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Mapping of Von Hippel-Lindau disease to chromosome 3p confirmed by genetic linkage analysis
Journal of the Neurological Sciences, 100 (1990) 27-30
Elsevier
27
JNS 03418
Mapping of Von Hippel-Lindau disease to chromosome 3p confirmed by genetic linkage analysis E.R. Maher l, E. Bentley ~, J.R.W. Yates ~, D. Barton l, A. Jennings 2, I.W. Fellows 3, M.A. Ponder 4, B.A.J. Ponder 4, C. Benjamin 5, R. Harris 5 and M.A. Ferguson-Smith ~ ~Department of Pathology, Cambridge University, Tennis Court Road. Cambridge (U.K.), 2Hallamshire Hospital SheJ~eld (U.K.), 3UniversityHospital, Nottingham (U.K.), 4Cancer Research Campaign, Human Cancer Genetics Research Group, Department of Pathology, Tennis Court Road, Cambridge (U.K.) and 5Department of Medical Genetics, Universityof Manchester, Manchester (U.K.)
(Received 14 March, 1990) (Revised, received 15 June, 1990) (Accepted 22 June, 1990) Key words: Von Hippel-Lindau disease; Chromosome 3p; Genetic linkage analysis; Mapping
Summary Genetic linkage studies were performed in 12 British families with von Hippel-Lindau disease (VHL) using RFLPs at three loci (DNF 15$2, THRB, RAF 1) on the short arm of chromosome 3. Linkage was detected between the VHL disease locus and RAF 1 with a maximum lod score of 3.88 at a recombination fraction of 0.05 (confidence interval 0.003-0.18). Multipoint linkage analysis suggested that the most likely location for the VHL disease locus is telomeric to THRB. These results confirm earlier reports localizing the VHL gene to the short arm of chromosome 3, and provide no evidence for genetic heterogeneity. Introduction Von Hippel-Lindau (VHL) disease is a dominantly inherited familial cancer syndrome with variable expression. The most frequent complications are haemangioblastomas of the central nervous system and retina, renal cell carcinoma, phaeochromocytoma and renal, pancreatic and epididymal cysts (Melmon and Rosen 1964; Horton et al. 1976; Lamiell et al. 1989). Although most patients with VHL disease present before age 40 years, an important minority present late and prolonged ophthalmological and radiological screening of at risk relatives is indicated (Maher et al. 1990a). The identification of polymorphic DNA probes closely linked to the locus for VHL disease would allow presymptomatic and prenatal diagnosis, and optimise the efficiency of screening programmes by concentrating resources on those relatives shown to be at high risk. Recently, Seizinger et al. (1988) mapped the gene for VHL disease to the short arm of chromosome 3 by genetic linkage analysis, and further studies are required to confirm this localization. We report
Correspondence to: Dr. E.R. Maher, Departmentof Medical Genetics,
Level 1, Addenbrooke's Hospital, Hills Road, Cambridge CB2 2QQ, U.K.
the results of genetic linkage studies in 12 British families with VHL disease.
Patients and methods Patient details
Families with VHL disease were ascertained for the purpose of molecular genetic studies by contacting specialists in Neurology, Neurosurgery, Medical Genetics, Nephrology, Ophthalmology and Urology throughout Great Britain. The 12 families consisted of 92 individuals, including 52 affected patients who satisfied standard diagnostic criteria for VHL disease (Melmon and Rose 1964). All 12 families included an individual with a retinal or CNS haemangioblastoma, and renal cell carcinoma had occurred in 8 families. Only 1 patient had developed a phaeochromocytoma. One family has been reported in detail elsewhere (Jennings et al. 1988) and 46 affected patients from the other 11 families were included in a previous study of the clinical features of VHL disease (Maher et al. 1990a). D N A studies
Restriction fragment length polymorphisms (RFLPs) were studied at three loci on the short arm of chromosome 3
0022-510X/90/$03.50 © 1990Elsevier Science Publishers B.V. (BiomedicalDivision)
28 26
RAF1
25 24
THRB
23 22 21.3
DNF15S2 21.2 14.3 14.1 13
12
CEN
Fig. 1. Short arm chromosome3 showing physical locations of marker loci.
