Retinoblastoma is a malignancy arising in the retina, mostly diagnosed in youn children; two-thirds of all cases of retinoblastoma are diagnosed before age 2 years, and 95% before age 5 years. Between 25–30% of Retinoblastoma cases are heritable, having a germline mutation of the RB1 gene. The heritable cases are more likely to be bilateral (both eyes) and younger age, compared to the 70-75% of nonheritable cases. For children with germline mutation of RB1, approximately 25% are inherited from an affected parent, while 75% are thought to have occurred in utero at the time of conception. (Source: National Cancer Institute).

The penetrance of the RB1 mutation in retinoblastoma is thought to be dependent on concurrent genetic modifiers, in particular MDM2 and MDM4. In a family-based association analyses of 212 mutation carriers in 70 retinoblastoma families, Castéra L et al, 2010 reported a strong association between the MDM2 309G allele and incidence of bilateral or unilateral retinoblastoma among members of retinoblastoma families (p<0.001). de Oliveira Reis AH et al, 2012 reported findings that suggest that MDM2 and MDM4 polymorphisms may influence development and/or survival in RB.

See also: Retinoblastoma - clinical resources (12)

Literature Analysis

Mouse over the terms for more detail; many indicate links which you can click for dedicated pages about the topic.

Tag cloud generated 10 March, 2017 using data from PubMed, MeSH and CancerIndex

Mutated Genes and Abnormal Protein Expression (24)

How to use this data tableClicking on the Gene or Topic will take you to a separate more detailed page. Sort this list by clicking on a column heading e.g. 'Gene' or 'Topic'.

RB1 13q14.2 RB, pRb, OSRC, pp110, p105-Rb, PPP1R130 Germline
-RB1 mutation in Retinoblastoma
MYCN 2p24.3 NMYC, ODED, MODED, N-myc, bHLHe37 Amplification
-MYCN Amplification in Retinoblastoma
RBL2 16q12.2 Rb2, P130 -RBL2 and Retinoblastoma
MDM2 12q14.3-q15 HDMX, hdm2, ACTFS -MDM2 and Retinoblastoma
MDM4 1q32 HDMX, MDMX, MRP1 -MDM4 and Retinoblastoma
PRB1 12p13.2 PM, PMF, PMS, PRB1L, PRB1M -PRB1 and Retinoblastoma
E2F3 6p22 E2F-3 -E2F3 and Retinoblastoma
RBL1 20q11.2 PRB1, p107, CP107 -RBL1 and Retinoblastoma
KIF14 1q32.1 -KIF14 and Retinoblastoma
CDH11 16q21 OB, CAD11, CDHOB, OSF-4 -CDH11 and Retinoblastoma
DEK 6p22.3 D6S231E -DEK and Retinoblastoma
PRB2 12p13.2 Ps, cP7, IB-9, PRPPRB1 -PRB2 and Retinoblastoma
CDK6 7q21-q22 MCPH12, PLSTIRE -CDK6 and Retinoblastoma
CDH13 16q23.3 CDHH, P105 -CDH13 and Retinoblastoma
CD82 11p11.2 R2, 4F9, C33, IA4, ST6, GR15, KAI1, SAR2, TSPAN27 -CD82 and Retinoblastoma
DDX1 2p24 DBP-RB, UKVH5d Amplification
-DDX1 Amplification in Retinoblastoma cell lines
OTX2 14q22.3 CPHD6, MCOPS5 -OTX2 and Retinoblastoma
RXRA 9q34.3 NR2B1 -RXRA and Retinoblastoma
MIRLET7E 19q13.41 LET7E, let-7e, MIRNLET7E, hsa-let-7e -MicroRNA let-7e and Retinoblastoma
CACNA1G 17q22 NBR13, Cav3.1, Ca(V)T.1 -CACNA1G and Retinoblastoma
RCVRN 17p13.1 RCV1 -RCVRN and Retinoblastoma
NEUROG1 5q23-q31 AKA, ngn1, Math4C, bHLHa6, NEUROD3 -NEUROG1 and Retinoblastoma
HLA-C 6p21.3 HLC-C, D6S204, PSORS1, HLA-JY3 -HLA-C and Retinoblastoma
HLA-B 6p21.3 AS, HLAB, SPDA1 -HLA-B and Retinoblastoma

Note: list is not exhaustive. Number of papers are based on searches of PubMed (click on topic title for arbitrary criteria used).

Recurrent Chromosome Abnormalities

Selected list of common recurrent structural abnormalities

Abnormality Type Gene(s)
i(6p10) in RetinoblastomaIsochromosome

This is a highly selective list aiming to capture structural abnormalies which are frequesnt and/or significant in relation to diagnosis, prognosis, and/or characterising specific cancers. For a much more extensive list see the Mitelman Database of Chromosome Aberrations and Gene Fusions in Cancer.

i(6p10) in Retinoblastoma

Gain of the short arm of chromosome 6, usually through isochromosome 6p formation, is present in approximately 50% of retinoblastoma tumors. The minimal region of gain maps to chromosome band 6p22. Paderova et al (2007) suggested these may be due to translocations.

Horsthemke B, Greger V, Becher R, Passarge E
Mechanism of i(6p) formation in retinoblastoma tumor cells.
Cancer Genet Cytogenet. 1989; 37(1):95-102 [PubMed] Related Publications
Isochromosome (6p) represents a highly characteristic cytogenetic abnormality of human retinoblastoma (RB) cells and may be important for tumor progression. To elucidate the mechanism by which this abnormal chromosome is formed, 24 RB tumors and three cell lines were studied by means of DNA polymorphisms specific for the short arm and the long arm of chromosome 6. Our results indicate that mitotic nondisjunction leading to trisomy 6 precedes the isochromosome formation. The isochromosome may then be formed by transverse division of the centromere or intrachromosomal chromatid exchange.

Paderova J, Orlic-Milacic M, Yoshimoto M, et al.
Novel 6p rearrangements and recurrent translocation breakpoints in retinoblastoma cell lines identified by spectral karyotyping and mBAND analyses.
Cancer Genet Cytogenet. 2007; 179(2):102-11 [PubMed] Related Publications
Gain of the short arm of chromosome 6, usually through isochromosome 6p formation, is present in approximately 50% of retinoblastoma tumors. The minimal region of gain maps to chromosome band 6p22. Two genes, DEK and E2F3, are implicated as candidate oncogenes. However, chromosomal translocations have been overlooked as a potential mechanism of activation of oncogenes at 6p22 in retinoblastoma. Here, we report combined spectral karyotyping), 4',6-diamidino-2-phenylindole banding, mBAND, and locus-specific fluorescence in situ hybridization analyses of four retinoblastoma cell lines, RB1021, RB247c, RB383, and Y79. In RB1021 and RB247c, 6p undergoes structural rearrangements involving a common translocation breakpoint at 6p22. These data imply that 6p translocations may represent another mechanism of activation of 6p oncogene(s) in a subset of retinoblastomas, besides the copy number increase. In addition to 6p22, other recurrent translocation breakpoints identified in this study are 4p16, 11p15, 17q21.3, and 20q13. Common regions of gain map to chromosomal arms 1q, 2p, 6p, 17q, and 21q.

