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.     

Fluorescent resonance energy transfer (FRET) based detection of a multiplex ligation-dependent probe amplification assay (MLPA) product

A fluorescent resonance energy transfer (FRET)-based hybridization assay for detecting multiplex ligation-dependent probe amplification (MLPA) products has been developed, extending the diagnostic power of the technique and demonstrating the possibility of combining MLPA with microarrays for the detection of multiple mutations. FRET is one of the most commonly used detection techniques for hybridization assays. To investigate the applicability of FRET based detection of MLPA products, a sandwich assay was designed to detect gene copy number by exploiting an immobilized probe labeled with an acceptor dye, Alexa Fluor 555, which hybridises to specific PCR amplicons, followed by hybridization of a second probe labeled with the donor dye, Alexa Fluor 488. Following excitation of the Alexa Fluor 488, a FRET signal was produced only if a DNA sequence specific to the BRCA1 exon 13 was present in the test sample. We have verified this assay on a DNA sample of a patient carrying a heterozygous BRCA1 exon 13 deletion using male genomic DNA as control. Here we demonstrate that the DNA sample containing the heterozygous deletion generated a considerably reduced FRET signal as compared to the control male human DNA. Our results show that the FRET design presented in this study can differentiate between reduced copy numbers any genomic DNA sequence after MLPA analysis, and the reported format is applicable to multiplex detection of MLPA products, using microarrays, or optical biosensor arrays, and future work will focus on the demonstration of this.     

Rapid identification and characterization of Penicillium marneffei using multiplex ligation-dependent probe amplification (MLPA) in paraffin-embedded tissue samples

Penicillium marneffei infection is a deadly disease and early diagnosis leads to prompt and appropriate antifungal therapy. To develop a sensitive method to diagnose P. marneffei infection, a multiplex ligation-dependent probe amplification (MLPA) assay was adapted. This method can rapidly and specifically detect P. marneffei DNA in cultured cells and paraffin-embedded tissue samples. Three pairs of probes were designed for amplifying the internally (intergenic) transcribed spacer (ITS) region of P. marneffei rRNA using a systematic phylogenetic analysis. These three probe sets produced three amplicons of 198, 166, and 152 bp, respectively, specific for P. marneffei. In contrast, there was only one 198 bp amplicon produced for Talaromyces stipitatus, and one 152 bp amplicon for P. funiculosum, T. intermedius and T. derxii. The probes did not amplify any other reference strains. An array of 40 P. marneffei strains isolated from human patients, bamboo rat, and the local environment was tested by using MLPA, and all were positively identified. Most importantly, P. marneffei in paraffin-embedded tissue specimens from infected human patients was positively amplified by MLPA. The sensitivity and specificity of the MLPA assay could be a useful tool for prompt diagnosis, pathogen characterization, and epidemiological studies of fungal infections.     

A modified multiplex ligation-dependent probe amplification method for the detection of 22q11.2 copy number variations in patients with congenital heart disease

Background

Copy number variations (CNVs) of chromosomal region 22q11.2 are associated with a subset of patients with congenital heart disease (CHD). Accurate and efficient detection of CNV is important for genetic analysis of CHD. The aim of the study was to introduce a novel approach named CNVplex®, a high-throughput analysis technique designed for efficient detection of chromosomal CNVs, and to explore the prevalence of sub-chromosomal imbalances in 22q11.2 loci in patients with CHD from a single institute.

Results

We developed a novel technique, CNVplex®, for high-throughput detection of sub-chromosomal copy number aberrations. Modified from the multiplex ligation-dependent probe amplification (MLPA) method, it introduced a lengthening ligation system and four universal primer sets, which simplified the synthesis of probes and significantly improved the flexibility of the experiment. We used 110 samples, which were extensively characterized with chromosomal microarray analysis and MLPA, to validate the performance of the newly developed method. Furthermore, CNVplex® was used to screen for sub-chromosomal imbalances in 22q11.2 loci in 818 CHD patients consecutively enrolled from Shanghai Children’s Medical Center. In the methodology development phase, CNVplex® detected all copy number aberrations that were previously identified with both chromosomal microarray analysis and MLPA, demonstrating 100% sensitivity and specificity. In the validation phase, 22q11.2 deletion and 22q11.2 duplication were detected in 39 and 1 of 818 patients with CHD by CNVplex®, respectively. Our data demonstrated that the frequency of 22q11.2 deletion varied among sub-groups of CHD patients. Notably, 22q11.2 deletion was more commonly observed in cases with conotruncal defect (CTD) than in cases with non-CTD (P < 0.001). With higher resolution and more probes against selected chromosomal loci, CNVplex® also identified several individuals with small CNVs and alterations in other chromosomes.

