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Genetics of retinoblastoma: A study
ELSEV1ER
Genetics of Retinoblastoma: A Study Emilia Mateu, Francisco S inchez, Carmen N ijera, Magdalena Beneyto, Victoria Castell, Miguel Hermindez, Inmaculada Serra, and Ffilix Prieto
ABSTRACT: We have analyzed 43 families with either familial retinoblastoma (RB) (four kindreds), bilateral sporadic RB (10 individuals), or unilateral sporadic RB (29 individuals). Genetic studies focused on karyotype analysis, loss of heterozygosity of intragenic polymorphisms, and search for point mutations. We have been able to identify the genetic defect underlying the disease in eight cases. Deletions have been found in three patients with sporadic RB, two bilateral in one of which karyotyping had previously detected an interstitial deletion of chromosome I3 affecting (q13-q31) and one unilateral, Five different point mutations were responsible for three cases of bilateral sporadic RB, one case of unilateral sporadic RB, and one case of bilateral familial RB. The low frequency of constitutional mutations found h~ our study has led us to review and evaluate the possibilities and limitations of the present genetic analyses on RB and to access the different factors influencing the detection of mutations causing the disease, because genetic counseling is mainly based on mutation identification. © Elsevier Science Inc. 1997
INTRODUCTION Retinoblastoma (RB), the prototype of disease caused by m u t a t i o n in a tumor s u p p r e s s o r gene, is the most c o m m o n intraocular m a l i g n a n c y in children, w i t h a w o r l d w i d e incidence of between i in 13,500 and i in 25,000 live births. The disease is either unifocal or multifocal a n d unilateral or bilateral. The average age at diagnosis is 12 months for bilateral and 18 months for unilateral cases, w i t h most of the cases being diagnosed before the age of three. RB shows no variation between races, countries, or levels of industrialization. N a u m o v a and Sapienza [1] found that there is no significant difference in the n u m b e r of males and females w h o develop unilateral sporadic diseases but that there is a significant bias in bilateral sporadic cases t o w a r d males. This fact appears to result from the failure of a p r o p o r t i o n of affected males to have daughters. These authors propose that males with bilateral spo-
From the Unidad de Gen~tica y Diagn6stico Prenatal (E. M., M. B., F. P.), Unidad de Oncolog& Pedidtrica (V. C.), Departamento de Anatomfa Patol6gica (M. H.), Servicio de Oflalmologfa Pedidtica (L S.), Hospital La Fe, Valencia, Spain; and Departamento de Gen~tiea (F. S., C. N.), Facultad de Ciencias Biol6gicas, Universidad de Valencia, Valencia, Spain. Address reprint requests to: Dr. F~lix Prieto, Unidad de Gen~tica y Diagn6stico Prenatal, Hospital La Fe, Avda. Campanar 21, 46009 Valencia, Spain. E. Mateu and F. Sdnchez are equal first authors. Received August 6, 1996; accepted October 11, 1996. Cancer Genet Cytogenet95:40-50 (1997) © Elsevier Science Inc.. 1997 655 Avenue of the Americas, New York, NY 10010
radic disease are p r e s u m e d to carry a defective imprinting gene on their X chromosome. The p r i m a r y m e c h a n i s m in the d e v e l o p m e n t of retinoblastoma is the loss or inactivation of both alleles of the gene; thus, the retinoblastoma gene is a recessive gene at the cellular level [2].
Clinical Aspects Common signs and s y m p t o m s i n c l u d e leukokoria (cat'seye reflex), strabismus, low-vision orbital cellulitis, unilateral mydriasis, and heterochromia. Early diagnosis and treatment is of p r i m a r y importance in the survival of retinoblastoma patients. There are several effective methods for treatment of retinoblastoma tumors d e p e n d i n g on the presentation, size, and histopathology of the tumor. Patients with hereditary retinoblastoma have an increased risk of developing n o n o c u l a r tumors (mainly osteosarcomas) in later life [3-5]. The risk is estimated at 5%, whereas it is m u c h greater (35%}, in patients w h o received irradiation therapy [6].
Genetics Retinoblastoma (RB) occurs in both familial and sporadic forms. Most cases of RB are sporadic and the great majority of t h e m are because of somatic mutations (60% of the total RB). Hereditary p r e d i s p o s i t i o n to RB is caused by a germline m u t a t i o n at the RB1 locus. In the familial form, w h i c h represents 10% to 20% of all cases, the tumor phenotype segregates as a d o m i n a n t trait with 90% pane°
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Genetics of Retinoblasto~.a trance [2]. Forty percent of patients have the hereditary form of RB and "de nowY' germline mutations are three times more frequent than mutations transmitted from an affected parent. It is generally acceptect that all bilateral sporadic cases are inherited or caused by new mutations in the germ cells of the parents. Although most patients with hereditary familial RB have bilateral disease, occasionally there are unilaterally affected individuals. In 1971 Knudson [7], based on statistical calculations, suggested that two mutational events are required for RB tumor development ("two-hit hypothesis"). The first of these can be inherited through the germline or can be somatically acquired, whereas the second occurs somatically in both cases and leads to a tumor that is doubly defective at the RB locus. This second event could be identical in hereditary and nonhereditary cases but the probability of its occurring must be higher in hereditary cases. The second wild type chromosome allele may be lost or inactivated by several possible chromosomal mechanisms including mitotic nondisjunction with loss of the wild type chromosome; mitotic nondisjur~ction with reduplication of the mutant chromosome; mitotic recombination; gene conversion; deletion, or point mutation [8]. All these mechanisms lead to the homozygosity or hemizygosity of the RB1 locus in the retinal cell. The detection of only one allele variant in the tumor (homozygous or heterozygous) when two forms are present in constitutional cells (heterozygous) is indicative of loss of heterozygosity (LOH). Loss of heterozygosity has been used to suggest the presence of tumor suppressor genes in a wi:le variety of tumors. In sporadic cases, there is no differential susceptibility to somatic mutation between the homologous copies of the gene. However, it is of interest that 90% of "de novo" mutations occur in the paternal germline [9, 10]. Although genomic imprinting is one mechanism that might explain the unusual imbalance in mutation and retention of the paternal allele, the explanation with the most facticity is that far more cell divisions occur between embryonic development and meiosis in males than in females. It has not, however, been possible to attribute this to a paternal age effect [11].
Cytogenetics Detection of constitutional deletion on the long arm of chromosome 13 in some patients with RB suggested that the RB predisposition gene (RB1) was located in chromosome band 13q14 [12, 13]. Such deletions are rare (<5% of patients). Insertions and translocations involving band 13q14 have also been observed [14], but are much less frequent than deletions. Most patients with a deletion of 13q14 are mentally and physically retarded. The genes responsible for these defects are yet to be identified because, to date, the gene for esterase D (ESD) is the only gene known to be neighbor to the RB1 gene. Deletions of 13q14 are also rare in RB tumor cells. Only 10% of RB tumors have monosomy of the 13q14 region [15]. In contrast, tetrasomy 6p and trisomy lq are frequent occurrences [16, 17].
41
RB1 Gene Structure and Function In 1986 Friend et al. [18] cloned a cDNA corresponding to the RB1 gene. The 4.7 kb transcript detected by their cloned cDNA is derived from the 27 exons of the RB1 gene, which spans 180 kb. The largest intron spans more than 60 kb and the smallest has only 80 pb. The complete genomic sequence of the RB1 gene was reported by Toguchida et al. [19]. RB1 encodes a nuclear protein of 110 kd, pRB [20], playing a role in the regulation of the cell division cycle, and is found to be ubiquitously expressed in normal human tissues [21]. The phosphorylation of pRB is cell cycle-dependent. The protein is underphosphorylated in the phases of the cell cycle Go and G1, and highly phosphorylated during S and G2 [22-24]. It is also known that pRB interacts with oncoproteins such as SV40 large T antigen, adenovirus EIA and papilloma virus E7 [25-27]. The oncoprotein binding sites of pRB (aminoacids 393-572 and 646-772) overlap with the positions of naturally occurring mutations of the RB1 gene [28], suggesting that these protein domains are important for the normal function of pRB.
Linkage Analysis The regional assignment of the polymorphic enzyme Es D to chromosome band 13q14 [13] and demonstration of linkage of hereditary RB and ESD [29], provided confirmation that the locus for hereditary RB was located in this chromosomal region. In familial cases, the segregation of the disease-causing gene can be followed indirectly by linkage with polymorphic DNA markers. Wiggs et al. [30] isolated five unique DNA sequences from the introns of RB1 (RS2.0, PR0.6, R0.6, P0.3, and HS0.5) that, in combination, were very useful in gene carrier detection. The most useful of these probes was RS2.0, which identified a variable number of tandem repeats (VNTRs) of 53 bp with eight different alleles. Approximately 70% of individuals are heterozygous for this probe and the majority of the 30% of the remaining families are informative for one of the four other probes. Other informative intragenic markers that have been used are RB1.20 [31] and M1.8 [32]. In cases without familial antecedents, a study of RFLPs in DNA obtained from tumoral tissue and in constitutive DNA from lymphocytes could be carried out to verify if the loss of constitutional heterozygosity, which occurs in 70% of the RB, has happened. Moreover, using RFLPs and polymorphic microsatellites in the RB1 gene, the parental origin of new germline mutations could, in some cases, be determined [9].
Germline and Somatic Mutations Various deletions, insertions, and point mutations at the RB1 locus have been characterized in germline and constitutional DNA. In addition, hypermethylation of the CpG island at the 5'-end of the RB1 gene was observed in DNA from tumors [33]. Gross structural alterations as detectable by Southern blot hybridization are present in only 15% of patients with bilateral RB [34]. Multiple PCR and high-resolution
42 gel electrophoresis may detect small RB1 deletions that appear to make up another 35% of germline mutations. Point mutations exist in approximately 50% of patients with hereditary RB. Because there is no apparent hot spot for mutations within RB1, it is necessary to screen all the 27 exons and adjacent intronic sequences. Methods for prescreening the amplified product such as RNase protection assay [35], single strand conformation polymorphism (SSCP) [36], heteroduplex analysis (HDA) [37], have made the analysis more rapid than direct sequencing of amplified gene segments [38]. Nonsense and frameshift mutations constitute the largest number of detected nucleotide changes whereas missense mutations appear to be less common. The majority of changes were C ~ T transitions occurring in CGA codons specifying the arginine amino acid [39--41]. As a result of these changes, a TGA stop codon is generated. Out of 46 arginine codons, 14, distributed in only 10 different exons, are encoded by CGA. Some of these sites can be studied using restriction enzymes alone. No point mutation has been identified in exons 25-27 although two CGA codons are located within this region. Furthermore, none of the serine or threonine residues, where phosphorylation occurs, were mutated. The difficulties in mutation analysis of large genes with an extensive mutational heterogeneity, which result from the fact that not all mutations are detected by current mutation scanning techniques, are reflected in low rates of mutation detection in large groups of patients with RB. Other hypotheses also explain this low number of mutations. Causative mutations could be located outside the screened regions as occurs in one-half of all cases of severe hemophilia A [42] or a gene distinct from RB1 could be involved in RB development. A retinoblastoma related gene, referred to as RB2 and located on chromosome 16q12.2 [43] has, in fact, been cloned, The mutation type or position along the gene have not yet been related to the gravity of the disease. Blanquet et al. [40], report however, some patients carrying mutations in exon 19 who have also developed nonocular tumors.
Predictive DNA Diagnosis The great size of the RB1 gene and its multiple dispersed exons, complicate molecular screening for prenatal and presymptomatic diagnosis and for carrier detection. Using linkage analysis one can determine those individuals in RB families (15% of the total) who have inherited the predisposing mutation and those who have not. For those who, unequivocally, have not inherited the mutant gene, repeat ophthalmologic screening of subsequent generations is not necessary. Moreover, owing to reduced penetrance of mutations, some clinically unaffected relatives of patients might carry and transmit the mutation; identification of such carriers is important for accurate risk assessment. Of the 85% of RB patients without a family history, many will carry "new" germline mutations. The problem is that linkage analysis might not be available in these cases. It is accepted that bilateral and multifocal cases are inherited although Bona~ti-Pellie et al. [44] suggest that
E. Mateu et al. some sporadic bilateral forms of the disease could be nonhereditary. Only 15% of patients with unilateral RB have the inheritable type but, by chance, develop a tumor in only one eye. Only the existence of the two mutations in a tumor together with its absence in constitutional DNA guarantee the noninheritance of the disease. Taking into account that today enucleation is not practiced where avoidable, the lack of fresh tumor prevents its study. The objective of this work is to describe our experience in the genetic study of RB, either cytogenetic or molecular. The study was established to evaluate the possibilities and limitations, with regard to the capacity to carry out differential diagnosis between hereditary and nonhereditary cases, and prenatal and presymptomatic diagnosis, in a mainly clinical hospital laboratory.
