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Mutation spectrum of RB1 gene in unilateral retinoblastoma cases from Tunisia and correlations with clinical features.

ABSTRACT: 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.

SUBMITTER: Ayari-Jeridi H

PROVIDER: S-EPMC4300092 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Mutation spectrum of RB1 gene in unilateral retinoblastoma cases from Tunisia and correlations with clinical features.

Ayari-Jeridi Hajer H Moran Kimberly K Chebbi Amel A Bouguila Hédi H Abbes Imen I Charradi Khaoula K Benammar-Elgaaïed Amel A Ganguly Arupa A

PloS one 20150120 1

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 ...[more]

PMID: 25602518

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Project description:Retinoblastoma (RB) is a rare childhood malignant disorder caused by the biallelic inactivation of RB1 gene. Early diagnosis and identification of carriers of heritable RB1 mutations can improve disease outcome and management. In this study, mutational analysis was conducted on fifty-nine matched tumor and peripheral blood samples from 18 bilateral and 41 unilateral unrelated RB cases by a combinatorial approach of Multiplex Ligation-dependent Probe Amplification (MLPA) assay, deletion screening, direct sequencing, copy number gene dosage analysis and methylation assays. Screening of both blood and tumor samples yielded a mutation detection rate of 94.9% (56/59) while only 42.4% (25/59) of mutations were detected if blood samples alone were analyzed. Biallelic mutations were observed in 43/59 (72.9%) of tumors screened. There were 3 cases (5.1%) in which no mutations could be detected and germline mutations were detected in 19.5% (8/41) of unilateral cases. A total of 61 point mutations were identified, of which 10 were novel. There was a high incidence of previously reported recurrent mutations, occurring at 38.98% (23/59) of all cases. Of interest were three cases of mosaic RB1 mutations detected in the blood from patients with unilateral retinoblastoma. Additionally, two germline mutations previously reported to be associated with low-penetrance phenotypes: missense-c.1981C>T and splice variant-c.607+1G>T, were observed in a bilateral and a unilateral proband, respectively. These findings have implications for genetic counselling and risk prediction for the affected families. This is the first published report on the spectrum of mutations in RB patients from Singapore and shows that further improved mutation screening strategies are required in order to provide a definitive molecular diagnosis for every case of RB. Our findings also underscore the importance of genetic testing in supporting individualized disease management plans for patients and asymptomatic family members carrying low-penetrance, germline mosaicism or heritable unilateral mutational phenotypes.

Project description:Retinoblastoma (Rb) is a primary intraocular malignant tumor that occurs primarily in children, and results from loss-of-function mutations in the RB transcriptional corepressor 1 (RB1) gene. Genetic testing forms the basis of genetic counseling for affected families, as well as for clinical management of this disease. The aim of this study was to identify germline RB1 mutations and correlate the identified mutations with the clinical features of Rb patients. Genomic DNA was isolated from peripheral blood of 180 unrelated Rb patients and their parents (118 unilaterally and 62 bilaterally affected probands). Mutations in the RB1 gene, including the promoter region and exons 1-27 with ﬂanking intronic sequences, were identified by Sanger sequencing. The samples with negative sequencing results were further subjected to methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) to detect gross deletions or duplications. Sixty-three distinct mutations were identified in 75 of the 180 (41.7%) probands. Of the 75 patients carrying RB1 mutations, 56 developed bilateral Rb, while 19 developed unilateral Rb. The total detection rates for bilateral and unilateral Rb were 90.3% (56/62) and 16.1% (19/118), respectively. Among the 75 patients, the spectrum of mutation types comprised 29.3% (22/75) nonsense mutations, 22.7% (17/75) splicing mutations, 17.3% (13/75) small insertions/deletions, 16.0% (12/75) large deletions/duplications, and 13.3% (10/75) missense mutations, while only 1% (1/75) of the mutations were in the promoter region of the RB1 gene. Age at diagnosis was significantly different (p < 0.01) between patients with positive and negative test results for germline RB1 mutations. A c.2359C > T mutation (p.R787X) was identified in identical twins, but one child was affected bilaterally and the other unilaterally. Of the five patients with deletion of the entire RB1 gene, the deletion of two patients was inherited from unaffected parents. In conclusion, in this study, we provide a comprehensive spectrum of RB1 germline mutations in Chinese Rb patients, and describe the correlations among RB1 mutations, age at diagnosis, and laterality; moreover, we report that the clinical features of individuals carrying an identical mutation in the RB1 gene were highly variable, indicating that the pathogenesis of Rb is more complicated than currently believed.

