Betaína anidra para o tratamento de pacientes com homocistinúria com deficiências ou defeitos da cistationina-betasintetase, não responsivos a piridoxina (B6) / Anidra betaine for the treatment of patients with homocystinuria with cystathionine-betasynthetase deficiencies or defects, unresponsive to pyridoxine (B6)

INTRODUÇÃO: A homocistinúria ocorre por conta de mutações em enzimas envolvidas no metabolismo da metionina, sendo elas: deficiência da cistationina-beta-sintetase (CBS), deficiência da 5,10- metilenotetrahidrofolato redutase (MTHFR) ou defeito no cofator do metabolismo da cobalamina (cbl). É uma condição clínica que acomete um pequeno número de pessoas e designa um grupo de doenças metabólicas hereditárias, e em função de decorrer de mutações em enzimas do metabolismo da metionina, a homocistinúria, é uma doença progressiva, sendo mais grave a evolução quanto mais precocemente se manifestam os sintomas. Por ser uma doença progressiva e sistêmica, o quadro clínico da homocistinúria é caracterizado por complicações que podem atingir diversos sistemas biológicos. Algumas das principais manifestações clínicas são o deslocamento das lentes dos olhos para baixo (ectopia lentis), osteoporose, retardo mental, convulsões e, principalmente, fenômenos tromboembólicos. Os fenômenos tromboembólicos revestem-se da maior importância, por determinarem a ocorrência de eventos cardiovasculares sérios, como infarto do miocárdio e acidentes vasculares encefálicos muito precocemente na vida, com impacto significativo na morbimortalidade. PERGUNTA DE PESQUISA: O uso de betaína anidra como terapia complementar no tratamento de pacientes com homocistinúria com deficiências ou defeitos da cistationina-betasintetase, não responsivos a piridoxina (B6) é eficaz, seguro e custo-efetivo em relação às terapias disponíveis no SUS? EVIDÊNCIAS CIENTÍFICAS: Em revisão sistemática da literatura, o demandante selecionou 1 ECR e 04 séries de casos e 02 estudos observacionais com subgrupo de interesse. No relatório de avaliação crítica foi realizada nova busca nas bases de dados e foram excluídas da análise as quatro séries de casos pela limitação metodológica do tipo de estudo e o número muito baixo de pacientes. Observou-se que na maioria dos estudos, o uso de betaína levou a redução pelo menos numérica dos níveis de homocisteína. Destaca-se que em ECR, essa redução foi significativa para o grupo betaína frente ao placebo.) Ainda, foi observado aumento também pelo menos numérico da cisteína e da metionina em pacientes em uso de betaína.Entretanto, no ECR, o aumento da metionina no grupo betaína apesar de numericamente maior não apresentou significância estatística na comparação frente ao placebo. Os valores de densidade mineral óssea avaliados não foram observadas diferenças significativas entre betaína e placebo, apesar de pacientes em uso de betaína apresentarem resultados numericamente melhores. Em termos de segurança, a betaína foi considerada tolerável, sem relatos de disfunção hepática, renal ou de medula óssea e nenhum relato de efeito indesejado. Ainda, não foram reportados eventos adversos ou intolerância ao tratamento. AVALIAÇÃO ECONÔMICA: A análise econômica realizada pelo demandante baseou-se em modelo de custoutilidade e os resultados são apresentados na forma de custo incremental por ano de vida ganho e por QALY.O curso da doença foi simulado utilizando o modelo de Markov. O modelo incluiu três estados de saúde:Pacientes sem evento cardiovascular, pacientes com evento cardiovascular e morte. O horizonte temporal foi de 20 anos. Para um paciente viver um ano livre da ocorrência de eventos cardiovasculares (1 AVL de ECV) a RCEI foi de R$ 492.124,91. Para um paciente viver um ano com a qualidade de saúde plena (1 QALY) a RCEI foi de R$ 1.525.349,65 milhões. ANÁLISE DE IMPACTO ORÇAMENTÁRIO: Foi realizada pelo demandante, análise de impacto orçamentário (AIO) tendo como população alvo pacientes com homocistinúria clássica não responsivos à piridoxina (vitamina B6) O demandante criou dois cenários, um baseado em dados epidemiológicos e outro baseado em demanda aferida.A demanda epidemiológica , resulta em um investimento incremental, acumulados em 5 anos de R$ R$ 11.025.966 e de R$ 3.570.275 para a demanda aferida considerando um Market-share de 50% a 90% no quinto ano. MONITORAMENTO DO HORIZONTE TECNOLÓGICO: No horizonte considerado nesta análise, não se detectou nenhuma tecnologia potencial para o tratamento de pacientes com homocistinúria clássica, não responsivos a