The effect of diet-induced weight loss on circulating homocysteine levels in people with obesity and type 2 diabetes.

BACKGROUND/AIMS: Having type 2 diabetes (T2D) in combination with being overweight results in an additional increase in cardiovascular disease (CVD) risk. In addition, T2D and obesity are associated with increased levels of total homocysteine (tHcy), possibly contributing to the CVD risk. Weight loss dieting has positive effects on several CVD risk factors, but whether it affects tHcy remains unclear. Therefore, the aim of this study was to determine the effect of a calorie restricted diet on tHcy in overweight people with T2D. METHODS: In this post-hoc analysis of the POWER study, adults with T2D and a BMI greater than 27 kg/m² were included from the outpatient diabetes clinic of the Erasmus Medical Center, Rotterdam. The patients were subjected to a very low-calorie diet with fortified meal replacements for 20 weeks. Before and after this intervention, blood samples were collected to measure tHcy and other CVD risk factors like glycaemic and lipid parameters. RESULTS: 161 overweight participants with T2D were included, with a mean age of 54 years (range 26-74), mean weight of 104.6 ± 19.9 kg and mean HbA1c of 62.7 ± 14.3 mmol/mol. At baseline, men displayed higher tHcy than women, and tHcy level was positively correlated with body weight and triglyceride levels, while it was negatively correlated with renal function and HDL cholesterol. During the intervention, bodyweight was reduced by a mean of 9.7% (from 104.6 ± 19.9 to 94.5 ± 18.1 kg p < 0.001), and all measured glycaemic and lipid blood parameters improved significantly. However, tHcy remained unchanged (from 12.1 ± 4.1 to 12.1 ± 4.2 umol/L, p = 0.880). The change in tHcy during the intervention was negatively associated with the change in weight and BMI (p = 0.01 and p = 0.008, respectively). People who lost < 10 kg (n = 92) had a mean tHcy change of -0.47 umol/L, while people who lost more than ≥ 10 kg (n = 69) had a mean tHcy change of 0.60 umol/L (p = 0.021). CONCLUSION: In conclusion, our data show that a calorie restricted diet does not affect tHcy in people with T2D and obesity, despite the use of meal replacements fortified with folic acid and vitamin B12. Our data showed a negative correlation between change in tHcy levels and weight loss, suggesting that people who lost more weight (> 10 kg) showed an increase in tHcy. Future studies should explore the potential increase in tHcy induced by weight loss dieting and target the question if tHcy reduction strategies during weight loss could be clinically beneficial.

Homocysteine and methylmalonic acid in Phenylketonuria patients.

Hyperhomocysteinemia and vitamin B12 deficiency have been reported in patients with phenylketonuria. In this study, total homocysteine (tHcy) and methylmalonic acid (MMA) levels were analyzed in samples from 25 phenylketonuria (PKU) patients. Comparisons were made between pre- and post-treatment values (n= 3); on treatment values, between periods with high and normal/low phenylalanine (Phe) levels (n= 20); and in women before, during and after pregnancy (n= 3). THcy levels decreased after treating PKU with metabolic formula (p=0.014). Except for a pregnant woman before pregnancy, none of the patients had tHcy values above the normal range. In fact, tHcy was < 5 µmol/L in 34% of the samples. We observed a decrease in Phe, tHcy, and tyrosine levels during pregnancy. MMA levels did not differ significantly, with values remaining in the normal range. These data indicate that there was no B12 deficiency in patients who adhere to the diet. In conclusion, in PKU patients treated with metabolic formula, tHcy is frequently not elevated, remaining even in the lower normal range in some patients. Thus, clinical follow-up and adherence to dietary treatment are crucial to prevent B12 deficiency.

Integration of metabolomics with genomics: Metabolic gene prioritization using metabolomics data and genomic variant (CADD) scores.

