A chronic GM(2) gangliosidosis variant with a HEXA splicing defect: Quantitation of HEXA mRNAs in normal and mutant fibroblasts

Over 72 mutations have been identified in the HEXA gene of which only four (T538C, A590C, G805A, and C1495T) are believed to cause a chronic form of Tay-Sachs disease (TSD). We identified a novel HEXA mutation (IVS7, -7 G --> A) leading to chronic TSD in a Canadian patient of English ancestry, The second allele in this patient was the exon 11 4-bp insertion mutation (del 1277TATC), which is the most frequent TSD allele in Ashkenazi Jews. The IVS7, -7 G --> A mutation introduces a new 3' splice acceptor Site 5 bp upstream of the normal intron 7 splice acceptor site, The mutation leads to reduction of steady-state levels of HEXA mRNA by more than 80%, Two mRNA species are produced by the IVS7, -7 G --> A allele; a normal nRNA species and an mRNA lacking exon 8, No mRNA species that was spliced at the upstream 3' splice acceptor site was detected. We used competitive PCR to quantitate mRNA species in fibroblasts obtained from this patient, We compared the amounts of three identified mRNA species to HEXA mRNA levels in cells from normal individuals and from individuals heterozygous for del 1277TATC. The steady-state level of HEXA mRNA in cells from a normal individual was 17.3 pg/mu g RNA, An individual heterozygous for del 1277TATC produced 8.7 pg of normal HEXA mRNA/mu g RNA. The HEXA mRNA species with the insertion mutation was present in patient cells at 4.8% of the level of normal HEXA nRNA in homozygous normal cells, In fibroblasts from the patient carrying the IVS7, -7 G --> A mutation, the steady-state level of exon 8-deleted HEXA mRNA was 5.9% the level of that produced by homozygous normal cells. The level of normal HEXA nRNA in this patient's cells was 10.4%.