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.     

Evaluation of point mutations in dystrophin gene in Iranian Duchenne and Becker muscular dystrophy patients: introducing three novel variants

Duchenne and Becker muscular dystrophies (DMD and BMD) are X-linked neuromuscular diseases characterized by progressive muscular weakness and degeneration of skeletal muscles. Approximately two-thirds of the patients have large deletions or duplications in the dystrophin gene and the remaining one-third have point mutations. This study was performed to evaluate point mutations in Iranian DMD/BMD male patients. A total of 29 DNA samples from patients who did not show any large deletion/duplication mutations following multiplex polymerase chain reaction (PCR) and multiplex ligation-dependent probe amplification (MLPA) screening were sequenced for detection of point mutations in exons 50–79. Also exon 44 was sequenced in one sample in which a false positive deletion was detected by MLPA method. Cycle sequencing revealed four nonsense, one frameshift and two splice site mutations as well as two missense variants.     

Gross deletions/duplications in <Emphasis Type="Italic">PROS1</Emphasis> are relatively common in point mutation-negative hereditary protein S deficiency

Hereditary protein S (PS) deficiency is an autosomal disorder caused by mutations in the PS gene (PROS1). Conventional PCR-based mutation detection identifies PROS1 point mutations in approximately 50% of the cases. To verify if gross copy number variations (CNVs) are often present in point mutation-negative hereditary PS deficiency we used multiplex ligation-dependent probe amplification (MLPA) as a detection tool in samples from individuals with a high probability of having true PS deficiency. To this end, DNA samples from nine PS deficient probands with family members (seven type I and two type III) and nine isolated probands (three type I and six type III), in whom PROS1 mutations were not found by DNA sequencing, were evaluated. An independent quantitative PCR (qPCR) was performed to confirm the findings of the MLPA assay. Family members were also tested when DNA was available. Gross abnormalities of PROS1 were found in six out of eighteen probands. In three probands complete deletion of the gene was detected. Two probands had a partial deletion involving different parts of the gene (one from exon 4 through 9 and another from exon 9 through 11). One family showed a duplication of part of PROS1. qPCR analysis was in accordance with these results. In conclusion, this study substantiates that gross gene abnormalities in PROS1 are relatively common in hereditary PS deficient patients and that MLPA is a useful tool for direct screening of CNVs in PROS1 point mutation-negative individuals.     

Improved Detection of Deletions and Duplications in the DMD Gene Using the Multiplex Ligation-Dependent Probe Amplification (MLPA) Method

The multiplex ligation-dependent probe amplification (MLPA) assay is the most powerful tool in screening for deletions and duplications in the dystrophin gene in patients with Duchenne and Becker muscular dystrophy (DMD/BMD). The efficacy of the assay was validated by testing 20 unrelated male patients with DMD/BMD who had already been screened by multiplex PCR (mPCR). We detected two duplications that had been missed by mPCR. In one DMD patient showing an ambiguous MLPA result, a novel mutation (c.3808_3809insG) was identified. MLPA improved the mutation detection rate of mPCR by 15 %. The results of our study (1) confirmed MLPA to be the method of choice for detecting DMD gene rearrangements in DMD/BMD patients, (2) showed that ambiguous MLPA amplification products should be verified by other methods, and (3) indicated that the MLPA method could be used in screening even for small mutations located in the probe-binding regions.     

Point mutations and polymorphisms in the human dystrophin gene identified in genomic DNA sequences amplified by multiplex PCR

Summary About one third of Duchenne muscular dystrophy (DMD) patients have no gross DNA rearrangements in the dystrophin gene detectable by Southern blot analysis or multiplex exon amplification. Presumably, in these cases, the deficiency is caused by minor structural lesions of the dystrophin gene. However, to date, only a single human DMD case has been described where a point mutation, producing a stop codon, accounts for the DMD phenotype. To screen for microheterogeneities in the dystrophin gene, we applied analysis by chemical mismatch cleavage to thirteen exons amplified in multiplex sets by the polymerase chain reaction. This analysis covers approximately 20% of the dystrophin-coding sequence. Sixty DMD patients without detectable deletions or duplications were investigated, leading to the identification of two point mutations and four polymorphisms with a frequency higher than 5%. Both point mutations are frameshift mutations in exons 12 and 48, respectively, and are closely followed by stop codons, thus explaining the functional deficiency of the dystrophin gene products in both patients.     