MATERIALS AND METHODS Patients and DNA Samples We examined 43 families with either familial RB (four kindreds), bilateral sporadic (10 individuals), or unilateral sporadic RB (29 individuals), referred to us by pediatric oncologic units in Spain. Some of these families had been diagnosed as RB prior to the initiation of this work, for which the tumoral samples were paraffin-embedded. The other families were diagnosed during the present study, and DNA from fresh tumoral tissue was obtained from 5 of these patients. In all cases constitutional DNA was obtained. DNA from white cells and fresh tumoral tissue was extracted using standard phenol/chloroform procedures [45]. DNA from 10 ~m thick paraffin-embedded tumor sections was prepared according to the method of Shibata et al. [46], and modified by Onadim and Cowell [47]. First, individual paraffin sections were dissolved in 500 I~1 of xylene. The tissue was recovered by centrifugation and then washed twice in ethanol and desiccated from 30 rain to 2 hours. The DNA extraction solution consisted of 100 mmol/1 Tris-C1, 4 mmol/1 EDTA pH 8.0, and 0.5 mg/ml proteinase K. After between 12 and 18 hours at 37°C, this solution was boiled for seven rain and 1 to 10 ~1 used for PCR.
Cytogenetic Analysis The constitutional karyotype was carried out in lymphocytes of whole blood stimulated with phytohemaglutinin [48]. Chromosomes were identified by G- and R-banding [49, 50]. The tumoral cells were obtained by direct aspiration of the vitreus fluid through the cornea and the crystalline or directly from the tumor in the cases in which the eye was enucleated. In the latter cases, the tissue was broken up into small fragments of 1-2 mm and added to Hanks' balanced saline solution. Using a Pasteur pipette, the fragments were aspirated repeatedly until cells were separated from the tumor. The cells were subjected to hypotonic shock with sodium citrate (0.95 g/dl without previous treatment with colchicine), and were then fixed in Carnoy solution (3:1 methanol-acetic) and the slides stained with Giemsa (10%). The metaphases were photographed, dis-
Genetics of Retinoblastoma
Table I
43
P o l y m c r p h i c markers u s e d in this study
Polymorphic marker BamHI RBi2 KpnI RBi4 XbaI RsaF
RB1.20 RB1.26
Type
Method of analysis
Localization
Reference
RFLP Microsatellite RFLP Microsatellite RFLP VNTR Microsatellite RFLPd
PCR~ PCR Southern h PCR PCR~ Southern b PCR PCR
Intron 1 Intron 2 Intron 4 Intron 4 Intron 17 Intron 17 Intron 20 Intron 25
[32, 62] [19] [30] [19] [30, 63] [30] [31, 64] [31]
~These polymorphic markers were also studied in fresh tumoral tissue by Southern blot and hybridization with p123M1.8 and p88PR0.6 probes. bThe p95HS0.5 and 1:,68RS2.0probes have been employed detecting KpnI RFLP and RsaI VNTR, respectively. CGenomicDNA is di~;estedwith the restriction enzyme RsaI in order to detect the VNTRpolymorphism. dWe have observed that the polymorphic nucleotide change described by Yandel and Dryja [31] originates a new DraI site.
colored w i t h Carnoy solution, and the c h r o m o s o m e s were identified b y G-banding [49].
Hybridization to cDNA Probes Ten ng of genomic DNA from p e r i p h e r a l b l o o d leukocytes a n d tumoral tissue (from only fresh tumors) were digested w i t h 80 U of HindIII. The resulting fragments were separated on 0.8% agarose-gel, a n d blotted onto n y l o n membrane [51]. The filters were h y b r i d i z e d overnight at 42°C w i t h the p4.95BT probe labeled w i t h digoxigenin. After removal of the u n b o u n d probe, the b a n d s were detected using a c h e m i l u m i n e s c e n t substrate for alkaline phosphatase [52].
Analysis with Polymorphic Markers The p o l y m o r p h i c markers used in this study are s h o w n in Table 1. Three of the RFLPs were s t u d i e d by PCR-amplification of the fragments, and the digestion of the p r o d u c t was electrophoresed in p o l y a c r y l a m i d e or agarose gel and detected by silver-staining [41] with slight modifications, or e t h i d i u m bromide. The RFLP located in intron 4, a n d the VNTR marker were detected by Southern blotting. The analysis of microsatellite markers was carried out by PCR amplification, electrophoresis in polyacrylamide gel, a n d detection by silver-staining.
Mutation Analysis: SSCPs and Restriction Enzyme PCR primers used for the amplification of all 27 exons a n d the p r o m o t o r region of the RB1 gene were reported by Hogg et al. [53]. They were selected in intronic sequences, at least 50 b p u p s t r e a m or d o w n s t r e a m of the exon b o u n d aries. Promotor region and exons 1, 2, 5, 9, 15, 16, 21, 24, and 26 have not yet been studied. A p p r o x i m a t e l y 100 ng of genomic DNA were amplified in a 50 ~1 reaction mixtu:re containing 50 m M KC1, 10 m M Tris (pH 8.4), 1.5 m M ~v~gCl2, 0.2 m M of each dNTP, 50 p m o l of both primers, and 1 U Taq polymerase. Amplification conditions consisted of 40 cycles of denaturing at 94°C for i rain, annealing at 50 to 62°C for 1 min, and extension at 72°C for 2 min. The reaction was initiated by 10 rain incubation at 96°C and e n d e d with 10 rain incubation at 72°C.
Fifteen microliters of the PCR p r o d u c t were digested overnight w i t h 5 U of the a p p r o p r i a t e restriction enzyme in order to limit the size of the PCR fragments to <230 bp. Five microliters of the digested PCR p r o d u c t were m i x e d with 10 ~1 95% F o r m a m i d e , 20 m M NaOH, 0.05% Brom o p h e n o l Blue, and 0.05% xylene cyanol. Samples were d e n a t u r e d at 96°C for 10 m i n and i m m e d i a t e l y cooled in ice for 5 m i n to m i n i m i z e renaturation before being run on 10% n o n d e n a t u r i n g p o l y a c r y l a m i d e gel in TBE 1 × buffer at a constant temperature and voltage overnight. The temperature, voltage, and percentage of glycerol were standardized for each exon. DNA was visualized by silver-staining. The exon fragments that h a d p r e v i o u s l y been reported as containing CGA codons whose m u t a t i o n C-~T alters a restriction enzyme site [39], were subjected to a p p r o p r i a t e restriction enzyme digestion, using 15 ~1 of PCR reaction p r o d u c t and 10 U of the enzyme, and were run in a 2% agarose gel.
Sequencing Sequence analysis was performed b y use of Sequenase. Polymerase chain reaction (PCR) products were purified from u n i n c o r p o r a t e d primers and dNTPs by ultrafiltration using a Microcon 100 filtration unit. Sequencing reactions were electrophoresed on an automatic sequencer.
RESULTS The karyotype in l y m p h o c y t e s of p e r i p h e r a l b l o o d was s t u d i e d in 43 patients w i t h RB, although in only one case was a constitutional defect consisting of an interstitial deletion of c h r o m o s o m e 13, affecting to the genetic material b e t w e e n the q13-q31 bands detected (Fig. 1). The patient h a d bilateral retinoblastoma, d y s m o r p h i c features, and mental retardation. He was the first child of a couple both w i t h n o r m a l p h e n o t y p e and karyotype. The cytogenetic study carried out by direct m e t h o d in the tumoral cells of four patients m a d e clear that it is difficult to obtain the karyotype in these cells, In two cases the study was unsuccessful, one of the others s h o w e d eleven metaphases with the karyotype 47,XX,del(1)(p34),add(10)
44
E. Mateu et al.
G
R Figure 1 Chromosome pairs 13 with del(13)(q13q31) m a constitutional karyotype of a patient with retinoblastoma. G and R banding.
(q26),-19,+der(19)t(1;19)(q12;p13)×2 (Fig. 2) and the last case s h o w e d six metaphases w i t h the karyotype 48,XX, - 3,i(6)(p10), + 9, + 2mar. The analysis of the allele segregation with all p o l y m o r p h i c markers a p p o i n t e d in Table 1 was performed in constitutional DNA from the 43 families. F r o m this analysis the presence of a heterozygotic deletion in three males with sporadic RB was deduced, Two of these were bilateral cases and the other unilateral. In all cases the chromosome deleted was the paternal one. In one of the bilateral cases, the deletion was also detected by cytogenetic analysis, whereas in tile other patient, the loss of the paternal allele
was observed with RBi2 and RB1.20, which were the only fully informative markers (Fig. 3). In the patient with unilateral RB we have detected loss of paternal allele with the markers p123M1.8, p95HSO.5, p88PR0.6, and RB1.26, whereas RBi2 and RB1.20 markers were heterozygotic. The analysis with p o l y m o r p h i c markers in unaffected relatives of another 8 patients (1 bilateral and 7 unilateral cases) s h o w e d that one or two siblings of each patient share h a p l o t y p e with him/her. The p o l y m o r p h i c marker RB1.20, based on the repetition of CTTT(T) [31], detected the existence of a large unc o m m o n allele described by Toguchida et al. [19], w h i c h is related to the infrequent alleles of the Mbo II RFLP described by Wiggs et al. [30] because RB1.20 alleles scored as large contain e x p a n d e d repeat sequences including one or more internal MboII sites. We have found at least three different variants for this large allele, with the difference of a small n u m b e r of repetitions, in the constitutional DNA of a patient with unilateral RB and in his unaffected father and grandmother, as well as in the three affected members of another family with unilateral familial RB (Fig. 4), and in 3 (2.5%) of 120 unrelated and unaffected i n d i v i d u a l s in a local study. In the 7 sporadic cases (4 unilateral and 3 bilateral) in w h i c h we were able to study the tumoral tissue, analysis revealed a heterozygotic deletion in 4 patients with sporadic RB (1 unilateral and 3 bilateral cases). No deletion was observed in constitutional DNA of these patients, but in one of t h e m we detected a p o i n t m u t a t i o n in exon 8. The other 3 unilateral tumors s t u d i e d s h o w e d homozygotic deletion comprising all the RB1 gene.
Figure 2 Karyotype of retinoblastoma tumor cell: 47,XX,del(1)(p34),add(10)(q26),- 19,+ der(19)t(l;19)(q12; q13) ×2. The arrows indicate chromosomal aberrations.
5
12
18
Genetics of Retinoblastom~
45
H y b r i d i z a t i o n w i t h cDNA probes has confirmed the existence of deletions in the same cases referred to above. A l t h o u g h no extra b a n d was f o u n d in any case, the suspicion of deletion was established by a decrease in the hyb r i d i z a t i o n signal because of a lower dose. The screening of muta~:ions in the RB1 gene by SSCPs analysis a n d digestion of the exon fragments containing a CGA codon with the suitable restriction enzyme has shown the existence, in leucocytary DNA, of a n o m a l o u s bands in 3 patients w i t h bilateral sporadic RB (Fig. 5A), in one with unilateral sporadic RB (Fig. 5B), and in another of bilateral familial RB. These alterat:[ons were not found in their unaffected relatives, thus we a s s u m e d that they were the mutations p r e d i s p o s i n g to RB, raised "de novo" in sporadic cases. The subsequent sequencing of each of these exons w i t h a n o m a l o u s b a n d s has characterized the mutations, s h o w n in Table 2. In our study we have also identified n u c l e o t i d e changes outside the coding region, in intronic sequences a m p l i f i e d on both sides of the exons. Two of these changes
were located in intron 3, at postion +37 (A to T transversion) and +45 (C to T transition), a n d the other changes were found in intron 4 at p o s i t i o n +23 (G to T transversion), and in intron 19 at postion - 7 7 (A to G transition).
DISCUSSION Cytogenetic studies made in constitutional DNA of patients w i t h RB s h o w e d that about 3% of t h e m present interstitial deletions in 13q14 [54]. In our samples, 1 (2.3%) of 43 patients shows this alteration. Cytogenetic defects detected in tumoral cells tend to be secondary alterations found in neoplasias in a d v a n c e d stages. Of these alterations, the i(6p) a n d the extra material in the large arms of c h r o m o s o m e 1 are the most frequently found in RB [16]. Both alterations were found in two of our patients. The fact that the two 13 c h r o m o s o m e s were n o r m a l in these two patients, in both p e r i p h e r a l b l o o d and tumoral tissue, suggests that in most cases the p r i m a r y
Figure 3 (A) Pedigree of a family with bilateral sporadic retinoblastoma showing the segregation of polymorphic markers within the RB1 gene. The numbers beside each chromosome represent the allelic form of each locus. In the affected family member, the RBi2 and RB1.20 paternal alleles are lost. A question mark sign denotes that we cannot infer the hemizygous or homozygous state. (B) Electrophoretic pattern obtained with microsatellite markers RBi4 (a) and RB1.20 (b) in DNA samples from each family member.
