Hereditary predisposition to retinoblastoma is caused by germ line mutations in the RB1 gene. Genetic counseling of affected individuals and accurate risk prediction for their families requires identification of the disease causing mutation. Furthermore, the nature of a mutation can determine genetic penetrance, disease presentation and prognosis. We describe, and functionally characterize here, a novel mutant allele of RB1 present in the germ line of a patient with sporadic bilateral retinoblastoma. The mutation generates an operational splice acceptor site resulting in a predicted protein product with loss of 81 amino acids from its carboxy terminus. We demonstrate that the aberrantly spliced transcript is present in substantial amounts in peripheral blood of the patient and present evidence that the predicted protein product displays partial loss of activity reflecting in degree and presentation that of the partially penetrant RB1 missense mutant R661W. This infers that disease with reduced expressivity and incomplete penetrance may arise in individuals that carry the mutation and predicts such presentation for similar mutations with found in sporadic cases in the past. © 2004 Wiley-Liss, Inc.KEY WORDS: retinoblastoma; reduced expressivity; penetrance; splicing; RB1; pRB INTRODUCTIONRetinoblastoma, a childhood tumor of the eye, is caused by inactivation in the developing human retina of both alleles of the RB1 tumor suppressor gene (MIM# 180200) (reviewed in DiCiommo et al., 2000). In hereditary retinoblastoma (accounting for roughly 50% of cases) one RB1 allele is mutated in the germ line as either a novel or an inherited mutation. In this instance disease usually presents at an early age, with multiple tumor foci in both eyes, a high incidence of disease recurrence, increased lifetime risk for secondary malignancies and transmission to kindred with a penetrance of greater 95%. In its nonhereditary form mutational inactivation of both RB1 alleles DOI: 10.1002/humu.9305 2 Sánchez-Sánchez et al.arises from somatic events in a single retinal cell. Here disease presentation is far milder disease featuring late onset, single tumor foci in one eye only and absence of recurrence and secondary tumor development. In rare instance hereditary retinoblastoma can present in a form reminiscent to the non-hereditary disease (reduced expressivity), and often in this instance shows reduced penetrance in kindred (low penetrance).The product of the human RB1 gene (p110RB/pRB) is a nuclear phospho-protein composed of 928 amino acids. Through interacting with cellular proteins pRB influences cell proliferation, but also other types of cell behavior (reviewed in Kaelin, 1999;Zheng and Lee, 2001). Most noted is the function of pRB to control gene transcription via E2F (reviewed in Harbour and Dean, 2000;Stevens and La Thangue, 2003). Through association with specific members of this transcription factor family pRB represses genes required for cell cycle progression and proliferation, and by attracting additional chromatin modifyin...