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Genetic Basis of Neuromuscular Disorders
Published in Maher Kurdi, Neuromuscular Pathology Made Easy, 2021
On the other hand, multiplex ligation-dependent probe amplification (MLPA) is another technique widely used for the detection of copy number change, deletions, or duplications in single genes. For example, MLPA could be useful for a screening of patients who could benefit from Exondys 51 and have an exon 51 skipping in the DMD gene. The MLPA assay is a modified form of multiplex polymerase chain reaction where all targeted exons are hybridized into two specially modified oligonucleotide probes that will bind targets adjacent to each other. This binding is sequence specific and highly sensitive to mismatches. Following successful ligation of the two probes, the amplification of that is detected on DNA analyzers. Because MLPA uses a dedicated reference for every gene, the MLPA targets can be quantified and a deletion or duplication is detected.
Bannayan–Riley–Ruvalcaba Syndrome
Published in Dongyou Liu, Handbook of Tumor Syndromes, 2020
Gabriela Maria Abreu Gontijo, Clóvis Antônio Lopes Pinto
The diagnosis of BRRS is made clinically, despite the absence of specific diagnostic criteria. BRRS patients harboring a PTEN mutation are grouped in the PHTS assortment. Mutations and/or deletions within PTEN are identified by genetic testing. Detection of such alterations confirms the PHTS diagnosis and permits predictive testing and prenatal diagnosis among patients’ relatives [14]. Several methods are presently employed to detect PTEN deletions. These include multiplex ligation-dependent probe amplification (MLPA) (the preferred method), Southern blotting, monochromosomal hybrid analysis, real-time polymerase chain reaction (PCR), and semiquantitative multiplex PCR [14]. In order to optimize yield, the best order of PTEN testing should be first sequencing complete PTEN coding exons 1–9 and flanking intronic regions. The second step, if no pathogenic modification is found, would be analysis for deletion/duplication [30]. A more accurate and comprehensive assessment of chromosomal abnormalities can be achieved combining chromosomal microarray analysis and conventional cytogenetics in order to identify anomalies in chromosomes [31].
Genetic and genomic investigations
Published in Angus Clarke, Alex Murray, Julian Sampson, Harper's Practical Genetic Counselling, 2019
Multiplex ligation-dependent probe amplification (MLPA) is an application of the polymerase chain reaction (PCR) in which it is not the patient's DNA that is amplified by PCR but, instead, carefully designed probe sequences. Two DNA probes are designed to recognise and anneal to immediately adjacent sequences in a gene of interest; once in place, they are ligated together by a DNA ligase. The two probes carry an additional sequence, not complementary to human DNA sequences, including PCR primer binding sites and additional ‘stuffer sequences’ designed so that the PCR product is of a very specific size. The PCR reaction of the probe sequence, not human sequence, can then proceed, yielding a quantity of product that relates directly to the number of copies of the target DNA in the patient's genome (Figure 5.1).
Histological Evaluation of Products of Conception, Who Benefits from It?
Published in Fetal and Pediatric Pathology, 2023
Haleh Soltanghoraee, Arash Mohazzab, Azadeh Soltani, Soheila Ansaripour, Maryam Tavakoli, Maryam Rafati, Amir Hassan Zarnani, Saeed Reza Ghaffari
Samples were received in normal saline without any fixative, in a cool box with ice. They were examined macroscopically and the size of the gestational sac and embryo, if present, was recorded. Then 2–3 samples of chorionic villi, membranes, umbilical cord or embryo (if included) were taken and transferred to separate microtubes, and were stored in −20 °C for probable use in genetic tests. There were also 2–3 samples submitted from the sac, embryo and decidua which were prepared for histology. All macroscopic evaluations were performed by an experienced perinatal pathologist. After fixation in 10% buffered formalin, the tissue was processed in an automated tissue processor and embedded in paraffin, with 4-5 micron sections stained with H&E. All samples were assessed microscopically by the same pathologist who evaluated them macroscopically at first. Immunohistochemistry staining for confirmation of histiocytic origin of intervilli cells in CIUE group was performed using CD 68 antibodies (mouse monoclonal antibodies, clone KP1, EDTA pretreatment buffer, incubation, DAB staining, Diagnostic Biosystem, Netherlands). Some of the samples were referred to the genetics lab for MLPA (Multiplex Ligation-dependent Probe Amplification) per their practitioner’s order.
α- and β-Globin Gene Mutations in Individuals with Hemoglobinopathies in the Chattogram and Sylhet Regions of Bangladesh
Published in Hemoglobin, 2023
Tamanna Kabir, Saeed Anwar, Jarin Taslem Mourosi, Shanjida Akter, Mohammad Jakir Hosen
One of the significant strengths of this study was that it could characterize (or at least partially characterize) all the index subjects using the designed PCR-based assays designed (Tables 4 and 5). However, to be fully confirmed, the PCR assays we designed can detect all the major or commonly occurring Hb mutations in the population, further studies using advanced techniques, e.g. direct sequencing, comparative genomic hybridization (CGH), or multiplex ligation-dependent probe amplification (MLPA), are needed. Studies involving siblings and parental samples could also be advantageous [23]. Outcomes of this study and similar molecular epidemiological studies can be extended to families at elevated risk for genetic counseling, and families with rare and new mutations to better understand the diseases in the country.
Confirmatory test versus screening test analyses for fetal mosaic variations; a large scale study
Published in Alexandria Journal of Medicine, 2022
Seyed Akbar Moosavi, Behnam Hasannejad-Asl, Masoumeh Kourosh Arami, Mahsa Nasuti, Mehmet Cemal Oguz, Abdol-Hossain Naseri
Amniocentesis and Chorionic Villus Sampling (CVS) are routine invasive PTs offered to a pregnant female with an increased risk of carrying a child with a nonmosaic genetic anomaly [5,6]. To detect a chromosomal abnormality, initially, the fetal cells are separated from their matrix, cultured in an appropriate medium, harvested, and then analyzed by karyotyping technique at the metaphasic stage [7,8]. In some clinics, molecular tests such as Fluorescent in Situ Hybridization (FISH), Multiplex Ligation-dependent Probe Amplification (MLPA), array-Comparative Genomic Hybridization (a-CGH), and next-generation sequencing (NGS), are also performed along with karyotyping to avoid misdiagnosis and increase the sensitivity and accuracy of the PTs [9]. Recently, Quantitative Fluorescents polymerase-chain reaction (QF-PCR) have been introduced as a rapid screening molecular test for prenatal in which their results for the nonmosaic genetic test were compatible with the gold standard karyotype method [10,11].