(see Fig. 1): DNF15S2 an anonymous D N A segment mapped to the 3p21 region (Carritt et al. 1986); T H R B (formerly ERBA2) thyroid hormone receptor beta gene which has been localized to 3p22-24.1 (Drabkin et al. 1988); and RAF1 the homologue of the murine leukaemia (v-RAF- 1) oncogene which localizes to 3p25 (Teyssier et al. 1986). Details of probes, enzymes and allele frequencies are given in Table 1. D N A was extracted from peripheral blood lymphocytes by standard methods and 5-10/tg digested with the appropriate restriction enzyme under the manufacturers' recommended conditions. D N A fragments were resolved by electrophoresis on 0.8~o agarose gel and transferred to Nylon membranes (Hybond N, Amersham) by Southern blotting (Maniatis 1984). After hybridization with 32p_ labelled probes autoradiographs were developed with intensifying screens for 2 - 7 days at - 70 °C.
Linkage analysis Genetic linkage analysis between VHL disease and the marker loci was performed using the computer programs L I N K A G E (Lathrop and Lalouel 1984) and L I P E D (Ott 1974). Age at onset corrections were made using 8 agedependent penetrance classes based on the age at onset of 152 cases of VHL disease, e.g., penetrances at ages 20-24, 35-39 and > 60 years were set at 0.36, 0.85 and 0.95 respectively (Maher et al. 1990a). A lod score > 3 was taken as indicating significant linkage. Confidence limits were determined by taking values of the recombination fractions corresponding to a lod score one unit less than the maximum (Conneally et al. 1985). Genetic heterogeneity testing was performed using the H O M O G computer program (Ott 1985). Recombination fraction (theta) is a measure of genetic distance between two loci, the smaller the recombination fraction the nearer the loci are. The recombination fractions between DNF15S2 and T H R B , and between T H R B and RAF were taken as 0.3 and 0.13, respectively (Vance et al. 1990). Recombination fractions were assumed to be equal in males and females. Haldane's mapping functions were applied to convert recombination fractions into mapping distances.
Results Combined two point lod scores for the 12 families are given in Table 2. Four families informative at the D N F 15S2 locus showed no evidence of linkage to VHL disease. Four families informative for T H R B gave a maximum lod score 0.98 at a recombination fraction of 0.17. Significant linkage between the VHL disease and RAF 1 loci was detected (see Table 3) with a maximum lod score of 3.88 at a recombination fraction of 0.05 (CI 0.003-0.18). Multipoint analysis suggested that the most likely location
TABLE 1 DETAILS OF RFLPs STUDIED IN 12 FAMILIES WITH VHL DISEASE Locus
Probe
Restriction enzyme
Allele size (kb)
Allele frequency
PIC
DNF 15$2
H3H2
HindlII
pBH302
HindlIl
RAFI
p627
Taq 1
p627
BglI
0.46 0.54 0.33 0.67 0.74 0.26 0.54 0.46
0.37
THRB
2.3 2.0 7.0 5.5 6.8 6.3 4.0 3.3
PIC = polymorphisminformation content.
0.34 0.31 0.37
29 TABLE 2 TOTAL P A I R W l S E L OD SCORES F O R L I N K A G E B E T W E E N VHL D I S E A S E A N D C H R O M O S O M E 3p LOCI (DNF15S2, THRB, RAF1) I N 12 F A M I L I E S W I T H VHL D I S E A S E Locus
Recombination fraction
DNF15S2 THRB RAF 1
0.0
0.001
0.01
0.05
0.1
0.2
0.3
0.4
- m - oo - oo
- 7.3 - 3.3 2.6
- 4.2 - 1.2 3.56
- 2.1 0.3 3.88
- 1.18 0.81 3.63
- 0.44 0.96 2.72
- 0.14 0.66 1.58
- 0.03 0.23 0.51
TABLE 3 P A I R W I S E LOD SCORES B E T W E E N RAF1 A N D VHL D I S E A S E IN 8 I N F O R M A T I V E F A M I L I E S Family
Recombination fraction
No. 0.0
0.01
0.05
0.1
0.2
0.3
0.4
2 3 4 5 7 8 11 12
0.99 - ~ 0.67 0.76 1.62 0.11 0.25 0.24
0.97 - 1.0 0.66 0.74 1.58 0.11 0.24 0.26
0.88 -0.35 0.60 0.68 1.45 0.09 0.21 0.33
0.76 -0.11 0.52 0.59 1.27 0.07 0.16 0.36
0.53 0.06 0.35 0.41 0.89 0.04 0.09 0.33
0.30 0.09 0.19 0.23 0.49 0.02 0.04 0.22
0.09 0.07 0.06 0.07 0.13 0.0 0.0 0.07
Total
- m
3.56
3.88
3.63
2.72
1.58
0.51
for the VHL disease locus is telomeric to THRB. The likely orientation of the VHL locus with respect to RAF1 could not be established (see Fig. 2). The most likely location of the VHL disease gene, distal to T H R B in the THRB-RAF1
THRB
DNF15S2
RAF1
4
,-1
i
0
0
I
20
40
60
80
,
I
100
cM Fig. 2. Multipoint linkage analysis for the VHL disease locus in 12 families.
