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.     

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.     

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.     

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.     

Preparing long probes by an asymmetric polymerase chain reaction-based approach for multiplex ligation-dependent probe amplification

To clearly discriminate the results of simultaneous screening and quantification of up to 40 different targets–DNA sequences, long probes from 100 to 500 nt, rather than smaller or similar-sized synthetic ones, were adopted for multiplex ligation-dependent probe amplification (MLPA). To prepare the long probes, asymmetric polymerase chain reaction (PCR) was employed to introduce non-complementary stuffers in between the two parts of the MLPA probe with specially designed primers, then restriction enzymes were selected to digest the double-stranded DNAs, and finally polyacrylamide gel electrophoresis was used to purify the single-stranded DNAs (i.e., the long probes). By using this approach, 12 long probes were prepared and used to identify genetically modified (GM) maize. Our experimental results show that the prepared long probes were in full accordance with the designed ones and could be assembled in 4-, 7-, and 10-plex MLPA analysis without losing result specificity and accuracy, showing they were as effective and reliable in MLPA analysis as those prepared with M13-derived vectors. This novel asymmetric PCR-based approach does not need expensive equipment, special reagents, or complicated operations when compared with previous methods. Therefore, our new approach could make MLPA analysis more independent, efficient, and economical.     

Improved molecular diagnosis of dystrophin gene mutations using the multiplex ligation-dependent probe amplification method

Mutation detection in the DMD gene defective in Duchenne (DMD) and Becker muscular dystrophies (BMD) is complicated by the presence of 79 exons. The majority of recognized mutations are, however, copy number changes of individual exons, which traditionally have been identified by three common multiplex polymerase chain reaction (PCR) assays and/or Southern blotting. Here we report the use of the newly developed quantitative assay multiplex ligation-dependent probe amplification (MLPA) to determine the copy number of each of the 79 DMD exons in 182 males and 14 carrier females referred to our diagnostic facility on the clinical suspicion of DMD or BMD. The MLPA method confirmed all previously recognized mutations and identified an additional 28, including four point mutations. Also, the assay reliably identified 7 carrier females, which are usually not easily recognized. In our hands the method is highly reproducible, easy to handle, and has increased our mutation pick-up rate by a total of 33%.     

Multiplex ligation-dependent probe amplification using a completely synthetic probe set

The recent development of multiplex ligation-dependent probe amplification (MLPA) has provided an efficient and reliable assay for dosage screening of multiple loci in a single reaction. However, a drawback to this method is the time-consuming process of generating a probe set by cloning in single-stranded bacteriophage vectors. We have developed a synthetic probe set to screen for deletions in a region spanning 18.5 Mb within chromosome 3q. In a pilot study, we tested 15 synthetic probes on 4 control samples and on 2 patients previously found to possess a heterozygous deletion in the region 3q26-q28. These synthetic probes detected deletions at all previously known deleted loci. Furthermore, using synthetic probes, the variability of results within samples was similar to that reported for commercially available M13-derived probes. Our results demonstrate that this novel approach to MLPA provides a generic solution to the difficulties of probe development by cloning; such synthetically generated probes may be used to screen a large number of loci in a single reaction. We conclude that the use of synthetic probes for MLPA is a rapid, robust, and efficient alternative for research (and potentially diagnostic) deletion and duplication screening of multiple genomic loci.     

Single universal primer multiplex ligation-dependent probe amplification with sequencing gel electrophoresis analysis

In this study, a novel single universal primer multiplex ligation-dependent probe amplification (SUP-MLPA) technique that uses only one universal primer to perform multiplex polymerase chain reaction (PCR) was developed. Two reversely complementary common sequences were designed on the 5′ or 3′ end of the ligation probes (LPs), which allowed the ligation products to be amplified through only a single universal primer (SUP). SUP-MLPA products were analyzed on sequencing gel electrophoresis with extraordinary resolution. This method avoided the high expenses associated with capillary electrophoresis, which was the commonly used detection instrument. In comparison with conventional multiplex PCR, which suffers from low sensitivity, nonspecificity, and amplification disparity, SUP-MLPA had higher specificity and sensitivity and a low detection limit of 0.1 ng for detecting single crop species when screening the presence of genetically modified crops. We also studied the effect of different lengths of stuffer sequences on the probes for the first time. Through comparing the results of quantitative PCR, the LPs with different stuffer sequences did not affect the ligation efficiency, which further increased the multiplicity of this assay. The improved SUP–MLPA and sequencing gel electrophoresis method will be useful for food and animal feed identification, bacterial detection, and verification of genetic modification status of crops.     