The integration of metabolomics data with sequencing data is a key step towards improving the diagnostic process for finding the disease-causing genetic variant(s) in patients suspected of having an inborn error of metabolism (IEM). The measured metabolite levels could provide additional phenotypical evidence to elucidate the degree of pathogenicity for variants found in genes associated with metabolic processes. We present a computational approach, called Reafect, that calculates for each reaction in a metabolic pathway a score indicating whether that reaction is deficient or not. When calculating this score, Reafect takes multiple factors into account: the magnitude and sign of alterations in the metabolite levels, the reaction distances between metabolites and reactions in the pathway, and the biochemical directionality of the reactions. We applied Reafect to untargeted metabolomics data of 72 patient samples with a known IEM and found that in 81% of the cases the correct deficient enzyme was ranked within the top 5% of all considered enzyme deficiencies. Next, we integrated Reafect with Combined Annotation Dependent Depletion (CADD) scores (a measure for gene variant deleteriousness) and ranked the metabolic genes of 27 IEM patients. We observed that this integrated approach significantly improved the prioritization of the genes containing the disease-causing variant when compared with the two approaches individually. For 15/27 IEM patients the correct affected gene was ranked within the top 0.25% of the set of potentially affected genes. Together, our findings suggest that metabolomics data improves the identification of affected genes in patients suffering from IEM.

Postauthorization safety study of betaine anhydrous.

Patient registries for rare diseases enable systematic data collection and can also be used to facilitate postauthorization safety studies (PASS) for orphan drugs. This study evaluates the PASS for betaine anhydrous (Cystadane), conducted as public private partnership (PPP) between the European network and registry for homocystinurias and methylation defects and the marketing authorization holder (MAH). Data were prospectively collected, 2013-2016, in a noninterventional, international, multicenter, registry study. Putative adverse and severe adverse events were reported to the MAH's pharmacovigilance. In total, 130 individuals with vitamin B6 nonresponsive (N = 54) and partially responsive (N = 7) cystathionine beta-synthase (CBS) deficiency, as well as 5,10-methylenetetrahydrofolate reductase (MTHFR; N = 21) deficiency and cobalamin C (N = 48) disease were included. Median (range) duration of treatment with betaine anhydrous was 6.8 (0-9.8) years. The prescribed betaine dose exceeded the recommended maximum (6 g/day) in 49% of individuals older than 10 years because of continued dose adaptation to weight; however, with disease-specific differences (minimum: 31% in B6 nonresponsive CBS deficiency, maximum: 67% in MTHFR deficiency). Despite dose escalation no new or potential risk was identified. Combined disease-specific treatment decreased mean ± SD total plasma homocysteine concentrations from 203 ± 116 to 81 ± 51 µmol/L (p < 0.0001), except in MTHFR deficiency. Recommendations for betaine anhydrous dosage were revised for individuals ≥ 10 years. PPPs between MAH and international scientific consortia can be considered a reliable model for implementing a PASS, reutilizing well-established structures and avoiding data duplication and fragmentation.

Cystathionine ß-synthase deficiency in the E-HOD registry-part I: pyridoxine responsiveness as a determinant of biochemical and clinical phenotype at diagnosis.

Cystathionine ß-synthase (CBS) deficiency has a wide clinical spectrum, ranging from neurodevelopmental problems, lens dislocation and marfanoid features in early childhood to adult onset disease with predominantly thromboembolic complications. We have analysed clinical and laboratory data at the time of diagnosis in 328 patients with CBS deficiency from the E-HOD (European network and registry for Homocystinurias and methylation Defects) registry. We developed comprehensive criteria to classify patients into four groups of pyridoxine responsivity: non-responders (NR), partial, full and extreme responders (PR, FR and ER, respectively). All groups showed overlapping concentrations of plasma total homocysteine while pyridoxine responsiveness inversely correlated with plasma/serum methionine concentrations. The FR and ER groups had a later age of onset and diagnosis and a longer diagnostic delay than NR and PR patients. Lens dislocation was common in all groups except ER but the age of dislocation increased with increasing responsiveness. Developmental delay was commonest in the NR group while no ER patient had cognitive impairment. Thromboembolism was the commonest presenting feature in ER patients, whereas it was least likely at presentation in the NR group. This probably is due to the differences in ages at presentation: all groups had a similar number of thromboembolic events per 1000 patient-years. Clinical severity of CBS deficiency depends on the degree of pyridoxine responsiveness. Therefore, a standardised pyridoxine-responsiveness test in newly diagnosed patients and a critical review of previous assessments is indispensable to ensure adequate therapy and to prevent or reduce long-term complications.

In silico and in vivo models for Qatari-specific classical homocystinuria as basis for development of novel therapies.

