Lessons Learned from Inherited Metabolic Disorders of Sulfur-Containing Amino Acids Metabolism.

The metabolism of sulfur-containing amino acids (SAAs) requires an orchestrated interplay among several dozen enzymes and transporters, and an adequate dietary intake of methionine (Met), cysteine (Cys), and B vitamins. Known human genetic disorders are due to defects in Met demethylation, homocysteine (Hcy) remethylation, or cobalamin and folate metabolism, in Hcy transsulfuration, and Cys and hydrogen sulfide (H2S) catabolism. These disorders may manifest between the newborn period and late adulthood by a combination of neuropsychiatric abnormalities, thromboembolism, megaloblastic anemia, hepatopathy, myopathy, and bone and connective tissue abnormalities. Biochemical features include metabolite deficiencies (e.g. Met, S-adenosylmethionine (AdoMet), intermediates in 1-carbon metabolism, Cys, or glutathione) and/or their accumulation (e.g. S-adenosylhomocysteine, Hcy, H2S, or sulfite). Treatment should be started as early as possible and may include a low-protein/low-Met diet with Cys-enriched amino acid supplements, pharmacological doses of B vitamins, betaine to stimulate Hcy remethylation, the provision of N-acetylcysteine or AdoMet, or experimental approaches such as liver transplantation or enzyme replacement therapy. In several disorders, patients are exposed to long-term markedly elevated Met concentrations. Although these conditions may inform on Met toxicity, interpretation is difficult due to the presence of additional metabolic changes. Two disorders seem to exhibit Met-associated toxicity in the brain. An increased risk of demyelination in patients with Met adenosyltransferase I/III (MATI/III) deficiency due to biallelic mutations in the MATIA gene has been attributed to very high blood Met concentrations (typically >800 µmol/L) and possibly also to decreased liver AdoMet synthesis. An excessively high Met concentration in some patients with cystathionine ß-synthase deficiency has been associated with encephalopathy and brain edema, and direct toxicity of Met has been postulated. In summary, studies in patients with various disorders of SAA metabolism showed complex metabolic changes with distant cellular consequences, most of which are not attributable to direct Met toxicity.

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.

The clinical presentation of cobalamin-related disorders: From acquired deficiencies to inborn errors of absorption and intracellular pathways.

This review gives an overview of clinical characteristics, treatment and outcome of nutritional and acquired cobalamin (Cbl; synonym: vitamin B12) deficiencies, inborn errors of Cbl absorption and intracellular trafficking, as well as methylenetetrahydrofolate dehydrogenase (MTHFD1) and methylene tetrahydrofolate reductase (MTHFR) deficiencies, which impair Cbl-dependent remethylation. Acquired and inborn Cbl-related disorders and MTHFR deficiency cause multisystem, often severe disease. Failure to thrive, neurocognitive or psychiatric symptoms, eye disease, bone marrow alterations, microangiopathy and thromboembolic events are characteristic. The recently identified MTHFD1 defect additionally presents with severe immune deficiency. Deficient Cbl-dependent enzymes cause reduced methylation capacity and metabolite toxicity. Further net-effects of perturbed Cbl function or reduced Cbl supply causing oxidative stress, altered cytokine regulation or immune functions are discussed.

Ocular Pseudoexfoliation Syndrome Linkage to Cardiovascular Disease.

