Detection of Large Gene Rearrangements in X-linked Genes by Dosage Analysis: Identification of Novel α-Galactosidase A (GLA) Deletions Causing Fabry Disease

For most Mendelian disorders, targeted genome sequencing is an effective method to detect causative mutations. However, sequencing PCR-amplified exonic regions and their intronic boundaries can miss large deletions or duplications and mutations that lead to PCR failures in autosomal dominant disorders and in heterozygote detection for X-linked diseases. Here, a method is described for detecting large (450 bp) deletions/duplications in the X-linked a-galactosidase A (GLA) gene, which cause Fabry disease. Briefly, multiplex PCR mixtures were designed to amplify each GLA exon and an unrelated internal control exon to normalize GLA exonic amplicon peak heights. For each normalized GLA amplicon, the normal control female to male peak-height ratios were 1.8 to 2.2 (expected 2.0), whereas the expected ratios for deletions or duplications would be similar to 1.0 or 3.0, respectively. Using this method, three novel deletions, c.369+3_547+954del4096insT, c.194+2049_369+773del2619insCG, and c.207_369+651del814ins231, were detected in unrelated women with signs and/or symptoms suggestive of Fabry disease, but no sequencing-detectable mutations. The deletions were confirmed by sequencing their respective GLA RT-PCR products. This method can identify gene rearrangements that may be cryptic to genomic DNA sequencing and can be readily adapted to other X-linked or autosomal dominant genes. Hum Mutat 32:688-695, 2011. (C) 2011 Wiley-Liss, Inc.