Probe-specific mixed-model approach to detect copy number differences using multiplex ligation-dependent probe amplification (MLPA)

Background  

MLPA method is a potentially useful semi-quantitative method to detect copy number alterations in targeted regions. In this paper, we propose a method for the normalization procedure based on a non-linear mixed-model, as well as a new approach for determining the statistical significance of altered probes based on linear mixed-model. This method establishes a threshold by using different tolerance intervals that accommodates the specific random error variability observed in each test sample.     

Designing a simple multiplex ligation-dependent probe amplification （MLPA） assay for rapid detection of copy number variants in the genome

Copy number variants （CNVs） are pervasive in the human genome and are responsible for many Mendelian diseases and genomic disorders. The detection of CNVs is an essential element of a complete mutation screening strategy. Many techniques have been developed for gene dosage testing. Multiplex ligation-dependent probe amplification （MLPA） is a robust, easy and flexible technique that can detect both deletions and duplications for more than 40 loci in one assay. It has been widely used in research and diagnostic laboratories. We routinely develop our own MLPA assays for quick validation of array comparative genomic hybridization （CGH） findings. Here we discuss the general principles and critical aspects of MLPA assay development and validation using all synthetic MLPA probes. We believe that MLPA will play important roles in the rapid detection of genomic disorders associated with genomic imbalances, the confirmation of pathogenic mutations involving exonic deletions/duplications, CNV genotyping and population frequency analysis of CNVs.     

Methylation-specific ligation detection reaction (msLDR): a new approach for multiplex evaluation of methylation patterns

A new sensitive method for multiplex gene-specific methylation analysis was developed using a ligation-based approach combined with a TaqMan-based detection and readout employing universal reporter probes. The approach, termed methylation-specific Ligation Detection Reaction (msLDR), was applied to test 16 loci in 8 different colorectal cancer cells in parallel. These loci encode immune regulatory genes involved in T-cell and natural killer cell activation, whose silencing is associated with the development or progression of colorectal cancer. Parallel analysis of HLA-A, HLA-B, STAT1, B2M, LMP2, LMP7, PA28α, TAP1, TAP2, TAPBP, ULBP2 and ULBP3 by msLDR in eight colorectal cancer cell lines showed preferential methylation at the HLA-B, ULBP2 and ULBB3 loci, but not at the other loci. MsLDR was found to represent a suitable and sensitive method for the detection of distinct methylation patterns as validated by conventional bisulphite Sanger sequencing and COBRA analysis. Since gene silencing by epigenetic mechanisms plays a central role during transformation of a normal differentiated somatic cell into a cancer cell, characterization of the gene methylation status in tumours is a major topic not only in basic research, but also in clinical diagnostics. Due to a very simple workflow, msLDR is likely to be applicable to clinical samples and thus comprises a potential diagnostic tool for clinical purposes.     

Altered methylation at microRNA-associated CpG islands in hereditary and sporadic carcinomas: a methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA)-based approach

MicroRNAs (miRNAs) are small noncoding RNAs that contribute to tumorigenesis by acting as oncogenes or tumor suppressor genes and may be important in the diagnosis, prognosis and treatment of cancer. Many miRNA genes have associated CpG islands, suggesting epigenetic regulation of their expression. Compared with sporadic cancers, the role of miRNAs in hereditary or familial cancer is poorly understood. We investigated 96 colorectal carcinomas, 58 gastric carcinomas and 41 endometrial carcinomas, occurring as part of inherited DNA mismatch repair (MMR) deficiency (Lynch syndrome), familial colorectal carcinoma without MMR gene mutations or sporadically. Methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) assays were developed for 11 miRNA loci that were chosen because all could be epigenetically regulated through the associated CpG islands and some could additionally modulate the epigenome by putatively targeting the DNA methyltransferases or their antagonist retinoblastoma-like 2 (RBL2). Compared with the respective normal tissues, the predominant alteration in tumor tissues was increased methylation for the miRNAs 1-1, 124a-1, 124a-2, 124a-3, 148a, 152 and 18b; decreased methylation for 200a and 208a; and no major change for 373 and let-7a-3. The frequencies with which the individual miRNA loci were affected in tumors showed statistically significant differences relative to the tissue of origin (colorectal versus gastric versus endometrial), MMR proficiency versus deficiency and sporadic versus hereditary disease. In particular, hypermethylation at miR-148a and miR-152 was associated with microsatellite-unstable (as opposed to stable) tumors and hypermethylation at miR-18b with sporadic disease (as opposed to Lynch syndrome). Hypermethylation at miRNA loci correlated with hypermethylation at classic tumor suppressor promoters in the same tumors. Our results highlight the importance of epigenetic events in hereditary and sporadic cancers and suggest that MS-MLPA is an excellent choice for quantitative analysis of methylation in archival formalin-fixed, paraffin-embedded samples, which pose challenges to many other techniques commonly used for methylation studies.     

