the molecular genetics of retinoblastoma and wilms' tumor

Volume 7, Issue 2 (1987) 153

THE MOLECULAR GENETICS OF RETINOBLASTOMA AND W1LMS' TUMOR

Authors: John Cowell ICRF Laboratory of Molecular Genetics Department of HematoIogy/Oncology institute of Child Health London, England

Jon Pritchard Department of Hcmatology/Oncology Hospital for Sick Children London, England

Referee: Robert 5. Sparkes Department of Medicine UCLA Center [or the Health Sciences LOS Angeles, California

I. INTRODUCTION

Cancers in adults are often sporadic, mostly affect people in later life, and may arise, it is thought, as a result of continued exposure to environmental carcinogens. However the familial occurrence of some tumors, e.g., carcinoma of the coital associated with polyposis coil and medullary carcinoma of the thyroid, strongly suggests a genetic component in the development of these tumors.~ There is equally compelling evidence for a genetic component in pathogenesis of some children's tumors, especially Wilms' tumor and retinoblastoma. Detailed genetic analysis of patients with both of these cancers, which predominantly affect young children, has led to the identification of specific chromosome regions thought to be the site of the predisposition loci.

Although the exact nature of the genetic defect associated with the predisposition to Wilms' tumor and retinoblastoma is unknown, these loci, for convenience, will be referred to as "genes". Recently, molecular-biological techniques have been used in an attempt to isolate and characterize these cancer-predisposition genes. The approaches being used to isolate specific DNA sequences together with the observations leading to the identification of the critical regions are described in this article. It should be stressed, however, that at present these experiments are only in the preliminary stages.

A. Two-Hit Hypothesis Pedigree analysis in cancer families often demonstrates that the mode of transmis-

sion is dominant. However, at the cellular level inheritance is apparently recessive since not all cells become malignant. Alternatively, reduced penetrance with dominant inheritance would also account for this observation. A solution to this apparent paradox was suggested by Knudson ~ when he proposed the "two-hit hypothesis". From a study of retinoblastoma and Wiims' tumor, he suggested that the susceptibility gene (first hit) is in the germ line and is a prezygotic event, present in all cells. However, a second event at the homologous locus (or another independent locus) is required to initiate tumor formation. This second hit is therefore a somatic event and is expressed only in the progenitor tumor cell and its progeny. In nonhereditary sporadic forms of some cancers both "hits" are postzygotie, somatic events.

154 CRC Critical Reviews in Oncology/Hematology

In this theory the hit can be any disturbance of the genetic material, such as a point mutation or a chromosome deletion, inversion, or translocation. This theory has been more or less confirmed by the molecular-biological studies described in this article. It accounts for the observation that the hered!tary forms are most often multifocal and bilateral (in paired organs) and have an earlier age of onset than sporadic forms of the same tumor. Although there have been some criticisms (see Reference 1 for review) the "two-hit" theory probably provides the best working hypothesis to date for the origin of retinoblastoma and Wilms' tumor.

B. Genetic Analysis of Cancer Predisposition I. Retinoblastoma

Retinoblastoma is an eye tumor of young children occurring in both sporadic and hereditary forms. In Western populations retinoblastoma accounts for l to 2~ of neoplasms in children; it has an incidence of about I case per 20,000 live births. Early estimates 3 suggested that 60% of retinoblastoma were sporadic and the remaining 40% were hereditary. If the definition of hereditary cases was restricted to those where there were affected individuals in at least two generations, then this figure of 40~ would apl:lear to be an overestimate. In our own study in Britain, only about 24% of patients has a positive family history. But, the likelihood of multiple, sporadic, postzygotic double hits is extremely small. Thus all patients with bilateral tumors should probably be considered as kereditary cases, even in the absence of a family history. 3 If this assumption is correct then in some populations a figure approaching 40~ is probably accurate, although in the British population the percentage of hereditary cases may prove to be even higher.

