American Journal of Medical Genetics 39:196-200 (1991)
Characterization of Deletions at the Retinoblastoma Locus in Patients With Bilateral Retinoblastoma Kai Kloss, Petra Wahrisch, Valerie Greger, Elmar Messmer, Henning Fritze, Wolfgang Hopping, Eberhard Passarge, and Bernhard Horsthemke Znstitut fur Humangenetik (K.K., P.W., V.G., E.P., B.H.) and Zentrum fur Augenheilkunde (E.M., H.F., W.H.), Universitatsklinikum Essen, Germany
DNA samples from 92 unrelated patients with bilateral retinoblastoma were analyzed by Southern blot hybridization with cDNA and genomic clones of the retinoblastoma (RB-1) gene. Qualitative and quantitative evaluation of the Southern blot patterns showed a deletion of all or part of the RB-1 gene in 15 patients. Deletion hot spots were not detected. The study shows that 16% of germ cell mutations are detectable by Southern blot hybridization, but that densitometric analysis is required in most cases. KEY WORDS: RB-1 gene, Southern blot patterns, cDNA
RNase protection assays and direct sequencing of enzymatically amplified gene fragments have successfully been employed [Dunn et al., 1988; Yandell et al., 19891. However, the first method is not generally applicable, and the second is too laborious for rapid diagnostic testing. Since most studies on RB-1 gene deletions have been performed on tumor cells, which carry either a germ line and a somatic mutation or 2 somatic mutations [Dryja et al., 1986; F’riend et al., 1986; Bookstein et al., 1988; Goddard et al., 19881, we asked what proportion of germ line mutations can be detected by Southern blot hybridization. Here we describe the analysis of 92 lymphocyte DNA samples from patients with bilateral retinoblastoma.
MATERIALS AND METHODS Patients INTRODUCTION Two hundred seventy patients who had been treated Approximately 40% of patients with retinoblastoma for bilateral retinoblastoma a t the Department of Ophcarry a germ line mutation at the retinoblastoma (RB-1) thalmology, University of Essen, between 1968 and locus on chromosome 13 [Vogel, 19791. Most of these 1982 were asked for follow-up examinations. Diagnosis patients develop bilateral tumors. Non-hereditary reti- had been established by current ophthalmologic critenoblastoma occurs in 60% of patients and is always ria. Ninety-nine patients came, and peripheral blood unilateral. Predictive DNA diagnosis in sibs and off- was obtained from 92 unrelated patients. Twenty-six spring of patients helps to detect tumor development in patients (28%) had one or more affected relatives; 66 carriers or to avoid frequent ophthalmologic examina- patients (72%) were isolated cases. Patient 395 had an tions under general anesthesia in children not a t risk. In osteosarcoma in the paranasal sinus. Patient 406 was families with 2 or more affected individuals, the risk slightly retarded. Cytogenetic analysis was not perassessment can be based on direct detection of the muta- formed. tion [Horsthemke et al., 1987al or genetic segregation DNA Analysis analysis with linked DNA markers [Cavenee et al., 1986; Greger et al., 1988; Wiggs et al., 1988; Scheffer et DNA was isolated and analyzed by Southern blot hyal., 1989; Goddard et al., 19901. bridization as described before [Horsthemke et al., In contrast, predictive DNA diagnosis in isolated 1987bl. Blots were exposed to preflashed films for one to cases requires direct identification of the mutation. 3 days. Each DNA sample was studied in a t least 2 While gross structural alterations can easily be detected independent experiments. The RB-1 cDNA clone [Friend by Southern blot hybridization, point mutations are dif- et al., 19861 and the genomic subclones p123 and p68 ficult to identify and require different techniques. [Wiggs et al., 19881 were provided by Dr. T.P. Dryja, Boston. The genomic RB-1 fragment H3-8 [Lalande et al., 19841was from Dr. S.A. Latt, Boston. 9 D l l (D13S2) Received for publication January 18, 1990; revision received and L32 (D8S48) were used as internal hybridization April 17, 1990. standards. 9 D l l [Cavenee et al., 19841 was provided by Address reprint requests to Dr. Bernhard Horsthemke, Institut Dr. W.K. Cavenee, Montreal. L32 was obtained by mifiir Humangenetik, Universitatsklinikum Essen, Hufelandstrasse crodissection of 8q [Liidecke et al., 19891.Relative inten5 5 , D-4300 Essen 1, Germany. 0 1991 Wiley-Liss, Inc.
