Identification of Recombinant Alleles Using Quantitative Real-Time PCR: Implications for Gaucher Disease

Abstract

Pseudogenes, resulting from duplications of functional genes, contribute to the functional complexity of their parental genes. The glucocerebrosidase gene (GBA), located in a gene-rich region on chromosome 1q 21, is mutated in Gaucher disease. The presence of contiguous, highly homologous pseudogenes for both GBA and metaxin 1 at this locus increases the likelihood of DNA rearrangement. We describe a facile method to identify and analyze recombinant alleles in patients with Gaucher disease. Genomic DNA from 20 patients with recombinant GBA alleles and five controls was evaluated to identify DNA rearrangements or copy number variation using six probes specific for either the GBA gene or pseudogene. Quantitative real-time PCR was performed on genomic DNA, and Southern blot analyses using HincII together with sequencing confirmed the real-time results. Both GBA fusions and duplications could be detected. Different sites of crossover were identified, and alleles resulting from gene conversion could be distinguished from reciprocal recombinant alleles. Quantitative real-time PCR is a sensitive and rapid method to detect fusions and duplications in patients with recombinant GBA alleles. This technique is more sensitive, faster, and cheaper than Southern blot analysis, and can be used in diagnostic laboratories, and to detect other recombinant alleles within the genome.