RESUMEN
Homocystinuria (HCU) due to cystathionine beta-synthase (CBS) deficiency is the most common inborn error of sulfur amino acid metabolism. Recent work suggests that missense pathogenic mutations-regardless of their topology-cause instability of the C-terminal regulatory domain, which likely translates into CBS misfolding, impaired assembly, and loss of function. However, it is unknown how instability of the regulatory domain translates into cellular CBS turnover and which degradation pathways are involved in CBS proteostasis. Here, we developed a human HEK293-based cellular model lacking intrinsic CBS and stably overexpressing wild-type (WT) CBS or its 10 most common missense HCU mutants. We found that HCU mutants, except the I278T variant, expressed similarly or better than CBS WT, with some of them showing impaired oligomerization, activity and response to allosteric activator S-adenosylmethionine. Cellular stability of all HCU mutants, except P49L and A114V, was significantly lower than the stability of CBS WT, suggesting their increased degradation. Ubiquitination analysis of CBS WT and two representative CBS mutants (T191M and I278T) showed that proteasomal degradation is the major pathway for CBS disposal, with a minor involvement of lysosomal-autophagic and endoplasmic reticulum-associated degradation (ERAD) pathways for HCU mutants. Proteasomal inhibition significantly increased the half-life and activity of T191M and I278T CBS mutants. Lysosomal and ERAD inhibition had only a minor impact on CBS turnover, but ERAD inhibition rescued the activity of T191M and I278T CBS mutants similarly as proteasomal inhibition. In conclusion, the present study provides new insights into proteostasis of CBS in HCU.
Asunto(s)
Cistationina betasintasa , Homocistinuria , Mutación Missense , Proteolisis , Cistationina betasintasa/genética , Cistationina betasintasa/metabolismo , Cistationina betasintasa/química , Humanos , Homocistinuria/genética , Homocistinuria/metabolismo , Células HEK293 , Complejo de la Endopetidasa Proteasomal/metabolismo , Complejo de la Endopetidasa Proteasomal/genética , Ubiquitinación , Degradación Asociada con el Retículo EndoplásmicoRESUMEN
Cystathionine beta-synthase-deficient homocystinuria (HCU) is a life-threatening disorder of sulfur metabolism. HCU can be treated by using betaine to lower tissue and plasma levels of homocysteine (Hcy). Here, we show that mice with severely elevated Hcy and potentially deficient in the folate species tetrahydrofolate (THF) exhibit a very limited response to betaine indicating that THF plays a critical role in treatment efficacy. Analysis of a mouse model of HCU revealed a 10-fold increase in hepatic levels of 5-methyl -THF and a 30-fold accumulation of formiminoglutamic acid, consistent with a paucity of THF. Neither of these metabolite accumulations were reversed or ameliorated by betaine treatment. Hepatic expression of the THF-generating enzyme dihydrofolate reductase (DHFR) was significantly repressed in HCU mice and expression was not increased by betaine treatment but appears to be sensitive to cellular redox status. Expression of the DHFR reaction partner thymidylate synthase was also repressed and metabolomic analysis detected widespread alteration of hepatic histidine and glutamine metabolism. Many individuals with HCU exhibit endothelial dysfunction. DHFR plays a key role in nitric oxide (NO) generation due to its role in regenerating oxidized tetrahydrobiopterin, and we observed a significant decrease in plasma NOx (NO2 + NO3) levels in HCU mice. Additional impairment of NO generation may also come from the HCU-mediated induction of the 20-hydroxyeicosatetraenoic acid generating cytochrome CYP4A. Collectively, our data shows that HCU induces dysfunctional one-carbon metabolism with the potential to both impair betaine treatment and contribute to multiple aspects of pathogenesis in this disease.
