Array painting using microdissected chromosomes to map chromosomal breakpoints

Molecular characterization of breakpoints of chromosomal rearrangements is a successful strategy for the identification of candidate disease genes. Mapping translocation breakpoints and rearranged chromosomal boundaries is labor intensive and/or time consuming. Here, we present a novel and rapid procedure to map such chromosomal breakpoints by hybridizing amplified microdissection derived DNA of aberrant chromosomes to arrays containing genomic clones. We illustrate the potential of the technique by molecularly delineating the breakpoints in five small supernumerary marker chromosomes (sSMC) and mapping the breakpoints of five different chromosomal translocations.     

How to narrow down chromosomal breakpoints in small and large derivative chromosomes – a new probe set

Here a new fluorescence in situ hybridization (FISH-) based probe set is presented and its possible applications are highlighted in 34 exemplary clinical cases. The so-called pericentric-ladder-FISH (PCL-FISH) probe set enables a characterization of chromosomal breakpoints especially in small supernumerary marker chromosomes (sSMC), but can also be applied successfully in large inborn or acquired derivative chromosomes. PCL-FISH was established as 24 different chromosome-specific probe sets and can be used in two- up multicolor-FISH approaches. PCL-FISH enables the determination of a chromosomal breakpoint with a resolution between 1 and ～10 megabasepairs and is based on locus-specific bacterial artificial chromosome (BAC) probes. Results obtained on 29 sSMC cases and five larger derivative chromosomes are presented and discussed. To confirm the reliability of PCL-FISH, eight of the 29 sSMC cases were studied by array-comparative genomic hybridization (aCGH); the used sSMC-specific DNA was obtained by glass-needle based microdissection and DOP-PCR-amplification. Overall, PCL-FISH leads to a better resolution than most FISH-banding approaches and is a good tool to narrow down chromosomal breakpoints.     

Paint-assisted microdissection-FISH: Rapid and simple mapping of translocation breakpoints in the embryonal rhabdomyosarcoma cell line RD.

BACKGROUND: Spectral karyotyping and multiple fluorophore fluorescence in situ hybridisation (M-FISH) facilitate identification of inter-chromosomal rearrangements, but are of low cytogenetic resolution in mapping translocation breakpoints. Reverse chromosome painting yields increased cytogenetic information but isolation of aberrant chromosomes is technically difficult. We have developed the technique of paint-assisted microdissection FISH (PAM-FISH), which enables microdissection of aberrant chromosomes to be carried out easily and rapidly using relatively simple apparatus. METHODS: A selected chromosome paint is hybridised to abnormal metaphases to label a chromosome of interest, which is then microdissected, amplified, labelled by polymerase chain reaction (PCR), and reverse painted onto extended normal metaphases. RESULTS: PAM-FISH was used to reassess structural chromosomal abnormalities identified by molecular cytogenetics in the rhabdomyosarcoma cell line RD. PAM-FISH improved the analysis of virtually all structural abnormalities, identifying six novel translocations and indicating that seven previously described rearrangements were in fact not present in RD. Accuracy of the breakpoint mapping obtained was confirmed by bacterial artificial chromosome-FISH. CONCLUSIONS: PAM-FISH is ideally suited to analysis of tumour metaphases as it is not affected by poor chromosome morphology. Reagents generated by PAM-FISH are also suitable for other investigations, such as mapping using sequence tagged-site PCR or genomic microarrays. PAM-FISH is technically straightforward and could readily be adopted in a routine cytogenetics laboratory for accurate high-throughput analysis of chromosome breakpoints.     

Multicolor chromosome banding (MCB) with YAC/BAC-based probes and region-specific microdissection DNA libraries

Multicolor chromosome banding (MCB) allows the delineation of chromosomal regions with a resolution of a few megabasepairs, i.e., slightly below the size of most visible chromosome bands. Based on the hybridization of overlapping region-specific probe libraries, chromosomal subregions are hybridized with probes that fluoresce in distinct wavelength intervals, so they can be assigned predefined pseudo-colors during the digital imaging and visualization process. The present study demonstrates how MCB patterns can be produced by region-specific microdissection derived (mcd) libraries as well as collections of yeast or bacterial artificial chromosomes (YACs and BACs, respectively). We compared the efficiency of an mcd library based approach with the hybridization of collections of locus-specific probes (LSP) for fluorescent banding of three rather differently sized human chromosomes, i.e., chromosomes 2, 13, and 22. The LSP sets were comprised of 107 probes specific for chromosome 2, 82 probes for chromosome 13, and 31 probes for chromosome 22. The results demonstrated a more homogeneous coverage of chromosomes and thus, more desirable banding patterns using the microdissection library-based MCB. This may be related to the observation that chromosomes are difficult to cover completely with YAC and/or BAC clones as single-color fluorescence in situ hybridization (FISH) experiments showed. Mcd libraries, on the other hand, provide high complexity probes that work well as region-specific paints, but do not readily allow positioning of breakpoints on genetic or physical maps as required for the positional cloning of genes. Thus, combinations of mcd libraries and locus-specific large insert DNA probes appear to be the most efficient tools for high-resolution cytogenetic analyses.     

