Cystathionine beta-synthase (CBS)-deficient homocystinuria (HCU) is the most common inborn error of sulfur amino acid metabolism. The pyridoxine non-responsive form of the disease manifests itself by massively increasing plasma and tissue concentrations of homocysteine, a toxic intermediate of methionine metabolism that is thought to be the major cause of clinical complications including skeletal deformities, connective tissue defects, thromboembolism and cognitive impairment. The current standard of care involves significant dietary interventions that, despite being effective, often adversely affect quality of life of HCU patients, leading to poor adherence to therapy and inadequate biochemical control with clinical complications. In recent years, the unmet need for better therapeutic options has resulted in development of novel enzyme and gene therapies and exploration of pharmacological approaches to rescue CBS folding defects caused by missense pathogenic mutations. Here, we review scientific evidence and current state of affairs in development of recent approaches to treat HCU.

• MeSH
• cystathionin-beta-synthasa genetika   metabolismus   MeSH
• homocystinurie * farmakoterapie   genetika   metabolismus   MeSH
• kvalita života MeSH
• lidé MeSH
• missense mutace MeSH
• tromboembolie * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Research Support, N.I.H., Extramural MeSH

Elevated total plasma homocysteine (hyperhomocysteinemia) is a marker of cardiovascular, thrombotic, and neuropsychological disease. It has multiple causes, including the common nutritional vitamin B12 or folate deficiency. However, some rare but treatable, inborn errors of metabolism (IEM) characterized by hyperhomocysteinemia can be missed due to variable presentations and the lack of awareness. The aim of this study is to identify undiagnosed IEM in adults with significantly elevated homocysteine using key existing clinical data points, then IEM specific treatment can be offered to improve outcome. We conducted a retrospective study with data mining and chart review of patients with plasma total homocysteine >30 μmol/L over a two-year period. We offer biochemical and genetic testing to patients with significant hyperhomocysteinemia without a clear explanation to diagnose IEM. We identified 22 subjects with significant hyperhomocysteinemia but no clear explanation. Subsequently, we offered genetic testing to seven patients and diagnosed one patient with classic homocystinuria due to cystathionine beta-synthase deficiency. With treatment, she lowered her plasma homocysteine and improved her health. This study stresses the importance of a thorough investigation of hyperhomocysteinemia in adults to identify rare but treatable IEM. We propose a metabolic evaluation algorithm for elevated homocysteine levels.

• MeSH
• dospělí MeSH
• homocystein MeSH
• homocystinurie * diagnóza   genetika   MeSH
• hyperhomocysteinemie * diagnóza   genetika   MeSH
• kyselina listová MeSH
• lidé MeSH
• retrospektivní studie MeSH
• vitamin B 12 terapeutické užití   MeSH
• vrozené poruchy metabolismu * diagnóza   genetika   MeSH
• Check Tag
• dospělí MeSH
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH

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
• cystathionin-beta-synthasa genetika   metabolismus   MeSH
• fenotyp MeSH
• genetické testování metody   MeSH
• genotyp MeSH
• homocystinurie genetika   MeSH
• lidé MeSH
• missense mutace * MeSH
• Saccharomyces cerevisiae - proteiny genetika   MeSH
• Saccharomyces cerevisiae MeSH
• testy genetické komplementace metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

BACKGROUND: Severe methylenetetrahydrofolate reductase (MTHFR) deficiency is a rare inborn defect disturbing the remethylation of homocysteine to methionine (<200 reported cases). This retrospective study evaluates clinical, biochemical genetic and in vitro enzymatic data in a cohort of 33 patients. METHODS: Clinical, biochemical and treatment data was obtained from physicians by using a questionnaire. MTHFR activity was measured in primary fibroblasts; genomic DNA was extracted from cultured fibroblasts. RESULTS: Thirty-three patients (mean age at follow-up 11.4 years; four deceased; median age at first presentation 5 weeks; 17 females) were included. Patients with very low (<1.5%) mean control values of enzyme activity (n = 14) presented earlier and with a pattern of feeding problems, encephalopathy, muscular hypotonia, neurocognitive impairment, apnoea, hydrocephalus, microcephaly and epilepsy. Patients with higher (>1.7-34.8%) residual enzyme activity had mainly psychiatric symptoms, mental retardation, myelopathy, ataxia and spasticity. Treatment with various combinations of betaine, methionine, folate and cobalamin improved the biochemical and clinical phenotype. During the disease course, patients with very low enzyme activity showed a progression of feeding problems, neurological symptoms, mental retardation, and psychiatric disease while in patients with higher residual enzyme activity, myelopathy, ataxia and spasticity increased. All other symptoms remained stable or improved in both groups upon treatment as did brain imaging in some cases. No clear genotype-phenotype correlation was obvious. DISCUSSION: MTHFR deficiency is a severe disease primarily affecting the central nervous system. Age at presentation and clinical pattern are correlated with residual enzyme activity. Treatment alleviates biochemical abnormalities and clinical symptoms partially.

