RB1 and CDKN2A functional defects resulting in retinoblastoma

Multiplex methylation-sensitive PCR was employed in studying the methylation of the RB1 and CDKN2A/p16 promoter regions in 52 retinoblastomas. Aberrant methylation inactivating RB1 was detected in 14 (27%) tumors. Methylation of p16 was for the first time observed in retinoblastoma (9 tumors, 17%). Both promoters proved to be methylated in two tumors. In four tumors, aberrant methylation was combined with structural defects of both RB1 alleles. Aberrant methylation of the p16 promoter was the second mutation event in two tumors and was not accompanied by RB1 defects in one tumor. Complex testing for RB1 mutations, loss of heterozygosity, and functional inactivation of the two genes revealed a molecular defect in at least one allele in 51 (98%) tumors.     

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.     

RB1 and CDKN2A Functional Defects Resulting in Retinoblastoma

Multiplex methylation-sensitive PCR was employed in studying the methylation of the RB1 and CDKN2A/p16 promoter regions in 52 retinoblastomas. Aberrant methylation inactivating RB1 was detected in 14 (27%) tumors. Methylation of p16 was for the first time observed in retinoblastoma (9 tumors, 17%). Both promoters proved to be methylated in two tumors. In four tumors, aberrant methylation was combined with structural defects of both RB1 alleles. Aberrant methylation of the p16 promoter was the second mutation event in two tumors and was not accompanied by RB1 defects in one tumor. Complex testing for RB1 mutations, loss of heterozygosity, and functional inactivation of the two genes revealed molecular defects in at least one allele in 51 (98%) tumors.     

Mutation Spectrum of RB1 Gene in Unilateral Retinoblastoma Cases from Tunisia and Correlations with Clinical Features

Retinoblastoma, an embryonic neoplasm of retinal origin, is the most common primary intraocular malignancy in children. Somatic inactivation of both alleles of the RB1 tumor suppressor gene in a retinal progenitor cell through diverse mechanisms including genetic and epigenetic modifications, is the crucial event in initiation of tumorigenesis in most cases of isolated unilateral retinoblastoma. We analyzed DNA from tumor tissue and from peripheral blood to determine the RB1 mutation status and seek correlations with clinical features of 37 unrelated cases of Tunisian origin with sporadic retinoblastoma. All cases were unilateral except one who presented with bilateral disease, in whom no germline coding sequence alteration was identified. A multi-step mutation scanning protocol identified bi-allelic inactivation of RB1 gene in 30 (81%) of the samples tested. A total of 7 novel mutations were identified. There were three tumors without any detectable mutation while a subset contained multiple mutations in RB1 gene. The latter group included tumors collected after treatment with chemotherapy. There were seven individuals with germline mutations and all presented with advanced stage of tumor. There was no difference in age of onset of RB based on the germline mutation status. Thus 20% of the individuals with sporadic unilateral RB in this series carried germline mutations and indicate the importance of genetic testing all children with sporadic retinoblastoma. These findings help to characterize the spectrum of mutations present in the Tunisian population and can improve genetic diagnosis of retinoblastoma.     

Marked differences in unilateral isolated retinoblastomas from young and older children studied by comparative genomic hybridization

Although it is established that the loss of function of both alleles of the RB1 gene is a prerequisite for the development of retinoblastoma, little is known about the genetic events that are required for tumor progression. We used comparative genomic hybridization (CGH) to search for DNA copy number changes in isolated unilateral retinoblastomas. From a series of 66 patients with retinoblastomas with somatic mutations in both RB1 alleles, tumor samples from 13 children with the youngest (2.0-9.8 months) and 13 with the oldest (36.2-84.1 months) age at operation were studied. Loss at 13q14, the location of RB1, was demonstrated in two tumors only. Recurring chromosome imbalances included gains at 6p (11/26), 1q (10/26), 2p (4/26), and 17q (4/26), gains of the entire chromosome 19 (3/26), and losses at 16q (9/26). A commonly gained region at 1q32 was identified. Increased dosage of GAC1, a candidate oncogene located in 1q32, was found in two of four tumors by Southern blot analysis. Comparison of the CGH findings revealed that retinoblastomas from children with an older age at operation showed significantly more frequent (13/13 cases vs 4/13 cases; P = 0.0005) and more complex genetic abnormalities (median, 5 changes/abnormal tumor vs median, 1.5 changes/abnormal tumor; P = 0.003) than retinoblastomas from children with a young age at operation. Gains at 1q, 2p, 17q, of the entire chromosome 19 and losses of 16q were restricted to the older age group. Our results suggest that the progression of retinoblastomas from older patients follows mutational pathways different from those of younger patients.     

