Inverted Duplication Research Articles

MICRODISSECTION AND DOP‐PCR‐BASED REVERSE CHROMOSOME PAINTING AS A FAST AND RELIABLE STRATEGY IN THE ANALYSIS OF VARIOUS STRUCTURAL CHROMOSOME ABNORMALITIES

Reverse chromosome painting has become a powerful tool in clinical genetics for the characterization of cytogenetically unclassifiable aberrations. In this report, the application of a sensitive and rapid procedure for the complete and precise identification of four different de novo structural chromosome abnormalities is presented. These chromosome rearrangements include a marker derived from chromosome 3(cen–q11), an interstitial deletion of chromosome 13 [del(13)(q14q22)], an unbalanced translocation [46,XY,−4,+der(4)t(4;8)(p15.2;p21.1)] leading to Wolf–Hirschhorn syndrome, and a partial inverted duplication in conjunction with a partial deletion of chromosome 5p [46,XX,−5,+der(5)(:p13–p15.1::p15.1–qter)] which is responsible for the manifestation of the cri-du-chat syndrome. The importance of a fast and reliable evaluation of complex chromosome aberrations in pre- and postnatal diagnosis with regard to comprehensive genetic counselling is emphasized.

Formation of Extrachromosomal Circular Amplicons with Direct or Inverted Duplications in Drug-Resistant Leishmania tarentolae

Selection for methotrexate resistance in Leishmania spp. is often associated with amplification of the H locus short-chain dehydrogenase-reductase gene ptr1 as part of extrachromosomal elements. Extensive sequences are always coamplified and often contain inverted duplications, most likely formed by the annealing of inverted repeats present at the H locus. By gene targeting mediated by homologous recombination, several repeated sequences were introduced in the vicinity of ptr1. Selection for methotrexate resistance in these transfectants led to ptr1 amplification as part of small circles with direct or inverted duplications whether the integrated sequences consisted of direct or inverted repeats. Hence, for a region to he amplified in L. tarentolae during drug selection, a drug resistance gene is required and must be flanked by (any) homologous repeated sequences. The distance between these repeats and their orientation will determine the length of the amplicon and whether it contains direct or inverted duplications.

Familial partial trisomy 8p without dysmorphic features and only mild mental retardation

We report on a mother and her two sons who had a direct duplication of chromosome region 8p22-8p23.1 without dysmorphic features and only mild mental retardation. The patients have been studied using G banding, chromosome painting, and FISH using cosmid probes specific for the region 8p23.1-8pter. Comparison of the phenotypes of our patients and of published patients with an inversion duplication of the short arm of chromosome 8 indicates that trisomy for chromosome band 8p21 causes the more severe clinical picture in the latter.

Four new cases of inverted terminal duplication: A modified hypothesis of mechanism of origin

We present 4 recently diagnosed cases of inverted tandem duplication with involvement of the respective terminal band. Based on these 4 cases and review of the literature, the term "inverted terminal duplication" is proposed to designate specifically the type of inverted tandem duplication which involves the terminal band. A modification of the previous hypothesis of mechanism of origin is advanced. It is speculated further that a telomeric deletion of a meiotic chromosome followed by a U-type reunion of the chromatids, considered to be the first steps of the proposed mechanism of origin, may not be a rare gonadal event.

Location in the source chromosome of the 180-kb minichromosome of Leishmania major and characterization of the novel junction

The 180-kb LD1 minichromosome of Leishmania major (M180) is a large inverted duplication which arises spontaneously from a megabasic chromosome. In this work this locus has been located in the source chromosome at a telomeric position. Both the novel junction created in the middle of the minichromosome, as well as its counterpart region in the source chromosome were cloned and sequenced. Two inverted repeats, which could give rise to two imperfect stem-loops, and an A + T-rich DNA sequence were the only particular features observed in this region. A putative model for the formation of the minichromosome is proposed.

Partial trisomy of the short arm of chromosome 18 due to inversion duplication and direct duplication

We report one patient with a de novo inversion duplication 18 (pter-->cen) and two cases of direct tandem duplication 18 (pter-->cen), one due to maternal inheritance and the other arising as mosaicism of unknown origin. The duplications are demonstrated by high resolution banding. They were verified by in situ hybridization with a paint specific for chromosome 18 and with DNA probe LI.84 specific for the centromere region of chromosome 18. FISH with the genomic DNA probe pHRR68 specific for 18p11.32 revealed a subtle deletion concomitantly involved in the case of inversion duplication 18p. The patients exhibit slight developmental delay/moderate mental retardation and only a few dysmorphic features. The literature on trisomy 18p is reviewed and the present cases are compared to it.