piridoxina (B6). Porém, cabe informar que existem duas tecnologias com estudos de fase 1/2 em andamento para homocistinúria clássica, independentemente da responsividade à piridoxina, a pegtarviliase e a pegtibatinase. CONSIDERAÇÕES FINAIS: A análise da evidência clínica apresentada pelo demandante sugere que a tecnologia proposta apresenta efetividade superior quando usada em combinação às alternativas disponíveis atualmente no SUS. Os estudos avaliando betaína são estudos com baixo número amostral,suscetíveis a vieses importantes e há ausência de um ensaio clínico randomizado que avalie a betaína anidra em um número de pacientes maior.O fato de ser uma doença rara corrobora para não termos evidências robustas dessa tecnologia. O modelo de custo-efetividade/utilidade construído pelo demandante utilizou uma abordagem considerando apenas três estados de transições e um horizonte temporal de 20 anos, porém não justificou essa escolha que difere do recomendado nas diretrizes brasileiras. Não foi referida a utilização de taxa de desconto. O modelo adotado considerou manifestações clínicas relacionadas apenas a eventos cardiovasculares, porém a homocistinúria têm outras manifestações clínicas importantes que podem impactar diretamente na qualidade de vida dos pacientes que não estão avaliadas no modelo. Os dados de custos dos medicamentos consideraram PMVG DE 17% sendo mais adequado a utilização de valores considerando 18% de imposto. Como a homocistinúria acomete pacientes pediátricos seria importante apresentar análises de sensibilidade ou justificar o valor do tratamento para não superestimar os custos já que o medicamento avaliado é calculado por peso do paciente. O demandante apresentou uma AIO adequada em relação ao market share e considerou a fragilidade dos dados da população ­alvo apresentando uma análise por demanda aferida e por demanda epidemiológica. A demanda aferida calculada apresenta dados mais perto da realidade dos pacientes diagnosticados no Brasil com homocistinúria. RECOMENDAÇÃO PRELIMINAR DA CONITEC: Os membros do Plenário, presentes na 113ª Reunião Ordinária da Conitec, no dia 05 de outubro de 2022, deliberaram que deliberaram que a matéria fosse disponibilizada em consulta pública com recomendação preliminar desfavorável à incorporação ao SUS da betaína anidra para o tratamento de pacientes com homocistinúria com deficiências ou defeitos da cistationina-beta- sintetase, não responsivos a piridoxina (B6). CONSULTA PÚBLICA: Foram recebidas 121 contribuições, sendo 07 pelo formulário técnico-científico e 114 pelo formulário sobre experiência ou opinião. Todas as contribuições de cunho técnico-científico recebidas foram contra a recomendação inicial da Conitec. A maioria dos respondentes do formulário de experiência e opinião manifestou-se favorável à incorporação da betaína anidra. Entre as opiniões favoráveis, destacaram-se os temas sobre direito à saúde e o papel essencial do medicamento avaliado para o tratamento dos pacientes não responsivos à piridoxina. Os participantes também mencionaram o aumento da qualidade de vida, o fato de o medicamento se destinar ao tratamento de uma doença rara, ser um medicamento recomendado por profissionais de saúde, o tratamento da doença demandar a associação do medicamento com outras tecnologias, a segurança em relação à padronização na produção do medicamento e se tratar de uma questão de sobrevivência. Em relação à experiência com a betaína anidra, como efeitos positivos ou facilidades, os respondentes relataram o controle da doença, boa resposta terapêutica, prevenção de manifestações clínicas da doença e ação anticoagulante. Em contraponto, reportaram a dificuldade de acesso ao medicamento. RECOMENDAÇÃO FINAL DA CONITEC: Pelo exposto, o Plenário da Conitec, em sua 12ª Reunião Extraordinária, no dia 29 de novembro de 2022, deliberou, por unanimidade, recomendar a não incorporação da betaína anidra para o tratamento de pacientes com homocistinúria com deficiências ou defeitos da cistationina-beta-sintetase, não responsivos a piridoxina (B6). Por fim, foi assinado o Registro de Deliberação nº 788/2022. DECISÃO: Não incorporar, no âmbito do Sistema Único de Saúde - SUS, a betaína anidra para o tratamento de pacientes com homocistinúria com deficiências ou defeitos da cistationina-betasintetase, não responsivos a piridoxina (B6), conforme protocolo estabelecido pelo Ministério da Saúde, conforme a Portaria nº 176, publicada no Diário Oficial da União nº 238, seção 1, página 231, em 20 de dezembro de 2022.

