- Email: [email protected]
Susceptibility of skin fibroblasts from patients with retinoblastoma to transformation by murine sarcoma virus
137
Cancer Letters, 18 (1983) 137-142 Elsevier Scientific Publishers Ireland Ltd.
SUSCEPTIBILITY OF SKIN FIBROBLASTS RETINOBLASTOMA TO TRANSFORMATION VIRUS
MICHIKO MIYAKIa
FROM PATIENTS WITH BY MURINE SARCOMA
, NORIKO AKAMATSUa, TETSUO ONOa and MASAO S. SASAKIb
aDepartment of Biochemistry, The Tokyo Metropolitan Institute of Medical Science, 3-18-22 Honkomagome, Bunkyoku, Tokyo 113 and bRadiation Biology Center, Kyoto University, Yoshida-Konoecho, Sakyoku, Kyoto 606 (Japan) (Received 3 June 1982) (Revised version received 6 November 1982) (Accepted 17 November 1982)
SUMMARY
Cultured skin fibroblasts from patients with retinoblastoma (RB) of different etiology have been studied for their susceptibility to transformation by murine sarcoma virus. The cells from patients with a deletion in chromosome 13 (13q-) and those from sporadic unilateral cases due to somatic mutation were as sensitive as normal cells. However, the cells from familial cases showed an extremely high sensitivity to transformation. Moreover, in familial cases the susceptibility was significantly higher in bilaterally affected patients than in unilateral cases. These findings suggest that the heritable RB gene is different from 13q- and its degree of expression is also manifest at the cellular level.
INTRODUCTION
Retinoblastoma is a malignant eye tumor of infants and young children and occurs either by inheriting germinal mutation of autosomal dominant mode of expression with a limited degree of penetrance and expression, or by somatic mutation in embryonic retinal cells [16]. A small class of germinal mutations, called l3q-, has been demonstrated to involve an interstitial deletion of chromosome 13 including band q14. Sporadic nonhereditary RB due to somatic mutation is mostly unilateral, while the hereditary form and those with 13q- are either unilateral or bilateral. The difference in the expression as manifested by tumor laterality led Knudson [ 3-51 to the hypothesis that RB is caused by 2 mutational events; in the hereditary form, one mutation is inherited via germinal cells and the second 0304-3835/83/0000-0000/$03.00 o 1983 EIsevier Scientific Publishers Ireland Ltd. - Published and Printed in Ireland
138
occurs in somatic cells, whereas in the non-hereditary form, both mutations occur in somatic cells. To explain different expression of the RB gene, he assumed that the second mutation process was distributed according to the Poisson scheme. However, Matsunaga [ 6-81 has proposed inherited host resistance; the different expression of the RB gene among carriers is caused by multifactorial inheritance with 2 thresholds, and thus tumor formation in the less sensitive group with hereditary unilateral cases is suppressed twice that in the most susceptible group with bilateral cases. The cellular manifestation of the RB gene and its relevance to the development of tumors are of particular interest in the etiology of RB. Here, we describe a distinct difference in the susceptibility to viral transformation between different groups of RB and the finding which suggests that the expression of the heritable RB gene is constitutively determined at the cellular level. MATERIALS
AND METHODS
Fibroblast strains were established from biopsies of 19 patients with RB, 3 unaffected family members of RB patients and 2 normal individuals. The cells were cultivated in Eagle’s minimum essential medium supplemented with heat-inactivated 13% fetal bovine serum and passaged as described [ 121. The virus was obtained from the culture medium of rat cell 78Al transformed by Moloney MSV (MLV). The 78Al virus sample contained Moloney MSV, XC’ ecotropic MLV and XC+ dualtropic MLV; the former MLV replicates only in mouse cells and the latter MLV replicates both in mouse cells and in non-mouse cells [ 111. The titer of virus suspension used in the present experiment was 160 FFUIO.2 ml when assayed on S’L- mink cells and 20 FFLJIO.2 ml when assayed on normal mink cells. At 20 h after seeding of 8 X lo4 cells per 35 mm plastic dish, the cells were treated with 4 pg/ml polybrene for 1 h and infected by 0.2 ml of Moloney 78Al-MSV (MLV). After cultivation for 11 days the foci of transformed cells were counted. Values are averages of 2 or 3 independent experiments, and each experiment contained PL78 (skin fibroblasts from a patient with familial adenomatosis coli) as a high control, and C4 (skin fibroblasts from a normal individual) as a low control, the susceptibilities to viral transformation of which have previously been described [ 111. RESULTS
AND DISCUSSION
Table 1 summarizes the clinical and karyotypic data of patients studied in this experiment. The cultured skin fibroblasts from patients with RB and those from normal individuals were infected by Moloney murine sarcoma virus of rat 78Al cells, 78Al-MSV(MLV), and the foci of transformed cells were counted. Figure 1 shows the number of foci produced in the fibroblast strains from different groups of RB, the number indicating the susceptibility of fibroblasts to viral transformation.
