Multiplex Ligation-dependent Probe Amplification Technology Research Articles

Lessons learned from the first national population-based genetic carrier-screening program for Duchenne muscular dystrophy

PurposeTo summarize the results of first year implementation of pan-ethnic screening testing for Duchenne muscular dystrophy (DMD) and present the ensuing challenges. MethodsData acquisition for this study was performed by retrospective search of Ministry of Health registry for reports of all laboratories performing genetic screening tests. DMD testing was performed by multiplex ligation-dependent probe amplification technology. In case of single-exon deletion, sequencing of the specific exon was performed to rule out underlying single-nucleotide variant. ResultsOf overall 85,737 DMD tests, 82 clinically significant findings were noted (0.095%, or 1:1,046 women). In addition, 80 findings with uncertain clinical significance were detected (0.093%, or 1:1072), as well as 373 cases (0.4%, or 1:230) of single-exon deletions subsequently identified as false positives because of underlying single-nucleotide variant, mostly variants in exon 8 in North African Jewish population, and in exon 48 in Arab Muslim population. ConclusionInterpretation of population-based DMD carrier screening is complex, occasionally requiring additional genetic testing methods and ethical considerations. Multicenter data registry, including ethnic origin and familial segregation in selected cases, is crucial for optimal definition of the results during genetic counseling and informed decisions regarding prenatal testing.

Advances of multiplex ligation-dependent probe amplification technology in molecular diagnostics.

Multiplex ligation-dependent probe amplification (MLPA) is a multiplex copy number analysis tool which is routinely used to detect large mutations in genetic diseases. With continuous modifications, MLPA has been extended for the detection of DNA methylation variation, single nucleotide polymorphisms, chromosome abnormalities and other forms of genomic variation. The combination with other techniques has even enlarged the application of MLPA in molecular diagnostics of various human diseases. In this review, the principle of MLPA-based techniques as well as their main and latest applications in clinical detection are described. It is believed that with improved automation, increased multiplexing, lower cost and the combination with other technologies, MLPA will play an increasingly important role in molecular diagnosis of human disease.

Genetic analysis of 1051 Chinese families with Duchenne/Becker Muscular Dystrophy

BackgroundDuchenne Muscular Dystrophy (DMD) is the most common muscle disease in children, and there are no effective therapies for DMD or Becker Muscular Dystrophy (BMD). Currently, targeted gene therapy treatments have emerged. As a result, genetic diagnosis is the basis of treatment. In addition, genetic and prenatal diagnosis significantly reduces their incidence rates. This study combines the application of multiplex ligation-dependent probe amplification technology (MLPA) and “next-generation” sequencing technology (NGS) as the most economical and efficient method of diagnosis. Therefore, in the diagnosis of DMD/BMD, patients’ MLPA data are first used to detect DMD gene deletions or duplications, and NGS and Sanger sequencing are then applied to exclude MLPA-negative samples. Meanwhile, polymerase chain reaction (PCR) is used to detect single exon deletions to exclude false-positives in MLPA caused by point mutations.MethodsIn this study, we recruited 1051 proband families of DMD from 2016 to 2018 and had access to information that could identify individual participants during or after data collection. Patients who were diagnosed with DMD were first tested by MLPA. MLPA results with single exon deletions were validated with PCR amplification and Sanger sequencing. The negative results of MLPA were further analysed with NGS and validated by Sanger sequencing. For novel missense mutations, phenotype-genotype correlations were analysed using PolyPhen2 and mutation taster. All methods were performed in accordance with the relevant guidelines and regulations.ResultsDMD mutations were identified in 1029 families (97.91%, 1029/1051). Large deletions, duplications, and small mutations accounted for 70.41% (740/1051), 8.28% (87/1051), and 19.12% (201/1051) of all cases, respectively. There were 205 small mutation types, 53 of which were novel. The rate of de novo mutations was 39.45% (187/474) and was higher in large duplications (49.53%, 157/317). Among 68 asymptomatic patients (< 3 years old) with unexplained persistent hyperCKaemia upon conventional physical examination, 63 were diagnosed as DMD/BMD according to genetic diagnosis.ConclusionOur results expand the spectrum of DMD mutations, which could contribute to the treatment of DMD/BMD and provide an effective diagnosis method. Thus, the combination of MLPA, NGS and Sanger sequencing is of great significance for family analysis, gene diagnosis and gene therapy.