Long-term functional correction of cystathionine ß-synthase deficiency in mice by adeno-associated viral gene therapy.

Cystathionine ß-synthase (CBS) deficiency is a recessive inborn error of sulfur metabolism characterized by elevated blood levels of total homocysteine (tHcy). Patients diagnosed with CBS deficiency are currently treated by a combination of vitamin supplementation and restriction of foods containing the homocysteine precursor methionine, but the effectiveness of this therapy is limited due to poor compliance. A mouse model for CBS deficiency (Tg-I278T Cbs-/- ) was used to evaluate a potential gene therapy approach to treat CBS deficiency utilizing an AAVrh.10-based vector containing the human CBS cDNA downstream of the constitutive, strong CAG promoter (AAVrh.10hCBS). Mice were administered a single dose of virus and followed for up to 1 year. The data demonstrated a dose-dependent increase in liver CBS activity and a dose-dependent decrease in serum tHcy. Liver CBS enzyme activity at 1 year was similar to Cbs+/- control mice. Mice given the highest dose (5.6 × 1011 genomes/mouse) had mean serum tHcy decrease of 97% 1 week after injection and an 81% reduction 1 year after injection. Treated mice had either full- or substantial correction of alopecia, bone loss, and fat mass phenotypes associated with Cbs deficiency in mice. Our findings show that AAVrh.10-based gene therapy is highly effective in treating CBS deficiency in mice and supports additional pre-clinical testing for eventual use human trials.

Glucose-induced decrease of cystathionine ß-synthase mediates renal injuries.

Exogenous hydrogen sulfide (H2 S) protects kidneys from diabetic injuries in animal models. In order to explore the role of endogenous H2 S in diabetic nephropathy, we determined the renal H2S producing enzymes in vivo and in vitro. In diabetic mice, H2 S levels in blood and kidney were decreased while cystathionine ß-synthase (CBS), mainly located in mouse renal proximal convoluted tubules (PCT), was reduced selectively. In cultured mouse PCT cells treated with high glucose, CBS protein and activity was reduced while ubiquitinated CBS was increased, which was abolished by a proteasome inhibitor MG132 at 1 hour; high glucose drove CBS colocalized with proteasome 26S subunit ATPase6, indicating an involvement of ubiquitination proteasome degradation. At 48 hours, high glucose also selectively decreased CBS protein, concentration-dependently, but increased the ubiquitination of CBS; silence of CBS by siRNA increased nitrotyrosine, a marker for protein oxidative injury. Nitrotyrosine was also increased by high glucose treatments. The increases of nitrotyrosine either by cbs-siRNA or by glucose were restored by GYY4137, indicating that the H2 S donor may protect kidney from oxidative injury induced by CBS deficiency. In diabetic kidneys, ubiquitinated CBS and nitrotyrosine were increased but restored by GYY4137. The treatment also ameliorated albuminuria and renal morphologic changes in diabetic mice. Our findings suggest that high glucose induces reduction of renal CBS protein and activity in vivo and in vitro that is critical to the pathogenesis of diabetic kidney disease.

Immunological Feature and Transcriptional Signaling of Ly6C Monocyte Subsets From Transcriptome Analysis in Control and Hyperhomocysteinemic Mice.

Background: Murine monocytes (MC) are classified into Ly6Chigh and Ly6Clow MC. Ly6Chigh MC is the pro-inflammatory subset and the counterpart of human CD14++CD16+ intermediate MC which contributes to systemic and tissue inflammation in various metabolic disorders, including hyperhomocysteinemia (HHcy). This study aims to explore molecule signaling mediating MC subset differentiation in HHcy and control mice. Methods: RNA-seq was performed in blood Ly6Chigh and Ly6Clow MC sorted by flow cytometry from control and HHcy cystathionine ß-synthase gene-deficient (Cbs-/-) mice. Transcriptome data were analyzed by comparing Ly6Chigh vs. Ly6Clow in control mice, Ly6Chigh vs. Ly6Clow in Cbs-/- mice, Cbs-/- Ly6Chigh vs. control Ly6Chigh MC and Cbs-/- Ly6Clow vs. control Ly6Clow MC by using intensive bioinformatic strategies. Significantly differentially expressed (SDE) immunological genes and transcription factor (TF) were selected for functional pathways and transcriptional signaling identification. Results: A total of 7,928 SDE genes and 46 canonical pathways derived from it were identified. Ly6Chigh MC exhibited activated neutrophil degranulation, lysosome, cytokine production/receptor interaction and myeloid cell activation pathways, and Ly6Clow MC presented features of lymphocyte immunity pathways in both mice. Twenty-four potential transcriptional regulatory pathways were identified based on SDE TFs matched with their corresponding SDE immunological genes. Ly6Chigh MC presented downregulated co-stimulatory receptors (CD2, GITR, and TIM1) which direct immune cell proliferation, and upregulated co-stimulatory ligands (LIGHT and SEMA4A) which trigger antigen priming and differentiation. Ly6Chigh MC expressed higher levels of macrophage (MΦ) markers, whereas, Ly6Clow MC highly expressed lymphocyte markers in both mice. HHcy in Cbs-/- mice reinforced inflammatory features in Ly6Chigh MC by upregulating inflammatory TFs (Ets1 and Tbx21) and strengthened lymphocytes functional adaptation in Ly6Clow MC by increased expression of CD3, DR3, ICOS, and Fos. Finally, we established 3 groups of transcriptional models to describe Ly6Chigh to Ly6Clow MC subset differentiation, immune checkpoint regulation, Ly6Chigh MC to MΦ subset differentiation and Ly6Clow MC to lymphocyte functional adaptation. Conclusions: Ly6Chigh MC displayed enriched inflammatory pathways and favored to be differentiated into MΦ. Ly6Clow MC manifested activated T-cell signaling pathways and potentially can adapt the function of lymphocytes. HHcy reinforced inflammatory feature in Ly6Chigh MC and strengthened lymphocytes functional adaptation in Ly6Clow MC.

