Alpha-galactosidase A gene rearrangements causing Fabry disease. Identification of short direct repeats at breakpoints in an Alu-rich gene

Fabry disease, an inborn error of glycosphingolipid catabolism, results from mutations in the X-linked gene encoding the lysosomal enzyme, alpha-galactosidase A (EC 3.2.1.22). Six alpha-galactosidase A gene rearrangements that cause Fabry disease were investigated to assess the role of Alu repetitive elements and short direct and/or inverted repeats in the generation of these germinal mutations. The breakpoints of five partial gene deletions and one partial gene duplication were determined by either cloning and sequencing the mutant gene from an affected hemizygote, or by polymerase chain reaction amplifying and sequencing the genomic region containing the novel junction. Although the alpha-galactosidase A gene contains 12 Alu repetitive elements (representing approximately 30% of the 12-kilobase (kb) gene or approximately 1 Alu/1.0 kb), only one deletion resulted from an Alu-Alu recombination. The remaining five rearrangements involved illegitimate recombinational events between short direct repeats of 2 to 6 base pairs (bp) at the deletion or duplication breakpoints. Of these rearrangements, one had a 3' short direct repeat within an Alu element, while another was unusual having two deletions of 1.7 kb and 14 bp separated by a 151-bp inverted sequence. These findings suggested that slipped mispairing or intrachromosomal exchanges involving short direct repeats were responsible for the generation of most of these gene rearrangements. There were no inverted repeat sequences or alternating purine-pyrimidine regions which may have predisposed the gene to these rearrangements. Intriguingly, the tetranucleotide CCAG and the trinucleotide CAG (or their respective complements, CTGG and CTG) occurred within or adjacent to the direct repeats at the 5' breakpoints in three and four of the five alpha-galactosidase A gene rearrangements, respectively, suggesting a possible functional role in these illegitimate recombinational events. These studies indicate that short direct repeats are important in the formation of gene rearrangements, even in human genes like alpha-galactosidase A that are rich in Alu repetitive elements.     

AluElements: Repetitive DNA as Facilitators of Chromosomal Rearrangement

Alu repeats are the most common type of repetitive DNA sequences dispersed throughout the human genome. Technical advances in the field of cytogenetics and molecular biology have facilitated the analysis of epithelial tumors and hematologic malignancies which has led to the observation of Alu elements in and near sites often involved in chromosomal rearrangements. Repair mechanisms of double strand breaks (DSB) such as homol-ogous recombination (HR) may rely on the sequence homology of Alu repeats, potentially leading to chromosomal rearrange-ments. Databases have confirmed the strong association between Alu repeats, specifically the 26 bp consensus sequence and chro-mosomal regions involved in deletions and translocations. Although the Alu repetitive sequence is a potential "hotspot" during homologous recombination, there are other cellular mech-anisms that may play a more prominent role in the initiation of chromosomal rearrangements.     

The Contribution of Alu Elements to Mutagenic DNA Double-Strand Break Repair

Alu elements make up the largest family of human mobile elements, numbering 1.1 million copies and comprising 11% of the human genome. As a consequence of evolution and genetic drift, Alu elements of various sequence divergence exist throughout the human genome. Alu/Alu recombination has been shown to cause approximately 0.5% of new human genetic diseases and contribute to extensive genomic structural variation. To begin understanding the molecular mechanisms leading to these rearrangements in mammalian cells, we constructed Alu/Alu recombination reporter cell lines containing Alu elements ranging in sequence divergence from 0%-30% that allow detection of both Alu/Alu recombination and large non-homologous end joining (NHEJ) deletions that range from 1.0 to 1.9 kb in size. Introduction of as little as 0.7% sequence divergence between Alu elements resulted in a significant reduction in recombination, which indicates even small degrees of sequence divergence reduce the efficiency of homology-directed DNA double-strand break (DSB) repair. Further reduction in recombination was observed in a sequence divergence-dependent manner for diverged Alu/Alu recombination constructs with up to 10% sequence divergence. With greater levels of sequence divergence (15%-30%), we observed a significant increase in DSB repair due to a shift from Alu/Alu recombination to variable-length NHEJ which removes sequence between the two Alu elements. This increase in NHEJ deletions depends on the presence of Alu sequence homeology (similar but not identical sequences). Analysis of recombination products revealed that Alu/Alu recombination junctions occur more frequently in the first 100 bp of the Alu element within our reporter assay, just as they do in genomic Alu/Alu recombination events. This is the first extensive study characterizing the influence of Alu element sequence divergence on DNA repair, which will inform predictions regarding the effect of Alu element sequence divergence on both the rate and nature of DNA repair events.     