Asunto(s)
Homocistinuria , Hígado , Oxidación-Reducción , Tetrahidrofolato Deshidrogenasa , Tetrahidrofolatos , Animales , Homocistinuria/metabolismo , Homocistinuria/tratamiento farmacológico , Homocistinuria/genética , Ratones , Tetrahidrofolatos/metabolismo , Hígado/metabolismo , Tetrahidrofolato Deshidrogenasa/metabolismo , Tetrahidrofolato Deshidrogenasa/genética , Betaína/metabolismo , Betaína/farmacología , Homocisteína/metabolismo , Ratones Endogámicos C57BL , Cistationina betasintasa/metabolismo , Cistationina betasintasa/genética , Carbono/metabolismo , Masculino , Ácido Fólico/metabolismo , FemeninoRESUMEN
BACKGROUND: Cystathionine ß-synthase (CBS)-deficient homocystinuria (HCU) is an inherited disorder of sulfur amino acid metabolism with varying severity and organ complications, and a limited knowledge about underlying pathophysiological processes. Here we aimed at getting an in-depth insight into disease mechanisms using a transgenic mouse model of HCU (I278T). METHODS: We assessed metabolic, proteomic and sphingolipidomic changes, and mitochondrial function in tissues and body fluids of I278T mice and WT controls. Furthermore, we evaluated the efficacy of methionine-restricted diet (MRD) in I278T mice. RESULTS: In WT mice, we observed a distinct tissue/body fluid compartmentalization of metabolites with up to six-orders of magnitude differences in concentrations among various organs. The I278T mice exhibited the anticipated metabolic imbalance with signs of an increased production of hydrogen sulfide and disturbed persulfidation of free aminothiols. HCU resulted in a significant dysregulation of liver proteome affecting biological oxidations, conjugation of compounds, and metabolism of amino acids, vitamins, cofactors and lipids. Liver sphingolipidomics indicated upregulation of the pro-proliferative sphingosine-1-phosphate signaling pathway. Liver mitochondrial function of HCU mice did not seem to be impaired compared to controls. MRD in I278T mice improved metabolic balance in all tissues and substantially reduced dysregulation of liver proteome. CONCLUSION: The study highlights distinct tissue compartmentalization of sulfur-related metabolites in normal mice, extensive metabolome, proteome and sphingolipidome disruptions in I278T mice, and the efficacy of MRD to alleviate some of the HCU-related biochemical abnormalities.
Asunto(s)
Cistationina betasintasa , Modelos Animales de Enfermedad , Homocistinuria , Hígado , Metabolómica , Ratones Transgénicos , Proteómica , Esfingolípidos , Animales , Ratones , Homocistinuria/metabolismo , Homocistinuria/genética , Proteómica/métodos , Cistationina betasintasa/metabolismo , Cistationina betasintasa/deficiencia , Cistationina betasintasa/genética , Hígado/metabolismo , Metabolómica/métodos , Esfingolípidos/metabolismo , Mitocondrias/metabolismo , Lipidómica/métodos , Proteoma/metabolismoRESUMEN
Homocystinuria is a rare disease caused by mutations in the CBS gene that results in a deficiency of cystathionine ß-synthase (CBS). CBS is an essential pyridoxal 5'-phosphate (PLP)-dependent enzyme in the transsulfuration pathway, responsible for combining serine with homocysteine to produce cystathionine, whose activity is enhanced by the allosteric regulator S-adenosylmethionine (SAM). CBS also plays a role in generating hydrogen sulfide (H2S), a gaseous signaling molecule with diverse regulatory functions within the vascular, nervous, and immune systems. In this study, we present the clinical and biochemical characterization of two novel CBS missense mutations that do not respond to pyridoxine treatment, namely c.689T > A (L230Q) and 215A > T (K72I), identified in a Chinese patient. We observed that the disease-associated K72I genetic variant had no apparent effects on the spectroscopic and catalytic properties of the full-length enzyme. In contrast, the L230Q variant expressed in Escherichia coli did not fully retain heme and when compared with the wild-type enzyme, it exhibited more significant impairments in both the canonical cystathionine-synthesis and the alternative H2S-producing reactions. This reduced activity is consistent with both in vitro and in silico evidence, which indicates that the L230Q mutation significantly decreases the overall protein's stability, which in turn, may represent the underlying cause of its pathogenicity.
Asunto(s)
Cistationina betasintasa , Homocistinuria , Mutación Missense , Cistationina betasintasa/genética , Cistationina betasintasa/química , Cistationina betasintasa/metabolismo , Homocistinuria/genética , Homocistinuria/metabolismo , Homocistinuria/enzimología , Humanos , Masculino , FemeninoRESUMEN
Pyridoxine-unresponsive homocystinuria has lifelong implications for health. In this issue, Perreault and colleagues present evidence that orally delivered engineered probiotic Escherichia Coli Nissle SYNB1353 is a promising candidate in reducing homocysteine, with successful trials in mice, monkeys, and humans. However, further probiotic optimization and safety assessments are required.