中国人群中抗肌萎缩蛋白基因突变类型和分布特点及其与临床症状的相关性

假性肥大型进行性肌营养不良症(Duchenne’s muscular dystrophy,DMD)是源于横纹肌的一种X-连锁隐性致死性遗传病,由编码抗肌营养不良蛋白(dystrophin)基因突变所致。为了探讨中国人群中DMD患者的dystrophin基因突变类型和分布特点及其与临床症状的相关性,文章采用Multiplex Ligation-Dependent Probe Amplification(MLPA)方法对720例DMD患者及其母亲和20例正常成年男性进行dystrophin基因分析。结果显示,检出率为64.9%(467/720),54.3%(391/720)的患者为基因缺失;10.6%(76/720)的患者为基因重复。累及Exon45-54缺失突变型占全部缺失型患者的71.9%(281/391);重复突变型累及Exon1-40占全部重复型患者82.9%(63/76);检出的患者中,DMD型和中间型营养不良症(Intermediate muscular dystrophy,IMD)型占90.6%(423/467),Becker型营养不良症(Becker muscular dystrophy,BMD)型占9.4%(44/467)。表明假肥大型肌营养不良症以dystrophin基因缺失突变为主,突变发生在整个基因中非均匀分布,存在突变热区,在缺失和重复的位置和片段长度与肌病的临床症状严重程度之间并不存在简单的相关关系。     

Novel mutations of dystrophin gene in DMD patients detected by rapid scanning in biplex exons DHPLC analysis

Mutations in the dystrophin gene result in both Duchenne and Becher muscular dystrophies (DMD and BMD). Approximately 65% of all mutations causing DMD are deletions (60%) or duplications (5%) of large segments of this gene, spanning one exon or more. Due to the large size of the dystrophin gene (79 exons), finding point mutations has been prohibitively expensive and laborious. Recent studies confirm the utility of pre-screening methods, as denaturing high-performance liquid chromatography (DHPLC) analysis in the identification of point mutations in the dystrophin gene, with an increment of mutation detection rate from 65% to more than 92%. Here we suggest an alternative and convenient method of DHPLC analysis in order to find mutations in a more rapid and less expensive way by introducing the analysis of 16 couples of dystrophin amplicons, in biplex exons DHPLC runs. Using this new protocol of biplex exons DHPLC screening, new mutations were identified in four male patients affected by DMD who had tested negative for large DNA rearrangements.     

The value of MLPA in Waardenburg syndrome

Waardenburg syndrome (WS) is an autosomal-dominant neurocristopathy characterized by sensorineural hearing loss, pigmentary abnormalities of the iris, hair, and skin, and is responsible for about 3% of congenital hearing loss. Point mutations in PAX3 have been identified in more than 90% of affected individuals with WS Type 1/WS Type 3. MITF point mutations have been identified in 10-15% of individuals affected with WS Type 2 (lacking dystopia canthorum). Multiplex ligation-dependent probe amplification (MLPA) is now a standard technology in the molecular genetics laboratory to detect copy number changes in targeted genes. We employed MLPA for PAX3 and MITF in a cohort of patients submitted with a diagnosis of WS1, 2 or 3 who were sequence negative for PAX3 and/or MITF. All coding exons of PAX3 and exons 1, 2, 3, and 10 of MITF were included in the MLPA assay. MLPA on 48 patients with WS 1 or 3 revealed 3 PAX3 whole gene deletions (2 WS1; 1 WS3), 2 PAX3 partial gene deletions [WS1, exon 1 and promoter (1st report); WS1, exons 5-9], and 1 partial MITF deletion ("WS1", exons 3-10) (6/48 approximately 12.5%). MLPA on 41 patients with WS2 and 20 patients submitted with a diagnosis of either WS1 or WS2 revealed no copy number changes. The detection of both partial and whole gene deletions of PAX3/MITF in this clinical cohort increases the mutation detection yield by at least 6% and supports integrating MLPA into clinical molecular testing primarily for patients with WS1 and 3.     

Digital Detection of Multiple Minority Mutants and Expression Levels of Multiple Colorectal Cancer-Related Genes Using Digital-PCR Coupled with Bead-Array

To simultaneously analyze mutations and expression levels of multiple genes on one detection platform, we proposed a method termed “multiplex ligation-dependent probe amplification–digital amplification coupled with hydrogel bead-array” (MLPA–DABA) and applied it to diagnose colorectal cancer (CRC). CRC cells and tissues were sampled to extract nucleic acid, perform MLPA with sequence-tagged probes, perform digital emulsion polymerase chain reaction (PCR), and produce a hydrogel bead-array to immobilize beads and form a single bead layer on the array. After hybridization with fluorescent probes, the number of colored beads, which reflects the abundance of expressed genes and the mutation rate, was counted for diagnosis. Only red or green beads occurred on the chips in the mixed samples, indicating the success of single-molecule PCR. When a one-source sample was analyzed using mixed MLPA probes, beads of only one color occurred, suggesting the high specificity of the method in analyzing CRC mutation and gene expression. In gene expression analysis of a CRC tissue from one CRC patient, the mutant percentage was 3.1%, and the expression levels of CRC-related genes were much higher than those of normal tissue. The highly sensitive MLPA–DABA succeeds in the relative quantification of mutations and gene expressions of exfoliated cells in stool samples of CRC patients on the same chip platform. MLPA–DABA coupled with hydrogel bead-array is a promising method in the non-invasive diagnosis of CRC.     

