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
C. Buesa et al., 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, Journal of lipid research, 37(11), 1996, pp. 2420-2432
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
.