RB-224 Band//
2
2
RBi2 Xpnl RBi4 Xbal P,sol RB1.20 RBI.26
2 1 1 2 2 1 1
4 1 1 2 2 2 1
RB-225 1 3 1 2 1 1 4
RB-227 ? del ? ? ? ? del ?
2 1 1 1 2 2 3
(o)
RB..226
2 1 1 1 2 2 3 1
2 2 1 1 2 2 1 1
A
1 3 1 2 1 1 4 1
B
46
E. Mateu et el.
A bp D
M
B bp
M
517"
m
39(
396'
344
344-
298298 '
1
2
2
3
3
4
4
5
5
d
u
d
u
d
u
d
u
d
u
Figure 4 Electrophoretic pattern obtained with the RB1.20 marker. (A) The arrows denote the uncommon allele (~450 bp), detected in several members (1 to 5) of two unrelated families. Common shorter alleles are in the 250330 bp range. (B) Intron 20 fragments of subjects showing the large allele was digested with MbolI. Digested (d) and undigested (u) samples were electrophoresed. The picture shows the difference between both patterns.
structural alterations occurring in the RB gene cannot be detected by cytogenetic analysis and one must resort to analysis with molecular biology techniques. In 3 (7%) of the 43 patients (2 bilateral and 1 unilateral sporadic), we identified large constitutional deletions using genomic probes or microsatellite markers detecting intragenic polymorphisms. The detection of these mutations in two bilateral sporadic cases ("a priori" presumptive hereditary form), suggests that in 14% of our hereditary cases (n = 14), the direct identification of deletions using intragenic polymorphisms is possible, which is in agreement with previous reports [34, 41, 55] based on hereditary RB. The development of microsatellite markers spanning the whole RB1 gene has greatly improved the technology applied to the detection of constitutional deletions because of its higher informativity. In our familial cases, the analysis of segregation with intragenic polymorphisms determined the inheritance of the mutation predisposing to RB by examining their coinheritance with DNA polymorphisms. In one of them, there is a discrepancy between DNA polymorphism and phenotype, which probably reflects low penetrance. Scheffer et el. [56] identified several kindreds with a low penetrance phenotype by similar analysis. DNA polymorphism should, according to Wiggs et el. [30], allow the genotype and the eventual phenotype of newborns in kindred with familial RB to be predecided. However, because of incomplete penetrance it is difficult in nonfamilial cases to determine the probability of carrying a tumor predisposing mutation within a RB gene when an unaffected sibling has an identical haplotype as the affected member. Thus, one of the
children of family 24 (Fig. 5B) had inherited identical alleles as his affected DZ twin who was diagnosed at 8 years old, although at 12 the former had not developed RB. The identification of a germline mutation in the affected child, an A-~C transversion in exon 14 (codon 447), not present in his DZ twin brother or his parents, was the definitive finding in the accurate determination of risk in all members with inherited associated alleles, or in the parents. Concerning the analysis of tumoral DNA we have had serious difficulties in the interpretation of results in DNA proceedings from paraffin-embedded tissues, because the DNA sample is, in many cases, insufficient for dosage valorization hybridizing with cDNA in Southern blotting. Moreover, the DNA is frequently degraded, making certain experimental procedures impossible, such as PCR amplification when PCR fragments are too long. On the other hand, in fresh or fixed tissue, false heterozygosity may show amplified from DNA of normal cells in some cases. At present, early diagnosis of RB patients leads to a reduction in the use of classic methods for treatment of RB, including enucleation, so it could be that the analysis of DNA extracted from tumor tissue may only be able to be performed in a few cases. With regard to this aspect, an important consideration about segregation analysis including tumoral DNA, is the fact that, in a patient known to have the hereditary form of RB, evaluation of the tumor for the RB1 gene alleles homozygosity may have important implications for genetic counseling. According to Dryja et el. [57] their comment that if the tumor has lost the allele present on one chromosome 13 homologue, one can assume that the chromosome 13 remaining in the tumor is the one
Genetics of Retinoblastoma
47
A
B
Figure 5 Mutation analysis by SSCPs. A) Pedigree of family fRB-33 and electrophoresis of DNA samples from father's, mother's, and patient's leukocyte and tumor. PCR fragment spanning exon 18 shows the existence of aberrant SSCP bands in the affected patient. B) Pedigree of family fRB-24 and result of SSCP analysis of exon 14 PCR product from leukocyte DNA, showing additional bands in the affected family member.
that harbors the mutant allele, and therefore predict which family members are at risk for the disease. Moreover, because RB tumors do not have normal RB1 allele, finding the germline mutation is most efficiently accomplished by first identifying the RB1 mutations in tumoral DNA [58]. Our results confirm, in general, the conclusions reported before by other authors, i.e., Cowell et al. [39], Blanquet et al. [40], and L o h m a n n et al. [41], concerning the spectrum of h u m a n germline mutations in the RBI gene that confers hereditary predisposition to RB. In agreement with these authors, we have observed that base substitution represents, in conjunction with deletion or insertion of one base, the most c o m m o n germline mutation identified in patients with hereditary RB. With regard to the base substitutions, the C to T transitions affecting CGAarg codons is
Table 2 Patient
the most frequent nucleotide change. Lohmann et al. [41] detected C--~T transitions in CGA codons in 29 (40.8%) of 71 patients with hereditary RB by mutational screening using HDA, SSCPs, or sequencing. Cowell et al. [39] identified this type of mutation, which results in the generation of premature stop codons, in 18 (16%) of 113 patients with bilateral RB by SSCP analysis and restriction enzymes. We have detected C to T transition in 2 (20%) of 10 patients with bilateral RB by mutational screening using SSCPs and restriction enzymes and, although we have not scanned all the exons of the RB1 gene, the remaining exons are not frequently reported as preferentially altered by this type of mutation. With regard to the local distribution of these mutations within a RB1 gene, our results are in agreement with Blanquet et al. [40, 59], who, by denaturant gradient gel electrophoresis (DGGE) analysis, were able to show the majority of C to T mutations in exon 8 and exon 18. Thus, the mutation in exon 8 (codon 255) was detected in a total of 7 (13%) of 54 affected individuals in which the germline mutation was characterized (3 bilateral and 4 familial), whereas the C-~T transition in codon 579 of exon 18 was identified in 3 (5.5%) of them [40]. Lohmann et al. [41], using a combination of different techniques of mutational screening, reported that the exons preferentially altered with C-~T mutations in CGA codons are, besides exons 8 and 18, the exon 10 (codon 320), exon 11 (codon 358), exon 14 (codon 445), exon 15 (codon 467), and exon 23 (codon 787). Curiously, Lohmann et al. [41] detected some of these mutations, concretely those in exon 10 and 14, only by sequencing and they were not detected using SSCPs or HDA. Moreover, these authors reported that the most frequent C to T transition in exon 8 is in codon 251, in agreement with Cowell et al. [39], who described that the same mutation and the change C-~T in exon 17 (codon 552) represent 73% of all C-~T mutations detected in the RB1 gene and 10 to 15% of the total mutations in hereditary RB. Neither in our analysis, nor previously noticed by Blanquet et al. [40], was the mutation in exon 17 (codon 552) found in any patient, even though this exon was analyzed by different methods. We suggest, in the light of all these findings, that detection of C-~T transitions in the RB1 gene could be influenced by several factors: population analyzed, technique, and local conditions in the ex-
Summary of germline point mutations in the RB1 gene Phenotype
Mutation
Position
Putative consequence
RB-190 RB-198
Bilateral sporadic Bilateral sporadic
Nonsense C-~T transition C-~T transition
Exon 8 (codon 255) Exon 18 (codon 579)
Arg-~Stop Arg-~Stop
RB-61 RB-210
Bilateral sporadic Bilateral familial
Frameshiff T insertion G deletion
Exon 20 (codon 667) Exon 19 (codon 617)
Stop 685 Stop 622
RB-142
Unilateral sporadic
Missense A-~C transversion
Exon 14 (codon 447)
Lys-oGln
48 perimental procedure. In spite of this, the scanning of C to T mutations in CGA codons of RB1 gene is the most efficient strategy for detecting germline mutations in RB patients. Other types of mutations, such as small length alterations (1-18 bp) can be identified by SSCPs, HDA, or highresolution fragment analysis, although if the PCR fragments are too long, the last technique is not able to detect this type of mutation, as L o h m a n n et al. have reported [41, 60]; However, we have only identified a deletion of 1 bp and an insertion of I bp, respectively, in one familial and one bilateral sporadic RB case using SSCP analysis foll o w e d b y direct sequencing w h e n an abnormal pattern was observed. Other small length alterations comprising more than one bp have not been found. It has been described that deletions of 1 bp are the most c o m m o n [60], however, we cannot exclude the possibility that deletions or insertions of several bp are being missed by SSCPs in our study. Both of the nucleotide changes detected in intron 3 have been described previously as p o l y m o r p h i c changes [31, 40]. The other intronic variants observed by us in intron 4 and intron 19 have not been reported before. The fact that these changes have been detected both in affected and unaffected relatives suggests its irrelevance in the pathogenesis of the disease. With the totality of procedures techniques performed, we have detected the responsible germline m u t a t i o n in 6 (42.8%) of 14 unrelated patients w i t h familial or bilateral sporadic RB, and in 2 (7%) of 29 cases with unilateral sporadic disease, rates very low in c o m p a r i s o n with the rate obtained by L o h m a n n et al. [41] in hereditary cases, w h o defined this p r o p o r t i o n as 83%. Our w h o l e results in bilateral sporadic and familial RB are more in agreement with the lower rates (56.5%) reported by Blanquet et al. [40]. It could be that w h e n the scanning of the w h o l e RB1 gene is finished, the frequency of detection of an R B I m u t a t i o n m a y reach a higher percentage, but obviously far from 83%. Likewise, our results in unilateral RB are, up to now, lower than Vogel's data [2], w h i c h suggest that 10 to 12% of unilateral sporadic cases are carriers of a germline mutation. On the other hand, Draper et al. [61] define this proportion at 2%. In summary, our study reflects that m o l e c u l a r analysis af RB patients to perform an accurate genetic counseling is feasible and profitable in familial form, even w i t h o u t identifying the responsible mutation. This objective is, however, h a r d l y accomplishable in sporadic cases, because of the low rates of mutational detection, extensive mutational heterogeneity, variability about the frequency of detection because of u n k n o w n variants, and the need to use several technical procedures. All of these factors are responsible for the high cost and difficulty of the study, w h i c h limit the i m p l e m e n t a t i o n of the analysis in clinical routine. There is a need for further investigation to clarify mutational m e c h a n i s m s in the RB1 gene a n d definitive spectrum in each population, in order to k n o w if the scientific achievements are converted into benefits for those at risk, and if it is possible to offer this s t u d y to all RB patients at different clinical laboratories.
E. Mateu et al. We should like to thank the families for their generous cooperation, and Pr.T.P. Dryja for providing the p4.95BT cDNA probe and genomic probes used in this study. This work was supported by the CICYT (SAF 92/0266). F. S~nchez is the recipient of a fellowship from the Consellerfa de Educaci6n y Ciencia de la Comunidad Valenciana. REFERENCES
1. Naumova A, Sapienza C (1994): The genetics of retinoblastoma, revisited, Am J Hum Genet 54:264-273. 2. Vogel F (1979): Genetics of retinoblastoma. Hum Genet 52:1-54.