PURPOSE OF REVIEW: Pseudoexfoliation syndrome (PEX) is a common cause of open-angle glaucoma that is characterized by stress-induced elastic microfibrillopathy related to an accumulation of matrix metalloproteinases. The accumulation of matrix metalloproteinases increases deposition of protein substance within ocular structures and other organs including the heart. Many studies have associated the presence of cardiovascular disease with pseudoexfoliation syndrome, but much debate exists between studies in terms of significant relationships. The following meta-analysis aims to relate pseudoexfoliation syndrome with certain cardiovascular events and disorders. A thorough literature review was performed to acquire information concerning PEX patients with certain cardiovascular disorders. Diseases considered included myocardial infarction, ischemic heart disease, angina, congestive heart failure, cardiomyopathy, aortic aneurysm, hypertension, and homocystinuria. Patients without evidence of pseudoexfoliation disease were the controls of our study. Multiple forest plots were created to compile and analyze collected data for statistical comparison. RECENT FINDINGS: From a literature review, 18 studies were selected for our analysis. Cardiovascular disorders that had a statistically significant association (within a 95 % confidence interval) with PEX included ischemic heart disease, aortic aneurysms, and homocystinuria. The association between ischemic heart disease and PEX was statistically significant (p = 0.045). Myocardial infarction, chronic ischemic heart disease, angina, and hypertension did not show a correlation of relationship with the presence of pseudoexfoliation. Patients with PEX are prone to present with ischemic heart disease in addition to abdominal aortic aneurysms and homocystinuria. Patients that present with PEX should be screened for these detrimental cardiovascular disorders.

Molecular and biochemical investigations of patients with intermediate or severe hyperhomocysteinemia.

A discrepancy has been identified between numbers of expected and identified patients with homocystinuria due to cystathionine beta-synthase (CBS) deficiency. Patients homozygous for the frequent c.833T>C (p.I278T) are most often responsive to vitamin B6, and can present with a total-homocysteine (tHcy) <100 µM on a normal diet. In Denmark, patients with tHcy <100 µM are not routinely sequenced for CBS(2) mutations. This study investigated the prevalence of CBS mutations and the common methylenetetrahydrofolate reductase (MTHFR) c.677C>T polymorphism in patients with tHcy ≥ 50 µM and the association with clinical manifestations. We studied a cohort of patients with intermediate and severe hyperhomocysteinemia (tHcy ≥ 50 µM) determined between 1996 and 2011. Among the 413 eligible patients, 184 (45%) patients agreed to participate in the present follow-up study. A MTHFR(3)c.677TT genotype was found in 49% of the patients. Eight patients were found to have mutations in CBS(2). Of those, two were homozygous for c.833T>C (p.I278T), and four were compound heterozygous for c.833T>C. One c.833T>C (p.I278T) compound heterozygote was identified by lowering the threshold for sequencing from tHcy at 100 µM to 50 µM. The most prominent clinical presentation among patients with a CBS(2) mutation was thrombosis presenting at a median age of 25 years. In case of arterial or venous thrombosis without any explanation in individuals below 40 years, tHcy should be part of the thrombophilia screening. When tHcy is between 50 and 100 µM genotyping for the MTHFR(3) c.677TT is relevant, and when tHcy >100 µM CBS should be genotyped.

Clinical presentation, gene analysis and outcomes in young patients with early-treated combined methylmalonic acidemia and homocysteinemia (cblC type) in Shandong province, China.

OBJECTIVES: To estimate the incidence of MMA on newborn screening in Shandong province from May 2011 to May 2014 and summarize the clinical presentation, biochemical features, mutation analysis, and treatment regime of early-treated patients with cblC disease. METHODS: Between May 2011 and May 2014, 35,291 newborns were screened for MMA in Jinan maternal and Child Care Hospital, Shandong province. The levels of C3, C3/C2, methionine and tHcy were measured. Most patients received treatment with intramuscular hydroxocobalamin after diagnosis. Metabolic parameters, clinical presentation and mental development were followed up. RESULTS: Nine patients were identified among 35,291 by newborn screening, giving an estimated incidence of 1:3920 live births for MMA, and all were classified as cblC disease. Among them, five patients received treatment with intramuscular hydroxocobalamin and two patients did not receive any treatment. One patient died of metabolic crises triggered by infection at the age of 38 days. Seven different mutations (c.609G>A, c.455_457delCCC, c.394C>T, c.445_446insA, c.658_660delAAG, c.452A>G and IVS1+1G>A) were detected. The mutations (c.455_457delCCC and IVS1+1G>A) are novel. Five patients who received treatment had favorable metabolic response, with both reduction of urine MMA and tHcy and increase of methionine. We obtained 7 records of DQ assessment. The five patients who received treatment presented with developmental delay and obvious neurological manifestations. In two patients who did not receive any treatment, case 8 presented with severe mental retardation and developmental delay, while case 9 had nearly normal DQ values at the age of 1(1/12)years. CONCLUSION: Our study characterized variable phenotypes of neurodevelopment in early-treated cblC patients diagnosed on newborn screening. The long-term outcomes of cblC disease are unsatisfactory in spite of conventional treatment and improvement of biochemical abnormalities. Although the number of patients is too small, the information provided in this work is of value in highlighting possible genotype-phenotype correlation that influences outcomes in cblC disease by future studies.

