Introduction

Cancer usually owes its existence to cancer-critical genes (Vogelstein and Kinzler, 1998), which are broadly classified into two main types i.e. “proto-oncogenes”, in which a gain-of-function mutation converts them to oncogenes, driving cells to become cancerous and; “tumor suppressor genes”, for whom a loss-of-function mutation creates cancer. While overexpression of oncogenes makes it easier to identify them, down-regulation or inactivation of tumor suppressor genes makes the job of hunting them down, tough. These tumor suppressor genes or antioncogenes are actually the most frequently mutated genes in cancers, except in case of leukemias and lymphomas (Knudson, 1993). Yet, only few tumor suppressor genes have been discovered and studied (Alberts, Johnson et al., 2002).

 

First tumor suppressor gene to be discovered was RB1, whose mutations were established as main cause of retinoblastoma (RB) (Corson and Gallie, 2007; MacPherson and Dyer, 2007). The challenge posed by antioncogenes with regards to their characterization is that, it’s their decreased expression rather than hyperactivity that leads to cancer. Thus, while identifying a gene that is being overexpressed is relatively easier; localization of a gene with diminished expression is tough and requires different strategies. RB1 gene could be discovered, as it was the sole reason for retinoblastoma (OMIM +180200). Since this discovery, retinoblastoma system has served as an important model for study of antioncogene-associated cancers. Over the years, a lot of progress has been made into the genetics of retinoblastoma so much so that the researchers have now established a full-fledged database of mutations present in the RB1 gene, which are capable of driving retinoblastoma (Corson and Gallie, 2007; MacPherson and Dyer, 2007). In the present review, an attempt has been made to revisit retinoblastoma, as a model of antioncogene-associated cancers. Following sections discuss RB1 gene, RB protein and retinoblastoma as well as spectrum of mutations related to them in light of latest findings and discoveries.

 

The RB1 Gene and RB Protein (pRB)

 

RB1 gene has significant sequence conservation across wide range of human ethnic groups and 5 primate species, evident by the fact that the nucleotide diversity of RB1 gene’s coding region is 52 times less than that of its non-coding regions (Sivakumaran, Shen et al., 2005). Phylogeny-based maximum likelihood analysis revealed that RB1 has extensive linkage disequillibrium over 174kb and possess only 4 unique recombination events, 2 in Africa and 1 each in Southwest Asia and Europe. RB1 gene is located at chromosomal locus 13q14.2 and spans about 180 kb in size, comprising of 27 exons. At its 5’ end RB1 gene has a normally unmethylated CpG island (Lohmann and Gallie, 2004). The promoter region of RB1 gene is unique in the sense that it contains binding motifs for transcription factors Sp1 and ATF but no TATA or CAAT box. The mRNA of this gene is 4.7 kb in size. RB1 gene was first isolated by the technique of “Chromosome Walking” (Lee, Bookstein et al., 1987).    

 

Open reading frame of RB1 gene encodes a 928 amino acid nucleophosphoprotein of weight 110 kD, called the ‘RB protein’ (GenBank NP_000312) (Lee, Shew et al., 1987). RB protein has several functional domains (Harbour, 2001). These functional domains have been meticulously divided into 4 types, Amino-terminal region, “A” domain, “B” domain and carboxy-terminal region. Domains A and B together form the largest domain in RB protein called Pocket Domain (amino acid 379-792). These domains interact to attain a particular active form capable of binding with several viral oncoproteins like SV-40 large T-antigen and cellular regulatory proteins that contain amino acid motif LXCXE and play vital role in cell cycle and cell proliferation. This pocket domain also interacts with E2F transcription factors. However since E2F lacks LXCXE motif, it requires help of both A/B pocket and C-terminal to bind with RB proteins (Knudsen and Wang, 1996). The carboxy-terminal domain (Amino acid 793-928) contains nuclear localization signal, alternative binding site for E2F, MDM2 (OMIM +164785) and c-Abl (OMIM +189980). The region of C-terminal, which binds to c-Abl tyrosine kinase, is also known as “C-pocket” (Knudsen and Wang, 1996). This region also serves as phosphorylation site for cyclin-dependant kinase (CDK) and docking site for Cdk 4 (OMIM +123829) / Cdk 6 (OMIM +603368). Similarly, the amino-terminal domain (amino acid 1-378) has six-consensus sequences serving as target sites for Cdk phosphorylation. The sites in RB protein, which serve the purpose of being phosphorylation targets, are often rich in 16 Serine/Threonine-Proline motifs (Knudsen and Wang, 1996). 

