Homocystinuria Research Articles

Long-term betaine therapy in a murine model of cystathionine beta-synthase deficient homocystinuria: Decreased efficacy over time reveals a significant threshold effect between elevated homocysteine and thrombotic risk

Classical homocystinuria (HCU) is caused by deficiency of cystathionine β-synthase and is characterized by connective tissue disturbances, mental retardation and cardiovascular disease. Treatment for pyridoxine non-responsive HCU typically involves lowering homocysteine levels with a methionine-restricted diet and dietary supplementation with betaine. Compliance with the methionine-restricted diet is difficult and often poor. Investigating optimization of the efficacy of long-term betaine treatment in isolation from a methionine-restricted diet is precluded by ethical considerations regarding patient risk. The HO mouse model of HCU developed in our laboratory, exhibits constitutive expression of multiple pro-inflammatory cytokines and a hypercoagulative phenotype both of which respond to short-term betaine treatment. Investigation of the effects of long-term betaine treatment in the absence of methionine-restriction in HO HCU mice revealed that the ability of betaine treatment to lower homocysteine diminished significantly over time. Plasma metabolite analysis indicated that this effect was due at least in part, to reduced betaine-homocysteine S-methyltransferase (BHMT) mediated remethylation of homocysteine. Western blotting analysis revealed that BHMT protein levels are significantly repressed in untreated HCU mice but are significantly induced in the presence of betaine treatment. The observed increase in plasma homocysteine during prolonged betaine treatment was accompanied by a significant increase in the plasma levels of TNF-alpha and IL-1beta and reversion to a hypercoagulative phenotype. Our findings are consistent with a relatively sharp threshold effect between severely elevated plasma homocysteine and thrombotic risk in HCU and indicate that the HO mouse model can serve as a useful tool for both testing novel treatment strategies and examining the optimal timing and dosing of betaine treatment with a view toward optimizing clinical outcome.

Cystathionine Beta-Synthase Deficiency Causes Fat Loss in Mice

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.

Estudio epidemiológico de las enfermedades metabólicas con homocistinuria en España

ObjectivesTo determine the prevalence of homocystinuria in Spain and to establish the measures and mechanisms to ensure its prevention, diagnosis and treatment. Material and methodsA national cross-sectional survey was conducted by means of a questionnaire sent to 35 hospitals in which children and adult patients are treated. ResultsUsing the questionnaires submitted by 25 physicians from 16 centres, 75 patients were identified: 41 transsulphuration defects (one deceased), 27 remethylation (six deaths) and 7 without a syndromic diagnosis. The age at diagnosis varied widely, and 18 cases had more than one sibling affected. The more severe clinical manifestations involved the patients with remethylation defects. There was a high percentage of cognitive impairment, followed by lens diseases. Almost half of the patients had neurological disorders. There was increased vascular involvement in CBS-deficient adults. The therapeutic options most used were, folic acid, hydroxycobalamin and betaine. ConclusionsIn view of these results and especially the small number of CBS deficiencies detected, we conclude that there is a need to introduce newborn screening for classical homocystinuria and ensure implementation of an appropriate diagnostic workup in all patients at risk.

Role of Homocysteine Measurement for Early Diagnosis of Vitamin B12 Deficiency in the First Days of Life

Background: Vitamin B12 (vit B12) deficiency is one of the major causes of megaloblastic anaemia and should be avoided as early as possible since a supplementation of mother and child can prevent neurological symptoms of the baby. Furthermore, the neurological symptoms of affected children are (partially) reversible. Elevated methylmalonic acid in urine and homocysteine (Hcy) in plasma are sensitive indicators. In the State of Qatar, extended newborn screening of classical homocystinuria was realized for all 73,994 neonates in last 4.5 years. Newborns with slightly elevated Hcy levels in dried blood spots (DBS) were followed up with regard to possible vit B12 deficiency. In addition, the propionylcarnitine (C3) levels were analysed. Methods: Determination of Hcy in DBS was performed using liquid chromatography electrospray tandem mass spectrometry. C3 levels were obtained from general newborn screening. The vit B12 levels in plasma were analysed spectrophotometrically. Results: In all, 117 cases with mildly elevated Hcy levels were found. 65 were diagnosed with vit B12 deficiency. Only 9 of these 65 newborns had abnormal C3 levels. No correlation was found in this group between Hcy and C3 levels. Conclusion: Extended neonatal screening of Hcy is a useful tool for early diagnosis and treatment of vit B12 deficiency.