or RAFl-pter intervals, is favoured 16 : 1 over a position in the DNF15S2-THRB interval. Formal heterogeneity testing using the H O M O G computer program (Ott 1985) was performed by combining our RAF1 linkage data with that of Seizinger et al. (1988). No evidence of genetic heterogeneity was found (P = 0.5) the most likely proportion of linked families being 1.0 (95~o confidence intervals 0.6-1.0).
Discussion Our results suggest that the VHL disease locus is situated distal to T H R B on the short arm of chromosome 3. This confirms the mapping of a locus for VHL disease to the short arm of chromosome 3 by Seizinger et al. (1988) who found a combined maximum lod score of 4.38 at a recombination fraction of 0.11 (CI0.04-0.23) between VHL disease and RAF1 in 9 families. Vance et al. (1990) have also reported linkage studies in two families with VHL disease: they found a peak lod score of 2.3 at zero recombination with RAF1. Multipoint analysis with RAF1 and THRB resulted in a peak lod score of 3.1, however they also could not resolve the position of VHL disease relative to these loci. Molecular studies on VHL disease tumours suggest that the VHL locus is telomeric to DNF15S2 (Tory et al. 1989). Recent data from Boston imply that the most likely localization for the VHL gene is telomeric to RAF1 (Sezinger et al. 1989). Crossovers between the VHL gene and RAF1 locus observed by us and others (Seizinger et al. 1988) have excluded mutations in the RAF1 gene from causing VHL disease. Currently available DNA markers are not close enough to allow reliable presymptomatic or prenatal diagnosis and the identification of closely linked probes flanking the VHL gene is a priority so that DNA probe data can be combined with age at onset data to estimate an individual's risk of developing VHL disease. This will enable screening protocols to be modified such that the frequency of screening is reduced in older relatives shown to be at low risk. Interfamilial variability in the predisposition to phaeo-
30 chromocytoma is well recognised in VHL disease (Green et al. 1986; Maher et al. 1990a; Lamiell et al. 1989). Despite this phenotypic variation, no evidence of genetic heterogeneity has been reported in families studied by us (n -- 12), Seizinger et al. (1988) (n = 9) or by Vance et al. (1990) (n = 2). However, our series contained only one small family in which phaeochromocytoma was apparent (family 4), and this aspect will warrant further study. The underlying biochemical defect in VHL disease is unknown, and the identification and characterization of the VHL gene will provide an insight into tumourigenesis in VHL disease and offer the possibility of developing therapies directed at the underlying abnormality. The VHL gene appears to function as a recessive 'tumour suppressor gene" (King et al. 1987; Tory et al. 1989; Maher et al. 1990b) and may also prove to have a role in the pathogenesis of more common nonfamilial tumours (Zbar et al. 1987). Acknowledgements We thank the numerous colleagues who referred their patients, and Dr, B. Carritt (pH3H2), Dr. W.E.C. Bradley (pBH302) and Dr. U.R. Rapp (p627) for supplying DNA probes. This work was supported by grants from the National Kidney Research Fund and the British Medical Association (Helen Tomkinson Award).