• MeSH
• ataxie genetika   MeSH
• betain terapeutické užití   MeSH
• dítě MeSH
• fenotyp MeSH
• genetické asociační studie metody   MeSH
• homocystinurie farmakoterapie   enzymologie   genetika   MeSH
• kyselina listová terapeutické užití   MeSH
• lidé MeSH
• mentální retardace genetika   MeSH
• methionin terapeutické užití   MeSH
• methylentetrahydrofolátreduktasa (NADPH2) nedostatek   genetika   MeSH
• mutace genetika   MeSH
• nemoci míchy genetika   MeSH
• psychotické poruchy farmakoterapie   enzymologie   genetika   MeSH
• retrospektivní studie MeSH
• svalová spasticita farmakoterapie   enzymologie   genetika   MeSH
• vitamin B 12 terapeutické užití   MeSH
• Check Tag
• dítě MeSH
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH

Severe 5,10-methylenetetrahydrofolate reductase (MTHFR) deficiency is caused by mutations in the MTHFR gene and results in hyperhomocysteinemia and varying severity of disease, ranging from neonatal lethal to adult onset. Including those described here, 109 MTHFR mutations have been reported in 171 families, consisting of 70 missense mutations, 17 that primarily affect splicing, 11 nonsense mutations, seven small deletions, two no-stop mutations, one small duplication, and one large duplication. Only 36% of mutations recur in unrelated families, indicating that most are "private." The most common mutation is c.1530A>G (numbered from NM_005957.4, p.Lys510 = ) causing a splicing defect, found in 13 families; the most common missense mutation is c.1129C>T (p.Arg377Cys) identified in 10 families. To increase disease understanding, we report enzymatic activity, detected mutations, and clinical onset information (early, <1 year; or late, >1 year) for all published patients available, demonstrating that patients with early onset have less residual enzyme activity than those presenting later. We also review animal models, diagnostic approaches, clinical presentations, and treatment options. This is the first large review of mutations in MTHFR, highlighting the wide spectrum of disease-causing mutations.

• MeSH
• databáze genetické MeSH
• homocystinurie genetika   MeSH
• katalytická doména MeSH
• lidé MeSH
• methylentetrahydrofolátreduktasa (NADPH2) chemie   nedostatek   genetika   metabolismus   MeSH
• modely nemocí na zvířatech MeSH
• mutace * MeSH
• novorozenec MeSH
• novorozenecký screening MeSH
• psychotické poruchy genetika   MeSH
• svalová spasticita genetika   MeSH
• věk při počátku nemoci MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• novorozenec MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Classical homocystinuria is caused by mutations in the cystathionine β-synthase (CBS) gene. Previous experiments in bacterial and yeast cells showed that many mutant CBS enzymes misfold and that chemical chaperones enable proper folding of a number of mutations. In the present study, we tested the extent of misfolding of 27 CBS mutations previously tested in E. coli under the more folding-permissive conditions of mammalian CHO-K1 cells and the ability of chaperones to rescue the conformation of these mutations. Expression of mutations in mammalian cells increased the median activity 16-fold and the amount of tetramers 3.2-fold compared with expression in bacteria. Subsequently, we tested the responses of seven selected mutations to three compounds with chaperone-like activity. Aminooxyacetic acid and 4-phenylbutyric acid exhibited only a weak effect. In contrast, heme arginate substantially increased the formation of mutant CBS protein tetramers (up to sixfold) and rescued catalytic activity (up to ninefold) of five out of seven mutations (p.A114V, p.K102N, p.R125Q, p.R266K, and p.R369C). The greatest effect of heme arginate was observed for the mutation p.R125Q, which is non-responsive to in vivo treatment with vitamin B(6). Moreover, the heme responsiveness of the p.R125Q mutation was confirmed in fibroblasts derived from a patient homozygous for this genetic variant. Based on these data, we propose that a distinct group of heme-responsive CBS mutations may exist and that the heme pocket of CBS may become an important target for designing novel therapies for homocystinuria.

