BACKGROUND: For the majority of rare clinical missense variants, pathogenicity status cannot currently be classified. Classical homocystinuria, characterized by elevated homocysteine in plasma and urine, is caused by variants in the cystathionine beta-synthase (CBS) gene, most of which are rare. With early detection, existing therapies are highly effective. METHODS: Damaging CBS variants can be detected based on their failure to restore growth in yeast cells lacking the yeast ortholog CYS4. This assay has only been applied reactively, after first observing a variant in patients. Using saturation codon-mutagenesis, en masse growth selection, and sequencing, we generated a comprehensive, proactive map of CBS missense variant function. RESULTS: Our CBS variant effect map far exceeds the performance of computational predictors of disease variants. Map scores correlated strongly with both disease severity (Spearman's ϱ = 0.9) and human clinical response to vitamin B6 (ϱ = 0.93). CONCLUSIONS: We demonstrate that highly multiplexed cell-based assays can yield proactive maps of variant function and patient response to therapy, even for rare variants not previously seen in the clinic.

• MeSH
• Cystathionine beta-Synthase genetics   metabolism   MeSH
• Phenotype MeSH
• Genetic Testing methods   MeSH
• Genotype MeSH
• Homocystinuria genetics   MeSH
• Humans MeSH
• Mutation, Missense * MeSH
• Saccharomyces cerevisiae Proteins genetics   MeSH
• Saccharomyces cerevisiae MeSH
• Genetic Complementation Test methods   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• Cystathionine beta-Synthase MeSH
• Saccharomyces cerevisiae Proteins MeSH

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.

• MeSH
• Betaine metabolism   MeSH
• Cystathionine beta-Synthase deficiency   MeSH
• Homocysteine metabolism   MeSH
• Homocystinuria diet therapy   drug therapy   MeSH
• Humans MeSH
• Methionine metabolism   MeSH
• Pyridoxine therapeutic use   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Systematic Review MeSH
• Names of Substances
• Betaine MeSH
• Cystathionine beta-Synthase MeSH
• Homocysteine MeSH
• Methionine MeSH
• Pyridoxine MeSH

Newborn screening (NBS) is justified if early intervention is effective in a disorder generally not detected early in life on a clinical basis, and if sensitive and specific biochemical markers exist. Experience with NBS for homocystinurias and methylation disorders is limited. However, there is robust evidence for the success of early treatment with diet, betaine and/or pyridoxine for CBS deficiency and good evidence for the success of early betaine treatment in severe MTHFR deficiency. These conditions can be screened in dried blood spots by determining methionine (Met), methionine-to-phenylanine (Met/Phe) ratio, and total homocysteine (tHcy) as a second tier marker. Therefore, we recommend NBS for cystathionine beta-synthase and severe MTHFR deficiency. Weaker evidence is available for the disorders of intracellular cobalamin metabolism. Early treatment is clearly of advantage for patients with the late-onset cblC defect. In the early-onset type, survival and non-neurological symptoms improve but the effect on neurocognitive development is uncertain. The cblC defect can be screened by measuring propionylcarnitine, propionylcarnitine-to-acetylcarnitine ratio combined with the second tier markers methylmalonic acid and tHcy. For the cblE and cblG defects, evidence for the benefit of early treatment is weaker; and data on performance of Met, Met/Phe and tHcy even more limited. Individuals homozygous or compound heterozygous for MAT1A mutations may benefit from detection by NBS using Met, which on the other hand also detects asymptomatic heterozygotes. Clinical and laboratory data is insufficient to develop any recommendation on NBS for the cblD, cblF, cblJ defects, glycineN-methyltransferase-, S-adenosylhomocysteinehydrolase- and adenosine kinase deficiency.