Gene Deletions in Retinoblastoma sities of hybridization signals were determined with the help of a SHIMADZU CS 9000 densitometer.
RESULTS DNA samples were analyzed by Southern blot hybridization with cDNA and genomic probes for the RB-1 gene. Since the gene has 27 exons, which are spread over 200 kb [McGee et al., 1989; Hong et al., 1989; T’Ang et al., 19891, the 4.7 kb cDNA was cut into 3 fragments with HpaI and EcoRI (Fig. 1).R0.6 and R3.8 detect exons 3-8 and 9-27, respectively. Since exons 3 and 4 did not give strong hybridization signals with probe R0.6, the genomic subclone H3-8, which contains exon 4, was used to detect deletions involving exon 4. The 0.3 kb EcoRIHpaI fragment at the 5’ end ofthe cDNA is very GC-rich and did not give clear Southern patterns. Therefore, the genomic subclone p123 was used for identifying deletions involving exon 1. For mapping breakpoints within intron 17, which spans approximately 90 kb, we used the genomic subclone p68. This probe detects a highly informative “variable number of tandem repeats” polymorphism on RsaI blots [Wiggs et al., 19881. Patients found to be heterozygous in this test were assumed to have 2 copies of this part of the gene. The other patients were studied by quantitative Southern blot hybridization.
9 D l l (D13S21, which maps to 13q22 [Cavenee et al., 19841, and L32 (D8S48),which maps to 8q24.1 [Ludecke et al., 19891, were used as internal hybridization standards for densitometric analysis. Abnormal Southern patterns were detected in 3 samples (441,380, and 369; Fig. 2). In 369, but not in 380 and 441, a n abnormal band was also detected in SacI digests. Quantitative studies in 380 (Fig. 3) and 441 (not shown) showed loss of genetic material adjacent to the abnormal gene fragment. This indicates that the abnormal bands do not represent neutral DNA polymorphisms, but deletion junction fragments. Densitometric evaluation of the Southern blots showed deletions in another 12 cases. Representative blots are shown in Figure 3. Thus, a total of 15 deletions were identified in 92 patients (16%). Four of the 15 patients (056,408,491, and 511) have a positive family history of retinoblastoma. There was no difference in the frequency or type of deletion in patients with and without a positive family history [4 of 26 patients (15%) vs. 11 of 66 patients (17%)1. In 5 patients (396, 406, 408,443, and 491) the entire RB-1 gene was deleted. In 2 patients (374 and 440) the 5’ end was deleted, and in 5 patients (386, 056, 441, 446, and 511) the 3’ end. Three patients (428, 369, and 380)
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Fig. 1. Restriction map of the RB-1 gene and 15 mutant alleles. The gene consists of 27 exons spread over 200 kb. The cDNA fragments R0.6 and R3.8 are indicated above the gene. The Hind111and SacI restriction map as well as the genomic subclones p123, H3-8, and p68 are shown below the gene. The deletions present in 15patients (numbers on the right) are indicated by a gap. The dashed lines represent breakpoint regions.
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Fig. 2. hstriction fragment patterns ofpatients 441,380,369, and a normal control (C) as analysed by Southern blot hybridization with the enzymes and probes given underneath the lanes.
had intragenic deletions. Deletion hot spots in the RB-1 gene were not detected (Fig. 1).
