Low frequency of oncogenic mutations in the core promoter region of the RB1 gene.
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DNA Mutational Analysis↗
Biomedical subjects
J M Rapaport
Publications and source records attributed to J M Rapaport.
Hypermethylation in the retinoblastoma gene is associated with unilateral, sporadic retinoblastoma.
We previously reported 9 unilateral, sporadic retinoblastomas with hypermethylation in the 5' region of the RB gene, and we found that CpG methylation in the RB promoter inhibits the binding of the retinoblastoma binding factor 1 (RBF-1) and the activating transcription factor (ATF)-like factors, thereby resulting in a considerable reduction in RB promoter activity. In this study, we screened for hypermethylation in 121 additional cases of retinoblastoma, and found 5 tumors with hypermethylation, including 4 unilateral, sporadic tumors, and one hereditary tumor. The hereditary tumor had a germline deletion of one allele, and the hypermethylation was an acquired, epigenetic change in the other allele. Another tumor had hypermethylation restricted to approximately 800 base pairs in the RB promoter region including the essential RBF-1 and ATF sites. The frequency of hypermethylation in unilateral, sporadic tumors was 9.3% combining our previous and present examinations (13 among 140), whereas the frequency was 1.0% in bilateral hereditary tumors (one among 101). The statistical analyses using the chi-square test indicated significant correlation between hypermethylation and unilateral, sporadic tumors (p < 0.05). These results suggest that hypermethylation in the RB gene is always an acquired, epigenetic change and causes about 9% of unilateral, sporadic tumors.
Blotting, Southern↗
Quantification of the paternal allele bias for new germline mutations in the retinoblastoma gene.
New germline mutations in the human retinoblastoma gene are known to arise preferentially on paternally derived chromosomes, but the magnitude of that bias has not been measured. We evaluated 49 cases with a new germline mutation and found that in 40 cases (82%) the mutation arose on the paternally derived allele. We also evaluated 48 cases likely to have a somatic initial mutation; in this group the initial mutation arose on paternal or maternal chromosomes with approximately equal frequency. There was no statistically significant difference in the average age of fathers of children with new paternal germline mutations from the average age of fathers of children with new maternal germline mutations or somatic initial mutations. Combining the data with that from previous reports from other groups, the proportion of new germline mutations arising on a paternally derived allele is 85% (based on 72 cases; 95% confidence interval = 76-93%). This number can be useful in the genetic counseling of some families with retinoblastoma.
Adult↗
Allele-specific hypermethylation of the retinoblastoma tumor-suppressor gene.
Inactivation of the retinoblastoma gene appears to have a fundamental role in the genesis of retinoblastoma, osteosarcoma, and other malignant tumors. The gene is generally inactivated because of loss-of-function mutations, although epigenetic phenomena, such as hypermethylation of the promoter region, could possibly have the same effect. We investigated the methylation pattern at the 5' end of the retinoblastoma gene, including its promoter region and exon 1, in DNA purified from 56 primary retinoblastomas. We found five tumors with evidence for hypermethylation, all from unilateral, simplex patients. No methylation abnormalities were detected in DNA purified from the leukocytes from these patients. It is interesting that in one of these tumors the hypermethylation was confined to one allele. There were no mutations in a 1,306-bp sequence including the hypermethylated region that might account for the allele-specific hypermethylation. We believe that the hypermethylation of the retinoblastoma gene that we found in these tumors corresponds to the allelic inactivation of the gene, and we speculate that erroneous hypermethylation without alteration of nucleotide sequence occasionally plays a role in the genesis of this cancer. If this is true, then retinoblastomas with hypermethylation might be treatable with chemotherapeutic agents that interfere with methylation of DNA.
Alleles↗
Parental origin of mutations of the retinoblastoma gene.
Retinoblastoma and osteosarcoma arise from cells that have lost both functional copies of the retinoblastoma gene. Using the cloned retinoblastoma gene and other linked polymorphic loci, it is possible to reconstruct the sequential loss of the two homologous gene copies that precedes the development of these tumours. In non-hereditary tumours, the loss of each of the two homologues occurs somatically; in hereditary cases, the initial mutation is in the germline. Recently, Toguchida et al. reported that the paternally derived copy is preferentially the first one to become mutant during the genesis of non-hereditary osteosarcomas. We report here a similar analysis of patients with retinoblastoma in which we find no such predilection for initial somatic mutations. In contrast, when an initial mutation was a new germline mutation, it was derived from the father, a result which is consistent with new germline mutations arising primarily during spermatogenesis.
Adult↗
Structure and expression of the murine retinoblastoma gene and characterization of its encoded protein.
We have isolated a cDNA clone of the murine homologue of the human retinoblastoma (Rb) susceptibility gene. DNA sequence analysis reveals a high degree of conservation with the human Rb sequence, both in the coding and in the noncoding regions. The predicted amino acid sequence of the mouse Rb protein shows 91% identity to that of the human protein. Both proteins were found to contain a peptide sequence reminiscent of a leucine-repeat motif ("leucine-zipper") that is also found in the myc, fos, and jun oncogenes. Synthetic peptide antiserum directed against a portion of the mouse Rb protein detects three proteins of 104-110 kDa in cells that were transiently transfected with a mouse Rb gene expression construct. In the mouse embryo the expression of Rb mRNA was ubiquitous, with maximal expression being observed around 13 days of gestation. In the embryo, the highest level of expression was observed in liver and brain. In contrast, the Rb gene was found to be expressed at a very low level in adult mouse liver with high levels being found in lung, thymus, and spleen. A shorter Rb transcript was detected in mouse testes.
