Applying and testing the conveniently optimized enzyme mismatch cleavage method to clinical DNA diagnosis

Establishing a simple and effective mutation screening method is one of the most compelling problems with applying genetic diagnosis to clinical use. Because there is no reliable and inexpensive screening system, amplifying by PCR and performing direct sequencing of every coding exon is the gold standard strategy even today. However, this approach is expensive and time consuming, especially when gene size or sample number is large. Previously, we developed CEL nuclease mediated heteroduplex incision with polyacrylamide gel electrophoresis and silver staining (CHIPS) as an ideal simple mutation screening system constructed with only conventional apparatuses and commercially available reagents. In this study, we evaluated the utility of CHIPS technology for genetic diagnosis in clinical practice by applying this system to screening for the COL2A1, WRN and RPS6KA3 mutations in newly diagnosed patients with Stickler syndrome (autosomal dominant inheritance), Werner syndrome (autosomal recessive inheritance) and Coffin-Lowry syndrome (X-linked inheritance), respectively. In all three genes, CHIPS detected all DNA variations including disease causative mutations within a day. Direct sequencing of all coding exons of these genes confirmed 100% sensitivity and specificity. We demonstrate high sensitivity, high cost performance and reliability of this simple system, with compatibility to all inheritance modes. Because of its low technology, CHIPS is ready to use and potentially disseminate to any laboratories in the world.