Exon skipping caused by an intronic insertion of a young Alu Yb9 element leads to severe hemophilia A

Short interspersed elements, such as Alu elements, have propagated to more than one million copies in the human genome. They affect the genome in several ways, caused by retrotransposition, recombination between elements, gene conversion, and alterations in gene expression. These events, including novel insertions into active genes, have been associated with a number of human disorders. Hemophilia A is an X-linked severe bleeding disorder and is caused by mutations in the Factor VIII gene. The spectrum of mutations includes point mutations, rearrangements, insertions, and deletions. Recently, an Alu retrotransposition event in a coding exon has been reported in a family with a severe form of hemophilia A. This was the first report of an Alu insertion in the Factor VIII gene. Here, we report a second Alu insertion event that lies in an intron of the same gene that causes exon skipping and the complete disruption of gene expression.     

Genetic recombination in a chromosomal translocation t(2;8)(p11;q24) of a Burkitt's lymphoma cell line, KOBK101

We analyzed a chromosomal translocation, t(2;8)(p11;q24), in a Burkitt's lymphoma cell line, KOBK101. The translocation reciprocally occurred between a site about 150 bp upstream from the J5 segment in the Ig kappa-encoding gene on chromosome 2 and the A-rich end of an Alu repetitive element located far downstream from the c-myc gene on chromosome 8. Short segments of both parental chromosomes were deleted at the rearrangement site. A sequence related to the heptamer recognition signal for the V-J recombination of Ig genes and a topoisomerase I-recognition sequence were detected at the breakpoints. The V-J recombination occurred on both chromosome 2 and the translocated chromosome 2p- at the J3 and J4 segments, respectively. The J region on the translocated chromosomes was mutated, as compared with that on the untranslocated chromosome, while the Alu element and its upstream sequence were conserved. These results suggest the following aspects to the chromosomal translocation of this cell line. A V-J recombination seems to have occurred at the proximal end of the J4 segment first, and then the translocation took place in the region between the J4 and J5 segments. The translocation may have been mediated by the functions of topoisomerase I and the Alu repetitive sequence located at the breakpoint, although the possibility cannot be ruled out that the recombination machinery for Ig gene rearrangements functioned irregularly.     

The involvement of Alu repeats in recombination events at the α-globin gene cluster: characterization of two α°-thalassaemia deletion breakpoints

Alu repetitive sequences are frequently involved in homologous and non-homologous recombination events in the α-cluster. Possible mechanisms involved in Alu-mediated recombination events are strand exchange, promoted by DNA pairing between highly homologous Alu repeats, and subsequent strand invasion. Alternatively, Alu sequences might play a more active role in recombinogenic processes in the α-cluster. We describe a novel 33-kb α°-thalassaemia deletion ––^DUTCH encompassing the α- and zeta-globin genes and pseudogenes in a kindred of Dutch-Caucasian origin. This deletion appears similar, although not identical, to the previously described ––^MEDII deletion. Cloning and sequencing of both the ––^DUTCH and ––^MEDII deletion breakpoints clearly indicate that the mechanism leading to these α°-thalassaemia deletions involves misalignment between the highly homologous tandemly arranged Alu repeats at both parental sides, which are normally 33 kb apart. Comparison of breakpoint positions along the Alu consensus sequence indicate the involvement of a 26-bp core sequence in two out of five α°-thalassaemia deletions. This sequence has been identified by others as a possible hotspot of recombination. These findings favour the idea that Alu repeats stimulate recombination events not only by homologous pairing, but also by providing binding sites for recombinogenic proteins. Received: 14 October 1996 / Revised: 14 November 1996     

Topoisomerase-I- and Alu-mediated genomic deletions of the APC gene in familial adenomatous polyposis

