Homocystinuria Research Articles

Leptin concentrations and SCD-1 indices in classical homocystinuria: Evidence for the role of sulfur amino acids in the regulation of lipid metabolism

BackgroundWe describe body composition, lipid metabolism and Stearoyl-CoA desaturase-1 (SCD-1) indices in patients with classical homocystinuria (HCU). MethodsEleven treated HCU patients and 16 healthy controls were included. Body composition and bone mineral density were assessed by dual X-ray absorptiometry. Sulfur amino acids (SAA) and their derivatives (total homocysteine, cysteine, methionine, S-adenosylmethionine, S-adenosylhomocysteine, and glutathione), lipids (free fatty acids, acylcarnitines, triglycerides and lipoproteins), glucose, insulin, leptin, adiponectin, and isoprostanes were measured in plasma. Insulin resistance was evaluated by HOMA-IR. To estimate liver SCD-1 activity, SCD-16 [16:1(n−7)/16:0] and SCD-18 [18:1(n−9)/18:0] desaturation indices were determined. ResultsIn HCU patients, SCD-16 index was significantly reduced (p=0.03). A trend of an association of SCD-16 index with cysteine was observed (r=0.624, p=0.054). HCU patients displayed lower lean mass (p<0.05), with no differences in fat mass percentage. Leptin and low-density lipoprotein concentrations were lower in HCU patients (p<0.05). Femur bone mineral density Z-scores were correlated with plasma cysteine (r=0.829; p=0.04) and total homocysteine (r=−0.829; p=0.04) in HCU patients. ConclusionsWe report alterations in leptin and SCD-1 in HCU patients. These results agree with previous findings from epidemiologic and animal studies, and support a role for SAA on lipid homeostasis.

H2S and homocysteine control a novel feedback regulation of cystathionine beta synthase and cystathionine gamma lyase in cardiomyocytes

Hydrogen sulfide (H2S), a cardioprotective gas, is endogenously produced from homocysteine by cystathionine beta synthase (CBS) and cystathionine gamma lyase (CSE) enzymes. However, effect of H2S or homocysteine on CBS and CSE expression, and cross-talk between CBS and CSE are unclear. We hypothesize that homocysteine and H2S regulate CBS and CSE expressions in a dose dependent manner in cardiomyocytes, and CBS deficiency induces cardiac CSE expression. To test the hypothesis, we treated murine atrial HL1 cardiomyocytes with increasing doses of homocysteine or Na2S/GYY4137, a H2S donor, and measured the levels of CBS and CSE. We found that homocysteine upregulates CSE but downregulates CBS whereas Na2S/GYY4137 downregulates CSE but upregulates CBS in a dose-dependent manner. Moreover, the Na2S-treatment downregulates specificity protein-1 (SP1), an inducer for CSE, and upregulates miR-133a that targets SP1 and inhibits cardiomyocytes hypertrophy. Conversely, in the homocysteine-treated cardiomyocytes, CBS and miR-133a were downregulated and hypertrophy was induced. In vivo studies using CBS+/−, a model for hyperhomocysteinemia, and sibling CBS+/+ control mice revealed that deficiency of CBS upregulates cardiac CSE, plausibly by inducing SP1. In conclusion, we revealed a novel mechanism for H2S-mediated regulation of homocysteine metabolism in cardiomyocytes, and a negative feedback regulation between CBS and CSE in the heart.

The c.797 G>A (p.R266K) cystathionine β-synthase mutation causes homocystinuria by affecting protein stability.

