Molecular basis of very long chain acyl-CoA dehydrogenase deficiency in three Israeli patients: Identification of a complex mutant allele with P65L and K247Q mutations, the former being an exonic mutation causing exon 3 skipping

Very long chain acyl-CoA dehydrogenase (VLCAD) deficiency is a life-threate ning disorder of mitochondrial fatty acid beta-oxidation. We identified fou r novel mutations in three unrelated patients, All patients had the severe childhood form of VLCAD deficiency with early onset and high mortality. Imm unoblot analysis revealed that VLCAD protein was undetectable in patients 2 and 3, whereas normal-size VLCAD protein and an aberrant form of VLCAD (4k Da smaller) were detected in patient 1, As expected, null mutations were fo und in patients 2 and 3: patient 2 is homozygous for a frameshift mutation, del 4 bp at 798-801, and patient 3 is homozygous for a nonsense mutation 6 5C>A(S22X), Patient 1 was homozygous for a complex mutant allele containing two alterations, including a 194C>T transition (P65L) and 739A>C transvers ion (K247Q); in the case of P65L, the amino acid change does not reduce enz yme activity. However, the nucleotide change resulted in exon 3 skipping, w hereas the latter K247Q mutation had a drastic effect on enzyme activity. W e verified these events by in vivo splicing experiments and transient expre ssion analysis of mutant cDNAs. The P65L mutation locates 11 bases upstream of a splice donor site of intron 3. This is an example of an exonic mutati on which affects exon-splicing. Hum Mutat 15:430-438, 2000. (C) 2000 Wiley- Liss, Inc.