Homocystinuria is a rare inborn error of methionine metabolism caused by cystathionine ß-synthase (CBS) deficiency. The prevalence of homocystinuria in Qatar is 1:1,800 births, mainly due to a founder Qatari missense mutation, c.1006C>T; p.R336C (p.Arg336Cys). We characterized the structure-function relationship of the p.R336C-mutant protein and investigated the effect of different chemical chaperones to restore p.R336C-CBS activity using three models: in silico, ΔCBS yeast, and CRISPR/Cas9 p.R336C knock-in HEK293T and HepG2 cell lines. Protein modeling suggested that the p.R336C induces severe conformational and structural changes, perhaps influencing CBS activity. Wild-type CBS, but not the p.R336C mutant, was able to restore the yeast growth in ΔCBS-deficient yeast in a complementation assay. The p.R336C knock-in HEK293T and HepG2 cells decreased the level of CBS expression and reduced its structural stability; however, treatment of the p.R336C knock-in HEK293T cells with betaine, a chemical chaperone, restored the stability and tetrameric conformation of CBS, but not its activity. Collectively, these results indicate that the p.R336C mutation has a deleterious effect on CBS structure, stability, and activity, and using the chemical chaperones approach for treatment could be ineffective in restoring p.R336C CBS activity.

Effect of BTD gene variants on in vitro biotinidase activity.

INTRODUCTION: Biotinidase deficiency (BD), an autosomal recessive disease, is classified into profound (activity <10%) or partial BD (activity 10-30%). The most frequent variant in patients worldwide is c.1330G > C (p.Asp444His), which is associated with partial BD. In vivo studies indicate that this variant reduces the biotinidase activity by 50%. The objective of this study was to evaluate the in vitro effect of p.Asp444His and of five novel variants identified among Brazilian individuals showing low activity of biotinidase in serum. METHODS: The variants c.119 T > C (p.Leu40Pro), c.479G > A (p.Cys160Tyr), c.664G > A (p.Asp222Asn), c.1330G > C (p.Asp444His), c.1337 T > C (p.Leu446Pro), c.1466A > G (p.Asn489Ser) and the wild type (wt) BTD gene were expressed in HEK 293 cells. Biotinidase activity was quantified by colorimetric method in cells homogenates and culture medium. The wtBTD activity was considered 100%. RESULTS: The p.Leu40Pro, p.Cys160Tyr and p.Leu446Pro variants were associated to impaired biotinidase activity (activity in cells: 33%, 14%, 0%, respectively; activity in medium: 7%, 0.3%, 2%, respectively) and undetectable amount of protein in intra and extracellular space. The p.Asn489Ser variant had these effects restricted to the extracellular space (activity in medium: 43%), and the p.Asp222Asn variant showed normal activity. The expression of p.Asp444His variant resulted in detectable protein and slightly reduced activity only in cells (activity in cells: 46%; activity in medium: 115%). CONCLUSION: Our findings suggest that the p.Leu40Pro, p.Cys160Tyr and p.Leu446Pro variants are deleterious; the p.Asn489Ser is probably related to a mild biochemical phenotype; and p.Asp222Asn variant is probably not deleterious. The p.Asp444His variant seems to code for a protein with variable activity.

Analysis of the Qatari R336C cystathionine ß-synthase protein in mice.

Classical homocystinuria is a recessive inborn error of metabolism caused by mutations in the cystathionine beta-synthase (CBS) gene. The highest incidence of CBS deficiency in the world is found in the country of Qatar due to the combination of high rates of consanguinity and the presence of a founder mutation, c.1006C>T (p.R336C). This mutation does not respond to pyridoxine and is considered severe. Here we describe the creation of a mouse that is null for the mouse Cbs gene and expresses human p.R336C CBS from a zinc-inducible transgene (Tg-R336C Cbs -/- ). Zinc-treated Tg-R336C Cbs -/- mice have extreme elevation in both serum total homocysteine (tHcy) and liver tHcy compared with control transgenic mice. Both the steady-state protein levels and CBS enzyme activity levels in liver lysates from Tg-R336C Cbs -/- mice are significantly reduced compared to that found in Tg-hCBS Cbs -/- mice expressing wild-type human CBS. Treatment of Tg-R336C Cbs -/- mice with the proteasome inhibitor bortezomib results in stabilization of liver CBS protein and an increase in activity to levels found in corresponding Tg-hCBS Cbs -/- wild type mice. Surprisingly, serum tHcy did not fully correct even though liver enzyme activity was as high as control animals. This discrepancy is explained by in vitro enzymatic studies of mouse liver extracts showing that p.R336C causes reduced binding affinity for the substrate serine by almost 7-fold and significantly increased dependence on pyridoxal phosphate in the reaction buffer. These studies demonstrate that the p.R336C alteration effects both protein stability and substrate/cofactor binding.