In those families showing dominant inheritance of retinoblastoma the penetrance of the gene has been assessed at 90o70. 3 in only two families in our study was there evidence that the expression of the gene had skipped a generation. This incomplete penetrance is illustrated in Figure I. In the first family, the expression of the tumor apparently misses a generation. In the s~cond family, several affected sibs are born to unaffected parents. In another family reported elsewhere, 4 a 2-year-old girl presented with a unilateral tumor in a branch of the family which had apparently not inherited the predisposition. Ophthalmological reexamination of the mother of this child revealed, in one eye, a retinal scar presumed to indicate a regressed tumor; she was in fact an affected gene carrier. Unfortunately the grandfather was not available for analysis but the possibility must exist that he also had a regre3sed tumor. Thus incomplete pen~.trance, in some cases, may be due to misdiagnosis or a missed diagnosis. Another possible explanation for incomplete penetrance is that the predisposition is carried in the form of a balanced chromosome translocation in the unaffected transmitting parent. s-s Those children receiving the unbalanced form of the rearrangement develop the tumor, whereas those inheriting the balanced form are unaffected carriers (see below). The third possible explanation for incomplete penetrance is that the unaffected transmitting parents are mosaics for the predisposition mutation; the parents carry the defect in the germ line but not in the cell lineage from which the retina originated.

Among retinoblastoma patients there is a subset with a deletion on the long arm of chromosome 13, designated 13q--? Approximately l out of 20 of all retinoblastoma patients have this chromosome deletion." Analysis of over 50 13q-deletions ~~ has shown that the extent of the deletion may vary from patient to patient. However, in virtually all cases chromosome region t3ql4 is either partly or entirely missing from one of the chromosome 13 homologues. These deletions are constitutional yet, for the most part, the only tumor these patients get is retinoblastoma. This observation strongly suggests that a gene or group of genes located in the 13q 14 region are impor-

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FIGURE I. Family pedigrees showing the segregation of the retinoblastoma pheaotype. (A) Incomplete penetrance with transmission of the retlnoblastoma gene by an unaffected family member (arrow); (B) transmission of the disease phenotype to two siblings by unaffected parents. (rll) Unilateral disease, (11) bilateral disease.

taut in the normal development of the retina, and that in their absence abnormal retinal ddvelopment and tumor formation arc likely to occur. Yunis and Ramsay a3 described a single patient with loss of only half of band 13q14 and suggested that the critical region was 13q14.2.

Studies of the enzyme esterase-D, whose genetic locus is in region 13q14 ~ have pro- vided additional evidence to implicate this chromosome region in the pathogenesis of retinoblastoma. Since only one of the homologous chromosomes is affected, 13q- patients have only 50% of normal esterase-D activity which can conveniently be meas- ured in red blood cells. 4 This technique has been used to identify deletion patients within retinohlastoma populations and also for prenatal diagnosis of deletion car- tiers. 's In a series of chromosome studies carried out in our laboratory, we have observed a number of patients with large deletions of region q12-q14 (Figure 2h). These deletion~ were associated with other congenital abnormalities including mild to severe mental r&ardation. Other patients have only small deletions and no phenotypic abnormalities other than retinoblastoma (Figure 2a). Two patients were of particular interest; one had 50% normal esterase-D activity and only a subband deletion of 13q14. The other patient had normal esterase-D levels but was shown to have a 13q14-13q22 deletion, similar to that reported by Sparkes et al. '~

The conclusion reached from these two patients is that the proximal breakpoint of the deletion must have been between the esterase-D and relinoblastoma loci. Since the loss of chromosomal material extends distally from region 13q14, the retinoblastoma locus must be located in the distal part and the esterase-D gene in the proximal part of this band. We have also identified a patient in whom there is 50% esterase-D activity but no obvious deletion of chromosomal material from q14. This finding suggests the occurrence of a submicroscopic deletion of the type reported by Benedict et al. ~'

156 CRC Critical Reviews in Oneology/Hematology

a b

#'4t

FIGURE 2. Chromosome 13 deIetions associated with retinoblastoma. ~a) del (13)(q14.1.q14.3l; (b) del (13)(q12-q141.