3. Francois J, De Sutter E, Coppieters R, De Bies S (1980): Late extraocular tumors in retinoblastoma survivors. Ophtalmologica (Basel) 181:93-99. 4. Abramson DH, Ellsworth RM, Kitchin DF, Tung G (1984): Second nonocular tumors in retinoblastoma survivors, are they radiation induced? Ophthalmology 91:1351-1355. 5. Draper GJ, Sanders BM, Kingston JE (1986): Second primary neoplasms in patients with retinoblastoma. Br J Cancer 53: 661-671. 6. Roarty JD, McLean IW, Zimmerman LE (1988): Incidence of second neoplasm in patients with bilaterial retinoblastoma. Ophthalmology 95:1583-1587. 7. Knudson AG (1971): Mutation and cancer: Statistical study of retinoblastoma. Proc Natl Acad Sci USA 68:820-823. 8. Cavenee WK, Dryja TP, Phillips RA, Benedict WF, Godbout R, Gallie BL, Murphree AL, Strong LC, White RL (1983): Expression of recessive alleles by chromosomal mechanisms in retinoblastoma. Nature 305:779-784. 9. Dryja TP, Mukai S, Petersen R, Rapaport JM, Walton D, Yandell DW (1989): Parental origin of mutations of the retinoblastoma gene. Nature 339:556-558. 10. Zhu X, Dunn JM, Phillips RA, Goddard AD, Paton KE, Becker A, Gallie BL (1989): Preferential germline mutation of the paternal allele in retinoblastoma. Nature 340:312-313. 11. Matsunaga E, Minoda K, Sasaki MS (1990): Parental age and seasonal variation in the births of children with sporadic retinoblastoma: A mutation-epidemiologic study. Hum Genet 84:155-158. 12. Franke U (1976): Retinoblastoma and chromosome 13. Cytogenet Cell Genet 16:131-134. 13. Sparkes RS, Sparkes NC, Wilson NG, Towner JW, Benedit WF, Murphree AL, Yunis JJ (1980): Regional assignment of genes for human esterase-D and retinoblastoma to chromosome band 13q14. Science 208:1042-1044. 14. Turleau C, de Grouchy U, Chavin-Coi IN F, Junien C, Seger J, Schieinger P, Leblanc A, Haye C (1985): Cytogenetic forms of retinoblastoma: Their incidence in a survey of 66 patients. Cancer Genet Cytogenet 16:321-334. 15. Squire J, Gallie BL, Phillips RA (1985): A detailed analysis of chromosomal changes inheritable and non-heritable retinoblastoma. Hum Genet 70:291-301. 16. Kusnetsova LE, Prigogina EL, Pogosianz HE, Belkina BM (1982): Similar chromosomal abnormalities in several retinoblastomas. Hum Genet 61:201-204. 17. Squire J, Phillips RA, Boyce S, Godbout R, Rogers B, Gallie BL (1984): Isochromosome 6p, a unique chromosomal abnormality in retinoblastoma: The verification by standard staining techniques, new densitometric methods, and somatic cell hybridisation. Hum Genet 66:46-53. 18. Friend SH, Bernards R, Rojelj S, Weinberg RA, Rapaport JM, Albert DM, Dryja TP (1986): A human DNA segment with properties of the gene that predisposes to retinoblastoma and osteosarcoma. Nature 323:643-646.
Genetics of Retinoblastoma
19. Toguchida J, McGee TL, Paterson JC, Eagle JR, Tucker S, Yandell DW, Dryja TP (1993): Complete genomic sequence of the human retinoblastoma susceptibility gene. Genomics 17: 535-543. 20. Lee WH, Shew JY, Hong FD, Sery TW, Donoso LA, Young LJ, Bookstein R, Lee YP (1987): The retinoblastoma susceptibility gene encodes a nuclear phosphoprotein associated with DNA binding activity. Nature 329:642-645. 21. Hong FD, Huang H-JS, Young L-JS, Oro A, Bookstein R, Lee YP, Lee W-H (1989): Structure of the human retinoblastoma gene. Proc Natl Acad Sci USA 86:5502-5506. 22. Buchkovich K, Duffy LA, Harlow E (1989): The retinoblastoma protein is phosphorylated during specific phases of the cell cycle. Cell 58:1097-1105. 23. Mihara K, Cao X-R, Yen A, Chandler S, Driscoll B, Murphree AL, T-Aug A, Fung Y-KT (1989): Cell cycle-depandent regulation of phosphorylat[on of the human retinoblastoma gene product. Science 246:1300-1303. 24. Riley DJ, Lee EYHP, Lee WH (1994): The retinoblastoma protein: More than a tumor suppressor. Annu Rev Cell Biol 10: 1-29. 25. DeCaprio JA, Ludlow )W, Figge J, Shew J-Y, Huang C-M, Lee W-H, Marsilio E, Paucha E, Livingston DM (1988): SV40 large tumour antigen forms a specific complex with the product of the retinoblastoma susceptibility gene. Cell 44:275-283. 26. Whyte P, Buchkovich K, Horowitz JM, Friend SH, Raybuck M, Weinberg RA, Harlouw E (1988): Association between an oncogene and an anti-oncogene: The adenovirus EIA proteins bind to the retinoblastoma gene product. Nature 334:124-129. 27. Dyson N, Howley PN, Munger K, Harlow E (1989): The human papilloma virus-16 E7 oncoprotein is able to bind to the retinoblastoma gene product. Science 243:934-936. 28. Hu K, Dyson N, Harlow E (1990): The regions of the retinoblastoma protein needed for binding to adenovirus EIA or SV4O large T antigen are common sites for mutation. EMBO J 9:1147-1155. 29. Sparkes RS, Murphree AL, Lingua RW, Sparkes MC, Field LL, Funderburk SJ, Benedict WF (1983): Gene for hereditary retinoblastoma assigned to human chromosome 13 by linkage to esterase-D. Science 217:971-973. 30. Wiggs J, NordenskjSld M, Yandell D, Rapaport J, Grondin V, Janson M, Werelius E, Petersen R, Craft A, Riedel K, Liberford R, Walton D, Wilson W, Dryja TP (1988): Prediction of the risk of hereditary retinoblastoma, using DNA polymorphisms within the rotinoblastoma gene. New Engl J Med 318:151-157. 31. Yandell DW, Dryja TP (1989): Detection of DNA sequence polymorphims by onzymatic amplification and direct genomic sequencing. Am J Hum Genet 45:547-555. 32. Bookstein R, Lai C-G, To H, Lee W-H (1990): PCR-based detection of a polyr~.orphic BamHI site in intron 1 of the human retinoblastom~ (RB) gene. Nucleic Acids Res 18:1666. 33. Greger V, Debus N, Lohmann D. HSppin W, Passarge E, Horsthemke B (1994:): Frequency and parental origin of hypermethylated RB3 alleles in retinoblastoma. Hum Genet 94:491-496. 34. Kloss K, W~hrisch P, Greger V, Messmer E, Fritze H, HSpping W, Passarge E, Horsthemke B (1991): Characterization of deletions at the retinoblastoma locus in patients with bilateral retinoblastoma. Am J Med Genet 39:196-200. 35. Dunn JM, Phillips RA, Becker AJ, Gallie BL (1988): Identification of germline and somatic mutations affecting the retinoblastoma gene. Science 241:1797-1800. 36. Orita M, Suzuki Y, Sekiya T, Hayashi K (1989): Rapid and sensitive detection cf point mutations and DNA polymorphisms using the polymerase chain reaction. Genomics 5:874-879.
49 37. Ganguly A, Rock MJ, Prockop DJ (1993): Conformation-sensitive gel electrophoresis for rapid detection of single-base differences in double-stranded PCR product and DNA fragments: Evidence for solvant-induced bends in DNA heteroduplexes. Proc Natl Acad Sci USA 90:10325-10329. 38. Yandell DW, Campbell TA, Dayton SH, Petersen R, Walton D, Little JB, McConkie-Rosell A, Buckley EG, Dryja TP (1989): Oncogenic point mutations in the human retinoblastoma gene: Their application to genetic counseling. New Eng J Med. 321:1689-1695. 39. Cowell JK, Smith G, Bia B (1994): Frequent constitutional C to T mutations in CGA-arginine codons in the RB1 gene produce premature stop codons in patients with bilateral (hereditary} retinoblastoma. Eur J Hum Genet 2:281-290. 40. Blanquet V, Turleau C, Gross-Morand MS, Beaufort CS, Doz F, Besmond C (1995): Spectrum of germline mutations in the Rbl gene: A study of 232 patients with hereditary and non hereditary retinoblastoma. Hum Mol Genet 4:383-388. 41. Lohmann DR, Brandt B, H6pping W, Passarge E, Horsthemke B (1996): The spectrum of RB1 germ-line mutations in hereditary retinoblastoma. Am J Hum Genet 58:940-949. 42. Naylor JA, Green PM, Rizza CR, Gianelli F (1993): Analysis of factor VIII mRNA reveals defects in every one of 28 haemophilia A patients. Hum Mol Genet 2:11-17. 43. Yeung RS, Bell DW, Testa JR, Mayol X, Baldi A, Giana X, Klinga-Levan K, Knudson AG, Giordano A (1993): The retinoblastoma-related gene, RB2, maps to human chromosome 16q12 and not chromosome 19. Oncogene 8:3465-3468. 44. Bona'iti-Pellie C, Chompret MF, Tournade MF, Lemerly J, Voute PA, Delamarre JF (1993): Excess of multifocal tumors in nephroblastoma: Implications for mechanisms of tumors development and genetic counseling. Hum Genet 91:373376. 45. Kunkel LM, Tantravah V, Eisanhard M, Latt SA (1982): Regional localization on the human X of DNA sequences cloned from flow shorted chromosomes. Nucleic Acids Res 10:1557-1571. 46. Shibata DK, Arnheim N, Martin WJ (1988): Detection of human papilloma virus in paraffin-embedded tissue using polimerase chain reaction. J Exp Med 167:225-230. 47. Onadim Z, Cowell JK (1991): Application of PCR-amplification of DNA from paraffin-embedded tissue sections to linkage analysis in familial retinoblastoma. J Med Genet 28:312316. 48. Moorhead PS, Nowell PC, Mellman WJ, Battips DM, Hungerford DA (1960): Chromosome preparations of leukocytes cultures from peripheral blood. Exp Cell Res 20:613-616. 49. Seabright M (1971): A rapid binding technique for human chromosome. Lancett II:971-972. 50. Dutrillaux B, Laurent C, Gilgenkrantz S, Frederic J, Carpentier J, Lejeune J (1974): Les translocations du chromosome X. Etude apr~s traitement par le BUDR et coloration par l'acridine orange. Helv Paediatr Acta [suppl] 34:19-31. 51. Southern EM (1975): Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98:503-517. 52. Bronstein I, Voyta JC, Lazzari KG, Murphy OJ, Edwards B, Kricka LJ (1990): Rapid and sensitive detection of DNA in Southern blots with chemiluminescence. BioTechniques 8: 310-314. 53. Hogg A, Onadim Z, Baird PN, Cowell JK (1992): Detection of heterozygous mutation in the RB1 gene in retinoblastoma patient using single-strand conformation polymorphism analysis and polymerase chain reaction sequencing. Oncogene 7:1445-1451. 54. Cowell JK and Hogg A (1992): Genetics and cytogenetics of retinoblastoma. Cancer Genet Cytogenet 64:1-41.
50
55. Blanquet V, Cr~au-Goldberg N, de Grouchy J, Turleau C (1991): Molecular detection of constitutional deletions in patients with retinoblastoma. Am J Med Genet 39:355-361. 56. Scheffer H, 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 mutations, revealed by combined use of markers within and flanking the RB1 gene. Am J Hum Genet 45:252-260. 57. Dryja TP, Cavenee WK, White R, Rapaport JM, Peterson R, Albert DM, Bruns GA (1984): Homozygosity of chromosome 13 in retinoblastoma. New Engl J Med 310:550-553. 58. Gallie BL, Dunn JM, Chan HSL, Hamel PA, Phillips RA (1991): The genetics of retinoblastoma. Relevance to the patient. Pediatr Clin North Am 38:299-315. 59. Blanquet V, Gross MS, Turleau C, Senamand-Beaufort C, Doz F, Besmond C (1994): Three novel germline mutations in the exon 8 and 18 of the retinoblastoma gene. Hum Mol Genet 7:1185-1186.
E. Mateu et al.
60. Lohmann DR, Brandt B, H6pping W, Pasarge E, Horsthemke B (1994): Spectrum of small length germline mutations in the RB1 gene. Hum Mol Genet 3:2187-2193. 61. Draper GJ, Sanders BM, Brownbill PA, Hawkins MM (1992): Pattern of risk of hereditary RB and applications to genetic counselling. Br J Cancer 66: 212-219. 62. Bookstein R, Lee EY, To H, Young LJ, Sery TW, Hayes RC, Friedmann T, Lee WH (1988): Human retinoblastoma susceptibility gene: Genomic organization and analysis of heterozygous intragenic deletion mutants. Proc Natl Acad Sci USA 85:2210-2214. 63. McGee TL, Cowley GS, Yandell DW, Dryja TP. (1990): Detection of the XbaI RFLP within the retinoblastoma locus by PCR. Nucleic Acids Res 18:207. 64. Brandt B, Greger V, Yandell D, Passarge E, Horsthemke B (1992): A simple and nonradioactive method for detecting the Rbl.20 DNA polymorphism in the retinoblastoma gene. Am J Hum Genet. 51:1450-1451.
