Entamoeba histolytica DNA methyltransferase (Ehmeth) is a nuclear matrix protein that binds EhMRS2, a DNA that includes a scaffold/matrix attachment region (S/MAR)

The protozoan parasite Entamoeba histolytica express a cytosine-5 DNA methyltransferase (Ehmeth) that belongs to the DNMT2 protein family. The biological function of members of this DNMT2 family is unknown. In the present study, we have demonstrated that Ehmeth is a nuclear matrix protein. Indeed, we showed by south-western analysis and yeast one-hybrid system that Ehmeth binds to EhMRS2, a DNA element which contains the eukaryotic consensus scaffold/matrix attachment regions (S/MAR) bipartite recognition sequences. S/MARs have been implicated in a variety of important functions, such as genome organization and gene expression. The methylation status of cytosine located within EhMRS2 was analyzed by bisulfite genomic sequencing. We observed the presence of methylated cytosine within the 3'-end of EhMRS2. These data provide the first evidence that a member of the DNMT2 family interacts with a S/MAR containing DNA element.     

In silico prediction of scaffold/matrix attachment regions in large genomic sequences

Scaffold/matrix attachment regions (S/MARs) are essential regulatory DNA elements of eukaryotic cells. They are major determinants of locus control of gene expression and can shield gene expression from position effects. Experimental detection of S/MARs requires substantial effort and is not suitable for large-scale screening of genomic sequences. In silico prediction of S/MARs can provide a crucial first selection step to reduce the number of candidates. We used experimentally defined S/MAR sequences as the training set and generated a library of new S/MAR-associated, AT-rich patterns described as weight matrices. A new tool called SMARTest was developed that identifies potential S/MARs by performing a density analysis based on the S/MAR matrix library (http://www.genomatix.de/cgi-bin/smartest_pd/smartest.pl). S/MAR predictions were evaluated by using six genomic sequences from animal and plant for which S/MARs and non-S/MARs were experimentally mapped. SMARTest reached a sensitivity of 38% and a specificity of 68%. In contrast to previous algorithms, the SMARTest approach does not depend on the sequence context and is suitable to analyze long genomic sequences up to the size of whole chromosomes. To demonstrate the feasibility of large-scale S/MAR prediction, we analyzed the recently published chromosome 22 sequence and found 1198 S/MAR candidates.     

DNA polymorphism and epigenetic marks modulate the affinity of a scaffold/matrix attachment region to the nuclear matrix

Mechanisms that regulate attachment of the scaffold/matrix attachment regions (S/MARs) to the nuclear matrix remain largely unknown. We have studied the effect of simple sequence length polymorphism (SSLP), DNA methylation and chromatin organization in an S/MAR implicated in facioscapulohumeral dystrophy (FSHD), a hereditary disease linked to a partial deletion of the D4Z4 repeat array on chromosome 4q. This FSHD-related nuclear matrix attachment region (FR-MAR) loses its efficiency in myoblasts from FSHD patients. Three criteria were found to be important for high-affinity interaction between the FR-MAR and the nuclear matrix: the presence of a specific SSLP haplotype in chromosomal DNA, the methylation of one specific CpG within the FR-MAR and the absence of histone H3 acetylated on lysine 9 in the relevant chromatin fragment.     

Molecular analysis of repetitive DNA elements from Entamoeba histolytica,which encode small RNAs and contain matrix/scaffold attachment recognition sequences

We have isolated two DNA elements-Eh MRS1 and Eh MRS2-from Entamoeba histolytica, which contain the eukaryotic consensus Scaffold/Matrix Attachment Region (S/MAR) bipartite recognition sequences. Both these sequences bind to high salt extractable nuclear proteins and insoluble nuclear matrix proteins in E. histolytica HM1:IMSS, suggesting that the predicted S/MAR recognition sequences may indeed function as scaffold attachment regions in E. histolytica. Sequence analysis shows that Eh MRS1 and Eh MRS2 contain internal tandem repeats ranging from units of 8-11bp and are themselves present as independent arrays of tandemly repeating units of approximately 1100bp each. Eh MRS1 and Eh MRS2 are localised on different chromosomes in E. histolytica HM1:IMSS. Both Eh MRS1 and Eh MRS2 also code for small molecular weight RNAs of unknown function. Thus, two unique sequences-Eh MRS1 and Eh MRS2-demonstrate very similar properties, suggesting that they belong to a superfamily of genomic elements, which may function as scaffold or matrix attachment sites in Entamoeba.     

