Patient understanding of genetic information influences reproductive decision making in retinoblastoma.

BACKGROUND: Retinoblastoma is the most common malignant tumour of the eye in childhood, with nearly all bilateral tumours and around 17% to 18% of unilateral tumours due to an oncogenic mutation in the RB1 gene in the germline. Genetic testing enables accurate risk assessment and optimal clinical management for the affected individual, siblings, and future offspring. MATERIAL AND METHODS: We carried out the first UK-wide audit of understanding of genetic testing in individuals with retinoblastoma. A total of 292 individuals aged 16 to 45 years were included. RESULTS: Patients with bilateral disease were significantly more likely to understand the implications of retinoblastoma for siblings and children. There was a significant association between not knowing the results of genetic testing or not understanding the implications and not having children, particularly in women. Surprisingly, this was also true for individuals treated for unilateral disease with a low risk of retinoblastoma for their offspring. CONCLUSION: We are concerned that individuals may be making life choices based on insufficient information regarding risks of retinoblastoma and reproductive options. We suggest that improvement in transition care is needed to enable individuals to make informed reproductive decisions and to ensure optimal care for children born at risk of retinoblastoma.

Heritable retinoblastoma and accelerated aortic valve disease.

Heritable retinoblastoma is associated with a germline mutation in the tumour suppressor gene RBI. The Rb protein (pRb) arises from the RB1 gene, which was the first demonstrated cancer susceptibility gene in humans. Second primary malignancies are recognised complications of retinoblastoma. Furthermore, pRb is implicated in valve remodelling in calcific aortic valve disease. We report a family with hereditary retinoblastoma and associated secondary primary malignancies. There are two interesting aspects to this family. The first is the concept of 'cancer susceptibility genes'; the RBI gene being the first reported in humans. A further feature of note is that two family members also have bicuspid aortic valves. We discuss a potential association between the gene defect responsible for retinoblastoma (with its associated propensity for further malignancies) and accelerated deterioration of the bicuspid aortic valve in the proband carrying this gene defect.

Retinoblastoma in Great Britain 1963-2002.

AIM: This paper describes the epidemiology and family history status of 1601 children with retinoblastoma in Great Britain diagnosed 1963-2002 and summarises the practical consequences for diagnosis and counselling of developments in molecular genetics. METHODS: Incidence rates were analysed according to year of diagnosis and tumour laterality. Cases were classified as heritable or non-heritable on the basis of laterality and family history of the disease. RESULTS: There were 998 unilateral cases, 581 bilateral and 22 of unknown laterality. Bilateral cases tended to be diagnosed at a younger age than unilateral. All bilateral cases are regarded as heritable, and 35% had a family history of the disease. 7% of the unilateral cases had a family history and are therefore heritable. Thus, at least (41%) of our cases are heritable. This is an underestimate, since these data on family history are incomplete. For unilateral cases aged below 1 year, the reported incidence rate increased significantly (p<0.0001) by about 2.5% per year; for the age group 1-4 years, the average increase was about 0.5% per year (not significant).

High level amplification of N-MYC is not associated with adverse histology or outcome in primary retinoblastoma tumours.

Twenty-five primary retinoblastoma tumours were analysed by real-time quantitative polymerase chain reaction to determine the genomic copy number of the N-MYC gene (2p24) relative to the copy number for REL, B2M, ALB, AF10 and MLL. Twenty-one of these tumours were shown by Comparative Genomic Hybridization to contain variable copy number increases of chromosomal material mapping to 2p. High level amplification (>30-fold) of N-MYC was found in three tumours, none of which showed adverse histological features and all patients are surviving at between 54 and 108 months post enucleation. Furthermore, the three tumours associated with metastasis and adverse patient outcome showed normal N-MYC copy number. Although high level amplification of N-MYC is an unfavourable prognostic indicator in neuroblastoma, these data show no evidence of a correlation between amplification of N-MYC and adverse outcome in retinoblastoma.

Dde I RFLP may falsify linkage analysis of hereditary retinoblastoma when using SSCP of p88PR0.6 region.

The polymorphic p88PR0.6 locus (Xba I RFLP) in intron 17 of the retinoblastoma gene is a DNA marker with high informative content frequently used for linkage analysis of familial retinoblastoma. We identified an unreported Dde I restriction fragment length polymorphism close to the polymorphic Xba I recognition site that interferes with the SSCP analysis of the PR0.6 region. We have named this new polymorphism RB1.17. Under most electrophoresis conditions, the single strand conformations reflect the Dde I genotype rather than that of Xba I. The chromosomal localization, allele frequencies, inheritance and PCR-based detection of the Dde I RFLP which is useful for linkage analysis itself are reported.