References Carritt, B., H.M. Welch and N.J. Parry-Jones (1986) Sequences homologous to the human D1S1 locus present on human chromosome 3. Am. J. Hum. Genet., 38: 428-436. Conneally, P.M., J.H. Edwards, K.K. Kidd et al. (1985) Report of the Committee on Methods of Linkage and Reporting. 8th Int. Workshop Human Gene Mapping. Cytogenet. Cell Genet., 40: 356-359. Drabkin, H.A., F.-T. Kao, I. Hartz et al. (1988) Localization of human ERBA2 to the 3p22-p24.1 region of chromosome 3 and variable deletion in small cell lung cancer. Proc. Natl. Acad. Sci. USA, 85: 9258-9262. Green, J.S., M.I. Bowmer and G.J. Johnson (1986) Von Hippel-Lindau disease in a Newfoundland kindred. Can. Med. Assoc. J., 134: 133-146. Horton, W.A., V. Wong and R. Eldridge (1976) Von Hippel-Lindau disease. Arch. Intern. Med., 136: 769-777.
Jennings, A.M., C. Smith, D.R. Cole, C. Jennings, J.R. Shortland, J.L. Williams and C.B. Brown (1988) Von Hippel-Lindau disease in a large British family: clinicopathological features and recommendations for screening and follow-up. Q. J. Med., 66: 233-249. King, C.R., R.N. Schimke, T. Arthur, B. Davoren and D. Collins (1987) Proximal 3p deletion in renal cell carcinoma cells from a patient with von Hippel-Lindau disease. Cancer Genet. Cytogenet., 27: 345-348. Lamiell, J.M., F.G. Salazar and Y.E. Hsia (1989) Von Hippel-Lindau disease affecting 43 members of a single kindred. Medicine, 68: 1-29. Lathrop, G.M. and J.M. Lalouel (1984) Easy calculation of lod scores on small computers. Am. J. Hum. Genet., 36: 460-465. Maher, E.R., J.R.W. Yates, R. Harries et al. (1990a) Clinical features and natural history of von Hippel-Lindau disease. Q. J. Med., in press. Maher, E.R., J.R.W. Yates and M.A. Ferguson-Smith (1990b) Statistical analysis of the two stage mutation model in von Hippel-Lindau disease, sporadic cerebellar haemangioblastoma and renal cell carcinoma. J. Med. Genet., 27: 311-314. Maniatis, T., E.F. Fritsch and J. Sambrook (1984) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Melmon, K.L. and S.W. Rosen (1964) Lindau's disease. Am. J. Med., 36: 595-617. Ott, J. (1974) Estimation of the recombination fraction in human pedigrees: efficient computation of the likelihood for human linkage studies. Am. J Hum. Genet., 8: 80-96. Ott, J. (1985) Analysis of Human Genetic Linkage. Johns Hopkins University Press, Baltimore, MD, pp. 105-119. Seizinger, B.R., G.A. Rouleau, L.J. Ozelius et al. (1988) Von HippelLindau disease maps to the region of chromosome 3 associated with renal cell carcinoma. Nature, 332: 268-269. Seizinger, B.R., G. Farmer, J. Haines et al. (1989) Isolating the gene(s) for von Hippel-Lindau disease and renal cell carcinoma (Abstract), Am. J. Hum. Genet., 45: A32. Teyssier, J.R., I. Henry, C. Dozier, D. Ferre, J.J. Adnet and M. Pulot (1986) Recurrent deletion of the short arm of chromosome 3 in human renal cell carcinoma: shift of the c-raf 1 locus. J. Natl. Cancer Inst., 77: 1187-1191. Tory, K., H. Brauch, M. Lineham et al. (1989) Specific genetic change in tumours associated with von Hippel-Lindau disease. J. Natl. Cancer Inst., 81: 1097-1101. Vance, J.M., K. Small, M.A. Jones, J.M. Stajich, L.H. Yamaoka, A.D. Roses, W.-Y. Hung and M.A. Pericak-Vance (1990) Confirmation of linkage in von Hippel-Lindau disease. Genomic, 6: 565-567. Zbar, B., H. Brauch, C. Talmadge and M. Lineham (1987) Loss of alleles of loci on the short arm of chromosome 3 in renal cell carcinoma. Nature, 327: 721-724.
Elsevier
27
JNS 03418
Mapping of Von Hippel-Lindau disease to chromosome 3p confirmed by genetic linkage analysis E.R. Maher l, E. Bentley ~, J.R.W. Yates ~, D. Barton l, A. Jennings 2, I.W. Fellows 3, M.A. Ponder 4, B.A.J. Ponder 4, C. Benjamin 5, R. Harris 5 and M.A. Ferguson-Smith ~ ~Department of Pathology, Cambridge University, Tennis Court Road. Cambridge (U.K.), 2Hallamshire Hospital SheJ~eld (U.K.), 3UniversityHospital, Nottingham (U.K.), 4Cancer Research Campaign, Human Cancer Genetics Research Group, Department of Pathology, Tennis Court Road, Cambridge (U.K.) and 5Department of Medical Genetics, Universityof Manchester, Manchester (U.K.)
