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Mouse models of cystathionine beta-synthase deficiency reveal significant threshold effects of hyperhomocysteinemia.

ABSTRACT: Untreated cystathionine beta-synthase (CBS) deficiency in humans is characterized by extremely elevated plasma total homocysteine (tHcy>200 microM), with thrombosis as the major cause of morbidity. Treatment with vitamins and diet leads to a dramatic reduction in thrombotic events, even though patients often still have severe elevations in tHcy (>80 microM). To understand the difference between extreme and severe hyperhomocysteinemia, we have examined two mouse models of CBS deficiency: Tg-hCBS Cbs(-/-) mice, with a mean serum tHcy of 169 microM, and Tg-I278T Cbs(-/-) mice, with a mean tHcy of 296 microM. Only Tg-I278T Cbs(-/-) animals exhibited strong biological phenotypes, including facial alopecia, osteoporosis, endoplasmic reticulum (ER) stress in the liver and kidney, and a 20% reduction in mean survival time. Metabolic profiling of serum and liver reveals that Tg-I278T Cbs(-/-) mice have significantly elevated levels of free oxidized homocysteine but not protein-bound homocysteine in serum and elevation of all forms of homocysteine and S-adenosylhomocysteine in the liver compared to Tg-hCBS Cbs(-/-) mice. RNA profiling of livers indicate that Tg-I278T Cbs(-/-) and Tg-hCBS Cbs(-/-) mice have unique gene signatures, with minimal overlap. Our results indicate that there is a clear pathogenic threshold effect for tHcy and bring into question the idea that mild elevations in tHcy are directly pathogenic.

SUBMITTER: Gupta S

PROVIDER: S-EPMC2653989 | biostudies-literature |

REPOSITORIES: biostudies-literature

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

Project description:Missense mutations in the cystathionine beta-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 microM compared to 16+/-13 microM 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.

| S-EPMC2630375 | biostudies-literature

Protein arginine hypomethylation in a mouse model of cystathionine β-synthase deficiency.

Project description:Accumulation of the homocysteine (Hcy) precursor S-adenosylhomocysteine (AdoHcy) may cause cellular hypomethylation in the setting of hyperhomocysteinemia because of cystathionine β-synthase (CBS) deficiency, an inborn error of metabolism. To test this hypothesis, DNA and protein arginine methylation status were assessed in liver, brain, heart, and kidney obtained from a previously described mouse model of CBS deficiency. Metabolite levels in tissues and serum were determined by high-performance liquid chromatography or liquid chromatography-electrospray ionization-tandem mass spectrometry. Global DNA and protein arginine methylation status were evaluated as the contents of 5-methyldeoxycytidine in DNA and of methylarginines in proteins, respectively. In addition, histone arginine methylation was assessed by Western blotting. CBS-deficient mice exhibited increased (>6-fold) Hcy and AdoHcy levels in all tissues examined compared with control levels. In addition, global DNA methylation status was not affected, but global protein arginine methylation status was decreased (10-35%) in liver and brain. Moreover, asymmetric dimethylation of arginine 3 on histone H4 (H4R3me2a) content was markedly decreased in liver, and no differences were observed for the other histone arginine methylation marks examined. Our results show that CBS-deficient mice present severe accumulation of tissue Hcy and AdoHcy, protein arginine hypomethylation in liver and brain, and decreased H4R3me2a content in liver. Therefore, protein arginine hypomethylation arises as a potential player in the pathophysiology of CBS deficiency.

| S-EPMC4021445 | biostudies-literature

Cystathionine β-synthase and methylenetetrahydrofolate reductase mutations in Mexican individuals with hyperhomocysteinemia.