Hairpin structures are the primary amplification products: a novel mechanism for generation of inverted repeats during gene amplification.

Early events of DNA amplification which occur during perturbed replication were studied by using simian virus 40 (SV40)-transformed Chinese hamster cells (CO60) as a model system. The amplification is observed shortly after carcinogen treatment, and the amplified sequences contain molecules organized as inverted repeats (IRs). SV40 amplification in vitro was studied by using extracts from carcinogen-treated CO60 cells. In the amplified DNA the SV40 origin region was rereplicated, while more distal sequences were not replicated even once. Using several experimental procedures such as sucrose gradients, "snap-back" assay, and two-dimensional gel electrophoresis, we show that the overreplicated DNA contains IRs which are synthesized de novo as hairpins or stem-loop structures which were detached from the template molecules. The fully replicated SV40 molecules synthesized by the HeLa extracts do not contain such IRs. We propose "U-turn replication" as a novel mechanism for gene amplification, accounting for the generation of extrachromosomal inverted duplications as a result of perturbed replication and template switching of the DNA polymerases.

Hairpin Structures Are the Primary Amplification Products: a Novel Mechanism for Generation of Inverted Repeats during Gene Amplification

Early events of DNA amplification which occur during perturbed replication were studied by using simian virus 40 (SV40)-transformed Chinese hamster cells (CO60) as a model system. The amplification is observed shortly after carcinogen treatment, and the amplified sequences contain molecules organized as inverted repeats (IRs). SV40 amplification in vitro was studied by using extracts from carcinogen-treated CO60 cells. In the amplified DNA the SV40 origin region was rereplicated, while more distal sequences were not replicated even once. Using several experimental procedures such as sucrose gradients, "snap-back" assay, and two-dimensional gel electrophoresis, we show that the overreplicated DNA contains IRs which are synthesized de novo as hairpins or stem-loop structures which were detached from the template molecules. The fully replicated SV40 molecules synthesized by the HeLa extracts do not contain such IRs. We propose "U-turn replication" as a novel mechanism for gene amplification, accounting for the generation of extrachromosomal inverted duplications as a result of perturbed replication and template switching of the DNA polymerases.

Partial trisomy and monosomy 8p due to inversion duplication

Fluorescent in situ hybridization with probes specific for a chromosomal subregion and chromosome-specific libraries (chromosome painting) are important new methods for assessing chromosome rearrangements. In this paper we present four patients with additional chromosomal material on chromosome 8p who have been studied using G-banding techniques, chromosome painting and FISH with cosmid probes specific for the region 8p23.1-->8pter. In all cases we found a partial inversion duplication of 8p along with a deletion of the region 8p23.1-->8pter.

Mutant woodchuck hepatitis virus genomes from virions resemble rearranged hepadnaviral integrants in hepatocellular carcinoma.

Although hepadnaviruses are implicated in the etiology of hepatocellular carcinoma, the pathogenic mechanisms involved remain uncertain. Clonally propagated integrations of hepadnaviral DNA into cellular DNA can be demonstrated in most virally induced hepatocellular carcinomas. Integration occurs at random sites in cellular DNA, but the highly preferred sites in viral DNA are adjacent to the directly repeated sequence DR1, less often DR2, or in the cohesive overlap region. Integrants invariably contain simple deletions or complex rearrangements that have been thought to occur after integration. We report here the detection of mutant woodchuck hepatitis virus (WHV) genomes cloned from virions in serum that are strikingly similar to the rearranged hepadnaviral genomes found previously as integrated sequences in cellular DNA. Of 102 cloned genomes studied, 2 had large inverted duplications, 1 a 219-nucleotide direct duplication, and 1 a 219-nucleotide deletion. Virus-virus DNA junctions occurred either adjacent to DR1 or DR2 or in the cohesive overlap region at preferred topoisomerase I cleavage sites. Since these sites are located in the single-stranded regions of the genome, cleavage by topoisomerase I would produce linear molecules that would be expected to be highly recombinogenic since this enzyme, possessing nicking and ligating activities, would remain covalently attached. Sucrose density gradient centrifugation coupled with polymerase chain reaction studies confirmed that the mutant WHV DNA forms resided in virions and did not represent free viral DNA released from infected cells or were unlikely to be an artifact of the cloning process. Thus, the finding in virions of mutant WHV DNA similar to WHV DNA integrated into cellular DNA suggests that the processes of mutation and integration are linked in some instances. Furthermore, the mutant genomes that are preferentially integrated into cellular DNA may have an etiologic role in hepatocarcinogenesis.

Identification of a defective transposable element in tobacco.