• MeSH
• arginin farmakologie   MeSH
• CHO buňky MeSH
• Cricetulus MeSH
• cystathionin-beta-synthasa genetika   metabolismus   MeSH
• fenotyp MeSH
• fibroblasty účinky léků   enzymologie   MeSH
• genetická predispozice k nemoci MeSH
• hem farmakologie   MeSH
• homocystinurie diagnóza   farmakoterapie   enzymologie   genetika   MeSH
• homozygot MeSH
• katalytická doména MeSH
• konformace proteinů MeSH
• lidé MeSH
• molekulární chaperony farmakologie   MeSH
• molekulární modely MeSH
• mutace * MeSH
• poruchy proteostázy diagnóza   farmakoterapie   enzymologie   genetika   MeSH
• sbalování proteinů MeSH
• substrátová specifita MeSH
• transfekce MeSH
• vztahy mezi strukturou a aktivitou MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• kazuistiky MeSH
• práce podpořená grantem MeSH

BACKGROUND: Cystathionine β-synthase (CBS) is released into plasma from organs expressing this enzyme. Decreased plasma CBS activity has been demonstrated in CBS-deficient patients with 16 different genotypes. The aim of this study was to determine plasma CBS activity in patients carrying 11 additional genotypes using two LC-MS/MS methods. Patients and methods CBS activity was measured in EDTA or heparin plasma using either a previously described or a newly developed LC-MS/MS method optimized for analysis of the reaction product, 3,3-(2)H2-cystathionine, as its butyl ester derivative. We analyzed plasma samples from 26 CBS-deficient patients with known genotypes and 57 controls. RESULTS: We developed a new LC-MS/MS method for simple and sensitive determination of CBS activity. Plasma CBS activity was low (i.e., 0.001-0.036 of the multiples of median control values, MoM) in patients homozygous for the prevalent Hispanic mutation c.572C>T (p.T191M) but was highly elevated (2.95 MoM) in a single patient homozygous for the c.1330G>A (p.D444N) mutation. Patients with the remaining nine genotypes exhibited decreased activities (0.00-0.22 MoM), which did not overlap with the controls (0.29-2.10 MoM). CONCLUSIONS: The determination of CBS activity in plasma is a rapid and non-invasive procedure for detecting a subgroup of CBS-deficient patients with distinct genotypes.

• MeSH
• chromatografie kapalinová MeSH
• cystathionin-beta-synthasa krev   genetika   MeSH
• cystathionin krev   MeSH
• dítě MeSH
• dospělí MeSH
• exprese genu MeSH
• genotyp MeSH
• homocystinurie krev   diagnóza   genetika   MeSH
• homozygot MeSH
• kojenec MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• mutace * MeSH
• předškolní dítě MeSH
• rodokmen MeSH
• senioři MeSH
• tandemová hmotnostní spektrometrie MeSH
• Check Tag
• dítě MeSH
• dospělí MeSH
• kojenec MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• mužské pohlaví MeSH
• předškolní dítě MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