Methionine/galactose ratio on newborn blood spots useful for reduction of false positives for homocystinuria and galactosemia by high-performance anion-exchange chromatography with pulsed amperometric detection

BackgroundMethionine (Met) in blood and urine is a useful diagnostic marker for homocystinuria (HCU). However, galactosemia could be misdiagnosed as HCU when Met is used as the sole marker, since elevated excretion of Met presents in both galactosemia and HCU. Use of a more specific diagnostic marker in addition to Met is therefore necessary for reduction of false positive results for HCU as well as confirmative diagnosis of HCU. MethodsChromatographic separation was performed using an anion-exchange column. The levels of Met and galactose (Gal) on blood were measured and Met/Gal ratios were calculated from blood spot samples from 300 normal volunteers, eight galactosemia patients, and three HCU patients. ResultsThe Met/Gal ratio ranged 0–4.95 for normal blood spots (n=300), 0–0.22 for galactosemia samples (n=8), and >1250 for HCU patient samples. ConclusionsSeparation, extraction, and deproteinization procedures were established for Met and Gal in blood spots. And Met/Gal ratio allowed HCU to clearly distinguish from galactosemia. As a way of second tier confirmative analysis, the ratio is the best way to reduce false positives. The assay is most appropriate to reduce false positives in labs that do not screen for galactosemia.

Folate, vitamin B12 and homocystine status in the elderly in North-eastern Iran

Cystathionine β-synthase (CBS) deficiency is the most common cause of homocystinuria. More than 130 pathogenic mutations, mostly in the Caucasian populations, have been described. Recently, our group reported a mutation analysis of Japanese homocystinuric patients. In the present paper, we report an expression study of several mutant CBS enzymes in Escherichia coli, i.e., R121H, G148R, G151R, S217F, H232D, R266G, 1591delTTCG, and K441X. All of the mutants except K441X exhibited severely decreased activity, and the capability to form tetramers of most mutants was severely impaired. The K441X mutant, on the other hand, exhibited relatively high activity (63% of the wild type activity). This was probably due to two factors. First, the high abundance of the full-length CBS protein, a likely K441Q mutant, which was produced through suppression of the amber termination codon by glutamine tRNA in E. coli. And second, the presence of a C-terminally truncated protein, which was previously shown to be constitutively activated. Patient-derived lymphocytes, however, showed no detectable CBS subunits. As previously hypothesized, the increased aggregation of mutant CBS subunits might be a common pathogenic mechanism in CBS deficiency.

A novel 110-bp insertion in a patient with homocysteinuria.

Homocysteinuria is an inherited recessive disorder caused by cystathionine beta-synthase (CBS) deficiency that has a wide spectrum of clinical manifestations, including ocular lens dislocation, skeletal disproportion, osteoporosis, vascular thrombosis, and central nervous system dysfunction [1]. The CBS enzyme catalyzes the synthesis of cystathionine from homocysteine and serine in the methionine pathway. This results in accumulation of homocysteine and methionine in plasma, leading to excretion of excessive urinary homocysteine. CBS deficiency has a worldwide incidence of 1/344,000 live births (range: 1/58,000-1/1000 live births) [2]. Human CBS is located in the cytoplasm of cells and is composed of 4 identical 63-kDa subunits. The CBS gene is located on the long (q) arm of chromosome 21 at position 22.3, encodes a protein consisting of 551 amino acids, and has 16 exons [3]. Herein we report a patient with homocysteinuria that was a carrier of compound heterozygote mutations in the CBS gene, 1 of which is novel, and a discussion of the clinical and molecular findings. A 14-year-old boy was referred to our hospital for evaluation of ocular symptoms that began approximately 1 year earlier. He was the first born of non-consanguineous parents that had lost 1 fetus. Family history of neurologic and thromboembolic disease was negative. Pregnancy and delivery were uneventful. The proband had neuromotor developmental delay. At the age of 10 years he had a clonic seizure of the left side. He had a marfanoid face and neurological examination showed left-sided central facial palsy, and mild left hemiparesis. Complete blood count results were normal. Cranial CT showed an area of low attenuation in the left frontoparietal region consistent with infarction. Prothrombotic work-up results were as follows: serum methionine level: 4.7 mg / dL (normal: 0.09-0.6 mg/dL); free homocysteine concentration: 50 mmol/L (normal: <12.5 mmol/L); urine homocysteine concentration: 360 mmol/L (normal: 5-15 mmol/L). The patient was diagnosed as homocysteinuria, and hydroxy cobalamin injections, vitamin B6, acetyl-salicylic acid, and a low-methionine diet were started and maintained.

