No abstract

Translocations and gross deletions are important causes of both cancer and inherited disease. Such gene rearrangements are nonrandomly distributed in the human genome as a consequence of selection for growth advantage and/or the inherent potential of some DNA sequences to be frequently involved in breakage and recombination. Using the Gross Rearrangement Breakpoint Database [GRaBD; www.uwcm.ac.uk/uwcm/mg/grabd/grabd.html] (containing 397 germ-line and somatic DNA breakpoint junction sequences derived from 219 different rearrangements underlying human inherited disease and cancer), we have analyzed the sequence context of translocation and deletion breakpoints in a search for general characteristics that might have rendered these sequences prone to rearrangement. The oligonucleotide composition of breakpoint junctions and a set of reference sequences, matched for length and genomic location, were compared with respect to their nucleotide composition. Deletion breakpoints were found to be AT-rich whereas by comparison, translocation breakpoints were GC-rich. Alternating purine-pyrimidine sequences were found to be significantly over-represented in the vicinity of deletion breakpoints while polypyrimidine tracts were over-represented at translocation breakpoints. A number of recombination-associated motifs were found to be over-represented at translocation breakpoints (including DNA polymerase pause sites/frameshift hotspots, immunoglobulin heavy chain class switch sites, heptamer/nonamer V(D)J recombination signal sequences, translin binding sites, and the chi element) but, with the exception of the translin-binding site and immunoglobulin heavy chain class switch sites, none of these motifs were over-represented at deletion breakpoints. Alu sequences were found to span both breakpoints in seven cases of gross deletion that may thus be inferred to have arisen by homologous recombination. Our results are therefore consistent with a role for homologous unequal recombination in deletion mutagenesis and a role for nonhomologous recombination in the generation of translocations.

Genomic rearrangements are a frequent source of instability, but the mechanisms involved are poorly understood. A 2.5-kbp poly(purine⅐pyrimidine) sequence from the human PKD1 gene, known to form non-B DNA structures, induced long deletions and other instabilities in plasmids that were mediated by mismatch repair and, in some cases, transcription. The breakpoints occurred at predicted non-B DNA structures. Distance measurements also indicated a significant proximity of alternating purine-pyrimidine and oligo(purine⅐pyrimidine) tracts to breakpoint junctions in 222 gross deletions and translocations, respectively, involved in human diseases. In 11 deletions analyzed, breakpoints were explicable by non-B DNA structure formation. We conclude that alternative DNA conformations trigger genomic rearrangements through recombination-repair activities. G ross chromosomal rearrangements are a common source of genetic instability (1). Thus, characterization of the underlying molecular mechanisms of mutagenesis is fundamental for our understanding of human disease. A hallmark of gross deletions is the presence of short homologous tracts (typically 2-8 bp) at the breakpoints (2), a finding that has prompted speculation as to the two distinct mechanisms postulated to be responsible for their formation. The slipped mispairing model (3) envisages that during lagging strand DNA synthesis, distantly located repeats are brought into close proximity by the looping out of the single-stranded region, thereby enabling the replication complex to ''jump'' from the proximal to the distal repeat and hence bypass the looped structure. Alternative models propose that various types of repetitive sequence elements may serve as substrates for intra-or intermolecular recombination (2, 4). Neither model is satisfactory; slipped mispairing is inconsistent with deletions greater than Ϸ500 bp and deletions manifesting Ͻ4-bp homologies (5-9), whereas the recombination model does not provide a rationale for the initiation of the process.Specific sequence motifs such as direct and inverted repeats, (RY⅐RY) n and (R⅐Y) n , in which R represents purine and Y represents pyrimidine, and four closely spaced G-rich direct repeats [i.e., (G⅐C) 3 ] undergo structural transitions from the orthodox right-handed B-helical duplex to higher energy state non-B structures (slipped hairpin͞loops, cruciforms, left-handed Z-helices, triplexes, and tetraplexes, respectively) (10-12) under torsional stress (negative supercoiling) in vivo.Early articles in bacteria and hamster cells reported isolated cases in which deletions could occur by a recombination-repair reaction mediated by cruciform structures forming at each breakpoint (13,14). Recently, the breakpoint junctions of the human constitutional translocations t(1;22), t(4;22), t(11;22), and t(17;22), which involve a common locus on chromosome 22q11.2, were found to coincide with large (Ͼ95 bp) cruciform structures (15-18), suggesting that this conformation may predispose specific loci to genomic rearrangements.The po...