Development and validation of a CGH microarray for clinical cytogenetic diagnosis.

PURPOSE: We developed a microarray for clinical diagnosis of chromosomal disorders using large insert genomic DNA clones as targets for comparative genomic hybridization (CGH). METHODS: The array contains 362 FISH-verified clones that span genomic regions implicated in over 40 known human genomic disorders and representative subtelomeric clones for each of the 41 clinically relevant human chromosome telomeres. Three or four clones from almost all deletion or duplication genomic regions and three or more clones for each subtelomeric region were included. We tested chromosome microarray analysis (CMA) in a masked fashion by examining genomic DNA from 25 patients who were previously ascertained in a genetic clinic and studied by conventional cytogenetics. A novel software package implemented in the R statistical programming language was developed for normalization, visualization, and inference. RESULTS: The CMA results were entirely consistent with previous cytogenetic and FISH findings. For clone by clone analysis, the sensitivity was estimated to be 96.7% and the specificity was 99.1%. Major advantages of this selected human genome array include the following: interrogation of clinically relevant genomic regions, the ability to test for a wide range of duplication and deletion syndromes in a single analysis, the ability to detect duplications that would likely be undetected by metaphase FISH, and ease of confirmation of suspected genomic changes by conventional FISH testing currently available in the cytogenetics laboratory. CONCLUSION: The array is an attractive alternative to telomere FISH and locus-specific FISH, but it does not include uniform coverage across the arms of each chromosome and is not intended to substitute for a standard karyotype. Limitations of CMA include the inability to detect both balanced chromosome changes and low levels of mosaicism.     

Construction of a 2-Mb resolution BAC microarray for CGH analysis of canine tumors

Recognition of the domestic dog as a model for the comparative study of human genetic traits has led to major advances in canine genomics. The pathophysiological similarities shared between many human and dog diseases extend to a range of cancers. Human tumors frequently display recurrent chromosome aberrations, many of which are hallmarks of particular tumor subtypes. Using a range of molecular cytogenetic techniques we have generated evidence indicating that this is also true of canine tumors. Detailed knowledge of these genomic abnormalities has the potential to aid diagnosis, prognosis, and the selection of appropriate therapy in both species. We recently improved the efficiency and resolution of canine cancer cytogenetics studies by developing a small-scale genomic microarray comprising a panel of canine BAC clones representing subgenomic regions of particular interest. We have now extended these studies to generate a comprehensive canine comparative genomic hybridization (CGH) array that comprises 1158 canine BAC clones ordered throughout the genome with an average interval of 2 Mb. Most of the clones (84.3%) have been assigned to a precise cytogenetic location by fluorescence in situ hybridization (FISH), and 98.5% are also directly anchored within the current canine genome assembly, permitting direct translation from cytogenetic aberration to DNA sequence. We are now using this resource routinely for high-throughput array CGH and single-locus probe analysis of a range of canine cancers. Here we provide examples of the varied applications of this resource to tumor cytogenetics, in combination with other molecular cytogenetic techniques.     

A novel chromatin immunoprecipitation and array (CIA) analysis identifies a 460-kb CENP-A-binding neocentromere DNA

Centromere protein A (CENP-A) is an essential histone H3-related protein that constitutes the specialized chromatin of an active centromere. It has been suggested that this protein plays a key role in the epigenetic marking and transformation of noncentromeric genomic DNA into functional neocentromeres. Neocentromeres have been identified on more than two-thirds of the human chromosomes, presumably involving different noncentromeric DNA sequences, but it is unclear whether some generalized sequence properties account for these neocentromeric sites. Using a novel method combining chromatin immunoprecipitation and genomic array hybridization, we have identified a 460-kb CENP-A-binding DNA domain of a neocentromere derived from the 20p12 region of an invdup (20p) human marker chromosome. Detailed sequence analysis indicates that this domain contains no centromeric alpha-satellite, classical satellites, or other known pericentric repetitive sequence motifs. Putative gene loci are detected, suggesting that their presence does not preclude neocentromere formation. The sequence is not significantly different from surrounding non-CENP-A-binding DNA in terms of the prevalence of various interspersed repeats and binding sites for DNA-interacting proteins (Topoisomerase II and High-Mobility-Group protein I). Notable variations include a higher AT content similar to that seen in human alpha-satellite DNA and a reduced prevalence of long terminal repeats (LTRs), short interspersed repeats (SINEs), and Alus. The significance of these features in neocentromerization is discussed.     

