations (232 vs. 287 pmol/l, P=0.013) were reduced whilst there was no significant difference in vitamin B6 levels between the two groups (5.8 vs. 6.44 microg/l). CONCLUSIONS: Increased homocysteine concentrations in CBS gene mutation carriers are associated with reduced concentrations of folic acid and vitamin B12 in blood. In view of the adverse effects of mild hyperhomocysteinemia, routine testing of vitamin status in parents of homocystinuria patients may be warranted. The causal relationship and pathophysiological consequences are uncertain; it is likely that CBS gene mutation carriers need higher doses of dietary vitamins.     

Plasma total homocysteine levels and the C677T mutation in the methylenetetrahydrofolate reductase (MTHFR) gene: a study in an Italian population with dementia

Hyperhomocysteinemia is a known risk factor for vascular disease and commonly occurs in the elderly. Several studies have shown an association between elevated plasma homocysteine levels and cognitive impairment, indicating that it may play a role in the pathophysiology of dementia. We studied plasma homocysteine, folate, vitamin B12 levels and the MTHFR C677T genotype in an Italian population of patients with dementia. We confirmed that elevated plasma tHcy (>14 micromol/l) is common in elderly subjects with dementia. Although we found a high prevalence of the MTHFR TT genotype (21.2%) the allele frequency is not over-represented relative to the control population. We also observed a high incidence of folate deficiency (38%) in subjects with dementia. Elevated homocysteine was associated with low plasma folate (<5.7 nmol/l) and the MTHFR TT genotype. Moderate to severe hyperhomocysteinemia (>26.1 nmol/l) was associated with a significantly lower MMSE score. Hyperhomocysteinemia may be neurotoxic by several different mechanisms affecting cognitive function. Further studies are needed to fully explore the potential of B vitamin supplementation to lower plasma homocysteine and improve cognitive function.     

Cystathionine beta synthase deficiency affects mouse endochondral ossification

Cystathionine beta synthase (CBS) is a crucial regulator of plasma concentrations of homocysteine. Severe hyperhomocysteinemia due to CBS deficiency confers diverse clinical manifestations, notably characteristic skeletal abnormalities. To investigate this aspect of hyperhomocysteinemia, we analyzed the skeleton of CBS-deficient mice, a murine model of severe hyperhomocysteinemia. Radiography, Alcian Blue/Alizarin Red S-stained whole skeletal preparations, and histological comparisons were used to determine the extent, pattern, and distribution of skeletal abnormalities in CBS-deficient mice. Disruption of the murine CBS gene leads to skeletal abnormalities, notably kyphoscoliosis, with temporal shortening of long bones due to impaired cartilage differentiation, albeit to differing degrees.     

Total homocysteine, B-vitamins and genetic polymorphisms in patients with classical phenylketonuria

Hyperhomocysteinemia has occasionally been reported in patients with phenylketonuria (PKU) and B-vitamin deficiency. In our study total homocysteine (tHcy) and B-vitamins were measured in treated PKU patients and healthy controls. In the patients, dietary parameters and genetic polymorphisms affecting the Hcy pathway were investigated to identify parameters modulating tHcy. A case control study including 37 PKU patients and 63 healthy controls was conducted. t-Tests for independent samples were used to test between groups. Multiple regressions with tHcy as dependent variable were calculated. Hardy-Weinberg expectations were tested against the observed distribution of genotypes applying the Chi-square goodness-of-fit method. THcy concentrations were not significantly different (p=0.059) while folate and cobalamin (Cbl) concentrations were significantly higher in PKU patients compared to controls. However, 29.7% of patients had tHcy concentrations >97th centile. THcy did not vary with age nor correlate with folate and Cbl concentrations probably due to high saturatory levels. The presence of genetic polymorphisms had no impact on tHcy. In conclusion, in PKU patients treated with amino acid mixtures enriched with B-vitamins, tHcy is not significantly higher than in healthy controls, but tHcy concentrations exceed the 97th centile in about one third of patients. Even higher B-vitamin saturation may be required to further decrease tHcy concentrations and factors generally influencing tHcy such as betaine are to be investigated in PKU patients in the future.     

Polymorphisms in the CBS gene associated with decreased risk of coronary artery disease and increased responsiveness to total homocysteine lowering by folic acid

Elevated total plasma homocysteine (tHcy) is an established risk factor for the development of vascular disease and neural tube defects. Total homocysteine levels can be lowered by folic acid supplements but individual response is highly variable. In this case-control study, involving 142 coronary artery disease (CAD) patients and 102 controls, we have typed six genetic polymorphisms in three homocysteine metabolizing genes and examined their relationship to the incidence of CAD, tHcy levels, and lowering of tHcy levels in response to folic acid supplementation. We found that two single nucleotide polymorphisms in the cystathionine beta synthase (CBS) gene, 699C --> T and 1080T --> C, are associated with decreased risk of CAD and increased responsiveness to the tHcy lowering effects of folic acid. Individuals homozygous for 699T were significantly underrepresented in CAD patients as compared to controls (4.9% vs 17.3%, P = 0.0015), as were individuals homozygous for the 1080C (29.6% vs 44.2%, P = 0.018). Additionally, 699T and 1080C homozygous individuals were the most responsive to folate supplementation. 699T homozygotes lowered tHcy levels 13.6% on average, compared to 4.8% lowering in 699C homozygotes (P = 0.009), while 1080C homozygotes lowered 12.9% compared to just 2.7% for 1080T homozygotes (P = 0.005). The two polymorphisms in CBS are third codon changes and would not be predicted to affect the underlying protein. However, there is strong linkage disequilibrium between these two positions, suggesting that they may also be linked to other as yet unidentified polymorphisms within the CBS gene. These observations suggest that specific CBS alleles are a risk factor for the development of vascular disease and that genetic information could be predictive of individual response to folic acid supplementation.