High folic acid consumption leads to pseudo-MTHFR deficiency, altered lipid metabolism, and liver injury in mice.

BACKGROUND: Increased consumption of folic acid is prevalent, leading to concerns about negative consequences. The effects of folic acid on the liver, the primary organ for folate metabolism, are largely unknown. Methylenetetrahydrofolate reductase (MTHFR) provides methyl donors for S-adenosylmethionine (SAM) synthesis and methylation reactions. OBJECTIVE: Our goal was to investigate the impact of high folic acid intake on liver disease and methyl metabolism. DESIGN: Folic acid-supplemented diet (FASD, 10-fold higher than recommended) and control diet were fed to male Mthfr(+/+) and Mthfr(+/-) mice for 6 mo to assess gene-nutrient interactions. Liver pathology, folate and choline metabolites, and gene expression in folate and lipid pathways were examined. RESULTS: Liver and spleen weights were higher and hematologic profiles were altered in FASD-fed mice. Liver histology revealed unusually large, degenerating cells in FASD Mthfr(+/-) mice, consistent with nonalcoholic fatty liver disease. High folic acid inhibited MTHFR activity in vitro, and MTHFR protein was reduced in FASD-fed mice. 5-Methyltetrahydrofolate, SAM, and SAM/S-adenosylhomocysteine ratios were lower in FASD and Mthfr(+/-) livers. Choline metabolites, including phosphatidylcholine, were reduced due to genotype and/or diet in an attempt to restore methylation capacity through choline/betaine-dependent SAM synthesis. Expression changes in genes of one-carbon and lipid metabolism were particularly significant in FASD Mthfr(+/-) mice. The latter changes, which included higher nuclear sterol regulatory element-binding protein 1, higher Srepb2 messenger RNA (mRNA), lower farnesoid X receptor (Nr1h4) mRNA, and lower Cyp7a1 mRNA, would lead to greater lipogenesis and reduced cholesterol catabolism into bile. CONCLUSIONS: We suggest that high folic acid consumption reduces MTHFR protein and activity levels, creating a pseudo-MTHFR deficiency. This deficiency results in hepatocyte degeneration, suggesting a 2-hit mechanism whereby mutant hepatocytes cannot accommodate the lipid disturbances and altered membrane integrity arising from changes in phospholipid/lipid metabolism. These preliminary findings may have clinical implications for individuals consuming high-dose folic acid supplements, particularly those who are MTHFR deficient.

Visual outcome and incidence of glaucoma in patients with microspherophakia.

INTRODUCTION: A number of ocular complications have been reported in microspherophakia. The literature however is limited to small case reports and the incidence of these complications is largely unknown. Our study describes a series of patients who presented to our hospital from 1998 to 2008. MATERIAL AND METHODS: Data on the clinical and surgical findings of patients presented to us from 1998 to 2008 with microspherophakia were retrieved from the medical records and the results analyzed. RESULTS: Thirty-six eyes of 18 patients were reviewed. The mean age at presentation was 16±10 years. All patients had varying degrees of lenticular myopia with a mean of -11.07±5.03 D. Glaucoma developed in 16 eyes (44.4%). Half of them had high IOP at presentation. Despite medical and surgical management IOP remained high in five eyes at the last follow-up. Sixteen eyes (44.4%) required lensectomy for dislocated crystalline lens. Lensectomy did not have any impact on the intraocular pressures. Homocysteinuria was the most common systemic association noted. CONCLUSION: Microspherophakia is associated with a high incidence of lenticular myopia, subluxation of the crystalline lens and glaucoma. Management of glaucoma is difficult with the IOP remaining high in spite of combined medical and surgical management.