In the Human Gene Mutation Database (www.hgmd.org), microdeletions and microinsertions causing inherited disease (both defined as involving < or = 20 bp of DNA) account for 8,399 (17%) and 3,345 (7%) logged mutations, in 940 and 668 genes, respectively. A positive correlation was noted between the microdeletion and microinsertion frequencies for 564 genes for which both microdeletions and microinsertions are reported in HGMD, consistent with the view that the propensity of a given gene/sequence to undergo microdeletion is related to its propensity to undergo microinsertion. While microdeletions and microinsertions of 1 bp constitute respectively 48% and 66% of the corresponding totals, the relative frequency of the remaining lesions correlates negatively with the length of the DNA sequence deleted or inserted. Many of the microdeletions and microinsertions of more than 1 bp are potentially explicable in terms of slippage mutagenesis, involving the addition or removal of one copy of a mono-, di-, or trinucleotide tandem repeat. The frequency of in-frame 3-bp and 6-bp microinsertions and microdeletions was, however, found to be significantly lower than that of mutations of other lengths, suggesting that some of these in-frame lesions may not have come to clinical attention. Various sequence motifs were found to be over-represented in the vicinity of both microinsertions and microdeletions, including the heptanucleotide CCCCCTG that shares homology with the complement of the 8-bp human minisatellite conserved sequence/chi-like element (GCWGGWGG). The previously reported indel hotspot GTAAGT and its complement ACTTAC were also found to be overrepresented in the vicinity of both microinsertions and microdeletions, thereby providing a first example of a mutational hotspot that is common to different types of gene lesion. Other motifs overrepresented in the vicinity of microdeletions and microinsertions included DNA polymerase pause sites and topoisomerase cleavage sites. Several novel microdeletion/microinsertion hotspots were noted and some of these exhibited sufficient similarity to one another to justify terming them "super-hotspot" motifs. Analysis of sequence complexity also demonstrated that a combination of slipped mispairing mediated by direct repeats, and secondary structure formation promoted by symmetric elements, can account for the majority of microdeletions and microinsertions. Thus, microinsertions and microdeletions exhibit strong similarities in terms of the characteristics of their flanking DNA sequences, implying that they are generated by very similar underlying mechanisms.

Although 20 years have elapsed since the first single basepair substitution underlying an inherited disease in humans was characterised at the DNA level, the initiative has only recently been taken to establish central database resources for pathological genetic variants. Disease-associated gene lesions are currently collected and publicised by the Human Gene Mutation Database (HGMD) in Cardiff, locus-specific mutation databases, and to some extent also by the Genome Database (GDB) and Online Mendelian Inheritance in Man (OMIM). To date, HGMD represents the only comprehensive and publicly available database of gene lesions underlying human inherited disease. By July 1999, HGMD contained over 18,000 different mutations from some 900 human genes, the majority being single basepair substitutions. In addition to its potential as an information resource for clinicians and genetic counsellors, HGMD has allowed molecular geneticists to address a variety of biological questions through meta-analysis of the collated data. HGMD also promises to assist research workers in optimising mutation search strategies for a given gene. A questionnaire sent out to, and answered by, the editors of 20 key journals revealed that human genetics journals are increasingly reluctant to publish mutation reports. Electronic data submission and publication facilities are therefore urgently required. The World Wide Web (WWW) provides an excellent medium within which to combine the centralised management of basic mutation data, including rigorous quality control, with the possibility of publishing additional mutation-related information. In response to these needs, HGMD has both instituted a collaboration with Springer-Verlag GmbH, Heidelberg, to potentiate free online submission and electronic publication of human gene mutation data and developed links with the curators of locus-specific mutation databases.