INVESTIGADORES
CASALE Cesar Horacio
artículos
Título:
Aberrantly spliced mRNAs of the 3-hydrixy-3-methylglutaryl coenzime A lyase (HL) gene with a donor splice-site point mutation produce hereditary HL deficiency
Autor/es:
BUESA C; PIE J; BARCELO A; MASCARO C; CASALS N; CASALE CH; HARO D; DURAN M; SMEITINK, J. A. M.; HEGARDT FG
Revista:
JLR PAPERS IN PRESS
Referencias:
Año: 1996 vol. 37 p. 2420 - 2432
ISSN:
0022-2275
Resumen:
A novel point mutation in the 3-hydroxy-3-methylglutaryl
coenzyme A lyase gene was found in a Turkish patient
with homozygous 3-hydroxy-3-methylglutaric 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
two 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 resulted from a G + C transversion
at the nucleotide +1 of an intron, which changed the
invariant GT dinucleotide of the 5 donor splice site. This was
associated with the occurrence of an alternative splicing,
which led to the skipping of the whole exon of 126 bp, and
also with the activation of one cryptic donor splice site in the
same intron. These aberrant spliced mRNAs are predicted to
encode two abnormal HMGCoA lyase proteins: the first results
in a protein with an internal deletion of42 amino acids,
whose enzyme activity is largely abolished, as the catalytic site
was completely removed; the second contains 17 missense
amino acids that precede a stop codon. Northern blot analysis
showed that the overall content of these aberrantly spliced
mRNAs in proband fibroblasts was the same as that found in
control fibroblasts. However, hardly any transcript was observed
corresponding to the inserted 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 proportion of the inserted mRNA with respect to the deleted
mRNA is only 1.2%. The father, mother, and two brothers
of the proband were heterozygous in the G -+ C mutation
in the + 1 nucleotide of the intron considered, while the two
alleles of another brother were free of the mutation.-