 

RB protein plays a very important role in Mammalian Cell Cycle regulation. In fact it acts like a brake in G₁ phase of cell cycle (Alberts, Johnson et al., 2002). During G₁, RB protein binds to E2F factor and blocks the transcription of S-phase genes, thereby acting as mediator of G₁-to-S phase checkpoint. Initiation of S-phase requires cyclin E proteins whose expression depends upon E2F transcription factors (Murray, 2004). When cells are supposed to divide, active Cdk 4 and Cdk 6 proteins along with cyclin D, phosphorylate and inactivate RB proteins, which allow its disassociation from E2F protein to take place (Sherr and Roberts, 1999). This free E2F now helps in transcription of S-phase genes which code for different proteins such as cyclin E, Origin Recognition Complex (ORCs), MCMs, DNA Polymerase a etc. (Sears and Nevins, 2002; Stevaux and Dyson, 2002), which in turn help in S phase initiation.   

 

In mammalian cells, E2F factors may be bound to RB proteins or its family members i.e. p107 (RBL1 gene) or p130 (RBL2 or RB2 gene) (Lipinski and Jacks, 1999). RBL1 and RBL2 genes are located at chromosomal loci 20q11.2 and 16q12.2 respectively (Claudio, Tonini et al., 2002). All these 3 proteins are also sometimes known as pocket proteins. Here, RBL1 and RBL2 genes are evolutionarily more closely related to each other than to RB1 gene (Ichimura, Hanafusa et al., 2000). It has been proposed that the proteins of RB family arose through tandem duplication of region comprising of domain A (Kim and Cho, 1997).     

 

Loss of normal RB protein due to mutations in RB1 gene causes abolition of G₁-to-S phase checkpoint thereby increasing the possibility of unchecked cellular proliferation or cancer (Lomazzi, Moroni et al., 2002). Though signal transduction systems mediated by p53 and pRB are involved in regulation of the G₁ checkpoint yet these two are arguably the most commonly down regulated pathways in human cancers (Kastan and Bartek, 2004). It has also been found that p53 suppresses transcription of RB1 gene by acting upon a cis-acting element (GGAAGTGA) present in RB1 promoter (Shiio, Yamamoto et al., 1992). Apart from cell cycle regulation, RB protein has been found to regulate additional processes such as the control of telomeric length (Garcia-Cao, Gonzalo et al., 2002), cell senescence (Narita, Nunez et al., 2003), apoptosis (Berry, Lu et al., 1996), angiogenesis (Claudio, Stiegler et al., 2001), differentiation mechanisms like adipogenesis, myogenesis or hematopoiesis (Claudio, Tonini et al., 2002) and embryonic pattern formation (Dahiya, Wong et al., 2001).    