Conclusions

CNVplex® is sensitive and specific in its detection of CNVs, and it is an alternative to MLPA for batch screening of pathogenetic CNVs in known genomic loci.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1590-5) contains supplementary material, which is available to authorized users.     

Multiplex ligation-dependent probe amplification (MLPA) detects large deletions in the MECP2 gene of Swedish Rett syndrome patients

Mutations in the methyl-CpG-binding protein-2 (MECP2) gene on Xq28 have been found to be a cause of Rett syndrome (RS). In a previous mutation screening, we found MECP2 mutations in 81% of Swedish classical Rett women. In this study, we have analyzed 22 patients for MECP2 deletions using multiplex-ligation-dependent probe amplification (MLPA). Clinically, 11 of the patients who were classical Rett women, 3 were forme fruste, 1 was congenital RS, and 7 were Rett variants. As inclusion criteria, we used DNA from patients in whom previous sequencing results showed no mutations in coding portions of the MECP2 gene. MLPA is a method based on multiplex PCR. In one PCR, as many as 40 probes are amplified with the same primers. The specificity of the amplification products is determined by the site-specific hybridization of each probe construct, prior to amplification. Each PCR product has a unique length, which makes it possible to identify it by size separation. In 3 of 11 (27%) classical Rett women, we detected large deletions in MECP2 using MLPA. All these patients had deletions covering two exons; in 2 cases the deletion involved exons 3 and 4 and, in one case, exons 1 and 2 were missing. In the forme fruste, congenital and Rett-variant patients, we found no large deletions. We have found that MLPA is useful when it comes to finding large deletions compromising whole exons in MECP2. Used as a complementary method to DNA sequencing, it revealed new MECP2 mutations in classical RS patients.     

Somatic mosaicism for copy-neutral loss of heterozygosity and DNA copy number variations in the human genome

Background

Somatic mosaicism denotes the presence of genetically distinct populations of somatic cells in one individual who has developed from a single fertilised oocyte. Mosaicism may result from a mutation that occurs during postzygotic development and is propagated to only a subset of the adult cells. Our aim was to investigate both somatic mosaicism for copy-neutral loss of heterozygosity (cn-LOH) events and DNA copy number variations (CNVs) in fully differentiated tissues.

Results

We studied panels of tissue samples (11–12 tissues per individual) from four autopsy subjects using high-resolution Illumina HumanOmniExpress-12 BeadChips to reveal the presence of possible intra-individual tissue-specific cn-LOH and CNV patterns.We detected five mosaic cn-LOH regions >5 Mb in some tissue samples in three out of four individuals. We also detected three CNVs that affected only a portion of the tissues studied in one out of four individuals. These three somatic CNVs range from 123 to 796 kb and are also found in the general population. An attempt was made to explain the succession of genomic events that led to the observed somatic genetic mosaicism under the assumption that the specific mosaic patterns of CNV and cn-LOH changes reflect their formation during the postzygotic embryonic development of germinal layers and organ systems.

Conclusions

Our results give further support to the idea that somatic mosaicism for CNVs, and also cn-LOHs, is a common phenomenon in phenotypically normal humans. Thus, the examination of only a single tissue might not provide enough information to diagnose potentially deleterious CNVs within an individual. During routine CNV and cn-LOH analysis, DNA derived from a buccal swab can be used in addition to blood DNA to get information about the CNV/cn-LOH content in tissues of both mesodermal and ectodermal origin. Currently, the real frequency and possible phenotypic consequences of both CNVs and cn-LOHs that display somatic mosaicism remain largely unknown. To answer these questions, future studies should involve larger cohorts of individuals and a range of tissues.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1916-3) contains supplementary material, which is available to authorized users.     

