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. (Cancer Sci 2006; 97: 400-405) V HL (MIM 193300) is an autosomal dominantly inherited disorder characterized by a predisposition to multiple neoplastic lesions, including retinal angiomas, central nervous system hemangioblastomas, pancreatic tumors, clear-cell renal carcinomas, PHEO, endolymphatic sac tumors, epididymal cystadenomas, and cystic lesions in various organs. (1,2) The gene responsible for VHL is located on chromosome 3p25 and was cloned as a VHL tumor suppressor. (3) Subsequently, the detection of germline VHL alterations as well as genotype-phenotype correlation analyses have been carried out extensively in various countries, and genetic testing based on the detection of germline alterations is now widely applied as a standard diagnostic procedure. (4-7) Previous studies demonstrated that approximately 70% of VHL families have relatively small intragenic mutations affecting nucleotides of up to 30 bp; such mutations are detectable by routine PCR-based sequencing of the three VHL coding exons. (4) However, standard sequencing analysis failed to detect germline alteration in the rest of the families. As the majority of these cases have been demonstrated to possess relatively large (Kb-Mb order) deletions involving the partial or whole VHL gene, various molecular genetic techniques other than sequencing, including pulsed-field gel electrophoresis, standard or quantitative Southern blot analysis, long-range or quantitative PCR, and fluorescence in situ hybri-dization, have been applied to identify such large deletions. ...

Alu-Alurecombination underlies the vast majority of largeVHLgermline deletions: Molecular characterization and genotype-phenotype correlations in VHL patients

“…In two previous VHL deletion studies, a reduced risk for renal cell carcinoma has been noted when the actin regulator gene HSPC300 was codeleted together with the VHL gene [Maranchie et al, 2004;Cascó n et al, 2007]. While a number of studies have characterized partial and complete germline deletions of the VHL gene by quantitative Southern blot, multiplex ligation-dependent probe amplification (MLPA), and/or quantitative real-time polymerase chain analyses (qPCR) [Stolle et al, 1998;Hes et al, 2000;Cybulski et al, 2002;Gallou et al, 2004;Maranchie et al, 2004;Hoebeeck et al, 2005;Casarin et al, 2006;Hattori et al, 2006;Cascó n et al, 2007;Hes et al, 2007;Huang et al, 2007;Ong et al, 2007] and while some of the breakpoints have been narrowed down more precisely, the exact breakpoint sequence has only been determined for one case, which was shown to result from Alu-Alu recombination [Casarin et al, 2006]. Alu-Alu recombination-mediated deletions have been described for various inherited disorders such as familial hypercholesterolemia and a-thalassemia [for reviews see Deininger and Batzer, 1999;Batzer and Deininger, 2002].…”

Clinical and molecular characterization of Brazilian families with von Hippel-Lindau disease: a need for delineating genotype-phenotype correlation

“…Furthermore, as the controls also presented the isoform II but with weaker intensity, thus, among unaffected individuals, the isoform II is presumed to be underexpressed or may not have any expression. Some studies have reported VHL patients with germline deletions of exon 2 [11,12], as well with splicing defects in the same region as the proband's mutation, in renal cancer cells [5]. Therefore, it is plausible that IVS 1-1G [ A likewise isoform II does not encode a functional VHL product, as it was not recognized by a splicing predictor database, suggesting its deleterious effect [24].…”

“…Practically all the mutation types may be detected by combining direct sequencing with quantitative Southern blotting [9]. More recently, qRT-PCR and MLPA have been efficiently shown to identify large VHL germline deletions and rearrangements [10][11][12]. There is an evident genotype-phenotype correlation among families with VHL disease, who are classified into type 1 (low-risk of PC) and type 2 (high-risk of PC), the former being associated mainly with protein truncating mutations and large deletions, the latter being caused essentially by missense mutations [7,13,14].…”

Familial neuroendocrine tumor syndromes: From genetics to clinical practice

“…Alternatively, the client may have a large deletion of the gene which cannot be detected by simple sequence analysis. In von Hippel-Lindau disease, about 30% of patients have a deletional mutation in the VHL gene, which can be detected by gene dosage analysis [7].…”