应用端粒区带涂染探针检测染色体微小结构重排

为了评估染色体端粒区带涂染探针在遗传诊断的应用价值,应用显微切割获得的11q、12q和22q等3个染色体端粒区涂染探针（11q23.3→qter,12q24.1→qter,22q13.1→qter）,通过荧光原位杂交技术分析两个疑有染色体末端微小易位的习惯性流产病例。结果显示,病例1和病例2分别为t(11;12)和t(11;22)长臂末端间的微小易位,结合G显带技术确定断裂位点位于11q23.3、12q24.1、22q13.1。结果表明特异性染色体端粒区带探针可以确诊染色体末端区域的微小结构异常,可作为一种检出隐匿易位携带者并确定断裂位点的方法。     

Interstitial deletion of chromosome 6q: Precise definition of the breakpoints by microdissection,DNA amplification,and reverse painting

Routine chromosomal analysis using GTG-banding alone showed a mosaic terminal deletion of 6q in a 14-week-old boy with developmental retardation, facial anomalies, agenesis of corpus callosum, cleft palate, hypotonia, short neck and pterygium colli, and minor anomalies of hands and feet. Discrepancies between the clinical findings on our patient and those described in the literature on patients having terminal deletions led to a more precise analysis of the karyotype. Reverse painting was performed on normal G-banded metaphases for exact determination of the breakpoints and on metaphases of the patient for evaluation of mosaicism. A DNA library that was obtained by microdissection of three deleted chromosomes 6 was used as a painting probe. Subsequent DNA amplification was performed with the help of topoisomerase-pretreated degenerate oligonucleotide primers. Unexpectedly, the hybridization pattern on normal metaphase chromosomes revealed an interstitial deletion with breakpoints at 6q25.1 and 6q27 instead of a terminal deletion. Hybridization on metaphases of the patient showed one deleted chromosome 6 in all metaphases analyzed at a higher resolution rather than mosaicism as previously assumed [karyotype, 46,XY,del(6)(q25.1→q27)]. We assume that in the single cases of 6q^– described in the literature the deletions are misclassified. This might be due to difficulties in distinguishing between interstitial and terminal deletions at 6q and in precisely defining chromosomal breakpoints after GTG-banding alone. Received: 29 November 1995 / Revised: 15 January 1996     

Genetic recombination in plants

Meiotic recombination generates novel allelic arrays on chromosomes. Recent experiments have revealed an extraordinarily nonrandom distribution of recombination breakpoints along the lengths of plant chromosomes; for example, recombination breakpoints often resolve within genic sequences, and thereby generate novel alleles. The mechanism by which recombination breakpoints are determined is an area of active investigation. In addition, recent developments are providing recombination-based technologies for creating targeted alterations in the architecture of plant genomes.     

Three autosomal chromosome translocations associated with repeated early embryonic loss (REEL) in the domestic horse (Equus caballus)

Repeated early embryonic loss (REEL) represents a considerable economic loss to the horse industry. Mares that experience REEL may be overlooked as potential carriers of a chromosome abnormality. Here we report three different autosomal translocations in Thoroughbred mares presented for chromosome analysis because of REEL. The karyotypes were 64,XX,t(1;21), 64,XX,t(16;22), and 64,XX,t(4;13), respectively. In order to confirm the chromosomes involved in the translocations, to map the breakpoints, and to determine if the translocations were reciprocal, genes surrounding the breakpoints were identified using existing maps and from the newly assembled horse genome sequence. Bacterial artificial chromosomes containing the genes of interest were identified and mapped to the translocation chromosomes by fluorescence in situ hybridization (FISH). FISH confirmed that the t(16;22) and t(4;13) translocations were reciprocal, while the t(1;21) was not. The breakpoints on horse chromosomes 1 and 16 appear to be the same or near breakpoints previously identified in translocations. These breakpoints are at the fusion boundary of human chromosomes 10 and 15 on horse chromosome 1 and at human chromosome 3p and 3q on horse chromosome 16. These sites may represent ancient breakpoints reused during equid evolution. Overall, chromosome abnormalities may have a greater influence on mare fertility than previously known. Thus, it is important to karyotype subfertile mares exhibiting REEL.     