 

The Origin of Retinoblastoma

 

Retinoblastoma originates in retinal cells through series of events, which have been characterized almost completely. Probing into origin of retinoblastoma has been rather challenging since in contrast to other cancer cells, retinoblastoma cells tend to show finite lifespan in vitro and are thus tough to culture artificially for study purposes (Seigel, 1999). Environmental factors haven’t been found to play critical role in origin of retinoblastoma (Buckley, 1992). Retinoblastoma generally exists in two forms, one hereditary and the other not. In the hereditary appearance, several tumors typically arise independently, affecting both eyes (bilateral manifestation); in the non-hereditary form only one eye is affected, and that too by only one tumor (unilateral manifestation). Most new retinoblastoma patients have no family history of this disease (Gallie and Phillips, 1984). As we discussed earlier, mutation or deletion affecting the RB1 gene or RB1 locus causes retinoblastoma. Knudson (1971) was the first person to put forth an oncogenesis model for retinoblastoma. Knudson (1971) noted that in hereditary predisposed individuals, retinoblastoma happened at an earlier age while in Non-hereditary type, the tumors were diagnosed at a slightly older age. Thus he concluded that, in hereditary retinoblastoma the predisposed individuals carried one mutation and for that reason in them, oncogenesis required only one more mutation while in absence of heritable tendency, 2 or more mutational events were required. He also went on to suggest that, retinoblastoma happened due to two mutational events viz. M₁ and M₂, which happened one after another.

 

Based upon the model put forth by Knudson (1971), we can draw resolved conclusions regarding the origin of retinoblastoma (Alberts, Johnson et al., 2002). In individuals who are suffering from hereditary form of retinoblastoma, a deletion or mutation occurs in one copy of the RB1 gene (M₁ event) in each cell of the body. Hence, these cells especially retinal cells are predisposed to becoming cancerous, but are not actually cancerous since they still retain one good and intact copy of the RB1 gene. In this case, the retinal cells that do become cancerous are actually defective in both copies of RB1 because a somatic mutation (M₂ event) has occurred, in addition to the original inherited mutation, and has eliminated the remaining good copy of the gene. On the other hand, in patients with the nonhereditary retinoblastoma, the noncancerous retinal cells exhibit no blemish in either copy of RB1, while the cancerous cells are again defective in both copies. Nonhereditary retinoblastomas require the coincidence of two somatic mutations (M₁ and M₂) in a single retinal cell, so as to destroy both copies of the RB1 gene.

 

Over the years, the model proposed by Knudson (1971) for retinoblastoma has been further refined and modified for accuracy (Knudson, 1971; Phillips and Gallie, 1984). According to this classical model (Figure 1), the first mutational event (M₁) in RB1 gene at chromosome 13q14 causes formation of a cell, which is heterozygous for this mutation. Such heterozygosity doesn’t have much effect on RB1 protein mediated pathways in cells since the cells still have one normal RB1 gene allele. This condition may exist in both heritable as well as non-heritable retinoblastoma with only difference being that in heritable case, the individuals would be heterozygous for this mutation in their somatic as well as germline cells while non-heritable cases would have this heterozygosity only in somatic retinal cells. Later however, the normal allele may be lost (M₂ mutational event) due to random chromosomal loss, duplication, mitotic recombination or gene based point mutations and deletions.

   

Figure 1. The Classical model of Origin of Retinoblastoma (Modified from Phillips & Gallie 1984).

  

This second mutational event in retinal cell is responsible for progression of RB tumor. These events are frequently followed by further genetic changes causing chromosomal instability (M_n) e.g. acquisition of isochromosome 6p or extra 1q. Such M_n events actually confer proliferative advantage to RB tumors. All these events are highly specific and imperative for transformation of normal immature retinal cells into cancerous retinoblastoma cells. Thus, retinoblastoma owes its origin solely to mutations in RB1 genes. One very important thing to bear in mind here is that, M₂ events have no effect on fully differentiated retinal cells thus; RB tumors arise only in immature retinal cells, not adult retinal cells (Gallie and Phillips, 1984).

 

From the above discussion, one question that arises is that in hereditary retinoblastoma predisposed individuals, all the cells in the body initially have one defective RB1 gene however, mutation in second RB1 gene can take place in retinal cells as well as other somatic cells yet we observe that RB1 mutations more frequently cause retinoblastoma and only on few occasions cause other cancers like carcinomas of lung or breast. An answer to this according to recent trends in research related to RB gene family is that, other body tissues are less susceptible to RB1 mutation-driven cancer than retinal cells because RBL1 and RBL2, the other members of RB gene family may compensate for RB1 loss in these tissues (Lipinski and Jacks, 1999).   

