ABERRANTLY SPLICED MESSENGER-RNAS OF THE 3-HYDROXY-3-METHYLGLUTARYL COENZYME-A LYASE (HL) GENE WITH A DONOR SPLICE-SITE POINT MUTATION PRODUCE HEREDITARY HL DEFICIENCY

A novel point mutation in the 3-hydroxy-3-methyl glutaryl coenzmne A l yase gene was found in a Turkish patient with homozygous 3-hydroxy-3-m ethylglutaric acidemia. Amplification by RT-PCR of the mRNA using six different pairs of oligonucleotides produced no differences in four of the fragments amplified with respect to the control, but generated tw o fragments of different size. One was representative of a deletion of 126 bp and the other of an insertion of 78 bp. These abnormal mRNAs r esulted from a G --> G transversion at the nucleotide + 1 of an intron , which changed the invariant GT dinucleotide of the 5' donor splice s ite. This was associated with the occurrence of an alternative splicin g, which led to the skipping of the whole exon of 126 bp, and also wit h the activation of one cryptic donor splice site in the same intron. These aberrant spliced mRNAs are predicted to encode two abnormal HMG- CoA lyase proteins: the first results in a protein with an internal de letion of 42 amino acids, whose enzyme activity is largely abolished, as the catalytic site was completely removed; the second contains 17 m issense amino acids that precede a stop codon. Northern blot analysis showed that the overall content of these aberrantly spliced mRNAs in p roband fibroblasts was the same as that found in control fibroblasts. However, hardly any transcript was observed corresponding to the inser ted mutated mRNA when it was examined by a specific probe. To quantify the relative proportion of the two mRNAs. a quantitative RT-PCR (the DNA-mimic PCR reaction) was carried out. Results show that the proport ion of the inserted mRNA with respect to the deleted mRNA is only 1.2% . The father, mother, and two brothers of the proband were heterozygou s in the G --> G mutation in the + 1 nucleotide of the intron consider ed, while the two alleles of another brother were free of the mutation .