Asunto(s)
Homocistinuria , Probióticos , Ratones , Humanos , Animales , Homocistinuria/genética , Homocistinuria/terapia , Escherichia coli/genética , Probióticos/uso terapéutico , Piridoxina , HomocisteínaRESUMEN
Mutations in MMACHC cause cobalamin C disease (cblC, OMIM 277400), the commonest inborn error of vitamin B12 metabolism. In cblC, deficient activation of cobalamin results in methylcobalamin and adenosylcobalamin deficiency, elevating methylmalonic acid (MMA) and total plasma homocysteine (tHcy). We retrospectively reviewed the medical files of seven cblC patients: three compound heterozygotes for the MMACHC (NM_015506.3) missense variant c.158T>C p.(Leu53Pro) in trans with the common pathogenic mutation c.271dupA (p.(Arg91Lysfs*14), "compounds"), and four c.271dupA homozygotes ("homozygotes"). Compounds receiving hydroxocobalamin intramuscular injection monotherapy had age-appropriate psychomotor performance and normal ophthalmological examinations. In contrast, c.271dupA homozygotes showed marked psychomotor retardation, retinopathy and feeding problems despite penta-therapy (hydroxocobalamin, betaine, folinic acid, l-carnitine and acetylsalicylic acid). Pretreatment levels of plasma and urine MMA and tHcy were higher in c.271dupA homozygotes than in compounds. Under treatment, levels of the compounds approached or entered the reference range but not those of c.271dupA homozygotes (tHcy: compounds 9.8-32.9 µM, homozygotes 41.6-106.8 (normal (N) < 14); plasma MMA: compounds 0.14-0.81 µM, homozygotes, 10.4-61 (N < 0.4); urine MMA: compounds 1.75-48 mmol/mol creatinine, homozygotes 143-493 (N < 10)). Patient skin fibroblasts all had low cobalamin uptake, but this was milder in compound cells. Also, the distribution pattern of cobalamin species was qualitatively different between cells from compounds and from homozygotes. Compared to the classic cblC phenotype presented by c.271dupA homozygous patients, c.[158T>C];[271dupA] compounds had mild clinical and biochemical phenotypes and responded strikingly to hydroxocobalamin monotherapy.
Asunto(s)
Proteínas Portadoras , Hidroxocobalamina , Fenotipo , Deficiencia de Vitamina B 12 , Vitamina B 12 , Humanos , Hidroxocobalamina/administración & dosificación , Hidroxocobalamina/uso terapéutico , Masculino , Femenino , Deficiencia de Vitamina B 12/genética , Deficiencia de Vitamina B 12/tratamiento farmacológico , Deficiencia de Vitamina B 12/sangre , Vitamina B 12/sangre , Preescolar , Proteínas Portadoras/genética , Estudios Retrospectivos , Oxidorreductasas/genética , Niño , Ácido Metilmalónico/sangre , Homocistinuria/tratamiento farmacológico , Homocistinuria/genética , Lactante , Mutación Missense , Homocigoto , Heterocigoto , Homocisteína/sangre , Adolescente , Errores Innatos del Metabolismo de los Aminoácidos/genética , Errores Innatos del Metabolismo de los Aminoácidos/tratamiento farmacológico , Errores Innatos del Metabolismo de los Aminoácidos/sangre , AdultoRESUMEN
BACKGROUND: Based on research, c.609G>A (p.W203X) is a universal mutation site for MMACHC in methylmalonic acidemia (MMA) combined with homocystinuria, cblC type (cblC disease), and c.467G>A (p.G156D) mutation in families with such disease have not yet been reported. To conduct clinical and molecular genetic analysis of a family with cblC disease. METHODS: This work followed the Declaration of Helsinki. All testing methods were performed under the informed consent of our children patients' parents. A second-generation cblC family with 5 members, was selected as the research subject, including sick siblings and parents and an older sister with normal phenotype, given newborn screening for acylcarnitine spectrum via liquid chromatography tandem mass spectrometry (LC-MS/MS), and diagnosed through combining urine organic acid with homocysteine detection via gas chromatography-mass spectrometry (GC-MS) with second-generation gene sequencing technology. The peripheral blood of five family members was collected for genomic DNA extraction, and the changes were screened in disease-related MMACHC sequence via PCR and direct DNA sequencing. RESULTS: The family conformed to the autosomal recessive inheritance, the proband and younger sister were cblC patients, diagnosed in February and at 22d given relevant treatment. The proband died, whereas the younger sister received follow-up treatment. Their parents and sister had normal phenotype. In 2 cases, there was compound heterozygous mutation in MMACHC called c.609G>A (p.W203X) nonsense mutation and c.467G>A (p.G156D) missense mutation in exon 4, while the father with normal phenotype had heterozygous mutation c.609G>A in exon 4 coding area. In its protein, the 203rd amino acid changed from tryptophan to a stop codon (p.W203 x). The normal mother and sister had a heterozygous mutation c.467G>A in exon 4 coding area. In its protein, the 156th amino acid changed from glycine to aspartic acid (p.G156D). CONCLUSIONS: The cblC family results from c.609G>A (p.W203X) and c.467G>A (p.G156D) compound heterozygous mutations in MMACHC, which has a pathogenic impact.
