Molecular characterization and investigation of pathomechanisms of methylmalonic aciduria: towards novel therapies

Abstract

Methylmalonic acidurias represent a group of rare inborn errors of metabolism caused by deficient activity of the mitochondrial enzyme methylmalonyl-CoA mutase (MUT). Deficiency of MUT results either from defects of the MUT apoenzyme itself, which is encoded by the MUT gene, or from defects of the intracellular synthesis of its cofactor, 5-deoxyadenosylcobalamin, and leads to the accumulation of methylmalonic acid and other toxic metabolites. Methylmalonic aciduria (MMAuria) typically presents in early infancy with life-threatening metabolic decompensations. Most surviving patients show failure to thrive and develop chronic renal failure linked to tubulointerstitial nephritis. Moreover, approximately half of them suffer from severe neurological impairment such as an extrapyramidal movement disorder. Although the symptoms observed in MMAuria are thought to be the result of the accumulation of toxic intermediary products, knowledge concerning pathomechanisms is scarce, and the evidence base and efficacy of current treatment strategies is not satisfactory. A fundamental limitation for pathophysiological studies is that durable animal models of MMAuria have not been established, i.e. full knockout mice display neonatal lethality. Given the severe phenotypic and pathological outcomes of MMAuria there is an imminent need to explore the pathomechanisms of MMAuria and search for novel therapeutic approaches. In this PhD thesis, this problem is tackled by 1) characterizing a cohort of MMAuria patients regarding their mutations and biochemical properties; 2) investigating functional consequences of MUT missense mutations on enzyme activity, cofactor binding, structural protein integrity, and thermal stability; 3) investigating the effect of the cobalamin cofactor of MUT and of screening-derived small molecule chaperones on MUT protein stability and enzymatic activity; and 4) validation and characterization of novel conditional Mut knock-out and constitutive Mut knock-in mice in order to study the recapitulation of MMAuria in these models. The analysis of MMAuria patients revealed a very diverse cohort with regard to mutations and mutation types, enzymatic and biochemical properties, and clinical phenotype. Further detailed analysis of 23 missense mutations derived from this cohort, allowed an in-depth categorization of these mutations into various classes of biochemical defects, identifying specific missense mutations which were judged to be of value in pursuing the aims of two follow-up studies: i. Identification of missense mutations that produce an unstable protein allowed addition of MMAuria to the list of misfolding disorders. We established MUT as a suitable candidate for pharmacological chaperone screening in which identified small molecules that can rescue unstable MUT protein via stabilization. ii. A missense allele which correlates to low residual enzyme activity and to an intermediate MMAuria clinical phenotype was selected in order to generate a novel knock-in mouse model which was successfully validated as an accurate model recapitulating human MMAuria and which may be further utilized as a tool in future studies on pathomechanisms and treatment of MMAuria.