Expanded newborn screening for inherited metabolic disorders and genetic characteristics in a southern Chinese population

To evaluate the incidence, disease spectrum, and genetic characteristics of inherited metabolic disorders (IMDs) of newborns in Quanzhou area, China. We analyze the expanded newborn screening results of IMDs detected by tandem mass spectrometry (MS/MS) during 5 years. Suspected positive patients were diagnosed through next-generation sequencing and validated by Sanger sequencing. In addition, multiplex ligation-dependent probe amplification technology has also been applied to assist in diagnosis of diseases with deletion or duplication mutations. A total of 364,545 newborns were screened, 130 IMDs were identified yielding an incidence of 1:2804. In addition, 9 cases of maternal disorders were also identified by our MS/MS newborn screening program. There were 42 newborns with amino acid disorders (1:8680), 39 with organic acid disorders (1:9347), and 49 with fatty acid oxidation disorders (1:7440). Unlike other studies, our study indicated that fatty acid oxidation disorder has the highest proportion (37.7%), particularly primary carnitine deficiency (PCD) with incidence up to 1:10,126 was the most common disorder in the region. The recurrent mutations of relatively common diseases like PCD, phenylalanine hydroxylase deficiency, short-chain acyl-CoA dehydrogenase deficiency, citrin deficiency, glutaric acidemia type I, isobutyryl-CoA dehydrogenase deficiency, and multiple acyl-CoA dehydrogenase deficiency in this region were also clearly elucidated. Therefore, our data indicated that IMDs are never uncommon in Quanzhou, the disease spectrum and genetic backgrounds were clearly elucidated, contributing to the treatment and prenatal genetic counseling of these disorders in this region.

Diversity of genetic events associated with MLH1 promoter methylation in Lynch syndrome families with heritable constitutional epimutation

PurposeConstitutional epimutations are an alternative to genetic mutations in the etiology of genetic diseases. Some of these epimutations, termed secondary, correspond to the epigenetic effects of cis-acting genetic defects transmitted to the offspring following a Mendelian inheritance pattern. In Lynch syndrome, a few families with such apparently heritable MLH1 epimutations have been reported so far. MethodsWe designed a long-range polymerase chain reaction next-generation sequencing strategy to screen MLH1 entire gene and applied it to 4 French families with heritable epimutations and 10 additional patients with no proven transmission of their epimutations. ResultsThis strategy successfully detected the insertion of an Alu element in MLH1 coding sequence in one family. Two previously unreported MLH1 variants were also identified in other epimutation carriers: a nucleotide substitution within intron 1 and a single-nucleotide deletion in the 5′-UTR. Detection of a partial MLH1 duplication in another family required multiplex ligation-dependent probe amplification technology. We demonstrated the segregation of these variants with MLH1 methylation and studied the functional consequences of these defects on transcription. ConclusionThis is the largest cohort of patients with MLH1 secondary epimutations associated with a broad spectrum of genetic defects. This study provides further insight into the complexity of molecular mechanisms leading to secondary epimutations.

Whole dystrophin gene analysis by next-generation sequencing: a comprehensive genetic diagnosis of Duchenne and Becker muscular dystrophy.