A putative defective transposable element has been identified in tobacco. This element has been found and characterised in two separate parts of the tobacco genome, specifically within the 3rd intron of the pollen-specific polygalacturonase gene (Npg1) and upstream of the endochitinase gene (Chn50). The element is ca. 0.4 kb in length and is bounded by conserved inverted repeats and putative target site duplications. It appears to fall into the category of non-autonomous transposable elements.

Elucidation of the centromere involvement in an inversion (13) by fluorescent in situ hybridisation.

A newborn infant with phenotypic features of trisomy for distal 13q was found to have recombinant inversion duplication involving the (13)(q22-->qter) region. Parental karyotypes showed that the mother had a normal 46,XX complement and the father had an apparently balanced pericentric inversion of a chromosome 13. Because of the unusual nature of the inversion, the exact position of the centromere on the father's inverted chromosome 13 was difficult to assign by GTG banding, even on prometaphase chromosomes. CBG and NOR banding were not informative in determining the location of the centromere. Fluorescent in situ hybridisation with an alpha satellite DNA probe for D13Z1/D21Z1 helped in confirming the exact position of the centromere in the rearranged paternal chromosome. Thus, the origin of the proband's abnormal chromosome 13 was clarified.

A large inverted duplicated DNA region associated with an amplified oncogene is stably maintained in a YAC.

In the polyoma virus (Py) transformed 3B rat cell line the Py oncogene and adjacent cellular DNA are amplified in arrays of very large inverted duplications. A region of the 3B amplified DNA was cloned as a 550 kb insert in a Yeast Artificial Chromosome (YAC) vector, designated y3B01. Analysis of the y3B01 cloned insert revealed it contained a large inverted duplicated DNA region which was approximately 400 kb in size (two palindromic arms of about 200 kb). At least 420 kb of the 550 kb YAC insert has been identified as being derived from the 3B amplified DNA and the amplicon in 3B cells is at least 220 kb in size. No DNA instability of the y3B01 YAC clone was detected. The y3B01 DNA replicated as efficiently as yeast chromosomes and was structurally stable in yeast cells during more than 30 cell divisions. Comparison of the restriction endonuclease maps of the inverted duplicated region of the y3B01 DNA insert and the amplified 3B genomic DNA did not reveal any gross differences suggesting that no rearrangements had occurred during or after the cloning into the YAC vector. These results suggest that large inverted duplications, which can show instability in prokaryotic cloning systems, can be stably cloned and maintained in YAC vectors in yeast.

Homologous recombination between direct repeat sequences yields P-glycoprotein containing amplicons in arsenite resistant Leishmania.

The protozoan parasite Leishmania often responds to drug pressure by amplifying part of its genome. At least two loci derived from the same 800 kb chromosome were amplified either as extrachromosomal circles or linear fragments after sodium arsenite selection. A 50 kb linear amplicon was detected in six independent arsenite mutants and revertants grown in absence of arsenite rapidly lost the amplicon and part of their resistance. The circular extrachromosomal amplicons, all derived from the H locus of Leishmania, were characterized more extensively. In all cases, direct repeated sequences appeared to be involved in the formation of circular amplicons. Most amplicons were generated after homologous recombination between two linked P-glycoprotein genes. This recombination event was, in two cases, associated with the loss of one allele of the chromosomal copy. A novel rearrangement point was found in a mutant where the amplicon was created by recombination between two 541 bp direct repeats surrounding the P-glycoprotein gene present at the H locus. It is also at one of these repeats that an H circle with large inverted duplications was formed. We propose that the presence of repeated sequences in the H locus facilitates the amplification of the drug resistance genes concentrated in this locus.

Do mutant woodchuck hepatitis viruses play a role in hepatocellular carcinogenesis?

Although hepadnaviruses are implicated in the aetiology of hepatocellular carcinoma, the pathogenic mechanisms involved remain uncertain. Clonally propagated integrations of hepadnaviral DNA into cellular DNA can be demonstrated in most virally induced hepatocellular carcinomas. Integration occurs at random sites in cellular DNA, but the highly preferred sites in viral DNA are adjacent to the directly repeated sequence, DR1, less often DR2, or in the cohesive overlap region. Integrants invariably contain simple deletions or complex rearrangements that have been thought to occur after integration. We report here the detection, in the serum of woodchucks with hepatocellular carcinoma, of mutant woodchuck hepatitis viruses that are strikingly similar to the rearranged genomes found previously as integrated sequences in cellular DNA. Of the four mutants studied, two had large inverted duplications, one a 219 nucleotide direct duplication, and one a 219 nucleotide deletion. Virus-virus DNA junctions occurred either adjacent to DR1 or DR2 or in the cohesive overlap region at topoisomerase I cleavage sites. Thus, it is possible that rearrangement of the hepadnavirus genome precedes integration of viral DNA into cellular DNA and that mutant genomes that are preferentially integrated into cellular DNA have an aetiological role in hepatocarcinogenesis.