Development of a high-density pericentromeric region BAC clone set for the detection and characterization of small supernumerary marker chromosomes by array CGH.

PURPOSE: Small supernumerary marker chromosomes are centric chromosomal segments that, by definition, cannot be characterized unambiguously by conventional chromosome banding. Marker chromosomes are of particular interest in clinical cytogenetics because they are nearly 10 times more frequent in individuals with mental retardation (0.426%) than in the normal population (0.043%). However, they are often found in only a small percentage of cells, making them difficult to detect and characterize in a diagnostic setting. We designed, constructed, and employed a bacterial artificial chromosome (BAC)-based microarray to demonstrate the utility of array-based comparative genomic hybridization (array CGH) for detecting and characterizing marker chromosomes in clinical diagnostic specimens. METHODS: We constructed a high-density microarray using 974 BAC clones that were mapped by fluorescence in situ hybridization and cover approximately 5 Mb of the most proximal unique sequence adjacent to the centromere on all 43 unique pericentromeric regions of the human genome (excluding the acrocentric short arms). This array was used to further characterize 20 previously identified marker chromosomes that were originally found with either conventional chromosome analysis or a targeted microarray. RESULTS: The enhanced coverage of this pericentromeric array not only identified the chromosomal origin of each marker in 15 cases, it also distinguished between the involvement of the short arm and/or the long arm of each chromosome, defined the sizes of many of the markers, and revealed complex rearrangements or multiple markers in single individuals. However, in five cases, the markers could not be identified by this assay, most likely because of very low levels of mosaicism and/or their small size and lack of detectable euchromatin. The expanded coverage of the pericentromeric regions represented on the array was adequate to refine the breakpoints in two-thirds of all cases in which a marker chromosome was identified by this assay. CONCLUSIONS: This study demonstrates the utility of array CGH in the detection and characterization of mosaic marker chromosomes. Because approximately one-third of the markers characterized in this study involved more unique sequence than that represented on this array, additional pericentromeric coverage may be even more valuable. We anticipate that this will allow detailed characterization of small supernumerary marker chromosomes that will greatly facilitate phenotype/genotype correlations and play a valuable role in the diagnosis and medical management of both pre- and postnatal cases in which marker chromosomes have been identified.     

Detection of deleted genomic DNA using a semiautomated computational analysis of GeneChip data

Genomic diversity within and between populations is caused by single nucleotide mutations, changes in repetitive DNA systems, recombination mechanisms, and insertion and deletion events. The contribution of these sources to diversity, whether purely genetic or of phenotypic consequence, can only be investigated if we have the means to quantitate and characterize diversity in many samples. With the advent of complete sequence characterization of representative genomes of different species, the possibility of developing protocols to screen for genetic polymorphism across entire genomes is actively being pursued. The large numbers of measurements such approaches yield demand that we pay careful attention to the numerical analysis of data. In this paper we present a novel application of an Affymetrix GeneChip to perform genome-wide screens for deletion polymorphism. A high-density oligonucleotide array formatted for mRNA expression and targeted at a fully sequenced 4.4-million-base pair Mycobacterium tuberculosis standard strain genome was adapted to compare genomic DNA. Hybridization intensities to 111,000 probe pairs (perfect complement and mismatch complement) were measured for genomic DNA from a clinical strain and from a vaccine organism. Because individual probe-pair hybridization intensities exhibit limited sensitivity/specificity characteristics to detect deletions, data-analytical methodology to exploit measurements from multiple probes in tandem locations across the genome was developed. The TSTEP (Tandem Set Terminal Extreme Probability) algorithm designed specifically to analyze the tandem hybridization measurements data was applied and shown to discover genomic deletions with high sensitivity. The TSTEP algorithm provides a foundation for similar efforts to characterize deletions in many hybridization measures in similar-sized and larger genomes. Issues relating to the design of genome content screening experiments and the implications of these methods for studying population genomics and the evolution of genomes are discussed.     

