Constitutional and somatic RB1 mutation spectrum in nonfamilial unilateral and bilateral retinoblastoma in India

An epidemiologic survey has indicated a comparatively high prevalence of retinoblastoma (Rb) in Asian countries. Recently, the development of preventive strategies in nonfamilial Rb has become a major goal. The present studies were designed for identification and characterization of constitutional and somatic RB1 gene mutations by conventional cytogenetics, fluorescent in situ hybridization (FISH) and polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP)-DNA sequencing. Of 34 patients 32 were nonfamilial and 2 were familial Rb. Maternal inheritance of del (13q14) was common. FISH was sensitive in detecting monoallelic RB1 deletion/deletion mosaicism as a first genetic hit in 20% of cases. Somatic and germline RB1 point mutations affected exons 3, 17, 20, and 21 and these were identified as novel mutations. Involvement of exon 20 as a predisposing mutation in sporadic unilateral retinoblastoma (URB) probably suggests the susceptibility of exon 20 to unknown etiologic factors in our population. A de novo RB1 deletion along with transmitted RB1 point mutation from an asymptomatic parent was identified as a unique predisposing RB1 mutation chimerism in a URB case that later evolved to bilateral retinoblastoma (BRB). The predisposing mutations such as del (13q), RB1 mono-allelic deletion and RB1 point mutation in sporadic Rb were de novo as well as transmitted mutations from asymptomatic/symptomatic parents. The RB1 mutation incidence was comparatively higher (25%) in nonfamilial Rb with emphasis on high prevalence in sporadic URB (18% versus 0%-9% in the literature series). The present studies demonstrated the efficacy of a multitechnique approach to detect various types of constitutional RB1 mutations such as RB1 deletion, deletion mosaicism, point mutation, mutation chimerism in patients of symptomatic/asymptomatic parents.     

Molecular-genetic analysis of two cases with retinoblastoma: Benefits for disease management

Effective counselling and management of retinoblastoma families using genetic information is presently practised in many parts of the world. We studied histopathological, chromosomal and molecular-genetic data of two retinoblastoma patients from India. The two patients, one with bilateral and the other with unilateral retinoblastoma, underwent complete ophthalmic examination, cytogenetic study, retinoblastoma gene (RB1) mutational analysis andRB1 promoter region methylation screening. In the bilateral retinoblastoma patient deletion of chromosome region 13q14 in peripheral blood lymphocytes and a hemizygous novel 8-bp deletion in exon 4 ofRB1 in tumour sample were observed. In the unilaterally affected patient CGA to TGA transition protein truncation mutations were observed in exons 8 and 14 ofRB1.     

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.     

Constitutional RB1-gene mutations in patients with isolated unilateral retinoblastoma.

In most patients with isolated unilateral retinoblastoma, tumor development is initiated by somatic inactivation of both alleles of the RB1 gene. However, some of these patients can transmit retinoblastoma predisposition to their offspring. To determine the frequency and nature of constitutional RB1-gene mutations in patients with isolated unilateral retinoblastoma, we analyzed DNA from peripheral blood and from tumor tissue. The analysis of tumors from 54 (71%) of 76 informative patients showed loss of constitutional heterozygosity (LOH) at intragenic loci. Three of 13 uninformative patients had constitutional deletions. For 39 randomly selected tumors, SSCP, hetero-duplex analysis, sequencing, and Southern blot analysis were used to identify mutations. Mutations were detected in 21 (91%) of 23 tumors with LOH. In 6 (38%) of 16 tumors without LOH, one mutation was detected, and in 9 (56%) of the tumors without LOH, both mutations were found. Thus, a total of 45 mutations were identified in tumors of 36 patients. Thirty-nine of the mutations-including 34 small mutations, 2 large structural alterations, and hypermethylation in 3 tumors-were not detected in the corresponding peripheral blood DNA. In 6 (17%) of the 36 patients, a mutation was detected in constitutional DNA, and 1 of these mutations is known to be associated with reduced expressivity. The presence of a constitutional mutation was not associated with an early age at treatment. In 1 patient, somatic mosaicism was demonstrated by molecular analysis of DNA and RNA from peripheral blood. In 2 patients without a detectable mutation in peripheral blood, mosaicism was suggested because 1 of the patients showed multifocal tumors and the other later developed bilateral retinoblastoma. In conclusion, our results emphasize that the manifestation and transmissibility of retinoblastoma depend on the nature of the first mutation, its time in development, and the number and types of cells that are affected.     

