Evaluation of DHPLC analysis in mutational scanning of Notch3, a gene with a high G C content英文电子书.pdf

518 ESCARY ET AL. HUMAN MUTATION 16:518–526 (2000) METHODS 1 2 3 1 Jean-Louis Escary, Michaelle Cécillon, Jacqueline Maciazek, Mark Lathrop, Elisabeth Tournier-Lasserve,2,3 and Anne Joutel2,3* 1Centre National de Génotypage, Evry, France 2Laboratoire de Cytogénétique, Hôpital Lariboisière, Paris, France 3INSERM EPI 99-21, Faculté de Médecine Lariboisière, Paris, France Communicated by Jean-Claude Kaplan Notch3 mutations cause CADASIL, an increasingly recognized cause of subcortical ischemic stroke and vascular dementia in human adults. In the absence of any specific diagnostic criteria, CADASIL diagnosis is based on mutational scanning of Notch3, which is a large gene composed of 33 exons with a high G-C content. In this study we examined the sensitivity of denaturing high performance liquid chromatography (DHPLC). First we established the theoretical opti- mal parameters, then we examined a large collection of amplicons in which we had previously identified distinct pathogenic mutations or polymorphisms. We further performed Notch3 mu- tational scanning in five patients suspected of CADASIL diagnosis in which previous scanning, including SSCP and heteroduplexes analysis, failed to detect any pathogenic mutation. DHPLC resolved 97% of mutations previously detected by sequencing and allowed identification of two novel pathogenic mutations: R607C and F984C. These data indicate that DHPLC is a sensitive screening method particularly suitable for epidemio-genetic screening of CADASIL. Hum Mutat 16:518–526, 2000. © 2000 Wiley-Liss, Inc. KEY WORDS: CADASIL; Notch3; DHPLC; cysteine; G-C content; stroke, subcortical ischemic; demen-