High-resolution genome-wide cytosine methylation profiling with simultaneous copy number analysis and optimization for limited cell numbers

Many genome-wide assays involve the generation of a subset (or representation) of the genome following restriction enzyme digestion. The use of enzymes sensitive to cytosine methylation allows high-throughput analysis of this epigenetic regulatory process. We show that the use of a dual-adapter approach allows us to generate genomic representations that includes fragments of <200bp in size, previously not possible when using the standard approach of using a single adapter. By expanding the representation to smaller fragments using HpaII or MspI, we increase the representation by these isoschizomers to more than 1.32 million loci in the human genome, representing 98.5% of CpG islands and 91.1% of refSeq promoters. This advance allows the development of a new, high-resolution version of our HpaII-tiny fragment Enrichment by Ligation-mediated PCR (HELP) assay to study cytosine methylation. We also show that the MspI representation generates information about copy-number variation, that the assay can be used on as little as 10ng of DNA and that massively parallel sequencing can be used as an alternative to microarrays to read the output of the assay, making this a powerful discovery platform for studies of genomic and epigenomic abnormalities.     

The effect of C(5) cytosine methylation at CpG sequences on mitomycin-DNA bonding profiles

Recent studies have documented that cytosine C(5) methylation of CpG sequences enhances mitomycin C (1) adduction. The reports differ on the extent and uniformity of 1 modification at the nucleotide level. We have determined the bonding profiles for mitomycin monoalkylation in two DNA restriction fragments where the CpG sequences were methylated. Three mitomycin substrates were used and two different enzymatic assays employed to monitor the extent of drug modification at the individual base sites. Drug DNA modification was accomplished with I and 10-decarbamoylmitomycin C (2) under reductive (Na2S2O4) condilions and with N-methyl-7-methoxyaziridinomitosene (3) under nonreductive conditions. The UvrABC incision assay permitted us to quantitate the sites of drug adduction, and the lambda-exonuclease stop assay provided a qualitative estimation of drug-DNA modification consistent with the UvrABC data. We learned that C(5) cytosine methylation (m5C) enhanced the extent of overall DNA modification. Using the UvrABC endonuclease assay, we found that modification by 1 increased 2.0 and 7.4 times for the two DNA restriction fragments. Analysis of the modification sites at the nucleotide sequence level revealed that guanine (G) was the only base modified and that the overall increased level of DNA adduction was due to enhanced modification of select m5CpG* (G* = mitomycin (mitosene) adduction sites) loci compared with CpG* sites: the largest differences reached two orders of magnitude. Significantly, not all CpG* sites underwent increased drug adduction upon C(5) cytosine methylation. The effect of C(5) cytosine methylation on the drug adduction profiles was less pronounced for G* sites located within dinucleotide sequences other than CpG*. We observed that DNA methylation often led to slightly diminished adduction levels at these sites. The different m5CpG* adduction patterns provided distinctive sequence-selective bonding profiles for 1-3. We have attributed the large differences in guanine reactivity to DNA structural factors created, in part, by C(5) cytosine methylation. The significance of these findings in cancer chemotherapy is briefly discussed.     

Origins and breakpoint analyses of copy number variations: up close and personal

Array-based methods have enabled the detection of many genomic gains and losses. These are stated as copy number variants (CNVs) and comprise up to 13% of the human genome. Based on their breakpoints and modes of formation CNVs are termed recurrent or nonrecurrent. Recurrent CNVs are flanked by low copy repeats and are of a fixed size. They arise as a result of misalignment during meiosis by a mechanism named nonallelic homologous recombination. Several of such recurrent CNVs have been linked to human diseases. Nonrecurrent CNVs, which are not flanked by low copy repeats, are of variable size and may arise via mechanisms like nonhomologous end joining and replication-based mechanisms described by the fork stalling and template switching and microhomology-mediated break-induced replication models. It is becoming clear that most disease-causing CNVs are nonrecurrent and generally arise via replication-based mechanisms. Furthermore, it is now appreciated that genomic features other than low copy repeats play a role in the formation of nonrecurrent CNVs. This review will discuss the different mechanisms of CNV formation and how high resolution analyses of CNV breakpoints have added to our knowledge of their precise structure.     