Genetics of Retinoblastoma: A Study Emilia Mateu, Francisco S inchez, Carmen N ijera, Magdalena Beneyto, Victoria Castell, Miguel Hermindez, Inmaculada Serra, and Ffilix Prieto
ABSTRACT: We have analyzed 43 families with either familial retinoblastoma (RB) (four kindreds), bilateral sporadic RB (10 individuals), or unilateral sporadic RB (29 individuals). Genetic studies focused on karyotype analysis, loss of heterozygosity of intragenic polymorphisms, and search for point mutations. We have been able to identify the genetic defect underlying the disease in eight cases. Deletions have been found in three patients with sporadic RB, two bilateral in one of which karyotyping had previously detected an interstitial deletion of chromosome I3 affecting (q13-q31) and one unilateral, Five different point mutations were responsible for three cases of bilateral sporadic RB, one case of unilateral sporadic RB, and one case of bilateral familial RB. The low frequency of constitutional mutations found h~ our study has led us to review and evaluate the possibilities and limitations of the present genetic analyses on RB and to access the different factors influencing the detection of mutations causing the disease, because genetic counseling is mainly based on mutation identification. © Elsevier Science Inc. 1997
INTRODUCTION Retinoblastoma (RB), the prototype of disease caused by m u t a t i o n in a tumor s u p p r e s s o r gene, is the most c o m m o n intraocular m a l i g n a n c y in children, w i t h a w o r l d w i d e incidence of between i in 13,500 and i in 25,000 live births. The disease is either unifocal or multifocal a n d unilateral or bilateral. The average age at diagnosis is 12 months for bilateral and 18 months for unilateral cases, w i t h most of the cases being diagnosed before the age of three. RB shows no variation between races, countries, or levels of industrialization. N a u m o v a and Sapienza [1] found that there is no significant difference in the n u m b e r of males and females w h o develop unilateral sporadic diseases but that there is a significant bias in bilateral sporadic cases t o w a r d males. This fact appears to result from the failure of a p r o p o r t i o n of affected males to have daughters. These authors propose that males with bilateral spo-
From the Unidad de Gen~tica y Diagn6stico Prenatal (E. M., M. B., F. P.), Unidad de Oncolog& Pedidtrica (V. C.), Departamento de Anatomfa Patol6gica (M. H.), Servicio de Oflalmologfa Pedidtica (L S.), Hospital La Fe, Valencia, Spain; and Departamento de Gen~tiea (F. S., C. N.), Facultad de Ciencias Biol6gicas, Universidad de Valencia, Valencia, Spain. Address reprint requests to: Dr. F~lix Prieto, Unidad de Gen~tica y Diagn6stico Prenatal, Hospital La Fe, Avda. Campanar 21, 46009 Valencia, Spain. E. Mateu and F. Sdnchez are equal first authors. Received August 6, 1996; accepted October 11, 1996. Cancer Genet Cytogenet95:40-50 (1997) © Elsevier Science Inc.. 1997 655 Avenue of the Americas, New York, NY 10010
radic disease are p r e s u m e d to carry a defective imprinting gene on their X chromosome. The p r i m a r y m e c h a n i s m in the d e v e l o p m e n t of retinoblastoma is the loss or inactivation of both alleles of the gene; thus, the retinoblastoma gene is a recessive gene at the cellular level [2].
Clinical Aspects Common signs and s y m p t o m s i n c l u d e leukokoria (cat'seye reflex), strabismus, low-vision orbital cellulitis, unilateral mydriasis, and heterochromia. Early diagnosis and treatment is of p r i m a r y importance in the survival of retinoblastoma patients. There are several effective methods for treatment of retinoblastoma tumors d e p e n d i n g on the presentation, size, and histopathology of the tumor. Patients with hereditary retinoblastoma have an increased risk of developing n o n o c u l a r tumors (mainly osteosarcomas) in later life [3-5]. The risk is estimated at 5%, whereas it is m u c h greater (35%}, in patients w h o received irradiation therapy [6].
Genetics Retinoblastoma (RB) occurs in both familial and sporadic forms. Most cases of RB are sporadic and the great majority of t h e m are because of somatic mutations (60% of the total RB). Hereditary p r e d i s p o s i t i o n to RB is caused by a germline m u t a t i o n at the RB1 locus. In the familial form, w h i c h represents 10% to 20% of all cases, the tumor phenotype segregates as a d o m i n a n t trait with 90% pane°
0165-4608/97/$17.00 PII S0165-4608(96)00387-1
Genetics of Retinoblasto~.a trance [2]. Forty percent of patients have the hereditary form of RB and "de nowY' germline mutations are three times more frequent than mutations transmitted from an affected parent. It is generally acceptect that all bilateral sporadic cases are inherited or caused by new mutations in the germ cells of the parents. Although most patients with hereditary familial RB have bilateral disease, occasionally there are unilaterally affected individuals. In 1971 Knudson [7], based on statistical calculations, suggested that two mutational events are required for RB tumor development ("two-hit hypothesis"). The first of these can be inherited through the germline or can be somatically acquired, whereas the second occurs somatically in both cases and leads to a tumor that is doubly defective at the RB locus. This second event could be identical in hereditary and nonhereditary cases but the probability of its occurring must be higher in hereditary cases. The second wild type chromosome allele may be lost or inactivated by several possible chromosomal mechanisms including mitotic nondisjunction with loss of the wild type chromosome; mitotic nondisjur~ction with reduplication of the mutant chromosome; mitotic recombination; gene conversion; deletion, or point mutation [8]. All these mechanisms lead to the homozygosity or hemizygosity of the RB1 locus in the retinal cell. The detection of only one allele variant in the tumor (homozygous or heterozygous) when two forms are present in constitutional cells (heterozygous) is indicative of loss of heterozygosity (LOH). Loss of heterozygosity has been used to suggest the presence of tumor suppressor genes in a wi:le variety of tumors. In sporadic cases, there is no differential susceptibility to somatic mutation between the homologous copies of the gene. However, it is of interest that 90% of "de novo" mutations occur in the paternal germline [9, 10]. Although genomic imprinting is one mechanism that might explain the unusual imbalance in mutation and retention of the paternal allele, the explanation with the most facticity is that far more cell divisions occur between embryonic development and meiosis in males than in females. It has not, however, been possible to attribute this to a paternal age effect [11].
Cytogenetics Detection of constitutional deletion on the long arm of chromosome 13 in some patients with RB suggested that the RB predisposition gene (RB1) was located in chromosome band 13q14 [12, 13]. Such deletions are rare (<5% of patients). Insertions and translocations involving band 13q14 have also been observed [14], but are much less frequent than deletions. Most patients with a deletion of 13q14 are mentally and physically retarded. The genes responsible for these defects are yet to be identified because, to date, the gene for esterase D (ESD) is the only gene known to be neighbor to the RB1 gene. Deletions of 13q14 are also rare in RB tumor cells. Only 10% of RB tumors have monosomy of the 13q14 region [15]. In contrast, tetrasomy 6p and trisomy lq are frequent occurrences [16, 17].
41
RB1 Gene Structure and Function In 1986 Friend et al. [18] cloned a cDNA corresponding to the RB1 gene. The 4.7 kb transcript detected by their cloned cDNA is derived from the 27 exons of the RB1 gene, which spans 180 kb. The largest intron spans more than 60 kb and the smallest has only 80 pb. The complete genomic sequence of the RB1 gene was reported by Toguchida et al. [19]. RB1 encodes a nuclear protein of 110 kd, pRB [20], playing a role in the regulation of the cell division cycle, and is found to be ubiquitously expressed in normal human tissues [21]. The phosphorylation of pRB is cell cycle-dependent. The protein is underphosphorylated in the phases of the cell cycle Go and G1, and highly phosphorylated during S and G2 [22-24]. It is also known that pRB interacts with oncoproteins such as SV40 large T antigen, adenovirus EIA and papilloma virus E7 [25-27]. The oncoprotein binding sites of pRB (aminoacids 393-572 and 646-772) overlap with the positions of naturally occurring mutations of the RB1 gene [28], suggesting that these protein domains are important for the normal function of pRB.
Linkage Analysis The regional assignment of the polymorphic enzyme Es D to chromosome band 13q14 [13] and demonstration of linkage of hereditary RB and ESD [29], provided confirmation that the locus for hereditary RB was located in this chromosomal region. In familial cases, the segregation of the disease-causing gene can be followed indirectly by linkage with polymorphic DNA markers. Wiggs et al. [30] isolated five unique DNA sequences from the introns of RB1 (RS2.0, PR0.6, R0.6, P0.3, and HS0.5) that, in combination, were very useful in gene carrier detection. The most useful of these probes was RS2.0, which identified a variable number of tandem repeats (VNTRs) of 53 bp with eight different alleles. Approximately 70% of individuals are heterozygous for this probe and the majority of the 30% of the remaining families are informative for one of the four other probes. Other informative intragenic markers that have been used are RB1.20 [31] and M1.8 [32]. In cases without familial antecedents, a study of RFLPs in DNA obtained from tumoral tissue and in constitutive DNA from lymphocytes could be carried out to verify if the loss of constitutional heterozygosity, which occurs in 70% of the RB, has happened. Moreover, using RFLPs and polymorphic microsatellites in the RB1 gene, the parental origin of new germline mutations could, in some cases, be determined [9].
Germline and Somatic Mutations Various deletions, insertions, and point mutations at the RB1 locus have been characterized in germline and constitutional DNA. In addition, hypermethylation of the CpG island at the 5'-end of the RB1 gene was observed in DNA from tumors [33]. Gross structural alterations as detectable by Southern blot hybridization are present in only 15% of patients with bilateral RB [34]. Multiple PCR and high-resolution
42 gel electrophoresis may detect small RB1 deletions that appear to make up another 35% of germline mutations. Point mutations exist in approximately 50% of patients with hereditary RB. Because there is no apparent hot spot for mutations within RB1, it is necessary to screen all the 27 exons and adjacent intronic sequences. Methods for prescreening the amplified product such as RNase protection assay [35], single strand conformation polymorphism (SSCP) [36], heteroduplex analysis (HDA) [37], have made the analysis more rapid than direct sequencing of amplified gene segments [38]. Nonsense and frameshift mutations constitute the largest number of detected nucleotide changes whereas missense mutations appear to be less common. The majority of changes were C ~ T transitions occurring in CGA codons specifying the arginine amino acid [39--41]. As a result of these changes, a TGA stop codon is generated. Out of 46 arginine codons, 14, distributed in only 10 different exons, are encoded by CGA. Some of these sites can be studied using restriction enzymes alone. No point mutation has been identified in exons 25-27 although two CGA codons are located within this region. Furthermore, none of the serine or threonine residues, where phosphorylation occurs, were mutated. The difficulties in mutation analysis of large genes with an extensive mutational heterogeneity, which result from the fact that not all mutations are detected by current mutation scanning techniques, are reflected in low rates of mutation detection in large groups of patients with RB. Other hypotheses also explain this low number of mutations. Causative mutations could be located outside the screened regions as occurs in one-half of all cases of severe hemophilia A [42] or a gene distinct from RB1 could be involved in RB development. A retinoblastoma related gene, referred to as RB2 and located on chromosome 16q12.2 [43] has, in fact, been cloned, The mutation type or position along the gene have not yet been related to the gravity of the disease. Blanquet et al. [40], report however, some patients carrying mutations in exon 19 who have also developed nonocular tumors.
Predictive DNA Diagnosis The great size of the RB1 gene and its multiple dispersed exons, complicate molecular screening for prenatal and presymptomatic diagnosis and for carrier detection. Using linkage analysis one can determine those individuals in RB families (15% of the total) who have inherited the predisposing mutation and those who have not. For those who, unequivocally, have not inherited the mutant gene, repeat ophthalmologic screening of subsequent generations is not necessary. Moreover, owing to reduced penetrance of mutations, some clinically unaffected relatives of patients might carry and transmit the mutation; identification of such carriers is important for accurate risk assessment. Of the 85% of RB patients without a family history, many will carry "new" germline mutations. The problem is that linkage analysis might not be available in these cases. It is accepted that bilateral and multifocal cases are inherited although Bona~ti-Pellie et al. [44] suggest that
E. Mateu et al. some sporadic bilateral forms of the disease could be nonhereditary. Only 15% of patients with unilateral RB have the inheritable type but, by chance, develop a tumor in only one eye. Only the existence of the two mutations in a tumor together with its absence in constitutional DNA guarantee the noninheritance of the disease. Taking into account that today enucleation is not practiced where avoidable, the lack of fresh tumor prevents its study. The objective of this work is to describe our experience in the genetic study of RB, either cytogenetic or molecular. The study was established to evaluate the possibilities and limitations, with regard to the capacity to carry out differential diagnosis between hereditary and nonhereditary cases, and prenatal and presymptomatic diagnosis, in a mainly clinical hospital laboratory.