 

Thus for cancer to originate in non-retinal somatic cells, more complex series of genetic changes other than changes in RB1 gene are required. Other types of cancer with whom RB1 gene mutations are associated are discussed in the following sections.

 

Retinoblastoma: An Overview

 

Retinoblastoma (RB) is an embryonic malignant neoplasm that originates in retinal cells due to mutations in RB1 gene. Unique anatomy of eye permits early diagnosis of RB1 tumors (Gallie and Phillips, 1984). The frequency of this cancer in U.S. has been found to be about 1 in 23,000 live births (Macklin, 1960). With regards to relationship of RB with ethnicity, research by Jensen and Miller (1971) has elucidates that probably retinoblastoma has 2.5 times greater mortality risk in blacks than in whites (Jensen and Miller, 1971). If we analyze the population genetics data for retinoblastoma, we find that there exists an incidence rate of 3.58 retinoblastoma cases from each million children below the age of 15 years (Pendergrass and Davis, 1980).

 

Retinoblastoma is mostly non-hereditary and shows its typical symptoms in early childhood (OMIM +180200). Retinoblastoma becomes grossly visible as a white papillary reflex in the eye boundary. Retinoblastoma has been reported to exhibit itself in 3 manners i.e. (1) Frank retinoblastoma, (2) Unilateral, Bilateral or Trilateral retinoblastoma and (3) Retinoma (Connolly, Payne et al., 1983). While not much is known about frank retinoblastoma, rests of retinoblastoma types are considerably characterized.

 

Out of the total cases of retinoblastoma, 55-65% are unilateral non-hereditary while 25-30% and 10-15% of hereditary retinoblastoma cases are bilateral and unilateral respectively (Devesa, 1975; Pendergrass and Davis, 1980). Unilateral retinoblastoma is characterized by presence of tumors in only one eye while tumors in both eyes represent bilateral retinoblastoma. Trilateral retinoblastoma is characterized by presence of neoplasm in pineal region of bilateral retinoblastoma patients (Brownstein, de Chadarevian et al., 1984). This type of neoplasm is also referred to as “Pinealoma”. This condition is very rare and is exclusively associated with bilateral retinoblastoma. Kivela (1999) had reported that trilateral retinoblastoma usually affects the second or third generation of a family having retinoblastoma (Kivela, 1999). Similarly it has also been found that, bilateral retinoblastoma increases the risk of osteogenic sarcoma by 500-fold in its patients (Abramson, Ellsworth et al., 1976). Retinoma on the other hand is a term, which describes benign lesions in retina (Gallie and Phillips, 1982). Translucent and grayish retinal mass protrusion, into vitreous fluid is a typical feature of retinoma. According to Gallie and Phillips (1982), retinoma represents either spontaneous regression of a retinoblastoma or a benign manifestation of RB1 gene, both in homozygous state.

 

As discussed in the preceding section, research has revealed that apart from RB1 gene-based mutations, chromosomal aberrations may also cause retinoblastoma. In such a scenario, patients with retinoblastoma also show various other symptoms e.g. Motegi et al. (1983) reported that, patients in whom retinoblastoma occurs due to interstitial deletion of 13q chromosomal region show appearance of midface, prominent eyebrows, broad nasal bridge, bulbous nose tip, large mouth and long philtrum (Motegi, Kaga et al., 1983). A number of other chromosomal aberrations have been reported with regards to retinoblastoma like, chromosomal translocation between X and 13^th chromosome with break point at 13q12.1 (Laquis, Rodriguez-Galindo et al., 2002) and chromosomal deletion from 13q14 up to 13q32 (Schocket, Beaverson et al., 2003).

