Detection of a rare oligo(A) repeat tract mutation (8As → 7As) in the sequence encoding the La/SS-B autoantigen

Several diseases are characterized by the presence of point mutations, which are amenable to molecular detection using a number of methods such as PCR. However, certain mutations are particularly difficult to detect due to factors such as low abundance and the presence of special (e.g., oligonucleotide repeat) sequences. The mutation 7A in the oligoA sequence of exon 7 of the gene encoding the La autoantigen is difficult to detect at the DNA level, and even at the RNA level, due to both its estimated low abundance and its differentiation from the wild-type 8A sequence. This article describes a technique in which amplification of the excess wild-type 8A La sequence is suppressed by a peptide nucleic acid (PNA) during a nested PCR step. Detection of the amplified 7A mutant form was then performed by simple electrophoresis following a final primer extension step with an infrared dye-labeled primer. This technique allowed us to detect the mutation in 3 of 7 individuals harboring serum immunoglobulin G (IgG) antibodies reactive with a neo-B cell epitope in the 7A mutant protein product. We propose that this method is a viable screening test for mutations in regions containing simple polynucleotide repeats.