John K. Cowell

Cytogenetic Changes in Wilms' Tumors

Cytogenetic analysis of 20 Wilms tumors using short-term culture techniques were undertaken. Chromosome abnormalities were detected in all tumors. In 19 of 20 cases only minor karyotypic changes were observed within cells with near-diploid chromosome numbers; only one tumor was predominantly hyperdiploid. Rearrangements involving chromosome 1 were the most frequently observed abnormality (in 25%) and often resulted in partial or complete trisomy for the long arm. In 20% of the tumors, abnormalities involving chromosomes 11 and 16 were present. The only other chromosomes frequently involved in structural or numerical changes were #12, and #18. Two discrete tumor foci within the same kidney differed cytogenetically, suggesting an independent origin for each focus. No correlation could be made between specific chromosome abnormalities and tumor stage or histologic subtype. Although constitutional deletion of chromosome region 11p13 has frequently been reported to predispose to Wilms tumor formation, only two tumors with deletions involving this region were observed. Chromosomes from tumors treated with chemotherapy prior to surgical removal and culture yielded findings similar to those in untreated tumor cells.

Frequent constitutional C to T mutations in CGA-arginine codons in the RB1 gene produce premature stop codons in patients with bilateral (hereditary) retinoblastoma.

We have shown previously that the most common point mutation in the RBI gene in retinoblastoma tumours is a C→T transition and that the majority of these occur in CGAarg codons. As a result of this mutation, a TGAstop codon is generated directly. We have analysed the 14 CGAarg codons in the RB1 gene for mutations in 113 patients with bilateral retinoblastoma. At 6 of these sites, C→T mutations in CGA codons alter a restriction enzyme site which makes their identification relatively straightforward. It was necessary, however, to analyse the other 8 CGA codons using single-strand conformation polymorphism (SSCP) analysis. A total of 18 C→T mutations were found, which represents 16% of all patients. Of these 13 (73%) were at two particular CGA codons in exon 8 (codon 251) and exon 17 (codon 552). During the course of the SSCP analysis, mutations were identified in 7 other individuals. Thus, 20–25% of all mutations can be identified by a relatively quick survey of the CGA codons in the RB1 gene, which has important implications for genetic screening programmes. All of the mutations in the RB1 gene in these bilaterally affected patients would be predicted to result in the absence of a functional protein.

Mapping genetic markers on human chromosome 19 using subchromosomal fragments in somatic cell hybrids

A series of mouse-human somatic cell hybrid lines (WILF) were derived from a hybrid that was originally thought to have chromosome 19 as its only human chromosome. In situ hybridization has been used to assess the amount of human material present in the different lines. All appear to contain different numbers of human chromosome fragments. A series of X-chromosome-specific DNA sequences hybridized against DNA from the lines revealed that material from the X long arm is present in several cases. Chromosome 19-specific DNA sequences used in a similar way show that fragmentation of this this chromosome has occurred with subsequent segregation of the fragments in different lines. The localization of these markers to various regions of chromosome 19, and their relation to the fragments observed in the WILF lines, is discussed.

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.

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.

Chromosome analysis of human neuroblastoma cell line TR14 showing double minutes and an aberration involving chromosome 1

The chromosomal analysis of a human neuroblastoma cell line derived from a pediatric patient is presented. The cell line has a modal chromosome number of 64 and contains large numbers of double minutes (DMs). The DMs are stable over many passages in vitro. A consistent feature of the karyotype is an abnormality involving the short arm of chromosome 1, which is consistent with previous reports from human neuroblastomas. The region distal to band 1p31 has homogeneously staining characteristics and as such this cell line represents a rare example where DMs and HSRs are constitutive features of the same cells. The possibility that such regions could arise from chromosome rearrangements is discussed.

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.

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.

An assessment of the usefulness of electrophoretic variants of esterase-D in the antenatal diagnosis of retinoblastoma in the United Kingdom

Fifty retinoblastoma families have been studied. In 41 it has been possible to determine the esterase-D phenotypes in all family members. Seven families were informative for the enzyme polymorphism and in all cases cosegregation of the retinoblastoma gene and esterase-D alleles was demonstrated, giving a lod score of 2.61. When combined with other published reports the cumulative lod score is 13.69 with no recombination in 45 meioses. In 10-15% of retinoblastoma families therefore, it is possible to offer prenatal diagnosis using the ESD protein polymorphism. The application of this test to the retinoblastoma population in the UK is limited by the low frequency of the rarer allele (0.116) and, as a result of genetic counseling, the smaller families generally associated with retinoblastoma.

The genetics of retinoblastoma.

Retinoblastoma (Rb) is a childrens eye cancer affecting 1:20,000 young children. Approximately 40% of cases have a genetic basis and 25-30% have a positive family history with the tumour phenotype segregating as an autosomal dominant mutation based on a single autosomal locus. Of the 70% apparently sporadic cases a proportion (20-30%) represent new germinal mutations. Although the Rb gene mutation shows high penetrance approximately 10% of gene carriers do not develop tumours (incomplete penetrance) so clearly only a predisposition to cancer is inherited. In a mathematical treatise of Rb Knudson (1971) demonstrated that, in hereditary cases, a single additional event was sufficient for tumorigenesis (the two-hit hypothesis), a theory which accounts for the fact that tumours in these patients are usually bilateral, multifocal and have an earlier age of onset compared with sporadic cases which are most often unilateral and unifocal. Mutations are defined as any disturbance of the gene which results in loss of function and includes deletions, translocations and point mutations. Early detection of tumours means that survival is almost guaranteed, but once the tumour has escaped the confines of the eye it is usually lethal. Identification of gene carriers, therefore, is essential for improved clinical management of the disease. The recent molecular cloning of the Rb gene has proved invaluable in establishing gene carrier status and also provides the opportunity to study early events in tumorigenesis.