Evolution versus constitution: differences in chromosomal inversion

We compared the chromosomal breakpoints of evolutionary conserved and constitutional inversions. Multicolor banding and human-specific bacterial artificial chromosomes were applied to map the breakpoints of constitutional pericentric inversions on human chromosomes 2 and 9. For the first time, we present a high-resolution analysis of the breakpoint regions, which are characterized by gene destitution, co-localization with fragile sites, multitude repeats as well as pseudogenes and, remarkably, a large sequence homology to the opposite breakpoint. In contrast, evolutionary inversion breakpoints lack such extensive cross-hybridizing regions and are often associated with fragile sites of the genome and low-copy repeats. These molecular characteristics gave evidence for different types of inversion formation and indicate that evolutionary inversions cannot originate from constitutional inversions like those of chromosomes 2 and 9. Finally, the constitutional inversion breakpoints were investigated on three different great ape species and on four test persons each bearing the same cytogenetically determined inversion on chromosomes 2 and 9, respectively. Our data indicate the existence of different molecular breakpoints for the two variant chromosomes.     

Molecular definition of high-resolution multicolor banding probes: first within the human DNA sequence anchored FISH banding probe set.

Fluorescence in situ hybridization (FISH) banding approaches are standard for the exact characterization of simple, complex, and even cryptic chromosomal aberrations within the human genome. The most frequently applied FISH banding technique is the multicolor banding approach, also abbreviated as m-band, MCB, or in its whole genomic variant multitude MCB (mMCB). MCB allows the differentiation of chromosome region-specific areas at the GTG band and sub-band level and is based on region-specific microdissection libraries, producing changing fluorescence intensity ratios along the chromosomes. The latter are used to assign different pseudocolors to specific chromosomal regions. Here we present the first bacterial artificial chromosome (BAC) array comparative genomic hybridization (aCGH) mapped, comprehensive, genome-wide human MCB probe set. All 169 region-specific microdissection libraries were characterized in detail for their size and the regions of overlap. In summary, the unique possibilities of the MCB technique to characterize chromosomal breakpoints in one FISH experiment are now complemented by the feature of being anchored within the human DNA sequence at the BAC level.     

The sex-determining gene tra of Drosophila: molecular cloning and transformation studies.

In Drosophila, the primary signal for sex determination is given by the ratio of X chromosomes to sets of autosomes (X:A). The primary signal is read by a key gene (Sxl) and transmitted down to the differentiation genes by the subordinate control genes tra, tra-2, ix and dsx. Mutations in tra transform chromosomal females (X/X; tra/tra) into sterile males (pseudomales). We have cloned the tra region by microdissection and chromosomal walking. We identified the gene using deficiency breakpoints, DNA aberrations in three different alleles of tra and by P-mediated transformation. A 3.8-kb fragment perfectly rescued the mutant phenotype of X/X; tra/tra flies, showing that it contained all the necessary information to restore female-specific functions in the mutant flies. We present evidence that most of the function of tra can be provided by a subsegment of 2 kb that is differentially transcribed or processed in males and females.     

The sites of radiation induced-breakage in human lymphocyte chromosomes, determined by quinacrine fluorescence

We have examined the distribution of 298 radiation-induced breakpoints in human chromosomes by quinacrine fluorescence. Very little damage was discovered which would not have been detectable with simple staining methods. The data suggest an excessive number of breaks in chromosomes 3 and a deficit in No. 16. The participation of chromosomes in rearrangements appears to be proportional to chromosome length. Within chromosome arms, breakpoints are selectively placed in telomeric regions.     

