CBS deficiency: clinical guidelines Andrew Morris, Viktor Kožich, Saikat Santra, Generoso Andria, Tawfeg Ben-Omran, Anupam Chakrapani, Ellen Crushell, Mick Henderson, Michel Hochuli, Martina Huemer, Miriam Janssen, Francois Maillot, Philip Mayne, Jenny McNulty, Tara Morrison, Siobhan O’Sullivan, Helene Ogier, Markéta Pavlíková, Isabel Tavares de Almeida, Sufin Yap, Henk Blom, Kimberly Chapman

Methyltetrahydrofolate Methionine Methyl group B12 Methyltetrahydrofolate Homocysteine Serine Cystathionine β-synthase Pyridoxal 5-phosphate Cystathionine Cysteine 2

CBS deficiency Adverse effects Brain Eye Skeleton Blood clotting Complete deficiency causes early-onset multisystem disease B6-responsive cases may be asymptomatic or only suffer adult-onset thromboembolism

CBS deficiency Problems Rare, maybe 3/million diagnosed globally, but many mild cases are undiagnosed Awareness is low Still many uncertainties about diagnosis & management Need evidence-based guidelines To help doctors with limited experience To highlight uncertainties & stimulate research

Evidence-based Guideline EHOD: European Network and Registry for Homocystinurias and Methylation Defects Team of paediatricians, adult physicians, biochemists, dietitians, a geneticist, a statistician Questions agreed, answers prepared & evidence graded (A: randomised controlled trials, B: cohort studies, C: case reports, D: expert opinion) Guideline reviewed (Wilcken, Leonard) Published in 2017, J Inherit Metab Dis 40:49-74, Open Access 5 years ago, the EU provided funding for EHOD, a European Network and Registry for Homocystinurias and Methylation Defects. One of the aims was to write guidelines for the management of classical homocystinuria. A team of specialists was assembled, the key questions were agreed and answers prepared with supporting evidence. Unfortunately, the evidence was generally poor: there were no randomised trials and most was based on case reports or expert opinion! The guidelines have now been published and they can be downloaded freely.

Clinical problems Eye Brain Lens dislocation Learning difficulties High Myopia Brain Learning difficulties Psychiatric & behaviour disorders Seizures Clumsiness Strokes Statements 1, 30 & 33: grades B-D

Clinical problems: Skeleton Knock knees Pigeon chest Tall Osteoporosis Scoliosis Statements 1 & 31: grades B-D

Clinical problems: Blood clots Deep Venous Thromboses Pulmonary Emboli Saggital sinus thrombosis Statements 1 & 31 grades B-D

Terminology Homocysteine Homocystine Cysteine Cystine Mixed disulfide At this stage, I need to say something about terminology. Homocysteine, spelt with an e before the i, has an SH or thiol group. This reacts readily with other thiol groups. Thus, two homocysteine molecules can react to form a dimer, which is known as homocystine. The amino acid, cysteine, also has a thiol group, allowing it to form a dimer, known as cystine. Alternatively, homocysteine & cysteine can react together to form a mixed disulphide. Mixed disulfide

TOTAL HOMOCYSTEINE Plasma proteins Hcy-SH FREE HOMOCYSTEINE Hcy-S-S-Cys HOMOCYSTEINE-CYSTEINE MIXED DISULFIDE Hcy-S-S-Hcy FREE HOMOCYSTINE Hcy-S-S-R OTHER DISULFIDES PROTEIN BOUND HOMOCYSTEINE Hcy │ S Cys Hcy │ S Cys If you measure the homocysteine level after breaking all the disulphide bonds, it is known as the total homocysteine concentration. If you measure the homocysteine level after breaking all the disulphide bonds, it is known as the total homocysteine concentration. Plasma proteins

Diagnosis of homocystinuria Plasma total homocysteine (tHcy) Free homocystine (in plasma or urine) unreliable tHcy may be normal in B6-responsive patients if taking multi-vitamins containing B6 (Plasma should be separated promptly) Statements 4-6, grades B-C

Other causes of high homocysteine Vitamin B12 or folate deficiencies Remethylation defects Renal failure CBS deficiency confirmed by mutation analysis or CBS assay (in plasma or fibroblasts) Statements 7-8, grades B-C

CBS deficiency: Newborn screening Blood methionine Outcomes Normal if good compliance Problem Some cases not detected Statement 12, grade C

Dietary restriction Methionine Betaine Homocysteine Pyridoxine Folic acid B12 Betaine Homocysteine Pyridoxine Cystathionine Cysteine 14

Treatment with Pyridoxine Presumed to stabilise the mutant CBS enzyme Low doses normalise homocysteine in some patients Others have partial response even on high doses Side effects Peripheral neuropathy with high doses (>900 mg/d) Recommended dose Lowest effective Up to 10mg/kg/d, max. 500mg/d Statements 17-18, grade D

Testing for B6 responsiveness We propose Test when patient stable, on normal protein intake Give folate & correct B12 if deficient Give pyridoxine 10mg/kg/day up to 500mg/day Continue for 6 weeks with at least 2 tHcy measurements before & on pyridoxine If detected by newborn screening, response very rare Pyridoxine 100mg/day for 2 weeks Statement 16, grade C-D

Low methionine diet Small, measured amounts of food containing methionine Methionine-free amino acid mixture (includes cystine but requirement unknown) Adequate energy from low protein foods most fruit & vegetables synthetic e.g. low-protein bread, biscuits, pasta Vitamin & mineral supplements Statements 21 & 24: grade D

Monitoring Statement 29, grade D Total Homocysteine Methionine Frequency depends on severity, treatment, age Blood spots can be used but most labs require liquid blood, separated promptly Also B12, folate Nutrition Bone density Statement 29, grade D

Homocysteine targets Normal IQ & no complications in Irish pts with free Homocystine <11 umol/l (Yap 1998 & 2001) We propose keeping tHcy <100 Safety of tHcy <120 based on few pts, up to 30yr Lower levels hard to achieve tHcy 120 fHcy 11 Statement 15, grade C

Low methionine diet Problems Diet & supplements relatively unpalatable Restrictions unpopular Expert dietitians required Introduction of diet harder after infancy Diet needed throughout life but compliance deteriorates

Betaine Dimethylglycine Methionine Betaine Homocysteine Roles Add to diet if unable to reach homocysteine targets Seldom achieves adequate control alone Side effects Taste, fishy odour at high doses Methionine rises, seldom toxic but brain swelling in few patients with methionine >1000 umol/l Statements 26 & 28, grades C-D

Minimise risk of thromboses During infections, travel, surgery & pregnancy Good hydration Compression stockings Early mobilisation Anticoagulants Contraceptives Avoid oestrogens (combined pill) Statements 36-39, grades C-D Statement 35, grade D

Future developments Few of our recommendations are controversial To be revised with evidence from EHOD register, mouse models etc Dietary treatment e.g. improved supplements, data on methionine content of foods, blood spot tHcy Novel treatments e.g. chaperones, ERT Primary need is earlier diagnosis Increased awareness Newborn screening