Remethylation, B12, folate, biotin, thiamine & pyridoxine disorders Diana Ballhausen Paediatric Metabolic Unit, University Children’s Hospital Lausanne (CHUV), Switzerland Modified from Andrew Morris, Manchester Outline • Homocystinurias – Classical homocystinuria – Remethylation defects • Cerebral folate deficiencies • Biotin disorders • Thiamine disorders • Pyridoxine disorders Homocystinurias Classical Homocystinuria (HCU) • CBS deficiency 1/300’000 – Commoner in Ireland 1/65’000 & Qatar 1/2’000 Remethylation defects • MTHFR deficiency • Defects of methylcobalamin synthesis – Esp. CblC disease – Incidence unknown, 1/100’000 in small USA study – Commoner than classical HCU in Southern Europe All are autosomal recessive Tetrahydrofolate Methionine Methyl- Methionine Methyl cobalamin synthase group Methyl- Homocysteine tetrahydrofolate Serine Cystathionine B6 β-synthase Cystathionine Cysteine Remethylation defects Tetrahydrofolate Methionine Methyl group Methyl- Homocysteine tetrahydrofolate Classical homocystinuria Cystathionine Cysteine Classic homocystinuria – clinics –Learning difficulties –Dislocated lenses –Marfanoid habitus –Unexplained thromboses –Psychoses (esp. if falling IQ, poor response to treatment) Marfan Classical syndrome Homocystinuria • Skeletal changes • Skeletal changes • Lens dislocation • Lens dislocation • Learning difficulties • Seizures etc • Aortic dilatation • Thromboembolism /dissection • Symptomatic • Specific treatment treatment esp CVS Remethylation Defects Serine Glycine ATP Tetrahydro- Methionine folate S-Adenosyl- 5,10-Methylene- Methionine tetrahydrofolate Me-B12 Methyl group Adenosyl- homocysteine MTHFR 5- Methyl- tetrahydrofolate Homocysteine AMP Pathogenesis • Homocysteine toxicity • Lack of S-adenosylmethionine (methyl-group donor) Methylene tetrahydrofolate reductase (MTHFR) deficiency Infantile onset (few months of age) • developmental slowing, microcephaly • encephalopathy – lethargy, apnoea, seizures • hydrocephalus Late onset (rare) • ataxia • schizophrenia • thromboembolism MTHFR polymorphism • c.677C>T • Common (10% population are homozygous) • This is NOT MTHFR deficiency! • Causes mild hyper-homocysteinaemia (15-50 mmol/l, n<15) • Possibly a risk factor for vascular disease Treatment? • Folic acid & B12 (may not be needed) Intracellular cobalamin metabolism Lysosome cblG Transcobalamin Homocysteine Methionine MethylCobalamin OHCbl OHCbl cblE cblF & J Cbl 2+ cblC cblD OHCbl 3+ Cbl 2+ Cbl 2+ Hcy + MMA cblA & B Hcy Adenosyl MMA Methylmalonyl-CoA Succinyl-CoA Cobalamin Defects of methylcobalamin synthesis Infantile onset (commonest) • Neurological problems: poor feeding, seizures • Megaloblastic anaemia Later onset • Developmental delay • Motor or psychiatric symptoms Cobalamin C (CblC) disease Infantile onset (most common) • Poor feeding, vomiting • Hypotonia, seizures, coma • Poor vision (retinopathy, maculopathy) • Megaloblastic anaemia ± pancytopenia ± Metabolic acidosis ± Haemolytic uraemic syndrome ± Interstitial pneumonia Cobalamin C (CblC) disease Later onset • Developmental delay • Deteriorating school work • Confusion / psychiatric symptoms • Subacute combined degeneration of cord – Spastic paraplegia, incontinence • Thromboembolism ± Megaloblastic anaemia DD of homocystinurias • Amino acids - urine or plasma (separated promptly), plasma total homocysteine • Raised plasma homocysteine & methionine • Blood count • Serum vit. B12, folate • Methylmalonate in plasma or urine • Enzyme assay, complementation studies on fibroblasts (Cbl disorders) or mutation analysis Diagnosistic pathway for hypermethioninemia I. Baric et al. , JIMD 2017; 40:5-20. Diagnosistic pathway for remethylation disorders M. Huemer et al. , JIMD 2017; 40:21-48. Homocystinuria: Newborn screening • Blood methionine • Secondary testing of homocysteine Problem : methionine often normal in milder patients Outcomes – Normal, if good compliance Monitoring • Requires liquid blood, separated promptly • Target plasma levels – Free homocystine <10 mmol/l prevents major complications ( total homocysteine <80-100 ) – Ideal: total homocysteine <50-60 mmol/l but little evidence for this & often hard to achieve in pyridoxine-unresponsive pts or remethylation defects Classical homocystinuria - treatment Strategy in early diagnosed patients • Folate supplements, correct B12 deficiency • Trial of pyridoxine (100-200 mg/d in infants) – 10-50% respond, often only partially • If negative: low methionine diet • Add betaine , if control inadequate (or deteriorates) Dietary restriction Methionine Folic acid B12 Betaine Homocysteine Pyridoxine Cystathionine Cysteine Low methionine diet • Methionine-free amino acid mixture • Low protein foods • Vitamin & mineral supplements Problems • Diet & supplements relatively