• MeSH
• Acetylcarnitine blood   MeSH
• Betaine therapeutic use   MeSH
• Homocystinuria diagnosis   MeSH
• Carnitine analogs & derivatives   blood   MeSH
• Methylmalonic Acid blood   MeSH
• Humans MeSH
• Methionine blood   MeSH
• Methylenetetrahydrofolate Reductase (NADPH2) deficiency   drug effects   MeSH
• Methylation MeSH
• Infant, Newborn MeSH
• Neonatal Screening * MeSH
• Practice Guidelines as Topic MeSH
• Check Tag
• Humans MeSH
• Infant, Newborn MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Systematic Review MeSH
• Names of Substances
• Acetylcarnitine MeSH
• Betaine MeSH
• Carnitine MeSH
• Methylmalonic Acid MeSH
• Methionine MeSH
• Methylenetetrahydrofolate Reductase (NADPH2) MeSH
• propionylcarnitine MeSH Browser

Protein misfolding due to missense mutations is a common pathogenic mechanism in cystathionine β-synthase (CBS) deficiency. In our previous studies, we successfully expressed, purified, and characterized nine CBS mutant enzymes containing the following patient mutations: P49L, P78R, A114V, R125Q, E176K, R266K, P422L, I435T, and S466L. These purified mutants exhibited full heme saturation, normal tetrameric assembly, and high catalytic activity. In this work, we used several spectroscopic and proteolytic techniques to provide a more thorough insight into the conformation of these mutant enzymes. Far-UV circular dichroism, fluorescence, and second-derivative UV spectroscopy revealed that the spatial arrangement of these CBS mutants is similar to that of the wild type, although the microenvironment of the chromophores may be slightly altered. Using proteolysis with thermolysin under native conditions, we found that the majority of the studied mutants is more susceptible to cleavage, suggesting their increased local flexibility or propensity for local unfolding. Interestingly, the presence of the CBS allosteric activator, S-adenosylmethionine (AdoMet), increased the rate of cleavage of the wild type and the AdoMet-responsive mutants, while the proteolytic rate of the AdoMet-unresponsive mutants was not significantly changed. Pulse proteolysis analysis suggested that the protein structure of the R125Q and E176K mutants is significantly less stable than that of the wild type and the other mutants. Taken together, the proteolytic data shows that the conformation of the pathogenic mutants is altered despite retained catalytic activity and normal tetrameric assembly. This study demonstrates that the proteolytic techniques are useful tools for the assessment of the biochemical penalty of missense mutations in CBS.

• MeSH
• Circular Dichroism MeSH
• Cystathionine beta-Synthase deficiency   genetics   metabolism   MeSH
• Escherichia coli metabolism   MeSH
• Protein Conformation MeSH
• Humans MeSH
• Mutation, Missense MeSH
• Models, Molecular MeSH
• Proteolysis MeSH
• S-Adenosylmethionine MeSH
• Protein Folding * MeSH
• Spectrophotometry, Ultraviolet MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• Cystathionine beta-Synthase MeSH
• S-Adenosylmethionine MeSH

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.

• MeSH
• Blood Chemical Analysis methods   standards   MeSH
• Chromatography, Liquid MeSH
• Cystathionine beta-Synthase deficiency   metabolism   MeSH
• Homocystinuria blood   diagnosis   enzymology   MeSH
• Immunoenzyme Techniques methods   standards   MeSH
• Calibration MeSH
• Plasma chemistry   enzymology   metabolism   MeSH
• Humans MeSH
• Pyridoxal Phosphate pharmacology   MeSH
• S-Adenosylmethionine pharmacology   MeSH
• Enzyme Stability MeSH
• Case-Control Studies MeSH
• Tandem Mass Spectrometry methods   standards   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Evaluation Study MeSH
• Research Support, Non-U.S. Gov't MeSH
• Validation Study MeSH
• Names of Substances
• Cystathionine beta-Synthase MeSH
• Pyridoxal Phosphate MeSH
• S-Adenosylmethionine MeSH

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

• MeSH
• Child MeSH
• Adult MeSH
• Homocystinuria complications   epidemiology   MeSH
• Incidence MeSH
• Middle Aged MeSH
• Humans MeSH
• Adolescent MeSH
• Young Adult MeSH
• Vascular Diseases diagnosis   epidemiology   etiology   MeSH
• Child, Preschool MeSH
• Check Tag
• Child MeSH
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Adolescent MeSH
• Young Adult MeSH
• Male MeSH
• Child, Preschool MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Case Reports MeSH
• Research Support, Non-U.S. Gov't MeSH
• Geographicals
• Czech Republic epidemiology   MeSH