DISCUSSION This is the first extensive molecular study of a series of unrelated patients with bilateral retinoblastoma to assess the frequency of gross structural germ line mutations at the RB-1 locus. A deletion of all or part of the RB-1 gene was found in 16% of patients irrespective of their family history. These results confirm and extend previous findings obtained in smaller patient groups [Horsthemke et al., 1987a; Wiggs et al., 1988;Scheffer et al., 19891. Assuming that most of the isolated cases are caused by de novo germ line mutations [Vogel,19791,our results mean that deletions are transmitted at the same rate as non-deletion mutations. Cytogenetically visible deletions that are the cause of mental and physical retardation in some patients, however, are rarely transmitted. Although cytogenetic analysis was not performed on the patients, the lack of physical or mental retardation in any of the patients except for 406 makes it unlikely that important genes other than the RB-1 gene were
deleted. However, we cannot exclude that some patients do have small visible deletions. Patient 406 will now be studied by chromosome analysis. Because of the lack of narrowly spaced DNA probes at either side of the RB-1 gene, the presence of extragenic hot spots for deletions cannot be excluded. Intragenic hot spots for deletions were not observed. Therefore, in each new patient the entire gene needs to be searched for the mutation. Our data indicate that 3 probes ( ~ 1 2 3H3-8, , and R3.8) are sufficient to detect most deletions. Recent cloning and sequencing studies have identified short repeats at different locations within the RB-1 gene, which may predispose to deletions [Canning and Dryja, 1989; Greger et al., 19901. On the other hand, Alu-DNA sequences, which are frequently found to be involved in deletions in the LDL-receptor gene [Lehrman et al., 1985; Horsthemke et al., 1987~1,do not appear to be involved in RB-1 gene deletions. The breakpoints in some of our patients will be cloned and sequenced in order to determine whether short repeats are involved in their deletions. Our study shows that densitometric analysis is important in the identification of RB-1 gene deletions. Abnormal restriction fragment patterns were observed only in 3 of 15deletions. Thus, most of the deletions (80%)would not have been detected by qualitative Southern blot analysis alone. As quantitative Southern blot hybridization is difficult, at least 2 independent experiments as well as densitometry are necessary. It should be noted, however, that Southern blot analysis is not sensitive enough to detect partial gene duplications (1.5-foldsignal strength difference compared to a 2-fold difference in deletions) unless abnormal restriction fragment patterns are generated. Thus, the frequency of gross struttural alterations may exceed 16%. We have found that small blots hybridized in a rotary oven give the best results. Appropriate internal hybridization standards have to be included in the analysis, and the film response has to be in the linear range. Most of the RB-1 gene fragments give good hybridization signals, which can reliably be analyzed by densitometry. Since exons 1to 4 do not give suitable signals with the cDNA probe, we used genomic subclones to detect deletions involving exons 1 and 4. Most germinal mutations at the RB-1 locus are obviously too small to be identified by Southern blot analysis and require different detection techniques, RNase protection and direct sequencing are useful in research and special diagnostic situations, but not generally applicable or too laborious for rapid diagnostic testing. Predictive DNA testing in retinoblastoma can effectively aid ophthalmologic diagnosis and genetic counseling. In the past 2 years we have performed 8 DNA tests in families with 2 or more affected members, 2 prenatally and 6 at birth. All children had a formal genetic risk of 50%.Direct detection of the genetic lesion was possible in 3 families. In 5 families, segregation analysis with linked DNA markers had to be used. Four children were identified as carriers, and in each of them retinoblastoma was diagnosed within the first 2 weeks after birth. The other 4 were found to have normal RB-1 alleles, and each of them is still tumor-free. Subse-
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Fig. 3. Quantitative Southern blot analysis of 5 different patients (408,379,385,429,380),and a normal control (C). R0.6, R3.8, p123, H3-8, and p68 represent RB-1 gene probes (see Fig. 1).9 D l l and L32 are internal hybridization standards. Representative blots for each experiment are shown. The RsaI polymorphism blot shown in E was evaluated qualitatively only (see text). 379 and 385 are heterozygous and assumed to have 2 copies of this part of the gene. Using quantitative Southern blot hybridizations (not shown), 408, but not 429, was found to have a deletion around p68. The blots shown in A-D, and F were analysed by densitometry. The relative hybridization intensities of the RB-1 gene fragments:standard (probes in parentheses) in the patients and normal controls are as follows: A) 7.O(R0.6):2.4(9Dll)= 0.23 (control:0.48), 6.8(R0.6):2.4(9D11) = 2.4 (control:5.5); B) 10.O(R3.8):2.4(9Dll) = 2.1 (control:5.5), 7.5(R3.8):2.4(9D11)= 0.9 (control:1.9),6.2(R3.8):2.4(9Dll) = 3.4 (control:7.6), 5.3(R3.8):2.4(9D11)= 1.1 (control:2.5);C) 6.1(p123):10.5(L32)= 0.8 (control:2.2); D) 1.5(H3-8):2.4(9D11)= 0.43 (control:0.88); F) 9.4(p68):2.4(9D11)= 1.0 (control:1.9).