5,10-Methylenetetrahydrofolate reductase (MTHFR) deficiency is the most common inherited disorder of folate metabolism and causes severe hyperhomocysteinaemia. To better understand the relationship between mutation and function, we performed molecular genetic analysis of 76 MTHFR deficient patients, followed by extensive enzymatic characterization of fibroblasts from 72 of these. A deleterious mutation was detected on each of the 152 patient alleles, with one allele harboring two mutations. Sixty five different mutations (42 novel) were detected, including a common splicing mutation (c.1542G>A) found in 21 alleles. Using an enzyme assay in the physiological direction, we found residual activity (1.7%-42% of control) in 42 cell lines, of which 28 showed reduced affinity for nicotinamide adenine dinucleotide phosphate (NADPH), one reduced affinity for methylenetetrahydrofolate, five flavin adenine dinucleotide-responsiveness, and 24 abnormal kinetics of S-adenosylmethionine inhibition. Missense mutations causing virtually absent activity were found exclusively in the N-terminal catalytic domain, whereas missense mutations in the C-terminal regulatory domain caused decreased NADPH binding and disturbed inhibition by S-adenosylmethionine. Characterization of patients in this way provides a basis for improved diagnosis using expanded enzymatic criteria, increases understanding of the molecular basis of MTHFR dysfunction, and points to the possible role of cofactor or substrate in the treatment of patients with specific mutations.

• MeSH
• aktivace enzymů MeSH
• alely MeSH
• alternativní sestřih MeSH
• exony MeSH
• fibroblasty metabolismus   MeSH
• genetické asociační studie * MeSH
• homocystinurie diagnóza   genetika   metabolismus   MeSH
• introny MeSH
• jednonukleotidový polymorfismus MeSH
• kinetika MeSH
• lidé MeSH
• methylentetrahydrofolátreduktasa (NADPH2) nedostatek   genetika   metabolismus   MeSH
• mutace MeSH
• psychotické poruchy diagnóza   genetika   metabolismus   MeSH
• stabilita proteinů MeSH
• svalová spasticita diagnóza   genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The prevalent c.903+469T>C mutation in MTRR causes the cblE type of homocystinuria by strengthening an SRSF1 binding site in an ESE leading to activation of a pseudoexon. We hypothesized that other splicing regulatory elements (SREs) are also critical for MTRR pseudoexon inclusion. We demonstrate that the MTRR pseudoexon is on the verge of being recognized and is therefore vulnerable to several point mutations that disrupt a fine-tuned balance between the different SREs. Normally, pseudoexon inclusion is suppressed by a hnRNP A1 binding exonic splicing silencer (ESS). When the c.903+469T>C mutation is present two ESEs abrogate the activity of the ESS and promote pseudoexon inclusion. Blocking the 3'splice site or the ESEs by SSOs is effective in restoring normal splicing of minigenes and endogenous MTRR transcripts in patient cells. By employing an SSO complementary to both ESEs, we were able to rescue MTRR enzymatic activity in patient cells to approximately 50% of that in controls. We show that several point mutations, individually, can activate a pseudoexon, illustrating that this mechanism can occur more frequently than previously expected. Moreover, we demonstrate that SSO blocking of critical ESEs is a promising strategy to treat the increasing number of activated pseudoexons.

• MeSH
• buněčné linie MeSH
• exony * MeSH
• ferredoxin-NADP-reduktasa genetika   metabolismus   MeSH
• HEK293 buňky MeSH
• homocystinurie enzymologie   genetika   MeSH
• kultivované buňky MeSH
• lidé MeSH
• megaloblastová anemie enzymologie   genetika   MeSH
• místa sestřihu RNA MeSH
• mutace * MeSH
• oligonukleotidy * MeSH
• regulační sekvence ribonukleových kyselin * MeSH
• sestřih RNA * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem 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
• alely MeSH
• betain farmakologie   terapeutické užití   MeSH
• cystathionin-beta-synthasa chemie   účinky léků   genetika   metabolismus   MeSH
• Escherichia coli metabolismus   MeSH
• glycerol farmakologie   MeSH
• homocystinurie farmakoterapie   genetika   metabolismus   MeSH
• jednonukleotidový polymorfismus fyziologie   MeSH
• konformace proteinů účinky léků   MeSH
• kyselina aminolevulová farmakologie   terapeutické užití   MeSH
• lidé MeSH
• ligandy MeSH
• molekulární chaperony farmakologie   terapeutické užití   MeSH
• multimerizace proteinu účinky léků   MeSH
• mutantní proteiny chemie   účinky léků   metabolismus   MeSH
• sbalování proteinů účinky léků   MeSH
• taurin farmakologie   terapeutické užití   MeSH
• vazba proteinů MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• práce podpořená grantem MeSH