Project description:BackgroundHyperhomocysteinemia, a thrombotic risk factor, may have several causes. Among the genetic causes of hyperhomocysteinemia, there are polymorphisms in the enzymes methylenetetrahydrofolate reductase (C677T) and cystathionine β-synthase (C699T, C1080T, and 844ins68). Although the frequency of hyperhomocysteinemia in our country is high, there is no evidence about the frequencies of these polymorphisms.MethodsWe analyzed 80 healthy individuals from several regions in our country. We evaluated the fasting and post-oral methionine load plasma Hcy and the genotypes in order to obtain the allele frequencies of the polymorphisms C677T of methylenetetrahydrofolate reductase and C699T, C1080T, and 844ins68 of the cystathionine β-synthase.ResultsNo individual had deficiency of folic acid, vitamins B12, or B6, but 80% had post-oral methionine load hyperhomocysteinemia. We found a significant increase in the Hcy plasma concentration associated with age and gender. Only the polymorphism C1080T was significantly associated with hyperhomocysteinemia.ConclusionThere is an association between fasting and post-oral methionine load plasma Hcy concentrations with the allelic frequencies of the polymorphisms C669T, 844ins68, and C1080T of the cystathionine β-synthase and C667T of the methylenetetrahydrofolate reductase in healthy Mexican individuals. As compared with individuals with normal fasting or post-oral methionine load Hcy plasma levels, only C1080T was significantly associated with hyperhomocysteinemia.

| S-EPMC7682208 | biostudies-literature

Cystathionine beta synthase expression in mouse retina.

Project description:Abstract Purpose: Cystathionine ?-synthase (CBS), a key enzyme in the transsulfuration metabolic pathway, converts homocysteine to cystathionine, which is converted to cysteine required for the synthesis of major retinal antioxidant glutathione (GSH). Enzyme activity assays suggest that CBS is present in human and pig retina, however recent studies reported that CBS is not expressed in mouse retina. We found this species difference puzzling. Given the plethora of studies using mouse retina as a model system, coupled with the importance of GSH in retina, we investigated CBS expression in mouse retina at the molecular and cell biological level.Wildtype (WT) mice or mice lacking the gene encoding CBS (cbs(-)(/)(-)) were used in these studies. RNA and protein were isolated from retinas and liver (positive control) for the analysis of cbs gene expression by RT-PCR and CBS protein expression by Western blotting, respectively. CBS was analyzed by immunofluorescence in retinal cryosections and primary retinal cells (ganglion, Müller, retinal pigment epithelial). CBS enzyme activity was measured in primary Müller cells.RT-PCR revealed robust cbs expression in WT liver, brain and retina. Western blotting detected CBS in retina, brain and liver of WT mice, but not in cbs(-)(/)(-) mice liver. In immunohistochemical studies, CBS was present abundantly in the ganglion cell layer of retina; it was detected also in primary isolations of Müller, RPE and ganglion cells. CBS activity was detected in Müller cells by fluorescent detection of H2S.We have compelling molecular evidence that CBS is expressed in mouse retina at the gene and protein level. Our immunofluorescence data suggest that it is present in several retinal cell types and the data from the enzyme activity assay suggest activity in Müller cells. These findings set the stage to investigate the role of CBS and the transsulfuration pathway in the generation of GSH in mouse retina.

| S-EPMC3731150 | biostudies-literature

Vascular presentation of cystathionine beta-synthase deficiency in adulthood.