Latest Publications

Zhang H, Zhong J, Bian Z, et al.
Long non-coding RNA CCAT1 promotes human retinoblastoma SO-RB50 and Y79 cells through negative regulation of miR-218-5p.
Biomed Pharmacother. 2017; 87:683-691 [PubMed] Related Publications
OBJECTIVE: To investigate the regulatory role and potential mechanism of long non-coding RNAs (lncRNA) in human retinoblastoma (RB).
METHODS: The lncRNA profile in RB tissues were analyzed by microarray and quantitative reverse transcription PCR (qRT-PCR). One of the identified lncRNAs (LncRNA CCAT1) was selected for further experiments. SO-RB50 and Y79 cells were transfected with negative control, siRNA targeting lncRNA CCAT1 (si-CCAT1) and si-CCAT1+miR218-5p inhibitor, respectively. lncRNA CCAT1 expression was measured by qRT-PCR. Cell proliferation, migration and invasion were detected by CCK8, wound scratching, and transwell assay, respectively. Apoptosis and cell cycle distribution were assessed by flow cytometry. Apoptosis- (cle-caspase-3, cle-caspase-9, Bax and Bcl-2) and cell cycle-related protein expression (cyclin B1, CDC2 and p-CDC2 (Thr161)) were analyzed by Western blot.
RESULTS: lncRNA CCAT1 expression in SO-RB50 and Y79 cells was significantly inhibited after si-CCAT1 transfection (P<0.01). Both RB cells exhibited significantly reduced proliferation, migration and invasion abilities, but markedly increased apoptosis at 48h after si-CCAT1 transfection (P<0.05 or 0.01). RB cells in si-CCAT1+miR218-5p inhibitor group had significantly higher proliferation, migration and invasion, but notably lower apoptosis compared with si-CCAT1 group at 24 and 48h after transfection (all P<0.05 or 0.01). si-CCAT1 significantly increased the expression of cle-caspase-3, cle-caspase-9, Bax, but decreased Bcl-2 expression (P<0.01). The proportion of G2/M SO-RB50 and Y79 cells in siCCAT1 group was significantly increased compared with negative control group (P<0.01). LncRNA CCAT1 interference significantly reduced the expression of cyclin B1, CDC2 and p-CDC2 (Thr161) (P<0.01).
CONCLUSION: LncRNA CCAT1 promotes the proliferation migration and invasion, and reduces cell apoptosis of SO-RB50 and Y79 cells, probably through negative modulation of miR-218-5p. Our study suggested lncRNA CCAT1 as a potential biomarker and therapeutic target for RB.

Singh U, Malik MA, Goswami S, et al.
Epigenetic regulation of human retinoblastoma.
Tumour Biol. 2016; 37(11):14427-14441 [PubMed] Related Publications
Retinoblastoma is a rare type of eye cancer of the retina that commonly occurs in early childhood and mostly affects the children before the age of 5. It occurs due to the mutations in the retinoblastoma gene (RB1) which inactivates both alleles of the RB1. RB1 was first identified as a tumor suppressor gene, which regulates cell cycle components and associated with retinoblastoma. Previously, genetic alteration was known as the major cause of its occurrence, but later, it is revealed that besides genetic changes, epigenetic changes also play a significant role in the disease. Initiation and progression of retinoblastoma could be due to independent or combined genetic and epigenetic events. Remarkable work has been done in understanding retinoblastoma pathogenesis in terms of genetic alterations, but not much in the context of epigenetic modification. Epigenetic modifications that silence tumor suppressor genes and activate oncogenes include DNA methylation, chromatin remodeling, histone modification and noncoding RNA-mediated gene silencing. Epigenetic changes can lead to altered gene function and transform normal cell into tumor cells. This review focuses on important epigenetic alteration which occurs in retinoblastoma and its current state of knowledge. The critical role of epigenetic regulation in retinoblastoma is now an emerging area, and better understanding of epigenetic changes in retinoblastoma will open the door for future therapy and diagnosis.

Francis JH, Levin AM, Abramson DH
Update on Ophthalmic Oncology 2014: Retinoblastoma and Uveal Melanoma.
Asia Pac J Ophthalmol (Phila). 2016 Sep-Oct; 5(5):368-82 [PubMed] Related Publications
PURPOSE: The aim of this study was to review peer-reviewed articles on ophthalmic oncology (specifically retinoblastoma and uveal melanoma) published from January to December 2014.
DESIGN: This study is a literature review.
METHODS: The terms retinoblastoma and uveal melanoma were used in a MEDLINE literature search. Abstracts were studied, and the most relevant articles were selected for inclusion and further in-depth review.
RESULTS: In retinoblastoma, more eyes are being salvaged due to intravitreal melphalan. The year 2014 marks a deepening in our understanding of the biological basis of the disease and the cell of origin. Knowledge on the genetic underpinnings of uveal melanoma has broadened to include other pathways, interactions, and potential therapeutic targets.
CONCLUSIONS: In 2014, there were valuable advancements in our knowledge of retinoblastoma and uveal melanoma. Some of these resulted in improved patient management.

Gui F, Hong Z, You Z, et al.
MiR-21 inhibitor suppressed the progression of retinoblastoma via the modulation of PTEN/PI3K/AKT pathway.
Cell Biol Int. 2016; 40(12):1294-1302 [PubMed] Related Publications
MicroRNA-21 (miR-21) was reported to act as an oncogene during the development of many human tumors. However, little was revealed about the function of miR-21 in retinoblastoma (RB). In this study, we examined the expression of miR-21 in RB tissues and explored the relationship between miR-21 and phosphatase and tensin homolog (PTEN)/phosphatidylinositol-3-OH kinase (PI3K)/AKT signal. Quantitative real-time PCR (qRT-PCR) results showed that the level of miR-21 in RB tissues was higher than that in retinal normal tissues. In Weri-Rb-1 cells, miR-21 inhibitor suppressed the expression of miR-21 and cell viability, but improved cell apoptotic rates by modulating the levels of PDCD4, Bax, and Bcl-2. Meanwhile, miR-21 inhibitor suppressed cell migration and invasion via inhibiting the protein levels of MMP2 and MMP9 and significantly affected the expression of PTEN, PI3K, and p-AKT. Taken together, miR-21 inhibitor suppressed cell proliferation, migration, and invasion via the PTEN/PI3K/AKT signal. These findings revealed the molecular basis of miR-21 functioning in the progression of RB and provided a new means for cell therapy in RB.

Croce CM
Alfred G. Knudson (1922-2016).
Nature. 2016; 536(7617):397 [PubMed] Related Publications

Mallipatna A, Marino M, Singh AD
Genetics of Retinoblastoma.
Asia Pac J Ophthalmol (Phila). 2016 Jul-Aug; 5(4):260-4 [PubMed] Related Publications
Retinoblastoma is a malignant retinal tumor that affects young children. Mutations in the RB1 gene cause retinoblastoma. Mutations in both RB1 alleles within the precursor retinal cell are essential, with one mutation that may be germline or somatic and the second one that is always somatic. Identification of the RB1 germline status of a patient allows differentiation between sporadic and heritable retinoblastoma variants. Application of this knowledge is crucial for assessing short-term (risk of additional tumors in the same eye and other eye) and long-term (risk of nonocular malignant tumors) prognosis and offering cost-effective surveillance strategies. Genetic testing and genetic counseling are therefore essential components of care for all children diagnosed with retinoblastoma. The American Joint Committee on Cancer has acknowledged the importance of detecting this heritable trait and has introduced the letter "H" to denote a heritable trait of all cancers, starting with retinoblastoma (in publication). In this article, we discuss the clinically relevant aspects of genetic testing and genetic counseling for a child with retinoblastoma.

