Short, direct repeats at the breakpoints of deletions of the retinoblastoma gene.

Abstract

We found deletions involving the retinoblastoma gene in 12 of49 tumors from patients with retinoblastoma or osteosarcoma. After mapping the deletion breakpoints, we found that no two breakpoints coincided. Thus, our data do not support the conclusions of others regarding the existence of a "hotspot" for deletion breakpoints in this gene. In 4 of the tumors, we sequenced 200 base pairs surrounding each deletion breakpoint. Three deletions had termini within pairs of short, direct repeats ranging in size from 4 to 7 base pairs. These results indicate that the "slipped mispairing" mechanism may predominate in the generation of deletions at this locus. Our review of deletion breakpoints at other genetic loci reveals that the nature of the sequences present at deletion breakpoints (short, direct repeats versus middle repetitive elements) varies according to the genetic locus under study.The retinoblastoma gene serves as a model for the study of recessive oncogenes. Mutations at the retinoblastoma locus play a role in the development of retinoblastoma, a malignant tumor arising in the eyes of young children, and possibly in the development of other human cancers, such as osteosarcoma (1-4), small cell lung carcinoma (5), and breast carcinoma (6, 7). The recent cloning (1, 2, 8) and restriction and exon mapping (9) of the retinoblastoma locus have allowed for a detailed analysis ofthe molecular basis of mutations that cause inactivation of the retinoblastoma gene and subsequent oncogenic transformation.Mutations of the retinoblastoma gene occur at a rate much higher than for most other human genes (10). Approximately 10-40o of the mutations are deletions detectable by Southern blotting (1)(2)(3)11). The data we present here address two questions concerning deletions that inactivate the retinoblastoma gene. We would like to know whether deletions occur preferentially at hotspots within the gene and whether specific DNA sequences within the gene predispose to them. To study these issues we mapped the deletion breakpoints in 12 tumors and sequenced the breakpoints in 4 of them. We present here the results of this study and discuss their relevance to the possible mechanisms for the generation of deletions of this gene.

Hotspot for deletions in the CGG repeat region of FMR1 in fragile X patients

“…Several studies on the mechanism of deletions have shown that -40% of the large deletions in human disorders are Hanked by very short direct repeats of 2-6 bp (37)(38)(39). It is proposed that these short repeats slip and mispair during replication, resulting in the formation of a loop between the two repeats (37,38).…”

“…It is proposed that these short repeats slip and mispair during replication, resulting in the formation of a loop between the two repeats (37,38). Subsequent excision of this loop removes both the sequences between the repeats as well as one of the repeats.…”

Mechanisms of oncogenesis in patients with familial retinoblastoma

“…(Cowell, 1992). All three mutations reported here are in different exons which contributes to the emerging pattern that no particular part of the gene is preferentially involved in mutation (Canning & Dryja, 1989;Dunn et al, 1989;). (1985) concluded that short direct repeats, of at least 2 bp, occurred more frequently than would be expected from random breakage and reunion.…”

The Role of Error-Prone Alternative Non-Homologous End-Joining in Genomic Instability in Cancer

“…Notably, microhomology sequences suggestive of ALT-NHEJ have been found at the recombination junctions of radiation-induced genomic rearrangements (Morris andThacker, 1993, Nohmi et al, 1999) implying that radiation-induced DSBs can be repaired by ALT-NHEJ. Moreover, microhomologies are frequently detected at the breakpoints of chromosomal deletions and translocations in human cancer cells (Canning and Dryja, 1989, Smanik et al, 1995, Wiemels and Greaves, 1999.…”

“…They mapped the deletion breakpoints and sequenced 200 base pairs surrounding each deletion breakpoint in DNA from 4 tumor samples. Three deletions had termini characterized by direct repeats ranging in size from 4 to 7 base pairs (Canning and Dryja, 1989). Recurrent chromosome translocations characterize leukemia and lymphoma and are specifically associated with their classification and prognosis.…”