 

Inheritance and Penetrance of Retinoblastoma

 

Retinoblastoma has a novel pattern of inheritance whose study paved way for discovery and study of other type of cancers such as Wilms tumor, neuroblastoma and medulloblastoma. Hereditary retinoblastoma mostly manifests bilateral tumors (Vogel, 1979). In pedigree analysis, retinoblastoma behaves in a dominant manner however this is interesting since RB1 gene-related tumor arise only in its homozygous form proving its recessive nature. Thus we may conclude that “susceptibility” to retinoblastoma is dominant but “victimization” to it is recessive. Statistics imply that retinoblastoma is inherited in 5-10% of cases, arises due to germinal mutations in 20-30% of cases and shows sporadic nature or occurrence due to somatic mutations in 60-70% of cases (Fitzgerald, Stewart et al., 1983). Research on inherited bilateral retinoblastoma has shown that, during the process of loss of heterozygosity (LOH) for RB1 mutation, “paternal chromosome” is preferentially retained (Dryja, Mukai et al., 1989; Zhu, Dunn et al., 1989). This phenomenon indicates that either RB1 mutations more commonly occur during spermatogenesis or the paternal chromosome is more susceptible to mutations or DNA repair deficiencies during embryogenesis. The susceptibility of RB1 gene to mutations during spermatogenesis has been proved by RFLP (Restriction Fragment Length Polymorphism) analysis of 13q14 locus (Dryja et al. 1989). This preferential transmission of mutant RB1 allele from fathers to their children has also been confirmed by segregation distortion analysis as well as epidemiologic studies (Munier, Spence et al., 1992). Recent analyses into this one-parent biased inheritance pattern have refuted the argument of spermatogenesis being responsible for preferential paternal chromosome retention. Girardet et al. (2000) have cited that this favoring of mutant RB1 allele inheritance may be occurring due to reasons other than spermatogenesis, like fertilization advantage, better motility of sperm bearing the mutant RB1 gene or presence of defective gene imprinting on human X chromosome (Girardet, McPeek et al., 2000). Defective imprinting of gene on X-chromosome has also been supported by other experiments (Naumova and Sapienza, 1994). Based upon these experiments a “Genome Imprinting” model has been proposed for inherited retinoblastoma. Incidence of “Mosaicism” has also been cited in support of inherited retinoblastoma (Sippel, Fraioli et al., 1998).

 

Other than above arguments, the phenomenon of irregularities in inheritance or incomplete penetrance of mutant RB1 gene has also been reported (Macklin, 1959). Incomplete penetrance in this case refers to the process during which individuals transmit mutant RB1 gene to their progeny without being affected themselves (Bia and Cowell, 1995). In nearly 20% of familial retinoblastoma cases, individuals either remain unaffected carriers (reduced penetrance) or victimize to unilateral RB/ benign retinoma (partial penetrance). This is often called Low Penetrance (LP) phenotype (Valverde, Alonso et al., 2005). Mutant low-penetrant RB alleles may be divided into two types i.e. those affecting the RB1 promoter region and those forming a truncated or mutant RB protein (Otterson, Chen et al., 1997). In this regards a study of RB pocket-binding proteins has suggested that, families with incomplete penetrance of familial retinoblastoma carry unstable, mutant RB alleles, which have temperature-sensitive pocket protein-binding activity (Otterson, Modi et al., 1999). Thus it has been concluded that low-penetrance retinoblastoma mutations are of two types; (1) Class 1 mutations, which cause reduction in amount of normal RB protein and (2) Class 2 mutations, which cause formation of impaired RB proteins (Harbour, 2001). Low-penetrance phenotype in retinoblastoma has also been associated with splicing mutations (Alonso et al. 2001) like skipping of exon 6 (Klutz, Brockmann et al., 2002). Richter et al. (2003) in a larger perspective, have suggested that probably unilateral-sporadic RB victims are founders of LP families (Richter, Vandezande et al., 2003).