Chronic hyperhomocysteinemia induces oxidative damage in the rat lung

Tissue accumulation of homocysteine occurs in classical homocystinuria, a metabolic disease characterized biochemically by cystathionine β-synthase deficiency. Vascular manifestations such as myocardial infarction, cerebral thrombosis, hepatic steatosis, and pulmonary embolism are common in this disease and poorly understood. In this study, we investigated the effect of chronic hyperhomocysteinemia on some parameters of oxidative stress (thiobarbituric acid-reactive substances, protein carbonyl content, 2',7'-dichlorofluorescein fluorescence assay, and total radical-trapping antioxidant potent) and activities of antioxidant enzymes (superoxide dismutase, catalase, and glutathione peroxidase) in the rat lung. Reduced glutathione content and glucose 6-phosphate dehydrogenase activity, as well as nitrite levels, were also evaluated. Wistar rats received daily subcutaneous injections of Hcy (0.3-0.6 μmol/g body weight) from the 6th to the 28th days-of-age and the control group received saline. One and 12 h after the last injection, rats were killed and the lungs collected. Hyperhomocysteinemia increased lipid peroxidation and oxidative damage to protein, and disrupted antioxidant defenses (enzymatic and non-enzymatic) in the lung of rats, characterizing a reliable oxidative stress. In contrast, this amino acid did not alter nitrite levels. Our findings showed a consistent profile of oxidative stress in the lung of rats, elicited by homocysteine, which could explain, at least in part, the mechanisms involved in the lung damage that is present in some homocystinuric patients.

Identification and functional analyses of CBS alleles in Spanish and Argentinian homocystinuric patients

Homocystinuria due to CBS deficiency is a rare autosomal recessive disorder characterized by elevated plasma levels of homocysteine (Hcy) and methionine (Met). Here we present the analysis of 22 unrelated patients of different geographical origins, mainly Spanish and Argentinian. Twenty-two different mutations were found, 10 of which were novel. Five new mutations were missense and five were deletions of different sizes, including a 794-bp deletion (c.532-37_736 + 438del794) detected by Southern blot analysis. To assess the pathogenicity of these mutations, seven were expressed heterologously in Escherichia coli and their enzyme activities were assayed in vitro, in the absence and presence of the CBS activators PLP and SAM. The presence of the mutant proteins was confirmed by Western blotting. Mutations p.M173del, p.I278S, p.D281N, and p.D321V showed null activity in all conditions tested, whereas mutations p.49L, p.P200L and p.A446S retained different degrees of activity and response to stimulation. Finally, a minigene strategy allowed us to demonstrate the pathogenicity of an 8-bp intronic deletion, which led to the skipping of exon 6. In general, frameshifting deletions correlated with a more severe phenotype, consistent with the concept that missense mutations may recover enzymatic activity under certain conditions.