Cystathionine beta-synthase deficiency heralded by cerebral sinus venous thrombosis and stroke.

BACKGROUND: Elevated plasma homocysteine is a risk factor for arterial and venous thromboses in adults. Homocysteine is increased in cystathionine beta-synthase deficiency, a treatable amino acid metabolic disorder that may be missed on newborn screening placing children at risk of thrombosis and strokes. PATIENT: We present a 3-year-old girl with normal newborn screening for cystathionine beta-synthase deficiency who developed a symptomatic cerebral venous sinus thrombosis. Subsequent testing revealed marked hyperhomocystinemia and genetic testing confirmed cystathionine beta-synthase deficiency. CONCLUSIONS: Current newborn screening is limited in its ability to detect cystathionine beta-synthase deficiency and although postanalytical interpretation may provide increased sensitivity, a normal newborn screening result should not replace the importance of physician surveillance.

Changes in bone mineral density and body composition of children with well-controlled homocystinuria caused by CBS deficiency.

Homocystinuria due to cystathionine ß-synthase (CBS) deficiency is an inherited disorder of the metabolism of methionine. Clinical manifestations include mental retardation, dislocation of the optic lens, vascular lesions, arterial and venous thromboembolism, skeletal abnormalities, and osteoporosis. Most homocystinuria patients diagnosed in adulthood have severe osteoporosis, and homocystinuria is frequently mentioned as a cause of osteoporosis. Good control of plasma homocysteine may prevent or delay some of these complications. However, the effectiveness of bone mineral density (BMD) gain or fracture prevention has not been addressed. Here, we describe changes in BMD and body composition in 5 CBS deficiency patients who were diagnosed at young age and were managed with good metabolic control. We found that the BMD of each region was within the normal range. BMD gain was adequate and the patients had no significant change in skeletal morphology.

[Epidemiological study of the metabolic diseases with homocystinuria in Spain]. / Estudio epidemiológico de las enfermedades metabólicas con homocistinuria en España.

OBJECTIVES: To determine the prevalence of homocystinuria in Spain and to establish the measures and mechanisms to ensure its prevention, diagnosis and treatment. MATERIAL AND METHODS: A national cross-sectional survey was conducted by means of a questionnaire sent to 35 hospitals in which children and adult patients are treated. RESULTS: Using the questionnaires submitted by 25 physicians from 16 centres, 75 patients were identified: 41 transsulphuration defects (one deceased), 27 remethylation (six deaths) and 7 without a syndromic diagnosis. The age at diagnosis varied widely, and 18 cases had more than one sibling affected. The more severe clinical manifestations involved the patients with remethylation defects. There was a high percentage of cognitive impairment, followed by lens diseases. Almost half of the patients had neurological disorders. There was increased vascular involvement in CBS-deficient adults. The therapeutic options most used were, folic acid, hydroxycobalamin and betaine. CONCLUSIONS: In view of these results and especially the small number of CBS deficiencies detected, we conclude that there is a need to introduce newborn screening for classical homocystinuria and ensure implementation of an appropriate diagnostic workup in all patients at risk.

Isolated remethylation disorders: do our treatments benefit patients?