Germline mutation in the adenomatous polyposis coli (APC) gene results in familial adenomatous polyposis (FAP), a heritable form of colorectal cancer. We have previously reported two novel mutations that delete exons 11 and 14 of the APC gene, respectively, at the cDNA level without any splice junction defects at the genomic level. We describe here the precise breakpoints of the two mutations and the possible mechanisms leading to the genomic rearrangement. The first rearrangement is most likely a topoisomerase-I-mediated non-homologous recombination resulting in a 2-kb deletion that deletes exon 11 of the APC gene. Both 5' and 3' breakpoints have two topoisomerase I recognition sites and runs of pyrimidines within the 10-bp sequences in their vicinity. Further, the 3' breakpoint has an adenine-thymidine-rich region. This is probably the first report of a topoisomerase-I-mediated germline mutation in a tumor suppressor gene. The second rearrangement is most likely an Alu-Alu homologous recombination resulting in a 6-kb deletion encompassing exon 14. The Alu elements at the 5' and 3' breakpoints include the 26-bp core sequence thought to stimulate recombination. In both rearrangements, partial sequences from the long interspersed nuclear element family are in the vicinity of the breakpoints. Other than serving as markers for regions of DNA damage, their precise role in the recombination events, if any, is unclear. Both deletions result in truncated APC proteins missing the beta-catenin- and axin-binding domains, resulting in severe polyposis and cancer.     

Conformational polymorphysm of G-rich fragments of DNA Alu-repeats. I. Noncanonical structures

We report results of the first systematic study of conformational polymorphism of G-rich DNA fragments of Alu-repeats. Alu retrotransposons are primate-specific short interspersed elements. Using the Alu sequence of the prooncogen bcl2 intron and the consensus AluS_x sequence as representative examples, we have determined characteristic Alu sites that are capable of adopting G-quadruplex (GQ) conformations (i.e., potential quadruplex sites—PQSAlu), and demonstrated by bioinformatics methods that these sites are Alu-specific in the human genome. Genomic frequencies of PQSAlu were assessed (~1/10000 bp). These sites were found to be characteristic of young (active) Alu families (Alu-Y). A recombinant DNA sequence bearing the Alu element of the human bcl2 gene (304 bp) and its PQS-mutant (Alu-PQS) were constructed. The formation of noncanonical structures in Alu_bcl2 dsDNA and their absence in the case of Alu-PQS have been shown using DMS-footprinting and atomic force microscopy (AFM). Expression vectors bearing wild-type and mutant Alu insertions in the promoter regions of the reporter gene have been prepared, and their regulatory effects have been compared during transfection of НЕК293 and HeLa cells. We suggest that the dynamic study of the spatial organization of Alu repeats may provide insight into the mechanisms of genomic rearrangements responsible for the development of many oncological and neurodegenerative diseases.     

An<Emphasis Type="Italic"> Alu</Emphasis>-mediated rearrangement as cause of exon skipping in Hunter disease

Hunter syndrome (Mucopolysaccharidosis type II), a rare X-linked lysosomal storage disorder, results from deleterious mutations in the iduronate-2-sulfatase ( IDS) gene located on Xq27.3-q28. Partial or complete deletions and large rearrangements have been extensively reported in the IDS gene as the basis of Hunter disease. The present report, however, is the first report on a Hunter patient in which Alu-mediated recombinations are implicated. Our patient showed the skipping of exon 8 at the cDNA level, without any splice-junction defects at the genomic level, where a new large rearrangement was identified instead. This new mutant allele consisted of an extensive deletion of IDS sequence of about 3 kb, as well as an additional inserted sequence of 157 bp. Two different computer programs were necessary to elucidate the nature of the insert. NCBI-BLAST query detected a single match for 126 bp out of 157 of the fragment that aligned exactly with a specific chromosomal region, Xq25-27.1, where an AluSg sequence is adjacent to an L1. Instead, the Repeat Masker program identified only 83 bp out of 157 of the insert, which was confirmed as an AluS. The observed homology between the AluSc sequence in the IDS intron 8 and the inserted AluS element, as well as the closeness of 26 bp Alu core sequence, considered to be a recombination hotspot, made us hypothesise upon the fact that both an Alu retrotransposition and an Alu-mediated deletion underlie the disease-producing rearrangement. We, therefore, now propose a mechanism that led to the large genomic deletion causing the production of the aberrant mRNA splicing.     