Duchenne/Becker muscular dystrophies are the most frequent inherited neuromuscular diseases caused by mutations of the dystrophin gene. However, approximately 30% of patients with the disease do not receive a molecular diagnosis because of the complex mutational spectrum and the large size of the gene. The introduction and use of next-generation sequencing have advanced clinical genetic research and might be a suitable method for the detection of various types of mutations in the dystrophin gene. To identify the mutational spectrum using a single platform, whole dystrophin gene sequencing was performed using next-generation sequencing. The entire dystrophin gene, including all exons, introns and promoter regions, was target enriched using a DMD whole gene enrichment kit. The enrichment libraries were sequenced on an Illumina HiSeq 2000 sequencer using paired read 100 bp sequencing. We studied 26 patients: 21 had known large deletion/duplications and 5 did not have detectable large deletion/duplications by multiplex ligation-dependent probe amplification technology (MLPA). We applied whole dystrophin gene analysis by next-generation sequencing to the five patients who did not have detectable large deletion/duplications and to five randomly chosen patients from the 21 who did have large deletion/duplications. The sequencing data covered almost 100% of the exonic region of the dystrophin gene by ≥10 reads with a mean read depth of 147. Five small mutations were identified in the first five patients, of which four variants were unreported in the dmd.nl database. The deleted or duplicated exons and the breakpoints in the five large deletion/duplication patients were precisely identified. Whole dystrophin gene sequencing by next-generation sequencing may be a useful tool for the genetic diagnosis of Duchenne and Becker muscular dystrophies.

The application of multiplex ligation-dependent probe amplification technology in diagnosis and prenatal diagnosis of α-thalassemia

To investigate the multiplex ligation-dependent probe amplification (MLPA) technology in the detection of gene deletion and prenatal diagnosis of α-thalassaemia. Phenotypes were analyzed by whole blood cell counting and hemoglobin component detection of peripheral blood samples from the subjects. The gene deletions and point mutations of α- thalassaemia were detected with regular gap-PCR and reverse dot blot (RDB) method. At last, the MLPA method was applied for detection of α-globin gene deletion. All the prenatal diagnosis samples were detected with both gap-PCR and MLPA method. α-thalassaemia phenotype was found in 75 samples from 1256 (628 couples) peripheral blood samples for pre-pregnancy or prenatal thalassemia gene screening. Among them, 71 samples carrying α-gene mutations and consistent with phenotypes were detected by routine methods. In the other 3 samples with no α-gene mutations detected and 1 sample with HbH phenotype but genotype of ﹣α(4.2)/αα were analyzed by MLPA and found each one samples of whole α-globin gene cluster deletion, respectively. Seventeen high risk couples were screened. Among the 17 prenatal diagnosis samples, 2 villus samples contaminated by exogenous DNA were confirmed by MLPA method. MLPA is an effective complement for α-thalassaemia gene deletion detection. The molecular diagnosis strategy and process of gap-PCR combined with MLPA for α- thalassaemia gene deletion detection can prevent the missing of gene deletion, and false-positive or false-negative misdiagnosis of α-thalassaemia in prenatal diagnosis.

Application of MLPA technology in molecular diagnosis of three inherited diseases

Objective To evaluate multiplex ligation-dependent probe amplification (MLPA) as a senetic diagnostic method to detect copy number variants (CNVs) in SMA,DiGeorge syndrome and CAH. Methods Genomic DNA was extracted from peripheral blood of 21 patients and their family members received in Shanghai Children′s Medical Centre during February 2010 to February 2011.The procedure of MLPA includes hybridization of probe and target sequence,ligation,PCR amplification,capillary electrophoresis and data collection.MPLA data was analyzed by GeneMarker® software.The MLPA results were compared with conventional PCR-enzyme method and chromosomal microarray analysis. Results All of the 3 probands in SMA pedigrees suffer homozygous loss of exon 7 and 8 of SMN1 gene (copy number state was zero),and their parents have heterozygous loss on exon 7 and exon 8,respectively.Heterozygous deletions encompassing TBX1 were detected in 3 DiGeorge syndrome patients (copy number state≈0.5).The result of MLPA was confirmed by genomic microarray analysis,and the variant was scaled as a 1.3 Mb deletion.Various deletions including pathogenetic gene CYP21A2,the pseudogene CYP21A1P and module gene C4B were detected in six of eight CAH patients. Conclusions As a complement and enhancement of conventional methods for detecting CNVs. MLPA is effective in relative quantitative detection of copy number variations of pathogenic gene in SMA,DiGeorge syndrome and CAH patients and carriers.(Chin J Lab Med,2013,36:136-141) Key words: Nucleic acid amplifecation techniques; Muscularatrophy; spinal; DiGeorge syndrome; Adrenal hyperplasia; congenital

