Retinoblastoma (RB) is a childhood retinal cancer caused by mutations in the RB1 gene. Molecular diagnosis is crucial for early detection and treatment. Current DNA diagnostic screening requires substantial amounts of tumour and blood samples. However current screening methods face the challenges of limited DNA templates from minute retinal tumours and too much blood samples drawn from young patients. In addition, the starting DNA template amount and quality are important to ensure confident detection of disease-causing mutations. As the majority of RB1 mutations are unique and distributed throughout the RB1 gene with no real hot spots, the entire gene needs to be thoroughly analysed.
This investigation proposes to enrich DNA samples using a whole genome amplification (WGA) step prior to RB1 mutation screening by RB1 gene-specific PCR amplification as well as high resolution melt (HRM) analysis and sequencing. It also identifies RB1 mutations in two RB patients and explores whether WGA and saliva products can be a source of DNA templates for RB1 analysis. In addition, this study was conducted based on the hypotheses that RB1 mutations were the underlying cause of the disease in the two patients, and that the products from WGA could be used specifically for RB1 gene analysis to overcome the constraint of insufficient DNA samples.
Two anonymised genomic DNA samples from two unrelated RB patients and five normal healthy DNA samples were used in this project. WGA kits were compared according to three criteria, namely amplification yield, product fragment size and whether DNA is amplifiable. Prior to and after amplification, the optical density of two normal samples was measured to determine the increase in DNA yield. The amplicons were subjected to gel electrophoresis to determine the product fragment size. Exons 6, 14 and 25 of the original and amplified samples undergone PCR, and were examined again using gel electrophoresis to ascertain that the amplicons were amplifiable. Mutation analysis using HRM was carried out with pre-existing primers for all 27 exons and the promoter of RB1. Samples from patients were analysed against 83 saliva DNAs extracted using Oragene•DNA (OG-500) Kit.
REPLI-g was observed to produce higher yield and products of reliable fragment size. Single distinct bands were also seen for exons amplified using REPLI-g, indicating that REPLI-g is more accurate and suitable in the amplification of DNA. Abnormal melt profiles were obtained for exon 6 in RB477 and exon 14 in RB572 for HRM. These exons were sequenced to determine the exact mutation. Exon 6 was found to have a splice-site mutation g.607+1G>T, while a point mutation, g.1363C>T (p.Arg455X) was identified in exon 14.
Both the uses of saliva as a non-invasive DNA source and the WGA approach for enriching DNA sample for application in RB1 gene analysis have never been reported for RB. Although HRM analysis has been used for other diseases, this is its first instance applied in work on RB1 gene. In short, this report offers novel and promising approaches which would contribute significantly to the molecular analysis of mutations in RB.