Natural history, with clinical, biochemical, and molecular characterization of classical homocystinuria in the Qatari population.

Classical homocystinuria (HCU) is the most common inborn error of metabolism in Qatar, with an incidence of 1:1800, and is caused by the Qatari founder p.R336C mutation in the CBS gene. This study describes the natural history and clinical manifestations of HCU in the Qatari population. A single center study was performed between 2016 and 2017 in 126 Qatari patients, from 82 families. Detailed clinical and biochemical data were collected, and Stanford-Binet intelligence, quality of life and adherence to treatment assessments were conducted prospectively. Patients were assigned to one of three groups, according to the mode of diagnosis: (a) late diagnosis group (LDG), (b) family screening group (FSG), and (c) newborn screening group (NSG). Of the 126 patients, 69 (55%) were in the LDG, 44 (35%) in the NSG, and 13 (10%) in the FSG. The leading factors for diagnosis in the LDG were ocular manifestations (49%), neurological manifestations (45%), thromboembolic events (4%), and hyperactivity and behavioral changes (1%). Both FSG and NSG groups were asymptomatic at time of diagnosis. NSG had significantly higher intelligence quotient, quality of life, and adherence values compared with the LDG. The LDG and FSG had significantly higher methionine levels than the NSG. The LDG also had significantly higher total homocysteine levels than the NSG and FSG. Regression analysis confirmed these results even when adjusting for age at diagnosis, current age, or adherence. These findings increase the understanding of the natural history of HCU and highlight the importance of early diagnosis and treatment. SYNOPSIS: A study in 126 Qatari patients with HCU, including biochemical, clinical, and other key assessments, reveals that patients with a late clinical diagnosis have a poorer outcome, hereby highlighting the importance of early diagnosis and treatment.

Newborn screening for homocystinurias: Recent recommendations versus current practice.

PURPOSE: To assess how the current practice of newborn screening (NBS) for homocystinurias compares with published recommendations. METHODS: Twenty-two of 32 NBS programmes from 18 countries screened for at least one form of homocystinuria. Centres provided pseudonymised NBS data from patients with cystathionine beta-synthase deficiency (CBSD, n = 19), methionine adenosyltransferase I/III deficiency (MATI/IIID, n = 28), combined remethylation disorder (cRMD, n = 56) and isolated remethylation disorder (iRMD), including methylenetetrahydrofolate reductase deficiency (MTHFRD) (n = 8). Markers and decision limits were converted to multiples of the median (MoM) to allow comparison between centres. RESULTS: NBS programmes, algorithms and decision limits varied considerably. Only nine centres used the recommended second-tier marker total homocysteine (tHcy). The median decision limits of all centres were ≥ 2.35 for high and ≤ 0.44 MoM for low methionine, ≥ 1.95 for high and ≤ 0.47 MoM for low methionine/phenylalanine, ≥ 2.54 for high propionylcarnitine and ≥ 2.78 MoM for propionylcarnitine/acetylcarnitine. These decision limits alone had a 100%, 100%, 86% and 84% sensitivity for the detection of CBSD, MATI/IIID, iRMD and cRMD, respectively, but failed to detect six individuals with cRMD. To enhance sensitivity and decrease second-tier testing costs, we further adapted these decision limits using the data of 15 000 healthy newborns. CONCLUSIONS: Due to the favorable outcome of early treated patients, NBS for homocystinurias is recommended. To improve NBS, decision limits should be revised considering the population median. Relevant markers should be combined; use of the postanalytical tools offered by the CLIR project (Collaborative Laboratory Integrated Reports, which considers, for example, birth weight and gestational age) is recommended. tHcy and methylmalonic acid should be implemented as second-tier markers.