MATERIALS AND METHODS Patients and DNA Samples We examined 43 families with either familial RB (four kindreds), bilateral sporadic (10 individuals), or unilateral sporadic RB (29 individuals), referred to us by pediatric oncologic units in Spain. Some of these families had been diagnosed as RB prior to the initiation of this work, for which the tumoral samples were paraffin-embedded. The other families were diagnosed during the present study, and DNA from fresh tumoral tissue was obtained from 5 of these patients. In all cases constitutional DNA was obtained. DNA from white cells and fresh tumoral tissue was extracted using standard phenol/chloroform procedures [45]. DNA from 10 ~m thick paraffin-embedded tumor sections was prepared according to the method of Shibata et al. [46], and modified by Onadim and Cowell [47]. First, individual paraffin sections were dissolved in 500 I~1 of xylene. The tissue was recovered by centrifugation and then washed twice in ethanol and desiccated from 30 rain to 2 hours. The DNA extraction solution consisted of 100 mmol/1 Tris-C1, 4 mmol/1 EDTA pH 8.0, and 0.5 mg/ml proteinase K. After between 12 and 18 hours at 37°C, this solution was boiled for seven rain and 1 to 10 ~1 used for PCR.
Cytogenetic Analysis The constitutional karyotype was carried out in lymphocytes of whole blood stimulated with phytohemaglutinin [48]. Chromosomes were identified by G- and R-banding [49, 50]. The tumoral cells were obtained by direct aspiration of the vitreus fluid through the cornea and the crystalline or directly from the tumor in the cases in which the eye was enucleated. In the latter cases, the tissue was broken up into small fragments of 1-2 mm and added to Hanks' balanced saline solution. Using a Pasteur pipette, the fragments were aspirated repeatedly until cells were separated from the tumor. The cells were subjected to hypotonic shock with sodium citrate (0.95 g/dl without previous treatment with colchicine), and were then fixed in Carnoy solution (3:1 methanol-acetic) and the slides stained with Giemsa (10%). The metaphases were photographed, dis-
Genetics of Retinoblastoma
Table I
43
P o l y m c r p h i c markers u s e d in this study
Polymorphic marker BamHI RBi2 KpnI RBi4 XbaI RsaF
RB1.20 RB1.26
Type
Method of analysis
Localization
Reference
RFLP Microsatellite RFLP Microsatellite RFLP VNTR Microsatellite RFLPd
PCR~ PCR Southern h PCR PCR~ Southern b PCR PCR
Intron 1 Intron 2 Intron 4 Intron 4 Intron 17 Intron 17 Intron 20 Intron 25
[32, 62] [19] [30] [19] [30, 63] [30] [31, 64] [31]
~These polymorphic markers were also studied in fresh tumoral tissue by Southern blot and hybridization with p123M1.8 and p88PR0.6 probes. bThe p95HS0.5 and 1:,68RS2.0probes have been employed detecting KpnI RFLP and RsaI VNTR, respectively. CGenomicDNA is di~;estedwith the restriction enzyme RsaI in order to detect the VNTRpolymorphism. dWe have observed that the polymorphic nucleotide change described by Yandel and Dryja [31] originates a new DraI site.
colored w i t h Carnoy solution, and the c h r o m o s o m e s were identified b y G-banding [49].
Hybridization to cDNA Probes Ten ng of genomic DNA from p e r i p h e r a l b l o o d leukocytes a n d tumoral tissue (from only fresh tumors) were digested w i t h 80 U of HindIII. The resulting fragments were separated on 0.8% agarose-gel, a n d blotted onto n y l o n membrane [51]. The filters were h y b r i d i z e d overnight at 42°C w i t h the p4.95BT probe labeled w i t h digoxigenin. After removal of the u n b o u n d probe, the b a n d s were detected using a c h e m i l u m i n e s c e n t substrate for alkaline phosphatase [52].
Analysis with Polymorphic Markers The p o l y m o r p h i c markers used in this study are s h o w n in Table 1. Three of the RFLPs were s t u d i e d by PCR-amplification of the fragments, and the digestion of the p r o d u c t was electrophoresed in p o l y a c r y l a m i d e or agarose gel and detected by silver-staining [41] with slight modifications, or e t h i d i u m bromide. The RFLP located in intron 4, a n d the VNTR marker were detected by Southern blotting. The analysis of microsatellite markers was carried out by PCR amplification, electrophoresis in polyacrylamide gel, a n d detection by silver-staining.
Mutation Analysis: SSCPs and Restriction Enzyme PCR primers used for the amplification of all 27 exons a n d the p r o m o t o r region of the RB1 gene were reported by Hogg et al. [53]. They were selected in intronic sequences, at least 50 b p u p s t r e a m or d o w n s t r e a m of the exon b o u n d aries. Promotor region and exons 1, 2, 5, 9, 15, 16, 21, 24, and 26 have not yet been studied. A p p r o x i m a t e l y 100 ng of genomic DNA were amplified in a 50 ~1 reaction mixtu:re containing 50 m M KC1, 10 m M Tris (pH 8.4), 1.5 m M ~v~gCl2, 0.2 m M of each dNTP, 50 p m o l of both primers, and 1 U Taq polymerase. Amplification conditions consisted of 40 cycles of denaturing at 94°C for i rain, annealing at 50 to 62°C for 1 min, and extension at 72°C for 2 min. The reaction was initiated by 10 rain incubation at 96°C and e n d e d with 10 rain incubation at 72°C.
Fifteen microliters of the PCR p r o d u c t were digested overnight w i t h 5 U of the a p p r o p r i a t e restriction enzyme in order to limit the size of the PCR fragments to <230 bp. Five microliters of the digested PCR p r o d u c t were m i x e d with 10 ~1 95% F o r m a m i d e , 20 m M NaOH, 0.05% Brom o p h e n o l Blue, and 0.05% xylene cyanol. Samples were d e n a t u r e d at 96°C for 10 m i n and i m m e d i a t e l y cooled in ice for 5 m i n to m i n i m i z e renaturation before being run on 10% n o n d e n a t u r i n g p o l y a c r y l a m i d e gel in TBE 1 × buffer at a constant temperature and voltage overnight. The temperature, voltage, and percentage of glycerol were standardized for each exon. DNA was visualized by silver-staining. The exon fragments that h a d p r e v i o u s l y been reported as containing CGA codons whose m u t a t i o n C-~T alters a restriction enzyme site [39], were subjected to a p p r o p r i a t e restriction enzyme digestion, using 15 ~1 of PCR reaction p r o d u c t and 10 U of the enzyme, and were run in a 2% agarose gel.
Sequencing Sequence analysis was performed b y use of Sequenase. Polymerase chain reaction (PCR) products were purified from u n i n c o r p o r a t e d primers and dNTPs by ultrafiltration using a Microcon 100 filtration unit. Sequencing reactions were electrophoresed on an automatic sequencer.
RESULTS The karyotype in l y m p h o c y t e s of p e r i p h e r a l b l o o d was s t u d i e d in 43 patients w i t h RB, although in only one case was a constitutional defect consisting of an interstitial deletion of c h r o m o s o m e 13, affecting to the genetic material b e t w e e n the q13-q31 bands detected (Fig. 1). The patient h a d bilateral retinoblastoma, d y s m o r p h i c features, and mental retardation. He was the first child of a couple both w i t h n o r m a l p h e n o t y p e and karyotype. The cytogenetic study carried out by direct m e t h o d in the tumoral cells of four patients m a d e clear that it is difficult to obtain the karyotype in these cells, In two cases the study was unsuccessful, one of the others s h o w e d eleven metaphases with the karyotype 47,XX,del(1)(p34),add(10)
44
E. Mateu et al.
G
R Figure 1 Chromosome pairs 13 with del(13)(q13q31) m a constitutional karyotype of a patient with retinoblastoma. G and R banding.
(q26),-19,+der(19)t(1;19)(q12;p13)×2 (Fig. 2) and the last case s h o w e d six metaphases w i t h the karyotype 48,XX, - 3,i(6)(p10), + 9, + 2mar. The analysis of the allele segregation with all p o l y m o r p h i c markers a p p o i n t e d in Table 1 was performed in constitutional DNA from the 43 families. F r o m this analysis the presence of a heterozygotic deletion in three males with sporadic RB was deduced, Two of these were bilateral cases and the other unilateral. In all cases the chromosome deleted was the paternal one. In one of the bilateral cases, the deletion was also detected by cytogenetic analysis, whereas in tile other patient, the loss of the paternal allele
was observed with RBi2 and RB1.20, which were the only fully informative markers (Fig. 3). In the patient with unilateral RB we have detected loss of paternal allele with the markers p123M1.8, p95HSO.5, p88PR0.6, and RB1.26, whereas RBi2 and RB1.20 markers were heterozygotic. The analysis with p o l y m o r p h i c markers in unaffected relatives of another 8 patients (1 bilateral and 7 unilateral cases) s h o w e d that one or two siblings of each patient share h a p l o t y p e with him/her. The p o l y m o r p h i c marker RB1.20, based on the repetition of CTTT(T) [31], detected the existence of a large unc o m m o n allele described by Toguchida et al. [19], w h i c h is related to the infrequent alleles of the Mbo II RFLP described by Wiggs et al. [30] because RB1.20 alleles scored as large contain e x p a n d e d repeat sequences including one or more internal MboII sites. We have found at least three different variants for this large allele, with the difference of a small n u m b e r of repetitions, in the constitutional DNA of a patient with unilateral RB and in his unaffected father and grandmother, as well as in the three affected members of another family with unilateral familial RB (Fig. 4), and in 3 (2.5%) of 120 unrelated and unaffected i n d i v i d u a l s in a local study. In the 7 sporadic cases (4 unilateral and 3 bilateral) in w h i c h we were able to study the tumoral tissue, analysis revealed a heterozygotic deletion in 4 patients with sporadic RB (1 unilateral and 3 bilateral cases). No deletion was observed in constitutional DNA of these patients, but in one of t h e m we detected a p o i n t m u t a t i o n in exon 8. The other 3 unilateral tumors s t u d i e d s h o w e d homozygotic deletion comprising all the RB1 gene.
Figure 2 Karyotype of retinoblastoma tumor cell: 47,XX,del(1)(p34),add(10)(q26),- 19,+ der(19)t(l;19)(q12; q13) ×2. The arrows indicate chromosomal aberrations.
5
12
18
Genetics of Retinoblastom~
45
H y b r i d i z a t i o n w i t h cDNA probes has confirmed the existence of deletions in the same cases referred to above. A l t h o u g h no extra b a n d was f o u n d in any case, the suspicion of deletion was established by a decrease in the hyb r i d i z a t i o n signal because of a lower dose. The screening of muta~:ions in the RB1 gene by SSCPs analysis a n d digestion of the exon fragments containing a CGA codon with the suitable restriction enzyme has shown the existence, in leucocytary DNA, of a n o m a l o u s bands in 3 patients w i t h bilateral sporadic RB (Fig. 5A), in one with unilateral sporadic RB (Fig. 5B), and in another of bilateral familial RB. These alterat:[ons were not found in their unaffected relatives, thus we a s s u m e d that they were the mutations p r e d i s p o s i n g to RB, raised "de novo" in sporadic cases. The subsequent sequencing of each of these exons w i t h a n o m a l o u s b a n d s has characterized the mutations, s h o w n in Table 2. In our study we have also identified n u c l e o t i d e changes outside the coding region, in intronic sequences a m p l i f i e d on both sides of the exons. Two of these changes
were located in intron 3, at postion +37 (A to T transversion) and +45 (C to T transition), a n d the other changes were found in intron 4 at p o s i t i o n +23 (G to T transversion), and in intron 19 at postion - 7 7 (A to G transition).
DISCUSSION Cytogenetic studies made in constitutional DNA of patients w i t h RB s h o w e d that about 3% of t h e m present interstitial deletions in 13q14 [54]. In our samples, 1 (2.3%) of 43 patients shows this alteration. Cytogenetic defects detected in tumoral cells tend to be secondary alterations found in neoplasias in a d v a n c e d stages. Of these alterations, the i(6p) a n d the extra material in the large arms of c h r o m o s o m e 1 are the most frequently found in RB [16]. Both alterations were found in two of our patients. The fact that the two 13 c h r o m o s o m e s were n o r m a l in these two patients, in both p e r i p h e r a l b l o o d and tumoral tissue, suggests that in most cases the p r i m a r y
Figure 3 (A) Pedigree of a family with bilateral sporadic retinoblastoma showing the segregation of polymorphic markers within the RB1 gene. The numbers beside each chromosome represent the allelic form of each locus. In the affected family member, the RBi2 and RB1.20 paternal alleles are lost. A question mark sign denotes that we cannot infer the hemizygous or homozygous state. (B) Electrophoretic pattern obtained with microsatellite markers RBi4 (a) and RB1.20 (b) in DNA samples from each family member.