139 TABLE 1 CLINICAL AND KARYOTYPIC
DATA OF FIBROBLAST
Heredity
Patient
Sex
Tumor laterality
Sporadic
RB RB RB RB RB RB RB RB RB RB RB
ilT 16T 32T 1AW 9T 1K 25T 28T 4lT 50T 51T
F M M M F M F M M F F
B B B B B U U U U U U
RB RB RB RB RB RB RB RB
15T 18T 38T 54T 14T 17T 36T 37T
F F F M M M M M
Familial
Normal
N 1KM N 3T N 35T N C2 NC4
F F F F F
Age at biopsy (years/months) 614 413 219 312 115 314 114 215 117 110 213
30/l 3816 4918 3317 o/11 419 45/o
1014
-
2119 3112
-
314
1116 2916
STRAINS Karyotype
46,XX,del(l3) 46,XY,del(13) 46,XY,del(l3) 46,XY,del(l3) 46,X,t (13;X) 46,XY 46,Xx 46,XY 46,XY 46,Xx 46,Xx 46,Xx 46,Xx 46,Xx 46,XY 46,XY 46,XY 46,XY 46,XY 46,Xx 46 ,XX 46 yx 46$X 46,Xx.
(q12q22) (q14q22) (q14) (q12q22) (q12;p22)
(mother of RB14T) (mother of RB17T)
(father of RB37T)
I
(unaffected family member of RB)
\ (normal)
The fibroblasts from familial bilateral cases exhibited 4-5-fold higher transformation frequencies than normal. These values were comparable to the highest frequencies for the cells from patients with familial adenomatosis coli [ 111. The susceptibilities of unilateral familial RB were about half that ol the bilateral cases but the mean value of this group was still twice that of normal individuals. The sporadic unilateral cases with normal karyotype showed rather low susceptibilities which were comparable to those of normal individuals. RB patients associated with chromosomal 13 anomalies in this experiment consisted of 4 cases with interstitial deletion of region involving band 13q14 and one case with reciprocal translocation between chromosome 13 and one of two X chromosomes. In case of the 13/X translocation, the translocated X was late replicating and hence the inactivation was supposed to spread over to the translocated chromosome 13 including critical region of q14 [ 141. All of the chromosome 13 anomalies including the 13/X translocation were herewith referred to as 13q-. As seen in Fig. 1, the patients
0 37 T
( OPL7 ‘8)
8 ;i: :
0 171r
o 54T 0 38T 015T 0 35T
OlT
OlKM
0 1AW
8% oc2
Normal
o 16T o 9T
0
I
Ef
018T
28T %T 1K
0 32T
Familial bilateral
L (1425)
:6lf
10)
Fig. 1. Susceptibilities of fibroblasts from patients with different groups of RB and normal individuals to transformation by murine sarcoma virus. Number of foci was determined as described in Materials and Methods. Each value is average of 2 or 3 independent experiments. The mean f S.E. for each RB group is indicated in parentheses at the bottom of each column.
