A Molecular Genetic Service for Retinoblastoma [email protected] [email protected].
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Transcript of A Molecular Genetic Service for Retinoblastoma [email protected] [email protected].
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Retinoblastoma
• Retinoblasts fail to differentiate - continue to divide, forming tumors in the retina.
• Typically presents in first 2-3 years of life.
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RB Terminology – ConfusingActual findings / Inheritance
Bilateral (40%) - Always genetic/Inherited
Unilateral (60%) -90% non-genetic (sporadic)
10% genetic (Inherited)
Trilateral Multifocal,
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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).
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Treatment
Depends on: – size and site of tumour– whether unilateral or bilateral
Usuallyintra-arterial chemoCryo, laser for recurrence
RadiotherapySurgery
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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
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Knudson’s two hit hypothesisTwo distinct mutagenic events necessary for the development of RBMutations in the RB1 gene.
2-stage process
1-stage process
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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
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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
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MLPA - Blood
Normal Control
Exon 13 Deletion
Whole Gene Deletion
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Bidirectional Sequence Analysis - Blood
Point mutations
Frameshift mutation
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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
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1. MLPA analysis - Tumour
Normal Control
- Heterozygous whole gene deletion
- Heterozygous deletion of promoter to exon 17
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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
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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
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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