The research of combining high resolution melting with multiplex ligation-dependent probe amplification technology in the mutation scanning for PAH gene

To explore the value of combining high resolution melting (HRM) with multiplex ligation-dependent probe amplification (MLPA) for detecting mutations underlying phenylketonuria. HRM was used for detecting small mutations in phenylalanine hydroxylase gene (PAH) of 26 phenylketonuria patients. The results were verified with DNA sequencing. MLPA was used for detecting potential deletions/duplications in the PAH gene. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis was performed for additional potential mutations. A total of 21 mutations were found in 44/52 alleles (84.62%), which included a dupEx4. Among the 21 types of mutation, 19 were reported previously, and the remaining two were novel mutations: c.584_585insA and IVS10+1G>T. In addition, the mutation of R243Q (25%) was the most common type in China. The study showed that the combination of HRM and MLPA could increase the detection rate for mutation in PKU. The study has added new information to the human mutation database of PAH and provided a basis for clinical diagnosis and prenatal counseling.

Rapid and high-throughput multiplex ligation-dependent probe amplification for diagnosis of chromosome aneuploidy

To assess the diagnostic value of multiplex ligation-dependent probe amplification (MLPA) for detection of common chromosome aneuploidy in amniotic fluid (AF) cells in order to obtain an accurate, rapid, cost-effective and high-throughput method in routine prenatal clinical practice. The MLPA test was performed on 500 AF samples by using kit P095 and the results were obtained by using analysis software RH-MLPA-v511. The results were compared with that from fluorescence in situ hybridization (FISH) and traditional karyotyping (TK). The technical critical issues were analyzed in routine diagnostic application. The absolute specificity and sensitivity of the MLPA test to detect the aneuploidy were 100%. For the 500 AF samples, the success rate of the MLPA tests was 97%. Among them 92% were finished within three working days and 5% required more days for repeating. The test failure rate was 3%. The results confirmed that for the 38 detectable aneuploid samples, the probe reliability weighted mean ratio values were more than 4SD compared to normal diploids and the 2 suspected trisomy samples were more than 2SD. In this study, authors analyzed hybridization efficiencies of 8 probes for chromosome 21, and the presence of a trisomy was considered if at least 4 of the 8 probes gave probe ratio of >1.3. Thedata suggested that MLPA is a rapid, simple and reliable method for large scale testing for aneuploidy of chromosomes 13, 18, 21, X, or Y in AF. The MLPA technology is complementary to AF culture and valuable for prenatal diagnosis.

Detection, Analysis and Clinical Validation of Chromosomal Aberrations by Multiplex Ligation-Dependent Probe Amplification in Chronic Leukemia

Current diagnostic screening strategies based on karyotyping or fluorescent in situ hybridization (FISH) for detection of chromosomal abnormalities in chronic lymphocytic leukemia (CLL) are laborious, time-consuming, costly, and have limitations in resolution. Multiplex ligation-dependent probe amplification (MLPA) can simultaneously detect copy number changes of multiple loci in one simple PCR reaction, making it an attractive alternative to FISH. To enhance the clinical robustness and further harness MLPA technology for routine laboratory operations, we have developed and validated a protocol for comprehensive, automatic data analysis and interpretation. A training set of 50 normal samples was used to establish reference ranges for each individual probe, for the calling of statistically significant copy number changes. The maximum normal ranges of 2 and 3 standard deviations (SD) are distributed between 0.82 and 1.18 (Mean ± 2SD, 95% CI, P = 0.05), and between 0.73 and 1.27 (Mean ± 3SD, 99% CI, P = 0.01), respectively. We found an excellent correlation between MLPA and FISH with 93.6% concordance (P<0.0001) from a testing cohort of 100 clinically suspected CLL cases. MLPA analyses done on 94/100 patients showed sensitivity and specificity of 94.2% and 92.9%, respectively. MLPA detected additional copy number gains on 18q21.1 and chromosome 19, and novel micro-deletions at 19q13.43 and 19p13.2 loci in six samples. Three FISH-failed samples were tested positive by MLPA, while three 13q- cases with a low percentage of leukemia cells (7%, 12% and 19%) were not detected by MLPA. The improved CLL MLPA represents a high-throughput, accurate, cost-effective and user-friendly platform that can be used as a first-line screening test in a clinical laboratory.

