A Molecular Genetic Service for Retinoblastoma

simon.ramsden@cmft.nhs.uk

Retinoblastoma

• Retinoblasts fail to differentiate - continue to divide, forming tumors in the retina.

• Typically presents in first 2-3 years of life.

RB Terminology – ConfusingActual 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 glaucomaExtraocular 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

Usuallyintra-arterial chemoCryo, laser for recurrence

RadiotherapySurgery

Second tumours

Virtually none for non-geneticNew tumours, not metastases Most commonly

osteosarcoma

other sarcoma

melanoma

Avoid X-rays

Regular skin checks if visually impaired

Knudson’s two hit hypothesisTwo distinct mutagenic events necessary for the development of RBMutations 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 (1st hit)

OR - 2 changes in tumour (1st and 2nd 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

G AG GTExon2 Exon391bp insertion

Case 1 - RNA Results

Transcript 1

Transcript 2

Genomic Level – Normal SequenceGenomic Level – Mutant Sequence

XAG

c.264+2518G>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 - Tumour4. 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 region2: 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

simon.ramsden@cmft.nhs.uk