Abstract
Mutation in VHL gene causes the von Hippel-Lindau (VHL) disease, a dominantly inherited familial cancer syndrome. The VHL mutation pattern includes point mutations, small deletions and large deletions. While most mutations can be identified during sequencing, large deletions often remain unnoticed in initial mutational screening. We evaluated the utility of a previously described real-time quantitative PCR (RQ-PCR) using SYBR Green for detection of larger deletions in the VHL gene and normalized the data using two reference genes with a normal copy number i.e., ZNF80 (3q13.31) and GPR15 (3q12.1). DNA sequencing was also done on all cases included in the study. SJNB-6 cell line demonstrating distal 3p loss was used as a positive control for deletion. Out of 21 individual cases included of VHL disease, 2 cases were found with partial deletion by RQ-PCR, with an exon 1 deletion, while PCR–sequencing identified 5 cases with base pair substitution and 1 with splice site variant which were not picked up by RQ-PCR. RQ-PCR proved to be fast, accurate and sensitive for identifying large deletions and can be incorporated into the routine work-up for detection of large deletions in VHL disease.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Lonser RR, Glenn GM, Walther M, Chew EY, Libutti SK, Linehan WM, Oldfield EH (2003) von Hippel-Lindau disease. Lancet 361:2059–2067
Maher ER (2004) Von Hippel-Lindau disease. Curr Mol Med 4:833–842
Hoebeeck J, Luijt RVD, Poppe B, Smet ED, Yigit N, Claes K, Zewald R, Jong GD, Paepe AD, Speleman F, Vandesompele J (2005) Rapid detection of VHL exon deletions using real-time quantitative PCR. Lab Invest 85:24–33
Yao M, Hattori K, Baba M, Yoshida M, Kondo K, Nakaigawa N, Kishida T, Kanno H (2011) Germline mutational study of Von Hippel-Lindau disease in Japan. Austral-Asian J Cancer 10(2):91–102
Meyer-Rochow GY, Smith JM, Richardson AL et al (2009) Denaturing High performance liquid chromatography detection of SDHB, SDHD, and VHL Germline mutations in pheochromocytoma. J Surg Res 157:55–62
Boaz RJ, Ramakant P, Ebenazer A, Pai R, Rajaratnam S, Abraham D, Paul MJ (2011) Role of cortical sparing adrenalectomy and novel variant of mutation in patient with von Hippel-Lindau disease. Indian J Endo Metab 15(4):S402–S405
Maher ER, Webster AR, Richards FM et al (1996) Phenotypic expression in von Hippel-Lindau disease: correlations with germline VHL gene mutations. J Med Genet 33:328–332
Stolle C, Glenn G, Zbar B et al (1998) Improved detection of germline mutations in the von Hippel-Lindau disease tumor suppressor gene. Hum Mutat 12:417–423
Zbar B, Kishida T, Chen F et al (1996) Germline mutations in the von Hippel-Lindau disease (VHL) gene in families from North America, Europe, and Japan. Hum Mutat 8:348–357
Di Maria E, Ciotti P (2004) Gene symbol: VHL Disease: von Hippel-Lindau syndrome. Hum Genet 115(2):171
Hes FJ, Hoppener JWM, Lips CJM (2003) Pheochromocytoma in von Hippel-Lindau disease. J Clin Endocr Metab 88:969–974
Lamiell JM, Salazar FG, Hsia YE (1989) von Hippel-Lindau disease affecting 43 members of a single kindred. Medicine (Baltimore) 68(1):1–29
Maher ER, Yates JR, Harries R, Benjamin C, Harris R, Moore AT, Ferguson-Smith MA (1990) Clinical features and natural history of von Hippel-Lindau disease. Q J Med 77(283):1151–1163
Vandesompele J, De Preter K, Pattyn F, et al (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 3(7):research0034.1–12
Bustin SA (2002) Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems. J Mol Endocrinol 29(1):23–39
Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29(9):2002–2007
Hattori K, Teranishi J, Stolle C, Yoshida M, Kondo K, Kishida T, Kanno H, Baba M, Kubota Y, Yao M (2006) Detection of germline deletions using real-time quantitative polymerase chain reaction in Japanese patients with von Hippel–Lindau disease. Cancer Sci 97(5):400–405
Sieber OM, Lamlum H, Crabtree MD et al (2002) Whole-gene APC deletions cause classical familial adenomatous polyposis, but not attenuated polyposis or ‘multiple’ colorectal adenomas. Proc Natl Acad Sci USA 99:2954–2958
Ciotti P, Garuti A, Gulli R, Ballestrero A, Bellone E, Mandich P (2009) Germline mutations in the von Hippel-Lindau gene in Italian patients. Eur J Med Genet 52:311–314
Hes FJ, Zewald R, Peeters T, Sijmons R, Links T et al (2000) Genotype-pheotype correlations in families with deletions in the von Hippel-Lindau (VHL) gene. Hum Genet 106:425–431
Huang JS, Huang CJ, Chen SK, Chien CC, Chen CW et al (2007) Associations between VHL genotype and clinical phenotype in familial von Hippel-Lindau disease. Eur J Clin Inves 37:492–500
Mandich P, Montera M, Bellone E, Trojani A, Daniele S et al (1998) Three novel mutations in the von Hippel-Lindau tumour suppressor gene in Italian patients. Hum Mutat 1:S268–S270
Murgia A, Martella M, Vinanzi C, Polli R, Perilongo G, Opocher G (2000) Somatic mosaicism in von Hippel-Lindau disease. Human Mutat 15(1): 114
Sgambati MT, Stolle C, Choyke PL, Walther MM, Zbar B, Linehan WM, Glenn GM (2000) Mosaicism in von Hippel-Lindau disease: lessons from kindreds with germline mutations identified in offspring with mosaic parents. Am J Hum Genet 66(1):84–91
Perne A, Zhang X, Lehmann LE, Groth M, Stuber F, Book M (2009) Comparison of multiplex ligation-dependent probe amplification and real-time PCR accuracy for gene copy number quantification using the β-defensin locus. Biotechniques 47:1023–1028
Sellner LN, Taylor GR (2004) MLPA and MAPH: new techniques for detection of gene deletions. Hum Mutat 23(5):413–419
Acknowledgments
We thank J Vandesompele from Belgium, for providing us the SJNB-6 (Positive control) cell line. This work was supported through a grant received from the council for Scientific and Industrial Research (CSIR), India, with the grant no. 27(0221)/10/EMR-II.
Conflict of interest
None.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Ebenazer, A., Rajaratnam, S. & Pai, R. Detection of large deletions in the VHL gene using a Real-Time PCR with SYBR Green. Familial Cancer 12, 519–524 (2013). https://doi.org/10.1007/s10689-013-9606-2
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10689-013-9606-2