Single cell detection of inherited retinoblastoma predisposition.

Retinoblastoma susceptibility is an autosomal dominantly inherited cancer predisposition which also confers a life-long increased risk for various non-ocular malignancies. We developed a protocol for single cell detection of this disorder which enables its preimplantation genetic diagnosis as an alternative to prenatal diagnosis with attendant pregnancy termination. The presented method detects the underlying mutation of the disease, a linked intragenic polymorphism (p88PR0.6) and an independent marker (D21S1411) for genetic fingerprinting allowing detection of contamination. The strategy is based on the combination of nested triplex polymerase chain reaction, single strand conformation polymorphism analysis by conventional polyacrylamide gel electrophoresis and fragment size determination with automated laser fluorescence.

The RB1 gene mutation in a child with ectopic intracranial retinoblastoma.

The RB1 gene mutation was investigated in a child with ectopic intracranial retinoblastoma using DNA obtained from both the pineal and retinal tumours of the patient. A nonsense mutation in exon 17 (codon 556) of the RB1 gene was found to be present homozygously in both the retinal and the pineal tumours. The same mutation was present heterozygously in the DNA from the constitutional cells of the patient, proving it to be of germline origin. The initial mutation was shown to have occurred in the paternally derived RB1 allele. The mutation is in an area of the gene that encodes the protein-binding region known as the 'pocket' region and has been detected in other cases of retinoblastoma.

Mechanisms of oncogenesis in patients with familial retinoblastoma.

In an analysis of mutations in the RB1 gene in three patients, selected at random, who had a positive family history of tumours, we identified mutations, in constitutional cells, involving exons 3, 13 and 17 of the RB1 gene. We used SSCP and PCR sequencing to screen affected individuals and other members of their families. In two cases the mutations were 2 bp and 1 bp deletions identified in exons 3 and 17 respectively. The third mutation was a 1 bp insertion in exon 13. All three mutations lead to the generation of downstream premature stop codons as a result of frameshift changes, although the mutation in exon 3 possibly affects the splicing mechanism. The sites within the RB1 gene where these mutations occur contain interspersed repetitive DNA sequences, direct and inverted repeat sequences and/or dyad symmetrical elements suggesting that these areas promote the appropriate local sequence environment for the generation of deletions and insertions in the RB1 gene.

Molecular mechanisms of oncogenic mutations in tumors from patients with bilateral and unilateral retinoblastoma.

The RB1 gene from 12 human retinoblastoma tumors has been analyzed exon-by-exon with the single-strand conformation polymorphism technique. Mutations were found in all tumors, and one-third of the tumors had independent mutations in both alleles neither of which were found in the germ line, confirming their true sporadic nature. In the remaining two-thirds of the tumors only one mutation was found, consistent with the loss-of-heterozygosity theory of tumorigenesis. Point mutations, the majority of which were C-->T transitions, were the most common abnormality and usually resulted in the conversion of an arginine codon to a stop codon. Small deletions were the second most common abnormality and most often created a downstream stop codon as the result of a reading frameshift. Deletions and point mutations also affected splice junctions. Direct repeats were present at the breakpoint junctions in the majority of deletions, supporting a slipped-mispairing mechanism. Point mutations generally produced DNA sequences which resulted in perfect homology with endogenous sequences which lay within 14 bp.

Oncogenic point mutations in exon 20 of the RB1 gene in families showing incomplete penetrance and mild expression of the retinoblastoma phenotype.

The retinoblastoma-predisposition gene, RB1, segregates as an autosomal dominant trait with high (90%) penetrance. Certain families, however, show an unusual low-penetrance phenotype with many individuals being unaffected, unilaterally affected, or with evidence of spontaneously regressed tumors. We have used single-strand conformation polymorphism analysis and PCR sequencing to study two such families. Mutations were found in exon 20 of RB1 in both cases. In one family a C----T transition in codon 661 converts an arginine (CGG) to a tryptophan (TGG) codon. In this family, incomplete penetrance and mild phenotypic expression were observed in virtually all patients, possibly indicating that single amino acid changes may modify protein structure/function such that tumorigenesis is not inevitable. In the second family the mutation in codon 675 is a G----T transversion that converts a glutamine (GAA) to a stop (TAA) codon. However, this mutation also occurs near a potential cryptic splice acceptor site, raising the possibility of alternative splicing resulting in a less severely disrupted protein.

Detection of heterozygous mutations in the RB1 gene in retinoblastoma patients using single-strand conformation polymorphism analysis and polymerase chain reaction sequencing.