Probe-free allele-specific copy number detection and analysis of tumors

Cancer development and progression frequently involve nucleotide mutations as well as amplifications and deletions of genomic segments. Quantification of allele-specific copy number is an important step in characterizing tumor genomes for precision medicine. Despite advances in approaches to high-throughput genomic DNA analysis, inexpensive and simple methods for analyzing complex nucleotide and copy number variants are still needed. Real-time polymerase chain reaction (PCR) methods for discovering and genotyping single nucleotide polymorphisms are becoming increasingly important in genetic analysis. In this study, we describe a simple, single-tube, probe-free method that combines SYBR Green I-based quantitative real-time PCR and quantitative melting curve analysis both to detect specific nucleotide variants and to quantify allele-specific copy number variants of tumors. The approach is based on the quantification of the targets of interest and the relative abundance of two alleles in a single tube. The specificity, sensitivity, and utility of the assay were demonstrated in detecting allele-specific copy number changes critical for carcinogenesis and therapeutic intervention. Our approach would be useful for allele-specific copy number analysis or precise genotyping.     

CNAmet: an R package for integrating copy number, methylation and expression data

SUMMARY: Gene copy number and DNA methylation alterations are key regulators of gene expression in cancer. Accordingly, genes that show simultaneous methylation, copy number and expression alterations are likely to have a key role in tumor progression. We have implemented a novel software package (CNAmet) for integrative analysis of high-throughput copy number, DNA methylation and gene expression data. To demonstrate the utility of CNAmet, we use copy number, DNA methylation and gene expression data from 50 glioblastoma multiforme and 188 ovarian cancer primary tumor samples. Our results reveal a synergistic effect of DNA methylation and copy number alterations on gene expression for several known oncogenes as well as novel candidate oncogenes. AVAILABILITY: CNAmet R-package and user guide are freely available under GNU General Public License at http://csbi.ltdk.helsinki.fi/CNAmet.     

GEAR: genomic enrichment analysis of regional DNA copy number changes

We developed an algorithm named GEAR (genomic enrichment analysis of regional DNA copy number changes) for functional interpretation of genome-wide DNA copy number changes identified by array-based comparative genomic hybridization. GEAR selects two types of chromosomal alterations with potential biological relevance, i.e. recurrent and phenotype-specific alterations. Then it performs functional enrichment analysis using a priori selected functional gene sets to identify primary and clinical genomic signatures. The genomic signatures identified by GEAR represent functionally coordinated genomic changes, which can provide clues on the underlying molecular mechanisms related to the phenotypes of interest. GEAR can help the identification of key molecular functions that are activated or repressed in the tumor genomes leading to the improved understanding on the tumor biology. AVAILABILITY: GEAR software is available with online manual in the website, http://www.systemsbiology.co.kr/GEAR/.     

Methylation detection oligonucleotide microarray analysis: a high-resolution method for detection of CpG island methylation

Methylation of CpG islands associated with genes can affect the expression of the proximal gene, and methylation of non-associated CpG islands correlates to genomic instability. This epigenetic modification has been shown to be important in many pathologies, from development and disease to cancer. We report the development of a novel high-resolution microarray that detects the methylation status of over 25 000 CpG islands in the human genome. Experiments were performed to demonstrate low system noise in the methodology and that the array probes have a high signal to noise ratio. Methylation measurements between different cell lines were validated demonstrating the accuracy of measurement. We then identified alterations in CpG islands, both those associated with gene promoters, as well as non-promoter-associated islands in a set of breast and ovarian tumors. We demonstrate that this methodology accurately identifies methylation profiles in cancer and in principle it can differentiate any CpG methylation alterations and can be adapted to analyze other species.     

De novo methylation of CpG island sequences in human fibroblasts overexpressing DNA (cytosine-5-)-methyltransferase.