The association between maternal nutrient intake during pregnancy and the risk of sporadic unilateral retinoblastoma among offspring

Previous studies have associated maternal diet during pregnancy with the development of sporadic unilateral retinoblastoma (RB), but few studies have focused on the role of individual nutrients. The aim of this study is to investigate the association between maternal nutrient intake during pregnancy and the development of sporadic unilateral RB in the offspring.A modified food frequency questionnaire, with additional questions on supplement use, was completed via a phone interview. Cases were recruited from hospitals and controls were comprised of friends and relatives of the patient without a history of cancer. Overall, 168 sporadic unilateral RB cases and 145 controls were included in case-control study. We performed logistic regression to estimate odds ratios (ORs) and 95 % confidence intervals (CI), adjusting for child’s age, child’s sex, parental race/ethnicity, maternal education, total calorie intake during pregnancy, maternal age at birth, maternal smoking during pregnancy, pre-pregnancy body mass index, maternal weight gain during pregnancy, paternal age at birth, and maternal multivitamin use in the year before pregnancy.In the adjusted model, the interquartile (IQR) increase in vitamin A intake, which was measured in retinol activity equivalent (RAE; OR: 0.64, 95 % CI: 0.46–0.90), and vitamin D intake (OR: 0.62, 95 % CI: 0.42–0.91) significantly reduced the risk of sporadic unilateral RB.These findings suggest that a higher intake of vitamins A and D can be a protective factor for sporadic unilateral RB. Further analyses in consideration of multi-exposures such as parental occupational exposures are warranted to discover the complex etiology of sporadic unilateral RB. In addition, the role of nutritional epigenetics for how maternal nutrient intake influences the risk of sporadic unilateral RB in the offspring still needs to be explored.     

Murine bilateral retinoblastoma exhibiting rapid-onset, metastatic progression and N-myc gene amplification

Human retinoblastoma is a pediatric cancer initiated by RB gene mutations in the developing retina. We have examined the origins and progression of retinoblastoma in mouse models of the disease. Retina-specific inactivation of Rb on a p130-/- genetic background led to bilateral retinoblastoma with rapid kinetics, whereas on a p107-/- background Rb mutation caused predominantly unilateral tumors that arose with delayed kinetics and incomplete penetrance. In both models, retinoblastomas arose from cells at the extreme periphery of the murine retina. Furthermore, late retinoblastomas progressed to invade the brain and metastasized to the cervical lymph nodes. Metastatic tumors lacking Rb and p130 exhibited chromosomal changes revealed by representational oligonucleotide microarray analysis including high-level amplification of the N-myc oncogene. N-myc was found amplified in three of 16 metastatic retinoblastomas lacking Rb and p130 as well as in retinoblastomas lacking Rb and p107. N-myc amplification ranged from 6- to 400-fold and correlated with high N-myc-expression levels. These murine models closely resemble human retinoblastoma in their progression and secondary genetic changes, making them ideal tools for further dissection of steps to tumorigenesis and for testing novel therapies.     

Parental origin of germ-line and somatic mutations in the retinoblastoma gene

Segregation analysis of polymorphic sites within the retinoblastoma (RB) gene and on chromosome 13, as well as the parental origin of the lost allele in the tumor, were analyzed in 24 families with RB patients. Four mutant alleles transmitted through the germ-line and seven de novo germ-line mutant alleles were identified in 11 patients with hereditary RB. Segregation analysis within the RB gene and on chromosome 13 was useful for DNA diagnosis of susceptibility to RB in relatives of hereditary patients, even if mutations were not identified. All seven de novo germ-line mutant alleles were paternally derived. The bias toward the paternal allele for de novo germ-line mutations of the RB gene was statistically significant. Seven paternal alleles and six maternal alleles were lost in 13 non-hereditary RB tumors with no bias in the parental origin of the somatic allele loss. These results suggest that the physical environment or a deficiency in DNA repair during spermatogenesis may be associated with significant risk factors for de novo germ-line mutations.     

Genomic imprinting of the human serotonin-receptor (HTR2) gene involved in development of retinoblastoma.