Several families segregating the autosomal dominant form of the hereditary retinoblastoma predisposition gene have been analysed for the causative mutation. We have used the single-strand conformation polymorphism (SSCP) technique to screen for mutations, exon by exon, in the RB1 gene in affected patients from these families. The SSCP technique has proved a rapid and simple technique which relies on the sequence-dependent migration of single-stranded DNA in a non-denaturing polyacrylamide gel. Oligonucleotide primers flanking all 27 exons and the promoter region of the RB1 gene are reported here. The polymerase chain reaction (PCR)-amplified products range in size from 212 to 625 bp and include a flanking intron sequence which allows detection of mutations in these regions. The sensitivity of SSCP is optimal when DNA fragments are approximately 200 bp long. Consequently, restriction enzyme sites for each amplified region were identified, reducing the size of the PCR products analysed to less than 250 bp. Bands with aberrant migration patterns were observed on SSCP gels in the lymphocyte DNA from two patients with bilateral, familial retinoblastoma. Sequence analysis of these DNA fragments revealed the causative mutations. These consisted of a 1-bp insertion of a T in the coding strand of exon 20 and a G----A mutation in the coding strand of exon 14. This approach has proved to be a powerful method for the rapid detection of germline mutations in the RB1 gene, a programme which can be extended to individuals with new mutations.

Follow-up of retinoblastoma patients having prenatal and perinatal predictions for mutant gene carrier status using intragenic polymorphic probes from the RB1 gene.

We have carried out presymptomatic prediction of mutant gene carrier status in ten individuals with a family history of retinoblastoma. In all cases standard linkage studies were employed using intragenic DNA probes which recognise restriction fragment length polymorphisms. In four cases foetal DNA samples were obtained by chorionic villus sampling, the remaining six were derived from either cord blood samples or venipuncture of neonates. We demonstrated that the mutant gene was inherited by only one of these patients who has subsequently developed bilateral tumours. Six of the other cases have now reached the age beyond which it might have been expected that tumours would develop and are all disease free. It must be concluded that repeated ophthalmological examination of these and future patients shown not to have inherited the mutant gene, is unnecessary.

Application of PCR amplification of DNA from paraffin embedded tissue sections to linkage analysis in familial retinoblastoma.

A family segregating for the retinoblastoma predisposition gene has been analysed using the polymerase chain reaction to exclude their son as being an affected gene carrier. The unusual feature of this family is that the affected child, who would ordinarily have been used to establish phase in a linkage study, died as a result of developing a second tumour some years ago. The only tissue available from this child was a paraffin embedded, formalin fixed histopathological specimen from the second tumour. It was possible to isolate DNA from this tissue and amplify the DNA flanking two polymorphic restriction enzyme sites to establish alleles which cosegregated with tumour predisposition. Archival material can now be used to offer families such as this prenatal screening to provide informed genetic counselling.

Genetic counselling in retinoblastoma: importance of ocular fundus examination of first degree relatives and linkage analysis.

We report an unusual family pedigree segregating the retinoblastoma predisposition gene. Expression of the phenotype in different individuals in this family ranges from asymptomatic gene carriers, regressed tumours, through unifocal to bilateral multifocal lesions. Because of the unusual pattern of inheritance in this family, initial genetic counselling at a local hospital did not take into account the possibility of incomplete penetrance of the gene, and complete ophthalmological examination of unaffected family members was not undertaken. We have used DNA probes from within the retinoblastoma predisposition gene for unequivocal identification of gene carriers. The subsequent demonstration of regressed tumours in founder members of the family confirmed the diagnosis of a dominantly inherited disease. The circumstances of the management of this family emphasises the need for specialist ophthalmic examination of first degree relatives and detailed genetic analysis of all such families with DNA probes.

Application of intragenic DNA probes in prenatal screening for retinoblastoma gene carriers in the United Kingdom.

Restriction fragment length polymorphisms (RFLPs) in 55 families affected by retinoblastoma have been studied using recombinant DNA probes derived from within the retinoblastoma predisposition gene. Only six families were uninformative for any of the DNA polymorphisms. The remaining 49 families can be offered prenatal screening. No obligate recombinations between any of the polymorphic loci and the retinoblastoma phenotype were observed. Four previously unknown cases of non-penetrance were identified. Prenatal testing for the inheritance of mutant alleles was performed in two cases and perinatal screening in two additional cases. One fetus inherited the normal allele from the affected parent and is therefore not at risk of retinoblastoma; the second fetus inherited the mutant allele and will require frequent screening for early detection of retinoblastoma. Both perinatal tests showed the absence of the mutant allele.