A genome wide association study between copy number variation （CNV） and human height in Chinese population

Copy number variation (CNV) is a type of genetic variation which may have important roles in phenotypic variability and disease susceptibility. To hunt for genetic variants underlying human height variation, we performed a genome wide CNV association study for human height in 618 Chinese unrelated subjects using Affymetrix 500K array set. After adjusting for age and sex, we found that four CNVs at 6p21.3, 8p23.3-23.2, 9p23 and 16p12.1 were associated with human height (with borderline significant p value: 0.013, 0.011, 0.024, 0.049; respectively). However, after multiple tests correction, none of them was associated with human height. We observed that the gain of copy number (more than 2 copies) at 8p23.3-23.2 was associated with lower height (normal copy number vs. gain of copy number; 161.2 cm vs. 153.7 cm, p = 0.011), which accounted for 0.9% of height variation. Loss of copy number (less than 2 copies) at 6p21.3 was associated with 0.8% lower height (loss of copy number vs. normal copy number: 154.5 cm vs. 161.1 cm, p = 0.013). Since no important genes influencing height located in CNVs at loci of 8p23.3-23.2 and 6p21.3, the two CNVs may cause the structural rearrangements of neighbored important candidate genes, thus regulates the variation of height. Our results expand our knowledge of the genetic factors underlying height variation and the biological regulation of human height.     

Comparison of multiplex ligation dependent probe amplification to immunohistochemistry for assessing HER-2/neu amplification in invasive breast cancer

The HER-2/neu transmembrane tyrosine kinase receptor is both a prognostic marker and a therapeutic target for breast cancer. Accurate determination of HER-2/neu status is a prerequisite for selecting breast tumors for HER-2/neu immunotherapy or for taxan based chemotherapy. Unfortunately, there is no consensus concerning how this determination should be reached. We compared assessment of HER-2/neu status using Multiplex ligation-dependent probe amplification (MLPA) and immunohistochemistry (IHC). The patient group comprised 60 Indonesian breast cancers patients. IHC was performed on paraffin sections using the CB11 antibody from Novocastra. Results were scored according to the Hercept test. For MLPA, DNA was extracted from frozen samples, PCR amplified with a probe set containing three hemi-primer sets for the HER-2 locus and another nine control probes spread over chromosome 17 and other chromosomes, and analyzed on a gene scanner. A ratio above two for at least two HER-2 locus probes compared to the control probes was regarded as amplification. IHC for HER-2/neu was negative in 36 cases, and 24 cases (40%) showed expression. Seven, eight and nine of the latter cases were 1+, 2+ and 3+ positive, respectively. Forty-seven cases showed no amplification by MLPA, and 13 cases (22%) were amplified. Comparison of IHC and MPLA showed that none of the 36 IHC-negative or seven IHC 1+ cases was amplified. Five of the eight (63%) 2+ cases were amplified, and eight of nine (89%) of the IHC 3+ tumors showed gene amplification by MLPA assay. For HER-2/neu, there is a good correlation between gene amplification detected by MLPA and overexpression by IHC in invasive breast cancer. It appears that MLPA can detect the HER-2 amplified cases in the IHC 2+ class. Because MLPA is quick and inexpensive, it is an attractive method for detecting HER-2/neu amplification in daily laboratory practice.     

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.     

Development of a rapid,reliable and simple multiplex PCR assay for early detection of transgenic plant materials

Multiplex PCR is a variant of conventional PCR which includes two or more pairs of primers in a single reaction to amplify corresponding genes simultaneously. In this study, a reliable multiplex PCR analysis protocol was established for simple and fast detection of transgenes in plant materials. Two pairs of primers, corresponding to neomycin phosphotransferase gene and 1-aminocyclopropane-1-carboxylate synthase gene, were selected for target and resident gene respectively. The method bypasses routine DNA extraction, requires only very little amount of plant tissue and produces reliable results as shown by successful discrimination of transformed and nontransformed tobacco, tomato and kumquat materials. The method facilitates early identification of transgenic buds when they are still quite small.