RB-224 Band//
2
2
RBi2 Xpnl RBi4 Xbal P,sol RB1.20 RBI.26
2 1 1 2 2 1 1
4 1 1 2 2 2 1
RB-225 1 3 1 2 1 1 4
RB-227 ? del ? ? ? ? del ?
2 1 1 1 2 2 3
(o)
RB..226
2 1 1 1 2 2 3 1
2 2 1 1 2 2 1 1
A
1 3 1 2 1 1 4 1
B
46
E. Mateu et el.
A bp D
M
B bp
M
517"
m
39(
396'
344
344-
298298 '
1
2
2
3
3
4
4
5
5
d
u
d
u
d
u
d
u
d
u
Figure 4 Electrophoretic pattern obtained with the RB1.20 marker. (A) The arrows denote the uncommon allele (~450 bp), detected in several members (1 to 5) of two unrelated families. Common shorter alleles are in the 250330 bp range. (B) Intron 20 fragments of subjects showing the large allele was digested with MbolI. Digested (d) and undigested (u) samples were electrophoresed. The picture shows the difference between both patterns.
structural alterations occurring in the RB gene cannot be detected by cytogenetic analysis and one must resort to analysis with molecular biology techniques. In 3 (7%) of the 43 patients (2 bilateral and 1 unilateral sporadic), we identified large constitutional deletions using genomic probes or microsatellite markers detecting intragenic polymorphisms. The detection of these mutations in two bilateral sporadic cases ("a priori" presumptive hereditary form), suggests that in 14% of our hereditary cases (n = 14), the direct identification of deletions using intragenic polymorphisms is possible, which is in agreement with previous reports [34, 41, 55] based on hereditary RB. The development of microsatellite markers spanning the whole RB1 gene has greatly improved the technology applied to the detection of constitutional deletions because of its higher informativity. In our familial cases, the analysis of segregation with intragenic polymorphisms determined the inheritance of the mutation predisposing to RB by examining their coinheritance with DNA polymorphisms. In one of them, there is a discrepancy between DNA polymorphism and phenotype, which probably reflects low penetrance. Scheffer et el. [56] identified several kindreds with a low penetrance phenotype by similar analysis. DNA polymorphism should, according to Wiggs et el. [30], allow the genotype and the eventual phenotype of newborns in kindred with familial RB to be predecided. However, because of incomplete penetrance it is difficult in nonfamilial cases to determine the probability of carrying a tumor predisposing mutation within a RB gene when an unaffected sibling has an identical haplotype as the affected member. Thus, one of the
children of family 24 (Fig. 5B) had inherited identical alleles as his affected DZ twin who was diagnosed at 8 years old, although at 12 the former had not developed RB. The identification of a germline mutation in the affected child, an A-~C transversion in exon 14 (codon 447), not present in his DZ twin brother or his parents, was the definitive finding in the accurate determination of risk in all members with inherited associated alleles, or in the parents. Concerning the analysis of tumoral DNA we have had serious difficulties in the interpretation of results in DNA proceedings from paraffin-embedded tissues, because the DNA sample is, in many cases, insufficient for dosage valorization hybridizing with cDNA in Southern blotting. Moreover, the DNA is frequently degraded, making certain experimental procedures impossible, such as PCR amplification when PCR fragments are too long. On the other hand, in fresh or fixed tissue, false heterozygosity may show amplified from DNA of normal cells in some cases. At present, early diagnosis of RB patients leads to a reduction in the use of classic methods for treatment of RB, including enucleation, so it could be that the analysis of DNA extracted from tumor tissue may only be able to be performed in a few cases. With regard to this aspect, an important consideration about segregation analysis including tumoral DNA, is the fact that, in a patient known to have the hereditary form of RB, evaluation of the tumor for the RB1 gene alleles homozygosity may have important implications for genetic counseling. According to Dryja et el. [57] their comment that if the tumor has lost the allele present on one chromosome 13 homologue, one can assume that the chromosome 13 remaining in the tumor is the one
Genetics of Retinoblastoma
47
A
B
Figure 5 Mutation analysis by SSCPs. A) Pedigree of family fRB-33 and electrophoresis of DNA samples from father's, mother's, and patient's leukocyte and tumor. PCR fragment spanning exon 18 shows the existence of aberrant SSCP bands in the affected patient. B) Pedigree of family fRB-24 and result of SSCP analysis of exon 14 PCR product from leukocyte DNA, showing additional bands in the affected family member.
that harbors the mutant allele, and therefore predict which family members are at risk for the disease. Moreover, because RB tumors do not have normal RB1 allele, finding the germline mutation is most efficiently accomplished by first identifying the RB1 mutations in tumoral DNA [58]. Our results confirm, in general, the conclusions reported before by other authors, i.e., Cowell et al. [39], Blanquet et al. [40], and L o h m a n n et al. [41], concerning the spectrum of h u m a n germline mutations in the RBI gene that confers hereditary predisposition to RB. In agreement with these authors, we have observed that base substitution represents, in conjunction with deletion or insertion of one base, the most c o m m o n germline mutation identified in patients with hereditary RB. With regard to the base substitutions, the C to T transitions affecting CGAarg codons is
Table 2 Patient
the most frequent nucleotide change. Lohmann et al. [41] detected C--~T transitions in CGA codons in 29 (40.8%) of 71 patients with hereditary RB by mutational screening using HDA, SSCPs, or sequencing. Cowell et al. [39] identified this type of mutation, which results in the generation of premature stop codons, in 18 (16%) of 113 patients with bilateral RB by SSCP analysis and restriction enzymes. We have detected C to T transition in 2 (20%) of 10 patients with bilateral RB by mutational screening using SSCPs and restriction enzymes and, although we have not scanned all the exons of the RB1 gene, the remaining exons are not frequently reported as preferentially altered by this type of mutation. With regard to the local distribution of these mutations within a RB1 gene, our results are in agreement with Blanquet et al. [40, 59], who, by denaturant gradient gel electrophoresis (DGGE) analysis, were able to show the majority of C to T mutations in exon 8 and exon 18. Thus, the mutation in exon 8 (codon 255) was detected in a total of 7 (13%) of 54 affected individuals in which the germline mutation was characterized (3 bilateral and 4 familial), whereas the C-~T transition in codon 579 of exon 18 was identified in 3 (5.5%) of them [40]. Lohmann et al. [41], using a combination of different techniques of mutational screening, reported that the exons preferentially altered with C-~T mutations in CGA codons are, besides exons 8 and 18, the exon 10 (codon 320), exon 11 (codon 358), exon 14 (codon 445), exon 15 (codon 467), and exon 23 (codon 787). Curiously, Lohmann et al. [41] detected some of these mutations, concretely those in exon 10 and 14, only by sequencing and they were not detected using SSCPs or HDA. Moreover, these authors reported that the most frequent C to T transition in exon 8 is in codon 251, in agreement with Cowell et al. [39], who described that the same mutation and the change C-~T in exon 17 (codon 552) represent 73% of all C-~T mutations detected in the RB1 gene and 10 to 15% of the total mutations in hereditary RB. Neither in our analysis, nor previously noticed by Blanquet et al. [40], was the mutation in exon 17 (codon 552) found in any patient, even though this exon was analyzed by different methods. We suggest, in the light of all these findings, that detection of C-~T transitions in the RB1 gene could be influenced by several factors: population analyzed, technique, and local conditions in the ex-
Summary of germline point mutations in the RB1 gene Phenotype
Mutation
Position
Putative consequence
RB-190 RB-198
Bilateral sporadic Bilateral sporadic
Nonsense C-~T transition C-~T transition
Exon 8 (codon 255) Exon 18 (codon 579)
Arg-~Stop Arg-~Stop
RB-61 RB-210
Bilateral sporadic Bilateral familial
Frameshiff T insertion G deletion
Exon 20 (codon 667) Exon 19 (codon 617)
Stop 685 Stop 622
RB-142
Unilateral sporadic
Missense A-~C transversion
Exon 14 (codon 447)
Lys-oGln
48 perimental procedure. In spite of this, the scanning of C to T mutations in CGA codons of RB1 gene is the most efficient strategy for detecting germline mutations in RB patients. Other types of mutations, such as small length alterations (1-18 bp) can be identified by SSCPs, HDA, or highresolution fragment analysis, although if the PCR fragments are too long, the last technique is not able to detect this type of mutation, as L o h m a n n et al. have reported [41, 60]; However, we have only identified a deletion of 1 bp and an insertion of I bp, respectively, in one familial and one bilateral sporadic RB case using SSCP analysis foll o w e d b y direct sequencing w h e n an abnormal pattern was observed. Other small length alterations comprising more than one bp have not been found. It has been described that deletions of 1 bp are the most c o m m o n [60], however, we cannot exclude the possibility that deletions or insertions of several bp are being missed by SSCPs in our study. Both of the nucleotide changes detected in intron 3 have been described previously as p o l y m o r p h i c changes [31, 40]. The other intronic variants observed by us in intron 4 and intron 19 have not been reported before. The fact that these changes have been detected both in affected and unaffected relatives suggests its irrelevance in the pathogenesis of the disease. With the totality of procedures techniques performed, we have detected the responsible germline m u t a t i o n in 6 (42.8%) of 14 unrelated patients w i t h familial or bilateral sporadic RB, and in 2 (7%) of 29 cases with unilateral sporadic disease, rates very low in c o m p a r i s o n with the rate obtained by L o h m a n n et al. [41] in hereditary cases, w h o defined this p r o p o r t i o n as 83%. Our w h o l e results in bilateral sporadic and familial RB are more in agreement with the lower rates (56.5%) reported by Blanquet et al. [40]. It could be that w h e n the scanning of the w h o l e RB1 gene is finished, the frequency of detection of an R B I m u t a t i o n m a y reach a higher percentage, but obviously far from 83%. Likewise, our results in unilateral RB are, up to now, lower than Vogel's data [2], w h i c h suggest that 10 to 12% of unilateral sporadic cases are carriers of a germline mutation. On the other hand, Draper et al. [61] define this proportion at 2%. In summary, our study reflects that m o l e c u l a r analysis af RB patients to perform an accurate genetic counseling is feasible and profitable in familial form, even w i t h o u t identifying the responsible mutation. This objective is, however, h a r d l y accomplishable in sporadic cases, because of the low rates of mutational detection, extensive mutational heterogeneity, variability about the frequency of detection because of u n k n o w n variants, and the need to use several technical procedures. All of these factors are responsible for the high cost and difficulty of the study, w h i c h limit the i m p l e m e n t a t i o n of the analysis in clinical routine. There is a need for further investigation to clarify mutational m e c h a n i s m s in the RB1 gene a n d definitive spectrum in each population, in order to k n o w if the scientific achievements are converted into benefits for those at risk, and if it is possible to offer this s t u d y to all RB patients at different clinical laboratories.
E. Mateu et al. We should like to thank the families for their generous cooperation, and Pr.T.P. Dryja for providing the p4.95BT cDNA probe and genomic probes used in this study. This work was supported by the CICYT (SAF 92/0266). F. S~nchez is the recipient of a fellowship from the Consellerfa de Educaci6n y Ciencia de la Comunidad Valenciana. REFERENCES
1. Naumova A, Sapienza C (1994): The genetics of retinoblastoma, revisited, Am J Hum Genet 54:264-273. 2. Vogel F (1979): Genetics of retinoblastoma. Hum Genet 52:1-54.