Asunto(s)
Errores Innatos del Metabolismo de los Aminoácidos , Homocistinuria , Recién Nacido , Niño , Humanos , Homocistinuria/complicaciones , Homocistinuria/diagnóstico , Homocistinuria/genética , Cromatografía Liquida , Espectrometría de Masas en Tándem , Errores Innatos del Metabolismo de los Aminoácidos/diagnóstico , Errores Innatos del Metabolismo de los Aminoácidos/genética , Mutación , Aminoácidos , Biología Molecular , Vitamina B 12 , Ácido Metilmalónico , OxidorreductasasRESUMEN
PURPOSE: Serine (Ser) and glycine (Gly) levels were reported to differ between patients with macular telangiectasia type 2 (MacTel) compared with healthy controls. Because they are closely related to methylation metabolism, this report investigates methylation-associated metabolite levels in patients with MacTel and retinal changes in monogenetic methylation disorders. METHODS: Prospective, monocentric study on patients with MacTel and healthy controls underwent a standardized protocol including a blood draw. Methylation-associated metabolite levels in plasma were determined using targeted quantitative metabolomics. Furthermore, patient records of cystathionine beta-synthase, methylenetetrahydrofolate reductase, and methylmalonic aciduria and homocystinuria type C protein (MMACHC) deficiency were screened for reported retinal changes. RESULTS: In total, 29 patients with MacTel and 27 healthy controls were included. Patients with MacTel showed lower plasma Ser ( P = 0.02 and P = 0.01) and Gly ( P = 0.11 and P = 0.11) levels than controls. Principal component analyses revealed that methylation-associated metabolite, especially homocysteine, contributed to a distinct clustering of patients with MacTel. No retinal changes were seen in cystathionine beta-synthase (n = 1) and methylenetetrahydrofolate reductase (n = 2) deficiency, while two patients with MMACHC (n = 4) deficiency displayed extensive macular dystrophy. CONCLUSION: Patients with MacTel show distinct clustering of methylation-associated metabolite compared with controls. Of the three homocystinurias, only MMACHC resulted in macular dystrophy, possibly due to distinct compensatory pathways.