 

Supplementary Complications of mutated RB1 gene

 

As discussed before, occasional mutations in RB1 gene may associate with a range of different physiological complications other than retinoblastoma. Survivors of retinoblastoma having germline RB1 mutations have been found to show susceptibility towards other tumors notably, osteosarcoma (37%) and soft-tissue sarcomas (16.8%) (Goodrich and Lee, 1993; Moll, Imhof et al., 2001) followed by melanomas (7.4%), brain tumors (4.5%), leukemia (2.4%) and non-Hodkin lymphoma (1.6%). RB1 gene has been found to serve as target for somatic mutations in mesenchymal tumors even among patients lacking any predisposition to retinoblastoma (Friend, Horowitz et al., 1987). It has been found that, nearly 2/3^rd of secondary tumors in retinoblastoma victims are mesenchymal in origin. Bulk of these mesencymal tumors consist of osteosarcoma followed by soft tissue sarcomas such as fibrosarcoma, leiomyosarcoma, liposarcoma etc. Relative risk imposed by germline mutations of RB1 gene in its victim is nearly 10⁵ for retinoblastoma, 10³ for osteosarcoma and 10 or less for small-cell carcinoma of lung (Knudson, 1992). Henson et al. (1994) has also found that retinoblastoma locus is preferably targeted for deletion mutations (Henson, Schnitker et al., 1994). Thus in case of cancers such as astrocytomas, mutation of p53 gene may start low-grade tumor formation however later mutations in retinoblastoma gene are required for its conversion into high-grade form. Retinoblastoma has also been found to associate with other autosomal disorders such as Fanconi anemia and Bloom syndrome (Gibbons, Scott et al., 1995). Sebaceous carcinoma of the eyelid, another eye-related cancer has been found to occur in victims of hereditary retinoblastoma irrespective of primary radiotherapy treatment (Kivela, Asko-Seljavaara et al., 2001).

 

On the cellular front, retinoblastoma has been found to resist death-receptor mediated apoptosis, possibly due to overmethylation-mediated epigenetic gene silencing of caspase-8 expression (Poulaki, Mitsiades et al., 2005). 

 

Mutational Spectrum of RB1 gene

 

Mutational spectrum of RB1 gene is appreciably diverse and fails to exhibit any distinct hotspots (Valverde et al. 2005). Bulk of RB1 mutations are confined between exons 1 to 25 (Lohmann and Gallie, 2004). Interestingly, the two terminal RB1 exons, 26 and 27 exhibit no mutations even after possessing two CGA codons, which serve the purpose of being hotspots for nonsense mutations (Cooper and Krawczak, 1990). Non-coding regions exhibit major sequence variations in RB1 gene such that coding sequences show 50-fold less nucleotide diversity than non-coding sequences (Sivakumaran et al. 2005). The mutational diversity of RB1 gene however is biased such that most reports correspond to nonsense mutations (42.4%), small insertions/deletions (27.3%) and splicing mutations (20.8%). Lesser share goes to missense (8.7%) and regulatory region/element mutations (0.7%). However, if we take away recurrent RB1 mutations from above analysis than, about 48.2% of distinct/new mutations are of small insertions/deletions type followed by splicing mutations (22.2%), nonsense (18.6%), missense (10%) and regulatory element mutations (1%) (Valverde et al. 2005).  Majority of germline mutations have been identified to be nonsense or frameshift mutations (Lohmann and Gallie 2004).