(Received 14 March, 1990) (Revised, received 15 June, 1990) (Accepted 22 June, 1990) Key words: Von Hippel-Lindau disease; Chromosome 3p; Genetic linkage analysis; Mapping
Summary Genetic linkage studies were performed in 12 British families with von Hippel-Lindau disease (VHL) using RFLPs at three loci (DNF 15$2, THRB, RAF 1) on the short arm of chromosome 3. Linkage was detected between the VHL disease locus and RAF 1 with a maximum lod score of 3.88 at a recombination fraction of 0.05 (confidence interval 0.003-0.18). Multipoint linkage analysis suggested that the most likely location for the VHL disease locus is telomeric to THRB. These results confirm earlier reports localizing the VHL gene to the short arm of chromosome 3, and provide no evidence for genetic heterogeneity. Introduction Von Hippel-Lindau (VHL) disease is a dominantly inherited familial cancer syndrome with variable expression. The most frequent complications are haemangioblastomas of the central nervous system and retina, renal cell carcinoma, phaeochromocytoma and renal, pancreatic and epididymal cysts (Melmon and Rosen 1964; Horton et al. 1976; Lamiell et al. 1989). Although most patients with VHL disease present before age 40 years, an important minority present late and prolonged ophthalmological and radiological screening of at risk relatives is indicated (Maher et al. 1990a). The identification of polymorphic DNA probes closely linked to the locus for VHL disease would allow presymptomatic and prenatal diagnosis, and optimise the efficiency of screening programmes by concentrating resources on those relatives shown to be at high risk. Recently, Seizinger et al. (1988) mapped the gene for VHL disease to the short arm of chromosome 3 by genetic linkage analysis, and further studies are required to confirm this localization. We report
Correspondence to: Dr. E.R. Maher, Departmentof Medical Genetics,
Level 1, Addenbrooke's Hospital, Hills Road, Cambridge CB2 2QQ, U.K.
the results of genetic linkage studies in 12 British families with VHL disease.
Patients and methods Patient details
Families with VHL disease were ascertained for the purpose of molecular genetic studies by contacting specialists in Neurology, Neurosurgery, Medical Genetics, Nephrology, Ophthalmology and Urology throughout Great Britain. The 12 families consisted of 92 individuals, including 52 affected patients who satisfied standard diagnostic criteria for VHL disease (Melmon and Rose 1964). All 12 families included an individual with a retinal or CNS haemangioblastoma, and renal cell carcinoma had occurred in 8 families. Only 1 patient had developed a phaeochromocytoma. One family has been reported in detail elsewhere (Jennings et al. 1988) and 46 affected patients from the other 11 families were included in a previous study of the clinical features of VHL disease (Maher et al. 1990a). D N A studies
Restriction fragment length polymorphisms (RFLPs) were studied at three loci on the short arm of chromosome 3
0022-510X/90/$03.50 © 1990Elsevier Science Publishers B.V. (BiomedicalDivision)
28 26
RAF1
25 24
THRB
23 22 21.3
DNF15S2 21.2 14.3 14.1 13
12
CEN
Fig. 1. Short arm chromosome3 showing physical locations of marker loci.
(see Fig. 1): DNF15S2 an anonymous D N A segment mapped to the 3p21 region (Carritt et al. 1986); T H R B (formerly ERBA2) thyroid hormone receptor beta gene which has been localized to 3p22-24.1 (Drabkin et al. 1988); and RAF1 the homologue of the murine leukaemia (v-RAF- 1) oncogene which localizes to 3p25 (Teyssier et al. 1986). Details of probes, enzymes and allele frequencies are given in Table 1. D N A was extracted from peripheral blood lymphocytes by standard methods and 5-10/tg digested with the appropriate restriction enzyme under the manufacturers' recommended conditions. D N A fragments were resolved by electrophoresis on 0.8~o agarose gel and transferred to Nylon membranes (Hybond N, Amersham) by Southern blotting (Maniatis 1984). After hybridization with 32p_ labelled probes autoradiographs were developed with intensifying screens for 2 - 7 days at - 70 °C.