quently, the frequency of ophthalmologic examinations has been reduced. The direct identification of a n RB-1 gene mutation as reported here will allow predictive testing also in sibs and offspring of some patients with isolated retinoblastoma.
ACKNOWLEDGMENTS Part of this work was supported by the Deutsche Forschungsgemeinschaft and the Deutsche Krebshilfe. We thank Drs. W.K. Cavenee, Montreal, T.P. Dryja, Boston, and S.A. Latt, Boston, for providing the DNA probes, and Birgit Brandt for expert technical assistance. REFERENCES Bookstein R, Lee EYHP, Young LJ,Sery TW, Hayes RC, Friedmann T, Lee WH (1988): Human retinoblastoma susceptibility gene: genomic organisation and analysis of heterozygous intragenic deletion mutants. Proc Natl Acad Sci USA 85:2210-2214. Canning S, Dryja TP (1989):Short direct repeats at the breakpoints of deletions of the retinoblastoma gene. Proc Natl Acad Sci USA 86:5044-5048. Cavenee WK. Leach R, Mohandas T, Pearson P, White R (1984):Isola-
tion and regional localization of DNA segments revealing polymorphic loci from human chromosome 13. Am J Hum Genet 36:lO-24. Cavenee WK, Murphree AL, Shull MM, Benedict WF, Sparkes RS, Kock E, Nordenskjold M (1986): Prediction of familial predisposition t o retinoblastoma. N Engl J Med 314:1201-1207. Dryja TP, Rapaport JM, Joyce JM, Petersen RA (1986): Molecular detection of deletions involving band q14 of chromosome 13 in retinoblastomas. Proc Natl Acad Sci USA 83:7391-7394. Dunn JM, PhilliDs RA. Becker AJ. Gallie BL (1988): Identification of germline and'somatic mutations affecting the retinoblastoma gene. Science 241:1797-1800. Friend SH, Bernards R, Rogelj S, Weinberg RA, Rapaport JM, Albert DM, DryjaTP (1986):A human DNA segment with properties ofthe gene that predisposes to retinoblastoma and osteosarcoma. Nature 323:643-646. Goddard AD, Balakier H, Canton M, Dunn J , Squire J, Reyes E, Becker A, Phillips RA, Gallie BL (1988): Infrequent genomic rearrangement and normal expression of the putative RB1 gene in retinoblastoma tumors. Mol Cell Biol 82082-2088. Goddard AD, Phillips RA, Greger V, Passarge E, Hopping W, Gallie BL, Horsthemke B (1990): Use of the RB1 cDNA as a diagnostic probe in retinoblastoma families. Clin Genet 37:117-126. Greger V, Kerst S, Messmer E, Hopping W, Passarge E, Horsthemke B (1988): Application of linkage analysis to genetic counselling in families with hereditary retinoblastoma. J Med Genet 25:217-221.
