*2.6.3 Chromosomal microarray (CMA)*

Chromosomal microarray analysis (CMA) is a technology used for the detection of clinically significant microdeletions or duplications and genetic rearrangement (including RB1), with a high sensitivity for submicroscopic aberrations [78]. It can be utilized in retinoblastoma patients with dysmorphic features, developmental delay, and/or other congenital anomalies [79, 80].

## **2.7 Genetic counseling**

*Retinoblastoma - Past, Present and Future*

of the protein [33].

**2.6 Genetic testing**

*2.6.1 Clinical context*

*2.6.2 Single-gene testing*

found in this group of patients [76].

same conclusion also applies to the offspring.

RB1 and MYCN genes [77].

counseling.

mutations are usually missense mutations. Moreover, a low level of RB protein production due to mutations in the promoter region can occur without total absence

Heritability of retinoblastoma is confirmed if a proband with retinoblastoma has a family history of retinoblastoma. In the absence of family history of RB, genetic testing is required to identify heterozygous germline RB1 pathogenic variants. This will allow early diagnosis and identification of potential carriers of the heritable RB1 mutations which eventually will improve disease management and family

The following staging system has been proposed to facilitate description of one's

For bilateral, unilateral familial, and unilateral multifocal retinoblastoma, peripheral blood DNA should be tested initially using sequence analysis and genetargeted deletion/duplication analysis of RB1. If blood testing did not reveal any mutation, molecular analysis of tumor DNA (if either eye was enucleated) should subsequently be done. Mosaicism can be assumed if germline mutation was not

H1 Bilateral retinoblastoma, trilateral retinoblastoma (retinoblastoma with intracranial CNS midline embryonic tumor), family history of retinoblastoma, or RB1 pathogenic variant identified in blood

For unilateral unifocal non-familial retinoblastoma, the chance of carrying RB1 germline mutation is 15%. And, with the high sensitivity of RB1 mutation detection techniques which reach up to (95%), testing blood from unilateral patients, extracted at the time of the first examination under anesthesia may confirm the diagnosis of germline RB1 mutation. If blood DNA testing did not reveal any mutation, molecular analysis of tumor DNA (if the eye was enucleated) should subsequently be done. If pathogenic variants were detected in the tissue, the blood DNA should be re-tested looking for these specific variants or any large rearrangements within the RB1 gene. If tumor tissue was unavailable, a negative blood result will reduce the chance of possessing a mutation to less than 1%. With such very low risk of RB development, the proband needs no additional exam under anesthesia and frequent examination in the clinic is sufficient. The

It has been previously reported by Rushlow et al. that retinoblastoma is not exclusively caused by mutations in RB1 gene; mutations in MYCN oncogene has been predicted to be responsible for 18% of cases diagnosed with non-familial unilateral RB in children less than 6 months of age. They reported group of patients who harbored mutations in MYCN amplification and no RB1 mutations. An additional 1.5% of patients with unilateral non-familial RB were found to have normal

genetic risk of possessing germline RB1 pathogenic variant: [74, 75]

H0 Normal RB1 alleles in blood tested with demonstrated high-sensitivity assays

H0\* Normal RB1 in blood with <1% residual risk for mosaicism

HX Unknown or insufficient evidence of a constitutional (germline) RB1 pathogenic variant

**8**

Genetic testing and counseling are essential parts of RB disease management; they help delineate heritable RB for non-heritable ones. The increased knowledge of molecular basis of RB allowed for better understanding and management of the disease. Genetic evaluation and counseling should be done in collaboration with a geneticist and genetic counselor [81]. The aim of the geneticist is to implement the proper test to detect the underlying disease causing mutation and communicate with the genetic counselor who will educate patients and their families about their condition in general, discuss anticipated risk of developing subsequent tumors, and construct the appropriate screening plan for the affected individual and their relatives.

At first encounter of a patient and family of retinoblastoma, detailed family history should be obtained and family pedigree should be drawn with special attention to relatives at risk. Then, genetic testing is offered after discussion about purpose, possible outcome, and limitations. Specimens are then collected and sent to a specialized laboratory for analysis. Another counseling session is arranged to convey and interpret the reported results. Accordingly, additional testing and counseling should be offered to the relatives at risk.