with 13q-, all sporadic but bilaterally affected, had susceptibilities as low as those of normal individuals. Although the mechanism of susceptibility of fibroblasts to viral transformation is not understood well, the cells from patients with heritable neoplasms have reported to show increased transformation frequencies [2,10,11,13,]. The present data are also consistent with these lines of experimental evidence. Qf particular interest is the fact that the viral transformation promoted by the heritable RB gene seems to correlate not simply to the presence of the RB gene itself but rather intimately with its expression. The susceptibility to viral transformation was higher in bilaterally affected patients than in unilaterally affected patients even though they were members of the same family and inherited the same RB gene. Although the nature of the RB gene is not clear, the RB gene and some other dominantly expressed cancer-predisposing genes have been described as possibly related to a type
141
of gene-controlling element [ 9,14,15]. It is thus tempting to correlate the differential susceptibility to viral transformation with the RB gene-controlled expression or integration of viral genomes, or enhancement of more general genetic rearrangement favoring carcinogenesis. The 13q- is a simple structural loss of a critical region and hence might not act as a gene-controlling element, as has been shown in this report by the lack of the ability to promote viral transformation. Preliminary experiments suggest that the susceptibility of RB fibroblasts to viral transformation is not due to the replication of helper virus, because the number of transformed foci was not parallel to the titer of MLV released from the infected cells to the culture medium. Further examinations are needed to clarify whether these RB fibroblasts have increased susceptibility to transformation by other pseudotypes of MSV and chemical carcinogens. ACKNOWLEDGEMENT
This work was supported in part by grants for cancer research from the Ministry of Education, Science and Culture of Japan, and from The Princess Takamatsu Cancer Research Fund.
REFERENCES 1 Ejima, Y., Sasaki, M.S., Kaneko, A., Tanooka, H., Hara, Y., Hieda, T. and Kinishita, Y. (1982) Possible inactivation of part of chromosome 13 due to 13/X translocation associated with retinoblastoma. Clin. Genet. 21, 357-361. 2 Klement, V., Freedman, M.H., McAllister, R.M., Nelson-Rees, W.A. and Heubner, R.J. (1971) Differences in susceptibility of human cells to mouse sarcoma virus. J. Natl. Cancer Inst., 47, 65-73. 3 Knudson, A.G. (1971) Mutation and cancer: statistical study of retinoblastoma. Proc. Natl. Acad. Sci. U.S.A., 68,820-823. 4 Knudson, A.G., Hethcote, H.W. and Brown, B.W. (1975) Mutation and childhood cancer: a probabilistic model for the incidence of retinoblastoma. Proc. Natl. Acad. Sci. U.S.A., 72, 5116-5120. 5 Knudson, A.G., Meadows, A.T., Nichols, W.W. and Hill, R. (1976) Chromosomal deletion and retinoblastoma. N. Engl. J. Med., 295, 1120-1123. 6 Matsunaga, E. (1979) Hereditary retinoblastoma: host resistance and age at onset. J. Natl. Cancer Inst., 63, 933-939. 7 Matsunaga, E. (1980) Hereditary retinoblastoma: host resistance and second primary tumors. J. Natl. Cancer Inst., 65, 47-51. 8 Matsunaga, E. (1980) Retinoblastoma: host resistance and 13q- chromosomal deletion. Hum. Genet., 56, 53-58. 9 McClintock, B. (1950) The origin and behaviour of mutable loci in maize. Proc. Natl. Acad. Sci. U.S.A., 36, 344-355. 10 Miller, R.M. and Todaro, G.J. (1969) Viral transformation of cells from persons at high risk of cancer. Lancet, I, 81-82. 11 Miyaki, M., Akamatsu, N., Rokutanda, M., Ono, T., Yoshikura, H., Sasaki, M.S., Tonomura, A. and Utsunomiya, J. (1980) Increased sensitivity of fibroblasts of skin from patients with adenomatosis coli and Peutz-Jeghers’ syndrome to transformation by murine sarcoma virus. Gann, 71, 797-803.
142 12 Miyaki, M., Akamatsu, N., Ono, T., Tonomura, A. and Utsunomiya, J. (1982) Morphologic transformation and chromosomal changes induced by chemical carcinogens in skin fibroblasts from patients with familial adenomatosis coli. J. Natl. Cancer Inst., 68,563-571. 13 Pfeffer, L.M. and Kopelovich, L. (1977) Differential genetic susceptibility of cultured human skin fibroblasts to transformation by Kirsten murine sarcoma virus. Cell, 10, 313-320. 14 Sasaki, M.S. (1982) Dominantly expressed procancer mutations and induction of chromosome rearrangements. Prog. Mutat. Res., 4, 75 -84. 15 Sasaki, MS., Tsunematsu, Y., Utsunomiya, J. and Utsumi, J. (1980) Site-directed chromosome rearrangements in skin fibroblasts from persons carrying genes for hereditary neoplasms. Cancer Res., 40,4796-4803. 16 Vogel, F. (1979) Genetics of retinoblastoma. Hum. Genet., 52, l-54.