Linkage analysis Genetic linkage analysis between VHL disease and the marker loci was performed using the computer programs L I N K A G E (Lathrop and Lalouel 1984) and L I P E D (Ott 1974). Age at onset corrections were made using 8 agedependent penetrance classes based on the age at onset of 152 cases of VHL disease, e.g., penetrances at ages 20-24, 35-39 and > 60 years were set at 0.36, 0.85 and 0.95 respectively (Maher et al. 1990a). A lod score > 3 was taken as indicating significant linkage. Confidence limits were determined by taking values of the recombination fractions corresponding to a lod score one unit less than the maximum (Conneally et al. 1985). Genetic heterogeneity testing was performed using the H O M O G computer program (Ott 1985). Recombination fraction (theta) is a measure of genetic distance between two loci, the smaller the recombination fraction the nearer the loci are. The recombination fractions between DNF15S2 and T H R B , and between T H R B and RAF were taken as 0.3 and 0.13, respectively (Vance et al. 1990). Recombination fractions were assumed to be equal in males and females. Haldane's mapping functions were applied to convert recombination fractions into mapping distances.
Results Combined two point lod scores for the 12 families are given in Table 2. Four families informative at the D N F 15S2 locus showed no evidence of linkage to VHL disease. Four families informative for T H R B gave a maximum lod score 0.98 at a recombination fraction of 0.17. Significant linkage between the VHL disease and RAF 1 loci was detected (see Table 3) with a maximum lod score of 3.88 at a recombination fraction of 0.05 (CI 0.003-0.18). Multipoint analysis suggested that the most likely location
TABLE 1 DETAILS OF RFLPs STUDIED IN 12 FAMILIES WITH VHL DISEASE Locus
Probe
Restriction enzyme
Allele size (kb)
Allele frequency
PIC
DNF 15$2
H3H2
HindlII
pBH302
HindlIl
RAFI
p627
Taq 1
p627
BglI
0.46 0.54 0.33 0.67 0.74 0.26 0.54 0.46
0.37
THRB
2.3 2.0 7.0 5.5 6.8 6.3 4.0 3.3
PIC = polymorphisminformation content.
0.34 0.31 0.37
29 TABLE 2 TOTAL P A I R W l S E L OD SCORES F O R L I N K A G E B E T W E E N VHL D I S E A S E A N D C H R O M O S O M E 3p LOCI (DNF15S2, THRB, RAF1) I N 12 F A M I L I E S W I T H VHL D I S E A S E Locus
Recombination fraction
DNF15S2 THRB RAF 1
0.0
0.001
0.01
0.05
0.1
0.2
0.3
0.4
- m - oo - oo
- 7.3 - 3.3 2.6
- 4.2 - 1.2 3.56
- 2.1 0.3 3.88
- 1.18 0.81 3.63
- 0.44 0.96 2.72
- 0.14 0.66 1.58
- 0.03 0.23 0.51
TABLE 3 P A I R W I S E LOD SCORES B E T W E E N RAF1 A N D VHL D I S E A S E IN 8 I N F O R M A T I V E F A M I L I E S Family
Recombination fraction
No. 0.0
0.01
0.05
0.1
0.2
0.3
0.4
2 3 4 5 7 8 11 12
0.99 - ~ 0.67 0.76 1.62 0.11 0.25 0.24
0.97 - 1.0 0.66 0.74 1.58 0.11 0.24 0.26
0.88 -0.35 0.60 0.68 1.45 0.09 0.21 0.33
0.76 -0.11 0.52 0.59 1.27 0.07 0.16 0.36
0.53 0.06 0.35 0.41 0.89 0.04 0.09 0.33
0.30 0.09 0.19 0.23 0.49 0.02 0.04 0.22
0.09 0.07 0.06 0.07 0.13 0.0 0.0 0.07
Total
- m
3.56
3.88
3.63
2.72
1.58
0.51
for the VHL disease locus is telomeric to THRB. The likely orientation of the VHL locus with respect to RAF1 could not be established (see Fig. 2). The most likely location of the VHL disease gene, distal to T H R B in the THRB-RAF1
THRB
DNF15S2
RAF1
4
,-1
i
0
0
I
20
40
60
80
,
I
100
cM Fig. 2. Multipoint linkage analysis for the VHL disease locus in 12 families.
or RAFl-pter intervals, is favoured 16 : 1 over a position in the DNF15S2-THRB interval. Formal heterogeneity testing using the H O M O G computer program (Ott 1985) was performed by combining our RAF1 linkage data with that of Seizinger et al. (1988). No evidence of genetic heterogeneity was found (P = 0.5) the most likely proportion of linked families being 1.0 (95~o confidence intervals 0.6-1.0).
