title: Detection of germline deletions using real-time quantitative polymerase chain reaction in Japanese patients with von Hippel-Lindau disease.
abstract: Germline mutations of the VHL gene are responsible for VHL. Approximately 70% of VHL families display small intragenic mutations detectable by sequencing, whereas partial- or whole-gene deletions have been described in the majority of the remaining families. For such large deletions, complex genetic techniques other than sequencing might have to be used. In this study, we describe an RQ-PCR assay with TaqMan fluorescent probes to detect germline VHL deletions. The RQ-PCR primer/probe sets were designed for the three VHL coding exons as well as for the 5' promoter and 3' untranslated regions. The RQ-PCR assay for 30 normal and 10 known VHL deletion control samples demonstrated high sensitivity and specificity. We then screened 29 individuals from 19 classical VHL families (16 type 1, 2 type 2A, and one type 2B) and one PHEO family, as well as four solitary suspected cases, none displaying any sequence changes, for VHL deletions by the RQ-PCR assay. We detected germline deletions in 17 (89%) classical families including 15 type 1, one type 2A, and one type 2B. We also identified two mutation carriers and two non-carriers in our family cohort. The one PHEO family and four solitary cases did not display any deletion patterns. These findings indicated that the TaqMan-based RQ-PCR assay is a simple and potent technique for the rapid, sensitive, and specific investigation of VHL genetic diagnoses that could be used profitably before more complex large-deletion detection techniques.
authors.affiliation: Department of Urology and Molecular Genetics, Yokohama City University Graduate School of Medicine, 3-9 Fukuura, Kanazawa-ku, Yokohama 236-0004, Japan.
authors.forename: Keiko
authors.initials: K
authors.lastname: Hattori
authors.forename: Jun-ichi
authors.initials: J
authors.lastname: Teranishi
authors.forename: Catherine
authors.initials: C
authors.lastname: Stolle
authors.forename: Minoru
authors.initials: M
authors.lastname: Yoshida
authors.forename: Kei-ichi
authors.initials: K
authors.lastname: Kondo
authors.forename: Takeshi
authors.initials: T
authors.lastname: Kishida
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Kanno
authors.forename: Masaya
authors.initials: M
authors.lastname: Baba
authors.forename: Yoshinobu
authors.initials: Y
authors.lastname: Kubota
authors.forename: Masahiro
authors.initials: M
authors.lastname: Yao
mesh_terms: D020413:3' Untranslated Regions; D015139:Blotting, Southern; D015331:Cohort Studies; D004247:DNA / Q000097:blood; D005091:Exons; D005190:Family; D005456:Fluorescent Dyes / Q000378:metabolism; D005820:Genetic Testing / Q000379:methods*; D018095:Germ-Line Mutation; D006801:Humans; D007564:Japan; D008957:Models, Genetic; D016133:Polymerase Chain Reaction / Q000379:methods*; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000378:metabolism; D051794:Von Hippel-Lindau Tumor Suppressor Protein / Q000235:genetics* / Q000378:metabolism; D006623:von Hippel-Lindau Disease / Q000175:diagnosis* / Q000235:genetics / Q000378:metabolism
chemical_list: D020413:3' Untranslated Regions; D005456:Fluorescent Dyes; D012333:RNA, Messenger; D004247:DNA; D051794:Von Hippel-Lindau Tumor Suppressor Protein; C497446:VHL protein, human