Classical familial homocystinuria in an adult presenting as an isolated lens subluxation

To report a case of late diagnosis of a classical familial homocystinuria based on an ophthalmologic examination. A 35-year-old male with Marfan-like phenotype complained of a progressive increase of myopia during the previous 2years. Ophthalmologic exploration showed a bilateral subluxation of the lens with inferior and nasal displacement. Biochemical study detected a profile of increased amino acid levels (homocysteinemia) consistent with suspected homocystinuria. Vascular and skeletal studies ruled out Marfan syndrome. Response to treatment demonstrated B(6)-non-responsive homocystinuria. Molecular study showed compound heterozygous T353N and D444N mutations of the cystathionine beta-synthase (CBS) gene, and also a C667T homozygous mutation of the methylenetetrahydrofolate-reductase (MTHFR) gene. Family study showed classical homocystinuria in his father and sister, although they did not present any systemic or ocular features of the disease. Homocystinuria is a metabolic disease usually presenting at an early age as vascular, skeletal and neuropsychiatric abnormalities, as well as ectopia lentis. Our case is atypical because of the absence of thromboembolism and the mild phenotype, in spite of being B(6)-non-responsive, and the association of a rare compound heterozygous mutation of the CBS gene and also an homozygous mutation of the MTHFR gene. It is necessary to rule out homocystinuria in patients with ectopia lentis, even in the absence of systemic symptoms.

Clinical and molecular findings of 13 families from Saudi Arabia and a family from Sudan with homocystinuria

Homocystinuria due to cystathionine beta synthase (CBS) deficiency results in elevated plasma homocysteine and methionine levels, which are associated with multiple organ pathologies, including vascular, respiratory, musculoskeletal, nervous, and ocular tissues. This autosomal recessive disorder is caused by homozygous or compound heterozygous mutations in the CBS gene encoding for the CBS. Although homocystinuria is observed in Arab and North African patients, their clinical presentations have not been described and molecular causes remained largely uninvestigated. In this study, we describe the clinical presentations of 22 homocystinuria patients from 13 Saudi Arabian families and 1 North African Sudanese family. Cardinal biochemical features of homocystinuria manifested in all patients, but heterogeneity of expression was observed for other associated phenotypes. One patient developed Legg-Calvé-Perthes disease that has not been previously described in homocystinuria. In the Saudi families, a novel nonsense mutation, p.Trp323X, and recurrent p.Arg336Cys and p.Gly153Arg mutations were identified in the CBS gene. The p.Trp323X mutation was found in 10 of the 13 unrelated Saudi families. In the Sudanese family, the p.Thr257Met mutation in the CBS gene, previously described in Italian and Spanish patients, was found. This study shows that the spectrum of CBS gene mutations in Saudi homocystinuria patients is quite different than the Arab patients from Qatar and Israel. This study is the only detailed phenotypic and genetic depiction of homocystinuria patients from Saudi Arabia and Sudan. The data are useful for diagnosis and management of Saudi patients.

Constitutive induction of pro-inflammatory and chemotactic cytokines in cystathionine beta-synthase deficient homocystinuria

Cystathionine beta-synthase (CBS) deficient homocystinuria (HCU) is an inherited metabolic defect that if untreated, typically results in cognitive impairment, connective tissue disturbances, atherosclerosis and thromboembolic disease. In recent years, chronic inappropriate expression of the inflammatory response has emerged as a major driving force of both thrombosis and atherosclerotic lesion development. We report here a characterization of the abnormalities in cytokine expression induced in both a mouse model of HCU and human subjects with the disease in the presence and absence of homocysteine lowering therapy. HCU mice exhibited highly significant induction of the pro-inflammatory cytokines Il-1alpha, Il-1beta and TNF-alpha. Similarly, in untreated/poorly compliant human subjects with HCU we observed constitutive induction of multiple pro-inflammatory cytokines (IL-1alpha, IL-6, TNF-alpha, Il-17 and IL-12(p70)) and chemotactic chemokines (fractalkine, MIP-1alpha and MIP-1beta) compared to normal controls. These HCU patients also exhibited significant induction of IL-9, TGF-alpha and G-CSF. The expression levels of anti-inflammatory cytokines were unaffected in both HCU mice and human subjects with the disease. In the human subjects, homocysteine lowering therapy was associated with either normalization or significant reduction of all of the pro-inflammatory cytokines and chemokines investigated. We conclude that HCU is a disease of chronic inflammation and that aberrant cytokine expression has the potential to contribute to multiple aspects of pathogenesis. Our findings indicate that anti-inflammatory strategies could serve as a useful adjuvant therapy for this disease.