Adenosine Kinase Deficiency: Report and Review.

Adenosine kinase (ADK) deficiency (OMIM [online mendelian inheritance in man]: 614300) is an autosomal recessive disorder of adenosine and methionine metabolism, with a unique clinical phenotype, mainly involving the central nervous system and dysmorphic features. Patients usually present early in life with sepsis-like symptoms, respiratory difficulties, and neonatal jaundice. Subsequently, patients demonstrate hypotonia and global developmental delay. Biochemically, methionine is elevated with normal homocysteine levels and the diagnosis is confirmed through molecular analysis of the ADK gene. There is no curative treatment; however, a methionine-restricted diet has been tried with variable outcomes. Herein, we report a 4-year-old Saudi female with global developmental delay, hypotonia, and dysmorphic features. Interestingly, she has a tall stature, developmental dysplasia of the hip, optic nerve gliosis, and tigroid fundus. We found a mutation not reported previously and we compared the current case with previously reported cases. We alert clinicians to consider ADK deficiency in any neonate presenting with global developmental delay, hypotonia, dysmorphic features, and high methionine levels.

S-adenosylhomocysteine induces inflammation through NFkB: A possible role for EZH2 in endothelial cell activation.

S-adenosylhomocysteine (SAH) can induce endothelial dysfunction and activation, contributing to atherogenesis; however, its role in the activation of the inflammatory mediator NFkB has not been explored. Our aim was to determine the role of NFkB in SAH-induced activation of endothelial cells. Furthermore, we examined whether SAH, as a potent inhibitor of S-adenosylmethionine-dependent methyltransferases, suppresses the function of EZH2 methyltransferase to contribute to SAH-induced endothelial cell activation. We found that excess SAH increases the expression of adhesion molecules and cytokines in human coronary artery endothelial cells. Importantly, this up-regulation was suppressed in cells expressing a dominant negative form of the NFkB inhibitor, IkB. Moreover, SAH accumulation triggers the activation of both the canonical and non-canonical NFkB pathways, decreases EZH2, and reduces histone 3 lysine 27 trimethylation. EZH2 knockdown recapitulated the effects of excess SAH on endothelial activation, i.e., it induced NFkB activation and the subsequent up-regulation of adhesion molecules and cytokines. Our findings suggest that suppression of the epigenetic regulator EZH2 by excess SAH may contribute to NFkB activation and the consequent vascular inflammatory response. These studies unveil new targets of SAH regulation, demonstrating that EZH2 suppression and NFkB activation mediated by SAH accumulation may contribute to its adverse effects in the vasculature.

Small aminothiol compounds improve the function of Arg to Cys variant proteins: effect on the human cystathionine ß-synthase p.R336C.

The key regulatory point of L-methionine (Met) and L-homocysteine (Hcy) degradation is catalyzed by cystathionine beta-synthase (CBS). CBS deficiency is caused by mutations in CBS gene, often resulting in protein misfolding. The prevalence of CBS deficiency in Qatar is 1/1800, ∼200-fold higher than the worldwide prevalence of 1/344 000. Almost all patients bear the CBS p.R336C variant. More than 20 years ago, it was shown in vitro that two unrelated protein variants with a substitution of an arginine (Arg) residue by cysteine (Cys) could be rescued by cysteamine (mercaptoethylamine), likely via formation of a disulfide between Cys and cysteamine, functionally mimicking the wild-type (WT) Arg side-chain. Based on these findings, we aimed to study whether cysteamine was able to improve the function of p.R336C CBS variant. Additionally, we tested the effect of mercaptoethylguanidine (MEG), a compound with a guanidino and a thiol function that may resemble Arg structure better than cysteamine. Three purified recombinant CBS proteins (p.R336C, p.R336H and WT) were pre-incubated with cysteamine, MEG or Cys (as negative control), and CBS activity and stability were measured. Pre-incubation with cysteamine and MEG increased the enzymatic activity of the p.R336C protein, which was absent upon pre-incubation with Cys. The WT and the p.R336H variant enzyme activity presented no increase with any of the tested compounds. Our results show that cysteamine and MEG are able to specifically improve the function of the CBS p.R336C variant, suggesting that any Arg-to-Cys substitution accessible to these small molecules may be converted back to a moiety resembling Arg.