Low-pass single-chromosome sequencing of human small supernumerary marker chromosomes (sSMCs) and <Emphasis Type="Italic">Apodemus</Emphasis> B chromosomes

Supernumerary chromosomes sporadically arise in many eukaryotic species as a result of genomic rearrangements. If present in a substantial part of species population, those are called B chromosomes, or Bs. This is the case for 70 mammalian species, most of which are rodents. In humans, the most common types of extra chromosomes, sSMCs (small supernumerary marker chromosomes), are diagnosed in approximately 1 of 2000 postnatal cases. Due to low frequency in population, human sSMCs are not considered B chromosomes. Genetic content of both B-chromosomes and sSMCs in most cases remains understudied. Here, we apply microdissection of single chromosomes with subsequent low-pass sequencing on Ion Torrent PGM and Illumina MiSeq to identify unique and repetitive DNA sequences present in a single human sSMC and several B chromosomes in mice Apodemus flavicollis and Apodemus peninsulae. The pipeline for sequencing data analysis was made available in Galaxy interface as an addition to previously published command-line version. Human sSMC was attributed to the proximal part of chromosome 15 long arm, and breakpoints leading to its formation were located into satellite DNA arrays. Genetic content of Apodemus B chromosomes was species-specific, and minor alterations were observed in both species. Common features of Bs in these Apodemus species were satellite DNA and ERV enrichment, as well as the presence of the vaccinia-related kinase gene Vrk1. Understanding of the non-essential genome elements content provides important insights into genome evolution in general.     

Nonrandom distribution of chromosomal aberrations induced by three chemicals

The G-band locations of 3244 breakpoints induced by cis-platinum (II) diamminedichloride (PDD), 1460 breakpoints induced by cytosine arabinoside (ara-C), and 1257 breakpoints induced by triethylenemelamine (TEM) in human lymphocyte chromosomes were identified. The breakpoints induced by each of these chemicals demonstrated a significantly nonrandom distribution within the human karyotype. The overall pattern of the interarm distribution was dependent upon the chemical used, but certain chromosomes arms exhibited similar responses to all 3 chemicals. Comparison of the frequencies of breakpoints within individual G-bands indicated that (1) certain bands were susceptible to damage induced by all 3 chemicals; (2) certain bands were resistant to damage by all 3 chemicals; (3) certain bands demonstrated variable susceptibility to induced damage dependent upon the chemical agent; and (4) other bands demonstrated near expected frequencies of damage (by length) to all 3 agents.     

Microdissection-derived murine mcb probes from somatic cell hybrids.

The multicolor-banding (mcb) technique is a fluorescence in situ hybridization (FISH)-banding approach, which is based on region-specific microdissection libraries producing changing fluorescence intensity ratios along the chromosomes. The latter are used to assign different pseudocolors to specific chromosomal regions. Here we present the first three available mcb-probe sets for the Mus musculus chromosomes 3, 6, and 18. In the present work, the creation of the microdissection libraries was done for the first time on mouse/human somatic cell hybrids. During creation of the mcb-probes, the latter enabled an unambiguous identification of the, otherwise in GTG-banding, hardly distinguishable murine chromosomes.     

黑麦B染色体端粒相关序列的克隆

利用显微切割技术,分离了黑麦（ＳｅｃａｌｅｃｅｒｅａｌＬ．）１０个Ｂ染色体短臂端部片段,并利用寡核苷酸引物（ＣＣＣＴＡＡＡ）３及新建立的二级单引物序列ＰＣＲ扩增法,扩增了黑麦Ｂ染色体端粒相关序列。染色体原位杂交实验将ＰＣＲ产物定位于Ｂ染色体短臂末端,多数Ａ染色体末端也显示清晰的杂交信号。部分ＰＣＲ产物克隆到ｐＵＣ１９载体中,对其中一个克隆子ｐｐ３的序列分析结果表明,它与玉米亚端部克隆子ｐＢＦ２６６部分区域的同源性为９２％。就目前资料检索,黑麦、玉米端粒相关序列具有高度同源性还未见报道。对这一实验设计在构建高密度ＲＦＬＰ图谱中的应用进行了探讨     

