Background: Mutations at splice junctions causing exon skipping are uncommon compared to exonic mutations, and two intronic mutations causing an aberrant phenotype have rarely been reported. Despite the high number of functional ABCA1 mutations reported to date, splice variants have been reported infrequently. We screened DNA from a 41 year-old male with low HDL-C (12 mg/dL [0.31 mmol/L]) and a family history of premature coronary heart disease (CHD) using polymerase chain reaction single-strand conformation polymorphism (SSCP) analysis.
Methods: Family members with low levels of HDL-C (n = 6) were screened by SSCP for mutations in ABCA1. Samples with altered SSCP patterns were sequenced directly using either an ABI 3700 or ABI3730Xl DNA Analyzer. To screen for splicing defects, cDNA was isolated from the proband's RNA and was sequenced as above. A series of minigenes were constructed to determine the contribution of normal and defective alleles.
Results: Two novel splice variants in ABCA1 were identified. The first mutation was a single base pair change (T->C) in IVS 7, 6 bps downstream from the exon7/intron7 junction. Amplification of cDNA and allelic subcloning identified skipping of Exon 7 that results in the elimination of 59 amino acids from the first extracellular loop of the ABCA1 protein. The second mutation was a single base pair change (G->C) at IVS 31 -1, at the intron/exon junction of exon 32. This mutation causes skipping of exon 32, resulting in 8 novel amino acids followed by a stop codon and a predicted protein size of 1496 AA, compared to normal (2261 AA). Bioinformatic studies predicted an impact on splicing as confirmed by in vitro assays of constitutive splicing.
Conclusion: In addition to carnitine-acylcarnitine translocase (CACT) deficiency and Hermansky-Pudlak syndrome type 3, this represents only the third reported case in which 2 different splice mutations has resulted in an aberrant clinical phenotype.