Corrigendum: Diagnosis of the accurate genotype of HKaa carriers in patients with thalassemia using multiplex ligation-dependent probe amplification combined with nested polymerase chain reaction.
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PROVIDER: S-EPMC7386347 | biostudies-literature | 2020 Jul
REPOSITORIES: biostudies-literature
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Corrigendum: Diagnosis of the accurate genotype of HKaa carriers in patients with thalassemia using multiplex ligation-dependent probe amplification combined with nested polymerase chain reaction.
Chinese medical journal 20200701 13
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Project description:BackgroundMultiplex ligation-dependent probe amplification (MLPA) was originally described as an efficient and reliable technique for gene dosage or DNA copy number variation (CNV) analysis. Due to its low cost, reliability, sensitivity, and relative simplicity, MLPA has rapidly gained acceptance in research and diagnostic laboratories, and fills the gap between genome-wide analysis and single gene analysis. A number of new applications have been developed shortly after the introduction of MLPA, including methylation-specific MLPA (MS-MLPA), the use of MLPA in SNP genotyping, copy number analysis in segmentally duplicated regions, etc. However, probe design is time consuming and error prone. Recently software has been developed to help human genomic MLPA probe selection and optimization. For other genomes and MS-MLPA, probe design remains a challenge.FindingsThis paper describes a number of new features added to the previous H-MAPD software, which include: 1) probe selection for MS-MLPA; 2) support of mouse and rat genomes; 3) a set of new stuffer sequences. In addition, a physical-chemical property verification tool was implemented to verify user defined probes.ConclusionsMAPD is a web-based tool which is freely available to non-commercial users. The previous H-MAPD software has been used by about 200 users from more than 30 countries. With the new features, the author hopes MAPD will bring more convenience to the MLPA community.
| S-EPMC2893534 | biostudies-literature
Project description:Gene amplification is an important mechanism for oncogene activation, a crucial step in carcinogenesis. Compared to female breast cancer, little is known on the genetic makeup of male breast cancer, because large series are lacking. Copy number changes of 21 breast cancer related genes were studied in 110 male breast cancers using multiplex ligation-dependent probe amplification. A ratio of >1.3 was regarded indicative for gene copy number gain and a ratio >2.0 for gene amplification. Data were correlated with clinicopathological features, prognosis and 17 genes were compared with a group of female breast cancers. Gene copy number gain of CCND1, TRAF4, CDC6 and MTDH was seen in >40 % of the male breast cancer cases, with also frequent amplification. The number of genes with copy number gain and several single genes were associated with high grade, but only CCND1 amplification was an independent predictor of adverse survival in Cox regression (p = 0.015; hazard ratio 3.0). In unsupervised hierarchical clustering a distinctive group of male breast cancer with poor prognosis (p = 0.009; hazard ratio 3.4) was identified, characterized by frequent CCND1, MTDH, CDC6, ADAM9, TRAF4 and MYC copy number gain. Compared to female breast cancers, EGFR (p = 0.005) and CCND1 (p = 0.041) copy number gain was more often seen in male breast cancer, while copy number gain of EMSY (p = 0.004) and CPD (p = 0.001) and amplification in general was less frequent. In conclusion, several female breast cancer genes also seem to be important in male breast carcinogenesis. However, there are also clear differences in copy number changes between male and female breast cancers, pointing toward differences in carcinogenesis between male and female breast cancer and emphasizing the importance of identifying biomarkers and therapeutic agents based on research in male breast cancer. In addition CCND1 amplification seems to be an independent prognosticator in male breast cancer.
| S-EPMC3413821 | biostudies-literature
Project description:AimTo develop a SULT1A1 multiplex ligation-dependent probe amplification assay and to investigate multi-ethnic copy number variant frequencies.MethodsA novel multiplex ligation-dependent probe amplification assay was developed and tested on 472 African-American, Asian, Caucasian, Hispanic and Ashkenazi Jewish individuals.ResultsThe frequencies of atypical total copy number (i.e., greater or less than two) were 38.7% for Hispanics, 38.9% for Ashkenazi Jewish, 43.2% for Caucasians, 53.6% for Asians and 64.1% for African-Americans. Heterozygous SULT1A1 deletion carriers (slow sulfators) were most common among Caucasians (8.4%), whereas African-Americans had the highest frequencies of three or more copies (rapid sulfators; 60.9%).ConclusionDifferent ethnic and racial populations have varying degrees of SULT1A1-mediated sulfation activity, which warrants further research and that may have utility for drug response prediction among SULT1A1-metabolized medications.