Li X, Yang L, Shuai T, et al.
MiR-433 inhibits retinoblastoma malignancy by suppressing Notch1 and PAX6 expression.
Biomed Pharmacother. 2016; 82:247-55 [PubMed] Related Publications
Retinoblastoma (RB) is the most frequent primary intraocular cancer. It has been demonstrated by previous studies that retinoblastoma is initiated primarily by the inactivation of the retinoblastoma Rb1 gene in retinal cells. However, additional genetic alterations than Rb1 mutation could play important roles in the process of transforming benign retinal cells into retinoblastoma tumor cells. In this study, we identified that microRNA miR-433 is one of such genetic factors. We found that the expression levels of miR-433 were downregulated in RB tissues. We also determined that miR-433 negatively regulated RB cell proliferation, migration and invasion, and induced cell cycle arrest and apoptosis of RB cells. We used bioinformatics method to predict and confirmed that Notch1 and PAX6 were miR-433 target genes in RB cells. Importantly, we demonstrated that restoration of Notch1 and PAX6 expression partially rescued the inhibition of cell proliferation and metastasis induced by miR-433 overexpression, suggesting that miR-433 regulates RB cell proliferation and metastasis through suppressing the expression of Notch1 and PAX6.

Fadakar P, Akbari A, Ghassemi F, et al.
Evaluation of SD-208, a TGF-β-RI Kinase Inhibitor, as an Anticancer Agent in Retinoblastoma.
Acta Med Iran. 2016; 54(6):352-8 [PubMed] Related Publications
Retinoblastoma is the most common intraocular tumor in children resulting from genetic alterations and transformation of mature retinal cells. The objective of this study was to investigate the effects of SD-208, TGF-β-RI kinase inhibitor, on the expression of some miRNAs including a miR-17/92 cluster in retinoblastoma cells. Prior to initiate this work, the cell proliferation was studied by Methyl Thiazolyl Tetrazolium (MTT) and bromo-2'-deoxyuridine (BrdU) assays. Then, the expression patterns of four miRNAs (18a, 20a, 22, and 34a) were investigated in the treated SD-208 (0.0, 1, 2 and 3 µM) and untreated Y-79 cells. A remarkable inhibition of the cell proliferation was found in Y-79 cells treated with SD-208 versus untreated cells. Also, the expression changes were observed in miRNAs 18a, 20a, 22 and 34a in response to SD-208 treatment (P<0.05). The findings of the present study suggest that the anti-cancer effect of SD-208 may be exerted due to the regulation of specific miRNAs, at least in this particular retinoblastoma cell line. To the best of the researchers' knowledge, this is the first report demonstrating that the SD-208 could alter the expression of tumor suppressive miRNAs as well as oncomiRs in vitro. In conclusion, the present data suggest that SD-208 could be an alternative agent in retinoblastoma treatment.

Chen C, Ma FW, Du CY, Wang P
Multiple Differential Networks Strategy Reveals Carboplatin and Melphalan-Induced Dynamic Module Changes in Retinoblastoma.
Med Sci Monit. 2016; 22:1508-15 [PubMed] Free Access to Full Article Related Publications
BACKGROUND Retinoblastoma (RB) is the most common malignant tumor of the eye in childhood. The objective of this paper was to investigate carboplatin (CAR)- and melphalan (MEL)-induced dynamic module changes in RB based on multiple (M) differential networks, and to generate systems-level insights into RB progression. MATERIAL AND METHODS To achieve this goal, we constructed M-differential co-expression networks (DCNs), assigned a weight to each edge, and identified seed genes in M DCNs by ranking genes based on their topological features. Starting with seed genes, a module search was performed to explore candidate modules in CAR and MEL condition. M-DMs were detected according to significance evaluations of M-modules, which originated from refinement of candidate modules. Further, we revealed dynamic changes in M-DM activity and connectivity on the basis of significance of Module Connectivity Dynamic Score (MCDS). RESULTS In the present study, M=2, a total of 21 seed genes were obtained. By assessing module search, refinement, and evaluation, we gained 18 2-DMs. Moreover, 3 significant 2-DMs (Module 1, Module 2, and Module 3) with dynamic changes across CAR and MEL condition were determined, and we denoted them as dynamic modules. Module 1 had 27 nodes of which 6 were seed genes and 56 edges. Module 2 was composed of 28 nodes and 54 edges. A total of 28 nodes interacted with 45 edges presented in Module 3. CONCLUSIONS We have identified 3 dynamic modules with changes induced by CAR and MEL in RB, which might give insights in revealing molecular mechanism for RB therapy.

Kooi IE, Mol BM, Massink MP, et al.
A Meta-Analysis of Retinoblastoma Copy Numbers Refines the List of Possible Driver Genes Involved in Tumor Progression.
PLoS One. 2016; 11(4):e0153323 [PubMed] Free Access to Full Article Related Publications
BACKGROUND: While RB1 loss initiates retinoblastoma development, additional somatic copy number alterations (SCNAs) can drive tumor progression. Although SCNAs have been identified with good concordance between studies at a cytoband resolution, accurate identification of single genes for all recurrent SCNAs is still challenging. This study presents a comprehensive meta-analysis of genome-wide SCNAs integrated with gene expression profiling data, narrowing down the list of plausible retinoblastoma driver genes.
METHODS: We performed SCNA profiling of 45 primary retinoblastoma samples and eight retinoblastoma cell lines by high-resolution microarrays. We combined our data with genomic, clinical and histopathological data of ten published genome-wide SCNA studies, which strongly enhanced the power of our analyses (N = 310).
RESULTS: Comprehensive recurrence analysis of SCNAs in all studies integrated with gene expression data allowed us to reduce candidate gene lists for 1q, 2p, 6p, 7q and 13q to a limited gene set. Besides the well-established driver genes RB1 (13q-loss) and MYCN (2p-gain) we identified CRB1 and NEK7 (1q-gain), SOX4 (6p-gain) and NUP205 (7q-gain) as novel retinoblastoma driver candidates. Depending on the sample subset and algorithms used, alternative candidates were identified including MIR181 (1q-gain) and DEK (6p gain). Remarkably, our study showed that copy number gains rarely exceeded change of one copy, even in pure tumor samples with 100% homozygosity at the RB1 locus (N = 34), which is indicative for intra-tumor heterogeneity. In addition, profound between-tumor variability was observed that was associated with age at diagnosis and differentiation grades.
INTERPRETATION: Since focal alterations at commonly altered chromosome regions were rare except for 2p24.3 (MYCN), further functional validation of the oncogenic potential of the described candidate genes is now required. For further investigations, our study provides a refined and revised set of candidate retinoblastoma driver genes.