Asunto(s)
Telangiectasia Retiniana , Humanos , Femenino , Masculino , Estudios Prospectivos , Telangiectasia Retiniana/diagnóstico , Telangiectasia Retiniana/metabolismo , Telangiectasia Retiniana/genética , Persona de Mediana Edad , Tomografía de Coherencia Óptica , Adulto , Anciano , Metilación , Errores Innatos del Metabolismo de los Aminoácidos/genética , Errores Innatos del Metabolismo de los Aminoácidos/complicaciones , Errores Innatos del Metabolismo de los Aminoácidos/metabolismo , Errores Innatos del Metabolismo de los Aminoácidos/diagnóstico , Angiografía con Fluoresceína/métodos , Glicina , Homocistinuria/genética , Homocistinuria/complicaciones , Homocistinuria/diagnósticoRESUMEN
Homocystinuria (HCU), an inherited metabolic disorder caused by lack of cystathionine beta-synthase (CBS) activity, is chiefly caused by misfolding of single amino acid residue missense pathogenic variants. Previous studies showed that chemical, pharmacological chaperones or proteasome inhibitors could rescue function of multiple pathogenic CBS variants; however, the underlying mechanisms remain poorly understood. Using Chinese hamster DON fibroblasts devoid of CBS and stably overexpressing human WT or mutant CBS, we showed that expression of pathogenic CBS variant mostly dysregulates gene expression of small heat shock proteins HSPB3 and HSPB8 and members of HSP40 family. Endoplasmic reticulum stress sensor BiP was found upregulated with CBS I278T variant associated with proteasomes suggesting proteotoxic stress and degradation of misfolded CBS. Co-expression of the main effector HSP70 or master regulator HSF1 rescued steady-state levels of CBS I278T and R125Q variants with partial functional rescue of the latter. Pharmacological proteostasis modulators partially rescued expression and activity of CBS R125Q likely due to reduced proteotoxic stress as indicated by decreased BiP levels and promotion of refolding as indicated by induction of HSP70. In conclusion, targeted manipulation of cellular proteostasis may represent a viable therapeutic approach for the permissive pathogenic CBS variants causing HCU.
Asunto(s)
Cistationina betasintasa , Homocistinuria , Humanos , Cistationina betasintasa/genética , Cistationina betasintasa/química , Cistationina betasintasa/metabolismo , Homocistinuria/tratamiento farmacológico , Homocistinuria/genética , Homocistinuria/metabolismo , Cistationina/metabolismo , Cistationina/uso terapéutico , Proteostasis , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas de Choque Térmico/metabolismoRESUMEN
Cystathionine ß-synthase (CBS) catalyzes the committing step in the transsulfuration pathway, which is important for clearing homocysteine and furnishing cysteine. The transsulfuration pathway also generates H2S, a signaling molecule. CBS is a modular protein with a heme and pyridoxal phosphate-binding catalytic core, which is separated by a linker region from the C-terminal regulatory domain that binds S-adenosylmethionine (AdoMet), an allosteric activator. Recent cryo-EM structures reveal that CBS exists in a fibrillar form and undergoes a dramatic architectural rearrangement between the basal and AdoMet-bound states. CBS is the single most common locus of mutations associated with homocystinuria, and, in this study, we have characterized three clinical variants (K384E/N and M391I), which reside in the linker region. The native fibrillar form is destabilized in the variants, and differences in their limited proteolytic fingerprints also reveal conformational alterations. The crystal structure of the truncated K384N variant, lacking the regulatory domain, reveals that the overall fold of the catalytic core is unperturbed. M391I CBS exhibits a modest (1.4-fold) decrease while the K384E/N variants exhibit a significant (â¼8-fold) decrease in basal activity, which is either unresponsive to or inhibited by AdoMet. Pre-steady state kinetic analyses reveal that the K384E/N substitutions exhibit pleiotropic effects and that the differences between them are expressed in the second half reaction, that is, homocysteine binding and reaction with the aminoacrylate intermediate. Together, these studies point to an important role for the linker in stabilizing the higher-order oligomeric structure of CBS and enabling AdoMet-dependent regulation.
Asunto(s)
Cistationina betasintasa , Mutación , Humanos , Regulación Alostérica/genética , Cristalografía por Rayos X , Cistationina betasintasa/química , Cistationina betasintasa/genética , Cistationina betasintasa/metabolismo , Homocisteína/metabolismo , Homocistinuria/enzimología , Homocistinuria/genética , Cinética , S-Adenosilmetionina/metabolismo , Conformación Proteica , Dominio CatalíticoRESUMEN
Classical homocystinuria is caused by pathogenic variants in the CBS gene leading to a deficiency of the vitamin B6-dependent enzyme cystathionine beta synthase. The disease is typically associated with high blood homocysteine concentrations. Clinical features include developmental delay/intellectual disability, psychiatric problems, thromboembolism, lens dislocation, and marfanoid habitus. We report on a child with classical homocystinuria presenting with acute episodes of dystonia and symmetrical basal ganglia abnormalities mimicking a mitochondrial disease. After starting treatment with vitamin B6, homocysteine levels rapidly normalized and dystonic episodes did not re-occur. Moreover, brain-imaging findings almost completely disappeared. The case illustrates that homocystinuria should be considered as a treatable differential diagnosis of dystonia.