| S-EPMC6021911 | biostudies-literature
Project description:Multiplex ligation-dependent probe amplification (MLPA) is a recently described method for detecting gross deletions or duplications of DNA sequences, aberrations which are commonly overlooked by standard diagnostic analysis. To determine the incidence of copy number variants in cancer predisposition genes from families in the Wessex region, we have analysed the hMLH1 and hMSH2 genes in patients with hereditary nonpolyposis colorectal cancer (HNPCC), BRCA1 and BRCA2 in families with hereditary breast/ovarian cancer (BRCA) and APC in patients with familial adenomatous polyposis coli (FAP). Hereditary nonpolyposis colorectal cancer (n=162) and FAP (n=74) probands were fully screened for small mutations, and cases for which no causative abnormality were found (HNPCC, n=122; FAP, n=24) were screened by MLPA. Complete or partial gene deletions were identified in seven cases for hMSH2 (5.7% of mutation-negative HNPCC; 4.3% of all HNPCC), no cases for hMLH1 and six cases for APC (25% of mutation negative FAP; 8% of all FAP). For BRCA1 and BRCA2, a partial mutation screen was performed and 136 mutation-negative cases were selected for MLPA. Five deletions and one duplication were found for BRCA1 (4.4% of mutation-negative BRCA cases) and one deletion for BRCA2 (0.7% of mutation-negative BRCA cases). Cost analysis indicates it is marginally more cost effective to perform MLPA prior to point mutation screening, but the main advantage gained by prescreening is a greatly reduced reporting time for the patients who are positive. These data demonstrate that dosage analysis is an essential component of genetic screening for cancer predisposition genes.
| S-EPMC2747696 | biostudies-other
Project description:To estimate the contribution of single and multi-exon NF1 gene copy-number changes to the NF1 mutation spectrum, we analysed a series of 201 Italian patients with neurofibromatosis type 1 (NF1). Of these, 138 had previously been found, using denaturing high-performance liquid chromatography or protein truncation test, to be heterozygous for intragenic NF1 point mutations/deletions/insertions, and were excluded from this analysis. The remaining 63 patients were analysed using multiplex ligation-dependent probe amplification (MLPA), which allows detection of deletions or duplications encompassing >or=1 NF1 exons, as well as entire gene deletions. MLPA results were validated using real-time quantitative PCR (qPCR) or fluorescent in situ hybridisation. MLPA screening followed by real-time qPCR detected a total of 23 deletions. Of these deletions, six were single exon, eight were multi-exon, and nine were of the entire NF1 gene. In our series, deletions encompassing >or=1 NF1 exons accounted for approximately 7% (14/201) of the NF1 gene mutation spectrum, suggesting that screening for these should now be systematically included in genetic testing of patients with NF1.
| S-EPMC2652822 | biostudies-literature
Project description: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.
| S-EPMC3291544 | biostudies-literature
Project description:BackgroundThe Duchenne muscular dystrophy (DMD) gene is located in the short arm of the X chromosome (Xp21). It spans 2.4 Mb of the human genomic DNA and is composed of 79 exons. Mutations in the Dystrophin gene result in DMD and Becker muscular dystrophy. In this study, the efficiency of multiplex ligation-dependent probe amplification (MLPA) over multiplex polymerase chain reaction (PCR) assays in an Iranian population was investigated.Materials and methodsMultiplex PCR assays and MLPA analysis were carried out in 74 patients affected with DMD.ResultsMultiplex PCR detected deletions in 51% of the patients with DMD. MLPA analysis could determine all the deletions detected by the multiplex PCR. Additionally, MLPA was able to identify one more deletion and duplication in patients without detectable mutations by multiplex PCR. Moreover, MLPA precisely determined the exact size of the deletions.ConclusionAlthough MLPA analysis is more sensitive for detection of deletions and duplications in the dystrophin gene, multiplex PCR might be used for the initial analysis of the boys affected with DMD in the Iranian population as it was able to detect 95% of the rearrangements in patients with DMD.
| S-EPMC3950794 | biostudies-literature
Project description: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. RESULTS: Through simulation studies we have shown that our proposed method outperforms two existing methods that are based on simple threshold rules or iterative regression. We have illustrated the method using a controlled MLPA assay in which targeted regions are variable in copy number in individuals suffering from different disorders such as Prader-Willi, DiGeorge or Autism showing the best performace. CONCLUSION: Using the proposed mixed-model, we are able to determine thresholds to decide whether a region is altered. These threholds are specific for each individual, incorporating experimental variability, resulting in improved sensitivity and specificity as the examples with real data have revealed.
| S-EPMC2492880 | biostudies-other
Project description: Not available
| S-EPMC6708679 | biostudies-literature
Project description: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.
| S-EPMC5494700 | biostudies-literature