1,1 2,1

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FIGURE 3. Segregation ~ ~he esterase-D alleles in a re- t[noblastoma family. The two electrophotetic varients for esterase.D, type 1 and type 2, were analyzed in relevant family members. The disease phenotype segregates with the type 2 allele,

Cases with deletions visible in standard chromosome preparations account fog tess than 5% of the total number of retinoblastoma patients. Nonetheless, it seems reason- able to postulate that the same region is involved in the hereditary nondeletion forms of retinoblastoma. Because esterase-D has two electrophorctic variants, type I and type 2, this hypothesis can be tested by investigating *.he linkage relationship between hereditary retinoblastoma and esterase-D as outlined in the pedigree in Figure 3, In one such series of experiments, Sparkes et al., t~ analyzed 35 retinoblastoma families. In the three informative cases it was possible to demonstrate linkage between retinoblastoma and estesase-D. Thus, it appears that region 13q14 is also important in the hereditary nondeletion forms of retinoblastoma, in a recent study, t9 it has been estimated that esterase-D typing is at least 90% accurate in the prediction of retinoblastoma gene carriers in informative families. Unfortunately this analysis has limited use for prenatal diagnosis since, as illustrated in Sparkes's series, only about I in 10 families are informative for the estesase-D polymorphism.

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Analysis of constitutive chromosome rearrangements associated with the tumor phenotype has also implicated chromosome region 13q14. ~'n'2a'zt In two cases, 6'2~ chromosome rearrangements have involved the translocation of chromosomal material from chromosome 13 to the X chromosome. Replication analysis of the rearrangement in fibrublast cultures showed that it is the translocated X chromosome which is inacti- vated. Thus, there is inactivation rather than physical deletion of one region of 13qi4. I-lowevar~ since the inactivation process can spread over considerable distances, the breakpoint in these cases does not necessarily occur within the critical gene. Other reports describe rearrangements involving translocation of region 13q14 to another site, which is neither an X chromosome nor heteroehromatlc material. This suggests that the rearrangement might involve breakpoints within the predisposition locus, x2 These types of rearrangements offer an opportunity for studying the DIqA sequences adjacent to the breakpoint, and thereby help to define the exact genetic defect under- lying predisposition.

2. Wilms' Tumor Wilms' tumor is a kidney tumor affecting about 1 in 10,000 live births and accounts

for 6% of nil pediatric neoplasms. The tumor is usually sporadic. Although the frequency of the hereditary form has perhaps been overstated in the past, occasional cases of familial Wilms' tumor have been recorded, occurring in less than 1% of all cases. However, a very definite constitutive genetic abnormality has been identified in a small subgroup of Wilms' tumor patients. These children also have aniridia (absence of ir- ises) which is often associated with abnormal gonadal development and mental retardation. This phenotype has been referred to as the AGR triad (consisting of aniridia, gonadal dysplasia, and mental retardation, ~3 and carries a 50% risk of development of Wilms' tumor, z'

Chromosome analysis of these patients has shown, almost without exception, a chromosome deletion on the short arm of chromosome 11. The extent of the deletion may vary but in each case, all or part of, chromososme band ! Ipl3 is missing from one of the chromosome I 1 homologues, zs In the case where small deletions have been reported, the associated GR (gonadal dysplasia and mental retardation) phenotypes may be absent. However, aniridia usually acts as a dominant marker of the syndrome. In some eases the deletions have proven to be so small that they have been missed by conventional chromosomal analysis but revealed by high resolution studies.

Thus, loss of chromosomal material from the short arm of chromosome 11 appears to confer a predisposition to the development of Wilms' tumor. As with retinoblastoma, these deletions are constitutive yet the patients rarely develop cancer other than Wilms' tumor. This implies that there are genes located in region llp13 which control normal kidney development and, if disturbed, give rise to abnormal development and tumor formation. Occasionally, patients with the AGR triad (aniridia, gonadal dyspslasia, and mental retardation) have developed gonadoblastoma. 2.'~7 An explana: tion for this occurrence may be that during the early stages of normal kidney development part of the cell lineage goes on to form the germ line tissue. It is possible that a genetic defect occurring at an early stage in this primitive cell lineage could give rise to abnormal gonadal development and thus cause tumor formation.

By studying ! Ip deletion patients, Junien et al., 2a were able to map the catalase gene to region 11p13. Individuals with the deletion may consequently have only 50% of normal catalase levels in their cells. In some patients with aniridia, however, there is a n obvious deletion, but normal catalase activity. In these cases the deletion can be shown to involve the distal part of band I lpl3 and only part of I lp14. This observation places the catalase gene in the proximal region of 1 lp13. Recently, Turleau et al., ~'


reported a patient with a deletion of region l l p l l -p l3 who had Wilms' tumor and 50~ catalase activity but no aniridia. These findings placed the aniridia locus in the distal region of band l lp13.