Epidemiological and genetic studies of retinoblastoma (RB) suggested that imprinted genes might be genetically linked to the RB gene. In this study, we found that the human serotonin-receptor, HTR2, gene, which had been mapped nearby the RB gene on chromosome 13, was expressed only in human fibroblasts with a maternal allele and not in cells without a maternal allele. The 5' genomic region of the human HTR2 gene was cloned by PCR-mediated method. Only the 5' region of the gene was methylated in cells with the maternal gene, and it was not methylated in cells without the maternal gene. A polymorphism of PvuII site of the gene was also found and useful for the segregation analysis in a family of a RB patient and for analysis of loss of heterozygosity on chromosome 13 in tumor and its parental origin. These results suggest that the human HTR2 gene might be affected by genomic imprinting and that exclusive expression of the maternal HTR2 gene may be associated with the delayed occurrence of RB, which had lost the maternal chromosome 13.     

Mutation risk associated with paternal and maternal age in a cohort of retinoblastoma survivors

Autosomal dominant conditions are known to be associated with advanced paternal age, and it has been suggested that retinoblastoma (Rb) also exhibits a paternal age effect due to the paternal origin of most new germline RB1 mutations. To further our understanding of the association of parental age and risk of de novo germline RB1 mutations, we evaluated the effect of parental age in a cohort of Rb survivors in the United States. A cohort of 262 Rb patients was retrospectively identified at one institution, and telephone interviews were conducted with parents of 160 survivors (65.3%). We classified Rb survivors into three groups: those with unilateral Rb were classified as sporadic if they had no or unknown family history of Rb, those with bilateral Rb were classified as having a de novo germline mutation if they had no or unknown family history of Rb, and those with unilateral or bilateral Rb, who had a family history of Rb, were classified as familial. We built two sets of nested logistic regression models to detect an increased odds of the de novo germline mutation classification related to older parental age compared to sporadic and familial Rb classifications. The modeling strategy evaluated effects of continuous increasing maternal and paternal age and 5-year age increases adjusted for the age of the other parent. Mean maternal ages for survivors classified as having de novo germline mutations and sporadic Rb were similar (28.3 and 28.5, respectively) as were mean paternal ages (31.9 and 31.2, respectively), and all were significantly higher than the weighted general US population means. In contrast, maternal and paternal ages for familial Rb did not differ significantly from the weighted US general population means. Although we noted no significant differences between mean maternal and paternal ages between each of the three Rb classification groups, we found increased odds of a survivor being in the de novo germline mutation group for each 5-year increase in paternal age, but these findings were not statistically significant (de novo vs. sporadic ORs 30-34 = 1.7 [0.7-4], ≥ 35 = 1.3 [0.5-3.3]; de novo vs. familial ORs 30-34 = 2.8 [1.0-8.4], ≥ 35 = 1.6 [0.6-4.6]). Our study suggests a weak paternal age effect for Rb resulting from de novo germline mutations consistent with the paternal origin of most of these mutations.     

Detection of mutations of the RB1 gene in retinoblastoma patients by using exon-by-exon PCR-SSCP analysis.

Most sporadic cases of retinoblastoma, malignant eye tumor of children, may require the identification of a mutation of the retinoblastoma gene (RB1 gene) for precise genetic counseling. We established a mutation detection system of and screened for the RB1 gene mutation in 24 patients with retinoblastoma--12 bilateral patients and 12 unilateral patients. Mutation analysis was performed by PCR-mediated SSCP analysis in the entire coding region and promoter region, as an initial screening method, followed by direct genomic sequencing. Possible oncogenic mutations were identified in 14 (58%) of 24 tumors, of which 6 were single base substitutions, 4 were small deletions, 3 were small insertions, and 1 was a complex alteration due to deletion-insertion. A constitutional somatic mosaicism was suggested in one bilateral patient. A majority (57%) of mutations were found in E1A binding domains, and all were presumed to truncate the normal gene products. The mutation analysis presented here may provide a basis for the screening system of RB1 gene mutations in retinoblastoma patients.     

Similar chromosomal abnormalities in several retinoblastomas

Summary The study of banded chromosomes of nine sporadic unilateral retinoblastomas revealed near diploid karyotypes with multiple numerical and (or) structural abnormalities in all tumors. An identical marker i(6p) was noted in cells of the modal class of six retinoblastomas. Extra copies of the short arm of chromosome 6 were observed in seven tumors: +i(6p) in 6 and +6q- in one. Less regular but repeated findings were a loss of one sex chromosome, and markers 1p+ and 17q+. The structure of these markers was not identical in different tumors. Abnormalities of chromosome 13 were not observed in tumor cells, nor in blood lymphocytes stimulated by PHA.