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%.     

Use of in silico tools for classification of novel missense mutations identified in dystrophin gene in developing countries

DMD gene which is composed of 79 exons is the largest known gene located on X chromosome (Xp21). Point mutations in the dystrophin gene are responsible for 30–35% of cases with DMD/BMD. Mutation analysis of all the exons of the DMD gene is costly in developing countries, therefore, a few of the exons are selected to be analyzed routinely in clinical laboratories. In this study, direct sequencing was used for detection of point mutations in 10 exons of dystrophin gene in patients affected with DMD without detectable large rearrangements. Freely available programs were used to predict the damaging effects of the mutations. Point mutations were successfully detected in three patients. Three novel mutations, two missense mutations located on nonconservative domains and a single nucleotide deletion, were detected. Missense mutations were predicted to change splicing efficiency. Detection of point mutations by DNA analysis followed by prediction of the pathogenecity by using bioinformatic tool might be an asset to provide proper diagnosis or genetic counseling to patients and their family.     

Multiplex ligation-dependent probe amplification of LDLR enhances molecular diagnosis of familial hypercholesterolemia

Autosomal dominant (AD) familial hypercholesterolemia [FH; Mendelian Inheritance in Man (MIM) 143890] typically results from mutations in the LDL receptor gene (LDLR), which are now commonly diagnosed using exon-by-exon screening methods, such as exon-by-exon sequence analysis (EBESA) of genomic DNA (gDNA). However, many patients with FH have no LDLR mutation identified by this method. Part of the diagnostic gap is attributable to the genetic heterogeneity of AD FH, but another possible explanation is inadequate sensitivity of EBESA to detect certain mutation types, such as large deletions or insertions in LDLR. Multiplex ligation-dependent probe amplification (MLPA) is a new method that detects larger gDNA alterations that are overlooked by EBESA. We hypothesized that some FH patients with no LDLR mutation detectable by EBESA would have an abnormal LDLR MLPA pattern. In 70 unrelated FH patients, 44 had LDLR mutations detected by EBESA, including missense, RNA splicing, nonsense, or small deletion mutations, and 5 had the APOB R3500Q mutation. Among the remaining 21 AD FH patients with no apparent LDLR mutation, we found abnormal LDLR MLPA patterns in 12 and then demonstrated the deleted sequence in 5 of these. These findings indicate that MLPA may be a useful new adjunctive tool for the molecular diagnosis of FH.     

High-Throughput Amplicon-Based Copy Number Detection of 11 Genes in Formalin-Fixed Paraffin-Embedded Ovarian Tumour Samples by MLPA-Seq

Whilst next generation sequencing can report point mutations in fixed tissue tumour samples reliably, the accurate determination of copy number is more challenging. The conventional Multiplex Ligation-dependent Probe Amplification (MLPA) assay is an effective tool for measurement of gene dosage, but is restricted to around 50 targets due to size resolution of the MLPA probes. By switching from a size-resolved format, to a sequence-resolved format we developed a scalable, high-throughput, quantitative assay. MLPA-seq is capable of detecting deletions, duplications, and amplifications in as little as 5ng of genomic DNA, including from formalin-fixed paraffin-embedded (FFPE) tumour samples. We show that this method can detect BRCA1, BRCA2, ERBB2 and CCNE1 copy number changes in DNA extracted from snap-frozen and FFPE tumour tissue, with 100% sensitivity and >99.5% specificity.     

Multiplex Ligation-dependent Probe Amplification (MLPA) et sondes « à façon » entièrement synthétiques. Guide pratique,recommandations et expérience au CHU de Grenoble

Multiplex Ligation-dependent Probe Amplification (MLPA) is a powerful technique for gene dosage quantification. This technique is based on the ligation followed by PCR amplification of two adjacently hybridising oligonucleotides. Its reliability and effectiveness have been proven and is now well established. Besides the commercial kits are distributed by the patentee, MRC-Holland?, it makes it possible to utilise custom-designed MLPA probes for a targeted use of this technique. This strategy has opened the field of MLPA for various applications ranging from diagnostic of rarest pathologies to confirmation of a CGH-detected abnormalities. However, it may be difficult to develop without a solid experience. Here, we detail the guidelines and recommendations for designing custom MLPA probes and illustrate their use in our laboratory.     