Liver transplant as a curative treatment in a pediatric patient with classic homocystinuria: A case report.

We report a patient with homocystinuria and hyperoxaluria who was cured of homocystinuria-related disease following liver transplant. The patient was diagnosed with homocystinuria as a newborn and was treated with dietary modifications and supplements. At 22 months, he passed a calcium oxalate stone and was found to have numerous bilateral kidney stones. Genetic testing confirmed primary hyperoxaluria, type 1. He underwent preemptive liver transplant at age four to treat primary hyperoxaluria. Following transplant, his serum methionine and homocysteine levels normalized, thus, demonstrating resolution of homocystinuria. Methionine and homocysteine levels remained normal 6 years later. Homocystinuria is associated with ophthalmologic, skeletal, neurologic, and thromboembolic complications. As cystathionine beta-synthase resides in the liver, transplant was hypothesized to be an effective treatment. Primary hyperoxaluria generally progresses to chronic kidney disease and is treated with combined kidney-liver transplant at the time of end stage kidney disease. Given this patient's dual diagnoses, we proceeded with preemptive liver transplantation. Three prior cases of patients with homocystinuria treated with liver transplantation have been reported. In all cases, transplant resolved metabolic effects. However, our case represents a pediatric patient without disease-related complications prior to transplant. This case supports liver-targeted gene therapies as an effective treatment for homocystinuria.

Hyperhomocysteinemia: an instigating factor for periodontal disease.

Hyperhomocysteinemia (HHcy) affects bone remodeling, since a destructive process in cortical alveolar bone has been linked to it; however, the mechanism remains at large. HHcy increases proinflammatory cytokines viz. TNF-α, IL-1b, IL-6, and IL-8 that leads to a cascade that negatively impacts methionine metabolism and homocysteine cycling. Further, chronic inflammation decreases vitamins B12, B6, and folic acid that are required for methionine homocysteine homeostasis. This study aims to investigate a HHcy mouse model (cystathionine ß-synthase deficient, CBS+/-) for studying the potential pathophysiological changes, if any, in the periodontium (gingiva, periodontal ligament, cement, and alveolar bone). We compared the periodontium side-by-side in the CBS+/- model with that of the wild-type (C57BL/6J) mice. Histology and histomorphometry of the mandibular bone along with gene expression analyses were carried out. Also, proangiogenic proteins and metalloproteinases were studied. To our knowledge, this research shows, for the first time, a direct connection between periodontal disease during CBS deficiency, thereby suggesting the existence of disease drivers during the hyperhomocysteinemic condition. Our findings offer opportunities to develop diagnostics/therapeutics for people who suffer from chronic metabolic disorders like HHcy.

High-methionine diet in skeletal muscle remodeling: epigenetic mechanism of homocysteine-mediated growth retardation.

Epigenetic DNA methylation (1-carbon metabolism) is crucial for gene imprinting/off-printing that ensures epigenetic memory but also generates a copious amount of homocysteine (Hcy), unequivocally. That is why during pregnancy, expectant mothers are recommended "folic acid" preemptively to avoid birth defects in the young ones because of elevated Hcy levels (i.e., hyperhomocysteinemia (HHcy)). As we know, children born with HHcy have several musculoskeletal abnormalities, including growth retardation. Here, we focus on the gut-dysbiotic microbiome implication(s) that we believe instigates the "1-carbon metabolism" and HHcy causing growth retardation along with skeletal muscle abnormalities. We test our hypothesis whether high-methionine diet (HMD) (an amino acid that is high in red meat), a substrate for Hcy, can cause skeletal muscle and growth retardation, and treatment with probiotics (PB) to mitigate skeletal muscle dysfunction. To test this, we employed cystathionine ß-synthase, CBS deficient mouse (CBS+/-) fed with/without HMD and with/without a probiotic (Lactobacillus rhamnosus) in drinking water for 16 weeks. Matrix metalloproteinase (MMP) activity, a hallmark of remodeling, was measured by zymography. Muscle functions were scored via electric stimulation. Our results suggest that compared to the wild-type, CBS+/- mice exhibited reduced growth phenotype. MMP-2 activity was robust in CBS+/- and HMD effects were successfully attenuated by PB intervention. Electrical stimulation magnitude was decreased in CBS+/- and CBS+/- treated with HMD. Interestingly; PB mitigated skeletal muscle growth retardation and atrophy. Collectively, results imply that individuals with mild/moderate HHcy seem more prone to skeletal muscle injury and its dysfunction.