Mutations in the cystathionine beta-synthase (CBS) gene are the cause of classical homocystinuria, the most common inborn error in sulfur metabolism. The c.797 G>A (p.R266K) mutation in CBS was originally described in several Norwegian pyridoxine responsive CBS deficient patients, and heterologous gene expression studies have shown that the protein has near wild-type levels of enzyme activity. Here, we characterize a transgenic mouse lacking endogenous Cbs and expressing p.R266K human CBS protein from a zinc inducible metallothionein promoter (Tg-R266K Cbs-/- ). Unlike mice expressing other mutant CBS alleles, the Tg-R266K transgene is unable to efficiently rescue neonatal lethality of Cbs-/- on a C57BL/6J background. On a C3H/HeJ background, zinc-induced Tg-R266K Cbs-/- mice express CBS mRNA, but have very low levels of CBS protein and enzyme activity, resulting in extreme elevations in serum total homocysteine (tHcy). Treatment with pyridoxine did not have any appreciable effect on tHcy, indicating this allele is not pyridoxine responsive in mice. However, treatment with the proteasome inhibitor bortezomib resulted in an 97% reduction in tHcy and a 2381% increase in liver CBS activity. These studies show that the p.R266K mutation causes increased proteasomal degradation in vivo, and that treatments that stabilize the protein can be used to reverse its effect.

Cystathionine β-synthase deficiency: Of mice and men.

Cystathionine β-synthase (CBS) deficiency (Online Mendelian Inheritance in Man [OMIM] 236,200) is an autosomal recessive disorder that is caused by mutations in the CBS gene. It is the most common inborn error of sulfur metabolism and is the cause of classical homocystinuria, a condition characterized by very high levels of plasma total homocysteine and methionine. Although recognized as an inborn error of metabolism over 60years ago, these is still much we do not understand related to how this specific metabolic defect gives rise to its distinct phenotypes. To try and answer these questions, several groups have developed mouse models on CBS deficiency. In this article, we will review various mouse models of CBS deficiency and discuss how these mouse models compare to human CBS deficient patients.

Abstract 146: Deficiency of Cystathionine beta Synthase Alters Blood Brain Barrier Integrity and Exacerbates Cerebral Ischemia Reperfusion Injury in Mice

Hyperhomocysteinemia is a risk factor for stroke; however the mechanisms by which elevated homocysteine leads to stroke are poorly defined. In a murine model of cystathionine beta synthase (CBS) deficiency, we investigated whether mild to severe elevation in plasma total homocysteine (tHcy) increases susceptibility to cerebral infarction and if this phenotype is associated with loss of blood brain barrier (BBB) integrity. We studied male Cbs -/- mice conditionally expressing a zinc-inducible mutated human CBS (I278T) transgene along with Cbs+/- and Cbs+/+ littermates at 10-14 weeks of age. The human transgene was allowed to express only until weaning to overcome the early mortality of Cbs-/- mice. Experimental stroke was induced with middle cerebral artery occlusion for one hour followed by 24 hours of reperfusion. TTC stained coronal sections were used to quantify the infarcted area. BBB integrity was assessed prior to injury using a standard Evan’s blue (EB) infusion method. Leakage of EB was quantified in brain homogenates and normalized to brain weight. Mild to severe increases in plasma tHcy were observed in Cbs +/- and Cbs -/- mice (6.1±0.3 and 309±18 μM, respectively) compared with Cbs+/+ littermates (3.1±0.6 μM, P&lt;0.01). Both Cbs +/- and Cbs -/- mice exhibited significant increases in infarct size following ischemia-reperfusion injury as compared to Cbs+/+ mice (12.2±3% in Cbs+/+ mice vs. 35.1±7.7% in Cbs+/- and 27.7±7.8% in Cbs-/- mice, P&lt;0.05). A significant increase in EB extravasation was observed in Cbs+/- and Cbs -/- mice (3.8±0.8 and 4.8±0.2 μg/g, respectively) compared to Cbs+/+ mice (1.7±0.4 μg/g, P&lt;0.05). The similarity in extent of cerebral infarction observed in Cbs+/- and Cbs-/- mice suggests a threshold effect and that a mild increase in tHcy is enough to disrupt BBB integrity and produce a severe stroke phenotype in experimental murine models.

Engineering and Characterization of an Enzyme Replacement Therapy for Classical Homocystinuria.

