title: Applying and testing the conveniently optimized enzyme mismatch cleavage method to clinical DNA diagnosis.
abstract: 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.
authors.affiliation: Research Center for Child Mental Development, Kanazawa University, Ishikawa, Japan. niida@staff.kanazawa-u.ac.jp
authors.forename: Yo
authors.initials: Y
authors.lastname: Niida
authors.forename: Mondo
authors.initials: M
authors.lastname: Kuroda
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Mitani
authors.forename: Akiko
authors.initials: A
authors.lastname: Okumura
authors.forename: Ayano
authors.initials: A
authors.lastname: Yokoi
mesh_terms: D001168:Arthritis / Q000175:diagnosis* / Q000235:genetics; D001483:Base Sequence; D001681:Biological Assay; D002675:Child, Preschool; D038921:Coffin-Lowry Syndrome / Q000175:diagnosis* / Q000235:genetics; D024043:Collagen Type II / Q000235:genetics; D003240:Connective Tissue Diseases / Q000175:diagnosis* / Q000235:genetics; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005090:Exodeoxyribonucleases / Q000235:genetics; D005091:Exons; D006319:Hearing Loss, Sensorineural / Q000175:diagnosis* / Q000235:genetics; D006801:Humans; D007223:Infant; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D009154:Mutation; D009692:Nucleic Acid Heteroduplexes / Q000032:analysis*; D053484:RecQ Helicases / Q000235:genetics; D015203:Reproducibility of Results; D012163:Retinal Detachment / Q000175:diagnosis* / Q000235:genetics; D038744:Ribosomal Protein S6 Kinases, 90-kDa / Q000235:genetics; D012680:Sensitivity and Specificity; D016622:Silver Staining; D014898:Werner Syndrome / Q000175:diagnosis* / Q000235:genetics; D000071657:Werner Syndrome Helicase
chemical_list: C499177:COL2A1 protein, human; D024043:Collagen Type II; D009692:Nucleic Acid Heteroduplexes; D038744:Ribosomal Protein S6 Kinases, 90-kDa; C069330:ribosomal protein S6 kinase, 90kDa, polypeptide 3; D005090:Exodeoxyribonucleases; D004262:DNA Restriction Enzymes; D053484:RecQ Helicases; C098785:WRN protein, human; D000071657:Werner Syndrome Helicase