Cystathionine ß-synthase Deficiency Impairs Vision in the Fruit Fly, Drosophila melanogaster.

PURPOSE: Deficiency in Cystathionine ß-synthase (CBS) leads to an abnormal accumulation of homocysteine and results in classical homocystinuria, a multi-systemic disorder that affects connective tissue, muscles, the central nervous system, and the eyes. However, the genetic players and mechanisms underlying vision alterations in patients with homocystinuria are little understood. MATERIALS AND METHODS: The fruit fly, Drosophila melanogaster, is a useful system to investigate the genetic basis of several human diseases, but no study to date has used Drosophila as model of homocystinuria. Here, we use Drosophila genetic tools to down-regulate CBS expression and evaluate its behavioral response to light. RESULTS: We show that CBS-deficient flies do not display the normal stereotypical behavior of attraction towards a luminous source, known as phototaxis. This behavior cannot be attributed to a motor or olfactory deficiency, but it is most likely related to a lower visual acuity. CBS-deficient flies are overall smaller, but smaller eyes do not explain their lack of phototactic response. CONCLUSIONS: The vision phenotype of CBS knock-down flies is consistent with severe myopia in homocystinuria patients. We propose to use Drosophila as a model to investigate ocular manifestations underlying homocystinuria.

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

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

Altered circadian dynamics of Per2 after cystathionine-ß-synthase and/or cystathionine-γ-lyase pharmacological inhibition in serum-shocked NIH-3T3 cells.

Circadian clock genes are found in almost every cell that has a nucleus; they regulate the rhythmic nature of all processes that are cyclical. Among the genes controlled by the circadian clock, there are numerous factors that regulate key processes in the functioning of the cell. Disturbances in the functioning of the circadian clock are associated with numerous disorders. A recent study has shown the key role of H2S in regulating circadian rhythm. In this study, we investigated the in vitro effect of pharmacological inhibition of cystathionine-ß-synthase (CBS) and/or cystathionine-γ-lyase (CSE) on the circadian dynamics of Per2 expression in serum-shocked NIH-3T3 cells. Alternatively, Cbs and Cse were knocked down by transfection with siRNA. The 48-h treatment of serum-shocked NIH-3T3 cells with 1 mM dl-propargylglycine (PAG), a specific CSE inhibitor, significantly decreased the amplitude and baseline expression of Per2. During exposure to an effective CBS and CSE inhibitor (aminooxyacetic acid [AOAA]), the amplitude of oscillation and baseline expression of Per2 significantly increased. Incubation of NIH-3T3 cells with both inhibitors also significantly increased the amplitude and baseline expression of Per2 messenger RNA (mRNA). siCbs or siCse knockdowan significantly reduced the baseline and amplitude of oscillation of Per2. In conclusion, we showed that CBS/CSE/H2S pathway participates in the regulation of the circadian clock system. PAG and AOAA, change the general expression and dynamics of Per2 genes, but the increase of amplitude and overall Per2 mRNA level due to exposure to AOAA is probably caused by factors other than CBS and CSE activity.

Proteomic exploration of cystathionine ß-synthase deficiency: implications for the clinic.

Introduction: Homocystinuria due to cystathionine ß-synthase (CBS) deficiency, the most frequent inborn error of sulfur amino acid metabolism, is characterized biochemically by severely elevated homocysteine (Hcy) and related metabolites, such as Hcy-thiolactone and N-Hcy-protein. CBS deficiency reduces life span and causes pathological abnormalities affecting most organ systems in the human body, including the cardiovascular (thrombosis, stroke), skeletal/connective tissue (osteoporosis, thin/non-elastic skin, thin hair), and central nervous systems (mental retardation, seizures), as well as the liver (fatty changes), and the eye (ectopia lentis, myopia). Molecular basis of these abnormalities were largely unknown and available treatments remain ineffective. Areas covered: Proteomic and transcriptomic studies over the past decade or so, have significantly contributed to our understanding of mechanisms by which the CBS enzyme deficiency leads to clinical manifestations associated with it. Expert opinion: Recent findings, discussed in this review, highlight the involvement of dysregulated proteostasis in pathologies associated with CBS deficiency, including thromboembolism, stroke, neurologic impairment, connective tissue/collagen abnormalities, hair defects, and hepatic toxicity. To ameliorate these pathologies, pharmacological, enzyme replacement, and gene transfer therapies are being developed.