Cancer Letters, 18 (1983) 137-142 Elsevier Scientific Publishers Ireland Ltd.
SUSCEPTIBILITY OF SKIN FIBROBLASTS RETINOBLASTOMA TO TRANSFORMATION VIRUS
MICHIKO MIYAKIa
FROM PATIENTS WITH BY MURINE SARCOMA
, NORIKO AKAMATSUa, TETSUO ONOa and MASAO S. SASAKIb
aDepartment of Biochemistry, The Tokyo Metropolitan Institute of Medical Science, 3-18-22 Honkomagome, Bunkyoku, Tokyo 113 and bRadiation Biology Center, Kyoto University, Yoshida-Konoecho, Sakyoku, Kyoto 606 (Japan) (Received 3 June 1982) (Revised version received 6 November 1982) (Accepted 17 November 1982)
SUMMARY
Cultured skin fibroblasts from patients with retinoblastoma (RB) of different etiology have been studied for their susceptibility to transformation by murine sarcoma virus. The cells from patients with a deletion in chromosome 13 (13q-) and those from sporadic unilateral cases due to somatic mutation were as sensitive as normal cells. However, the cells from familial cases showed an extremely high sensitivity to transformation. Moreover, in familial cases the susceptibility was significantly higher in bilaterally affected patients than in unilateral cases. These findings suggest that the heritable RB gene is different from 13q- and its degree of expression is also manifest at the cellular level.
INTRODUCTION
Retinoblastoma is a malignant eye tumor of infants and young children and occurs either by inheriting germinal mutation of autosomal dominant mode of expression with a limited degree of penetrance and expression, or by somatic mutation in embryonic retinal cells [16]. A small class of germinal mutations, called l3q-, has been demonstrated to involve an interstitial deletion of chromosome 13 including band q14. Sporadic nonhereditary RB due to somatic mutation is mostly unilateral, while the hereditary form and those with 13q- are either unilateral or bilateral. The difference in the expression as manifested by tumor laterality led Knudson [ 3-51 to the hypothesis that RB is caused by 2 mutational events; in the hereditary form, one mutation is inherited via germinal cells and the second 0304-3835/83/0000-0000/$03.00 o 1983 EIsevier Scientific Publishers Ireland Ltd. - Published and Printed in Ireland
138
occurs in somatic cells, whereas in the non-hereditary form, both mutations occur in somatic cells. To explain different expression of the RB gene, he assumed that the second mutation process was distributed according to the Poisson scheme. However, Matsunaga [ 6-81 has proposed inherited host resistance; the different expression of the RB gene among carriers is caused by multifactorial inheritance with 2 thresholds, and thus tumor formation in the less sensitive group with hereditary unilateral cases is suppressed twice that in the most susceptible group with bilateral cases. The cellular manifestation of the RB gene and its relevance to the development of tumors are of particular interest in the etiology of RB. Here, we describe a distinct difference in the susceptibility to viral transformation between different groups of RB and the finding which suggests that the expression of the heritable RB gene is constitutively determined at the cellular level. MATERIALS
AND METHODS
Fibroblast strains were established from biopsies of 19 patients with RB, 3 unaffected family members of RB patients and 2 normal individuals. The cells were cultivated in Eagle’s minimum essential medium supplemented with heat-inactivated 13% fetal bovine serum and passaged as described [ 121. The virus was obtained from the culture medium of rat cell 78Al transformed by Moloney MSV (MLV). The 78Al virus sample contained Moloney MSV, XC’ ecotropic MLV and XC+ dualtropic MLV; the former MLV replicates only in mouse cells and the latter MLV replicates both in mouse cells and in non-mouse cells [ 111. The titer of virus suspension used in the present experiment was 160 FFUIO.2 ml when assayed on S’L- mink cells and 20 FFLJIO.2 ml when assayed on normal mink cells. At 20 h after seeding of 8 X lo4 cells per 35 mm plastic dish, the cells were treated with 4 pg/ml polybrene for 1 h and infected by 0.2 ml of Moloney 78Al-MSV (MLV). After cultivation for 11 days the foci of transformed cells were counted. Values are averages of 2 or 3 independent experiments, and each experiment contained PL78 (skin fibroblasts from a patient with familial adenomatosis coli) as a high control, and C4 (skin fibroblasts from a normal individual) as a low control, the susceptibilities to viral transformation of which have previously been described [ 111. RESULTS
AND DISCUSSION
Table 1 summarizes the clinical and karyotypic data of patients studied in this experiment. The cultured skin fibroblasts from patients with RB and those from normal individuals were infected by Moloney murine sarcoma virus of rat 78Al cells, 78Al-MSV(MLV), and the foci of transformed cells were counted. Figure 1 shows the number of foci produced in the fibroblast strains from different groups of RB, the number indicating the susceptibility of fibroblasts to viral transformation.