Consensus recommendations for the diagnosis, treatment and follow-up of inherited methylation disorders.

Inherited methylation disorders are a group of rarely reported, probably largely underdiagnosed disorders affecting transmethylation processes in the metabolic pathway between methionine and homocysteine. These are methionine adenosyltransferase I/III, glycine N-methyltransferase, S-adenosylhomocysteine hydrolase and adenosine kinase deficiencies. This paper provides the first consensus recommendations for the diagnosis and management of methylation disorders. Following search of the literature and evaluation according to the SIGN-methodology of all reported patients with methylation defects, graded recommendations are provided in a structured way comprising diagnosis (clinical presentation, biochemical abnormalities, differential diagnosis, newborn screening, prenatal diagnosis), therapy and follow-up. Methylation disorders predominantly affect the liver, central nervous system and muscles, but clinical presentation can vary considerably between and within disorders. Although isolated hypermethioninemia is the biochemical hallmark of this group of disorders, it is not always present, especially in early infancy. Plasma S-adenosylmethionine and S-adenosylhomocysteine are key metabolites for the biochemical clarification of isolated hypermethioninemia. Mild hyperhomocysteinemia can be present in all methylation disorders. Methylation disorders do not qualify as primary targets of newborn screening. A low-methionine diet can be beneficial in patients with methionine adenosyltransferase I/III deficiency if plasma methionine concentrations exceed 800 µmol/L. There is some evidence that this diet may also be beneficial in patients with S-adenosylhomocysteine hydrolase and adenosine kinase deficiencies. S-adenosylmethionine supplementation may be useful in patients with methionine adenosyltransferase I/III deficiency. Recommendations given in this article are based on general principles and in practice should be adjusted individually according to patient's age, severity of the disease, clinical and laboratory findings.

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

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 µmol/L. Nevertheless, we recommend keeping the concentration below 100 µmol/L because levels fluctuate and the complications associated with high levels are so serious.

Paediatric reference values for total homocysteine, tryptophan, tyrosine and phenylalanine in blood spots.

Determining blood concentrations of the amino acids homocysteine, tryptophan, tyrosine and phenylalanine in children is of value in the clinical practice. Over the past decades, the use of blood spot samples to examine amino acid concentrations is increasing rapidly. In children, the use of blood spot samples is especially of relevance, as this method is much less invasive than venous blood sampling. Currently, no paediatric reference values for amino acids in blood spots are available. The aim of the current study was to establish reference values for blood spot concentrations of total homocysteine, tryptophan, tyrosine and phenylalanine in school-age children. Dried blood spots were obtained in a community sample of 104 healthy children, aged 6-12 years old (52% males). Blood spot concentrations of total homocysteine, tryptophan, tyrosine and phenylalanine were determined by positive electrospray liquid chromatography-tandem mass spectrometry. Parents of participants completed questions regarding demographic characteristics. Our sample consisted of healthy children from various ethnic backgrounds, with varying levels of socioeconomic status, in line with the composition of the Dutch society. Blood spot concentrations of total homocysteine, tryptophan, tyrosine and phenylalanine were similar in males and females, and independent of age. In conclusion, paediatric reference values for blood spot concentrations of total homocysteine, tryptophan, tyrosine and phenylalanine were established, which could be of use in the clinical practice.

Adenosine kinase deficiency: expanding the clinical spectrum and evaluating therapeutic options.

BACKGROUND: Adenosine kinase deficiency is a recently described defect affecting methionine metabolism with a severe clinical phenotype comprising mainly neurological and hepatic impairment and dysmorphism. METHODS: Clinical data of 11 additional patients from eight families with adenosine kinase deficiency were gathered through a retrospective questionnaire. Two liver biopsies of one patient were systematically evaluated. RESULTS: The main clinical symptoms are mild to severe liver dysfunction with neonatal onset, muscular hypotonia, global developmental retardation and dysmorphism (especially frontal bossing). Hepatic involvement is not a constant finding. Most patients have epilepsy and recurrent hypoglycemia due to hyperinsulinism. Major biochemical findings are intermittent hypermethioninemia, increased S-adenosylmethionine and S-adenosylhomocysteine in plasma and increased adenosine in urine. S-adenosylmethionine and S-adenosylhomocysteine are the most reliable biochemical markers. The major histological finding was pronounced microvesicular hepatic steatosis. Therapeutic trials with a methionine restricted diet indicate a potential beneficial effect on biochemical and clinical parameters in four patients and hyperinsulinism was responsive to diazoxide in two patients. CONCLUSION: Adenosine kinase deficiency is a severe inborn error at the cross-road of methionine and adenosine metabolism that mainly causes dysmorphism, brain and liver symptoms, but also recurrent hypoglycemia. The clinical phenotype varies from an exclusively neurological to a multi-organ manifestation. Methionine-restricted diet should be considered as a therapeutic option.