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.     

A comprehensive,sensitive and economical approach for the detection of mutations in the <Emphasis Type="Italic">RB1</Emphasis> gene in retinoblastoma

Retinoblastoma (Rb) is the most common primary intraocular malignancy in children. It is brought about by the mutational inactivation of both alleles of RB1 gene in the developing retina. To identify the RB1 mutations, we analysed 74 retinoblastoma patients by screening the exons and the promoter region of RB1. The strategy used was to detect large deletions/duplications by fluorescent quantitative multiplex PCR; small deletions/insertions by fluorescent genotyping of RB1 alleles, and point mutations by PCR-RFLP and sequencing. Genomic DNA from the peripheral blood leucocytes of 74 Rb patients (53 with bilateral Rb, 21 with unilateral Rb; 4 familial cases) was screened for mutations. Recurrent mutations were identified in five patients with bilateral Rb, large deletions in 11 patients (nine with bilateral Rb and two with unilateral Rb), small deletions/insertions were found in 12 patients all with bilateral Rb, and point mutations in 26 patients (14 nonsense, six splice site, five substitution and one silent change). Three mutations were associated with variable expressivity of the disease in different family members. Using this method, the detection rates achieved in patients with bilateral Rb were 44/53 (83%) and with unilateral Rb, 5/21 (23.8%). This approach may be feasible for clinical genetic testing and counselling of patients.     

A splicing mutation in RB1 in low penetrance retinoblastoma

The pediatric eye-tumor retinoblastoma is widely held as a paradigm of human cancer genetics and has been a model system for both the two-hit hypothesis of dominantly inherited cancer as well as for the concept of tumor-specific loss of constitutional heterozygosity to achieve expression of the tumorigenic phenotype. Familial retinoblastoma is usually inherited as an autosomal dominant disease with high penetrance and expressivity. In a small but significant number of families, however, retinoblastoma is inherited with greatly reduced penetrance and expressivity. In these families, retinoblastoma tumors occur relatively late, are often unilateral, and unaffected carriers may exist. We have identified a mutation in such a family that exhibited extremely low penetrance and expressivity. This mutation appeared to affect splicing of the mutant allele such that both a normal length RB1 mRNA and a truncated RB1 mRNA were expressed from the same allele. Received: 7 March 1997 / Accepted: 29 April 1997     

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.     

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.     

The spectrum of RB1 germ-line mutations in hereditary retinoblastoma.

We have searched for germ-line RB1 mutations in 119 patients with hereditary retinoblastoma. Previous investigations by Southern blot hybridization and PCR fragment-length analysis had revealed mutations in 48 patients. Here we report on the analysis of the remaining 71 patients. By applying heteroduplex analysis, nonisotopic SSCP, and direct sequencing, we detected germ-line mutations resulting in premature termination codons or disruption of splice signals in 51 (72%) of the 71 patients. Four patients also showed rare sequence variants. No region of the RB1 gene was preferentially involved in single base substitutions. Recurrent transitions were observed at most of the 14 codons within the RB1. No mutation was observed in exons 25-27, although this region contains two CGA codons. This suggests that mutations within the 3'-terminal region of the RB1 gene may not be oncogenic. When these data were combined with the results of our previous investigations, mutations were identified in a total of 99 (83%) of 119 patients. The spectrum comprises 15% large deletions, 26% small length alterations, and 42 % base substitutions. No correlation between the location of frameshift or nonsense mutations and phenotypic features, including age at diagnosis, the number of tumor foci, and manifestation of nonocular tumors was observed.     