Misfolding and aggregation of mutant enzymes have been proposed to play role in the pathogenesis of homocystinuria due to cystathionine β-synthase (CBS) deficiency. Chemical chaperones have been recently shown to facilitate proper assembly of several CBS mutants. To asses the number of patients that may respond to chaperone therapy, we examined the effect of selected CBS ligands and osmolytes on assembly and activity of 27 CBS mutants that represent 70% of known CBS alleles. The mutant enzymes were expressed in a bacterial system, and their properties were assessed by native Western blotting and sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) assay, respectively. We studied the chaperoning activity of δ-aminolevulinic acid (δ-ALA)-a heme precursor-and of three osmolytes betaine, 2-aminoethanesulfonic acid (taurine), and glycerol. Fourteen mutants responded by at least 30% increase in the amount of correctly assembled tetramers and enzymatic activity to the coexpressional presence of either 0.5 mM δ-ALA, 100 mM betaine, and/or 750 mM glycerol. Eight of these mutants (p.R266K, p.P49L, p.R125Q, p.K102N, p.R369C, p.V180A, p.P78R, p.S466L) were rescuable by all of these three substances. Four mutants showed increased formation of tetramers that was not accompanied by changes in activity. Topology of mutations appeared to determine the chaperone responsiveness, as 11 of 14 solvent-exposed mutations were substantially more responsive than three of 13 buried mutations. This study identified chaperone-responsive mutants that represent 56 of 713 known patient-derived CBS alleles and may serve as a basis for exploring pharmacological approaches aimed at correcting misfolding in homocystinuria.

• MeSH
• Alleles MeSH
• Betaine pharmacology   therapeutic use   MeSH
• Cystathionine beta-Synthase chemistry   drug effects   genetics   metabolism   MeSH
• Escherichia coli metabolism   MeSH
• Glycerol pharmacology   MeSH
• Homocystinuria drug therapy   genetics   metabolism   MeSH
• Polymorphism, Single Nucleotide physiology   MeSH
• Protein Conformation drug effects   MeSH
• Aminolevulinic Acid pharmacology   therapeutic use   MeSH
• Humans MeSH
• Ligands * MeSH
• Molecular Chaperones pharmacology   therapeutic use   MeSH
• Protein Multimerization drug effects   MeSH
• Mutant Proteins chemistry   drug effects   metabolism   MeSH
• Protein Folding drug effects   MeSH
• Taurine pharmacology   therapeutic use   MeSH
• Protein Binding MeSH
• Structure-Activity Relationship MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Evaluation Study MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Betaine MeSH
• Cystathionine beta-Synthase MeSH
• Glycerol MeSH
• Aminolevulinic Acid MeSH
• Ligands * MeSH
• Molecular Chaperones MeSH
• Mutant Proteins MeSH
• Taurine MeSH

Misfolding of mutant enzymes may play an important role in the pathogenesis of cystathionine beta-synthase (CBS) deficiency. We examined properties of a series of 27 mutant variants, which together represent 70% of known alleles observed in patients with homocystinuria due to CBS deficiency. The median amount of SDS-soluble mutant CBS polypeptides in the pellet after centrifugation of bacterial extracts was increased by 50% compared to the wild type. Moreover, mutants formed on average only 12% of tetramers and their median activity reached only 3% of the wild-type enzyme. In contrast to the wild-type CBS about half of mutants were not activated by S-adenosylmethionine. Expression at 18 degrees C substantially increased the activity of five mutants in parallel with increasing the amounts of tetramers. We further analyzed the role of solvent accessibility of mutants as a determinant of their folding and activity. Buried mutations formed on average less tetramers and exhibited 23 times lower activity than the solvent exposed mutations. In summary, our results show that topology of mutations predicts in part the behavior of mutant CBS, and that misfolding may be an important and frequent pathogenic mechanism in CBS deficiency.

• MeSH
• Cystathionine beta-Synthase chemistry   deficiency   genetics   MeSH
• Escherichia coli genetics   MeSH
• Homocystinuria enzymology   genetics   MeSH
• Catalytic Domain genetics   MeSH
• Catalysis MeSH
• Protein Structure, Quaternary MeSH
• Humans MeSH
• Models, Molecular MeSH
• Protein Multimerization MeSH
• Mutation * MeSH
• Mutant Proteins chemistry   genetics   metabolism   MeSH
• Cold Temperature MeSH
• Solubility MeSH
• Protein Folding MeSH
• Enzyme Stability MeSH
• Protein Structure, Tertiary MeSH
• Blotting, Western MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Cystathionine beta-Synthase MeSH
• Mutant Proteins MeSH