Methylation-specific multiplex ligation-dependent probe amplification analysis of subjects with chromosome 15 abnormalities

Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are neurodevelopmental disorders caused by loss of expression of imprinted genes from the 15q11-q13 region. They arise from similar defects in the region but differ in parent of origin. There are two recognized typical 15q11-q13 deletions depending on size and several diagnostic assays are available but each has limitations. We evaluated the usefulness of a methylation-specific multiplex ligation-dependent probe amplification (MLPA) kit consisting of 43 probes to detect copy number changes and methylation status in the region. We used the MLPA kit to genotype 82 subjects with chromosome 15 abnormalities (62 PWS, 10 AS and 10 individuals with other chromosome 15 abnormalities) and 13 with normal cytogenetic findings. We developed an algorithm for MLPA probe analysis which correctly identified methylation abnormalities associated with PWS and AS and accurately determined copy number in previously assigned genetic subtypes including microdeletions of the imprinting center. Furthermore, MLPA analysis identified copy number changes in those with distal 15q deletions and ring 15s. MLPA is a relatively simple, cost-effective technique found to be useful and accurate for methylation status, copy number and analysis of genetic subtype in PWS and AS, as well as other chromosome 15 abnormalities.     

Rapid analysis of Saccharomyces cerevisiae genome rearrangements by multiplex ligation-dependent probe amplification

Aneuploidy and gross chromosomal rearrangements (GCRs) can lead to genetic diseases and the development of cancer. We previously demonstrated that introduction of the repetitive retrotransposon Ty912 onto a nonessential chromosome arm of Saccharomyces cerevisiae led to increased genome instability predominantly due to increased rates of formation of monocentric nonreciprocal translocations. In this study, we adapted Multiplex Ligation-dependent Probe Amplification (MLPA) to analyze a large numbers of these GCRs. Using MLPA, we found that the distribution of translocations induced by the presence of Ty912 in a wild-type strain was nonrandom and that the majority of these translocations were mediated by only six translocation targets on four different chromosomes, even though there were 254 potential Ty-related translocation targets in the S. cerevisiae genome. While the majority of Ty912-mediated translocations resulted from RAD52-dependent recombination, we observed a number of nonreciprocal translocations mediated by RAD52-independent recombination between Ty1 elements. The formation of these RAD52-independent translocations did not require the Rad51 or Rad59 homologous pairing proteins or the Rad1-Rad10 endonuclease complex that processes branched DNAs during recombination. Finally, we found that defects in ASF1-RTT109-dependent acetylation of histone H3 lysine residue 56 (H3K56) resulted in increased accumulation of both GCRs and whole-chromosome duplications, and resulted in aneuploidy that tended to occur simultaneously with GCRs. Overall, we found that MLPA is a versatile technique for the rapid analysis of GCRs and can facilitate the genetic analysis of the pathways that prevent and promote GCRs and aneuploidy.     