Deficiency of 5,10-methylenetetrahydrofolate reductase (MTHFR), the very rare methionine synthase reductase (CblE) and methionine synthase (CblG) defects, and the recently identified CblD-variant-1 defect are primary remethylation defects characterized by an isolated defect in methionine synthesis without methylmalonic aciduria. The clinical signs are mainly neurological, and hematological signs are seen in CblE, CblG, and CblD-variant-1 defects. Patients with neonatal or early-onset disease exhibit acute neurological distress. Infants and children have unspecific mental retardation, often with acquired microcephaly. Without appropriate therapy, they may experience acute or rapidly progressive neurological deterioration, which may be fatal. Adolescents and adults show normal development or mild developmental delay initially and then experience rapid neurological or behavioral deterioration. A few patients may have signs of subacute combined degeneration of the spinal cord. Adults may be asymptomatic or present with isolated thromboembolism. All patients with suspected remethylation disorders should receive emergency treatment with parenteral administration of hydroxocobalamin and folate supplements combined with betaine orally. The long-term treatment of CblE, CblG, and CblD-variant-1 defects consists of parenterally administered hydroxocobalamin and orally administered folate and betaine supplements, whereas patients with MTHFR deficiency require long-term oral folate and betaine supplements. Long-term oral methionine therapy should also be considered. Early treatment may lead to a favorable outcome with developmental recovery and prevention of further neurological deterioration. In contrast, most late-treated patients have severe and irreversible neuromotor impairments. Hematological abnormalities are easily corrected.

Cystathionine gamma-Lyase-deficient mice require dietary cysteine to protect against acute lethal myopathy and oxidative injury.

Cysteine is considered a nonessential amino acid in mammals as it is synthesized from methionine via trans-sulfuration. However, premature infants or patients with hepatic failure may require dietary cysteine due to a lack of cystathionine gamma-lyase (CTH), a key trans-sulfuration enzyme. Here, we generated CTH-deficient (Cth(-/-)) mice as an animal model of cystathioninemia/cystathioninuria. Cth(-/-) mice developed normally in general but displayed hypercystathioninemia/hyperhomocysteinemia though not hypermethioninemia. When fed a low cyst(e)ine diet, Cth(-/-) mice showed acute skeletal muscle atrophy (myopathy) accompanied by enhanced gene expression of asparagine synthetase and reduced contents of glutathione in livers and skeletal muscles, and intracellular accumulation of LC3 and p62 in skeletal myofibers; they finally died of severe paralysis of the extremities. Cth(-/-) hepatocytes required cystine in a culture medium and showed greater sensitivity to oxidative stress. Cth(-/-) mice exhibited systemic vulnerability to oxidative injury, which became more prominent when they were fed the low cyst(e)ine diet. These results reveal novel roles of trans-sulfuration previously unrecognized in mice lacking another trans-sulfuration enzyme cystathionine beta-synthase (Cbs(-/-)). Because Cbs(-/-) mice display hyperhomocysteinemia and hypermethioninemia, our results raise questions against the homocysteine-based etiology of CBS deficiency and the current newborn screening for homocysteinemia using Guthrie's method, which detects hypermethioninemia.

[Present role of homocysteine in clinical medicine]. / Homocisteína en la clínica humana.

There has been a great interest in the last decades about the clinical significance of elevated total plasma homocysteine (tHcy), and especially its possible association with an increased cardiovascular risk. Measurement of tHcy is clearly indicated when homocystinuria is suspected in young or adult patients (in the presence of a severe, atypical or progressive myopia with ectopia lentis and/or venous thromboembolism and/or severe, premature or atypical atherosclerotic vascular disease) and in the evaluation of vitamin B12 and/or folic acid deficiencies. The current evidence does not support either the screening measurement of tHcy or the treatment with vitamin B12 and/or folic acid supplementation in patients with cardiovascular disease. It is important to remember that it remains to be proved whether the long-term administration of folic acid at pharmacological doses is safe.

Homocystinuria due to cystathionine beta synthase deficiency.

A two year-old male child presented with cutis marmorata congenita universalis, brittle hair, mild mental retardation, and finger spasms. Biochemical findings include increased levels of homocysteine in the blood-106.62 micromol/L (normal levels: 5.90-16 micromol/L). Biochemical tests such as the silver nitroprusside and nitroprusside tests were positive suggesting homocystinuria. The patient was treated with oral pyridoxine therapy for three months. The child responded well to this therapy and the muscle spasms as well as skin manifestations such as cutis marmorata subsided. The treatment is being continued; the case is reported here because of its rarity. Homocysteinuria arising due to cystathionine beta-synthase (CBS) deficiency is an autosomal recessive disorder of methionine metabolism that produces increased levels of urinary homocysteine and methionine It manifests itself in vascular, central nervous system, cutaneous, and connective tissue disturbances and phenotypically resembles Marfan's syndrome. Skin manifestations include malar flush, thin hair, and cutis reticulata / marmorata.