Asymmetric dimethylarginine in children with homocystinuria or phenylketonuria

Plasma concentration of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide (NO) synthesis from L-arginine and a cardiovascular risk factor, was found to be elevated in plasma of homocysteinemic adults. Enhanced cardiovascular risk due to homocystinuria and impaired renal function has been found in patients with phenylketonuria (PKU) on protein-restricted diet. However, it is still unknown whether ADMA synthesis is also elevated in children with homocystinuria due to cystathionine beta-synthase deficiency (classical homocystinuria), and whether ADMA may play a role in phenylketonuria in childhood. In the present study, we investigated the status of the L-arginine/NO pathway in six young patients with homocystinuria, in 52 young phenylketonuria patients on natural protein-restricted diet, and in age- and gender-matched healthy children serving as controls. ADMA in plasma and urine was determined by GC-MS/MS. The NO metabolites nitrate and nitrite in plasma and urine, and urinary dimethylamine (DMA), the dimethylarginine dimethylaminohydrolase (DDAH) metabolite of ADMA, were measured by GC-MS. Unlike urine ADMA excretion, plasma ADMA concentration in patients with homocystinuria was significantly higher than in controls (660±158 vs. 475±77 nM, P=0.035). DMA excretion rate was considerably higher in children with homocystinuria as compared to controls (62.2±24.5 vs. 6.5±2.9 μmol/mmol creatinine, P=0.068), indicating enhanced DDAH activity in this disease. In contrast and unexpectedly, phenylketonuria patients had significantly lower ADMA plasma concentrations compared to controls (512±136 vs. 585±125 nM, P=0.009). Phenylketonuria patients and controls had similar L-arginine/ADMA molar ratios in plasma. Urinary nitrite excretion was significantly higher in phenylketonuria as compared to healthy controls (1.7±1.7 vs. 0.7±1.2 μmol/mmol creatinine, P=0.003). Our study shows that the L-arginine/NO pathway is differently altered in children with phenylketonuria and homocystinuria. Analogous to hyperhomocysteinemic adults, elevated ADMA plasma concentrations could be a cardiovascular risk factor in children with homocystinuria. In phenylketonuria, the L-arginine/NO pathway seems not be altered. Delineation of the role of ADMA in childhood phenylketonuria and homocystinuria demands further investigation.

Vascular complications of cystathionine β-synthase deficiency: future directions for homocysteine-to-hydrogen sulfide research.

Homocysteine (Hcy), a cardiovascular and neurovascular disease risk factor, is converted to hydrogen sulfide (H(2)S) through the transsulfuration pathway. H(2)S has attracted considerable attention in recent years for many positive effects on vascular health and homeostasis. Cystathionine β-synthase (CBS) is the first, and rate-limiting, enzyme in the transsulfuration pathway. Mutations in the CBS gene decrease enzymatic activity, which increases the plasma Hcy concentration, a condition called hyperhomocysteinemia (HHcy). Animal models of CBS deficiency have provided invaluable insights into the pathological effects of transsulfuration impairment and of both mild and severe HHcy. However, studies have also highlighted the complexity of HHcy and the need to explore the specific details of Hcy metabolism in addition to Hcy levels per se. There has been a relative paucity of work addressing the dysfunctional H(2)S production in CBS deficiency that may contribute to, or even create, HHcy-associated pathologies. Experiments using CBS knockout mice, both homozygous (-/-) and heterozygous (+/-), have provided 15 years of new knowledge and are the focus of this review. These murine models present the opportunity to study a specific mechanism for HHcy that matches one of the etiologies in many human patients. Therefore, the goal of this review was to integrate and highlight the critical information gained thus far from models of CBS deficiency and draw attention to critical gaps in knowledge, with particular emphasis on the modulation of H(2)S metabolism. We include findings from human and animal studies to identify important opportunities for future investigation that should be aimed at generating new basic and clinical understanding of the role of CBS and transsulfuration in cardiovascular and neurovascular disease.

