Dataset Information

Spectrum of germline mutations in RB1 in Chinese patients with retinoblastoma: Application of targeted next-generation sequencing.

ABSTRACT:

Purpose

Retinoblastoma (RB) is a pediatric ocular malignancy due to biallelic inactivation of the RB1 gene. Genetic testing is critically important for treatment decisions for this disease. Targeted next-generation sequencing (NGS) has been demonstrated to be an effective strategy for discovering all types of mutations in the RB1 gene. The aim of this study is the application of targeted NGS in a cohort of Chinese patients with retinoblastoma to identify germline mutations in the RB1 gene.

Methods

Blood samples were collected from 149 unrelated probands with retinoblastoma (62 bilaterally and 87 unilaterally) and their parent(s). Genomic DNA was analyzed with custom panel-based targeted NGS, and the panel was designed to include exons 1-27 of the RB1 gene with flanking intronic sequences. Single nucleotide variations (SNVs) and small insertions/deletions (InDels) identified were confirmed with Sanger sequencing. If the Sanger sequencing of a low-frequency variant (LFV) detected with targeted NGS was negative, PCR-based deep NGS was conducted for added confirmation. Copy number variations (CNVs) detected with targeted NGS were confirmed with multiplex ligation-dependent probe amplification (MLPA).

Results

Overall, 74 germline mutations were detected in 48.3% of the probands (72/149, 56 bilateral and 16 unilateral cases). The total detection rate in the bilateral cases was 90.3% (56/62). These mutations included 64 SNVs and InDels (25 nonsense, 20 splicing, ten frameshift, eight missense, and one synonymous variants) and ten CNVs. All CNVs were confirmed with MLPA. Twenty-four (32.4%, 24/74) variants detected were novel, including nine splicing, six frameshift, five missense, and four nonsense variants. Eight LFVs (10.8%, 8/74) were found with targeted NGS; six of which were identified with Sanger sequencing, and two were identified with PCR-based deep NGS (13.16% and 3.000% mutant rates, respectively).

Conclusions

This study expanded the spectrum of germline mutations in RB1 using targeted NGS technology, which is a cost-saving and efficient method for genetic sequencing of retinoblastoma and may improve the molecular diagnosis of retinoblastoma.

SUBMITTER: Zou Y

PROVIDER: S-EPMC7803294 | biostudies-literature | 2021

REPOSITORIES: biostudies-literature

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Publications

Spectrum of germline mutations in RB1 in Chinese patients with retinoblastoma: Application of targeted next-generation sequencing.

Zou Yihua Y Li Jiakai J Hua Peiyan P Liang Tingyi T Ji Xunda X Zhao Peiquan P

Molecular vision 20210106

<h4>Purpose</h4>Retinoblastoma (RB) is a pediatric ocular malignancy due to biallelic inactivation of the <i>RB1</i> gene. Genetic testing is critically important for treatment decisions for this disease. Targeted next-generation sequencing (NGS) has been demonstrated to be an effective strategy for discovering all types of mutations in the <i>RB1</i> gene. The aim of this study is the application of targeted NGS in a cohort of Chinese patients with retinoblastoma to identify germline mutations ...[more]

PMID: 33456302

Similar Datasets

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: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

Spectrum of germline mutations in RB1 in Chinese patients with retinoblastoma: Application of targeted next-generation sequencing.

Purpose

Methods

Results

Conclusions

Publications

Spectrum of germline mutations in RB1 in Chinese patients with retinoblastoma: Application of targeted next-generation sequencing.

Similar Datasets

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