Zhang Y, Xue C, Zhu X, et al.
Suppression of microRNA-125a-5p upregulates the TAZ-EGFR signaling pathway and promotes retinoblastoma proliferation.
Cell Signal. 2016; 28(8):850-60 [PubMed] Related Publications
Retinoblastoma is the most common intraocular malignancy that occurs during childhood; however, the mechanism underlying retinoblastoma proliferation and progression remains unclear. MicroRNAs (miRNAs) play an important role in the regulation of a myriad of biological processes in various types of cancer. In this study, we performed microarray analysis followed by qRT-PCR using four classes of retinoblastoma tissues with increasing cTNM classification stages to identify crucial miRNAs whose expression was correlated with retinoblastoma progression. miR-125a-5p was downregulated, and its expression levels were inversely correlated with cell proliferation in retinoblastoma compared with adjacent non-tumor retinal tissues. The overexpression of miR-125a-5p significantly suppressed cell proliferation and tumor formation in retinoblastoma. We further identified the transcriptional co-activator with PDZ binding motif (TAZ) as a direct target of miR-125a-5p. Importantly, TAZ levels were inversely correlated with miRNA-125a-5p expression, and TAZ promoted retinoblastoma cell proliferation. Moreover, the overexpression of miR-125a-5p led to a decrease in TAZ expression and downstream EGFR signaling pathway activation both in vitro and vivo. Finally, TAZ overexpression in retinoblastoma cells overexpressing miR-125a-5p restored retinoblastoma cell proliferation and EGFR pathway activation. Taken together, our data demonstrated that miR-125a-5p functions as an important tumor suppressor that suppresses the EGFR pathway by targeting TAZ to inhibit tumor progression in retinoblastoma. Thus, the miR-125a-5p/TAZ/EGFR axis may be a potential therapeutic target for retinoblastoma.

Zhang Y, Wu D, Xia F, et al.
Downregulation of HDAC9 inhibits cell proliferation and tumor formation by inducing cell cycle arrest in retinoblastoma.
Biochem Biophys Res Commun. 2016; 473(2):600-6 [PubMed] Related Publications
Histone deacetylase 9 (HDAC9) is a member of class II HDACs, which regulates a wide variety of normal and abnormal physiological functions. Recently, HDAC9 has been found to be overexpressed in some types of human cancers. However, the role of HDAC9 in retinoblastoma remains unclear. In this study, we found that HDAC9 was commonly expressed in retinoblastoma tissues and HDAC9 was overexpressed in prognostically poor retinoblastoma patients. Through knocking down HDAC9 in Y79 and WERI-Rb-1 cells, the expression level of HDAC9 was found to be positively related to cell proliferation in vitro. Further investigation indicated that knockdown HDAC9 could significantly induce cell cycle arrest at G1 phase in retinoblastoma cells. Western blot assay showed downregulation of HDAC9 could significantly decrease cyclin E2 and CDK2 expression. Lastly, xenograft study in nude mice showed that downregulation of HDAC9 inhibited tumor growth and development in vivo. Therefore, our results suggest that HDAC9 could serve as a novel potential therapeutic target in the treatment of retinoblastoma.

Delhiwala KS, Vadakkal IP, Mulay K, et al.
Retinoblastoma: An update.
Semin Diagn Pathol. 2016; 33(3):133-40 [PubMed] Related Publications
Retinoblastoma is the most common ocular malignancy in children, and is initiated by mutation of the RB1 gene. The tumor may be unilateral or bilateral and can be inherited. Overall survival, eye salvage, and preservation of vision are largely dependent on the stage of disease at presentation. Despite a recently enhanced understanding of the etiology of retinoblastoma, the mortality associated with it remains high worldwide. This may relate to a continuing lack of awareness of the lesion by laypersons, and unavailability of modern treatment facilities. Adverse outcomes are also caused by the occurrence of secondary malignancies after treatment of retinoblastoma in childhood. Early diagnosis, multidisciplinary treatment, and genetic counseling are all priorities in the management of this tumor.

Eloy P, Dehainault C, Sefta M, et al.
A Parent-of-Origin Effect Impacts the Phenotype in Low Penetrance Retinoblastoma Families Segregating the c.1981C>T/p.Arg661Trp Mutation of RB1.
PLoS Genet. 2016; 12(2):e1005888 [PubMed] Free Access to Full Article Related Publications
Retinoblastoma (Rb), the most common pediatric intraocular neoplasm, results from inactivation of both alleles of the RB1 tumor suppressor gene. The second allele is most commonly lost, as demonstrated by loss of heterozygosity studies. RB1 germline carriers usually develop bilateral tumors, but some Rb families display low penetrance and variable expressivity. In order to decipher the underlying mechanisms, 23 unrelated low penetrance pedigrees segregating the common c.1981C>T/p.Arg661Trp mutation and other low penetrance mutations were studied. In families segregating the c.1981C>T mutation, we demonstrated, for the first time, a correlation between the gender of the transmitting carrier and penetrance, as evidenced by Fisher's exact test: the probability of being unaffected is 90.3% and 32.5% when the mutation is inherited from the mother and the father, respectively (p-value = 7.10(-7). Interestingly, a similar correlation was observed in families segregating other low penetrance alleles. Consequently, we investigated the putative involvement of an imprinted, modifier gene in low penetrance Rb. We first ruled out a MED4-driven mechanism by MED4 methylation and expression analyses. We then focused on the differentially methylated CpG85 island located in intron 2 of RB1 and showing parent-of-origin-specific DNA methylation. This differential methylation promotes expression of the maternal c.1981C>T allele. We propose that the maternally inherited c.1981C>T/p.Arg661Trp allele retains sufficient tumor suppressor activity to prevent retinoblastoma development. In contrast, when the mutation is paternally transmitted, the low residual activity would mimic a null mutation and subsequently lead to retinoblastoma. This implies that the c.1981C>T mutation is not deleterious per se but needs to be destabilized in order to reach pRb haploinsufficiency and initiate tumorigenesis. We suggest that this phenomenon might be a general mechanism to explain phenotypic differences in low penetrance Rb families.

Chang Y, Li B, Xu X, et al.
Lentivirus-Mediated Knockdown of Astrocyte Elevated Gene-1 Inhibits Growth and Induces Apoptosis through MAPK Pathways in Human Retinoblastoma Cells.
PLoS One. 2016; 11(2):e0148763 [PubMed] Free Access to Full Article Related Publications
PURPOSE: To explore expression and function of astrocyte elevated gene-1 (AEG-1) in human retinoblastoma (RB).
METHODS: The expression of AEG-1 in histological sections of human RBs and in RB cell lines was examined using immunohistochemical staining and RT-PCR and Western blotting respectively. We knocked down AEG-1 gene levels by AEG-1-siRNA lentivirus transfection of human RB cell lines SO-RB50 and Y79, and using an MTT assay, we assessed the role of AEG-1 on RB cell proliferation. The biological significance of lentivirus transfection induced AEG-1 down-regulation was examined by assessing the apoptosis rate in the transfected RB cells by Annexin V-APC staining and flow cytometry. We additionally measured the expression of Bcl-2, Bax, cleaved-caspase-3 and caspase-3, and the phosphorylation and non-phosphorylation alternation of MAPKs.
RESULTS: AEG-1 expression was detected to be strongly positive in the histological slides of 35 out of 54 (65%) patients with RB. AEG-1 expression increased significantly (P<0.05) with tumor stage. In the RB cell lines SO-RB50, Y79 and WERI-RB1 as compared with retinal pigment epithelium cells, expression of AEG-1 mRNA and AEG-1 protein was significantly higher. In AEG-1-siRNA lentivirus transfected cell cultures as compared with negative control lentivirus transfected cell cultures, levels of AEG-1 mRNA and of AEG-1 protein (P<0.05) and cell growth rates (P<0.01) were significantly lower, and apoptosis rate (P<0.001), Bax/Bcl-2 ratio and cleaved-caspase-3 protein level were significantly increased. The P-ERK/ERK ratio was significantly decreased in the AEG-1-siRNA lentivirus transfected cell lines.
CONCLUSIONS: Expression of AEG-1 was associated with RB, in histological slides of patients and in cell culture experiments. Lentivirus transfection induced knockdown of AEG-1 had a tumor suppressive effect, potentially by tumor cell apoptosis induction through inhibition of ERK.