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Greger V, Passarge E, Horsthemke B (1990): Somatic mosaicism in a patient with bilateral retinoblastoma. Am J Hum Genet 46:11871193. Hong FD, Huang HJS, To H, Young USY, Oro A, Bookstein R, Lee EYHP, Lee WH (1989): Structure of the human retinoblastoma gene. Proc Natl Acad Sci USA 865502-5506. Horsthemke B, Barnert HJ, Greger V, Passarge E, Hopping W (1987a): Early diagnosis in hereditary retinoblastoma by detection of molecular deletions at gene locus. Lancet i:511-512. Horsthemke B, Greger V, Barnert HJ, Hopping W, Passarge E (1987b): Detection of submicroscopic deletions and a DNA polymorphism a t the retinoblastoma locus. Hum Genet 76:257-261. Horsthemke B, Beisiegel U, Dunning A, Havinga JR, Williamson R, Humphries S (1987~): Unequal crossing-over between two alu-repetive DNA sequences in the low-density-lipoprotein-receptorgene. Eur J Biochem 164:77-81. Lalande M, Dryja TP, Schreck RR, Shipley J , Flint A, Latt SA (1984): Isolation of human chromosome 13 DNA sequences cloned from flow-sorted libraries. Cancer Genet Cytogenet 13:283-295. Lehrman MA, Schneider WE, Sudhof TC, Brown MS, Goldstein JL, Russell DW (1985):Mutation in LDL receptor: alu-alu recombination deletes exons encoding transmembrane and cytoplasmic domaines. Science 227:140-145. Ludecke HJ, Senger G, Claussen U, Horsthemke B (1989): Cloning
defined regions of the human genome by microdissection of banded chromosomes and enzymatic amplification. Nature 338:348-350. McGee T, Yandell DW, Dryja TP (1989): Structure and partial genomic sequence of the human retinoblastoma susceptibility gene. Gene 80:119-128. Scheffer H, te Meerman GJ, Kruize YCM, van den Berg AHM, Penninga DP, Tan KEWP, der Kinderen DJ, Buys CHCM (1989): Linkage analysis of families with hereditary retinoblastoma: Nonpenetrance of mutation, revealed by combined use of markers within and flanking the RBI gene. Am J Hum Genet 45:252-460. T'Ang A, Wu KJ, Hashimoto T, Liu WH, Takahashi R, Shi XH, Mihara K, Zhang FH, Chen YY, Du C, Quian J , Lin YG, Murphree AL, Qiu WR, ThompsonT, Benedict WF, Fung YKT (1989):Genomic organization of the human retinoblastoma gene. Oncogene 4:401-407. Vogel F (1979): Genetics of retinoblastoma. Hum Genet 52:l-54. Wiggs J,Nordenskjold M, Yandell D, Rapaport J, Grondin V, Janson M, Werelius B, Petersen R, Craft A, Riedel K, Liberfarb R, Walton D, Wilson W, Dryja TP (1988): Prediction of the risk of hereditary retinoblastoma, using DNA polymorphisms within the retinoblastoma gene. N Engl J Med 318:151-157. Yandell DW, Campbell TA, Dayton SH, Petersen R, Walton D, Little JB, McConkie-Rose11 A, Buckley EG, Dryja TP (1989): Oncogenic point mutations in the human retinoblastoma gene: Their application to genetic counseling. N Engl J Med 321:1689-1695.
Characterization of Deletions at the Retinoblastoma Locus in Patients With Bilateral Retinoblastoma Kai Kloss, Petra Wahrisch, Valerie Greger, Elmar Messmer, Henning Fritze, Wolfgang Hopping, Eberhard Passarge, and Bernhard Horsthemke Znstitut fur Humangenetik (K.K., P.W., V.G., E.P., B.H.) and Zentrum fur Augenheilkunde (E.M., H.F., W.H.), Universitatsklinikum Essen, Germany
DNA samples from 92 unrelated patients with bilateral retinoblastoma were analyzed by Southern blot hybridization with cDNA and genomic clones of the retinoblastoma (RB-1) gene. Qualitative and quantitative evaluation of the Southern blot patterns showed a deletion of all or part of the RB-1 gene in 15 patients. Deletion hot spots were not detected. The study shows that 16% of germ cell mutations are detectable by Southern blot hybridization, but that densitometric analysis is required in most cases. KEY WORDS: RB-1 gene, Southern blot patterns, cDNA
RNase protection assays and direct sequencing of enzymatically amplified gene fragments have successfully been employed [Dunn et al., 1988; Yandell et al., 19891. However, the first method is not generally applicable, and the second is too laborious for rapid diagnostic testing. Since most studies on RB-1 gene deletions have been performed on tumor cells, which carry either a germ line and a somatic mutation or 2 somatic mutations [Dryja et al., 1986; F’riend et al., 1986; Bookstein et al., 1988; Goddard et al., 19881, we asked what proportion of germ line mutations can be detected by Southern blot hybridization. Here we describe the analysis of 92 lymphocyte DNA samples from patients with bilateral retinoblastoma.