139 TABLE 1 CLINICAL AND KARYOTYPIC
DATA OF FIBROBLAST
Heredity
Patient
Sex
Tumor laterality
Sporadic
RB RB RB RB RB RB RB RB RB RB RB
ilT 16T 32T 1AW 9T 1K 25T 28T 4lT 50T 51T
F M M M F M F M M F F
B B B B B U U U U U U
RB RB RB RB RB RB RB RB
15T 18T 38T 54T 14T 17T 36T 37T
F F F M M M M M
Familial
Normal
N 1KM N 3T N 35T N C2 NC4
F F F F F
Age at biopsy (years/months) 614 413 219 312 115 314 114 215 117 110 213
30/l 3816 4918 3317 o/11 419 45/o
1014
-
2119 3112
-
314
1116 2916
STRAINS Karyotype
46,XX,del(l3) 46,XY,del(13) 46,XY,del(l3) 46,XY,del(l3) 46,X,t (13;X) 46,XY 46,Xx 46,XY 46,XY 46,Xx 46,Xx 46,Xx 46,Xx 46,Xx 46,XY 46,XY 46,XY 46,XY 46,XY 46,Xx 46 ,XX 46 yx 46$X 46,Xx.
(q12q22) (q14q22) (q14) (q12q22) (q12;p22)
(mother of RB14T) (mother of RB17T)
(father of RB37T)
I
(unaffected family member of RB)
\ (normal)
The fibroblasts from familial bilateral cases exhibited 4-5-fold higher transformation frequencies than normal. These values were comparable to the highest frequencies for the cells from patients with familial adenomatosis coli [ 111. The susceptibilities of unilateral familial RB were about half that ol the bilateral cases but the mean value of this group was still twice that of normal individuals. The sporadic unilateral cases with normal karyotype showed rather low susceptibilities which were comparable to those of normal individuals. RB patients associated with chromosomal 13 anomalies in this experiment consisted of 4 cases with interstitial deletion of region involving band 13q14 and one case with reciprocal translocation between chromosome 13 and one of two X chromosomes. In case of the 13/X translocation, the translocated X was late replicating and hence the inactivation was supposed to spread over to the translocated chromosome 13 including critical region of q14 [ 141. All of the chromosome 13 anomalies including the 13/X translocation were herewith referred to as 13q-. As seen in Fig. 1, the patients
0 37 T
( OPL7 ‘8)
8 ;i: :
0 171r
o 54T 0 38T 015T 0 35T
OlT
OlKM
0 1AW
8% oc2
Normal
o 16T o 9T
0
I
Ef
018T
28T %T 1K
0 32T
Familial bilateral
L (1425)
:6lf
10)
Fig. 1. Susceptibilities of fibroblasts from patients with different groups of RB and normal individuals to transformation by murine sarcoma virus. Number of foci was determined as described in Materials and Methods. Each value is average of 2 or 3 independent experiments. The mean f S.E. for each RB group is indicated in parentheses at the bottom of each column.