The gene order thus appears to be centromere-catalase-Wilms' tumor-aniridia-telo- mere. However, Narahara et al. 24 have suggested that the catalase locus is distal to the aniridia/Wilms' tumor loci. They reported two patients with the AGR triad but only one of whom developed Wilms' tumor. In the Wilms' tumor patient with AGR triad (WAGR) the breakpoints were reported as I Ipl 1-pl3. The patient who did not develop a tumor apparently had a deletion pl 1.2-p13.05. In these asssignments, Narahara et al., assigned the Wilms' tumor locus to the distal-most part of the band, and since both patients had aniridia this gene was assigned to a more proximal position. In fact, a more likely explanation is that both patients were WAGR deletion carriers but that one did not develop the tumor, a circumstance consitent with the 50% risk of Wilms' tumor associated with the deletion. The second patient also had a complex rearrangement making identification of exact breakpoints difficult. A similar misidentification was reported by Ladda et al2 ~ When reanalyzed by Francke et al.fl I with high resolution banding, the abnormality proved to be consistent with other reports.

The deleted region can also be carried in the form of a balanced chromosome translocation. Yunis and Ramsay ~2 reported one family in which the I lpl3 region was translocated to chromosome 2 in one of the parents. The one child who inherited the unbalanced form (i.e., the deleted chromosome 11) manifested the AGR triad and developed Wilms' tumor.

C. Chromosome Analysis of Tumor Cells The identification of particular regions associated with predisposition to a specific

cancer, whether by chromsome analysis or linkage between the tumor phenotype and other genes, indicates the sites of the predisposition loci. If these regions contain genes whicl~ are involved in the genesis of the specific tumors, it might be expected that, as predicted by the Knudson hypothesis, in the sporadic forms of the disease abnormalities involving the same regions might be observed but confined to the tumor cells. With this in mind, several groups have undertaken karyotypic analysis of tumor cells from retinoblastomas and Wilms' tumors.

I. Retinoblastoma In an analysis of 10 tumors, Gardner et al. 33 observed many chromosomal changes

but only two of these changes involved chromosome 13. Similarly, Kusnetsova et al. 34 demonstrated a low incidence of chromosome 13 abnormalities in retinoblastomas. ~5 By contrast, Hashem and Khalifa ~6 tentatively identified deletions in 4 out of 5 retJ- noblastomas consistent with D-group deletions, although detailed chromosome banding was not performed. The only convincing report showing a high frequency of chromosome 13 deletions in tumor cells comes from Balaban-Malenbaum et al., 37 in which 13q14 was deleted in all of 5 tumors. A large study by Benedict, et al., 38 showed deletion of either all or part of region q14 on chromosome 13 in 6 out of 15 tumors.

Another chromosome abnormality observed in these various studies is the presence of an isochromosome 6p. 34,3~'3s'~ The suggestion has been made that perhaps this is another chromososme abnormality implicated in tumorigenesis. However, changes observed in the tumors may simply be consequences of the transformed state of the cells and are most likely to reflect tumor progression rather than initiation. It should be pointed out that chromosome analysis of tumor cells is difficult and the resolution obtained is often quite poor. In tumors with apparently normal copies of chromosome 13, a small deletion cannot be excluded but measurements of esterase-D on the tumor

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cells may be informative. Benedict, et al., 1' identified a patient with reduced levels of esterase-D but there was no cytologically detectable deletion. They concluded that there was a submicroscopic deletion. No esterase-D activity was found in the tumor cells, although a single copy of chromosome 13 was observed. It was suggested that the normal copy of chromosome 13 had been lost and the deleted chromosome 13 was retained.

2. Wilms' Tumor Chromososme analysis of Wilms' tunlors has also yielded conflicting reports, with

evidence both for and against the presence of abnormalities involving chromosome 11. Kaneko et al. 4' reported a tumor with deletion of region llp13-p14, although the finding could not be confirmed in a later study. '~ Slater and de Kraker" showed multiple abnormalities of chromosome I I in Wilms' tumors. Douglass et al." analyzed 14 Wilms' tumors of which 6 showed rearrangements (deletions and translocations) of l ip, and 7 showed abnormalities involving chromosome 1. Slater et al. 45 also found that l ip was the chromosome arm most frequently involved in rearrangement, although there were often many other abnormalities.