Homocystinuria due to loss of cystathionine beta-synthase (CBS) causes accumulation of homocysteine and depletion of cysteine. Current treatments are suboptimal, and thus the development of an enzyme replacement therapy based on PEGylated human truncated CBS (PEG-CBS) has been initiated. Attenuation of potency was observed, which necessitated a screen of several PEG-CBS conjugates for their efficacy to correct and maintain the plasma metabolite profile of murine homocystinuria after repeated administrations interrupted with washouts. We found that CBS coupling with maleimide PEG inconsistently modified the enzyme. In contrast, the PEG-CBS conjugate with 20 kDa N-hydroxysuccinimide-PEG showed very little loss of potency likely due to a reproducible PEGylation resulting in species modified with five PEGs per subunit on average. We developed assays suitable for monitoring the extent of CBS PEGylation and demonstrated a sustainable partial normalization of homocystinuria upon continuous PEG-CBS administration via osmotic pumps. Taken together, we identified the PEG-CBS conjugate suitable for manufacturing and clinical development.

Histone Deacetylase 3 Supports Cystathionine β‐synthase Mediated Bone formation by Controlling Cytokine Signaling and Matrix Remodeling

Cystathionine beta‐synthase (CBS) is a crucial enzyme in homocysteine (Hcy) metabolism. CBS deficiency cause hyperhomocysteinemia (HHcy) which confers diverse clinical manifestations, notably characteristic skeletal abnormalities. However, several clinical trials demonstrated that despite reduction in Hcy levels, bone disease outcome remained unaffected, thus the mechanism of bone formation is poorly defined. Therefore, we hypothesize that CBS as a novel molecular regulator in osteoblasts which promotes bone development. To test this hypothesis, we used 8–12 weeks old mice in our study; wild type mice (C57BL/6, WT) and CBS+/− mice. Silencing CBS function in osteoblast by either inhibitor hydroxylamine (HA) or CRISPR/CAS‐9 mediated gene knockout in vitro and bone marrow derived CBS‐deficient (CBS+/−) osteoblast causes reduced osteoblast differentiation and mineralization and increased osteoclast activity. Bone marrow derived CBS‐deficient (CBS+/−) osteoblasts exhibited exaggerated oxidative stress mechanism and further increased the expression of inflammatory cytokine and matrix‐degrading genes (IL‐6, MMP‐9, MMP‐13) and reduced expression of genes related to extracellular matrix production, bone development (ALP, Col1A, RUNX2, OSTERIX, OCN, OPN). ELISA assay identified among others IL‐6 to be present in plasma of CBS(+/−) mice but not in plasma of wild type mice (C57BL/6, WT). Furthermore, histone deacetylase activity 3 also decreased under oxidative stress mechanism via phosphorylation dependent manner. This leads to activation and acetylation of nuclear factor Kb (NF‐kB). This increased inflammatory cytokine production further suppresses the osteoblast differentiation and paracrine activation of osteoclasts and bone resorption via IL‐6 signaling. This increased resorptive activity is further confirmed with immunostaining of tibia cross section by TRAP‐activity and increased osteoclasts number in bone tissue of CBS+/− mice. Blockade of HDAC3 activation in CBS‐deficient (CBS+/−) osteoblast by RGFP566 (HDAC3 Inhibitor) leads to the remodelling of histone landscapes in genome and thereby accelerates histone acetylation via recognising the acetylated lysine residue in histone (H3K27ac) of chromatin. This eventually resulted in transcriptional activation of MMP9,‐13 and IL‐6 gene expression. Thus, CBS controls the temporal and spatial expression of tissue‐remodeling genes and inflammatory responses in osteoblast to ensure proper bone development. In conclusion, we demonstrate that CBS control the osteoblasts balances, osteoblast differentiation/mineralization and osteoclastgenesis via HDAC3 dependent manner during bone development.Support or Funding InformationThis work is financial supported from National Institute of health grant AR‐067667 and HL‐107640‐NT are greatly acknowledged.

Cystathionine beta-synthase deficiency alters hepatic phospholipid and choline metabolism: Post-translational repression of phosphatidylethanolamine N-methyltransferase is a consequence rather than a cause of liver injury in homocystinuria.

