A Molecular Genetic Service for Retinoblastoma

Retinoblastoma Retinoblasts fail to differentiate - continue to divide, forming tumors in the retina. Typically presents in first 2-3 years of life.

RB Terminology – Confusing Actual findings / Inheritance Bilateral (40%) - Always genetic/Inherited Unilateral (60%) - 90% non-genetic (sporadic) 10% genetic (Inherited) Trilateral Multifocal,

Tumour Staging and Prognosis Intraocular stage (leukocoria) squint acute glaucoma Extraocular stage (neglected with necrosis) Whilst confined to the retina – cure rates up to 95%. Extraocular spread carries very poor prognosis (5-10% cure rate).

Treatment Depends on: –size and site of tumour –whether unilateral or bilateral Usually intra-arterial chemo Cryo, laser for recurrence Radiotherapy Surgery

Second tumours Virtually none for non-genetic New tumours, not metastases Most commonly osteosarcoma other sarcoma melanoma Avoid X-rays Regular skin checks if visually impaired

Knudson’s two hit hypothesis Two distinct mutagenic events necessary for the development of RB Mutations in the RB1 gene. 2-stage process 1-stage process

EUA Avoidance: Examination under anesthetic (EUA) and clinical visits until the age of 7. MOLECULAR GENETIC RESULTS Guide Treatment: Sporadic unilateral patients - risk of a tumour developing in the second eye - less likely to undergo external beam radiotherapy. Prenatal/preimplantation diagnosis

Mutation Detection Either - 1 change in blood (1 st hit) OR - 2 changes in tumour (1 st and 2 nd hits) If mutation found in blood – GENETIC RB - Accurate test for relatives

MLPA - Blood Normal Control Exon 13 Deletion Whole Gene Deletion

Bidirectional Sequence Analysis - Blood Point mutations Frameshift mutation

Exon2 Exon3 91bp insertion Exon3Exon2 GAGGTExon2Exon3 91bp insertion Case 1 - RNA Results Transcript 1 Transcript 2 Genomic Level – Normal SequenceGenomic Level – Mutant Sequence XA G c G>A CE NC H2O The novel exon contained a premature termination codon

1. MLPA analysis - Tumour Normal Control - Heterozygous whole gene deletion - Heterozygous deletion of promoter to exon 17

3. Bidirectional Sequence Analysis - Tumour Blood Tumour 12 Loss of Heterozygosity of Allele 2 4. Loss of Heterozygosity Analysis - Tumour

4. RB1 Promoter Hypermethylation Assay U M U M Control Band Methylated/Unmethylated band 1: Heterozygous hypermethylation of the RB1 promoter region 2: Normal unmethylated RB1 promoter region

Role of geneticist in RB Convey complex genetic information. Discuss implications of testing. Genetic test results….. Risk to family members Guide treatment EUA avoidance Secondary tumours PND/PGD