Gene scanning by amplicon melting analysis and SNP detection by unlabeled probe melting analysis

Abstract

Chronic Granulomatous Disease (CGD) is an inherited disorder affecting immune system cells. The most common defects in CGD are mutations in CYBB that encodes the gp91-phox protein component of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase system in phagocytes. The syndrome associated with these mutations is called X-linked CGD and accounts for ~65% of CGD cases. The next most common mutation is a GT deletion in NCF1 that encodes the p47-phox protein of the NADPH oxidase system. This autosomal recessive defect is found in ~25% of CGD cases. High resolution melting analysis (HRMA) of amplicons was used to develop a mutation scanning assay for 13 CYBB exons and a control exon. HRMA of an unlabeled probe was used for a SNP detection assay for the GT deletion in NCF1. Specimen DNA was extracted on a MagNA Pure Compact and quantified on the NanoDrop 8000 spectrophotometer. For the mutation scanning assay, pre-coated 96-well PCR plates were prepared with M13 tailed CYBB exon specific primers using a Nanodrop Express.PCR was performed on a C1000 thermal cycler followed by HRMA on the LightScanner. Cycle sequencing was done only on those rare exons that contained variations. Sequence analysis was accomplished on a 16-capillary 3130xl Genetic Analyzer. For the autosomal recessive CGD assay, primers were designed to amplify an 80 base pair region covering the end of intron 1 and the start of exon 2 of NCF1 where the GT deletion is located. An unlabeled probe was designed to match the wild-type sequence. Asymmetric PCR and melting analysis were performed on the LightScanner 32 with appropriate controls. The mutation scanning assay allowed negative patient results to be reported in ~3 hours. Specimens identified with variations could then be cycle sequenced, purified, and sequenced by capillary electrophoreses. Following data analysis, the specific variations could be reported within 6 hours. If no variations were identified in the CYBB mutation scanning assay, the unlabeled probe assay could be run to determine the NCF1 GT deletion genotype and results were known within 2 hours. These assays have provided methods for the reliable and timely analysis of patient specimens without the need for extensive sequencing or the use of labeled oligos. Data from a number of clinical specimens are provided as examples.