Pharmacological activation and genetic manipulation of cystathionine beta-synthase alter circulating levels of homocysteine and hydrogen sulfide in mice

Hydrogen sulfide (H 2S) is a recently discovered gasotransmitter found in mammalian tissues and blood. Treatment with H 2S donor molecules has shown promising results in preclinical models of inflammatory and cardiovascular diseases. Augmentation of H 2S levels thus holds promise as a novel therapeutic approach for treatment of disease in man. Cystathionine β-synthase (CBS) has been shown to catalyze H 2S production in vitro. CBS enzyme activity is allosterically regulated by the endogenous activator S-adenosyl methionine. This mode of regulation suggests the possibility for designing a small molecule activator of CBS to enhance H 2S production. This hypothesis, however, has not been directly tested in vivo. We show here that CBS contributes significantly to endogenous H 2S production in mice: adenovirus mediated over expression of CBS in the liver significantly increased circulating levels of H 2S, whereas CBS deficiency resulted in reduced levels. We demonstrate that CBS enzyme from endogenous sources can be activated by S-adenosyl methionine to a greater extent compared to recombinant enzyme, suggesting greater potential for activation than previously anticipated. Importantly, we show that circulating H 2S levels are increased by pharmacological activation of CBS in vivo; i.e. in the presence of the endogenous activator. Together, our data demonstrate that CBS activity partially regulates endogenous H 2S in mice, and suggest that pharmacological activation of CBS is a promising approach for enhancing endogenous production of H 2S for the treatment of cardiovascular and other diseases.

X-ray imaging of differential vascular density in MMP-9−/−, PAR-1−/+, hyperhomocysteinemic (CBS−/+) and diabetic (Ins2−/+) mice

Although protease activated receptor-1 (PAR-1) and matrix metalloproteinase-9 (MMP-9) play significant role in vascular remodelling in hyperhomocysteinemia (HHcy due to cystathionine beta synthase deficiency, CBS−/+) and diabetes, mechanism remains nebulous. We hypothesized that differential vascular density and remodelling in different vascular beds in HHcy and diabetes were responsible for an adaptive metabolic homeostasis during the pathogenesis. To test this hypothesis, vascular density in mice lacking PAR-1, MMP-9, CBS and Insulin-2 gene mutant (Ins2−/+, Akita) was measured and compared with wild type (WT, C57BL/6J) mice. The vascular density was detected by x-ray angiography using KODAK 4000 MM image station, using barium sulphate as contrasting agent. The % vascular density in the hearts of WT, CBS−/+ (HHcy), MMP-9−/−, PAR-1−/+ and Ins2−/+ (type-1 diabetes) was 100 ± 2.8, 85 ± 3.3, 90 ± 3.3, 95 ± 3.8 and 73 ± 1.7, respectively. The vascular density in CBS−/+ and Akita hearts decreased while it was increased in lungs of CBS−/+ and MMP-9−/−.There was decreased vascular density in liver and kidney of Akita mice. Vascular density in brain, kidney and mesentery was decreased in CBS−/+ mice. These findings support the notation that metabolic derangement in diabetes and HHcy causes the chronic decline and/or rarefaction in vascular density.

Determination of cystathionine beta‐synthase activity in human plasma by LC‐MS/MS: potential use in diagnosis of CBS deficiency