Classical homocystinuria (HCU) due to inactivating mutation of cystathionine β-synthase (CBS) is a poorly understood life-threatening inborn error of sulfur metabolism. A previously described cbs-/- mouse model exhibits a semi-lethal phenotype due to neonatal liver failure. The transgenic HO mouse model of HCU exhibits only mild liver injury and recapitulates multiple aspects of the disease as it occurs in humans. Disruption of the methionine cycle in HCU has the potential to impact multiple aspect of phospholipid (PL) metabolism by disruption of both the Kennedy pathway and phosphatidylethanolamine N-methyltransferase (PEMT) mediated synthesis of phosphatidylcholine (PC). Comparative metabolomic analysis of HO mouse liver revealed decreased levels of choline, and choline phosphate indicating disruption of the Kennedy pathway. Alterations in the relative levels of multiple species of PL included significant increases in PL degradation products consistent with enhanced membrane PL turnover. A significant decrease in PC containing 20:4n6 which primarily formed by the methylation of phosphatidylethanolamine to PC was consistent with decreased flux through PEMT. Hepatic expression of PEMT in both the cbs-/- and HO models is post-translationally repressed with decreased levels of PEMT protein and activity that inversely-correlates with the scale of liver injury. Failure to induce further repression of PEMT in HO mice by increased homocysteine, methionine and S-adenosylhomocysteine or depletion of glutathione combined with examination of multiple homocysteine-independent models of liver injury indicated that repression of PEMT in HCU is a consequence rather than a cause of liver injury. Collectively, our data show significant alteration of a broad range of hepatic PL and choline metabolism in HCU with the potential to contribute to multiple aspects of pathogenesis in this disease.

Newborn screening for remethylation disorders and vitamin B12 deficiency-evaluation of new strategies in cohorts from Qatar and Germany.

Newborn screening is a precondition for early diagnosis and successful treatment of remethylation disorders and classical homocystinuria (cystathionine-ß-synthase deficiency). Newborn screening for classical homocystinuria using total homocysteine measurement in dried blood spots has been very successfully performed for many years for newborns from Qatar. A new optimized newborn screening strategy for remethylation disorders and homocystinuria was developed and evaluated for newborns from Qatar using total homocysteine measurement as first-tier and methionine, methionine-phenylalanine-ratio and propionylcarnitine as second-tiers. Proposed cut-offs were also retrospectively evaluated in newborn screening samples of 12 patients with remethylation disorders and vitamin B12 deficiency from Qatar and Germany. Over a 12 months period, the proposed strategy led to a decrease in the recall rate in homocysteine screening for Qatar from 1.09% to 0.68%, while allowing for additional systematic inclusion of remethylation disorders and vitamin B12 deficiency into the screening panel for Qatar. In the evaluated period the applied strategy would have detected all patients with classical homocystinuria identified by the previous strategy and in addition 5 children with maternal nutritional vitamin B12 deficiency and one patient with an isolated remethylation disorder. Additional retrospective evaluation of newborn screening samples of 12 patients from Germany and Qatar with remethlyation disorders or vitamin B12 deficiency showed that all of these patients would have been detected by the cut-offs used in the proposed new strategy. In addition, an adapted strategy for Germany using methionine, methionine-phenylalanine-ratio and propionylcarnitine as first-tier, and homocysteine as a second-tier test was also positively evaluated retrospectively. The proposed strategy for samples from Qatar allows inclusion of remethylation disorders and vitamin B12 deficiency in the screening panel, while lowering the recall rate. An adapted second-tier strategy is presented for screening in Germany and will be prospectively evaluated over the next years in a pilot project named "Newborn Screening 2020".

Potential Pharmacological Chaperones for Cystathionine Beta-Synthase-Deficient Homocystinuria.

