Oncogene (1998) 16, 3211 ± 3213 1998 Stockton Press All rights reserved 0950 ± 9232/98 $12.00 http://www.stockton-press.co.uk/onc SHORT REPORT A novel missense mutation in patients from a retinoblastoma pedigree showing only mild expression of the tumor phenotype John K Cowell and Britta Bia Department of Neurosciences, Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, Ohio 44195, USA We have used single strand conformation polymorphism (Knudson, 1971; Comings, 1973; Cavenee et al., 1983, analysis to study the 27 exons of the RB1 gene in 1985; Dunn et al., 1988, 1989; Yandell et al., 1989; individuals from a family showing `mild' expression of Hogg et al., 1993). Since the inactivation of the normal the retinoblastoma phenotype. In this family aected gene in predisposed individuals in random, this event individuals developed unilateral tumors and, as a result follows a Poisson distribution (Knudson, 1971). Thus, of linkage analysis, unaected mutation carriers were within pedigrees where the majority of aected also identi®ed within the pedigree. A single band shift individuals have multiple tumors in both eyes, there using SSCP was identi®ed in exon 21 which resulted in a are, on rare occasions, individuals who only develop a missense mutation converting a cys?arg at nucleotide single tumor in one eye or who, since they do not position 28 in the exon. The mutation destroyed an NdeI experience the second mutation at all, are unaected restriction enzyme site. Analysis of all family members carriers of the germline mutation. This phenomenon is demonstrated that the missense mutation co-segregated referred to as incomplete penetrance and occurs at an with patients with tumors or who, as a result of linkage estimated frequency of 8 ± 10% (Draper et al., 1992). analysis had been predicted to carry the predisposing Typically these variant forms of the disease phenotype mutation. These observations point to another region of occur within families where the other aected members the RB1 gene where mutations only modify the function have the more severe form of the disease. However, of the gene and raise important questions for genetic there have been a few reports of families where having counseling in families with these distinctive phenotypes. a `mild' phenotype is the rule rather than the exception (Macklin et al., 1960; Connelly et al., 1983; Onadim et Keywords: retinoblastoma; missense mutation; incom- al., 1990) and linkage studies have identi®ed `at risk' plete penetrance; SSCP individuals who are unaected or have unilateral or regressed tumors (Onadim et al., 1992). It has now become clear that the majority of Mutational analysis of the RB1 gene has shown that mutations identi®ed in individuals with the severe patients with the hereditary form of retinoblastoma phenotype usually generate premature stop codons (Rb) carry germline mutations within the gene (Hogg (Blanquet et al., 1995; Liu et al., 1995; Cowell et al., et al., 1992; Onadim et al., 1993; Blanquet et al., 1993, 1994; Lohman et al., 1996), either as a result of single 1995; Lohmann et al., 1994, 1996; Shimizu et al., 1994). base pair mutations or following frame-shifts induced Hereditary cases are de®ned as individuals who have a by insertions and deletions. Mutations aecting splice family history of tumors or individuals without a sites have also been reported in about 30% of cases family history but who have bilateral and/or multifocal (Hogg et al., 1992; Liu et al., 1995; Lohmann et al., tumors usually with an early age of onset. By contrast, 1994) which are presumed to result in a non-functional individuals who present without a family history and protein. In contrast to the type of mutation seen in have only unilateral, unifocal tumors are generally these families, those pedigrees exhibiting a mild considered not to carry germline mutations. While this phenotype appear to carry a very dierent type of distinction holds true in the vast majority of cases there mutation such as missense mutations (Onadim et al., is some overlap since approximately 2% of unilateral 1992) or mutations in the promoter region (Sakai et al., cases have a family history (Draper et al., 1992) and we 1991; Cowell et al., 1996). The prediction in these cases have shown recently that unilateral cases who have a is that the mutation only compromises the function of particularly early age of onset may also carry germline the protein such that enough functional protein is mutations (Cowell and Cragg, 1996). Within the produced in the developing retinal cells that tumor- hereditary group it is now clear that both alleles of igenicity does not occur. Evidence supporting this the RB1 gene must be inactivated for tumorigenesis, conclusion was presented recently