Utility of MLPA in mutation analysis and carrier detection for Duchenne muscular dystrophy

CONTEXT:

Multiplex ligation probe amplification (MLPA) is a new technique to identify deletions and duplications and can evaluate all 79 exons in dystrophin gene in patients with Duchenne muscular dystrophy (DMD). Being semi-quantitative, MLPA is also effective in detecting duplications and carrier testing of females; both of which cannot be done using multiplex PCR. It has found applications in diagnostics of many genetic disorders.

AIM:

To study the utility of MLPA in diagnosis and carrier detection for DMD.

MATERIALS AND METHODS:

Mutation analysis and carrier detection was done by multiplex PCR and MLPA and the results were compared.

RESULTS AND CONCLUSIONS:

We present data showing utility of MLPA in identifying mutations in cases with DMD/BMD. In the present study using MLPA, we identified mutations in additional 5.6% cases of DMD in whom multiplex PCR was not able to detect intragenic deletions. In addition, MLPA also correctly confirmed carrier status of two obligate carriers and revealed carrier status in 6 of 8 mothers of sporadic cases.     

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.     

Determination of the exon structure of the distal portion of the dystrophin gene by vectorette PCR

The structure of the 3' one-third of the dystrophin gene has not previously been established. We have used vectorette PCR on a yeast artificial chromosome containing part of the human dystrophin gene to determine that there are 20 exons in this region and to characterize adjacent intron sequences of each one. Combined with previous information on the remainder of the gene, this study shows that the coding sequence is distributed between 79 exons. We have used PCR between exons to measure the distances that separate the more closely clustered exons. Vectorette PCR products were used as probes on Southern blots to assign all the 3' exons to genomic HindIII fragments that are commonly detected in the analysis of dystrophin gene deletions. The results will be useful for determining the effect of genomic deletions on the translational reading frame, for setting up genomic PCR assays to confirm point mutations, for analyzing splice site mutations, and for investigating potential cis-acting elements involved in tissue-specific alternative splicing. Vectorette PCR using primers derived from cDNA sequence represents an efficient and widely applicable method for establishing gene structure and obtaining intron sequence flanking exons, starting from a genomic clone and a cDNA sequence.     

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.     

Use of epitope libraries to identify exon-specific monoclonal antibodies for characterization of altered dystrophins in muscular dystrophy.

The majority of mutations in Xp21-linked muscular dystrophy (MD) can be identified by PCR or Southern blotting, as deletions or duplications of groups of exons in the dystrophin gene, but it is not always possible to predict how much altered dystrophin, if any, will be produced. Use of exon-specific monoclonal antibodies (mAbs) on muscle biopsies from MD patients can, in principle, provide information on both the amount of altered dystrophin produced and, when dystrophin is present, the nature of the genetic deletion or point mutation. For this purpose, mAbs which recognize regions of dystrophin encoded by known exons and whose binding is unaffected by the absence of adjacent exons are required. To map mAbs to specific exons, random "libraries" of expressed dystrophin fragments were created by cloning DNAseI digestion fragments of a 4.3-kb dystrophin cDNA into a pTEX expression vector. The libraries were then used to locate the epitopes recognized by 48 mAbs to fragments of 25-60 amino acids within the 1,434-amino-acid dystrophin fragment used to produce the antibodies. This is sufficiently detailed to allow further refinement by using synthetic peptides and, in many cases, to identify the exon in the DMD (Duchenne MD) gene which encodes the epitope. To illustrate their use in dystrophin analysis, a Duchenne patient with a frameshift deletion of exons 42 and 43 makes a truncated dystrophin encoded by exons 1-41, and we now show that this can be detected in the sarcolemma by mAbs up to and including those specific for exon 41 epitopes but not by mAbs specific for exon 43 or later epitopes.     

Direct electrochemical genosensing for multiple point mutation detection of Mycobacterium tuberculosis during the development of rifampin resistance

We present a robust and simple method for the direct detection of multiple point mutations in the Mycobacterium tuberculosis rpoB gene during the development of rifampin (RIF) resistance using an electrochemical genosensor. The device contained five different capture probes which are designed to hybridize with several sequence segments within the bacterial rpoB gene hotspot region. Point mutations were detected by monitoring the guanine oxidation with differential pulse voltammetry after hybridization between PCR amplicons and inosine modified capture probes at graphite surface. Changes in the peak voltage corresponding to guanine oxidation provide an electrochemical signal for hybridization that can be used to determine the presence of point mutations conferring rifampin resistance. The analytical parameters (sensitivity, selectivity and reproducibility) were evaluated. High selective discrimination against point mutation of bacteria at hot-spot region was observed. Several mutations were detected at several parts of the amplicon from 21 positive samples.