3. Francois J, De Sutter E, Coppieters R, De Bies S (1980): Late extraocular tumors in retinoblastoma survivors. Ophtalmologica (Basel) 181:93-99. 4. Abramson DH, Ellsworth RM, Kitchin DF, Tung G (1984): Second nonocular tumors in retinoblastoma survivors, are they radiation induced? Ophthalmology 91:1351-1355. 5. Draper GJ, Sanders BM, Kingston JE (1986): Second primary neoplasms in patients with retinoblastoma. Br J Cancer 53: 661-671. 6. Roarty JD, McLean IW, Zimmerman LE (1988): Incidence of second neoplasm in patients with bilaterial retinoblastoma. Ophthalmology 95:1583-1587. 7. Knudson AG (1971): Mutation and cancer: Statistical study of retinoblastoma. Proc Natl Acad Sci USA 68:820-823. 8. Cavenee WK, Dryja TP, Phillips RA, Benedict WF, Godbout R, Gallie BL, Murphree AL, Strong LC, White RL (1983): Expression of recessive alleles by chromosomal mechanisms in retinoblastoma. Nature 305:779-784. 9. Dryja TP, Mukai S, Petersen R, Rapaport JM, Walton D, Yandell DW (1989): Parental origin of mutations of the retinoblastoma gene. Nature 339:556-558. 10. Zhu X, Dunn JM, Phillips RA, Goddard AD, Paton KE, Becker A, Gallie BL (1989): Preferential germline mutation of the paternal allele in retinoblastoma. Nature 340:312-313. 11. Matsunaga E, Minoda K, Sasaki MS (1990): Parental age and seasonal variation in the births of children with sporadic retinoblastoma: A mutation-epidemiologic study. Hum Genet 84:155-158. 12. Franke U (1976): Retinoblastoma and chromosome 13. Cytogenet Cell Genet 16:131-134. 13. Sparkes RS, Sparkes NC, Wilson NG, Towner JW, Benedit WF, Murphree AL, Yunis JJ (1980): Regional assignment of genes for human esterase-D and retinoblastoma to chromosome band 13q14. Science 208:1042-1044. 14. Turleau C, de Grouchy U, Chavin-Coi IN F, Junien C, Seger J, Schieinger P, Leblanc A, Haye C (1985): Cytogenetic forms of retinoblastoma: Their incidence in a survey of 66 patients. Cancer Genet Cytogenet 16:321-334. 15. Squire J, Gallie BL, Phillips RA (1985): A detailed analysis of chromosomal changes inheritable and non-heritable retinoblastoma. Hum Genet 70:291-301. 16. Kusnetsova LE, Prigogina EL, Pogosianz HE, Belkina BM (1982): Similar chromosomal abnormalities in several retinoblastomas. Hum Genet 61:201-204. 17. Squire J, Phillips RA, Boyce S, Godbout R, Rogers B, Gallie BL (1984): Isochromosome 6p, a unique chromosomal abnormality in retinoblastoma: The verification by standard staining techniques, new densitometric methods, and somatic cell hybridisation. Hum Genet 66:46-53. 18. Friend SH, Bernards R, Rojelj S, Weinberg RA, Rapaport JM, Albert DM, Dryja TP (1986): A human DNA segment with properties of the gene that predisposes to retinoblastoma and osteosarcoma. Nature 323:643-646.
Genetics of Retinoblastoma
19. Toguchida J, McGee TL, Paterson JC, Eagle JR, Tucker S, Yandell DW, Dryja TP (1993): Complete genomic sequence of the human retinoblastoma susceptibility gene. Genomics 17: 535-543. 20. Lee WH, Shew JY, Hong FD, Sery TW, Donoso LA, Young LJ, Bookstein R, Lee YP (1987): The retinoblastoma susceptibility gene encodes a nuclear phosphoprotein associated with DNA binding activity. Nature 329:642-645. 21. Hong FD, Huang H-JS, Young L-JS, Oro A, Bookstein R, Lee YP, Lee W-H (1989): Structure of the human retinoblastoma gene. Proc Natl Acad Sci USA 86:5502-5506. 22. Buchkovich K, Duffy LA, Harlow E (1989): The retinoblastoma protein is phosphorylated during specific phases of the cell cycle. Cell 58:1097-1105. 23. Mihara K, Cao X-R, Yen A, Chandler S, Driscoll B, Murphree AL, T-Aug A, Fung Y-KT (1989): Cell cycle-depandent regulation of phosphorylat[on of the human retinoblastoma gene product. Science 246:1300-1303. 24. Riley DJ, Lee EYHP, Lee WH (1994): The retinoblastoma protein: More than a tumor suppressor. Annu Rev Cell Biol 10: 1-29. 25. DeCaprio JA, Ludlow )W, Figge J, Shew J-Y, Huang C-M, Lee W-H, Marsilio E, Paucha E, Livingston DM (1988): SV40 large tumour antigen forms a specific complex with the product of the retinoblastoma susceptibility gene. Cell 44:275-283. 26. Whyte P, Buchkovich K, Horowitz JM, Friend SH, Raybuck M, Weinberg RA, Harlouw E (1988): Association between an oncogene and an anti-oncogene: The adenovirus EIA proteins bind to the retinoblastoma gene product. Nature 334:124-129. 27. Dyson N, Howley PN, Munger K, Harlow E (1989): The human papilloma virus-16 E7 oncoprotein is able to bind to the retinoblastoma gene product. Science 243:934-936. 28. Hu K, Dyson N, Harlow E (1990): The regions of the retinoblastoma protein needed for binding to adenovirus EIA or SV4O large T antigen are common sites for mutation. EMBO J 9:1147-1155. 29. Sparkes RS, Murphree AL, Lingua RW, Sparkes MC, Field LL, Funderburk SJ, Benedict WF (1983): Gene for hereditary retinoblastoma assigned to human chromosome 13 by linkage to esterase-D. Science 217:971-973. 30. Wiggs J, NordenskjSld M, Yandell D, Rapaport J, Grondin V, Janson M, Werelius E, Petersen R, Craft A, Riedel K, Liberford R, Walton D, Wilson W, Dryja TP (1988): Prediction of the risk of hereditary retinoblastoma, using DNA polymorphisms within the rotinoblastoma gene. New Engl J Med 318:151-157. 31. Yandell DW, Dryja TP (1989): Detection of DNA sequence polymorphims by onzymatic amplification and direct genomic sequencing. Am J Hum Genet 45:547-555. 32. Bookstein R, Lai C-G, To H, Lee W-H (1990): PCR-based detection of a polyr~.orphic BamHI site in intron 1 of the human retinoblastom~ (RB) gene. Nucleic Acids Res 18:1666. 33. Greger V, Debus N, Lohmann D. HSppin W, Passarge E, Horsthemke B (1994:): Frequency and parental origin of hypermethylated RB3 alleles in retinoblastoma. Hum Genet 94:491-496. 34. Kloss K, W~hrisch P, Greger V, Messmer E, Fritze H, HSpping W, Passarge E, Horsthemke B (1991): Characterization of deletions at the retinoblastoma locus in patients with bilateral retinoblastoma. Am J Med Genet 39:196-200. 35. Dunn JM, Phillips RA, Becker AJ, Gallie BL (1988): Identification of germline and somatic mutations affecting the retinoblastoma gene. Science 241:1797-1800. 36. Orita M, Suzuki Y, Sekiya T, Hayashi K (1989): Rapid and sensitive detection cf point mutations and DNA polymorphisms using the polymerase chain reaction. Genomics 5:874-879.
49 37. Ganguly A, Rock MJ, Prockop DJ (1993): Conformation-sensitive gel electrophoresis for rapid detection of single-base differences in double-stranded PCR product and DNA fragments: Evidence for solvant-induced bends in DNA heteroduplexes. Proc Natl Acad Sci USA 90:10325-10329. 38. Yandell DW, Campbell TA, Dayton SH, Petersen R, Walton D, Little JB, McConkie-Rosell A, Buckley EG, Dryja TP (1989): Oncogenic point mutations in the human retinoblastoma gene: Their application to genetic counseling. New Eng J Med. 321:1689-1695. 39. Cowell JK, Smith G, Bia B (1994): Frequent constitutional C to T mutations in CGA-arginine codons in the RB1 gene produce premature stop codons in patients with bilateral (hereditary} retinoblastoma. Eur J Hum Genet 2:281-290. 40. Blanquet V, Turleau C, Gross-Morand MS, Beaufort CS, Doz F, Besmond C (1995): Spectrum of germline mutations in the Rbl gene: A study of 232 patients with hereditary and non hereditary retinoblastoma. Hum Mol Genet 4:383-388. 41. Lohmann DR, Brandt B, H6pping W, Passarge E, Horsthemke B (1996): The spectrum of RB1 germ-line mutations in hereditary retinoblastoma. Am J Hum Genet 58:940-949. 42. Naylor JA, Green PM, Rizza CR, Gianelli F (1993): Analysis of factor VIII mRNA reveals defects in every one of 28 haemophilia A patients. Hum Mol Genet 2:11-17. 43. Yeung RS, Bell DW, Testa JR, Mayol X, Baldi A, Giana X, Klinga-Levan K, Knudson AG, Giordano A (1993): The retinoblastoma-related gene, RB2, maps to human chromosome 16q12 and not chromosome 19. Oncogene 8:3465-3468. 44. Bona'iti-Pellie C, Chompret MF, Tournade MF, Lemerly J, Voute PA, Delamarre JF (1993): Excess of multifocal tumors in nephroblastoma: Implications for mechanisms of tumors development and genetic counseling. Hum Genet 91:373376. 45. Kunkel LM, Tantravah V, Eisanhard M, Latt SA (1982): Regional localization on the human X of DNA sequences cloned from flow shorted chromosomes. Nucleic Acids Res 10:1557-1571. 46. Shibata DK, Arnheim N, Martin WJ (1988): Detection of human papilloma virus in paraffin-embedded tissue using polimerase chain reaction. J Exp Med 167:225-230. 47. Onadim Z, Cowell JK (1991): Application of PCR-amplification of DNA from paraffin-embedded tissue sections to linkage analysis in familial retinoblastoma. J Med Genet 28:312316. 48. Moorhead PS, Nowell PC, Mellman WJ, Battips DM, Hungerford DA (1960): Chromosome preparations of leukocytes cultures from peripheral blood. Exp Cell Res 20:613-616. 49. Seabright M (1971): A rapid binding technique for human chromosome. Lancett II:971-972. 50. Dutrillaux B, Laurent C, Gilgenkrantz S, Frederic J, Carpentier J, Lejeune J (1974): Les translocations du chromosome X. Etude apr~s traitement par le BUDR et coloration par l'acridine orange. Helv Paediatr Acta [suppl] 34:19-31. 51. Southern EM (1975): Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98:503-517. 52. Bronstein I, Voyta JC, Lazzari KG, Murphy OJ, Edwards B, Kricka LJ (1990): Rapid and sensitive detection of DNA in Southern blots with chemiluminescence. BioTechniques 8: 310-314. 53. Hogg A, Onadim Z, Baird PN, Cowell JK (1992): Detection of heterozygous mutation in the RB1 gene in retinoblastoma patient using single-strand conformation polymorphism analysis and polymerase chain reaction sequencing. Oncogene 7:1445-1451. 54. Cowell JK and Hogg A (1992): Genetics and cytogenetics of retinoblastoma. Cancer Genet Cytogenet 64:1-41.
50
55. Blanquet V, Cr~au-Goldberg N, de Grouchy J, Turleau C (1991): Molecular detection of constitutional deletions in patients with retinoblastoma. Am J Med Genet 39:355-361. 56. Scheffer H, 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 mutations, revealed by combined use of markers within and flanking the RB1 gene. Am J Hum Genet 45:252-260. 57. Dryja TP, Cavenee WK, White R, Rapaport JM, Peterson R, Albert DM, Bruns GA (1984): Homozygosity of chromosome 13 in retinoblastoma. New Engl J Med 310:550-553. 58. Gallie BL, Dunn JM, Chan HSL, Hamel PA, Phillips RA (1991): The genetics of retinoblastoma. Relevance to the patient. Pediatr Clin North Am 38:299-315. 59. Blanquet V, Gross MS, Turleau C, Senamand-Beaufort C, Doz F, Besmond C (1994): Three novel germline mutations in the exon 8 and 18 of the retinoblastoma gene. Hum Mol Genet 7:1185-1186.
E. Mateu et al.
60. Lohmann DR, Brandt B, H6pping W, Pasarge E, Horsthemke B (1994): Spectrum of small length germline mutations in the RB1 gene. Hum Mol Genet 3:2187-2193. 61. Draper GJ, Sanders BM, Brownbill PA, Hawkins MM (1992): Pattern of risk of hereditary RB and applications to genetic counselling. Br J Cancer 66: 212-219. 62. Bookstein R, Lee EY, To H, Young LJ, Sery TW, Hayes RC, Friedmann T, Lee WH (1988): Human retinoblastoma susceptibility gene: Genomic organization and analysis of heterozygous intragenic deletion mutants. Proc Natl Acad Sci USA 85:2210-2214. 63. McGee TL, Cowley GS, Yandell DW, Dryja TP. (1990): Detection of the XbaI RFLP within the retinoblastoma locus by PCR. Nucleic Acids Res 18:207. 64. Brandt B, Greger V, Yandell D, Passarge E, Horsthemke B (1992): A simple and nonradioactive method for detecting the Rbl.20 DNA polymorphism in the retinoblastoma gene. Am J Hum Genet. 51:1450-1451.