Asunto(s)
Distonía , Trastornos Distónicos , Homocistinuria , Niño , Humanos , Homocistinuria/complicaciones , Homocistinuria/diagnóstico , Homocistinuria/genética , Distonía/diagnóstico , Distonía/etiología , Cistationina betasintasa , Piridoxina/uso terapéutico , Vitamina B 6/uso terapéutico , HomocisteínaRESUMEN
In early-onset (EO) cblC deficiency (MMACHC), hydroxocobalamin dose-intensification (OHCBL-DI) improved biochemical and clinical outcome. In mammals, Cobalamin is reduced, in a reaction mediated by MMACHC. Pathogenic variants in MMACHC disrupt the synthesis pathway of methyl-cobalamin (MetCbl) and 5'-deoxy-adenosyl-cobalamin (AdoCbl), cofactors for both methionine synthase (MS) and methyl-malonyl-CoA mutase (MCM) enzymes. In 5 patients (pts.), with EO cblC deficiency, biochemical and clinical responses were studied following OHCbl-DI (mean ± SD 6,5 ± 3,3 mg/kg/day), given early, before age 5 months (pts. 1, 2, 3 and 4) or lately, at age 5 years (pt. 5). In all pts., total homocysteine (tHcy), methyl-malonic acid (MMA) and Cob(III)alamin levels were measured. Follow-up was performed during 74/12 years (pts. 1, 2, 3), 33/12 years (pt. 4) and 34/12 years (pt. 5). OHCbl was delivered intravenously or subcutaneously. Mean ± SD serum Cob(III)alamin levels were 42,2 × 106 ± 28, 0 × 106 pg/ml (normal: 200-900 pg/ml). In all pts., biomarkers were well controlled. All pts., except pt. 5, who had poor vision, had central vision, mild to moderate nystagmus, and with peri-foveolar irregularity in pts. 1, 2 and 4, yet none had the classic bulls' eye maculopathy and retinal degeneration characteristic of pts. with EO cblC deficiency. Only pt. 5, had severe cognitive deficiency. Both visual and cognitive functions were better preserved with early than with late OHCBL-DI. OHCBL-DI is suggested to bypass MMACHC, subsequently to be rescued by methionine synthase reductase (MSR) and adenosyl-transferase (ATR) to obtain Cob(I)alamin resulting in improved cognitive and retinal function in pts. with EO cblC deficiency.
Asunto(s)
Disfunción Cognitiva , Homocistinuria , Degeneración Macular , Deficiencia de Vitamina B 12 , Preescolar , Humanos , Lactante , Masculino , Disfunción Cognitiva/tratamiento farmacológico , Homocistinuria/tratamiento farmacológico , Homocistinuria/genética , Hidroxocobalamina/uso terapéutico , Degeneración Macular/tratamiento farmacológico , Mamíferos , Oxidorreductasas , Vitamina B 12/metabolismo , Deficiencia de Vitamina B 12/tratamiento farmacológicoRESUMEN
Classical homocystinuria (HCU) is a rare inborn error of amino acid metabolism characterized by accumulation of homocysteine, an intermediate product of methionine metabolism, leading to significant systemic toxicities, particularly within the vascular, skeletal, and ocular systems. Most patients require lifelong dietary therapy with severe restriction of natural protein to minimize methionine intake, and many patients still struggle to maintain healthy homocysteine levels. Since eliminating methionine from the diet reduces homocysteine levels, we hypothesized that an enzyme that can degrade methionine within the gastrointestinal (GI) tract could help HCU patients maintain healthy levels while easing natural protein restrictions. We describe the preclinical development of CDX-6512, a methionine gamma lyase (MGL) enzyme that was engineered for stability and activity within the GI tract for oral administration to locally degrade methionine. CDX-6512 is stable to low pH and intestinal proteases, enabling it to survive the harsh GI environment without enteric coating and to degrade methionine freed from dietary protein within the small intestine. Administering CDX-6512 to healthy non-human primates following a high protein meal led to a dose-dependent suppression of plasma methionine. In Tg-I278T Cbs-/- mice, an animal model that recapitulates aspects of HCU disease including highly elevated serum homocysteine levels, oral dosing of CDX-6512 after a high protein meal led to suppression in serum levels of both methionine and homocysteine. When animals received a daily dose of CDX-6512 with a high protein meal for two weeks, the Tg-I278T Cbs-/- mice maintained baseline homocysteine levels, whereas homocysteine levels in untreated animals increased by 39%. These preclinical data demonstrate the potential of CDX-6512 as an oral enzyme therapy for HCU.
