Spectrum of RB1 Mutations in Argentine Patients: 20-years Experience in the Molecular Diagnosis of Retinoblastoma

ABSTRACT

Background: Retinoblastoma is a hereditary cancer of childhood caused by mutations in the RB1 tumor suppressor gene. An early diagnosis is critical for survival and eye preservation, thus identification of RB1 mutations is important for unequivocal diagnosis of hereditary retinoblastoma and risk assessment in relatives.

Methods: We studied 144 families for 20 years, performing methodological changes to improve detection of mutation. Segregation analysis of polymorphisms, MLPA, FISH and cytogenetic assays were used for detection of “at risk haplotypes” and large deletions. Small mutations were identified by heteroduplex/DNA sequencing.

Results: At risk haplotypes were identified in 11 familial and 26 sporadic cases, being useful for detection of asymptomatic carriers, risk exclusion from relatives and uncovering RB1 recombinations. Ten large deletions (eight whole gene deletions) were identified in six bilateral/familial and four unilateral retinoblastoma cases. Small mutations were identified in 29 cases (four unilateral retinoblastoma patients), being the majority nonsense/frameshift mutations. Genotype-phenotype correlations confirm that the retinoblastoma presentation is related to the type of mutation, but some exceptions may occur and it is crucial to be considered for genetic counseling. Three families included second cousins with retinoblastoma carrying different haplotypes, which suggest independent mutation events.

Conclusion: This study enabled us to obtain information about molecular and genetic features of patients with retinoblastoma in Argentina and correlate them to their phenotype.     

Sarcoma and familial retinoblastoma

Background: Retinoblastoma is the most common malignant ocular tumour of childhood. It results from mutations in the retinoblastoma gene, RB1, which may be sporadic or heritable. Only 10?25% of patients have a family history of retinoblastoma and can be assumed to have a heritable RB1 mutation. A small proportion of the remaining patients may have a heritable mutation despite the lack of relatives with retinoblastoma. Heritable RB1 mutations are associated with an increased risk of sarcoma. Described herein are three patients with a past history of retinoblastoma, no family history of retinoblastoma, and a first‐degree relative with sarcoma. Mutation analyses were performed on DNA samples from these patients to test for heritable RB1 mutations. Methods: A genomic DNA analysis of RB1 gene. Results: Heritable mutations in the RB1 gene were identified in two of the three cases. In these two cases, Bayesian risk calculation indicated that the chance of the affected relatives having the familial RB1 mutation was greater than 90%. In the case without an identified mutation, Bayesian risk analysis indicated that the chance of there being an unidentified familial RB1 mutation was low (16%) but could still be clinically significant in managing the family. Conclusion: The risk of non‐ocular malignancies and the availability of genetic testing for heritable RB1 mutations have important clinical implications for the management of children with retinoblastoma.     

108例视网膜母细胞瘤Rb基因突变的特征

目的；研究RB患者肿瘤及体细胞内Rb基因的存在状态、细微结构、Rb基因突变的特征、起源与传递。 方法；DNA分子杂交、SSCP分析、DNA序列测定。结果：108例RB肿瘤标本中80例(?4％)存在Rb基因点突变，其中44例为纯合型，20例有二个独立发生的杂合型点突变，16例只检出一个杂合型点突变。通过对比分析RB患者肿瘤与白细胞DNA、RB患者家庭成员白细胞DNARlo基因的结构，揭示了Rb基因点突变的起源与遗传特征。 结论：Rb基因是与RB肿瘤形成关系最为密切的基因。RB肿瘤发生需二次突变事件导致Rb基因的二个等位基因失活，第一次突变事件突出表现为点突变；第二次突变事件主要是LOH，其次是点突变。 （中华眼底病杂志，1997，13：12-16）     

Retinoblastoma in twins: Risk assessment of genotypic variants

Purpose:To describe methods of risk assessment in twins with retinoblastoma (RB).Methods:A case series of four RB probands with a twin sibling. Family status, clinical presentation, and RB1 germline status-based risk assessment were analyzed.Results:Two pairs had a positive family history (unilateral and bilateral RB in one of the parents (#1 and #2, respectively) and two pairs (#3 and #4) were sporadic. One of the familial twins (#1) had a high risk (90%) of manifesting RB in the twin. The other case (#2) with an absent RB1 germline mutation in the twin had a 0% risk of developing RB. Among sporadic cases of twins (#3), genetic testing did not identify a germline mutation (tumor sample unavailable) in the proband which downgraded the risk of germline mutation from 15% to <1%. The twin never developed RB (5 years of age at last follow-up). Pathogenic mosaicism for germline RB1 mutation (c.1723C>T) could be identified (tumor tissue available) in the proband (# 4). Identical germline mutation (and RB tumor) was also noted in the twin. In each case, there was concordance between the assessed risk and manifestation of RB.Conclusion:Assessment of risk of RB in a twin presents with a unique challenge. Depending upon the genotype variant, the risk of developing RB can vary from 0% to 90%. In addition to family history, clinical manifestation in the proband, zygosity status, and RB1 germline status are critical in formulating risk-appropriate surveillance guidelines.     