D. Identification of the Predisposition Locus The accurate pinpointing of cancer predisposition loci by the chromosome studies

just described has attracted several groups to attempt isolation of the responsible genetic material. The availability of restriction enzymes makes it possible to isolate DNA fragments. These fragments can then be mapped to regions of specific chromosomes using in situ hybridization or by using panels of somatic cell hybrids coz~taining different overlapping human chromosome deletions (see Reference 46). It would be naive, however, to assume that this approach will rapid:y lead to the isolation of the predisposition loci.

The human genome contains 3 x l09 base pairs (bp). Chromosome 11, 2.4% of the total 47 contains 7.2 x 10' bp while band l lp13, representing about 4~ of the chromosome, has 2.8 x 106 bp. On average, 40 kilobases (4 x l04 bp) of DNA can be cloned in a single cosmid. Thus, isolation of all of the DNA in band lip13 would require approximately 70 cosmids "laid end to end" Similar calculations can be applied to chromosome region 13ql4. If bacteriophage vectors, whose maximum size insert is only 15 to 20 kilobases of DNA, are used, the number of individual clones required would be considerably higher. To clone all of chromosome l 1, 1800 linearly arranged cosmids would be required. Also, sampling statistics dictate that 7600 independent clones would have to be isolated in order to achieve the number with 99% certainty. Such a task is formidable but not impossible.

The first step would be to obtain individual clones from the l l pl3 region. Taking random chromosome-I l-specific clones, l out of 25 should be from the deletion region. Other sequences from I Ipl3 could be generated by "chromosome walking", the principle of which is outlined in Figure 4. From a starting clone, the end region has to be identified, subcloned, or isolated fram gels, and then used as a probe back to the original library to find adjacent clones. A series of such single steps result in a "walk". An additonal problem is that the direction of the walk cannot be controlled. Thus walking has to be undertaken in both directions, which theoretically doubles the amount of work. This type of experiment for mapping of 70 cosmids would take a lifetime. To date, there have been few, if any, reports of chromosome walks in the human genome beyond the most adjacent clones.

I. Chromosome Walking The concept of a completely representative chromosome-specific library is critical to

160 CI~C Critical Reviews in Oncology/Hernatology

a i * i t i i l J i i I i I I

b L ~ . ~ I 2 3

I . 4 I I 5 I

6 7 . . . . I �9 ,.

8 I I

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FIGURE 4. Principle of chromosome walking. A length of DNA with vertical strokes representing cleavage sites for a particular restriction enzyme is shown in Cal. Partial digestion with a restriction enzyme produces a variety of overlapping fragments, examples of which are shown in (b). Starting from fragment 1, the end region is isolated following restriction enzyme digest analysis and used as a probe ( l } to find ether'fragments with the same sequence. In this example, fragments 4 and 6 have partial sequence homology, Fragment 4 represents a piece of DNA with only one short region in common with clone 1, whereas fragment 6 is virtually the same as fragment I and would extend the walk only a short distance. Therefore. clone 4 is used to extend the walk. If the dista|most region of clone 4 is now isolated and used in a similar way to probe for homologous sequences, clones 7 and 8 will be recognized, but clone 7 will be used to extend the wa lk because of its lesser overlap with c/one 4.

the understanding o f chromosome walking, I f the D N A used to prepare a library has been digested to complet ion with a particular restriction enzyme and then cloned, there will be fragments o f DNA which are both too small and too large to be cloned because o f the condit ions determined by the vector. Hence, the whole chromosome is not rep- resented in the library. A complete digest also does not generate the overlapping fragments essential for chromosome walking.

In order to isolate unique sequence probes, a second round of screening was undertaken in which D N A from individual clones was isolated, digested with a restriction enzyme, and probed with total human DNA. In this experiment human bands which failed to hybridize were presumed to be unique sequences. These sequences were then isolated frOm gels and used as probes against a panel o f somatic cell hybrids designed to identify chromosome 13 probes. In both cases a proport ion o f the clones isolated were from human chromosome 13. These clones were sublocaiized using two somatic cell hybrids where the min imum region o f overlap was region 13qi2-q22. In all, five such clones were isolated. Two of these clones were further sublocalized s= within region

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I3q12-q22 by analyzing the intensity of hybridization to DNA from a patient with a 13q deletion. In this experiment the intensity of hybridization was half that expected when compared with that of a control nonchromosome 13 probe.