Asunto(s)
Homocistinuria , Humanos , Ratones , Animales , Homocistinuria/tratamiento farmacológico , Homocistinuria/genética , Metionina/metabolismo , Homocisteína , Cistationina betasintasa/genética , Cistationina betasintasa/metabolismo , Racemetionina , Tracto Gastrointestinal/metabolismoRESUMEN
OBJECTIVES: To study the biochemical, clinical and molecular characteristics of 5,10- methylenetetrahydrofolate reductase (MTHFR) deficiency in Pakistani patients from a single center. METHODS: Medical charts, urine organic acid chromatograms, plasma methionine and Hcys levels, and molecular testing results of MTHFR gene of patients presenting at the Biochemical Genetics Clinic, AKUH from 2016 to 2022 were reviewed. RESULTS: Neonatal MTHFR deficiency was found in five patients. The median (IQR) age of symptom onset and diagnosis were 18 (8.5-22) and 26 (16.5-31) days. The median lag between symptom onset and diagnosis was 8 (4.5-12.5) days. The median age of treatment initiation and duration of treatment were 26 (16.5-49) and 32 (25.5-54) days. The most common clinical features were lethargy, poor feeding, and seizures. The MTHFR gene sequencing revealed homozygous variants p.K510K, p.R567*, and p.R157W. Renal insufficiency manifesting as elevated serum creatinine and responding to betaine therapy was noted in one patient. This has not been previously reported in neonatal MTHFR deficiency and may reflect engagement of alternate pathways of remethylation. Adult onset MTHFR deficiency was found in six patients, with a heterogeneous neurological presentation. The median lag between symptoms onset and diagnosis was 7 (3-11) years. MTHFR gene sequencing revealed homozygous variant p.A195V in five patients from one family and p.G261V in the other. Two of the five reported variants are novel that include p.R157W and p.G261V. CONCLUSIONS: Eleven patients of this rare disorder from a single center indicate the need for clinical awareness and appropriate biochemical evaluation to ensure optimal outcomes.
Asunto(s)
Homocistinuria , Trastornos Psicóticos , Adulto , Humanos , Homocistinuria/diagnóstico , Homocistinuria/genética , Homocistinuria/tratamiento farmacológico , Metilenotetrahidrofolato Reductasa (NADPH2)/genética , Pakistán , Trastornos Psicóticos/diagnóstico , Trastornos Psicóticos/tratamiento farmacológico , Trastornos Psicóticos/genéticaRESUMEN
Classic homocystinuria is an inborn error of metabolism caused mainly by missense mutations leading to misfolded and/or unstable human cystathionine ß-synthase (CBS) protein, causing the accumulation of excess total homocysteine (tHcy) in tissues. Previously, it has been shown that certain missense containing human CBS proteins can be functionally rescued in mouse models of CBS deficiency by treatment with proteasome inhibitors. The rescue by proteasome inhibitors is thought to work both by inhibiting the degradation of misfolded CBS protein and by inducing the levels of heat-shock chaperone proteins in the liver. Here we examine the effectiveness of two FDA approved protease inhibitors, carfilzomib and bortezomib, on various transgenic mouse models of human CBS deficiency. Our results show that although both drugs are effective in inducing the liver chaperone proteins Hsp70 and Hsp27, and are effective in inhibiting proteasome function, bortezomib was somewhat more robust in restoring the mutant CBS function. Moreover, there was no significant correlation between proteasome inhibition and CBS activity, suggesting that some of bortezomib's effects are via other mechanisms. We also test the use of low-doses of bortezomib and carfilzomib on various mouse models for extended periods of time and find that while low-doses are less toxic, they are also less effective at restoring CBS function. Overall, these results show that while restoration of mutant CBS function is possible with proteasome inhibitors, the exact mechanism is complicated and it will likely be too toxic for long-term patient treatment.