Characterization of an inversion duplication of the short arm of chromosome 8 by fluorescent in situ hybridization.

A de novo chromosome aberration in a woman with severe mental retardation and minor anomalies has been characterized cytogenetically. The patient's karyotype was described as 46, XX, inv dup (8)(p12-->p23.1). Previous Southern blot dosage studies with the marker locus D8S7 demonstrated that the patient was monosomic for this locus, suggesting that the rearrangement generated a duplication-deficiency chromosome. We have reinvestigated this patient using fluorescent in situ hybridization with chromosome 8 cosmids and an Alu-PCR product specific for 8p. These studies have confirmed directly that the duplicated chromosome also has undergone deletion.

Gene amplification accompanied by the loss of a chromosome containing the native allele and the appearance of the amplified DNA at a new chromosomal location.

The organization of amplified DNA in mammalian cells in the form of inverted repeats rather than tandem repeats was first observed and studied in the 3B rat cell line. The structure and chromosomal location of the amplified inverted duplications in this cell line have been further analyzed by cloning, long-range mapping, and fluorescence in situ hybridization. The amplification unit is at least 450 kilobases in size and all of the amplicons are located in a single chromosomal location of approximately 10 or 11 megabases. No heterogeneity in either size or molecular structure is detected between the 3B amplicons, indicating that the 20- to 40-fold amplification occurred in a single event and not through a series of events, which would result in heterogeneity among the amplicons. Thus the amplification in 3B cells may reflect more closely the situation seen in tumors containing amplified oncogenes/protooncogenes than the amplifications present in cell lines after multiple selections with cytotoxic drugs. The progenitor Rat-2 cell line contains three alleles of the region of DNA that is amplified in 3B cells; two are located on the two normal homologues of rat chromosome 2 and the third is at the equivalent position on a marker chromosome, der(3)t(2;3). 3B cells contain only one of the two normal homologues of chromosome 2 in addition to chromosome der(3)t(2;3). All of the amplified DNA is located on a new marker chromosome, M2, whose amplified DNA region does not resemble chromosome 2. These results are consistent with the amplification model proposed by Passananti et al. [Passananti, C., Davies, B., Ford, M. & Fried, M. (1987) EMBO J. 6, 1697-1703], in which the excision from a chromosome of the DNA to be amplified results in the loss of rearrangement of that chromosome. In this model the excised DNA can be amplified extrachromosomally during a single S phase before becoming stabilized by integration into a chromosome, probably at a different location to that of its unamplified allele.

Apparent Opitz BBBG syndrome with a partial duplication of 5p

We describe a patient with a paracentric inversion and partial duplication of chromosome 5p. In addition this patient presented with a malformation pattern consistent with Opitz BBBG syndrome. This implies that the gene responsible for this single gene defect may be located within this duplicated region.

Risk Determination in Cases with an Extra Minute Chromosome: Prenatal Diagnosis

The interpretation of an extra minute chromosome (EMC) detected in utero presents both diagnostic and prognostic problems. Two EMC cases are presented and a flow-chart guideline is proposed for the determination of developmental risk. A prenatally detected EMC was familially inherited through a normal phenotype father. The pregnancy was continued and a normal baby boy was delivered. At one year his development was within normal ranges. The second EMC case was a mentally retarded girl examined at 4 years and 7 months of age. The EMC was identified with a combination of various staining techniques to be an inversion duplication (15). The importance of differential diagnosis of EMCs by DA/DAPI staining is emphasized.

Direct and inverted DNA repeats associated with P-glycoprotein gene amplification in drug resistant Leishmania.

The H circle of Leishmania species contains a 30 kb inverted duplication separated by two unique DNA segments, a and b. The corresponding H region of chromosomal DNA has only one copy of the duplicated DNA. We show here that the chromosomal segments a and b are flanked by inverted repeats (198 and 1241 bp) and we discuss how these repeats could lead to formation of H circles from chromosomal DNA. Selection of Leishmania tarentolae for methotrexate resistance indeed resulted in the de novo formation of circles with long inverted duplication, but two mutants selected for arsenite resistance contained new H region plasmids without such duplications. One of these plasmids appears due to a homologous recombination between two P-glycoprotein genes with a high degree of sequence homology. Our results show how the same DNA region in Leishmania may be amplified to give plasmids with or without long inverted duplications and apparently by different mechanisms.