ProSeeK: A web server for MLPA probe design

BackgroundThe technological evolution of platforms for detecting genome-wide copy number imbalances has allowed the discovery of an unexpected amount of human sequence that is variable in copy number among individuals. This type of human variation can make an important contribution to human diversity and disease susceptibility. Multiplex Ligation-dependent Probe Amplification (MLPA) is a targeted method to assess copy number differences for up to 40 genomic loci in one single experiment. Although specific MLPA assays can be ordered from MRC-Holland (the proprietary company of the MLPA technology), custom designs are also developed in many laboratories worldwide. After our own experience, an important drawback of custom MLPA assays is the time spent during the design of the specific oligonucleotides that are used as probes. Due to the large number of probes included in a single assay, a number of restrictions need to be met in order to maximize specificity and to increase success likelihood.ResultsWe have developed a web tool for facilitating and optimising custom probe design for MLPA experiments. The algorithm only requires the target sequence in FASTA format and a set of parameters, that are provided by the user according to each specific MLPA assay, to identify the best probes inside the given region.ConclusionTo our knowledge, this is the first available tool for optimizing custom probe design of MLPA assays. The ease-of-use and speed of the algorithm dramatically reduces the turn around time of probe design. ProSeeK will become a useful tool for all laboratories that are currently using MLPA in their research projects for CNV studies.

Mutation screening of fumarate hydratase by multiplex ligation-dependent probe amplification: detection of exonic deletion in a patient with leiomyomatosis and renal cell cancer

Hereditary leiomyomatosis and renal cell cancer (HLRCC) is a syndrome predisposing to cutaneous and uterine leiomyomatosis as well as renal cell cancer and uterine leiomyosarcoma. Heterozygous germline mutations in the fumarate hydratase (FH, fumarase) gene are known to cause HLRCC. On occasion, no FH mutation is detected by direct sequencing, despite the evident HLRCC phenotype in a family. In the present study, to investigate whole gene or exonic deletions and amplifications in FH mutation-negative patients, we used multiplex ligation-dependent probe amplification technology. The study material comprised 7 FH mutation-negative HLRCC patients and 12 patients affected with HLRCC-associated phenotypes, including papillary RCC, early-onset RCC, uterine leiomyomas, or uterine leiomyosarcoma. A novel FH mutation, a deletion of FH exon 1 that encodes the mitochondrial signal peptide, was detected in one of the HLRCC patients (1/7). The patient with the FH mutation displayed numerous painful cutaneous leiomyomas and papillary type renal cell cancer. Our finding, together with the two patients with whole FH gene deletion who had been detected previously, suggests that exonic or whole-gene FH deletions are not a frequent cause of HLRCC syndrome. ©2008 Elsevier Inc. All rights reserved.

Molecular diagnosis utility of multiplex ligation-dependent probe amplification

Multiplex ligation-dependent probe amplification (MLPA) is a novel method that uses multiple pairs of oligonucleotide probes to hybridize targeted genomic DNA regions; products from ligation reaction and polymerase chain reaction amplification are then analyzed by capillary electrophoresis. MLPA has been broadly used to detect copy number variation from single nucleotide substitutions to several megabase nucleotide deletions/duplications to chromosomal aneuploidy, methylation pattern analysis for genetic imprinting disorders and malignant tumors, and gene expression profiles in various genetic conditions. The technical aspects of the MLPA assay are reviewed, its applications in molecular diagnosis are discussed, and information is presented about quality assurance, quality control and future possibilities. More than 200 MLPA-related publications have been reviewed. MLPA offers significant advantages and is an attractive alternative to many current molecular and cytogenetic diagnostic techniques for broad applications. For its robustness and potential high throughput, it is anticipated that MLPA technology will be more widely used in the molecular diagnostic laboratory as a routine test for many genetic disorders.