When to measure plasma homocysteine and how to place it in context: The homocystinurias.

This review presents clinical patterns that should trigger homocysteine measurement in blood, as well as the further diagnostic work-up focused on inborn errors of metabolism and disorders of vitamin B12 (cobalamin) absorption and supply. The numerous conditions (e.g. cardiovascular disease, Alzheimer's disease) for which mild-to-moderate hyperhomocysteinaemia caused by genetic polymorphisms or acquired reasons is considered a risk factor are beyond the scope of this review.Homocysteine is a sulphur-containing amino acid, which is derived from the amino acid methionine. Homocysteine is either trans-sulphurated to form cystathionine and then cysteine, or re-methylated to methionine. The trans-sulphuration reaction depends on the enzyme cystathionine beta synthase and its cofactor vitamin B6. The re-methylation reaction not only involves the enzymes methionine synthase and methionine synthase reductase but also depends on the cofactor cobalamin and on the provision of methyl groups from the folate cycle. Because the homocysteine-methionine cycle provides for the vast majority of methyl groups in the body, it is central to numerous pathways that depend on methyl group supply, such as creatine synthesis or DNA methylation. Based on this premise, the severity of clinical presentations of inborn errors of metabolism, such as classical homocystinuria or the cobalamin C (cblC) defect, affecting this pathway is unsurprising.

A cautionary tale of pyridoxine toxicity in cystathionine beta-synthase deficiency detected by two-tier newborn screening highlights the need for clear pyridoxine dosing guidelines.

Classic homocystinuria is due to deficiency of cystathionine beta-synthase (CBS), a pyridoxine-dependent enzyme that, depending on the molecular variants, may be co-factor responsive. Elevated methionine is often used as the primary analyte to detect CBS deficiency (CBSD) on newborn screening (NBS), but is limited by increased detection of other biochemical disorders with less clear clinical significance such as methionine aminotransferase (MAT) I/III heterozygotes. Our state has implemented a two-tier NBS algorithm for CBSD that successfully reduced the number of MATI/III heterozygotes, yet effectively detected a mild, co-factor responsive form of CBSD. After initial diagnosis, newborns with CBSD often undergo a pyridoxine challenge with high-dose pyridoxine to determine responsiveness. Here we describe our NBS-identified patient with a mild form of pyridoxine responsive CBSD who developed respiratory failure and rhabdomyolysis consistent with pyridoxine toxicity during a pyridoxine challenge. This case highlights the need for weight-based dosing and duration recommendations for pyridoxine challenge in neonates.

Telomere length and mtDNA copy number in human cystathionine ß-synthase deficiency.

Telomere shortening and mitochondrial DNA (mtDNA) copy number are associated with human disease and a reduced life span. Cystathionine ß-synthase (CBS) is a housekeeping enzyme that catalyzes the first step in metabolic conversion of homocysteine (Hcy) to cysteine. Mutations in the CBS gene cause CBS deficiency, a rare recessive metabolic disease, manifested by severe hyperhomocysteinemia (HHcy) and thromboembolism, which ultimately reduces the life span. However, it was not known whether telomere shortening or mtDNA is involved in the pathology of human CBS deficiency. We quantified leukocyte telomere length (TL), mtDNA copy number, and plasma Hcy levels in CBS-/- patients (n = 23) and in sex- and age-matched unaffected CBS+/+ control individuals (n = 28) 0.08-57 years old. We found that TL was significantly increased in severely HHcy CBS-/- female patients but unaffected in severely HHcy CBS-/- male patients, relative to the corresponding CBS+/+ controls who had normal plasma Hcy levels. In multiple regression analysis TL was associated with CBS genotype in women but not in men. MtDNA copy number was not significantly affected by the CBS-/- genotype. Taken together, these findings identify the CBS gene as a new locus in human DNA that affects TL in women and illustrate a concept that a housekeeping metabolic gene can be involved in telomere biology. Our findings suggest that neither telomere shortening nor reduced mtDNA copy number contribute to the reduced life span in CBS-/- patients.

Cystathionine ß-synthase deficiency: different changes in proteomes of thrombosis-resistant Cbs-/- mice and thrombosis-prone CBS-/- humans.

