Abstract Experimental conditions for detection of germ- line deletions of the von Hippel-Lindau (VHL) gene by means of long polymerase chain reaction have been estab- lished. Primers were designed to analyse the VHL gene in three overlapping fragments: 12.5 kb in length containing promoter and exons 1 and 2; 8.7 kb in length containing exons 2 and 3; and 16 kb in length containing exons 2 and 3 and the 3’ untranslated region. Using the described pro- cedure, it was possible to detect large deletions in four of five cases with such mutations previously detected by Southern blotting and in 5 of 11 unrelated Polish VHL pa- tients in whom constitutional VHL gene mutations were not found by sequencing. Introduction von Hippel-Lindau (VHL) disease is a dominantly inher- ited disorder characterised by occurrence of haeman- gioblastomas of the central nervous system and retina, re- nal cell carcinomas and pheochromocytomas, pancreatic cysts, tumours and endolymphatic sac tumours (Maher and Kaelin 1997). The VHL gene was cloned in 1993 (Latif et al. 1993). Inactivating mutations of the VHL gene have been detected by different investigators in 39–80% of VHL families (Whaley et al. 1994; Crossey et al. 1994; Chen et al. 1995; Clinical Research Group for Japan 1995; Glavac et al. 1996; Maher et al. 1996; Zbar et al. 1996). Recently, Stolle et al. (1998) reported technical im- provements in detection of germline VHL mutations allow- ing identification of abnormalities in up to 100% of VHL families. The critical investigation in enhancing mutation detection was the use of quantitative Southern blotting to detect large deletions (Stolle et al. 1998). However, South- ern blotting is not a procedure of first choice for most lab- oratories performing routine molecular diagnosis, because this technique is complex, time consuming, technically de- manding and, thus, expensive. Therefore, we have devel- oped a long polymerase chain reaction (PCR)-based method for the detection of VHL gene deletions, which avoids the need for Southern blotting in many cases. Material and methods Patients Studies were performed on a series of 16 patients affected by VHL disease diagnosed on a basis of pedigree and clinical criteria. In five cases (C1–C4 – from Dr. E.R. Maher; R – from Dr. S. Richard), large deletions of the VHL gene were identified previously by means of Southern blotting (C1 – deletion of exons 1 and 2; C2 – deletion of exon 1; C3 – deletion of exon 2; C4 – deletion of exon 3; R – dele- tion of exon 3). The remaining 11 cases (S1–S11) were selected from a series of 23 unrelated VHL patients from the Hereditary Can- cer Center, Szczecin, after sequencing of the coding region failed to identify a germline VHL mutation. Additionally, studies were ex- tended to relatives of Polish VHL patients (S1, S2, S3, S5) with dele- tions identified by means of long PCR. Long template PCR Large PCR fragments were amplified in a Perkin-Elmer 9600 ther- mal cycler in final volume of 25 μl that contained 1 × buffer 3 and Cezary Cybulski · Karol Krzystolik · Eamonn R. Maher · Stephane Richard · Grzegorz Kurzawski · Jacek Gronwald · Jan Lubiński Long polymerase chain reaction in detection of germline deletions in the von Hippel-Lindau tumour suppressor gene Hum Genet (1999) 105 : 333–336 © Springer-Verlag 1999 Digital Object Identifier (DOI) 10.1007/s004399900137 Received: 25 May 1999 / Accepted: 27 July 1999 / Published online: 9 September 1999 ORIGINAL INVESTIGATION C. Cybulski (✉) · K. Krzystolik · G. Kurzawski · Jacek Gronwald · J. Lubiński Department of Genetics and Pathology, Pomeranian Medical Academy, Szczecin ul. Powstanców Wlkp. 72, Poland e-mail: cezaryc@friko6.onet.pl, Tel.: +48-91-4828450, Fax: +48-91-4828450 C. Cybulski I Department of Pediatrics, Pomeranian Medical Academy, Szczecin, Poland E.R. Maher Department of Pathology, Cambridge University School of Medicine, Cambridge, England S. Richard Sainte-Anne Hospital, Paris, France K. Krzystolik I Department of Ophtalmology, Pomeranian Medical Academy, Szczecin, Poland