García-Chequer AJ, Méndez-Tenorio A, Olguín-Ruiz G, et al.
Overview of recurrent chromosomal losses in retinoblastoma detected by low coverage next generation sequencing.
Cancer Genet. 2016; 209(3):57-69 [PubMed] Free Access to Full Article Related Publications
Genes are frequently lost or gained in malignant tumors and the analysis of these changes can be informative about the underlying tumor biology. Retinoblastoma is a pediatric intraocular malignancy, and since deletions in chromosome 13 have been described in this tumor, we performed genome wide sequencing with the Illumina platform to test whether recurrent losses could be detected in low coverage data from DNA pools of Rb cases. An in silico reference profile for each pool was created from the human genome sequence GRCh37p5; a chromosome integrity score and a graphics 40 Kb window analysis approach, allowed us to identify with high resolution previously reported non random recurrent losses in all chromosomes of these tumors. We also found a pattern of gains and losses associated to clear and dark cytogenetic bands respectively. We further analyze a pool of medulloblastoma and found a more stable genomic profile and previously reported losses in this tumor. This approach facilitates identification of recurrent deletions from many patients that may be biological relevant for tumor development.

Sun Z, Zhang A, Jiang T, et al.
MiR-145 suppressed human retinoblastoma cell proliferation and invasion by targeting ADAM19.
Int J Clin Exp Pathol. 2015; 8(11):14521-7 [PubMed] Free Access to Full Article Related Publications
MicroRNAs (miRNAs) play critical roles in retinoblastoma (RB) initiation and progression, aberrant expression of miR-145 had been frequently reported in cancer studies. However, the role and mechanism of its function in RB is still unclear. In this study, our data showed that miR-145 was downregulated in RB tissues and cell lines. Overexpression of miR-145 suppressed RB cell proliferation, migration and invasion in vitro. ADAM19 was identified as a direct target of miR-145. Silencing of ADAM19 significantly inhibited RB cell proliferation, migration and invasion. In addition, a reverse correlation between miR-145 and ADAM19 expression was noted in RB tissues. Taken together, these findings suggested that miR-145 functions as a tumor suppressor in RB by directly targeting ADAM19. miR-145 could be an anticancer therapeutic target for RB patients.

Krzemień W, Wojcieszak J, Zawilska JB
[Retinoblastoma: genetic background, modern diagnostic methods and therapies].
Przegl Lek. 2015; 72(7):358-65 [PubMed] Related Publications
Retinoblastoma is the most common intraocular eye tumor of the pediatric age. It develops on account of a mutation on chromosome 13 in the 13q14 locus. New studies additionally demonstrated changes in the expression of other genes classified as oncogenes and suppressor genes. The tumor occurs in two forms--heritable (genetic) and non-heritable (non-genetic, sporadic). The most common clinical features of retinoblastoma are leucocoria and strabismus, however, they are not that specific because may also occur in several other eye diseases, such as Coats disease and toxocarosis. The diagnosis of retinoblastoma requires an indirect ophthalmoscopic examination. In addition, imaging techniques such as ultrasonography (USG), magnetic resonance imaging (MRI) and, less commonly, computer tomography (CT) are used. Biopsy is contraindicated because of the risk of spreading cancer cells to the adjacent tissues and possibility of a metastasis development. Currently, the stage of the disease and the therapy prognosis are classified by the International Intraocular Retinoblastoma Classification. At present, chemotherapy is the standard treatment of retinoblastoma. During the last decades new therapies have been introduced, such as transpupillary thermotherapy (TTT), cryotherapy, brachytherapy, limiting the use of teletherapy and the number of performed enucleations. Patients with therapy-induced remission of retinoblastoma should undergo routine examinations because of the increased risk of subsequent neoplasms and other possible complications.

Chen P, Yu N, Zhang Z, et al.
Thrombospondin-1 might be a therapeutic target to suppress RB cells by regulating the DNA double-strand breaks repair.
Oncotarget. 2016; 7(5):6105-20 [PubMed] Free Access to Full Article Related Publications
Retinoblastoma (RB) arises from the retina, and its growth usually occurs under the retina and toward the vitreous. Ideal therapy should aim to inhibit the tumor and protect neural cells, increasing the patient's life span and quality of life. Previous studies have demonstrated that Thrombospondin-1 (TSP-1) is associated with neurogenesis, neovascularization and tumorigenesis. However, at present, the bioactivity of TSP-1 in retinoblastoma has not been defined. Herein, we demonstrated that TSP-1 was silenced in RB cell lines and clinical tumor samples. HDAC inhibitor, Trichostatin A (TSA), could notably transcriptionally up-regulate TSP-1 in RB cells, WERI-Rb1 cells and Y79 cells. Moreover, we found human recombinant TSP-1 (hTSP-1) could significantly inhibit the cell viability of RB cells both in vitro and in vivo. Interestingly, hTSP-1 could significantly induce the expression of γ-H2AX, a well-characterized in situ marker of DNA double-strand breaks (DSBs) in RB cells. The DNA NHEJ pathway in WERI-Rb1 cells could be significantly inhibited by hTSP-1. A mutation in Rb1 might be involved in the hTSP-1-medicated γ-H2AX increasing in WERI-Rb1 cells. Furthermore, hTSP-1 could inhibit RB cells while promoting retinal neurocyte survival in the neuronal and retinoblastoma cell co-culture system. As such, TSP-1 may become a therapeutic target for treatment of retinoblastoma.

Quiñonez-Silva G, Dávalos-Salas M, Recillas-Targa F, et al.
"Monoallelic germline methylation and sequence variant in the promoter of the RB1 gene: a possible constitutive epimutation in hereditary retinoblastoma".
Clin Epigenetics. 2016; 8:1 [PubMed] Free Access to Full Article Related Publications
BACKGROUND: Retinoblastoma is a malignant tumor of the retina in children <5 years of age and occurs after two mutations in the RB1 gene. The first mutation (M1) is germinal and confers predisposition to the hereditary type, which is transmitted as an autosomal dominant highly penetrant trait, so 90 % of carriers develop retinoblastoma; however, 10 % of carriers either do not develop the tumor or develop it unilaterally. Most mutations are point mutations. Inactivation of the RB1 gene is usually caused by mutations affecting the coding region. Silencing by methylation of the RB1 promoter has been observed in retinoblastoma tumors as a second mutation (M2) and is classified as somatic epimutation. Germline methylation of the RB1 gene promoter was studied in a particular pedigree of six generations from the paternal side, with incomplete penetrance and bias towards healthy male carriers and those affected with unilateral retinoblastoma.
RESULTS: The methylation status of the 27 CpGs dinucleotides that constitute the core of the RB1 gene promoter, analyzed by cloning and genomic sequencing after DNA sodium bisulfite conversion, demonstrated a monoallelic methylation pattern which coincides with a c. [-187T > G; -188T > G] sequence variant that is found in peripheral blood lymphocytes and tumor DNA. Unexpectedly, it was the mother who transmitted this variant to two more generations. Microsatellite markers of D chromosome showed a biparental contribution of both D13 chromosomes to the retinoblastoma phenotype, conferring double heterozygosity in the affected cases.
CONCLUSIONS: The monoallelic genetic-epigenetic finding, the sequence variant, and methylation suggest a constitutive epimutation and probably a genetic-epigenetic hereditary predisposition for retinoblastoma in this family.