MATERIALS AND METHODS Patients INTRODUCTION Two hundred seventy patients who had been treated Approximately 40% of patients with retinoblastoma for bilateral retinoblastoma a t the Department of Ophcarry a germ line mutation at the retinoblastoma (RB-1) thalmology, University of Essen, between 1968 and locus on chromosome 13 [Vogel, 19791. Most of these 1982 were asked for follow-up examinations. Diagnosis patients develop bilateral tumors. Non-hereditary reti- had been established by current ophthalmologic critenoblastoma occurs in 60% of patients and is always ria. Ninety-nine patients came, and peripheral blood unilateral. Predictive DNA diagnosis in sibs and off- was obtained from 92 unrelated patients. Twenty-six spring of patients helps to detect tumor development in patients (28%) had one or more affected relatives; 66 carriers or to avoid frequent ophthalmologic examina- patients (72%) were isolated cases. Patient 395 had an tions under general anesthesia in children not a t risk. In osteosarcoma in the paranasal sinus. Patient 406 was families with 2 or more affected individuals, the risk slightly retarded. Cytogenetic analysis was not perassessment can be based on direct detection of the muta- formed. tion [Horsthemke et al., 1987al or genetic segregation DNA Analysis analysis with linked DNA markers [Cavenee et al., 1986; Greger et al., 1988; Wiggs et al., 1988; Scheffer et DNA was isolated and analyzed by Southern blot hyal., 1989; Goddard et al., 19901. bridization as described before [Horsthemke et al., In contrast, predictive DNA diagnosis in isolated 1987bl. Blots were exposed to preflashed films for one to cases requires direct identification of the mutation. 3 days. Each DNA sample was studied in a t least 2 While gross structural alterations can easily be detected independent experiments. The RB-1 cDNA clone [Friend by Southern blot hybridization, point mutations are dif- et al., 19861 and the genomic subclones p123 and p68 ficult to identify and require different techniques. [Wiggs et al., 19881 were provided by Dr. T.P. Dryja, Boston. The genomic RB-1 fragment H3-8 [Lalande et al., 19841was from Dr. S.A. Latt, Boston. 9 D l l (D13S2) Received for publication January 18, 1990; revision received and L32 (D8S48) were used as internal hybridization April 17, 1990. standards. 9 D l l [Cavenee et al., 19841 was provided by Address reprint requests to Dr. Bernhard Horsthemke, Institut Dr. W.K. Cavenee, Montreal. L32 was obtained by mifiir Humangenetik, Universitatsklinikum Essen, Hufelandstrasse crodissection of 8q [Liidecke et al., 19891.Relative inten5 5 , D-4300 Essen 1, Germany. 0 1991 Wiley-Liss, Inc.
Gene Deletions in Retinoblastoma sities of hybridization signals were determined with the help of a SHIMADZU CS 9000 densitometer.
RESULTS DNA samples were analyzed by Southern blot hybridization with cDNA and genomic probes for the RB-1 gene. Since the gene has 27 exons, which are spread over 200 kb [McGee et al., 1989; Hong et al., 1989; T’Ang et al., 19891, the 4.7 kb cDNA was cut into 3 fragments with HpaI and EcoRI (Fig. 1).R0.6 and R3.8 detect exons 3-8 and 9-27, respectively. Since exons 3 and 4 did not give strong hybridization signals with probe R0.6, the genomic subclone H3-8, which contains exon 4, was used to detect deletions involving exon 4. The 0.3 kb EcoRIHpaI fragment at the 5’ end ofthe cDNA is very GC-rich and did not give clear Southern patterns. Therefore, the genomic subclone p123 was used for identifying deletions involving exon 1. For mapping breakpoints within intron 17, which spans approximately 90 kb, we used the genomic subclone p68. This probe detects a highly informative “variable number of tandem repeats” polymorphism on RsaI blots [Wiggs et al., 19881. Patients found to be heterozygous in this test were assumed to have 2 copies of this part of the gene. The other patients were studied by quantitative Southern blot hybridization.