Detection of DNA copy number alterations in cancer by array comparative genomic hybridization

Over the past few years, various reliable platforms for high-resolution detection of DNA copy number changes have become widely available. Together with optimized protocols for labeling and hybridization and algorithms for data analysis and representation, this has lead to a rapid increase in the application of this technology in the study of copy number variation in the human genome in normal cells and copy number imbalances in genetic diseases, including cancer. In this review, we briefly discuss specific technical issues relevant for array comparative genomic hybridization analysis in cancer tissues. We specifically focus on recent successes of array comparative genomic hybridization technology in the progress of our understanding of oncogenesis in a variety of cancer types. A third section highlights the potential of sensitive genome-wide detection of patterns of DNA imbalances or molecular portraits for class discovery and therapeutic stratification.     

Detection of multiple gene amplifications in glioblastoma multiforme using array-based comparative genomic hybridization

We have used a new method of genomic microarray to investigate amplification of oncogenes throughout the genome of glioblastoma multiforme (GBM). Array-based comparative genomic hybridization (array CGH) allows for simultaneous examination of 58 oncogenes/amplicons that are commonly amplified in various human cancers. Amplification of multiple oncogenes in human cancers can be rapidly determined in a single experiment. Tumor DNA and normal control DNA were labeled by nick translation with green- and red-tagged nucleotides, respectively. Instead of hybridizing to normal metaphase chromosomes in conventional comparative genomic hybridization (CGH), the probes of the mixed fluorescent labeled DNA were applied to genomic array templates comprised of P1, PAC, and BAC clones of 58 target oncogenes. The baseline for measuring deviations was established by performing a series of independent array CGH using test and reference DNA made from normal individuals. In the present study, we examined fourteen GBMs (seven cell lines and seven tumours) with CGH and array CGH to reveal the particular oncogenes associated with this cancer. High-level amplifications were identified on the oncogenes/amplicons CDK4, GLI, MYCN, MYC, MDM2, and PDGFRA. The highest frequencies of gains were detected on PIK3CA (64.3%), EGFR (57.1%), CSE1L (57.1%), NRAS (50%), MYCN (42.9%), FGR (35.7%), ESR (35.7%), PGY1 (35.7%), and D17S167 (35.7%). These genes are suggested to be involved in the GBM tumorigenesis.     

Target sites for SINE integration in Brassica genomes display nuclear matrix binding activity

Short interspersed nuclear elements (SINEs) are ubiquitous components of complex animal and plant genomes. SINEs are believed to be important players in eukaryotic genome evolution. Studies on SINE integration sites have revealed non-random integration without strict nucleotide sequence requirements for the integration target, suggesting that the targeted DNA might assume specific secondary structures or protein associations. Here, we report that S1 SINE elements in the genomes of Brassica show an interesting preference for matrix attachment regions (MARs). Ten cloned genomic regions were tested for their ability to bind the nuclear matrix both before and after a SINE integration event. Eight of the genomic regions targeted by S1 display strong affinity for the nuclear matrix, while two show weaker binding. The SINE S1 did not display any matrix-binding capacity on its own in either non-methylated or methylated forms. In vivo, an integrated S1 is methylated while the surrounding genomic regions may remain undermethylated or undergo methylation. However, tested genomic regions containing methylated'S1, with or without methylated flanking genomic sequences, were found to vary in their ability to bind the matrix in vitro. These results suggest a possible molecular basis for a preferential targeting of SINEs to MARs and a possible impact of the integration events upon gene and genome function. This revised version was published online in July 2006 with corrections to the Cover Date.     