La[ande et al. ~( have also created a chromosome-13-specific phage library, but from flow-sorted chromosomes. In their case, the lambda clones which did not hybridize with total human DNA were isolated as presumptive unique sequences. Again, these clones were tested against a panel of somatic cell hybrids to determine whether they were from chromosome 13 and sublocalized using the same deletion hybrids. Two clones were tentatively assigned to region 13q14 by hybridization intensity experiments and in one case also by in situ hybridization.

3. Homozygosity in Tumor Cells The two-hit hypothesis predicts that the manifestation of retinobiastoma may rep-

resent a recessive state. Chromosome analysis from sporadic tumors supports the view that disturbance of region 13ql4 may be important iu a proportion of tumors. The recessive nature of the retinoblastoma gene in sporadic tumors was also demonstrated by Benedict et a l? ' A patient was shown to have 50~ ESD activity in red blood cells but had no obvious chromosome deletion in peripheral blood lymphocytes. It was concluded that there was a submicroscopic deletion in the 13q14 region in this patient. Analysis of the tumor cells from this patient showed that only one copy of chromosome 13 was retained and that there was no ESD activity. Thus, the lost chromosome was the nondeleted one. Hence, the tumor cells were nullisomic for region 13ql4. Us- ing chromosome-13-specific probes evidence has now been presented suggesting that the same region is involved in tumorigenesis in sporadic forms of the disease. The probes isolated by Cayenne et a i r ~ and Dryja et at. ~J from the q12-q22 region of chromosome 13 were shown to exhibit restriction fragment length polymorphisms (RFLPs) in the genelal population. Thus in heterozygotes it is possible to distinguish between the homologous copies of human chromosome 13.

In a series of retinoblastoma patients, both groups compared the RFLPs derived from the normal and from tumor tissue. In many cases where the patient was shown to be hetcrozygous in nontumor tissue they were homozygous in the tumor material. The implication of these observations is that, during the development of the retina, precursor tumor cells have lost one copy of chromosome 13 and duplicated the other. This is in keeping with the idea that the retinoblastoma gene is recessive acting in the tumor cells and that the chromosome carrying the normal gene has been lost. In two families Cayenne ctal. 5~ showed that, while the affected patients were constitutionally heterozygotes, the tumor coils were homozygous for a particular allele. By analyzing the parents in the same way, they were able to show that it was the chromosome from the affected, transmitting parent which was retained in the tumors in the children.

The recessive nature of the Wilms' tumor gene was also suggested by a comparison of genetic probes from normal and tumor tissue from the same patient. Three groups studied this independently, sT"~' Orkin et al. ~7 used polymorphisms in the beta-globin complex and the H-ras gene which are on chromosome 11. Of seven Wilms' patients who were heterozygous for any of the probes only one was also heterozygoas in the tumor. Fearon et a l? B and Koufos et al. s9 observed a similar phenomenon but with a higher frequency; four out of six and five out of seven, respectively, of cases of Wilms' tumor were heterozygous for the probes. In all cases the normal tissue used was either peripheral blood lymphocytes or normal kidney taken from adjacent to the tumor. Many other loci on other chromosomes were also analyzed and whenever they were hetcrozygous in (he normal tissue, they were also heterozygous in the tumor. This finding suggests that the genetic reorganizatior~ in the tumor is restricted to chromosome 11.


1 2 3 4 5 6

i il

q

t n t n t n FIGURE 5. Generation of homozygosity in Wilms' ta- mors. Constitutive DNA prepared from lymphocytes of three Wilms' tumor patients were digested with Taq I and probed with a calcitonin eDNA probe (lanes 2, ~,, and 6). All three individuals were heterozygous for the two restriction fragments 8 and 6.5 kilobases. By conttasl, analysis of the tumor DNA (lanes 1, 3, and 5) show homozygosity for either the lower band only (weakly shown in lane 1) or the upper band only (lanes 3 and 5), indicating horaozygosity for the region of chromosome t I containing the calclton[n gene.