Amino Acid Sequence↗
Molecular detection of deletions involving band q14 of chromosome 13 in retinoblastomas.
DNA fragments from a locus spanning 29 kilobases within chromosome band 13q14 detected deletions in 3 retinoblastomas out of 37 such tumors examined. Somatically occurring, homozygous deletions spanning at least 25 kilobases were detected in retinoblastomas from two unrelated patients. These deletions are bounded by the esterase D locus proximally. In a third patient, both tumor cells and leukocytes have a deletion of one chromosome 13 homolog, with one end of the deletion localized to a 1.55-kilobase fragment within the cloned region. It is likely that the cloned locus is within a few hundred kilobases of the retinoblastoma gene (i.e., the locus governing predisposition to such tumors) and that the deletions detected also involve the retinoblastoma gene. Further, it may be possible to base a successful approach to the isolation of the retinoblastoma gene on this assumed physical proximity of the two loci.
Chromosome Deletion↗
Chromosome 13 homozygosity in osteosarcoma without retinoblastoma.
We provide evidence that some human osteosarcomas arise subsequent to the development of homozygosity at loci on the long arm of chromosome 13. The resulting chromosome 13q homozygosity allows the phenotypic expression of any recessive allele on that chromosome. Clinical evidence suggests that it is the retinoblastoma locus within 13q14 that is involved in the formation of these bone tumors.
Alleles↗
Homozygosity of chromosome 13 in retinoblastoma.
We studied the frequency of chromosome 13 homozygosity in tumor tissue obtained directly from eyes harboring retinoblastomas. The data indicate that approximately half of all retinoblastomas are homozygous for large portions of 13q, that the homozygosity occurs in vivo and not as an event secondary to culture of the tumor cells, that chromosome 13 homozygosity is not correlated with the degree of histopathologic differentiation of the tumor, and that the homozygosity occurs in both sporadic and hereditary retinoblastomas. The development of chromosome 13 homozygosity may represent a fundamental event in the oncogenesis of a considerable number of retinoblastomas. This finding may have implications for the genetic counseling of patients with hereditary retinoblastoma. It may also be important in understanding the mechanism of oncogenesis of other tumors, especially hereditary tumors.
Alleles↗
Chromosome 13 restriction fragment length polymorphisms.
The gene locus for hereditary retinoblastoma is on human chromosome 13, band q14. With this gene localization in mind, we cloned DNA fragments from this chromosome. Three of the fragments identify restriction fragment length polymorphisms. These three fragments are from the region 13q12-13q22, the chromosome region which contains the retinoblastoma locus. We expect that these restriction fragment length polymorphisms will be linked to the retinoblastoma locus, and that they will serve in certain retinoblastoma families as predictors of retinoblastoma gene carriers. They will also be useful in studies of other gene loci thought to be on chromosome 13.
Alleles↗
Linkage of genes for human esterase D and hereditary retinoblastoma.
Determinations of esterase D isoenzymes in the members of a family with hereditary retinoblastoma gave results consistent with linkage between the loci for esterase D and retinoblastoma. This gene linkage allows the detection of gene carrier states in certain kindreds. We used this technique to predict that a 6-month-old boy carries the tumor-predisposing retinoblastoma gene.
Adult↗
Low incidence of deletion of the esterase D locus in retinoblastoma patients.
Esterase D was quantitatively measured in the red blood cells from three patients from three separate kindreds who had abnormalities of chromosome 13. The esterase D activity was proportional to the number of copies of the q14 region of chromosome 13 present. These findings confirm published data localizing the esterase D gene to chromosome band 13q14, a region which is important in the etiology of retinoblastoma. Fifty-one additional retinoblastoma patients not known to have any chromosomal defect also underwent esterase D determination. In none of these patients did the esterase D measurement detect a 13q14 deletion. The normal esterase D levels in this series of 51 retinoblastoma patients suggest that deletion of an esterase D locus is infrequent in retinoblastoma patients. It must be noted that patients who are mosaics, with a 13q14 deletion in only a fraction of all somatic cells, could possibly have normal red blood cell esterase D levels. Further study is necessary to determine if esterase D determination of all retinoblastoma patients is a worthwhile clinical tool.
Adolescent↗
A human DNA segment with properties of the gene that predisposes to retinoblastoma and osteosarcoma.
The genomes of various tumour cells contain mutant oncogenes that act dominantly, in that their effects can be observed when they are introduced into non-malignant cells. There is evidence for another class of oncogenes, in which tumour-predisposing mutations are recessive to wild-type alleles. Retinoblastoma is a prototype biological model for the study of such recessive oncogenes. This malignant tumour, which arises in the eyes of children, can be explained as the result of two distinct genetic changes, each causing loss of function of one of the two homologous copies at a single genetic locus, Rb, assigned to the q14 band of human chromosome 13. Mutations affecting this locus may be inherited from a parent, may arise during gametogenesis or may occur somatically. Those who inherit a mutant allele at this locus have a high incidence of non-ocular, second tumours, almost half of which are osteosarcomas believed to be caused by the same mutation. Here we describe the isolation of a complementary DNA segment that detects a chromosomal segment having the properties of the gene at this locus. The gene is expressed in many tumour types, but no RNA transcript has been found in retinoblastomas and osteosarcomas. The cDNA fragment detects a locus spanning at least 70 kilobases (kb) in human chromosome band 13q14, all or part of which is frequently deleted in retinoblastomas and osteosarcomas.
Chromosome Deletion↗