The origin of chromosome imbalances in neuroblastoma

Many recurrent large-scale chromosome abnormalities associated with poor clinical outcomes have been identified in neuroblastoma, a pediatric tumor that accounts for 15% of childhood cancer deaths. We have previously used high-resolution oligonucleotide array comparative genomic hybridization to map 461 chromosome breakpoints leading to large-scale chromosome imbalances in 56 primary neuroblastoma tumors and cell lines. Here, we analyze the distribution of DNA sequence elements and genomic landmarks found within these breakpoint intervals and in 15,800 randomly generated intervals of similar size. The most consistent finding was that neuroblastoma chromosome breakpoints occur preferentially in GC-rich regions of the genome. It is not unsurprising that these regions have fewer (AT)(n) microsatellite repeat sequences. In addition, chromosome breakpoints occurring in neuroblastoma also appeared to be preferentially associated with ancestral chromosome breakpoint regions on several chromosomes, suggesting that such sites also act as hotspots for chromosome rearrangement in somatic cells. Very little evidence for the enrichment of Alu and other types of repeats in breakpoint intervals was obtained. Overall, our results are consistent with a mechanistic model involving nonhomologous end joining of DNA double-strand breaks that have been generated in a nonrandom manner.     

Detection of single clone deletions using array CGH: identification of submicroscopic deletions in the 22q11.2 deletion syndrome as a model system

Constitutional submicroscopic DNA copy number alterations have been shown to cause numerous medical genetic syndromes, and are suspected to occur in a portion of cases for which the causal events remain undiscovered. Array comparative genomic hybridization (array CGH) allows high-throughput, high-resolution genome scanning for DNA dosage aberrations and thus offers an attractive approach for both clinical diagnosis and discovery efforts. Here we assess this capability by applying array CGH to the analysis of copy number alterations in 44 patients with a phenotype of the 22q11.2 deletion syndrome. Twenty-five patients had the deletion on chromosome 22 characteristic of this syndrome as determined by fluorescence in situ hybridization (FISH). The array measurements were in complete concordance with the FISH analysis, supporting their diagnostic utility. These data show that a genome-scanning microarray has the level of sensitivity and specificity required to prospectively interrogate and identify single copy number aberrations in a clinical setting. We demonstrate that such technology is ideally suited for microdeletion syndromes such as 22q11.2.     

Application of dual-genome oligonucleotidearray-based comparative genomic hybridization to the molecular diagnosis of mitochondrial DNA deletion and depletion syndromes

PurposeMitochondrial disorders constitute a group of clinically and genetically heterogeneous diseases for which molecular diagnosis has been a challenge. The current procedures for diagnosis of mitochondrial DNA deletion and depletion syndromes based on Southern analysis and quantitative polymerase chain reaction are particularly inefficient for determining important parameters of deletion endpoints and percent heteroplasmy. We have developed an improved approach for routine analyses of these disorders in a clinical laboratory.MethodsA custom-designed oligonucleotide array-based comparative genomic hybridization platform was developed to provide both tiled coverage of the entire 16.6-kb mitochondrial genome and high-density coverage of nuclear genes involved in mitochondrial biogenesis and function, for quick evaluation of mitochondrial DNA deletion and depletion.ResultsFor initial validation, the performance of this array was characterized in 20 samples with known mitochondrial DNA deletions and 12 with apparent depletions. All previously known deletions were clearly detected and the break points were correctly identified by the oligonucleotide array-based comparative genomic hybridization, within the limits of resolution of the array. The extent of mitochondrial DNA depletion and the percentage of deletion heteroplasmy were estimated using an automated computational approach that gave results comparable to previous methods. Conclusions from subsequent application of this approach with >300 new clinical samples have been in 100% concordance with those from standard methods. Finally, for one sample, we were able to identify an intragenic deletion in a nuclear gene that was responsible for the observed mitochondrial DNA depletion.ConclusionWe conclude that this custom array is capable of reliably detecting mitochondrial DNA deletion with elucidation of the deletion break points and the percentage of heteroplasmy. In addition, simultaneous detection of the copy number changes in both nuclear and mitochondrial genomes makes this dual genome array of tremendous value in the diagnoses of mitochondrial DNA depletion syndromes.     