Spontaneous chromosomal rearrangements in cultivated and wild barleys

Summary Four of 1,240 cultivated barley lines collected from different regions of the world and 3 of 120 lines of wild barley, Hordeum spontaneum C. Koch, carry spontaneous reciprocal translocations. Break-point positions and rearrangements in the interchanged chromosomes have been examined by both test crosses and Giemsa banding techniques. The four translocation lines in cultivated barley were all of Ethiopian origin and have the same translocation involving chromosomes 2 and 4. The breakpoints are at the centromeres of both chromosomes, resulting in interchanged chromosomes 2S+4S and 2L+4L (S=short arm, L=long arm). A wild barley line, Spont.II, also has translocated chromosomes 2 and 4 which are broken at the centromeres. The resultant chromosomes are, however, 2S+4L and 2L+4S. Another wild barley line, Spont.S-4, has interchanged chromosomes with breakpoints in the short arm of chromosome 3 and the long arm of chromosome 7. In addition, this line has a paracentric inversion in the short arm of chromosome 7 that includes a part of nucleolar constriction, resulting in two tandemly arranged nucleolar constrictions. The third wild barley line, Spont.S-7, has interchanged chromosomes with breakpoints in the long arms of both chromosomes 3 and 6. The translocated chromosome 3 is metacentric and the translocated chromosome 6 has a long arm similar in length to the long arm of chromosome 7.     

Structural analysis of aberrant chromosomes that occur spontaneously in diploid Saccharomyces cerevisiae: retrotransposon Ty1 plays a crucial role in chromosomal rearrangements.

The structural analysis of aberrant chromosomes is important for our understanding of the molecular mechanisms underlying chromosomal rearrangements. We have identified a number of diploid Saccharomyces cerevisiae clones that have undergone loss of heterozygosity (LOH) leading to functional inactivation of the hemizygous URA3 marker placed on the right arm of chromosome III. Aberrant-sized chromosomes derived from chromosome III were detected in approximately 8% of LOH clones. Here, we have analyzed the structure of the aberrant chromosomes in 45 LOH clones with a PCR-based method that determines the ploidy of a series of loci on chromosome III. The alterations included various deletions and amplifications. Sequencing of the junctions revealed that all the breakpoints had been made within repeat sequences in the yeast genome, namely, MAT-HMR, which resulted in intrachromosomal deletion, and retrotransposon Ty1 elements, which were involved in various translocations. Although the translocations involved different breakpoints on different chromosomes, all breakpoints were exclusively within Ty1 elements. Some of the resulting Ty1 elements left at the breakpoints had a complex construction that indicated the involvement of other Ty1 elements not present at the parental breakpoints. These indicate that Ty1 elements are crucially involved in the generation of chromosomal rearrangements in diploid yeast cells.     

Preferential Breakpoints in the Recovery of Broken Dicentric Chromosomes in Drosophila melanogaster

We designed a system to determine whether dicentric chromosomes in Drosophila melanogaster break at random or at preferred sites. Sister chromatid exchange in a Ring-X chromosome produced dicentric chromosomes with two bridging arms connecting segregating centromeres as cells divide. This double bridge can break in mitosis. A genetic screen recovered chromosomes that were linearized by breakage in the male germline. Because the screen required viability of males with this X chromosome, the breakpoints in each arm of the double bridge must be closely matched to produce a nearly euploid chromosome. We expected that most linear chromosomes would be broken in heterochromatin because there are no vital genes in heterochromatin, and breakpoint distribution would be relatively unconstrained. Surprisingly, approximately half the breakpoints are found in euchromatin, and the breakpoints are clustered in just a few regions of the chromosome that closely match regions identified as intercalary heterochromatin. The results support the Laird hypothesis that intercalary heterochromatin can explain fragile sites in mitotic chromosomes, including fragile X. Opened rings also were recovered after male larvae were exposed to X-rays. This method was much less efficient and produced chromosomes with a strikingly different array of breakpoints, with almost all located in heterochromatin. A series of circularly permuted linear X chromosomes was generated that may be useful for investigating aspects of chromosome behavior, such as crossover distribution and interference in meiosis, or questions of nuclear organization and function.     

Chromosomal aberrations: formation,identification and distribution

Chromosomal aberrations (CA) are the microscopically visible part of a wide spectrum of DNA changes generated by different repair mechanisms of DNA double strand breaks (DSB). The method of fluorescence in situ hybridisation (FISH) has uncovered unexpected complexities of CA and this will lead to changes in our thinking about the origin of CA. The inter- and intrachromosomal distribution of breakpoints is generally not random. CA breakpoints occur preferentially in active chromatin. Deviations from expected interchromosomal distributions of breakpoints may result from the arrangement of chromosomes in the interphase nucleus and/or from different sensitivities of chromosomes with respect to the formation of CA. Telomeres and interstitial telomere repeat like sequences play an important role in the formation of CA. Subtelomeric regions are hot spots for the formation of symmetrical exchanges between homologous chromatids and cryptic aberrations in these regions are associated with human congenital abnormalities.