Cystathionine ß-synthase (CBS)-deficient patients are prone to vascular thrombosis. In contrast, Cbs-/- mice show no abnormalities in blood coagulation. To identify molecular basis underlying these disparately different thrombotic phenotypes, we analyzed plasma proteomes of Cbs-/- vs. Cbs+/+ mice (8-month-old, 12/group, sex-matched) and CBS-/- vs. CBS+/+ humans (37 ± 7-year-old, 10-14/group, sex-matched) using label-free mass spectrometry. We identified 117 and 41 differentiating plasma proteins in Cbs-/- mice and CBS-/- humans, respectively. Twenty-one proteins were shared between CBS-/- humans and Cbs-/- mice, with sixteen changed in the opposite direction. Proteins involved in blood coagulation and complement/coagulation cascades represented a greater fraction of the differentiating proteins in CBS-/- patients (51%) than in Cbs-/- mice (21%). Top canonical pathways, identified by Ingenuity Pathways Analysis, such as LXR/RXR, FXR/RXR activation (- log[P-value] = 30-31) and atherosclerosis signaling (- log[P-value] = 10-11) were similarly affected in Cbs-/- mice and CBS-/- humans. The Coagulation System was affected stronger in CBS-/- humans than in Cbs-/- mice (- log[P-value] = 15 vs. 10, respectively) while acute phase response and complement system were affected stronger in Cbs-/- mice (- log[P-value] = 33 and 22, respectively) than in humans (- log[P-value] = 22 and 6, respectively). Other pathways, including IL-7 signaling and B cell development were affected only in Cbs-/- mice. Taken together, our findings suggest that differences in these processes, in particular in the Coagulation System, could account for the thrombotic phenotype in CBS-/- patients and the absence of thrombosis in Cbs-/- mice. Overall, our findings suggest that Cbs-/- mice have a better adaptive response to protect from prothrombotic effects of hyperhomocysteinemia than CBS-/- humans.

Analysis of Total Thiols in the Urine of a Cystathionine ß-Synthase-Deficient Mouse Model of Homocystinuria Using Hydrophilic Interaction Chromatography.

Homocysteine and related thiols (cysteine, cysteinylglycine, and glutathione) in the urine of a cystathionine ß-synthase (CBS)-deficient mouse model were quantified using hydrophilic interaction chromatography with fluorescence detection. Urine samples were incubated with tris(2-carboxyethyl) phosphine to reduce disulfide bonds into thiols. After deproteinization, thiols were fluorescently derivatized with ammonium 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate (SBD-F). Homocysteine, cysteine, cysteinylglycine, and glutathione in mouse urine were analyzed using an amide-type column with a mobile phase of acetonitrile/120 mM ammonium formate buffer (pH 3.0) (81:19). The developed method was well-validated. Thiol concentrations in the urine of CBS-wild type (-WT), -heterozygous (-Hetero), and -knockout (-KO) mice were quantified using the developed method. As expected, total homocysteine concentration in CBS-KO mice was significantly higher than that in CBS-WT and CBS-Hetero mice. The developed method shows promise for diagnoses in preclinical and clinical studies.

Memantine Protects From Exacerbation of Ischemic Stroke and Blood Brain Barrier Disruption in Mild But Not Severe Hyperhomocysteinemia.

Background Hyperhomocysteinemia is a risk factor for ischemic stroke; however, a targeted treatment strategy is lacking partly because of limited understanding of the causal role of homocysteine in cerebrovascular pathogenesis. Methods and Results In a genetic model of cystathionine beta synthase (CBS) deficiency, we tested the hypothesis that elevation in plasma total homocysteine exacerbates cerebrovascular injury and that memantine, a N-methyl-D-aspartate receptor antagonist, is protective. Mild or severe elevation in plasma total homocysteine was observed in Cbs+/- (6.1±0.3 µmol/L) or Cbs-/- (309±18 µmol/L) mice versus Cbs+/+ (3.1±0.6 µmol/L) mice. Surprisingly, Cbs-/- and Cbs+/- mice exhibited similar increases in cerebral infarct size following middle cerebral artery ischemia/reperfusion injury, despite the much higher total homocysteine levels in Cbs-/- mice. Likewise, disruption of the blood brain barrier was observed in both Cbs+/- and Cbs-/- mice. Administration of the N-methyl-D-aspartate receptor antagonist memantine protected Cbs+/- but not Cbs-/- mice from cerebral infarction and blood brain barrier disruption. Our data suggest that the differential effect of memantine in Cbs+/- versus Cbs-/- mice may be related to changes in expression of N-methyl-D-aspartate receptor subunits. Cbs-/-, but not Cbs+/- mice had increased expression of NR2B subunit, which is known to be relatively insensitive to homocysteine. Conclusions These data provide experimental evidence that even a mild increase in plasma total homocysteine can exacerbate cerebrovascular injury and suggest that N-methyl-D-aspartate receptor antagonism may represent a strategy to prevent reperfusion injury after acute ischemic stroke in patients with mild hyperhomocysteinemia.

Classical homocystinuria: From cystathionine beta-synthase deficiency to novel enzyme therapies.