Classical homocystinuria (HCU) is the most common loss-of-function inborn error of sulfur amino acid metabolism. HCU is caused by a deficiency in enzymatic degradation of homocysteine, a toxic intermediate of methionine transformation to cysteine, chiefly due to missense mutations in the cystathionine beta-synthase (CBS) gene. As with many other inherited disorders, the pathogenic mutations do not target key catalytic residues, but rather introduce structural perturbations leading to an enhanced tendency of the mutant CBS to misfold and either to form nonfunctional aggregates or to undergo proteasome-dependent degradation. Correction of CBS misfolding would represent an alternative therapeutic approach for HCU. In this review, we summarize the complex nature of CBS, its multi-domain architecture, the interplay between the three cofactors required for CBS function [heme, pyridoxal-5'-phosphate (PLP), and S-adenosylmethionine (SAM)], as well as the intricate allosteric regulatory mechanism only recently understood, thanks to advances in CBS crystallography. While roughly half of the patients respond to treatment with a PLP precursor pyridoxine, many studies suggested usefulness of small chemicals, such as chemical and pharmacological chaperones or proteasome inhibitors, rescuing mutant CBS activity in cellular and animal models of HCU. Non-specific chemical chaperones and proteasome inhibitors assist in mutant CBS folding process and/or prevent its rapid degradation, thus resulting in increased steady-state levels of the enzyme and CBS activity. Recent interest in the field and available structural information will hopefully yield CBS-specific compounds, by using high-throughput screening and computational modeling of novel ligands, improving folding, stability, and activity of CBS mutants.

Growth Patterns in the Irish Pyridoxine Nonresponsive Homocystinuria Population and the Influence of Metabolic Control and Protein Intake.

A low methionine diet is the mainstay of treatment for pyridoxine nonresponsive homocystinuria (HCU). There are various guidelines for recommended protein intakes for HCU and clinical practice varies. Poor growth has been associated with low cystine levels. This retrospective review of 48 Irish pyridoxine nonresponsive HCU patients assessed weight, height, body mass index (BMI), protein intake, and metabolic control up to 18 years at nine set time points. Patients diagnosed through newborn screening (NBS) were compared to late diagnosed (LD) patients. At 18 years the LD group (n = 12, mean age at diagnosis 5.09 years) were heavier (estimated effect +4.97 Kg, P = 0.0058) and taller (estimated effect +7.97 cm P = 0.0204) than the NBS group (n = 36). There was no difference in growth rate between the groups after 10 years of age. The HCU population were heavier and taller than the general population by one standard deviation with no difference in BMI. There was no association between intermittently low cystine levels and height. Three protein intake guidelines were compared; there was no difference in adult height between those who met the lowest of the guidelines (Genetic Metabolic Dietitians International) and those with a higher protein intake.

M39 - Investigating The Role of Treatable Genetic Diseases In Schizophrenia And Bipolar Disorder Populations And Its Clinical Diagnostic Impact