unpalatable • Introduction of diet harder after newborn period • Diet needed throughout life, but compliance deteriorates Betaine • Up to 150 mg/kg/d in 2x (max. 9 g/d) • Trimethylglycine, natural derivative of choline Dimethylglycine Methionine Betaine Homocysteine Methyltransferase Betaine Homocysteine • Lowers homocysteine • Methionine rises, cerebral oedema in 2 pts with methionine >1’000 mmol/l Betaine • Some dislike taste, bad compliance • Seldom achieves optimal levels as monotherapy • 1st line in countries without dietitian support – with low-protein diet • 1st line in remethylation disorders Remethylation Defects Treatment • Betaine 150-250 mg/kg/d – Lowers homocysteine & raises methionine • Folinic acid (in MTHFR deficiency) – Folate cannot enter brain • Hydroxocobalamin IM 1 mg daily (in Cbl defects) Outcomes • Significant handicap except for mild forms with prospective treatment Summary: Classical homocystinuria • Childhood onset: learning difficulties lens dislocation skeletal abnormalities • Later onset: thromboembolism • Treatment: pyridoxine (10-50% respond) low methionine diet betaine • Newborn screening improves outcome for pyridoxine-unresponsive patients Summary: Remethylation defects • Infantile onset – Feeding problems, hypotonia, seizures – Megaloblastic anaemia (cobalamin defects) • Later onset – Developmental delay – Motor or psychiatric symptoms • Treatment betaine folinic acid (esp. MTHFR deficiency) vitamin B 12 IM (cobalamin defects) • Outcomes disappointing Outline • Homocystinurias – Classical homocystinuria – Remethylation defects • Cerebral folate deficiencies • Biotin disorders • Thiamine disorders • Pyridoxine disorders Folate Deficiency • Folate: essential role in nucleic acid biosynthesis, one carbon and amino acid metabolism • Dietary intake from fruits and vegetables • Uptake at intestinal brush border • In blood: total folate in serum or plasma • In CSF: active form 5-methyltetrahydrofolate (5- MTHF), sample snap frozen and stored at -70°C, no rostro-caudal gradient , but age-related normal values (decrease with age) • Clinics: megaloblastic anaemia +/- neurology Cerebral Folate Deficiency: primary causes Disorders of folate metabolism: MTHFR = methylenetetrahydrofolate reductase ( most frequent ) DHFR = dihydrofolate reductase (extremely rare) MTHFS = 5-10-Methenyl-tetrahydrofolate synthase (link to purine metabolism) Disorders of folate transport: Fra = folate receptor alpha S. Pompe et al. , JIMD (2019) 42:655-672. Cerebral Folate Deficiency: secondary causes DHPR = dihydropteridine reductase AADC = aromatic L-amino acid decarboxylase S. Pompe et al. , JIMD (2019) 42:655-672. Disorders with low blood total folate and low CSF 5-MTHF Disorders of folate transport PCFT1 = proton-coupled folate transporter 1 Intestinal folate absorption and transport from blood to brain S. Pompe et al. , JIMD (2019) 42:655-672. Outline • Homocystinurias – Classical homocystinuria – Remethylation defects • Cerebral folate deficiencies • Biotin disorders • Thiamine disorders • Pyridoxine disorders Biotine disorders X X X 1. Biotinidase deficiency 2. Holocarboxylase synthetase deficiency: PCC = propionyl CoA carboxylase MCC = methylcrotonyl CoA carboxylase PC = pyruvate carboxylase ACC = acetyl CoA carboxylase Biotinidase deficiency • Disorder of biotin recycling • Manifestation after 3 months ( biotin reserve ) • Exemplary disease for newborn screening! • Skin eruptions, alopecia • Lactic acidosis, hyperventilation • Vomiting, dehydration • Leucopenia, abnormal T-lymphocyte function • Ataxia, hypotonia, psychomotor retardation • Hearing and/or visual loss • Treatment: life-long biotin (CAVE: compliance! ) – Potential interference with immunoassays! Holocarboxylase synthetase deficiency • Manifestation after hours/weeks • Vomiting, hypotonia, lethargy, seizures • Skin eruptions, alopecia • Lactic acidosis with ketosis, hyperammonemia • psychomotor retardation • Coma, death • Treatment: life-long biotin – Potential interference with immunoassays! Outline • Homocystinurias – Classical homocystinuria – Remethylation defects • Cerebral folate deficiencies • Biotin disorders • Thiamine disorders • Pyridoxine disorders Thiamine disorders • Thiamine (=vitamin B1): cofactor of many cellular processes, most importantly energy production • Humans: no thiamine biosynthesis • Most important dietary sources: whole grains, meat, eggs • Kidneys: regulate body thiamine homeostasis • Disorders of 3 thiamine transporters – Thiamine transporter-1: encoded by SLC19A2 – Thiamine transporter-2: encoded by SLC19A3 – Mitochondrial thiamine transporter: encoded by SLC25A19 SLC19A2 Deficiency = thiamine transporter 1 Progressive development of clinical triad: 1. Megaloblastic anaemia 2. Non-autoimmune diabetes