Yang Y, Mei Q
miRNA signature identification of retinoblastoma and the correlations between differentially expressed miRNAs during retinoblastoma progression.
Mol Vis. 2015; 21:1307-17 [PubMed] Free Access to Full Article Related Publications
PURPOSE: Retinoblastoma (RB) is a common pediatric cancer. The study aimed to uncover the mechanisms of RB progression and identify novel therapeutic biomarkers.
METHODS: The miRNA expression profile GSE7072, which includes three RB samples and three healthy retina samples, was used. After data normalization using the preprocessCore package, differentially expressed miRNAs (DE-miRs) were selected by the limma package. The targets of the DE-miRs were predicted based on two databases, followed by construction of the miRNA-target network. Pathway enrichment analysis was conducted for the targets of the DE-miRNAs using DAVID. The CTD database was used to predict RB-related genes, followed by clustering analysis using the pvclust package. The correlation network of DE-miRs was established. MiRNA expression was validated in another data set, GSE41321.
RESULTS: In total, 24 DE-miRs were identified whose targets were correlated with the cell cycle pathway. Among them, hsa-miR-373, hsa-miR-125b, and hsa-miR-181a were highlighted in the miRNA-target regulatory network; 14 DE-miRs, including hsa-miR-373, hsa-miR-125b, hsa-miR-18a, hsa-miR-25, hsa-miR-20a, and hsa-let-7 (a, b, c), were shown to distinguish RB from healthy tissue. In addition, hsa-miR-25, hsa-miR-18a, and hsa-miR-20a shared the common target BCL2L11; hsa-let-7b and hsa-miR-125b targeted the genes CDC25A, CDK6, and LIN28A. Expression of three miRNAs in GSE41321 was consistent with that in GSE7072.
CONCLUSIONS: Several critical miRNAs were identified in RB progression. Hsa-miR-373 might regulate RB invasion and metastasis, hsa-miR-181a might involve in the CDKN1B-mediated cell cycle pathway, and hsa-miR-125b and hsa-let-7b might serve as tumor suppressors by coregulating CDK6, CDC25A, and LIN28A. The miRNAs hsa-miR-25, hsa-miR-18a, and hsa-miR-20a might exert their function by coregulating BCL2L1.

Borysov SI, Nepon-Sixt BS, Alexandrow MG
The N Terminus of the Retinoblastoma Protein Inhibits DNA Replication via a Bipartite Mechanism Disrupted in Partially Penetrant Retinoblastomas.
Mol Cell Biol. 2015; 36(5):832-45 [PubMed] Free Access to Full Article Related Publications
The N-terminal domain of the retinoblastoma (Rb) tumor suppressor protein (RbN) harbors in-frame exon deletions in partially penetrant hereditary retinoblastomas and is known to impair cell growth and tumorigenesis. However, how such RbN deletions contribute to Rb tumor- and growth-suppressive functions is unknown. Here we establish that RbN directly inhibits DNA replication initiation and elongation using a bipartite mechanism involving N-terminal exons lost in cancer. Specifically, Rb exon 7 is necessary and sufficient to target and inhibit the replicative CMG helicase, resulting in the accumulation of inactive CMGs on chromatin. An independent N-terminal loop domain, which forms a projection, specifically blocks DNA polymerase α (Pol-α) and Ctf4 recruitment without affecting DNA polymerases ε and δ or the CMG helicase. Individual disruption of exon 7 or the projection in RbN or Rb, as occurs in inherited cancers, partially impairs the ability of Rb/RbN to inhibit DNA replication and block G1-to-S cell cycle transit. However, their combined loss abolishes these functions of Rb. Thus, Rb growth-suppressive functions include its ability to block replicative complexes via bipartite, independent, and additive N-terminal domains. The partial loss of replication, CMG, or Pol-α control provides a potential molecular explanation for how N-terminal Rb loss-of-function deletions contribute to the etiology of partially penetrant retinoblastomas.

Aerts I, Lumbroso-Le Rouic L, Gauthier-Villars M, et al.
[Retinoblastoma update].
Arch Pediatr. 2016; 23(1):112-6 [PubMed] Related Publications
Retinoblastoma is the most common intraocular malignancy of infancy with an incidence of 1/15,000 births. Sixty percent of retinoblastomas are unilateral, with a median age at diagnosis of 2 years, and in most cases they are not hereditary. Retinoblastoma is bilateral in 40% of cases, with an earlier median age at diagnosis of 1 year. All bilateral and multifocal unilateral forms are hereditary and are part of a genetic cancer predisposition syndrome. All children with a bilateral or familial form, and 10-15% of children with a unilateral form, constitutionally carry an RB1 gene mutation. The two most frequent symptoms at diagnosis are leukocoria and strabismus. Diagnosis is made by fundoscopy, with ultrasound and magnetic resonance imaging (MRI) contributing both to diagnosis and assessment of the extension of the disease. Treatment of patients with retinoblastoma must take into account the various aspects of the disease (unilateral/bilateral, size, location), the risks for vision, and the possible hereditary nature of the disease. The main prognostic aspects are still early detection and adapted coverage by a multidisciplinary, highly specialized team. Enucleation is still often necessary in unilateral disease; the decision for adjuvant treatment is made according to the histological risk factors. The most important recent therapeutic advances concern conservative treatment, which is proposed for at least one of the two eyes in most bilateral cases: laser alone or in combination with chemotherapy, cryotherapy, or brachytherapy. Recently, the development of new conservative techniques of treatment, such as intra-arterial selective chemotherapy perfusion and intravitreal injections, aims at preserving visual function in these children and decreasing the number of enucleations and the need for external beam radiotherapy. The vital prognosis related to retinoblastoma is now excellent in industrialized countries, but long-term survival is still related to the development of secondary tumors, mainly secondary sarcoma. Retinoblastoma requires multidisciplinary care as well as a long-term specialized follow-up. Early counseling of patients and their family concerning the risk of transmission of the disease and the risk of development of secondary tumors is necessary.

Wang QL, Chen X, Zhang MH, et al.
Identification of hub genes and pathways associated with retinoblastoma based on co-expression network analysis.
Genet Mol Res. 2015; 14(4):16151-61 [PubMed] Related Publications
The objective of this paper was to identify hub genes and pathways associated with retinoblastoma using centrality analysis of the co-expression network and pathway-enrichment analysis. The co-expression network of retinoblastoma was constructed by weighted gene co-expression network analysis (WGCNA) based on differentially expressed (DE) genes, and clusters were obtained through the molecular complex detection (MCODE) algorithm. Degree centrality analysis of the co-expression network was performed to explore hub genes present in retinoblastoma. Pathway-enrichment analysis was performed using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Validation of hub gene expression in retinoblastoma was performed by reverse transcription-polymerase chain reaction (RT-PCR) analysis. The co-expression network based on 221 DE genes between retinoblastoma and normal controls consisted of 210 nodes and 3965 edges, and 5 clusters of the network were evaluated. By assessing the centrality analysis of the co-expression network, 21 hub genes were identified, such as SNORD115-41, RASSF2, and SNORD115-44. According to RT-PCR analysis, 16 of the 21 hub genes were differently expressed, including RASSF2 and CDCA7, and 5 were not differently expressed in retinoblastoma compared to normal controls. Pathway analysis showed that genes in 2 clusters were enriched in 3 pathways: purine metabolism, p53 signaling pathway, and melanogenesis. In this study, we successfully identified 16 hub genes and 3 pathways associated with retinoblastoma, which may be potential biomarkers for early detection and therapy for retinoblastoma.