Background Many rare genetic syndromes are known to phenotypically manifest with psychiatric symptoms that can be indistinguishable from primary psychiatric disorders. While the majority of ongoing psychiatric genetic research has been dedicated to the identification and characterization of genes involved in primary psychiatric disorders, little research has been done to determine the extent to which rare genetic variants contribute to the overall psychiatric disease load. Within schizophrenia and bipolar populations, we are conducting the first study of its kind to determine the prevalence of four treatable genetic syndromes (Niemann Pick disease type C (NPC), Wilson disease, acute intermittent porphyria (AIP), and homocystinuria (HOM)) manifesting as primary psychiatric disorders. Methods We are screening 1323 schizophrenia and 1200 bipolar disorder samples, along with 980 sex- and age-matched healthy controls, all with available DNA and extensive phenotype data. We are using a matrix-type pooled targeted deep sequencing of the genes NPC1, NPC2, ATP7B, HMBS, and CBS to screen for the four genetic diseases. Pathogenic variants within the targeted genes will be identified using an in-house analytic pipeline with quality control, variant discovery designed specifically for identifying variants in the matrix pooled targeted sequencing approach, and functionality prediction programs (i.e. Polyphen, SIFT, Scone) to determine variant pathogenicity. Sanger sequencing will be used to validate identified mutations and decrease false positive calls. Results We hypothesize that a significant sub-population of patients with psychiatric disorders have underlying rare genetic conditions. Preliminary screening of 1023 schziophrenia patients for possible pathogenic variants resulted in the identification of 11 previously known pathogenic variants from the ClinVar database, with 2 pathogenic variants for NPC, 2 for HOM, and 7 for WD. Additionally, 34 variants were predicted to be pathogenic based on whether three or more pathogenicity prediction softwares (PolyPhen2, SIFT, MutationTaster and Condel) identified the variant to be damaging, of which 3 are predicted nonsense mutations. Discussion Identification of known and predicted pathogenic variants within the schizophrenia and bipolar populations strongly suggests the role of rare genetic diseases within psychiatric populations as hypothesized. Screening for treatable genetic diseases, such as NPC, WD, AIP and HOM, within schizophrenia and bipolar samples could provide a possible explanation for severe treatment resistance and treating the genetic condition can effectively “cure” patients of their otherwise difficult-to-treat psychiatric symptoms. Ultimately, this proof-of-principle study will lead to the development of molecular diagnostic tools for detection of underlying genetic disorders in psychiatric patients and will allow for precision medicine.

A Clinically Relevant Variant of the Human Hydrogen Sulfide-Synthesizing Enzyme Cystathionine β-Synthase: Increased CO Reactivity as a Novel Molecular Mechanism of Pathogenicity?

The human disease classical homocystinuria results from mutations in the gene encoding the pyridoxal 5′-phosphate- (PLP-) dependent cystathionine β-synthase (CBS), a key enzyme in the transsulfuration pathway that controls homocysteine levels, and is a major source of the signaling molecule hydrogen sulfide (H2S). CBS activity, contributing to cellular redox homeostasis, is positively regulated by S-adenosyl-L-methionine (AdoMet) but fully inhibited upon CO or NO• binding to a noncatalytic heme moiety. Despite extensive studies, the molecular basis of several pathogenic CBS mutations is not yet fully understood. Here we found that the ferrous heme of the reportedly mild p.P49L CBS variant has altered spectral properties and markedly increased affinity for CO, making the protein much more prone than wild type (WT) CBS to inactivation at physiological CO levels. The higher CO affinity could result from the slightly higher flexibility in the heme surroundings revealed by solving at 2.80-Å resolution the crystallographic structure of a truncated p.P49L. Additionally, we report that p.P49L displays impaired H2S-generating activity, fully rescued by PLP supplementation along the purification, despite a minor responsiveness to AdoMet. Altogether, the results highlight how increased propensity to CO inactivation of an otherwise WT-like variant may represent a novel pathogenic mechanism in classical homocystinuria.

Targeting Cystathionine Beta-Synthase Misfolding in Homocystinuria by Small Ligands: State of the Art and Future Directions

Classical homocystinuria (HCU) is the most common loss-of-function inborn error of sulfur amino acids metabolism. HCU is caused by a deficiency in enzymatic degradation of homocysteine, a toxic intermediate of methionine transformation to cysteine, chiefly due to missense mutations in the cystathionine betasynthase (CBS) gene. As with many other inherited disorders, the pathogenic mutations do not target key catalytic residues, but rather introduce structural perturbations leading to an enhanced tendency of the mutant CBS to misfold and either to form non-functional aggregates or to undergo proteasome-dependent degradation. Thus correction of CBS misfolding represents an alternative therapeutic approach for HCU. In this review, we summarize the complex nature of CBS, its multidomain architecture, the interplay between the three cofactors required for CBS function (heme, pyridoxal-5'-phosphate (PLP) and S-adenosyl-L-methionine) as well as the intricate allosteric regulatory mechanism only recently explained thanks to advances in CBS crystallography. While roughly half of the patients responds to treatment with a PLP precursor pyridoxine, many studies suggested usefulness of small chemicals, such as chemical and pharmacological chaperones or proteasome inhibitors, rescuing mutant CBS activity in cellular and animal models of HCU. Non-specific chemical chaperones and proteasome inhibitors assist in mutant CBS folding process and/or prevent its rapid degradation, thus resulting in increased steady state levels of the enzyme and CBS activity. Recent increased interest in the field and available structural information will hopefully yield CBS-specific compounds by using high-throughput screening and computational modeling of novel ligands improving folding, stability and activity of CBS.