In our own laboratory we have used the calcitonin gene (CALCI) which is on chromosome 11 to investigate the same phenomenon. To date, we have identified five individuals with Wilms' tumors who were heterozygous in somatic cells but homozygous in the tumors (Figure 5). One possible explanation for these observations is that a single copy of chromosome 11 was lost in the tumor. This possibility was excluded in two ways. First, in some of the tumors karyotypic analysis showed that two copies of chromosome 11 were present and, secondly, dosage studies in the tumors showed that in the homozygous band there was twice as much signal as in either of the heterozygous bands.

The generation of homozygosity can be explained in several ways. In its simplest form the whole of the chromosome carrying the recessive mutation is duplicated and the normal copy lost. Whether the duplication or loss occurs first is not clear. This

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mechanism would most likely follow a mitotic nondisjunctional event, An alternative explanation is that the homozygosity is generated as a result of mitotic recombination. Evidence for this mechanism has been presented by Cavenee et al. ++ and Raizis et al. +~

Mitotic recombination would also account for the observations that only some Wilms' tumors show homozygosity for a particular probe. The probes used (Hras, insulin, beta-globin) are all !ocated at the distal tip of l lp. some distance away from the l lp13 predisposition locus, Thus, if the crossing-over event occurred between the probe and locus it would remain heterozygous for the probe, but it would become homozygous for the predisposition locus. Thus, it might be expected that the closer the probe was situated to the 1 lp13 region the less tikely it would remain heterozygous. In our limited series, all tumors tested are homozygous for the ealcltonin gene which is closer to the 1 lp13 region than the other genes tested.

E. Gene Tracking Even in the absence of any specific knowledge about the nature of the genetic defect

responsible for the predisposition of cancer, its inheritance can be followed through families using anonymous DNA sequences derived from the same chromosomal region as the predisposition locus and exhibiting a RFLP. When individuals are heterozygous for that RFLP band pattern it is possible to follow the inheritance of the region in which the probe is located. If the predisposition can be shown to cosegregate with a particular band it can be tracked through the family. +

The ability to track genes in this way depends on close linkage between probe and locus. Recombination between the two destroys the predictive ability of the system. In order to be able to determine the degree of linkage either many families or a few large families must be analyzed to establish the linkage distance between probe and gene. Clearly, the fewer detected recombination events the better the predictive value of the system. The prediction can be improved by having different probes flanking the locus of interest since the chances of two recombination events separating both of the probes from the locus is then very low.

The identification of RFLPs is not a trivial matter. It usually involves analyzing the band patterns produced by a large number of restriction enzymes, although experience has now shown that certain enzymes such as Taq I and Msp I yield polymorphic variants more commonly than other restriction enzymes. +' Even the identification of a RFLP does not necessarily allow the gene to be tracked in all families since the phenotype must be shown to segregate with the more rare allele in the family. If both parents are homozygous for an allele the family is described as uninformative for that particular enzyme/probe combination.

The opportunity to track the predisposition in Wilms' tumor is very limited since only less tb.an I~ of cases are inherited, which means that linkage values would be difficuk to determine and there would be limited demand for the test. For retinoblastoma, on the other hand, there are many families that could benefit from prenatal diagnosis. Several probes isolated from region ql2-22 of chromosome 13 have been tested, sv~' and in one case a particular allele was shown to cosegregate with the tumor phenotype. The number of individuals involved, however, was too small to be able to establish the linkage distance accurately. Subsequent analysis of a larger number of families .+ has shown that none of these probes are sufficiently well linked to the tumor phenotype to be of practical use in genetic counseling. Flanking probes, however, have proved useful in this respect. Cavenee et al. 6t* have recently been able to successfully predict whether offspring born to retinoblastoma gene carriers would develop the tumor.