Relative quantification of 40 nucleic acid sequences by multiplex ligation-dependent probe amplification

We describe a new method for relative quantification of 40 different DNA sequences in an easy to perform reaction requiring only 20 ng of human DNA. Applications shown of this multiplex ligation-dependent probe amplification (MLPA) technique include the detection of exon deletions and duplications in the human BRCA1, MSH2 and MLH1 genes, detection of trisomies such as Down’s syndrome, characterisation of chromosomal aberrations in cell lines and tumour samples and SNP/mutation detection. Relative quantification of mRNAs by MLPA will be described elsewhere. In MLPA, not sample nucleic acids but probes added to the samples are amplified and quantified. Amplification of probes by PCR depends on the presence of probe target sequences in the sample. Each probe consists of two oligonucleotides, one synthetic and one M13 derived, that hybridise to adjacent sites of the target sequence. Such hybridised probe oligonucleotides are ligated, permitting subsequent amplification. All ligated probes have identical end sequences, permitting simultaneous PCR amplification using only one primer pair. Each probe gives rise to an amplification product of unique size between 130 and 480 bp. Probe target sequences are small (50–70 nt). The prerequisite of a ligation reaction provides the opportunity to discriminate single nucleotide differences.     

Search for subtelomeric imbalances by multiplex ligation-dependent probe amplification in Silver-Russell syndrome

Chromosomal aberrations are typically associated with primordial growth retardation, psychomotoric constrictions, and dysmorphisms. Since these features may be present in patients with Silver-Russell syndrome (SRS) and chromosomal disturbances are also detected in a subgroup of SRS patients, we screened a cohort of 45 SRS patients for cryptic subtelomeric imbalances. Submicroscopic deletions/duplications in the telomere regions are meanwhile well known to cause a broad spectrum of conspicuous phenotypes, characterized by mental retardation and multiple further congenital anomalies. We hypothesize that SRS might represent at the mild end of the broad phenotypic range of subtelomeric imbalances. Screening of the patients was performed by multiplex ligation-dependent probe amplification (MLPA), a technique that has already been shown to be effective and reliable for measuring copy numbers. We excluded pathogenetically relevant copy number variations in the subtelomeres in our SRS patient cohort, but one patient carried an apathogenic polymorphic Yq deletion. It can therefore be concluded that this type of chromosomal aberration does not belong to the genetic causes of SRS and it is not necessary to include this test in the diagnostic algorithm of the disease.     

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.     

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.     

Detection of colorectal cancer genes using a dye-free method combining barcode-base multiplex ligation-dependent probe amplification and pyrosequencing

Early detection of abnormal expression levels of cancer-related genes is crucial for reducing cancer mortality. Here, we describe a dye-free multiplex bioassay for simultaneous and quantitative analysis of the expression levels of multiple genes from one sample in a single assay, based on Sequence-tagged Multiplex ligationdependent probe Amplification (MLPA) coupled with pyrosequencing (termed as “SMAP”). Each pair of MLPA probes, containing a designed barcode, represents a gene of interest; thus, the use of various dyes to label different genes was avoided. The unique three-base barcode design on the probes, which can be decoded by pyrosequencing, enables individual quantification of the expression levels of six genes. Moreover, a new carryover contamination prevention system based on the use of restriction endonucleases was developed for PCR-based diagnostic screening assays. SMAP analysis revealed significant differences between the expression levels of CRC-related genes in the tumor tissues and normal tissues from a CRC patient. For PCR-based diagnostic screening assays, 0.5 U of the FokI restriction endonuclease was sufficient for the removal 0.01 pmol of PCR contamination. The ability to analyze the expression levels of a greater number of cancer-related genes would improve diagnostic sensitivity and efficacy. SMAP is amenable to the detection of an increased number of genes by lengthening the artificially designed barcodes; thus our method provides a promising means for cancer diagnostics and improving the treatment options available to cancer patients.     

Molecular differences between ductal carcinoma in situ and adjacent invasive breast carcinoma: a multiplex ligation-dependent probe amplification study