Brain White Matter Changes in Four Patients with Classical Homocystinuria: Two of Them were Reversible

Brain White Matter Changes in Four Patients with Classical Homocystinuria: Two of Them were Reversible

Lack of global epigenetic methylation defects in CBS deficient mice.

Cystathionine β-synthase (CBS) deficiency is a recessive inborn error of metabolism in which patients have extremely elevated plasma total homocysteine and have clinical manifestations in the vascular, visual, skeletal, and nervous systems. Homocysteine is an intermediary metabolite produced from the hydrolysis of S-adenosylhomocysteine (SAH), which is a by-product of methylation reactions involving the methyl-donor S-adenosylmethionine (SAM). Here, we have measured SAM, SAH, DNA and histone methylation status in an inducible mouse model of CBS deficiency to test the hypothesis that homocysteine-related phenotypes are caused by inhibition of methylation due to elevated SAH and reduced SAM/SAH ratio. We found that mice lacking CBS have elevated cellular SAH and reduced SAM/SAH ratios in both liver and kidney, but this was not associated with alterations in the level of 5-methylcytosine or various histone modifications. Using methylated DNA immunoprecipitation in combination with microarray, we found that of the 241 most differentially methylated promoter probes, 89% were actually hypermethylated in CBS deficient mice. In addition, we did not find that changes in DNA methylation correlated well with changes in RNA expression in the livers of induced and uninduced CBS mice. Our data indicates that reduction in the SAM/SAH ratio, due to loss of CBS activity, does not result in overall hypomethylation of either DNA or histones.

Vision of correction for classic homocystinuria.

Inherited metabolic disorders are often characterized by the lack of an essential enzyme and are currently treated by dietary restriction and other strategies to replace the substrates or products of the missing enzyme. Patients with homocystinuria lack the enzyme cystathionine β-synthase (CBS), and many of these individuals do not respond to current treatment protocols. In this issue of the JCI, Bublil and colleagues demonstrate that enzyme replacement therapy (ERT) provides long-term amelioration of homocystinuria-associated phenotypes in CBS-deficient murine models. A PEGylated form of CBS provided long-term stability and, when used in conjunction with the methylation agent betaine, dramatically increased survival in mice fed a normal diet. The results of this study provide one of the first examples of ERT for a metabolic disorder and suggest that PEGylated CBS should be further explored for use in patients.

Effect of cadmium administration in hyperhomocysteinemic mice due to cystathionine beta synthase deficiency

Homocysteine, a sulfur-containing amino acid formed during the metabolism of methionine, is commonly slightly elevated in the plasma of the general population. Additionally, we previously found that cystathionine beta synthase-deficient mice, a murine model of hyperhomocysteinemia, exhibit altered activities of xenobiotic metabolizing enzymes (XME), which dispose of foreign chemicals, in the liver. Thus, hyperhomocysteinemia may result in susceptibility to xenobiotics like cadmium, a heavy-metal toxicant found in drinking water, atmospheric air, and food. Consequently, we exposed hyperhomocysteinemic mice to cadmium via their drinking water for one month to analyze the combined effects of hyperhomocysteinemia and cadmium exposure in liver. No difference in plasma homocysteine level was found after cadmium administration in control and hyperhomocysteinemic mice, but the glutathione level was significantly lower in exposed hyperhomocysteinemic mice compared to control mice, reflecting oxidative stress. We therefore analyzed the effect of Cd administration on hepatic XMEs known to be dysregulated in hyperhomocysteinemic mice: paraoxonase 1, a phase I XME, and NAD(P)H:quinone oxidoreductase, a phase II XME. Cadmium exposure negatively affected activity of paraoxonase 1, a calcium-dependent enzyme. Thus, we analyzed another calcium-dependent enzyme known to be dysregulated in liver of hyperhomocysteinemic mice, calpain, which was also significantly lower after cadmium administration. A comparison of the calculated affinities of cadmium docking versus calcium redocking suggested that cadmium ions may inhibit enzymatic activities by preventing the binding of calcium ions. Moreover, the increased NAD(P)H:quinone oxidoreductase activity observed after cadmium administration could indicate the presence of protective mechanisms in liver of mice. In conclusion, although cadmium administration had no effect on plasma homocysteine level, its effects on plasma glutathionine level suggest a susceptibility to cadmium in the condition of hyperhomocysteinemia, which could be countered by an increased NAD(P)H:quinone oxidoreductase activity.