Genetic defects in cystathionine beta-synthase (CBS), a key enzyme of organic sulfur metabolism, result in deficiency of CBS activity and a rare inborn error of metabolism called classical homocystinuria (HCU). HCU is characterized by massive accumulation of homocysteine, an intermediate of methionine metabolism, and multisystemic clinical symptoms. Current treatment options for HCU are very limited and often inefficient, partially due to a low patient compliance with very strict dietary regimen. Novel therapeutic approaches are needed to cope with the toxic accumulation of homocysteine and restoration of a healthy metabolic balance. Human CBS is a complex intracellular multimeric enzyme that relies on three cofactors (heme, pyridoxal-5'-phosphate and S-adenosylmethionine) for proper function. Engineering and chemical modification of human CBS yielded OT-58, a first-in-class enzyme therapy candidate for HCU. Pre-clinical testing of OT-58 showed its substantial efficacy in lowering plasma and tissue concentrations of homocysteine, improving metabolic balance and correcting clinical symptoms of HCU. In addition, OT-58 showed great safety and toxicity profile when administered to non-human primates. Overwhelmingly positive and extensive pre-clinical package propelled OT-58 into a first-in-human clinical trial, which started on January 2019. In a meantime, other enzyme therapies based on modified human cystathionine gamma-lyase or erythrocyte-encapsulated bacterial methionine gamma-lyase have shown efficacy in decreasing plasma homocysteine in HCU mice. In addition, gene therapy approaches using adenovirus or minicircle DNA have been evaluated in HCU. In this review, we summarize the current efforts developing novel therapies for HCU to address a high unmet medical need among HCU patients.

Cystathionine ß-synthase (CBS) deficiency suppresses erythropoiesis by disrupting expression of heme biosynthetic enzymes and transporter.

The reduced iron usage induced by the suppression of erythropoiesis is a major cause of the systemic iron overload in CBS knockout (CBS-/-) mice. However, the relevant mechanisms are unknown. Here, we examined changes in granulocyte/erythroid cell ratios, iron content, and expression of iron-metabolism proteins, including; two key enzymes involved in the heme biosynthetic pathway, ALAS2 (delta-aminolevulinate synthase 2) and FECH (ferrochelatase), a heme exporter from the cytosol and mitochondria, FLVCR (feline leukemia virus subgroup C cellular receptor) as well as EPO (erythropoietin), EPOR (erythropoietin receptor) and HIF-2α (hypoxia inducible factor-2 subunit α), in the blood, bone marrow or liver of CBS-/- (homozygous), CBS+/- (heterozygous) and CBS+/+ (Wild Type) mice. Our findings demonstrate that CBS deficiency can induce a significant reduction in the expression of ALAS2, FECH, FLVCR, HIF-2α, EPO, and EPOR as well as an increase in interleukin-6 (IL-6), hepcidin and iron content in the blood, bone marrow or liver of mice. We conclude that the suppression of erythropoiesis is mainly due to the CBS deficiency-induced disruption in the expression of heme biosynthetic enzymes and heme-transporter.

Serum Proteome Alterations in Human Cystathionine ß-Synthase Deficiency and Ischemic Stroke Subtypes.

Ischemic stroke induces brain injury via thrombotic or embolic mechanisms involving large or small vessels. Cystathionine ß-synthase deficiency (CBS), an inborn error of metabolism, is associated with vascular thromboembolism, the major cause of morbidity and mortality in affected patients. Because thromboembolism involves the brain vasculature in these patients, we hypothesize that CBS deficiency and ischemic stroke have similar molecular phenotypes. We used label-free mass spectrometry for quantification of changes in serum proteomes in CBS-deficient patients (n = 10) and gender/age-matched unaffected controls (n = 14), as well as in patients with cardioembolic (n = 17), large-vessel (n = 26), or lacunar (n = 25) ischemic stroke subtype. In CBS-deficient patients, 40 differentially expressed serum proteins were identified, of which 18 were associated with elevated homocysteine (Hcy) and 22 were Hcy-independent. We also identified Hcy-independent differentially expressed serum proteins in ischemic stroke patients, some of which were unique to a specific subtype: 10 of 32 for cardioembolic vs. large-vessel, six of 33 for cardioembolic vs. lacunar, and six of 23 for large-vessel vs. lacunar. There were significant overlaps between proteins affected by CBS deficiency and ischemic stroke, particularly the cardioembolic subtype, similar to protein overlaps between ischemic stroke subtypes. Top molecular pathways affected by CBS deficiency and ischemic stroke subtypes included acute phase response signaling and coagulation system. Similar molecular networks centering on NFκB were affected by CBS deficiency and stroke subtypes. These findings suggest common mechanisms involved in the pathologies of CBS deficiency and ischemic stroke subtypes.