with 13q-, all sporadic but bilaterally affected, had susceptibilities as low as those of normal individuals. Although the mechanism of susceptibility of fibroblasts to viral transformation is not understood well, the cells from patients with heritable neoplasms have reported to show increased transformation frequencies [2,10,11,13,]. The present data are also consistent with these lines of experimental evidence. Qf particular interest is the fact that the viral transformation promoted by the heritable RB gene seems to correlate not simply to the presence of the RB gene itself but rather intimately with its expression. The susceptibility to viral transformation was higher in bilaterally affected patients than in unilaterally affected patients even though they were members of the same family and inherited the same RB gene. Although the nature of the RB gene is not clear, the RB gene and some other dominantly expressed cancer-predisposing genes have been described as possibly related to a type
141
of gene-controlling element [ 9,14,15]. It is thus tempting to correlate the differential susceptibility to viral transformation with the RB gene-controlled expression or integration of viral genomes, or enhancement of more general genetic rearrangement favoring carcinogenesis. The 13q- is a simple structural loss of a critical region and hence might not act as a gene-controlling element, as has been shown in this report by the lack of the ability to promote viral transformation. Preliminary experiments suggest that the susceptibility of RB fibroblasts to viral transformation is not due to the replication of helper virus, because the number of transformed foci was not parallel to the titer of MLV released from the infected cells to the culture medium. Further examinations are needed to clarify whether these RB fibroblasts have increased susceptibility to transformation by other pseudotypes of MSV and chemical carcinogens. ACKNOWLEDGEMENT
This work was supported in part by grants for cancer research from the Ministry of Education, Science and Culture of Japan, and from The Princess Takamatsu Cancer Research Fund.
REFERENCES 1 Ejima, Y., Sasaki, M.S., Kaneko, A., Tanooka, H., Hara, Y., Hieda, T. and Kinishita, Y. (1982) Possible inactivation of part of chromosome 13 due to 13/X translocation associated with retinoblastoma. Clin. Genet. 21, 357-361. 2 Klement, V., Freedman, M.H., McAllister, R.M., Nelson-Rees, W.A. and Heubner, R.J. (1971) Differences in susceptibility of human cells to mouse sarcoma virus. J. Natl. Cancer Inst., 47, 65-73. 3 Knudson, A.G. (1971) Mutation and cancer: statistical study of retinoblastoma. Proc. Natl. Acad. Sci. U.S.A., 68,820-823. 4 Knudson, A.G., Hethcote, H.W. and Brown, B.W. (1975) Mutation and childhood cancer: a probabilistic model for the incidence of retinoblastoma. Proc. Natl. Acad. Sci. U.S.A., 72, 5116-5120. 5 Knudson, A.G., Meadows, A.T., Nichols, W.W. and Hill, R. (1976) Chromosomal deletion and retinoblastoma. N. Engl. J. Med., 295, 1120-1123. 6 Matsunaga, E. (1979) Hereditary retinoblastoma: host resistance and age at onset. J. Natl. Cancer Inst., 63, 933-939. 7 Matsunaga, E. (1980) Hereditary retinoblastoma: host resistance and second primary tumors. J. Natl. Cancer Inst., 65, 47-51. 8 Matsunaga, E. (1980) Retinoblastoma: host resistance and 13q- chromosomal deletion. Hum. Genet., 56, 53-58. 9 McClintock, B. (1950) The origin and behaviour of mutable loci in maize. Proc. Natl. Acad. Sci. U.S.A., 36, 344-355. 10 Miller, R.M. and Todaro, G.J. (1969) Viral transformation of cells from persons at high risk of cancer. Lancet, I, 81-82. 11 Miyaki, M., Akamatsu, N., Rokutanda, M., Ono, T., Yoshikura, H., Sasaki, M.S., Tonomura, A. and Utsunomiya, J. (1980) Increased sensitivity of fibroblasts of skin from patients with adenomatosis coli and Peutz-Jeghers’ syndrome to transformation by murine sarcoma virus. Gann, 71, 797-803.
142 12 Miyaki, M., Akamatsu, N., Ono, T., Tonomura, A. and Utsunomiya, J. (1982) Morphologic transformation and chromosomal changes induced by chemical carcinogens in skin fibroblasts from patients with familial adenomatosis coli. J. Natl. Cancer Inst., 68,563-571. 13 Pfeffer, L.M. and Kopelovich, L. (1977) Differential genetic susceptibility of cultured human skin fibroblasts to transformation by Kirsten murine sarcoma virus. Cell, 10, 313-320. 14 Sasaki, M.S. (1982) Dominantly expressed procancer mutations and induction of chromosome rearrangements. Prog. Mutat. Res., 4, 75 -84. 15 Sasaki, MS., Tsunematsu, Y., Utsunomiya, J. and Utsumi, J. (1980) Site-directed chromosome rearrangements in skin fibroblasts from persons carrying genes for hereditary neoplasms. Cancer Res., 40,4796-4803. 16 Vogel, F. (1979) Genetics of retinoblastoma. Hum. Genet., 52, l-54.