Validation of an HPLC method for total homocysteine quantification in plasma

IntroductionHomocysteine (Hcy) is a nonessential amino acid which links the methionine and the folate cycles. Hcy levels are increased in genetic disorders, such as classic homocystinuria and methylmalonic aciduria combined with homocystinuria. Other monogenic, multifactorial diseases and physiological conditions are also associated with high Hcy levels. The aim of this study is to validate a method to quantify Hcy in plasma. Material and methodsA method to quantify Hcy in plasma by HPLC was validated, by determining the following parameters: specificity, linearity, precision, accuracy, and detection and quantification limits according ICH and EMEA guidelines. Homocysteine was measured in 43 healthy individuals, 2 patients with high levels of methylmalonic acid, and a previously diagnosed patient with classic homocystinuria. ResultsThe method was able to identify and quantify Hcy without interferences. A linear behavior was observed in a range of 6–100μM with r2=0.9967. The precision and accuracy studies showed variation coefficients under 6%. The limits of detection and quantification were 3.12μM and 6.25μM, respectively. The 43 healthy individuals studied had normal Hcy levels. The patient with elevated urinary methylmalonic acid levels and the homocystinuria patient showed high Hcy levels. ConclusionThe method is valid for the quantification of Hcy in plasma as it fulfills the requirements for validation of analytical methods. Its introduction into diagnostic and follow up algorithms is important in genetic diseases where this amino acid is increased.

Classical homocystinuria and psychiatric disturbances – A case report

IntroductionClassical homocystinuria (cystathionine beta synthase deficiency) is a rare autosomal recessive disease of methionine metabolism that causes accumulation of homocysteine in the blood and cysteine deficiency. It is characterized by intellectual disability, ectopia lentis, skeleton abnormalities resembling Marfan syndrome and thromboembolic episodes. The majority of patients have psychiatric disturbances as depression, behavioral disorders, personality disorders, obsessive-compulsive disorder and, less commonly, bipolar disorder and psychosis.Objectives and aimsTo briefly review psychiatric disturbances in patients with homocystinuria and present a case report.MethodsLiterature research and analysis of patient's clinical data.ResultsA 22-year-old male was diagnosed with classical homocystinuria at age 4 due to intellectual disability and renal alterations. With aging, other problems emerged: epilepsy; postural tremor; dysesthesia; ectopia lentis; orofacial myofunctional disorder; asthma; and patellar instability. He went to a special education program. At age sixteen, he initiated Child Psychiatry consultations due to anxiety and behavioral changes, as difficulty in controlling impulses, establishing relationships and in the perception of the self. Nowadays, the patient is followed in psychiatric consultations, where he has demonstrated high difficulty to empathize. He is being treated with vitamin supplements; betaine; levetiracetam; clobazam; and propranolol, combined with a special diet.ConclusionsIt is not practical to screen every psychiatric patient for Homocystinuria, but this disease should be considered when there is a family history, early and/or acute onset, intellectual disability, atypical symptoms, unusual response to treatment, progressive cognitive change and other organic disturbances present in this disorder.Disclosure of interestThe authors have not supplied their declaration of competing interest.