Discussion Our results suggest that the VHL disease locus is situated distal to T H R B on the short arm of chromosome 3. This confirms the mapping of a locus for VHL disease to the short arm of chromosome 3 by Seizinger et al. (1988) who found a combined maximum lod score of 4.38 at a recombination fraction of 0.11 (CI0.04-0.23) between VHL disease and RAF1 in 9 families. Vance et al. (1990) have also reported linkage studies in two families with VHL disease: they found a peak lod score of 2.3 at zero recombination with RAF1. Multipoint analysis with RAF1 and THRB resulted in a peak lod score of 3.1, however they also could not resolve the position of VHL disease relative to these loci. Molecular studies on VHL disease tumours suggest that the VHL locus is telomeric to DNF15S2 (Tory et al. 1989). Recent data from Boston imply that the most likely localization for the VHL gene is telomeric to RAF1 (Sezinger et al. 1989). Crossovers between the VHL gene and RAF1 locus observed by us and others (Seizinger et al. 1988) have excluded mutations in the RAF1 gene from causing VHL disease. Currently available DNA markers are not close enough to allow reliable presymptomatic or prenatal diagnosis and the identification of closely linked probes flanking the VHL gene is a priority so that DNA probe data can be combined with age at onset data to estimate an individual's risk of developing VHL disease. This will enable screening protocols to be modified such that the frequency of screening is reduced in older relatives shown to be at low risk. Interfamilial variability in the predisposition to phaeo-
30 chromocytoma is well recognised in VHL disease (Green et al. 1986; Maher et al. 1990a; Lamiell et al. 1989). Despite this phenotypic variation, no evidence of genetic heterogeneity has been reported in families studied by us (n -- 12), Seizinger et al. (1988) (n = 9) or by Vance et al. (1990) (n = 2). However, our series contained only one small family in which phaeochromocytoma was apparent (family 4), and this aspect will warrant further study. The underlying biochemical defect in VHL disease is unknown, and the identification and characterization of the VHL gene will provide an insight into tumourigenesis in VHL disease and offer the possibility of developing therapies directed at the underlying abnormality. The VHL gene appears to function as a recessive 'tumour suppressor gene" (King et al. 1987; Tory et al. 1989; Maher et al. 1990b) and may also prove to have a role in the pathogenesis of more common nonfamilial tumours (Zbar et al. 1987). Acknowledgements We thank the numerous colleagues who referred their patients, and Dr, B. Carritt (pH3H2), Dr. W.E.C. Bradley (pBH302) and Dr. U.R. Rapp (p627) for supplying DNA probes. This work was supported by grants from the National Kidney Research Fund and the British Medical Association (Helen Tomkinson Award).
References Carritt, B., H.M. Welch and N.J. Parry-Jones (1986) Sequences homologous to the human D1S1 locus present on human chromosome 3. Am. J. Hum. Genet., 38: 428-436. Conneally, P.M., J.H. Edwards, K.K. Kidd et al. (1985) Report of the Committee on Methods of Linkage and Reporting. 8th Int. Workshop Human Gene Mapping. Cytogenet. Cell Genet., 40: 356-359. Drabkin, H.A., F.-T. Kao, I. Hartz et al. (1988) Localization of human ERBA2 to the 3p22-p24.1 region of chromosome 3 and variable deletion in small cell lung cancer. Proc. Natl. Acad. Sci. USA, 85: 9258-9262. Green, J.S., M.I. Bowmer and G.J. Johnson (1986) Von Hippel-Lindau disease in a Newfoundland kindred. Can. Med. Assoc. J., 134: 133-146. Horton, W.A., V. Wong and R. Eldridge (1976) Von Hippel-Lindau disease. Arch. Intern. Med., 136: 769-777.
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