Gao Y, Lu X
Decreased expression of MEG3 contributes to retinoblastoma progression and affects retinoblastoma cell growth by regulating the activity of Wnt/β-catenin pathway.
Tumour Biol. 2016; 37(2):1461-9 [PubMed] Related Publications
The aberrant expression of MEG3 has been found in some types of cancers; however, little is known concerning the function of MEG3 in retinoblastoma. To elucidate the roles of MEG3 in retinoblastoma, MEG3 expression was quantified in 63 retinoblastoma samples and corresponding nontumor tissues in this work. Moreover, retinoblastoma cell lines were transfected with pcDNA3.1-MEG3 or si-MEG3, after which proliferation, apoptosis, and expression of β-catenin were assayed. TOP-Flash reporter assay was also used to investigate the activity of the Wnt/β-catenin pathway. The results showed that MEG3 was downregulated in retinoblastoma tissues, and the level of MEG3 was negatively associated with IIRC stages and nodal or distant metastasis. More importantly, Kaplan-Meier survival analysis demonstrated that patients with low MEG3 expression had poorer survival and multivariate Cox regression analysis revealed that MEG3 was an independent prognostic factor in retinoblastoma patients. We also observed that MEG3 expression can be modulated by DNA methylation by using 5-aza-CdR treatment. In addition, overexpression of MEG3 suppressed proliferation, promoted apoptosis, and influences the activity of the Wnt/β-catenin pathway in retinoblastoma cell lines. Furthermore, we found that Wnt/β-catenin pathway activator rescued the anticancer effect of MEG3 in retinoblastoma. In conclusion, our study for the first time demonstrated that MEG3 was a tumor suppressor by negatively regulating the activity of the Wnt/β-catenin pathway in the progression of retinoblastoma and might serve as a prognostic biomarker and molecular therapeutic target.

Neriyanuri S, Raman R, Rishi P, et al.
Prenatal genetic diagnosis of retinoblastoma--clinical correlates on follow-up.
Indian J Ophthalmol. 2015; 63(9):741-2 [PubMed] Free Access to Full Article Related Publications
Retinoblastoma is the most common malignant intraocular tumor in pediatric age group if undetected leads to ocular mortality. Prenatal diagnosis is an emerging technology to detect fatal diseases in utero such that subsequent management is planned to reduce the ocular morbidity. We describe a case demonstrating the importance of prenatal diagnosis in a child with a strong family history of retinoblastoma and importance of a long-term clinical follow-up in these cases.

Akbari MT, Naderi A, Saremi L, et al.
Methionine synthase A2756G variation is associated with the risk of retinoblastoma in Iranian children.
Cancer Epidemiol. 2015; 39(6):1023-5 [PubMed] Related Publications
Association of epigenetic modifications with cancer has been widely studied. Gene-specific hypermethylation and global DNA hypomethylation are the most frequently observed patterns in great number of tumors. The methionine synthase (MTR) gene plays key role in maintaining adequate intracellular folate, methionine and normal homocysteine concentrations and, its polymorphism have been associated with the risk of retinoblastoma and other neoplasms. We evaluated the association of MTR A2756G polymorphism with the risk of retinoblastoma in an Iranian population. Totally, 150 retinoblastoma patients and 300 individuals with no family history of cancer as control were included in this study. Genotyping of the A2756G polymorphism was performed by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) using the restriction enzymes HaeIII. Our results showed that the "G" was the minor allele with a frequency of 31.7% and 20.3% in both retinoblastoma and control groups, respectively. The frequency of the 2756GG genotype (P=0.023) and 2756G allele (P=0.0001) were significantly higher in the patients than control group, respectively. Individual with the 2756GG genotype had a 2.99 fold increased risk for retinoblastoma. According to our results, the MTR A2756G polymorphism was associated with the risk of retinoblastoma in Iranian patients.

Mohd Khalid MK, Yakob Y, Md Yasin R, et al.
Spectrum of germ-line RB1 gene mutations in Malaysian patients with retinoblastoma.
Mol Vis. 2015; 21:1185-90 [PubMed] Free Access to Full Article Related Publications
PURPOSE: The availability of molecular genetic testing for retinoblastoma (RB) in Malaysia has enabled patients with a heritable predisposition to the disease to be identified, which thus improves the clinical management of these patients and their families. In this paper, we presented our strategy for performing molecular genetic testing of the RB1 gene and the findings from our first 2 years of starting this service.
METHODS: The peripheral blood of 19 RB probands, including seven bilateral and 12 unilateral cases, was obtained, and genomic DNA was extracted. Analysis of the RB1 exons and the promoter region was conducted first using PCR and direct sequencing. Next, multiplex ligation-dependent probe amplification (MLPA) analysis was performed for patients whom the first results were negative. For patients whom either the first or second method results were positive, parental samples were analyzed to determine the origin of the mutation.
RESULTS: Ten RB1 mutations were identified in ten (52.6%) of the 19 probands (seven bilateral and three unilateral cases), of which 30.0% (3/10) was identified with MLPA. The detection rates in the bilateral and unilateral cases were 100.0% (7/7) and 25.0% (3/12), respectively. Three new RB1 mutations were discovered, two in patients with bilateral RB and one in patient with unilateral RB. Interestingly, all mutations detected with the PCR-sequencing method were predicted to create a premature stop codon. Eight mutations were proven to be de novo while one mutation was inherited from the mother in a family with a positive history of RB.
CONCLUSIONS: Our results confirmed the heterogeneous nature of RB1 mutations and the predominantly de novo origin. The high prevalence of pathogenic truncating mutations was evident among local patients with RB. The combination of PCR sequencing and MLPA is recommended for sensitive identification of heritable RB cases.

