heme-arginate and Homocystinuria

Acute intermittent porphyria (AIP) is a rare metabolic disease caused by mutations within the hydroxymethylbilane synthase gene. Previous studies have reported increased levels of plasma total homocysteine (tHcy) in symptomatic AIP patients. In this study, we present long-term data for tHcy and related parameters for an AIP patient cohort (n = 37) in different clinical disease-states. In total, 25 patients (68%) presented with hyperhomocysteinemia (HHcy; tHcy > 15 μmol/L) during the observation period. HHcy was more frequent in AIP patients with recurrent disease receiving heme arginate, than in nonrecurrent (median tHcy: 21.6 μmol/L; range: 10-129 vs median tHcy: 14.5 μmol/L; range 6-77). Long-term serial analyses showed a high within-person tHcy variation, especially among the recurrent patients (coefficient of variation: 16.4%-78.8%). HHcy was frequently associated with low blood concentrations of pyridoxal-5'-phosphate and folate, while cobalamin concentration and the allele distribution of the methylene-tetrahydrofolate-reductase gene were normal. Strikingly, 6 out of the 9 recurrent patients who were later included in a regime of givosiran, a small-interfering RNA that effectively reduced recurrent attacks, showed further increased tHcy (median tHcy in 9 patients: 105 μmol/L; range 16-212). Screening of amino acids in plasma by liquid-chromatography showed co-increased levels of methionine (median 71 μmol/L; range 23-616; normal <40), suggestive of acquired deficiency of cystathionine-β-synthase. The kynunerine/tryptophan ratio in plasma was, however, normal, indicating a regular metabolism of tryptophan by heme-dependent enzymes. In conclusion, even if HHcy was observed in AIP patients receiving heme arginate, givosiran induced an aggravation of the dysregulation, causing a co-increase of tHcy and methionine resembling classic homocystinuria.

Topics: Acetylgalactosamine; Adult; Arginine; Cystathionine beta-Synthase; Female; Folic Acid; Heme; Homeostasis; Homocysteine; Homocystinuria; Humans; Hydroxymethylbilane Synthase; Hyperhomocysteinemia; Male; Methionine; Middle Aged; Porphyria, Acute Intermittent; Pyridoxal Phosphate; Pyrrolidines; Young Adult

Classical homocystinuria is caused by mutations in the cystathionine β-synthase (CBS) gene. Previous experiments in bacterial and yeast cells showed that many mutant CBS enzymes misfold and that chemical chaperones enable proper folding of a number of mutations. In the present study, we tested the extent of misfolding of 27 CBS mutations previously tested in E. coli under the more folding-permissive conditions of mammalian CHO-K1 cells and the ability of chaperones to rescue the conformation of these mutations. Expression of mutations in mammalian cells increased the median activity 16-fold and the amount of tetramers 3.2-fold compared with expression in bacteria. Subsequently, we tested the responses of seven selected mutations to three compounds with chaperone-like activity. Aminooxyacetic acid and 4-phenylbutyric acid exhibited only a weak effect. In contrast, heme arginate substantially increased the formation of mutant CBS protein tetramers (up to sixfold) and rescued catalytic activity (up to ninefold) of five out of seven mutations (p.A114V, p.K102N, p.R125Q, p.R266K, and p.R369C). The greatest effect of heme arginate was observed for the mutation p.R125Q, which is non-responsive to in vivo treatment with vitamin B(6). Moreover, the heme responsiveness of the p.R125Q mutation was confirmed in fibroblasts derived from a patient homozygous for this genetic variant. Based on these data, we propose that a distinct group of heme-responsive CBS mutations may exist and that the heme pocket of CBS may become an important target for designing novel therapies for homocystinuria.

Topics: Animals; Arginine; Catalytic Domain; CHO Cells; Cricetulus; Cystathionine beta-Synthase; Female; Fibroblasts; Genetic Predisposition to Disease; Heme; Homocystinuria; Homozygote; Humans; Models, Molecular; Molecular Chaperones; Mutation; Phenotype; Protein Conformation; Protein Folding; Proteostasis Deficiencies; Structure-Activity Relationship; Substrate Specificity; Transfection