Molecular genetic characterisation of multiple acyl-CoA dehydrogenation deficiency

This PhD dissertation was accepted by the Faculty of Health Sciences of the University of Aarhus, and defended on February 4, 2004.

Official opponents: Anders Børglum, Allan Lund and DMSc, Associate Professor Lis Hasholt.

Tutor: DMSc, PhD, Professor Niels Gregersen and MSc, PhD, Associate Professor Brage Storstein Andresen.

Correspondence: Rikke Katrine Jentoft Olsen, Research Unit for Molecular Medicine, Skejby Sygehus, Brendstrupgaardsvej 100, DK-8200 Aarhus N.
E-mail: rikke.olsen@mmf.au.dk

Dan Med Bull 2004;51:228.

ABSTRACT

Multiple acyl-CoA dehydrogenation deficiency (MADD) is an inherited metabolic disorder characterized by impaired oxidation of fatty acids and some amino acids. Marked clinical heterogeneity of MADD has been reported with phenotypes varying from neonatal lethal forms with (type I) or without (type II) congenital anomalies to variable milder forms presenting with recurrent symptoms of fasting-induced hypoketotic hypoglycaemia, liver failure and/or muscular symptoms (type III). In most cases, the disorder is due to an autosomal recessively inherited deficiency of electron transfer flavoprotein (ETF) or ETF ubiquinone oxidoreductase (ETFQO), encoded by the ETFA, ETFB and ETFDH genes.

To examine whether the different clinical forms of MADD could be explained by different mutations in the ETF/ETFDH genes that result in different levels of residual ETF/ETFQO activity, we have investigated the molecular genetic basis for disease development in 30 patients representing the clinical spectrum of MADD.

We have elucidated the structures of the ETFA, ETFB and ETFDH genes, and established methods for cDNA- and genomic DNA-based mutation detection in the three genes. In the 30 patients investigated, we have identified 40 different mutant alleles, of which 31 have not been reported previously. Our characterisation of these mutations and correlation of mutation type with enzymatic and clinical phenotype allowed us to demonstrate a clear relationship between the nature of the ETF/ETFDH mutations and the severity of disease. Patients with severe MADD disease (type I and type II) are homozygous or compound heterozygous for ETF/ETFDH mutations that result in no or very low amount of residual enzyme activity, whereas patients with variant and milder forms of MADD (type III) possess at least one allele with a missense mutation, which may result in considerable residual enzyme activity.

Preliminary in vitro cell studies of missense mutations identified in patients with type III disease indicate that they are temperature sensitive. This suggests that the effect of the ETF/ETFDH genotype in patients with milder forms of MADD, in whom residual enzyme activity allows modulation of the enzymatic phenotype, may be influenced by environmental factors like cellular temperature (fever). The findings raise the important concept that the enzymatic phenotype can be amenable to modulation in vivo also, and thereby further contribute to the variant clinical phenotype observed in this group of patients.

Not least our study has contributed to the understanding of the molecular basis for the heterogeneous expression of MADD, it has also allowed development of DNA-based diagnosis for the disorder, which has been used to perform the first DNA-based prenatal diagnoses of MADD.