Pathogenic validation of unique germline intronic variants of RB1 in retinoblastoma patients using minigenes

Precise identification of the pathogenic character of germline mutations in the retinoblastoma gene (RB1) is fundamental to provide genetic counselling to patients at risk of developing retinoblastoma. In contrast to bona fide oncogenic RB1 mutations like nonsense or frameshift mutations, and those affecting invariant dinucleotides at splice sites, intronic variants affecting less conserved splice motifs require additional analysis to ascertain whether splicing is altered. Although the frequency of these variations is low, their impact on genetic counselling is high, since they are usually associated with low penetrance phenotypes and unaffected carriers. In this work, we used minigene assays to study infrequent germline intronic variations for which functional data were not available. Using this approach, the aberrant splicing and the resulting oncogenic nature of three intronic RB1 mutations was established (c.501-15T>G, c.719-9C>G, c.2326-8T>A). Conversely, the intronic variant c.1961-12T>C was categorized by minigene assay as a very infrequent neutral polymorphism. To our knowledge this is the first report describing the use of minigene constructs to study the oncogenic character of intronic RB1 variants detected during mutational screening and show the utility of this approach to ascertain the oncogenic nature of unique RB1 intronic variants for which no previous functional and clinical data are available. Minigene assay can be especially useful when lymphocyte RNA is not available for study, or when aberrant mRNA can not be detected as a consequence of nonsense mediated decay. Since RB1 minigene are time-consuming assays, owing to the genomic organization of the RB1 gene, it should be welcome the design of new expression vectors that make this type of studies more straightforward.