国人视网膜母细胞瘤患者RB1基因突变的特性

目的:检测国人RB患者体细胞中RB1基因突变,分析我国RB1基因突变的特性,探讨RB1基因突变的分子生物学机制。方法:应用PCR—SSCP技术筛查28例RB患者及亲属的白细胞基因组DNA,测序分析确定突变。结果:共确定7例RB1基因生殖细胞性突变。结论:本组RB1基因生殖细胞性突变的方式为点突变和小缺失,这些突变改变了RB1基因的遗传信息,致使异常的RB1基因蛋白产生,导致视网膜母细胞瘤的发生。中国RB1基因突变方式主要是以微小突变为主,但复杂突变的比例相对较高。     

Retinoblastoma and hypochondroplasia: a case report of two germline mutations arising simultaneously

PURPOSE: To report a rare case of a patient with two germline mutations arising de novo resulting in bilateral retinoblastoma and hypochondroplasia. DESIGN: A brief review about retinoblastoma and hypochondroplasia; a case report with genetic mutational analysis results. CASE REPORT: We report a patient manifesting the clinical features of both bilateral retinoblastoma and hypochondroplasia. Genetic analysis revealed two germline mutations, a seven base-pair deletion in exon 12 (G70313-703129del) in one allele of the retinoblastoma gene (RB1) and the N540K (C1620C > A) mutation in one allele of the fibroblast growth factor 3 (FGFR3) gene, a frequent mutation in hypochondroplasia. Neither parent has a personal or family history of cancer or ocular tumors. Only the patient's mother is short in stature, and her genetic analysis revealed no FGFR3 mutations. CONCLUSIONS: Although the probability of both germline mutations occurring in a single individual is exceedingly low, the etiology and mechanism are unknown in this patient. To the best of our knowledge, this is the first report of two clinically distinct heritable germline mutations arising de novo in an individual.     

Risk assessment of recurrence in sporadic retinoblastoma using a molecular-based algorithm

Purpose: Most RB1 mutations are unique and distributed throughout the RB1 gene. Their detection can be time-consuming and the yield especially low in cases of conservatively-treated sporadic unilateral retinoblastoma (Rb) patients. In order to identify patients with true risk of developing Rb, and to reduce the number of unnecessary examinations under anesthesia in all other cases, we developed a universal sensitive, efficient and cost-effective strategy based on intragenic haplotype analysis.

Methods: This algorithm allows the calculation of the a posteriori risk of developing Rb and takes into account (a) RB1 loss of heterozygosity in tumors, (b) preferential paternal origin of new germline mutations, (c) a priori risk derived from empirical data by Vogel, and (d) disease penetrance of 90% in most cases. We report the occurrence of Rb in first degree relatives of patients with sporadic Rb who visited the Jules Gonin Eye Hospital, Lausanne, Switzerland, from January 1994 to December 2006 compared to expected new cases of Rb using our algorithm.

Results: A total of 134 families with sporadic Rb were enrolled; testing was performed in 570 individuals and 99 patients younger than 4 years old were identified. We observed one new case of Rb. Using our algorithm, the cumulated total a posteriori risk of recurrence was 1.77.

Conclusions: This is the first time that linkage analysis has been validated to monitor the risk of recurrence in sporadic Rb. This should be a useful tool in genetic counseling, especially when direct RB1 screening for mutations leaves a negative result or is unavailable.     

Exon—by—exon Screening for RB Germline Mutations Using Heteroduplex—SSCP Analysis

Objective: To disclose the nature of RBI germline mutations in Chinese and to develop a practical and effective way for mutational screening. Methods: Leukocyte DNA was prepared from 8 Chinese patients with hereditary retinoblastoma. PCR combined with nonisotopic heteroduplex-SSCP analysis was used to screen leukocyte DNA for RBI germline mutations, exon-by-exon, without the use of restriction endonuclease digestion. The mutations were finally identified by sequencing. In order to testify the effectiveness of this method, the same method was used to detect other 17 samples which have been previously analyzed by other methods. Results: Heterozygous germline mutations were detected in the leukocyte DNA of 6 out of 8 Chinese patients;G del/codon 46,T del/codon 131 ,CAGAA del/condon 257 -258, GCAgta→GCAgca/donor of exon 16, C@T/codon 661, and C→T/codon 787. Heteroduplex-SSCP analysis may detect RBI germline mutations in 68% (177 25) unselected patients, which is more effective than SSCP(56%) or heterodupl     

Hereditary Diffuse Infiltrating Retinoblastoma

Retinoblastoma is one of the most common childhood cancers. The diffuse infiltrating retinoblastoma is a rare subtype of this neoplasm. The majority of cases of diffuse infiltrating retinoblastoma are unilateral and occur sporadically. Herein we report on a family with three children affected by retinoblastoma, among them one girl with diffuse infiltrating retinoblastoma. This girl was diagnosed at the age of 8 years with a unilateral diffuse infiltrating retinoblastoma. By contrast, the two brothers became clinically apparent in the first 2 years of life with bilateral retinoblastoma. The parents were clinically unremarkable. Genetic analysis of RB1 gene was performed. The girl with diffuse infiltrating RB was found to be heterozygous for an oncogenic mutation in the RB1 gene that was also carried by both brothers and the father of the family. These results show that diffuse infiltrating retinoblastoma can develop on the background of a hereditary predisposition to retinoblastoma.     

A novel translocation t(11;13) (q21;q14.2) in a child with suprasellar primitive neuroectodermal tumor and retinoblastoma

Purpose: To report on a novel translocation related to a suprasellar primitive neuroectodermal tumor (sPNET) and retinoblastoma.

Design: Case report.

Methods: A 6-year-old girl underwent genetic testing after developing unilateral retinoblastoma subsequent to treatment (surgery, chemotherapy, and stem-cell rescue) for a sPNET found at 1 year of age.

Results: Genetic testing found the girl’s karyotype to be 46,XX,t(11;13)(q21;q14.2); a novel translocation not previously reported in patients with either retinoblastoma or sPNET.

Conclusions: Our patient had a novel translocation affecting the retinoblastoma 1 (RB1) gene, 46,XX,t(11;13)(q21;q14.2) resulting in the late development of unilateral retinoblastoma. Although she only developed unilateral retinoblastoma, her central nervous system was affected at a very early age. How her complex mutation resulted in retinoblastoma and antecedent sPNET remains unknown.