Project description:Several recent studies describing a solely vascular presentation of cystathionine beta-synthase (CBS) deficiency in adulthood prompted us to analyze the frequency of patients manifesting with vascular complications in the Czech Republic. Between 1980 and 2009, a total of 20 Czech patients with CBS deficiency have been diagnosed yielding an incidence of 1:311,000. These patients were divided into three groups based on symptoms leading to diagnosis: those with vascular complications, with connective tissue manifestation and with neurological presentation. A vascular event such as a clinical feature leading to diagnosis of homocystinuria was present in five patients, while two of them had no other symptoms typical for CBS deficiency at the time of diagnosis. All patients with the vascular manifestation were diagnosed only during the past decade. The median age of diagnosis was 29 years in the vascular, 11.5 years in the connective tissue and 4.5 years in the neurological group. The ratio of pyridoxine responsive to nonresponsive patients was higher in the vascular (4 of 5 patients) and connective tissue groups (6 of 7 patients) than in the neurological group (2 of 8 patients). Mutation c.833T>C (p.I278T) was frequent in patients with vascular (6/10 alleles) and connective tissue presentation (8/14 alleles), while it was not present in patients with neurological involvement (0/16 alleles). During the last decade, we have observed patients with homocystinuria diagnosed solely due to vascular events; this milder form of homocystinuria usually manifests at greater ages, has a high ratio of pyridoxine responsiveness/nonresponsiveness, and the mutation c.833T>C (p.I278T) is often present.

| S-EPMC3026685 | biostudies-literature

Hyperhomocysteinemia promotes inflammatory monocyte generation and accelerates atherosclerosis in transgenic cystathionine beta-synthase-deficient mice.

Project description:Hyperhomocysteinemia (HHcy) is an independent risk factor for cardiovascular disease. Monocytes display inflammatory and resident subsets and commit to specific functions in atherogenesis. In this study, we examined the hypothesis that HHcy modulates monocyte heterogeneity and leads to atherosclerosis.We established a novel atherosclerosis-susceptible mouse model with both severe HHcy and hypercholesterolemia in which the mouse cystathionine beta-synthase (CBS) and apolipoprotein E (apoE) genes are deficient and an inducible human CBS transgene is introduced to circumvent the neonatal lethality of the CBS deficiency (Tg-hCBS apoE(-/-) Cbs(-/-) mice). Severe HHcy accelerated atherosclerosis and inflammatory monocyte/macrophage accumulation in lesions and increased plasma tumor necrosis factor-alpha and monocyte chemoattractant protein-1 levels in Tg-hCBS apoE(-/-) Cbs(-/-) mice fed a high-fat diet. Furthermore, we characterized monocyte heterogeneity in Tg-hCBS apoE(-/-) Cbs(-/-) mice and another severe HHcy mouse model (Tg-S466L Cbs(-/-)) with a disease-relevant mutation (Tg-S466L) that lacks hyperlipidemia. HHcy increased monocyte population and selective expansion of inflammatory Ly-6C(hi) and Ly-6C(mid) monocyte subsets in blood, spleen, and bone marrow of Tg-S466L Cbs(-/-) and Tg-hCBS apoE(-/-) Cbs(-/-) mice. These changes were exacerbated in Tg-S466L Cbs(-/-) mice with aging. Addition of l-homocysteine (100 to 500 micromol/L), but not l-cysteine, maintained the Ly-6C(hi) subset and induced the Ly-6C(mid) subset in cultured mouse primary splenocytes. Homocysteine-induced differentiation of the Ly-6C(mid) subset was prevented by catalase plus superoxide dismutase and the NAD(P)H oxidase inhibitor apocynin.HHcy promotes differentiation of inflammatory monocyte subsets and their accumulation in atherosclerotic lesions via NAD(P)H oxidase-mediated oxidant stress.

| S-EPMC2783582 | biostudies-literature

Cystathionine beta-synthase deficiency causes fat loss in mice.

Project description:Cystathionine beta synthase (CBS) is the rate-limiting enzyme responsible for the de novo synthesis of cysteine. Patients with CBS deficiency have greatly elevated plasma total homocysteine (tHcy), decreased levels of plasma total cysteine (tCys), and often a marfanoid appearance characterized by thinness and low body-mass index (BMI). Here, we characterize the growth and body mass characteristics of CBS deficient TgI278T Cbs(-/-) mice and show that these animals have significantly decreased fat mass and tCys compared to heterozygous sibling mice. The decrease in fat mass is accompanied by a 34% decrease in liver glutathione (GSH) along with a significant decrease in liver mRNA and protein for the critical fat biosynthesizing enzyme Stearoyl CoA desaturase-1 (Scd-1). Because plasma tCys has been positively associated with fat mass in humans, we tested the hypothesis that decreased tCys in TgI278T Cbs(-/-) mice was the cause of the lean phenotype by placing the animals on water supplemented with N-acetyl cysteine (NAC) from birth to 240 days of age. Although NAC treatment in TgI278T Cbs(-/-) mice caused significant increase in serum tCys and liver GSH, there was no increase in body fat content or in liver Scd-1 levels. Our results show that lack of CBS activity causes loss of fat mass, and that this effect appears to be independent of low serum tCys.