A BC200-derived element and Z-DNA as structural markers in annexin I genes: Relevance to Alu evolution and annexin tetrad formation

We have identified two types of structural elements in genomic DNA for annexin I that provide physical evidence of genetic events leading to conserved changes in gene structure. The sequence upstream of the transcribed region in human annexin I contained a rare, Alu-like repetitive element with flanking direct repeats, probably derived from the active BC200 gene via germline retroposition. Nucleotide substitutions in this BC200 insert relative to the 7SL gene and its absence in rodent annexins I identified it as a recent primate pseudogene. Phylogenetic analysis showed that the BC200 gene represents a new clade of primate Alu evolution that branched near the time of appearance of the progenitor to the free left Alu monomer, FLAM-C. Separate analysis identified a Z-DNA motif in pigeon annexin I intron 7 that may represent the vestigial recombination site involved in primordial assembly of the annexin tetrad. These distinct structural features in annexin I genes provide insight into the evolution of Alu repeats and the mechanism of annexin tetrad formation.     

A large MSH2 Alu insertion mutation causes HNPCC in a German kindred

Hereditary non-polyposis colorectal cancer (HNPCC) syndrome is an autosomal, dominantly inherited disease accounting for about 1%–5% of all colorectal cancer cases. HNPCC predisposition is caused by germline mutations in at least five genes coding for DNA mismatch repair (MMR) proteins. More than 400 MMR gene mutations have been identified in HNPCC patients. About 90% of mutations affect the MLH1 and MSH2 genes. The mutational spectrum mainly includes point mutations and small deletions or insertions. Here, we report a large 184 base-pair Alu insertion mutation in exon 6 of the MSH2 gene in a German HNPCC family. The inserted sequence contains repetitive Alu sequence elements that present the highest homology with the old Alu J subfamily. The Alu J insertion was most likely derived from Alu-mediated recombination, since Alu J elements have been found close to the insertion site in adjacent introns, and since elements pivotal for Alu retrotransposition are missing. Our results suggest that the recombination event occurred at least one generation ago. This is the first report of an Alu insertion in the coding sequence of a MMR gene as the cause of HNPCC. Our data thus further extend the spectrum of MMR gene mutations causative for HNPCC.M. Kloor and C. Sutter contributed equally to this work     

'Illegitimate' recombination events in polyoma-transformed rat cells

In the LPT line of polyoma (Py)-transformed rat cells, amplification of the integrated viral DNA and of cell nucleotide sequences flanking the viral integration site, can be induced either spontaneously or by treatment with carcinogens. We show here that the amplified DNA includes interspersed viral and cellular sequences generated by 'illegitimate' recombination events. Genomic libraries have been prepared in phage lambda vectors from LPT cells treated with the inducing agent mitomycin C and from untreated LPT cells. Four phages, including viral-cell DNA recombinants, have been isolated from these libraries. Sequencing through the recombination sites revealed the following characteristics: (i) The crossover points map at four different positions in the viral DNA and at four different positions in the flanking cell DNA. (ii) There are very short homologous sequences of 1, 2, or 4 bp, at the recombination sites. (iii) Aside from the exchanges between the viral and the cellular DNA, no further rearrangements occurred around the new viral-cellular DNA junctions. (iv) Next to the recombination sites, there are blocks of homopurine-homopyrimidine sequences, which may assume a structure that differs from the Watson-Crick double helix. (v) Clustered homologous sequence blocks of up to 10 bp are present less than 200 bp away from the recombination sites. These homologies are not in register. Based on these results, we propose a model that may account for these recombination events and, more generally, for recombination events that occur during gene amplification in mammalian cells.     

The Role of Recombination in the Origin and Evolution of Alu Subfamilies

Alus are the most abundant and successful short interspersed nuclear elements found in primate genomes. In humans, they represent about 10% of the genome, although few are retrotransposition-competent and are clustered into subfamilies according to the source gene from which they evolved. Recombination between them can lead to genomic rearrangements of clinical and evolutionary significance. In this study, we have addressed the role of recombination in the origin of chimeric Alu source genes by the analysis of all known consensus sequences of human Alus. From the allelic diversity of Alu consensus sequences, validated in extant elements resulting from whole genome searches, distinct events of recombination were detected in the origin of particular subfamilies of AluS and AluY source genes. These results demonstrate that at least two subfamilies are likely to have emerged from ectopic Alu-Alu recombination, which stimulates further research regarding the potential of chimeric active Alus to punctuate the genome.     

