title: Screening for Alagille syndrome mutations in the JAG1 and NOTCH2 genes using denaturing high-performance liquid chromatography.
abstract: Mutations in the JAG1 gene and the NOTCH2 gene cause Alagille syndrome. At present, however, genetic testing of Alagille syndrome is not commonly applied in clinical settings because the currently available assays are technically and financially demanding, mainly because of the size of the genes. In the present study, we optimized the highly sensitive and specific mutation scanning method automated denaturing high-performance liquid chromatography (DHPLC) to analyze the entire coding region of JAG1 and NOTCH2. The coding region was amplified by 69 primer pairs, all of which have the same cycling conditions, aliquoted on a 96-well format PCR plate. In this manner, all the exons were simultaneously amplified using a single block in a thermal cycler. We then wrote a computer script to analyze each segment of JAG1 and NOTCH2 by DHPLC in a serial manner using conditions that were optimized for each amplicon. The implementation of this screening method for JAG1 and NOTCH2 will help medical geneticists confirm their clinical impressions and provide accurate genetic counseling to the patients with Alagille syndrome and their families.
authors.affiliation: Department of Pediatrics, Keio University School of Medicine, Tokyo, Japan.
authors.forename: Hazuki
authors.initials: H
authors.lastname: Samejima
authors.forename: Chiharu
authors.initials: C
authors.lastname: Torii
authors.forename: Rika
authors.initials: R
authors.lastname: Kosaki
authors.forename: Kenji
authors.initials: K
authors.lastname: Kurosawa
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Yoshihashi
authors.forename: Koji
authors.initials: K
authors.lastname: Muroya
authors.forename: Nobuhiko
authors.initials: N
authors.lastname: Okamoto
authors.forename: Yoriko
authors.initials: Y
authors.lastname: Watanabe
authors.forename: Tomoki
authors.initials: T
authors.lastname: Kosho
authors.forename: Michiru
authors.initials: M
authors.lastname: Kubota
authors.forename: Osamu
authors.initials: O
authors.lastname: Matsuda
authors.forename: Miwa
authors.initials: M
authors.lastname: Goto
authors.forename: Kosuke
authors.initials: K
authors.lastname: Izumi
authors.forename: Takao
authors.initials: T
authors.lastname: Takahashi
authors.forename: Kenjiro
authors.initials: K
authors.lastname: Kosaki
mesh_terms: D016738:Alagille Syndrome / Q000175:diagnosis*; D000483:Alleles; D002135:Calcium-Binding Proteins / Q000235:genetics*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004252:DNA Mutational Analysis; D005091:Exons; D005820:Genetic Testing / Q000379:methods*; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics*; D000072100:Jagged-1 Protein; D008565:Membrane Proteins / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D051883:Receptor, Notch2 / Q000235:genetics*; D000072099:Serrate-Jagged Proteins
chemical_list: D002135:Calcium-Binding Proteins; D036341:Intercellular Signaling Peptides and Proteins; C000606273:JAG1 protein, human; D000072100:Jagged-1 Protein; D008565:Membrane Proteins; C497764:NOTCH2 protein, human; D051883:Receptor, Notch2; D000072099:Serrate-Jagged Proteins