A parent-of-origin effect in two families with retinoblastoma is associated with a distinct splice mutation in the RB1 gene

Abstract

We have identified a splice-site mutation (IVS6+1G-->T) in the RB1 gene, in two unrelated families with incomplete-penetrance retinoblastoma. Analysis of RNA from white blood cells showed that this mutation causes skipping of exon 6. Although this deletion results in a frameshift, most carriers of the mutation did not develop retinoblastoma. Interestingly, the relative abundance of the resultant nonsense messenger RNA varies between members of the same family and is either similar to or considerably lower than the transcript level of the normal allele. Moreover, variation of relative transcript levels is associated with both the sex of the parent that transmitted the mutant allele and phenotypic expression: All eight carriers with similar abundance of nonsense and normal transcript have received the mutant allele from their mother, and only one of them has developed retinoblastoma; by contrast, all eight carriers with reduced abundance of the nonsense transcript have received the mutant allele from their father, and all but two them have retinoblastoma. After treatment with cycloheximide, the relative abundance of transcripts from paternally inherited mutant alleles was partly restored, thus indicating that posttranscriptional mechanisms, rather than transcriptional silencing, are responsible for low levels of mutant messenger RNA. Our data suggest that a specific RB1 mutation can be associated with differential penetrance, on the basis of the sex of the transmitting parent.

Figure 1

Pedigrees and segregation of IVS6+1G→T. Blackened symbols denote patients with bilateral retinoblastoma; half-blackened symbols denote patients with unilateral retinoblastoma. Plus signs (+) indicate presence of IVS6+1G; “mt” indicates presence of IVS6+1T; horizontal bar indicates individuals investigated by RNA analysis. A, Family G162. The frame encloses the branch of this family that has been described elsewhere (Czeizel and Gardonyi 1974). B, Family M12487. Individual II-2 died of retinoblastoma and may have had bilateral tumors (half-crosshatched, half-blackened symbol).

Figure 2

Results of RNA analysis. A, Genomic organization of RB1. Boxes indicate exons; arrowheads indicate location and orientation of primers that were used for RT-PCR and sequencing. B, Agarose gel electrophoresis of products obtained by RT-PCR with primers c438se and c1487as followed by PCR with primers c438se and c879as. “C” denotes normal control; “Ø” denotes a negative control. The DNA standard used was pUC19/MspI. C, Result of sequence analysis of the smaller products from M12487 V-1 and G162 V-7. The first premature termination codon is created 23 bp downstream from the exons 5–7 junction and is indicated by an asterisk (*). D, Results of quantitative analysis of fluorescent RT-PCR products obtained with primers c438se and c879as. Boxed numeric values below peaks are the peak integrals as determined by the Genotyper software; the ratio of individual peak integrals to the grand total is shown to the right of each peak. E, Graph with summary of results of fluorescent RT-PCR. Each circle indicates the ratio of the mutant peak integral to the grand total and represents the result of a single RT-PCR; vertical bars indicate the arithmetic means.