9 D l l (D13S21, which maps to 13q22 [Cavenee et al., 19841, and L32 (D8S48),which maps to 8q24.1 [Ludecke et al., 19891, were used as internal hybridization standards for densitometric analysis. Abnormal Southern patterns were detected in 3 samples (441,380, and 369; Fig. 2). In 369, but not in 380 and 441, a n abnormal band was also detected in SacI digests. Quantitative studies in 380 (Fig. 3) and 441 (not shown) showed loss of genetic material adjacent to the abnormal gene fragment. This indicates that the abnormal bands do not represent neutral DNA polymorphisms, but deletion junction fragments. Densitometric evaluation of the Southern blots showed deletions in another 12 cases. Representative blots are shown in Figure 3. Thus, a total of 15 deletions were identified in 92 patients (16%). Four of the 15 patients (056,408,491, and 511) have a positive family history of retinoblastoma. There was no difference in the frequency or type of deletion in patients with and without a positive family history [4 of 26 patients (15%) vs. 11 of 66 patients (17%)1. In 5 patients (396, 406, 408,443, and 491) the entire RB-1 gene was deleted. In 2 patients (374 and 440) the 5’ end was deleted, and in 5 patients (386, 056, 441, 446, and 511) the 3’ end. Three patients (428, 369, and 380)
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Fig. 1. Restriction map of the RB-1 gene and 15 mutant alleles. The gene consists of 27 exons spread over 200 kb. The cDNA fragments R0.6 and R3.8 are indicated above the gene. The Hind111and SacI restriction map as well as the genomic subclones p123, H3-8, and p68 are shown below the gene. The deletions present in 15patients (numbers on the right) are indicated by a gap. The dashed lines represent breakpoint regions.
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Fig. 2. hstriction fragment patterns ofpatients 441,380,369, and a normal control (C) as analysed by Southern blot hybridization with the enzymes and probes given underneath the lanes.
had intragenic deletions. Deletion hot spots in the RB-1 gene were not detected (Fig. 1).
DISCUSSION This is the first extensive molecular study of a series of unrelated patients with bilateral retinoblastoma to assess the frequency of gross structural germ line mutations at the RB-1 locus. A deletion of all or part of the RB-1 gene was found in 16% of patients irrespective of their family history. These results confirm and extend previous findings obtained in smaller patient groups [Horsthemke et al., 1987a; Wiggs et al., 1988;Scheffer et al., 19891. Assuming that most of the isolated cases are caused by de novo germ line mutations [Vogel,19791,our results mean that deletions are transmitted at the same rate as non-deletion mutations. Cytogenetically visible deletions that are the cause of mental and physical retardation in some patients, however, are rarely transmitted. Although cytogenetic analysis was not performed on the patients, the lack of physical or mental retardation in any of the patients except for 406 makes it unlikely that important genes other than the RB-1 gene were
deleted. However, we cannot exclude that some patients do have small visible deletions. Patient 406 will now be studied by chromosome analysis. Because of the lack of narrowly spaced DNA probes at either side of the RB-1 gene, the presence of extragenic hot spots for deletions cannot be excluded. Intragenic hot spots for deletions were not observed. Therefore, in each new patient the entire gene needs to be searched for the mutation. Our data indicate that 3 probes ( ~ 1 2 3H3-8, , and R3.8) are sufficient to detect most deletions. Recent cloning and sequencing studies have identified short repeats at different locations within the RB-1 gene, which may predispose to deletions [Canning and Dryja, 1989; Greger et al., 19901. On the other hand, Alu-DNA sequences, which are frequently found to be involved in deletions in the LDL-receptor gene [Lehrman et al., 1985; Horsthemke et al., 1987~1,do not appear to be involved in RB-1 gene deletions. The breakpoints in some of our patients will be cloned and sequenced in order to determine whether short repeats are involved in their deletions. Our study shows that densitometric analysis is important in the identification of RB-1 gene deletions. Abnormal restriction fragment patterns