**2. Genetics of retinoblastoma**

## **2.1 Introduction**

Retinoblastoma (RB) is the most common intraocular malignancy in children affecting 1 in 18,000 live births which occurs as a result of biallelic inactivation of RB1 gene [28]. Hereditary RB is due to heterozygous germline mutation in one copy of the RB1 gene, hence is inherited as an autosomal dominant trait. In this form, all body cells have a dysfunctional RB1 allele and, thus, are vulnerable to neoplasia. The non-hereditary form of RB is consequent to somatic mutations, which is known to affect both RB1 alleles in retinal cells [29].

In these patients, RB development requires a second, somatic, mutation in the same cells that renders the other allele nonfunctional. The cumulative incidence rates of non-ocular tumors reach up to 90% at 30 years in patients who were exposed to radiation vs. 68% at 32 years in patients without radiation exposure [30].

The aim of this chapter is to provide a valuable summary of retinoblastoma genetics that is essential for genetic counseling and estimation of short-term (multifocal and bilateral ocular tumors) and long-term (secondary tumors) risks with an overall improvement of healthcare planning and management of our patients.

#### **2.2 The RB1 gene and protein function**

The RB1 gene located on the long arm of chromosome 13 (13q14) is a negative regulator element in the cell cycle process and was the first tumor suppressor gene identified [31]. This gene codes for the RB protein which has multiple cellular functions; it prevents the dividing cells from uncontrollable cycles in the mitosis stage and has a role in genomic stability, apoptosis, and differentiation [32]. Inactivation of the RB protein is usually caused by deletions and nonsense mutations [33].

#### **2.3 Inheritance of retinoblastoma**

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 gene will be unaffected carriers [34].

More than 900 mutations have been identified in the RB1gene. Different types of mutations have been identified in RB1 gene; deletions and gene rearrangements represent majority of mutations (http://www.hgmd.cf.ac.uk/ac/gene. 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 and unifocal retinoblastoma [36].

#### **2.4 Epigenetics of RB**

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 physiological phenotypic traits [37].

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 pathogenesis of cancer [44].

#### *2.4.1 MicroRNAs in RB*

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].

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*History and Genetics of Retinoblastoma DOI: http://dx.doi.org/10.5772/intechopen.89035*

poor prognostic indicator [66].

translocations, and copy number alteration [68].

*2.5.3 Secondary malignant neoplasms*

*2.5.4 Low-penetrant retinoblastoma*

**2.5 Features of heritable RB**

*2.5.1 13q deletion syndrome*

*2.5.2 Trilateral RB*

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

Children with this syndrome may present with characteristic dysmorphic features, developmental delay, and intellectual disability. Interstitial chromosome deletion or translocation of region 13q14 was found in approximately 6% of patients with RB [67]. The larger the size of chromosomal deletion, the more severe the associations. Dysmorphic facial features include high and broad forehead, short nose, prominent philtrum, and a thick everted lower lip [67]. Karyotype or chromosomal microarray is usually performed to detect chromosomal deletions,

Trilateral retinoblastoma indicates the concomitant presence of a heritable retinoblastoma and a midline tumor or a pineoblastoma [12, 69]. Around 5–13% of patients with RB develop trilateral retinoblastoma [70]. Therefore, in children with heritable retinoblastoma, a brain magnetic resonance imaging with gadolinium

As patients with heritable RB age, the risk of non-ocular malignancies significantly increases. These tumors include osteosarcoma, soft tissue sarcoma, epithelial cancers, and melanoma. It has been suggested that a greater risk for second cancers occurs in patients with familial RB compared with those with a de novo RB1 mutation [72]. External beam radiation therapy for patients with heritable disease has a

In a typical "null" germline mutation, there is a 90% chance that patient will develop RB. In a few families, however, the penetrance is far less than 90% with subsequent reduced expressivity and an increased proportion of unilateral RB. Some patients will be carriers with no tumors [73]. These low-penetrant

contrast every 6 months is recommended until the age of 5 years [71].

further increased risk of developing second malignancies.

*2.4.2 DNA methylation*
