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