DNA copy-number analysis of the 22q11 deletion-syndrome region using array-CGH with genomic and PCR-based targets

Deletions and duplications of genomic segments commonly cause developmental disorders. The resolution and efficiency in diagnosing such gene-dosage alterations can be drastically increased using microarray-based comparative genomic hybridization (array-CGH). However, array-CGH currently relies on spotting genomic clones as targets, which confers severe limitations to the approach including resolution of analysis and reliable gene-dosage assessment of regions with high content of redundant sequences. To improve the methodology for analysis, we compared the use of genomic clones, repeat-free pools of amplified genomic DNA and cDNAs (single and pooled) as targets on the array. For this purpose, we chose q11.2 locus on chromosome 22 as a testing ground. Microdeletions at 22q11 cause birth defects collectively described as the DiGeorge/velocardiofacial syndrome. The majority of patients present 3 Mb typical deletions. Here, we report the construction of a gene-dosage array, covering 6 Mb of 22q11 and including the typically deleted region. We hybridized DNA from six DiGeorge syndrome patients to the array, and show that as little as 11.5 kb non-redundant, repeat-free PCR-generated sequence can be used for reliable detection of hemizygous deletions. By extrapolation, this would allow analysis of the genome with an average resolution of 25 kb. In the case of cDNAs our results indicate that 3.5 kb sequence is necessary for accurate identification of haploid/diploid dosage alterations. Thus, for regions rich in redundant sequences and repeats, such as 22q11, a specifically tailored array-CGH approach is good for gene copy number profiling.     

Non-hotspot-related breakpoints of common deletions in Sotos syndrome are located within destabilised DNA regions

Background: Sotos syndrome (SoS) is a disorder characterised by excessive growth, typical craniofacial features, and developmental retardation. It is caused by haploinsuffiency of NSD1 at 5q35. There is a 3.0 kb recombination hotspot in which the breakpoints of around 80% of SoS patients with a common deletion can be mapped. Objective: To identify deletion breakpoints located outside the SoS recombination hotspot. Methods: A screening system for the directly orientated segments of the SoS LCRs was developed for 10 SoS patients with a common deletion who were negative for the SoS hotspot. Deletion-junction fragments were analysed for DNA duplex stability and their relation to scaffold/matrix attachment regions (S/MARs). These features were compared with the SoS hotspot and recombination hotspots of other genomic disorders. Results: The breakpoint was mapped in four SoS patients, two with a deletion in the maternally derived chromosome. These breakpoint regions were located ∼2.5 kb, ∼9.6 kb, ∼27.2, and ∼27.7 kb telomeric to the SoS hotspot and were confined to 164 bp, 46 bp, 256 bp, and 124 bp, respectively. Two of the regions were mapped within Alu elements. All crossover events were found to have occurred within or adjacent to a highly destabilised DNA duplex with a high S/MAR probability. In contrast, the SoS hotspot and other genomic disorders'' recombination hotspots were mapped to stabilised DNA helix regions, flanked by destabilised regions with high probability of containing S/MAR elements. Conclusions: The data suggest that a specific chromatin structure may increase susceptibility for recurrent crossover events and thus predispose to recombination hotspots in genomic disorders.     

Frequent occurrence of deletions in primary mediastinal B-cell lymphoma

Primary mediastinal B-cell lymphoma (PMBCL) is a distinct subtype of diffuse large B-cell lymphoma. PMBCL has been previously studied with a variety of genomic techniques resulting in frequent detection of chromosomal gains; however, chromosomal losses have been rarely reported. This finding contrasts many other types of lymphoma, in which deletions are common. We hypothesize that segmental losses do exist but may have escaped detection by methods used in the previous studies. Using array comparative genomic hybridization to a tiling-resolution microarray encompassing the entire human genome, PMBCL samples were analyzed for genomic copy number alterations. An almost equal number of gains and losses of chromosomal material were detected throughout the genome (216 vs. 193, respectively). A selection of these DNA copy number alterations were confirmed by quantitative real-time PCR. Recurrent gains were detected at all previously reported regions of gain, including 9p seen in approximately 70% of cases. Recurrent chromosomal losses were observed at 1p, 3p, 4q, 6q, 7p, and 17p, with a novel event at 1p13.1-p13.2 representing the most frequent at 42% of cases analyzed. We conclude that consistent losses are present in the PMBCL genome. Given the similar frequency of losses to that of segmental gains of DNA, they are likely to play an important role in the pathogenesis of PMBCL.