[Disruption of amino acid metabolism in astrocyte and neurological disorders--possible implication of abnormal glia-neuron network in homocystineuria].

CBS is a vitamin B6-dependent transsulfuration enzyme needed to synthesize cysteine from methionine, catalyzing the condensation of serine with homocysteine to form cystathionine. A deficiency of CBS causes homocystinuria (MIM 236200), one of the most prevalent inborn errors, characterized by mental retardation, seizures, psychiatric disturbances, skeletal abnormalities and vascular disorders. Patients with CBS deficiency exhibit a major biochemical abnormality, hyperhomocysteinemia (HHcy), a condition associated with highly elevated plasma homocysteine levels. HHcy is recognized as a risk factor for several neurological diseases, such as cognitive impairment, dementia and Alzheimer's disease. Although the link between CBS deficiency and homocystinuria was first described over 40 years ago and mental retardation was the first clinical feature of the disease to be classified, very little is known about the role of CBS in the CNS. Here we show the regional and cellular distribution of CBS in the adult and developing mouse brain. In the adult mouse brain, CBS was expressed ubiquitously, but most intensely in the cerebellar molecular layer and hippocampal dentate gyrus. Immunohistochemical analysis revealed that CBS is preferentially expressed in cerebellar Bergmann glia and in astrocytes throughout the brain. At early developmental stages, CBS was expressed in neuroepithelial cells in the ventricular zone, but its expression changed to radial glial cells and then to astrocytes during the late embryonic and neonatal periods. Moreover, CBS was significantly accumulated in reactive astrocytes in the hippocampus after kainic acid-induced seizures, and cerebellar morphological abnormalities were observed in CBS-deficient mice. These results support the role of CBS in the development and maintenance of the CNS, and suggest that radial glia/astrocyte dysfunction might be involved in the complex neuropathological features associated with abnormal homocysteine metabolism.

[Hyperhomocysteinemia in chronic renal failure.]. / L'iperomocisteinemia nell'insufficienza renale cronica: aspetti clinici, nutrizionali e tossicità.

Chronic renal failure (CRF) is frequently associated with increased plasma levels of homocysteine (Hcy), an amino acid that can be considered a new uremic toxin according to recent evidence. Studies on Hcy described first homocystinuria, an inherited disease characterized by high plasma Hcy levels and premature cardiovascular disease, resulting in high mortal-ity rates. Hyperhomocysteinemia was then shown to be associated with cardiovascular events both in the general population and in CRF patients. Hcy is a sulfur amino acid derived from dietary methionine, an essential amino acid. Methionine is condensed with ATP to form S-adenosylmethionine (AdoMet), the universal methyl donor in transmethylation reactions. The AdoMet demethylated product is S-adenosylhomocysteine (AdoHcy), which is the direct precursor of Hcy in vivo. Hcy is toxic for the endothelium, it enhances vascular smooth muscle cell proliferation, increases platelet aggregation, and acts on the coagulation cascade and fibrinolysis. Several mechanisms have been discussed to explain Hcy toxicity. Hcy levels increase as renal function declines and progresses to ESRD; the causes of hyperhomocysteinemia are still unclear. Studies in humans show that renal metabolic extraction depends on renal plasma flow; in addition, an alteration of the extrarenal metabolic clearance, depending on uremic toxins, may occur. Among the consequences of hyperhomocysteinemia in renal failure are: impaired protein methylation, with altered protein repair processes; DNA hypomethylation, with an alteration in the allelic expression of genes regulated through methylation; and protein homocysteinylation. Further, this review is dealing with the 'reverse epidemiology' issue, outlining also the main Hcy-lowering strategies.