Cystathionine β-synthase (CBS) deficiency is usually confirmed by assaying the enzyme activity in cultured skin fibroblasts. We investigated whether CBS is present in human plasma and whether determination of its activity in plasma could be used for diagnostic purposes. We developed an assay to measure CBS activity in 20 μL of plasma using a stable isotope substrate - 2,3,3-(2)H serine. The activity was determined by measurement of the product of enzyme reaction, 3,3-(2)H-cystathionine, using LC-MS/MS. The median enzyme activity in control plasma samples was 404 nmol/h/L (range 66-1,066; n = 57). In pyridoxine nonresponsive CBS deficient patients, the median plasma activity was 0 nmol/ho/L (range 0-9; n = 26), while in pyridoxine responsive patients the median activity was 16 nmol/hour/L (range 0-358; n = 28); this overlapped with the enzyme activity from control subject. The presence of CBS in human plasma was confirmed by an in silico search of the proteome database, and was further evidenced by the activation of CBS by S-adenosyl-L-methionine and pyridoxal 5'-phosphate, and by configuration of the detected reaction product, 3,3-(2)H-cystathionine, which was in agreement with the previously observed CBS reaction mechanism. We hypothesize that the CBS enzyme in plasma originates from liver cells, as the plasma CBS activities in patients with elevated liver aminotransferase activities were more than 30-fold increased. In this study, we have demonstrated that CBS is present in human plasma and that its catalytic activity is detectable by LC-MS/MS. CBS assay in human plasma brings new possibilities in the diagnosis of pyridoxine nonresponsive CBS deficiency.

Cystathionine γ-Lyase-deficient Mice Require Dietary Cysteine to Protect against Acute Lethal Myopathy and Oxidative Injury

Cysteine is considered a nonessential amino acid in mammals as it is synthesized from methionine via trans-sulfuration. However, premature infants or patients with hepatic failure may require dietary cysteine due to a lack of cystathionine gamma-lyase (CTH), a key trans-sulfuration enzyme. Here, we generated CTH-deficient (Cth(-/-)) mice as an animal model of cystathioninemia/cystathioninuria. Cth(-/-) mice developed normally in general but displayed hypercystathioninemia/hyperhomocysteinemia though not hypermethioninemia. When fed a low cyst(e)ine diet, Cth(-/-) mice showed acute skeletal muscle atrophy (myopathy) accompanied by enhanced gene expression of asparagine synthetase and reduced contents of glutathione in livers and skeletal muscles, and intracellular accumulation of LC3 and p62 in skeletal myofibers; they finally died of severe paralysis of the extremities. Cth(-/-) hepatocytes required cystine in a culture medium and showed greater sensitivity to oxidative stress. Cth(-/-) mice exhibited systemic vulnerability to oxidative injury, which became more prominent when they were fed the low cyst(e)ine diet. These results reveal novel roles of trans-sulfuration previously unrecognized in mice lacking another trans-sulfuration enzyme cystathionine beta-synthase (Cbs(-/-)). Because Cbs(-/-) mice display hyperhomocysteinemia and hypermethioninemia, our results raise questions against the homocysteine-based etiology of CBS deficiency and the current newborn screening for homocysteinemia using Guthrie's method, which detects hypermethioninemia.

Mutation spectrum of MMACHC in Chinese patients with combined methylmalonic aciduria and homocystinuria

The cblC type of combined methylmalonic aciduria (MMA) and homocystinuria (HC) is the most common inborn error of vitamin B(12) metabolism and is caused by mutations in the MMACHC gene. To elucidate the spectrum of mutations that causes combined MMA and HC in Chinese patients, the MMACHC gene was sequenced in 79 unrelated Chinese patients. Sequence analysis identified 98.1% of disease alleles and found that all patients had at least one MMACHC mutation. A total of 24 mutations were identified. Out of the 24 mutations identified, 9 were novel ones, including missense mutations (c.365A>T and c.452A>G), nonsense mutations (c.315C>G and c.615C>A), deletions (c.99delA and c.277-3_c.303del30), duplications (c.248dupT and c.626dupT) and an insertion (c.445_446insA). The c.609G>A, c.658_660delAAG, c.482G>A, c.394C>T and c.80A>G mutations were the most common mutations and accounted for 80% of disease alleles. Haplotype analysis suggests that the spread of the c.80A>G, c.609G>A and c.658_660delAAG mutations in Chinese patients were caused by a founder effect. The results indicate that defects occurring in the MMACHC gene are the major cause of this disease in Chinese patients with combined MMA and HC, and direct mutation analysis can therefore be used as a rapid confirmatory diagnosis among these Chinese patients.

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.