We have shown previously that a variant allele of the short-chain acyl-CoA dehydrogenase ( SCAD ) gene, 625G-->A, is present in homozygous form in 7% of control individuals and in 60% of 135 patients with elevated urinary excretion of ethylmalonic acid (EMA). We have now characterized three disease-causing mutations (confirmed by lack of enzyme activity after expression in COS-7 cells) and a new susceptibility variant in the SCAD gene of two patients with SCAD deficiency, and investigated their frequency in patients with elevated EMA excretion. The first SCAD-deficient patient was a compound heterozygote for two mutations, 274G-->T and 529T-->C. These mutations were not present in 98 normal control alleles, but the 529T-->C mutation was found in one allele among 133 patients with elevated EMA excretion. The second patient carried a 1147C-->T mutation and the 625G-->A polymorphism in one allele, and a single point mutation, 511C-->T, in the other. The 1147C-->T mutation was not present in 98 normal alleles, but was detected in three alleles of 133 patients with elevated EMA excretion, consistently as a 625A-1147T allele. On the other hand, the 511C-->T mutation was present in 13 of 130 and 15 of 67 625G alleles, respectively, of normal controls and patients with elevated EMA excretion, and was never associated with the 625A variant allele. This over-representation of the haplotype 511T-625G among the common 625G alleles in patients compared with controls was significant ( P < 0.02), suggesting that the allele 511T-625G-like 511C-625A-confers susceptibility to ethylmalonic aciduria. Expression of the variant R147W SCAD protein, encoded by the 511T-625G allele, in COS-7 cells showed 45% activity at 37 degrees C in comparison with the wild-type protein, comparable levels of activity at 26 degrees C, and 13% activity when incubated at 41 degrees C. This temperature profile is different from that observed for the variant G185S SCAD protein, encoded by the 511C-625A allele, where higher than normal activity was found at 26 and 37 degrees C, and 58% activity was present at 41 degrees C. These results corroborate the notion that the 511C-625A variant allele is one of the possible underlying causes of ethylmalonic aciduria, and suggest that the 511C-->T mutation represents a second susceptibility variation in the SCAD gene. We conclude that ethylmalonic aciduria, a commonly detected biochemical phenotype, is a complex multifactorial/polygenic condition where, in addition to the emerging role of SCAD susceptibility alleles, other genetic and environmental factors are involved.

Research Unit for Molecular Medicine Faculty of Health Sciences and Aarhus University Hospital Skejby Sygehus 8200 Aarhus N Denmark

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