Complete Screening of 50 Patients with CHARGE Syndrome for Anomalies in the CHD7 Gene Using a Denaturing High-Performance Liquid Chromatography–Based Protocol : New Guidelines and a Proposal for Routine Diagnosis

Abstract

Ocular coloboma, heart malformation, choanal atresia, retardation of growth and/or development, genital hypoplasia, and ear anomalies associated with deafness (CHARGE) syndrome is a rare, usually sporadic, autosomal dominant disorder, caused by mutations within the CHD7 (chromodomain helicase DNA-binding protein 7) gene, in nearly 70% of cases. Because human CHD7 is relatively large (38 exons encoding a 300-kDa protein), genetic analysis requires cost-effective and time-consuming techniques. Herein, we propose an alternative screening method to quickly detect CHD7 mutations using mainly denaturing high-performance liquid chromatography. The entire coding region with exon-intron boundaries was amplified under the same experimental conditions. Each amplicon of the same CHD7 region was subjected to denaturing high-performance liquid chromatography analysis, and resulting chromatograms were compared within small series of patients. Because a CHD7 mutation differs generally from one patient to another, corresponding chromatograms exhibited a unique pattern that is significantly different from common polymorphisms. Only amplicons exhibiting a unique profile were subjected to DNA sequencing analysis. Intragenic rearrangements were investigated with only nine multiplex PCRs. In conclusion, using our protocol, we can quickly detect the right containing mutation amplicon and we provide a robust, rapid, and cheaper method to screen CHD7 microrearrangements or an entire deletion.