*2.4.2 DNA methylation*

*Retinoblastoma - Past, Present and Future*

**2.3 Inheritance of retinoblastoma**

gene will be unaffected carriers [34].

and unifocal retinoblastoma [36].

physiological phenotypic traits [37].

**2.4 Epigenetics of RB**

esis of cancer [44].

*2.4.1 MicroRNAs in RB*

Retinoblastoma is an autosomal dominant inherited disease. Thus, there is a 50% risk of inheriting a germline mutation for each child born to a patient with a germline RB1 mutation. However, 90% of the children with a germline RB1 mutation will develop retinoblastoma with an overall risk of having a child with hereditary retinoblastoma of 45%. The remaining 10% of children with the mutated RB1

More than 900 mutations have been identified in the RB1gene. Different types

php?gene=RB1). Additionally, de novo mutation is considered to be present in most children with heritable retinoblastoma, as a positive family history is elicited in only 10% of all affected children, which is then transmissible in subsequent generations. In these children, in whom a germline mutation is present with a negative family history, 30% have bilateral disease and 60% develop unilateral RB [35]. A germline RB1 mutation is present in 15% of children diagnosed with a unilateral RB [34]. The "2-hit" hypothesis, which was first proposed by Knudson, described two complementary mutations that are essential for the development of hereditary and non-hereditary forms of retinoblastoma. The first "hit," or mutation, in heritable retinoblastoma is a germline mutation affecting all body cells. The second mutation is a somatic mutation occurring in many retinoblasts with subsequent multifocal or bilateral lesion. On the other hand, the first and second mutations in non-heritable retinoblastoma occur somatically in a single retinoblast presenting as a unilateral

Epigenetics is the study of heritable changes occurring in gene activity and expression of a specific phenotype. These changes do not cause alterations in the DNA sequence, external and/or environmental factors might affect cellular and

RB1 gene has been linked to the regulation of numerous epigenetic processes. These processes include DNA methylation, histone modification, and microRNA regulation [38–43]. In addition, dysfunction of RB1 gene causes deregulations in many tumor suppressor pathways. Tumorigenesis requires this epigenetic deregulation against which new therapeutic options can be invented. Retinoblastoma was the first tumor discovered to be showing the actions of epigenetics on the pathogen-

MicroRNAs are small, conserved, single-stranded, and non-coding RNA that comprise 1–5% of the human genome and are involved in regulating at least 30% of protein-coding genes [45–49]. MicroRNAs play an essential role in the regulation of gene expression governing various cellular and metabolic pathways [50–56]. MicroRNAs' deregulation has been linked to the development of RB and other human diseases [57–59]. Thus, microRNA studies on RB have offered novel understandings of the disease mechanisms. Messenger RNA (mRNA), a large family of RNA molecules that convey genetic information from DNA to ribosome, was also studied in cases of RB. A three-fold increase was noted in mRNA levels of ACVR1C/ ALK7 in retinoblastomas invading the optic nerve. This suggests that ACVR1C/ SMAD2 pathway has a function in promoting invasion and growth of RB [60].

of mutations have been identified in RB1 gene; deletions and gene rearrangements represent majority of mutations (http://www.hgmd.cf.ac.uk/ac/gene.

**6**

DNA methylation involves the addition of methyl groups to the DNA molecule. This process can change the DNA segment activity without altering its sequence. When this segment is located in a gene promoter, DNA methylation usually acts to block gene transcription. The role for promoter methylation in retinoblastoma development was discovered when there was methylation of a CpG island (CpG 106) that overlapped the RB1 promoter [61]. This has resulted in a decreased gene expression confirming the epigenetic factor in retinoblastoma tumorigenesis [44, 62–64].

Methylation of DNA segment was also reported in tumor suppressor genes beyond RB1. These genes include RASSF1A (RAS-associated domain family 1A) that was methylated in 59% of tumors analyzed and adenomatous polyposis coli (APC) in 6% [65]. Furthermore, hypermethylation of O6-methylguanine-DNA methyltransferase (MGMT) was detected in 15% of RB tumors. This was associated with advanced-stage RB suggesting that the presence of methylated MGMT is a poor prognostic indicator [66].
