Hogg, Annette; (1993) A study of oncogenic mutations in the RB1 gene in individuals with retinoblastoma. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Mutation of the retinoblastoma gene (RB1) prediposes to the development of ocular tumours. This study has involved the pursuit, establishment and refinement of a technique for the study of RB1 mutations. Primers and conditions for the amplification and analysis by PCR of all 27 exons of RB1 and the promoter region have been described. The single strand conformation polymorphism (SSCP) technique proved to be the most useful and reliable method for the rapid screening of PCR products. A fast, reliable and reproducible system of sequencing directly from PCR products has also been developed which involves using streptavidin-coated magnetic beads and biotin- labelled PCR products. Using SSCP/sequencing technology, twenty mutations in Rb patients and their tumours were identified. 50% were point mutations, 30% were deletions ranging from 1bp to 22bp long and 15% were insertions of 1bp. Significantly, 80% of point mutations were in CpG dinucleotides, the majority of which were consistent with somatic deamination of methylated cytosines. 86% of CpG→TpG mutations converted CGA (arginine) codons to TGA (stop codons). These occurred in five of the 14 CGA (arginine codons) in RB1 and illustrates that these codons represent mutation hotspots. All the mutations occurred adjacent to sequence motifs which gave clues to the mechanism of mutation. More than 90% of the mutations in this study would be predicted to generate a premature stop codon. A missense mutation was found in one family which showed low penetrance and, unlike other mutations did not disturb the translational reading frame and produced a mutant mRNA. These results suggest the Rb phenotype is related to the type of mutation.

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