Recent studies showing a correlation between the levels of DNA (cytosine-5-)-methyltransferase (DNA MTase) enzyme activity and tumorigenicity have implicated this enzyme in the carcinogenic process. Moreover, hypermethylation of CpG island-containing promoters is associated with the inactivation of genes important to tumor initiation and progression. One proposed role for DNA MTase in tumorigenesis is therefore a direct role in the de novo methylation of these otherwise unmethylated CpG islands. In this study, we sought to determine whether increased levels of DNA MTase could directly affect CpG island methylation. A full-length cDNA for human DNA MTase driven by the cytomegalovirus promoter was constitutively expressed in human fibroblasts. Individual clones derived from cells transfected with DNA MTase (HMT) expressed 1- to 50-fold the level of DNA MTase protein and enzyme activity of the parental cell line or clones transfected with the control vector alone (Neo). To determine the effects of DNA MTase overexpression on CpG island methylation, we examined 12 endogenous CpG island loci in the HMT clones. HMT clones expressing > or = 9-fold the parental levels of DNA MTase activity were significantly hypermethylated relative to at least 11 Neo clones at five CpG island loci. In the HMT clones, methylation reached nearly 100% at susceptible CpG island loci with time in culture. In contrast, there was little change in the methylation status in the Neo clones over the same time frame. Taken together, the data indicate that overexpression of DNA MTase can drive the de novo methylation of susceptible CpG island loci, thus providing support for the idea that DNA MTase can contribute to tumor progression through CpG island methylation-mediated gene inactivation.     

Construction and use of spotted large-insert clone DNA microarrays for the detection of genomic copy number changes

Microarray-based comparative genomic hybridization has become a widespread method for the analysis of DNA copy number changes across the human genome. Initial methods for microarray construction using large-insert clones required the preparation of DNA from large-scale cultures. This rapidly became an expensive and time-consuming process when expanded to the number of clones needed for higher resolution arrays. To overcome this problem, several PCR-based strategies have been developed to enable array construction from small amounts of cloned DNA. Here, we describe the construction of microarrays composed of human-specific large-insert clones (40-200 kb) using a specific degenerate oligonucleotide PCR strategy. In addition, we also describe array hybridization using manual and automated procedures and methods for array analysis. The technology and protocols described in this article can easily be adapted for other species dependent on the availability of clone libraries. According to our protocols, the procedure will take approximately 3 days from labeling the DNA to scanning the hybridized slides.     

High resolution detection and analysis of CpG dinucleotides methylation using MBD-Seq technology

Methyl-CpG binding domain protein sequencing (MBD-seq) is widely used to survey DNA methylation patterns. However, the optimal experimental parameters for MBD-seq remain unclear and the data analysis remains challenging. In this study, we generated high depth MBD-seq data in MCF-7 cell and developed a bi-asymmetric-Laplace model (BALM) to perform data analysis. We found that optimal efficiency of MBD-seq experiments was achieved by sequencing ～100 million unique mapped tags from a combination of 500 mM and 1000 mM salt concentration elution in MCF-7 cells. Clonal bisulfite sequencing results showed that the methylation status of each CpG dinucleotides in the tested regions was accurately detected with high resolution using the proposed model. These results demonstrated the combination of MBD-seq and BALM could serve as a useful tool to investigate DNA methylome due to its low cost, high specificity, efficiency and resolution.     

DNA methylation in mycobacteria: Absence of methylation at GATC (Dam) and CCA/TGG (Dcm) sequences

Abstract The presence of 6-methyladenine and 5-methylcytosine at Dam (GATC) and Dcm (CCA/TGG) sites in DNA of mycobacterial species was investigated using isoschizomer restriction enzymes. In all species examined, Dam and Dcm recognition sequences were not methylated indicating the absence of these methyltransferases. On the other hand, high performance liquid chromatographic analysis of genomic DNA from Mycobacterium smegmatis and Mycobacterium tuberculosis showed significant levels of 6-methyladenine and 5-methylcytosine suggesting the presence of DNA methyltransferases other than Dam and Dcm. Occurrence of methylation was also established by a sensitive genetic assay.     