Effect of vitamin B12 and folic acid supplementation on biomarkers of endothelial function and inflammation among elderly individuals with hyperhomocysteinemia.

B-vitamin trials failed to demonstrate beneficial effects on cardiovascular outcomes, but hyperhomocysteinemia still stands out as an independent cardiovascular risk factor, particularly in elderly individuals. B-vitamins may influence early vascular dysfunction, such as endothelial dysfunction, or may have adverse effects, for example on inflammation. We investigated the effect of B-vitamins on endothelial function and inflammation within an interventional study. This study was conducted within the framework of the B-PROOF trial, which included 2919 hyperhomocysteinemic elderly individuals, who received daily vitamin B12 (500 µg) and folic acid (400 µg) or placebo for 2 years. Using an electrochemiluminescence platform, we measured intercellular adhesion molecule 1 (ICAM-1), vascular adhesion molecule 1 (VCAM-1), serum amyloid A (SAA), vascular endothelial growth factor (VEGF) and C-reactive protein (CRP) at baseline and follow-up in a subsample of 522 participants (271 intervention group; 251 placebo). Treatment effects were analyzed with ANCOVA. The participants had a mean age of 72 years, and 55% of them were male. At the 2-year follow-up, B-vitamins did not change the ICAM-1 (+36% change in the intervention group versus +32% change in the placebo group; p = 0.72), VCAM-1 (+27% vs +25%; p = 0.39), VEGF (-1% vs +4%; p = 0.40), SAA (+34% vs +38%; p = 0.85) or CRP levels (+26% vs +36%; p = 0.70) as compared to placebo. In conclusion, in elderly patients with hyperhomocysteinemia, vitamin B12 and folic acid are unlikely to influence either endothelial function or low-grade systemic inflammation. ClinicalTrials.gov Identifier: NCT00696514.

Inhibition of cellular methyltransferases promotes endothelial cell activation by suppressing glutathione peroxidase 1 protein expression.

S-adenosylhomocysteine (SAH) is a negative regulator of most methyltransferases and the precursor for the cardiovascular risk factor homocysteine. We have previously identified a link between the homocysteine-induced suppression of the selenoprotein glutathione peroxidase 1 (GPx-1) and endothelial dysfunction. Here we demonstrate a specific mechanism by which hypomethylation, promoted by the accumulation of the homocysteine precursor SAH, suppresses GPx-1 expression and leads to inflammatory activation of endothelial cells. The expression of GPx-1 and a subset of other selenoproteins is dependent on the methylation of the tRNA(Sec) to the Um34 form. The formation of methylated tRNA(Sec) facilitates translational incorporation of selenocysteine at a UGA codon. Our findings demonstrate that SAH accumulation in endothelial cells suppresses the expression of GPx-1 to promote oxidative stress. Hypomethylation stress, caused by SAH accumulation, inhibits the formation of the methylated isoform of the tRNA(Sec) and reduces GPx-1 expression. In contrast, under these conditions, the expression and activity of thioredoxin reductase 1, another selenoprotein, is increased. Furthermore, SAH-induced oxidative stress creates a proinflammatory activation of endothelial cells characterized by up-regulation of adhesion molecules and an augmented capacity to bind leukocytes. Taken together, these data suggest that SAH accumulation in endothelial cells can induce tRNA(Sec) hypomethylation, which alters the expression of selenoproteins such as GPx-1 to contribute to a proatherogenic endothelial phenotype.

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

Accumulation of the homocysteine (Hcy) precursor S-adenosylhomocysteine (AdoHcy) may cause cellular hypomethylation in the setting of hyperhomocysteinemia because of cystathionine ß-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.