 

Carriers of RB1 nonsense or frameshift mutations mostly victimize to bilateral retinoblastoma, rarely such mutations cause unilateral retinoblastoma (Lohmann, Gerick et al., 1997). Nonsense mutations cause formation of premature stop codon within the RB1 transcript thereby leading to formation of truncated pRB, which is incapable of regulating cell cycle and other such functions. In homozygous condition (RB1 ^{- / -}), this situation triggers retinoblastoma. In heterozygous condition (RB1 ^{+ / -}) though, site of premature stop codon within RB1 gene has little or no effect on phenotypic expression perhaps because posttranscriptional modifications cause degradation of mutant transcripts with premature stop codons coded by the mutant RB1 allele (a process termed as “Nonsense-mediated decay”) and the normal functions are carried out by translational product of other normal RB1 allele transcripts (Frischmeyer and Dietz, 1999). A remarkable thing to note here is that, premature stop codon’s presence in exons 26 or 27 (carboxy-terminal region) fails to trigger nonsense-mediated decay (Hentze and Kulozik, 1999) and even the truncated pRB exhibiting aberrations in C-terminal domain retain sufficient tumor suppressive activity to prevent retinoblastoma development. Conversely though, splice site mutations causing exon skipping mostly manifest unilateral retinoblastoma that is; they exhibit incomplete penetrance probably because a fraction of mutant transcripts with splice site mutations may be spliced into a normal mRNA, a phenomenon which has been observed in case of other genes (Boerkoel, Exelbert et al., 1995) and suspected, but not yet confirmed in case of RB1 gene.

 

As mentioned earlier RB1 gene fails to show any creditable hotspots however based upon repetition reports of various RB1 mutations, certain discrete hotspots encompassing highly recurrent mutations have been characterized (Valverde et al. 2005). These hotspots hold 12 nonsense, 3 splice-site and 2 missense mutations. Bulk of these recurrent mutations (79%) correspond to C>T transitions in CGA (arginine) codons. This hypermutability of CGA has been attributed to epigenetic - DNA hypermethylation changes (Mancini, Singh et al., 1997).

 

For certain other hotspots (nonsense, E137X and 3 splice site mutations), presence of short quasi-repeat sequence motifs has been cited as causative reason (Maki, 2002). These quasi repeats have been accused of causing replication errors like misinsertions, misalignment, base substitutions and frameshifts. In terms of RB1 exons, it has been found that exons 9, 10, 14, 17, 18, 20 and 23 are having a very high mutability (Richter et al. 2003; Valverde et al. 2005). Apart from these, exons 19 to 21 (Domain B) experience most missense substitutions (60%) while intronic sequences adjacent to exons 6, 12, 16, 17, 19 and 24 show high vulnerability to splice-site mutations. The above stated RB1 mutational hotspots exhibit maximal propensity towards nonsense and frameshift mutations. Most RB1 nonsense mutations (80%) correspond to C>T transitions or G:T mispairings (Kolodner and Marsischky, 1999). Imbalance between DNA methylation and DNA mismatch repair activity has been responsible for this extraneous load of nonsense mutations within RB1 gene (Lengauer, Kinzler et al., 1998).

 

Apart from the relatively apparent RB1 mutations there are certain types of alterations in this gene, which are hard to detect. Most noteworthy of these encompass mosaicism (Frequency of 10%) (Sippel, Fraioli et al., 1998), mutations in promoter region (Sakai, Ohtani et al., 1991) and a very interesting phenomenon called, “Epigenetic variation”, occurring due to hypermethylation of CpG islands in RB1 promoter (Greger, Passarge et al., 1989; Sakai, Toguchida et al., 1991). Epigenetic changes like DNA hypermethylation have been mostly recorded in cases of sporadic-unilateral retinoblastoma. 

 

Concluding Remarks

 

Retinoblastoma was established as a model of antioncogene-associated cancers, more than a decade ago. Research done on Retinoblastoma, helped in discovery of several mechanisms orchestrating the proliferation of cancers driven by mutations in tumour suppressor genes. Though, a lot of research has already been done; there still remains room for ample more especially regarding the culturing of human retinoblastoma cells in vitro and regarding the exploration of mutational hot-spots within the RB1 gene. If these questions are answered than much can be achieved in terms of retinoblastoma diagnostics as well as therapeutics. It is also expected that further research on retinoblastoma would reveal additional mechanisms involved in origination of cancers due to such genes as RB1.

 

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