Project description:Purpose:Retinoblastoma (RB) is a retinal tumor that most commonly occurs in children. Approximately 40% of RB patients carry germline mutations in the RB1 gene. RB survivors with germline mutations are at increased risk of passing on the disease to future offspring and of secondary cancer in adulthood. This highlights the importance of genetic testing in disease management and counseling. This study aimed to identify germline RB1 mutations and to correlate the mutations with clinical phenotypes of RB patients. Methods:Genomic DNA was extracted from peripheral blood mononuclear cells isolated from 52 RB patients (27 unilaterally and 25 bilaterally affected probands). Mutations in the RB1 gene, including the promoter and exons 1-27 with flanking intronic sequences, were identified by direct sequencing. The samples with negative test results were subjected to multiplex ligation-dependent probe amplification (MLPA) to detect any gross mutations. A correlation of germline RB1 mutations with tumor laterality or age at diagnosis was determined for RB patients. Age at diagnosis was examined in regard to genetic test results and the presence of extraocular tumor extension. Results:Germline RB1 mutations were detected in 60% (31/52) of patients. RB1 mutations were identified in 92% (22/25) of bilateral RB patients, and a high rate of germline RB1 mutations was found in unilateral RB cases (33% or 9/27). Whole gene and exon deletions were reported in five patients. Twenty-three distinct mutations as a result of base substitutions and small deletions were identified in 26 patients; seven mutations were novel. Nonsense and splicing mutations were commonly identified in RB patients. Furthermore, a synonymous mutation was detected in a patient with familial RB; affected mutation carriers in this family exhibited differences in disease severity. The types of germline RB1 mutations were not associated with age at diagnosis or laterality. In addition, patients with positive and negative test results for germline RB1 mutations were similar in age at diagnosis. The incidence of extraocular tumors was high in patients with heritable RB (83% or 5/6), particularly in unilateral cases (33% or 3/9); the mean age at diagnosis of these patients was not different from that of patients with intraocular tumors. Conclusions:This study provides a data set of an RB1 genotypic spectrum of germline mutations and clinical phenotypes and reports the distribution of disease-associated germline mutations in Thai RB patients who attended our center. Our data and the detection methods could assist in identifying a patient with heritable RB, establishing management plans, and informing proper counseling for patients and their families.

Project description:Demographics for breast cancers vary widely among nations. The frequency of germline mutations in breast cancers, which reflects the hereditary cases, has not been investigated adequately and accurately in highly-consanguineous Pakistani population. In the present discovery case series, germ-line mutations in twenty-seven breast cancer candidate genes were investigated in eighty-four sporadic breast cancer patients along with the clinical correlations. The germ-line variants were also assessed in two healthy gender-matched controls. The clinico-pathological features were evaluated by descriptive analysis and Pearson χ2 test (with significant p-value <0.05). The most frequent parameters associated with hereditary cancer cases are age and ethnicity. Therefore, the analyses were stratified on the basis of age (≤40 years vs. >40 years) and ethnicity. The breast cancer gene panel assay was carried out by BROCA, which is a genomic capture, massively parallel next generation sequencing assay on Illumina Hiseq2000 with 100bp read lengths. Copy number variations were determined by partially-mapped read algorithm. Once the mutation was identified, it was validated by Sanger sequencing. The ethnic analysis stratified on the basis of age showed that the frequency of breast cancer at young age (≤40 years) was higher in Sindhis (n = 12/19; 64%) in contrast to patients in other ethnic groups. Majority of the patients had stage III (38.1%), grade III (50%), tumor size 2-5 cm (54.8%), and invasive ductal carcinoma (81%). Overall, the analysis revealed germ-line mutations in 11.9% of the patients, which was not significantly associated with younger age or any particular ethnicity. The mutational spectrum was restricted to three genes: BRCA1, BRCA2, and TP53. The identified mutations consist of seven novel germ-line mutations, while three mutations have been reported previously. All the mutations are predicted to result in protein truncation. No mutations were identified in the remaining twenty-four candidate breast cancer genes. The present study provides the framework for the development of hereditary-based preventive and treatment strategies against breast cancers in Pakistani population.

Project description:PurposeRetinoblastoma (RB) is a rare childhood malignant disorder caused by the biallelic inactivation of the RB1 gene. Early diagnosis and identification of carriers of heritable mutations in RB1 can improve disease outcome and management. In this study, we present the spectrum of mutations in the RB1 gene in Vietnamese patients with RB.MethodsTumor RNA from 50 probands with RB, including 12 bilateral and 38 unilateral cases, was extracted. cDNA, after reverse transcription, was sequenced to identify the RNA mutation of the RB1 gene. At the genomic DNA level, mutational analysis of all RB1 exons, exon-intron boundaries, and the promoter region was conducted using PCR and direct sequencing. Multiplex ligation-dependent probe amplification (MLPA) analysis was performed for patients for whom the first two results were negative. For patients for whom either the sequencing or MLPA results were positive for a tumor mutation, patients' and their parents' blood DNA was analyzed to determine the germline mutation.ResultsForty-one different kinds of RB1 tumor mutations were identified in 41 probands (82.0%), including 11 of 12 bilateral cases (91.7%) and 30 of 38 unilateral cases (78.9%). The majority of the detected mutations were nonsense (15 different kinds), followed by frameshift (11 kinds), and splice site mutations (nine kinds). Each splice site mutation was confirmed to create a deletion of the corresponding exon with RNA sequencing. The single promoter mutation c.-197G>A was reported previously; however, both missense mutations identified in exon 6 (c.601G>C: p.A201P) and exon 22 (c.2264T>C: p.F755S) were novel. Gross deletions were detected with MLPA in three probands. The detection rate of germline mutations in bilateral and unilateral cases with mutations were 81.8% and 30.0%, respectively. Only one father out of the 20 parents tested was positive for a germline mutation.ConclusionsMutations in the RB1 gene in Vietnamese patients were heterogeneous and highly prevalent with pathogenic truncated mutations. With advancement in therapeutics, early detection of RB is important for eye salvation.

Dataset Information

Mutation spectrum of RB1 gene in unilateral retinoblastoma cases from Tunisia and correlations with clinical features.

Publications

Mutation spectrum of RB1 gene in unilateral retinoblastoma cases from Tunisia and correlations with clinical features.

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