The nucleotide sequence of a nodule-specific gene,Nms-25 of Medicago sativa: its primary evolution via exon-shuffling and retrotransposon-mediated DNA rearrangements

We present the primary structure of a nudule-specific gene, Nms-25 from Medicago sativa L. cultivar Nagyszénási. Analysis of the nucleotide sequence of Nms-25 revealed that this gene shows all the characteristics of an interrupted plant gene consisting of 13 exons and 12 introns. The promoter region of Nms-25 contains the common promoter elements of plant genes as well as motifs which are supposed to be involved in nodule-specific expression. There are two exon-like sequences in the gene named PE1 and PE2 which are not present in the cDNA clones of Medicago sativa cultivar Cardinal. Intron 9 carries a retrotransposon-like element, Tms1, which might be responsible for downstream deletion events in which a heptanucleotide, ATTAGCT, might have been involved. Most of the exons, exept 1, 12 and 13, are similar to each other both in length (54 bp) and sequence (up to 94% sequence similarity). All exons are interrupted by introns in the same phase (type I). It is suggested that exon-shuffling based on illegitimate recombination in which the ATTAGCT motif might have played an active role, and retrotransposon-mediated DNA rearrangements were the primary events in the molecular evolution of the Nms-25 gene. The nucleotide sequence of the genomic clone of Nms-25 from Medicago sativa is available from the EMBL/GenBank/DDBJ Nucleotide Sequence Databases under the accession number X13287.     

RAD59 is required for efficient repair of simultaneous double-strand breaks resulting in translocations in Saccharomyces cerevisiae

Exposure to ionizing radiation results in a variety of genome rearrangements that have been linked to tumor formation. Many of these rearrangements are thought to arise from the repair of double-strand breaks (DSBs) by several mechanisms, including homologous recombination (HR) between repetitive sequences dispersed throughout the genome. Doses of radiation sufficient to create DSBs in or near multiple repetitive elements simultaneously could initiate single-strand annealing (SSA), a highly efficient, though mutagenic, mode of DSB repair. We have investigated the genetic control of the formation of translocations that occur spontaneously and those that form after the generation of DSBs adjacent to homologous sequences on two, non-homologous chromosomes in Saccharomyces cerevisiae. We found that mutations in a variety of DNA repair genes have distinct effects on break-stimulated translocation. Furthermore, the genetic requirements for repair using 300bp and 60bp recombination substrates were different, suggesting that the SSA apparatus may be altered in response to changing substrate lengths. Notably, RAD59 was found to play a particularly significant role in recombination between the short substrates that was partially independent of that of RAD52. The high frequency of these events suggests that SSA may be an important mechanism of genome rearrangement following acute radiation exposure.     

A bacterial genome in flux: the twelve linear and nine circular extrachromosomal DNAs in an infectious isolate of the Lyme disease spirochete Borrelia burgdorferi

We have determined that Borrelia burgdorferi strain B31 MI carries 21 extrachromosomal DNA elements, the largest number known for any bacterium. Among these are 12 linear and nine circular plasmids, whose sequences total 610 694 bp. We report here the nucleotide sequence of three linear and seven circular plasmids (comprising 290 546 bp) in this infectious isolate. This completes the genome sequencing project for this organism; its genome size is 1 521 419 bp (plus about 2000 bp of undetermined telomeric sequences). Analysis of the sequence implies that there has been extensive and sometimes rather recent DNA rearrangement among a number of the linear plasmids. Many of these events appear to have been mediated by recombinational processes that formed duplications. These many regions of similarity are reflected in the fact that most plasmid genes are members of one of the genome's 161 paralogous gene families; 107 of these gene families, which vary in size from two to 41 members, contain at least one plasmid gene. These rearrangements appear to have contributed to a surprisingly large number of apparently non-functional pseudogenes, a very unusual feature for a prokaryotic genome. The presence of these damaged genes suggests that some of the plasmids may be in a period of rapid evolution. The sequence predicts 535 plasmid genes >/=300 bp in length that may be intact and 167 apparently mutationally damaged and/or unexpressed genes (pseudogenes). The large majority, over 90%, of genes on these plasmids have no convincing similarity to genes outside Borrelia, suggesting that they perform specialized functions.