Grotta S, D'Elia G, Scavelli R, et al.
Advantages of a next generation sequencing targeted approach for the molecular diagnosis of retinoblastoma.
BMC Cancer. 2015; 15:841 [PubMed] Free Access to Full Article Related Publications
BACKGROUND: Retinoblastoma (RB) is the most common malignant childhood tumor of the eye and results from inactivation of both alleles of the RB1 gene. Nowadays RB genetic diagnosis requires classical chromosome investigations, Multiplex Ligation-dependent Probe Amplification analysis (MLPA) and Sanger sequencing. Nevertheless, these techniques show some limitations. We report our experience on a cohort of RB patients using a combined approach of Next-Generation Sequencing (NGS) and RB1 custom array-Comparative Genomic Hybridization (aCGH).
METHODS: A total of 65 patients with retinoblastoma were studied: 29 cases of bilateral RB and 36 cases of unilateral RB. All patients were previously tested with conventional cytogenetics and MLPA techniques. Fifty-three samples were then analysed using NGS. Eleven cases were analysed by RB1 custom aCGH. One last case was studied only by classic cytogenetics. Finally, it has been tested, in a lab sensitivity assay, the capability of NGS to detect artificial mosaicism series in previously recognized samples prepared at 3 different mosaicism frequencies: 10, 5, 1 %.
RESULTS: Of the 29 cases of bilateral RB, 28 resulted positive (96.5 %) to the genetic investigation: 22 point mutations and 6 genomic rearrangements (four intragenic and two macrodeletion). A novel germline intragenic duplication, from exon18 to exon 23, was identified in a proband with bilateral RB. Of the 36 available cases of unilateral RB, 8 patients resulted positive (22 %) to the genetic investigation: 3 patients showed point mutations while 5 carried large deletion. Finally, we successfully validated, in a lab sensitivity assay, the capability of NGS to accurately measure level of artificial mosaicism down to 1 %.
CONCLUSIONS: NGS and RB1-custom aCGH have demonstrated to be an effective combined approach in order to optimize the overall diagnostic procedures of RB. Custom aCGH is able to accurately detect genomic rearrangements allowing the characterization of their extension. NGS is extremely accurate in detecting single nucleotide variants, relatively simple to perform, cost savings and efficient and has confirmed a high sensitivity and accuracy in identifying low levels of artificial mosaicisms.

Blixt MK, Shirazi Fard S, All-Ericsson C, Hallböök F
Adding another piece to the retinoblastoma puzzle.
Cell Death Dis. 2015; 6:e1957 [PubMed] Related Publications

Further References

MacCarthy A, Birch JM, Draper GJ, et al.
Retinoblastoma in Great Britain 1963-2002.
Br J Ophthalmol. 2009; 93(1):33-7 [PubMed] Related Publications
AIM: This paper describes the epidemiology and family history status of 1601 children with retinoblastoma in Great Britain diagnosed 1963-2002 and summarises the practical consequences for diagnosis and counselling of developments in molecular genetics.
METHODS: Incidence rates were analysed according to year of diagnosis and tumour laterality. Cases were classified as heritable or non-heritable on the basis of laterality and family history of the disease.
RESULTS: There were 998 unilateral cases, 581 bilateral and 22 of unknown laterality. Bilateral cases tended to be diagnosed at a younger age than unilateral. All bilateral cases are regarded as heritable, and 35% had a family history of the disease. 7% of the unilateral cases had a family history and are therefore heritable. Thus, at least (41%) of our cases are heritable. This is an underestimate, since these data on family history are incomplete. For unilateral cases aged below 1 year, the reported incidence rate increased significantly (p<0.0001) by about 2.5% per year; for the age group 1-4 years, the average increase was about 0.5% per year (not significant).

de Oliveira Reis AH, de Carvalho IN, de Sousa Damasceno PB, et al.
Influence of MDM2 and MDM4 on development and survival in hereditary retinoblastoma.
Pediatr Blood Cancer. 2012; 59(1):39-43 [PubMed] Related Publications
BACKGROUND: Retinoblastoma (RB) accounts for 3% of all childhood malignancies, with different incidences around the world. This malignancy results from loss-of-function of both RB1 alleles although other genes, like MDM2 and MDM4, have been proposed to be involved in tumor development.
PROCEDURE: We genotyped rs2279744T>G and rs937283A>G in MDM2, and rs4252668T>C and rs116197192G>A in MDM4, in 104 unrelated RB patients and 104 controls. Sixty-month survival Kaplan-Meier curves and χ(2)-tests were performed for estimating the putative effect of MDM2 and MDM4 alleles on disease progression and survival of RB patients.
RESULTS: MDM2 rs2279744G was significantly more frequent in controls, indicating an apparently protective effect on RB development. However, survival of patients who carried a constitutional RB1 mutation was significantly lower with rs2279744TG or GG than with rs2279744TT. Presence of rs2279744G and a constitutional RB1 mutation was sixfold more frequent in the 0-12 month age group than other age groups at onset of symptoms (P = 0.0401). MDM4 rs4252668C was present at a significantly higher frequency in controls while the frequency of MDM4 rs116197192G was significantly higher in RB patients, suggesting that this allele might increase the risk of developing RB.
CONCLUSION: Our results indicate that MDM2 and MDM4 polymorphisms may influence development and/or survival in RB.

Castéra L, Sabbagh A, Dehainault C, et al.
MDM2 as a modifier gene in retinoblastoma.
J Natl Cancer Inst. 2010; 102(23):1805-8 [PubMed] Related Publications
Variability in the age of onset and number of tumors is occasionally described among retinoblastoma patients, and possible genetic modifiers might lie in the pRB or p53 pathways, both of which are involved in the development of retinoblastoma. MDM2, which increases p53 and pRB catabolism, is therefore a prominent candidate. The minor allele of MDM2 that includes a 309T>G transversion (single-nucleotide polymorphism rs2279744) in the MDM2 promoter is known to enhance MDM2 expression. Its genetic transmission was studied in 326 individuals including 212 RB1 mutation carriers in 70 retinoblastoma families, and the marker genotype was tested for association with age at diagnosis and disease phenotype. In family-based association analyses, the MDM2 309G allele was found to be statistically significantly associated with incidence of bilateral or unilateral retinoblastoma among members of retinoblastoma families (Z = 3.305, two-sided exact P = .001) under a recessive model (ie, affected patients tend to be homozygous for the G allele); in transmission disequilibrium analyses using the recessive model, the association was also observed (estimated odds ratio = 4.0, 95% confidence interval = 1.3 to 12.0). The strong association of this genotype with retinoblastoma development designates MDM2 as the first modifier gene to be identified among retinoblastoma patients and suggests that enhancement of pRB haploinsufficiency and/or resistance to p53-mediated apoptosis is critical to tumor formation.

Di Fiore R, D'Anneo A, Tesoriere G, Vento R
RB1 in cancer: different mechanisms of RB1 inactivation and alterations of pRb pathway in tumorigenesis.
J Cell Physiol. 2013; 228(8):1676-87 [PubMed] Related Publications
Loss of RB1 gene is considered either a causal or an accelerating event in retinoblastoma. A variety of mechanisms inactivates RB1 gene, including intragenic mutations, loss of expression by methylation and chromosomal deletions, with effects which are species-and cell type-specific. RB1 deletion can even lead to aneuploidy thus greatly increasing cancer risk. The RB1gene is part of a larger gene family that includes RBL1 and RBL2, each of the three encoding structurally related proteins indicated as pRb, p107, and p130, respectively. The great interest in these genes and proteins springs from their ability to slow down neoplastic growth. pRb can associate with various proteins by which it can regulate a great number of cellular activities. In particular, its association with the E2F transcription factor family allows the control of the main pRb functions, while the loss of these interactions greatly enhances cancer development. As RB1 gene, also pRb can be functionally inactivated through disparate mechanisms which are often tissue specific and dependent on the scenario of the involved tumor suppressors and oncogenes. The critical role of the context is complicated by the different functions played by the RB proteins and the E2F family members. In this review, we want to emphasize the importance of the mechanisms of RB1/pRb inactivation in inducing cancer cell development. The review is divided in three chapters describing in succession the mechanisms of RB1 inactivation in cancer cells, the alterations of pRb pathway in tumorigenesis and the RB protein and E2F family in cancer.

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