Fo Oncogenes No cellular or viral oncogene has yet been assigned to human chromosome 13. How-

ever, in one retinoblastoma cell line, Y79, there is cytological evidence of gene amplification in the form of a homogeneously staining region (HSR) on the short arm of chromosome 1. ̀2 A similar HSR has been reported in cells from primary retinoblastoma tissue. ~5 In a neuroblastoma cell line, IMR 32, a homogeneously staining region was also observed on chromosome 1, and DNA probes have been isolated from it. 63 It has been shown that the c-myc related oncogene N-myc is amplified in this cell line and has been localized to the HSR in chromosome 1. ~',65 Retinoblastoma cell lines containing either a homogeneously staining region or another form of amplified DNA, double minutes (DMs; see Reference 66) also show amplification of N-myc and another unre- lated sequence from the IMR 32 "probe 8". 67"68 Lee et al. 69 have also shown amplification of N-myc in primary retinoblastomas. Both retinoblastoma and neuroblastoma are of neural origin, arising from the neuroectoderm and the neural crest, respectively. It is not yet clear whether the amplification of N-myc observed in these cells is causal in tumorigenesis or merely a consequence of it, possibly related to tumor progression.

The H-ras oncogene has been assigned by many authors to the short arm of chromosome 1 I. Despite early reports to the contrary, TM the oncogene appears to be located in the distal region of the short arm, J lp15" which is some considerable distance away from the putative Wilms' predisposition locus. Loss of H-ras oncogene occurred in one reported Wilms' tumor, although in this case was associated with a complex chromosome rearrangement. Our own studies n provide no evidence of amplified expression of H-ras in Wilms' tumors, although IGF II which is also located on the short arm of chromosome 11, shows high expression. This observation is more likely to reflect the state of differentiation of the tumor cells rather than any causative role for IGF II in tumorigenesis.

The conclusion from the available data is that the genetic predisposition to both retinoblastoma and Wilms' tumor are not associated with known altered oncogene expression, as appears might be the case in some lymphomas and leukaemias. Indeed it is hard to see how oncogenes, which apparently function by dominant expression of their gene products, fit into a theory of fumorigenesis which depends on the recessive nature of a genetic locus.

G. Second Tumors and Associated Malformations The most common second malignancy in retinoblastoma patients is osteosarcoma,

occurring in about 7 to 12~ of all patients. ~3'74 A recent report by Gilman et al. 7s demonstrated the association of a 13; 14 chromosome rearrangement with familial osteosarcoma. In this case the breakpoint was in band 13q 12. Preliminary studies using polymorphic DNA probes from chromosome 13 have suggested that homozygosity is generated in some osteosarcomas. 7~ These finding suggest that there may be several genes located on chromosome 13 which control the development of various tissues and which, if disturbed, give rise to tumor development. Alternatively, the same gene could be responsible which has different effects and different timing for expression in different tissues.

Nephroblastomatosis is considered a premalignant lesion often developing into Wilms' tumor. As yet no reports have been presented demonstrating homozygosity in these premalignant lesions, although it might be expected that disturbance of genetic material on chromosome I I might be important. Beckwith-Wiedemann syndrome (BWS) is an apparently sporadic disorder with multiple abnormalities including a high risk of the development of rare childhood tumors, in particular Wilms' tumor, hepa- toblastoma, rhabdomyosarcoma, and adrenal carcinoma. It is possible that all of these

Volume 7, Issue 2 (1987) 165

rare tumors might arise as a result of a developmental error brought about by a disturbance at a particular genetic locus. In a recent report, Koufos et el. '7 demonstrated homozygosity for 1 lp markers in two hepatoblastomas and two rhabdomyosarcomas. Since Wilms ' tumors are also known to become homozygous for I lp loci, it is interest- ing that all of the tumors associated with BWS are following a similar pattern of development. However, since the markers used are distributed throughout the short arm of chromosome 11, different loci m~y well be involved.

I t seems from studies on the pathology of Wilms' tumor and retinoblastoma that the mal ignant phenotype may arise as a result of abnormal differentiation during embryo- genesis. In deletion cases, it is the loss of genetic material which gives rise to tumor development. This suggests that a gene critical for normal development fails to act at the appropriate time. This genetic locus may provide a differentiation signal or may be the target for one. In either event the response is negative. The fact that disturbance of the genetic material in the same chromosome region may give rise to several different embryonic tumors might imply that in the deletion regions there are genes which control several aspects of development in different tissues. The type of tumor would de- pend on the t iming and exact location of the second hit. Thus studies in developmental biology, rather than oneogenesls, may therefore throw more light on the pediatric syndromes with a cancer predisposition.

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the molecular genetics of retinoblastoma and wilms' tumor

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