for one missense supporting Knudson's original two-hit hypothesis mutation (Kratzke et al., 1994). To determine whether (Knudson, 1971). Thus, the ®rst mutation is inherited missense/promoter mutations are commonly the cause and the mutation resulting the loss of function of the of mild Rb phenotypes we have analysed another large homologous normal gene occurs as a random somatic family with mild phenotypes and have identi®ed a event which must occur in retinal precursor cells novel missense mutation segregating with tumor predisposition. The family shown in Figure 1, Family H, showed the typical manifestations of a mild phenotype. To date, all of the individuals who have developed tumors, Correspondence: JK Cowell Received 1 August 1997; revised 19 January 1998; accepted 19 only had unilateral disease. Furthermore, during the January 1998 course of our genetic linkage analysis of pedigrees from Hereditary missense mutation in RB1 JK Cowell and B Bia 3212 the UK (Onadim et al., 1990), using the RS2.0 probe Since our ®rst report (Onadim et al., 1992) of an (Wiggs et al., 1988), we were also able to identify Arg?Trp missense mutation in a family with a low unaected individuals who were predicted to be penetrance phenotype, several other groups have since carriers of the mutation segregating in this pedigree reported similar observations. Thus, Lohmann et al. (Figure 1). Although, to illustrate the extent of the (1994) identi®ed the same codon-661 mutation in two mild phenotype the full pedigree for this family is families with low penetrance and we (unpublished shown in Figure 1, the part of the family descended observations) have identi®ed the same mutation in from individual I.2 was not available to us for the another family in our series where both the two DNA analysis. We were therefore only able to study aected children and the unaected father carried the the two families descended from individuals I.5 and I.6. same 661 mutation. This family was too small to be A full scan of the RB1 exons using SSCP (Hogg et al., classi®ed as a low penetrance family but provides a 1992) revealed a bandshift for exon 21 which, on cautionary note to those counseling families with sequencing, was shown to be a T?C change resulting multiple aected ospring born to unaected parents in a missense mutation, cys?arg at nucleotide where the interpretation might have been that germline position 28 in the exon. Sequencing exon 21 from the mosaicism was involved in the absence of the mutation family members available to us demonstrated that all being identi®ed. The codon 661 mutation appears to of the aected individuals (II.7, II.8 and II.9) as well as reduce the eciency of Rb protein function without the two patients, I.6 and III.1 who were identi®ed abolishing it (Kratzke et al., 1994). Other mutations using the RS2.0 RFLP as being a carrier of the mutant which have been identi®ed in low penetrance families gene, in fact carried the missense mutation. This involve a three base pair deletion in exon 16 (Lohmann mutation destroys an NdeI site and so the presence et al., 1994) and an in-frame deletion of exon 4 (Dryja of the mutation can be visualized directly after et al., 1993). Again the suggestion is that these digestion of the PCR product with that enzyme. This mutations compromise rather than abolish the analysis (Figure 2) provided independent con®rmation eciency of Rb protein function. Although we do of the presence of the mutation. Thus, the 214 bp PCR not have direct proof we suspect that the same is true product is normally digested into two fragments by for the exon 21 mutation reported here. With the NdeI, which are 141 bp and 73 bp long. As shown in exception of the exon 4 deletion, all of the mutations Figure 2 all individuals carrying the mutation retain reported from low penetrance families occur within the the 214 bp fragment, in addition to the two smaller `pocket' region (Hu et al., 1990) of the Rb protein fragments. (pRb) which is responsible for the speci®c protein- Figure 1 Pedigree from family H showing the low penetrance Rb phenotype. Unilaterally aected individuals are indicated by the half ®lled symbols. The allele sizes at the RS2.0 locus are given above the symbols for those individuals from whom DNA was available. The Rb predisposition is segregating with the 1.9 Kb allele. Unaected individuals predicted from this analysis to carry the mutation are indicated by the arrows. This suggestion was con®rmed for individuals I.6 and III.1 by DNA sequencing l.6 l.7 ll.6 II.7 II.8 ll.9 III.1 III.2 lll.3 lll.4 lll.5 M U ll.10 M — 214 — 141 — 73 Figure 2 PCR analysis of the exon 21 mutation in family H. The 214 bp PCR product (U) is normally digested by NdeI into two fragments 141 and 73 bp long.