| S-EPMC3214081 | biostudies-other

Guidelines for the diagnosis and management of cystathionine beta-synthase deficiency.

Project description:Cystathionine beta-synthase (CBS) deficiency is a rare inherited disorder in the methionine catabolic pathway, in which the impaired synthesis of cystathionine leads to accumulation of homocysteine. Patients can present to many different specialists and diagnosis is often delayed. Severely affected patients usually present in childhood with ectopia lentis, learning difficulties and skeletal abnormalities. These patients generally require treatment with a low-methionine diet and/or betaine. In contrast, mildly affected patients are likely to present as adults with thromboembolism and to respond to treatment with pyridoxine. In this article, we present recommendations for the diagnosis and management of CBS deficiency, based on a systematic review of the literature. Unfortunately, the quality of the evidence is poor, as it often is for rare diseases. We strongly recommend measuring the plasma total homocysteine concentrations in any patient whose clinical features suggest the diagnosis. Our recommendations may help to standardise testing for pyridoxine responsiveness. Current evidence suggests that patients are unlikely to develop complications if the plasma total homocysteine concentration is maintained below 120 ?mol/L. Nevertheless, we recommend keeping the concentration below 100 ?mol/L because levels fluctuate and the complications associated with high levels are so serious.

| S-EPMC5203861 | biostudies-literature

Hyperhomocysteinemia and dyslipidemia in point mutation G307S of cystathionine β-synthase-deficient rabbit generated using CRISPR/Cas9.

Project description:BackgroundCongenital hyper-homocysteinemia (HHcy) is caused by a defective cystathionine β-synthase (CBS) gene, and is frequently associated with dyslipdemia. The aim of this study was to further elucidate the effect of mutated CBS gene on circulating lipids using a rabbit model harboring a homozygous G307S point mutation in CBS.MethodsCRISPR/Cas9 system was used to edit the CBS gene in rabbit embryos. The founder rabbits were sequenced, and their plasma homocysteine (Hcy) and lipid profile were analyzed.ResultsSix CBS-knockout (CBS-KO) founder lines with biallelic modifications were obtained. Mutation in CBS caused significant growth retardation and high mortality rates within 6 weeks after birth. In addition, the 6-week old CBS-KO rabbits showed higher plasma levels of Hcy, triglycerides (TG), total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) compared to the age-matched wild-type (WT) controls. Histological analysis of the mutants showed accumulation of micro-vesicular cytoplasmic lipid droplets in the hepatocytes. However, gastric infusion of vitamin B and betaine complex significantly decreased the plasma levels of TG, TC and LDL-C in the CBS-KO rabbits, and alleviated hepatic steatosis compared to the untreated animals.ConclusionA CBSG307S rabbit model was generated that exhibited severe dyslipidemia when fed on a normal diet, indicating that G307S mutation in the CBS gene is a causative factor for dyslipidemia.

| S-EPMC7560309 | biostudies-literature

Cystathionine-β-Synthase Promotes Colon Carcinogenesis

Project description:Purpose: The goal of this study is to investigate the role of CBS enzyme in colorectal carcinogenesis Methods: RNA-Seq transcriptome analysis of CBS-overexpression in NCM356 cels compared to control vector cells

2017-12-07 | GSE91000 | GEO

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