Asunto(s)
Cistationina betasintasa , Homocistinuria , Humanos , Ratones , Animales , Cistationina betasintasa/genética , Cistationina betasintasa/metabolismo , Inhibidores de Proteasoma/farmacología , Inhibidores de Proteasoma/uso terapéutico , Homocistinuria/tratamiento farmacológico , Homocistinuria/genética , Homocistinuria/metabolismo , Bortezomib/farmacología , Bortezomib/uso terapéutico , Complejo de la Endopetidasa Proteasomal , Ratones TransgénicosRESUMEN
BACKGROUND: the deficiency of 5,10-Methylenetetrahydrofolate reductase (MTHFR) constitutes a rare and severe metabolic disease and is included in most expanded newborn screening (NBS) programs worldwide. Patients with severe MTHFR deficiency develop neurological disorders and premature vascular disease. Timely diagnosis through NBS allows early treatment, resulting in improved outcomes. METHODS: we report the diagnostic yield of genetic testing for MTHFR deficiency diagnosis, in a reference Centre of Southern Italy between 2017 and 2022. MTHFR deficiency was suspected in four newborns showing hypomethioninemia and hyperhomocysteinemia; otherwise, one patient born in pre-screening era showed clinical symptoms and laboratory signs that prompted to perform genetic testing for MTHFR deficiency. RESULTS: molecular analysis of the MTHFR gene revealed a genotype compatible with MTHFR deficiency in two NBS-positive newborns and in the symptomatic patient. This allowed for promptly beginning the adequate metabolic therapy. CONCLUSIONS: our results strongly support the need for genetic testing to quickly support the definitive diagnosis of MTHFR deficiency and start therapy. Furthermore, our study extends knowledge of the molecular epidemiology of MTHFR deficiency by identifying a novel mutation in the MTHFR gene.
Asunto(s)
Homocistinuria , Metilenotetrahidrofolato Reductasa (NADPH2) , Humanos , Recién Nacido , Metilenotetrahidrofolato Reductasa (NADPH2)/genética , Homocistinuria/diagnóstico , Homocistinuria/genética , Pruebas Genéticas , Diagnóstico PrecozRESUMEN
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.
Asunto(s)
Homocistinuria , Tromboembolia , Humanos , Homocistinuria/tratamiento farmacológico , Homocistinuria/genética , Homocistinuria/metabolismo , Cistationina betasintasa/genética , Cistationina betasintasa/metabolismo , Calidad de Vida , Mutación MissenseRESUMEN
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.
Asunto(s)
Homocistinuria , Hiperhomocisteinemia , Errores Innatos del Metabolismo , Humanos , Adulto , Femenino , Ácido Fólico , Estudios Retrospectivos , Vitamina B 12/uso terapéutico , Hiperhomocisteinemia/diagnóstico , Hiperhomocisteinemia/genética , Homocistinuria/diagnóstico , Homocistinuria/genética , Errores Innatos del Metabolismo/diagnóstico , Errores Innatos del Metabolismo/genética , HomocisteínaRESUMEN
BACKGROUND: Homocystinuria due to methylenetetrahydrofolate reductase (MTHFR) deficiency is a rare autosomal recessive disorder. The purpose of this study is to expand the mutation site of the MTHFR gene and provide genetic counseling for this family. METHODS: A couple came to our hospital for pre-pregnancy genetic counseling. We collected the family history and detailed clinical information, then performed whole-exome sequencing, and analyzed the pathogenicity of the candidate mutations. RESULTS: We found that the father of the proband had homocystinuria, the proband and his brother had low blood methionine levels at birth, and the brain MRI showed brain dysplasia. The third fetus was found to have a broadened triangle of the bilateral ventricle at 19 weeks of pregnancy. The compound heterozygous variants of c.602 A > C (p.His201Pro) and c.1316T > C (p.Leu439Pro) of the MTHFR gene in the first three fetuses were found by whole-exome sequencing. The heterozygous c.602 A > C variant of the MTHFR gene is a novel missense variant that has been submitted to the ClinVar with Variation ID 992,662. CONCLUSION: In consideration of the clinical phenotype, family history, and result of genetic testing, we speculated that both patients may have homocystinuria due to MTHFR deficiency. Homocystinuria due to MTHFR deficiency caused by compound heterozygous mutations composed of the MTHFR gene in this family may be associated with cerebral atrophy and cerebral dysplasia. The novel compound heterozygous mutations broaden the mutation spectrum of the MTHFR gene and enhance the application of genetic counseling and carrier screening in rare diseases.