Multiple displacement amplification as a pre-polymerase chain reaction (pre-PCR) to process difficult to amplify samples and low copy number sequences from natural environments

Microbial assessment of natural biodiversity is usually achieved through polymerase chain reaction (PCR) amplification. Deoxyribonucleic acid (DNA) sequences from natural samples are often difficult to amplify because of the presence of PCR inhibitors or to the low number of copies of specific sequences. In this study, we propose a non-specific preamplification procedure to overcome the presence of inhibitors and to increase the number of copies prior to carrying out standard amplification by PCR. The pre-PCR step is carried out through a multiple displacement amplification (MDA) technique using random hexamers as priming oligonucleotides and phi 29 DNA polymerase in an isothermal, whole-genome amplification reaction. Polymerase chain reaction amplification using specific priming oligonucleotides allows the selection of the sequences of interest after a preamplification reaction from complex environmental samples. The procedure (MDA-PCR) has been tested on a natural microbial community from a hypogean environment and laboratory assemblages of known bacterial species, in both cases targeting the small subunit ribosomal RNA gene sequences. Results from the natural community showed successful amplifications using the two steps protocol proposed in this study while standard, direct PCR amplification resulted in no amplification product. Amplifications from a laboratory assemblage by the two-step proposed protocol were successful at bacterial concentrations >or= 10-fold lower than standard PCR. Amplifications carried out in the presence of different concentrations of fulvic acids (a soil humic fraction) by the MDA-PCR protocol generated PCR products at concentrations of fulvic acids over 10-fold higher than standard PCR amplifications. The proposed procedure (MDA-PCR) opens the possibility of detecting sequences represented at very low copy numbers, to work with minute samples, as well as to reduce the negative effects on PCR amplifications of some inhibitory substances commonly found in environmental samples.     

SiDCoN: a tool to aid scoring of DNA copy number changes in SNP chip data

The recent application of genome-wide, single nucleotide polymorphism (SNP) microarrays to investigate DNA copy number aberrations in cancer has provided unparalleled sensitivity for identifying genomic changes. In some instances the complexity of these changes makes them difficult to interpret, particularly when tumour samples are contaminated with normal (stromal) tissue. Current automated scoring algorithms require considerable manual data checking and correction, especially when assessing uncultured tumour specimens. To address these limitations we have developed a visual tool to aid in the analysis of DNA copy number data. Simulated DNA Copy Number (SiDCoN) is a spreadsheet-based application designed to simulate the appearance of B-allele and logR plots for all known types of tumour DNA copy number changes, in the presence or absence of stromal contamination. The system allows the user to determine the level of stromal contamination, as well as specify up to 3 different DNA copy number aberrations for up to 5000 data points (representing individual SNPs). This allows users great flexibility to assess simple or complex DNA copy number combinations. We demonstrate how this utility can be used to estimate the level of stromal contamination within tumour samples and its application in deciphering the complex heterogeneous copy number changes we have observed in a series of tumours. We believe this tool will prove useful to others working in the area, both as a training tool, and to aid in the interpretation of complex copy number changes.     

Ribosomal genes in inbred mouse strains: interstrain and intrastrain variations of copy number and extent of methylation

Quantitative dot hybridization was used to estimate the rDNA copy number in brain tissues of five inbred mouse strains (AKR/JY, NZB/B1OrlY, CBA/CaLacY, 101/HY, and 129/JY), which were obtained from the collection of the Research Center of Biomedical Technologies (Y). In each strain, 9-12 mice aged 1-2 months were examined. The rDNA copy number per diploid genome in strains AKR (range 105-181, mean +/- SD 136 +/- 27) and NZB (129-169, 148 +/- 12) was significantly lower than in strains CBA (172-267, 209 +/- 31), 101 (179-270, 217 +/- 30), and 129 (215-310, 264 +/- 33). Mice of strain NZB were relatively homogeneous in this trait (CV = 8.1%). Strains AKR, CBA, 101, and 129 displayed significant between-group differences, CV varying from 12.5 to 19.9%. The same DNA specimens were digested with MspI or HpaII and used to estimate the extent of methylation of the 28S rDNA region. Regardless of the strain, all mice could be classed into two groups. One group (20 mice) had a methylated fraction accounting for less than 8% of rDNA and included all nine mice of strain NZB, seven out of nine mice of strain 101, and three out of ten mice of strain 129. In the other group (29 mice), the methylated fraction varied from 18 to 38%. A possible role of methylation and the genome dosage of ribosomal genes in phenotypic variation (quantitative trait variation) of inbred mouse strains is discussed.