Ductal carcinoma in situ (DCIS) accounts for approximately 20% of mammographically detected breast cancers. Although DCIS is generally highly curable, some women with DCIS will develop life-threatening invasive breast cancer, but the determinants of progression to infiltrating ductal cancer (IDC) are largely unknown. In the current study, we used multiplex ligation-dependent probe amplification (MLPA), a multiplex PCR-based test, to compare copy numbers of 21 breast cancer related genes between laser-microdissected DCIS and adjacent IDC lesions in 39 patients. Genes included in this study were ESR1, EGFR, FGFR1, ADAM9, IKBKB, PRDM14, MTDH, MYC, CCND1, EMSY, CDH1, TRAF4, CPD, MED1, HER2, CDC6, TOP2A, MAPT, BIRC5, CCNE1 and AURKA.There were no significant differences in copy number for the 21 genes between DCIS and adjacent IDC. Low/intermediate-grade DCIS showed on average 6 gains/amplifications versus 8 in high-grade DCIS (p=0.158). Furthermore, alterations of AURKA and CCNE1 were exclusively found in high-grade DCIS, and HER2, PRDM14 and EMSY amplification was more frequent in high-grade DCIS than in low/intermediate-grade DCIS. In contrast, the average number of alterations in low/intermediate and high-grade IDC was similar, and although EGFR alterations were exclusively found in high-grade IDC compared to low/intermediate-grade IDC, there were generally fewer differences between low/intermediate-grade and high-grade IDC than between low/intermediate-grade and high-grade DCIS.In conclusion, there were no significant differences in copy number for 21 breast cancer related genes between DCIS and adjacent IDC, indicating that DCIS is genetically as advanced as its invasive counterpart. However, high-grade DCIS showed more copy number changes than low/intermediate-grade DCIS with specifically involved genes, supporting a model in which different histological grades of DCIS are associated with distinct genomic changes that progress to IDC in different routes. These high-grade DCIS specific genes may be potential targets for treatment and/or predict progression.     

多重连接探针扩增方法在假肥大性肌营养不良产前基因诊断中的应用

假肥大性肌营养不良(Duchenne/Becker muscular dystrophy, DMD/BMD)是一种由于DMD基因突变导致的X连锁隐性致死性遗传病。目前没有有效的治疗方法。为建立一种既可以对携带者进行检测又可以进行产前基因诊断的方法, 文章联合应用多重连接探针扩增技术(Multiplex ligation-dependent probe amplification, MLPA)和短串联重复序列(Short tandem repeats , STR)为遗传标记连锁分析的方法对26例有高风险再生育患儿的假肥大性肌营养不良家系的孕妇通过羊水穿刺进行产前基因诊断。26例进行产前基因诊断的羊水标本中有7例诊断为男性患儿, 4例诊断为女性携带者。MLPA可以作为筛查DMD基因缺失和重复突变的首选方法。联合应用MLPA和STR连锁分析, 可以提高假肥大性肌营养不良的产前基因诊断率。     

Rapid and reliable determination of transgene zygosity in mice by multiplex ligation-dependent probe amplification

The ability to rapidly and unequivocally distinguish heterozygous from homozygous transgenic mice is an integral part of any breeding strategy. Here we describe a quick and simple protocol for determining the zygosity of transgenic mice at multiple loci in a single reaction. This involved the development of a multiplex ligation-dependent probe amplification (MLPA) probe mix to simultaneously measure common transgenic alleles such as Cre recombinase (Cre), neomycin (Neo), β-galactosidase (LacZ) and enhanced green fluorescent protein (eGFP), as well as loci specific to the X and Y chromosome to allow sexing. Each reaction required as little as 100 ng of genomic DNA isolated from a tail biopsy using a simple procedure. Normalization against autosomal control loci resulted in 100% call accuracy, with no ambiguous results. This probe mix can be easily implemented in any laboratory with access to a PCR machine and a DNA sequencer, and can be rapidly adapted to genotype any additional loci of interest.     

Precise H1N1 swine influenza detection using stuffer-free multiplex ligation-dependent probe amplification in conformation-sensitive capillary electrophoresis

The H1N1 influenza virus has spread worldwide to become pandemic. Here, we developed a new method to discriminate various types of influenza A, including H1N1, using stuffer-free multiplex ligation-dependent probe amplification based on a conformation-sensitive separation method, namely capillary electrophoresis-single-strand conformation polymorphism. Unlike conventional methods, our approach precisely detects five relevant gene markers permitting confirmation of infection.