Familial,EsD-linked,retinoblastoma with reduced penetrance and variable expressivity

We report an example of four generation familial retinoblastoma in which there are three distinct categories of RB gene expression: frank retinoblastoma, unilateral or bilateral; retinoma; and no visible evidence of retinal pathology other than normal degeneration with age. Two large sibships derived from matings informative for RB and EsD provide strong confirmatory evidence for tight linkage between these loci (P = 0.0002), and thus assignment of RB to chromosome 13q14. There is a striking difference (P less than 2%) in RB penetrance between the two principal generations, which suggests that an additional epistatic, host-resistance gene may also be segregating within the family.     

Radiation sensitivity of fibroblasts of bilateral retinoblastoma patients as determined by micronucleus induction in vitro

The radiation sensitivity of fibroblasts isolated from bilateral retinoblastoma (RB) patients was investigated using an in vitro micronucleus assay. Bilateral RB is an autosomal dominant disease associated with a single locus, RB-1; therefore, all cells in an affected individual carry the germ line mutation. The ability to identify gene carriers made it possible to study the effect of the RB-1 mutation in the heterozygous state on the sensitivity of the cells to chromosome breakage by gamma-rays. The micronucleus assay was chosen for this study since it is a quick and easy measure of chromosomal aberrations. The fibroblasts from bilateral RB patients did not differ systematically from the normal fibroblasts in either the spontaneous or the induced rates of micronucleus production. Thus, bilateral RB fibroblasts are not more sensitive to the clastogenic effects of gamma-radiation than the controls.     

Detection of small RB1 gene deletions in retinoblastoma by multiplex PCR and high-resolution gel electrophoresis

Summary Loss of function of both copies of the RB1 gene is a causal event in the development of retinoblastoma. The predisposition to this tumor can be inherited as an autosomal dominant trait. Direct detection of the genetic defect is important for presymptomatic DNA diagnosis and genetic counseling in families with hereditary retinoblastoma. We have used multiplex polymerase chain reaction and high-resolution polyacrylamide gel electrophoresis to detect RB1 gene deletions as small as one base pair. By using three independent sets of amplification reactions, which cover 26% of the RB1 gene coding region, we identified RB1 gene deletions in the DNA of peripheral blood cells in 3 out of 24 (12.5%) unrelated patients with hereditary retinoblastoma. In one case, formalin-fixed paraffin-embedded tumor material was also used to detect the mutation. Sequencing of the mutated alleles revealed deletions of 1, 3 and 10 base pairs. Each deleted region was flanked by direct repeats.     

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.     

X-radiation-induced chromosome breakage in retinoblastoma lymphocytes

We have examined the spontaneous and X-radiation-induced chromosomal damage in normal humans and in patients with retinoblastoma using the BudR-Giemsa technique in lymphocytes cultured for 48 h. 9 sporadic unilateral non-hereditary cases, 11 hereditary cases (8 bilateral sporadic and 3 unilateral hereditary cases) and 20 healthy individuals were studied simultaneously. No difference in the spontaneous frequency of chromatid and chromosome aberrations was observed between patients and controls. After treatment with 150 rad the frequency of chromosome exchange aberrations was higher in unilateral hereditary cases than the controls (42.0% +/- 5.3 and 22.3% +/- 2.6 respectively; p = 0.05). In bilateral sporadic retinoblastoma 2 different groups were observed. A hypersensitive group showed a significant increment in radiation-induced chromosomal exchange aberrations over the control group (46.2% +/- 5.4 and 24.2% +/- 2.1 respectively; p = 0.01). The other group had a chromosomal exchange frequency similar to normal individuals (26.5% +/- 2.0 and 24.2% +/- 0.4 respectively; p = 0.10). Sporadic unilateral non-hereditary retinoblastoma had an exchange chromosomal aberration frequency similar to control individuals (26.1% +/- 2.8 and 24.6% +/- 2.7 respectively; p greater than 0.10). These results suggest that: There is no relationship between spontaneous chromosome fragility and retinoblastoma. Sporadic unilateral non-hereditary retinoblastoma has normal chromosome sensitivity to X-irradiation. Some hereditary cases of retinoblastoma are sensitive to X-rays while others behave like normals. A mutation or a submicroscopic deletion at a DNA repair locus which is independent of the retinoblastoma gene may cause this radiosensitivity.