were observed only in 3 of 15deletions. Thus, most of the deletions (80%)would not have been detected by qualitative Southern blot analysis alone. As quantitative Southern blot hybridization is difficult, at least 2 independent experiments as well as densitometry are necessary. It should be noted, however, that Southern blot analysis is not sensitive enough to detect partial gene duplications (1.5-foldsignal strength difference compared to a 2-fold difference in deletions) unless abnormal restriction fragment patterns are generated. Thus, the frequency of gross struttural alterations may exceed 16%. We have found that small blots hybridized in a rotary oven give the best results. Appropriate internal hybridization standards have to be included in the analysis, and the film response has to be in the linear range. Most of the RB-1 gene fragments give good hybridization signals, which can reliably be analyzed by densitometry. Since exons 1to 4 do not give suitable signals with the cDNA probe, we used genomic subclones to detect deletions involving exons 1 and 4. Most germinal mutations at the RB-1 locus are obviously too small to be identified by Southern blot analysis and require different detection techniques, RNase protection and direct sequencing are useful in research and special diagnostic situations, but not generally applicable or too laborious for rapid diagnostic testing. Predictive DNA testing in retinoblastoma can effectively aid ophthalmologic diagnosis and genetic counseling. In the past 2 years we have performed 8 DNA tests in families with 2 or more affected members, 2 prenatally and 6 at birth. All children had a formal genetic risk of 50%.Direct detection of the genetic lesion was possible in 3 families. In 5 families, segregation analysis with linked DNA markers had to be used. Four children were identified as carriers, and in each of them retinoblastoma was diagnosed within the first 2 weeks after birth. The other 4 were found to have normal RB-1 alleles, and each of them is still tumor-free. Subse-
Gene Deletions in Retinoblastoma
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Fig. 3. Quantitative Southern blot analysis of 5 different patients (408,379,385,429,380),and a normal control (C). R0.6, R3.8, p123, H3-8, and p68 represent RB-1 gene probes (see Fig. 1).9 D l l and L32 are internal hybridization standards. Representative blots for each experiment are shown. The RsaI polymorphism blot shown in E was evaluated qualitatively only (see text). 379 and 385 are heterozygous and assumed to have 2 copies of this part of the gene. Using quantitative Southern blot hybridizations (not shown), 408, but not 429, was found to have a deletion around p68. The blots shown in A-D, and F were analysed by densitometry. The relative hybridization intensities of the RB-1 gene fragments:standard (probes in parentheses) in the patients and normal controls are as follows: A) 7.O(R0.6):2.4(9Dll)= 0.23 (control:0.48), 6.8(R0.6):2.4(9D11) = 2.4 (control:5.5); B) 10.O(R3.8):2.4(9Dll) = 2.1 (control:5.5), 7.5(R3.8):2.4(9D11)= 0.9 (control:1.9),6.2(R3.8):2.4(9Dll) = 3.4 (control:7.6), 5.3(R3.8):2.4(9D11)= 1.1 (control:2.5);C) 6.1(p123):10.5(L32)= 0.8 (control:2.2); D) 1.5(H3-8):2.4(9D11)= 0.43 (control:0.88); F) 9.4(p68):2.4(9D11)= 1.0 (control:1.9).
quently, the frequency of ophthalmologic examinations has been reduced. The direct identification of a n RB-1 gene mutation as reported here will allow predictive testing also in sibs and offspring of some patients with isolated retinoblastoma.
ACKNOWLEDGMENTS Part of this work was supported by the Deutsche Forschungsgemeinschaft and the Deutsche Krebshilfe. We thank Drs. W.K. Cavenee, Montreal, T.P. Dryja, Boston, and S.A. Latt, Boston, for providing the DNA probes, and Birgit Brandt for expert technical assistance. REFERENCES Bookstein R, Lee EYHP, Young LJ,Sery TW, Hayes RC, Friedmann T, Lee WH (1988): Human retinoblastoma susceptibility gene: genomic organisation and analysis of heterozygous intragenic deletion mutants. Proc Natl Acad Sci USA 85:2210-2214. Canning S, Dryja TP (1989):Short direct repeats at the breakpoints of deletions of the retinoblastoma gene. Proc Natl Acad Sci USA 86:5044-5048. Cavenee WK. Leach R, Mohandas T, Pearson P, White R (1984):Isola-
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