Human Molecular Genetics Advance Access originally published online on February 18, 2009
Human Molecular Genetics 2009 18(9):1612-1623; doi:10.1093/hmg/ddp079
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Protein misfolding is the molecular mechanism underlying MCADD identified in newborn screening
1 Department of Molecular Pediatrics, Children's Research Center, Dr. von Hauner Children's Hospital, Ludwig-Maximilians-University, Lindwurmstr. 4, Munich 80337, Germany 2 Department of Clinical Chemistry and Clinical Biochemistry, Ludwig-Maximilians-University, Munich 80336, Germany
* To whom correspondence should be addressed. Tel: +49 89 5160 2746; Fax: +49 89 5160 7792; Email: ania.muntau{at}med.uni-muenchen.de
Received January 13, 2009; Accepted February 16, 2009
Newborn screening (NBS) for medium-chain acyl-CoA dehydrogenase deficiency (MCADD) revealed a higher birth prevalence and genotypic variability than previously estimated, including numerous novel missense mutations in the ACADM gene. On average, these mutations are associated with milder biochemical phenotypes raising the question about their pathogenic relevance. In this study, we analyzed the impact of 10 ACADM mutations identified in NBS (A27V, Y42H, Y133H, R181C, R223G, D241G, K304E, R309K, I331T and R388S) on conformation, stability and enzyme kinetics of the corresponding proteins. Partial to total rescue of aggregation by co-overexpression of GroESL indicated protein misfolding. This was confirmed by accelerated thermal unfolding in all variants, as well as decreased proteolytic stability and accelerated thermal inactivation in most variants. Catalytic function varied from high residual activity to markedly decreased activity or substrate affinity. Mutations mapping to the β-domain of the protein predisposed to severe destabilization. In silico structural analyses of the affected amino acid residues revealed involvement in functionally relevant networks. Taken together, our results substantiate the hypothesis of protein misfolding with loss-of-function being the common molecular basis in MCADD. Moreover, considerable structural alterations in all analyzed variants do not support the view that novel mutations found in NBS bear a lower risk of metabolic decompensation than that associated with mutations detected in clinically ascertained patients. Finally, the detailed insight into how ACADM missense mutations induce loss of MCAD function may provide guidance for risk assessment and counseling of patients, and in future may assist delineation of novel pharmacological strategies.
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors.