MLPA Analyses Reveal a Spectrum of Dystrophin Gene Deletions/Duplications in Pakistani Patients Suspected of Having Duchenne/Becker Muscular Dystrophy: A Retrospective Study

Ansar, Zeeshan; Nasir, Asghar; Moatter, Tariq; Khan, Sara; Kirmani, Salman; Ibrahim, Shahnaz; Imam, Kahkashan; Ather, Anif; Samreen, Azra; Hasan, Zahra

doi:10.1089/gtmb.2018.0262

Cited by 11 publications

(10 citation statements)

References 29 publications

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“…Retrospective studies have been conducted on dystrophinopathy patients at Aga Khan University Hospital Karachi utilizing MLPA to screen dystrophin gene exons for deletions/ duplications. 11,12 But no study so far has focused on derivation of iPSCs from these patients for future invitro disease modeling.…”

Section: Discussionmentioning

confidence: 99%

Episomal reprogramming of Duchenne muscular dystrophy patients derived CD3+ T cells towards induced pluripotent stem cells

Zehravi¹,

Wahid

Ashraf³

2021

Pak J Med Sci

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Objective: To derive Duchenne muscular dystrophy patient specific induced pluripotent stem cells (iPSCs) from CD3+T cells of DMD patients using episomal reprogramming and characterization of these DMD-iPSCs using immunofluorescence to confirm their pluripotent state. Methods: DMD patients were selected based upon their clinical history and examination. Peripheral blood mononuclear cells were isolated from peripheral blood of DMD patients (n=3) by density gradient centrifugation and were used to generate DMD patient specific T cells (DMD-T cells) using rhIL-2, plate bound anti CD3 antibody and T cell specific media along with specific culture conditions that promote T cell expansion. CD3+ T cells were characterized by flow cytometry and reprogrammed using episomal plasmid to generate DMD-iPSCs. These DMD-iPSCs were characterized using immunofluorescence. The study was carried out at Dow Research Institute of Biotechnology and Biomedical Sciences during August 2017- July 2018 for a period of approximately 12 months. Results: The peripheral blood mononuclear cells (PBMNC) derived T cells appeared as suspended cell clumps morphologically were positive for the expression of CD3 and negative for CD31. The DMD patient specific iPSCs appeared as round, compact and flat colonies with well-defined edges morphologically and were positive for the expression of pluripotency markers OCT4, SSEA-4 and TRA-1-81 on immunofluorescence. Conclusion: CD3+ T cell derived DMD-iPSCs were obtained under feeder free and xeno-free culture conditions using episomal reprogramming. doi: https://doi.org/10.12669/pjms.37.2.3388 How to cite this:Zehravi M, Wahid M, Ashraf J. Episomal reprogramming of Duchenne muscular dystrophy patients derived CD3+ T cells towards induced pluripotent stem cells. Pak J Med Sci. 2021;37(2):---------. doi: https://doi.org/10.12669/pjms.37.2.3388 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Section: Discussionmentioning

confidence: 99%

Episomal reprogramming of Duchenne muscular dystrophy patients derived CD3+ T cells towards induced pluripotent stem cells

Zehravi¹,

Wahid

Ashraf³

2021

Pak J Med Sci

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“… 17 Ansar et al in 2019 found deletions in exon 42-52 (exon 46 most frequent) and duplications in exon 3-7 to be the most frequent in Pakistani patients; while global data reveals exon 45 as the most frequent deletion but exon 2 as the most frequent duplication. 18 …”

Section: Discussionmentioning

confidence: 99%

“…deletions in exon 42-52 (exon 46 most frequent) and duplications in exon 3-7 to be the most frequent in Pakistani patients; while global data reveals exon 45 as the most frequent deletion but exon 2 as the most frequent duplication. 18 Urine is a suitable non-invasive, painless and convenient source of DMD patients' cells for generating DMD-iPSCs. 19 Hence, in this study, patient-specific urine-derived DMD-iPSCs have been chosen as starting material and been differentiated towards patient-specific DMD-Cardiomyocytes, as has been demonstrated previously by others like Guan et al and Pioner et al 20,21 In this study, we have used Gibco's PSC Cardiomyocyte Differentiation Kit.…”

Section: A H E a D O F P R I N Tmentioning

confidence: 99%

Induced pluripotent stem cells derived cardiomyocytes from Duchenne Muscular Dystrophy patients in vitro

Ghori¹,

Wahid

2021

Pak J Med Sci

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Objective: This study aimed at the in vitro generation of DMD-cardiomyocytes from patient-specific induced pluripotent stem cells derived from a Pakistani patient for future work on DMD in vitro disease modeling and drug testing for efficacy and toxicity. Methods: This in vitro experimental study was carried out from December 2018 to January 2019 at Stem Cells and Regenerative Medicine Lab (SCRML) at Dow Research Institute of Biotechnology and Biomedical Sciences (DRIBBS), Dow University of Health Sciences (DUHS) Urine derived DMD-iPSCs were used which had been generated previously from a Pakistani DMD patient who had been selected through non-random purposive sampling. These were differentiated towards cardiomyocytes using Cardiomyocytes Differentiation media having specified growth factors and then the molecular characterization of the differentiated cells was done using immunofluorescence. Results: Pakistani patient’s DMD-Cardiomyocytes were generated and their identity was confirmed by positive immunofluorescence for the expression of cardiac markers NKX2-5 and TNNT-2. Conclusion: This study aimed for in vitro generation of DMD cardiomyocytes for future application in disease modeling, new drug testing for efficacy and toxicity, as well as for drug-testing for tailored personalized therapy. To the best of our knowledge, this was the first time DMD-Cardiomyocytes were generated from Pakistani DMD patients using their own induced pluripotent stem cells. doi: https://doi.org/10.12669/pjms.37.5.3104 How to cite this:Ghori FF, Wahid M. Induced pluripotent stem cells derived cardiomyocytes from Duchenne Muscular Dystrophy patients in vitro. Pak J Med Sci. 2021;37(5):---------. doi: https://doi.org/10.12669/pjms.37.5.3104 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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“…In parallel, researchers across many countries are collecting mutational data on DBMD patients across the world. These efforts supplement consolidation of patient data into a global registry like TREAT-NMD [ 57 , 58 , 59 , 60 , 61 , 62 , 63 , 64 , 65 , 66 , 67 ].…”

Section: Patient Registries and The Personalization Of Exon Skippimentioning

confidence: 99%

Advances in Genetic Characterization and Genotype–Phenotype Correlation of Duchenne and Becker Muscular Dystrophy in the Personalized Medicine Era

Sheikh

Yokota

2020

JPM

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Currently, Duchenne muscular dystrophy (DMD) and the related condition Becker muscular dystrophy (BMD) can be usually diagnosed using physical examination and genetic testing. While BMD features partially functional dystrophin protein due to in-frame mutations, DMD largely features no dystrophin production because of out-of-frame mutations. However, BMD can feature a range of phenotypes from mild to borderline DMD, indicating a complex genotype–phenotype relationship. Despite two mutational hot spots in dystrophin, mutations can arise across the gene. The use of multiplex ligation amplification (MLPA) can easily assess the copy number of all exons, while next-generation sequencing (NGS) can uncover novel or confirm hard-to-detect mutations. Exon-skipping therapy, which targets specific regions of the dystrophin gene based on a patient’s mutation, is an especially prominent example of personalized medicine for DMD. To maximize the benefit of exon-skipping therapies, accurate genetic diagnosis and characterization including genotype–phenotype correlation studies are becoming increasingly important. In this article, we present the recent progress in the collection of mutational data and optimization of exon-skipping therapy for DMD/BMD.

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MLPA Analyses Reveal a Spectrum of Dystrophin Gene Deletions/Duplications in Pakistani Patients Suspected of Having Duchenne/Becker Muscular Dystrophy: A Retrospective Study

Cited by 11 publications

References 29 publications

Episomal reprogramming of Duchenne muscular dystrophy patients derived CD3+ T cells towards induced pluripotent stem cells

Episomal reprogramming of Duchenne muscular dystrophy patients derived CD3+ T cells towards induced pluripotent stem cells

Induced pluripotent stem cells derived cardiomyocytes from Duchenne Muscular Dystrophy patients in vitro

Advances in Genetic Characterization and Genotype–Phenotype Correlation of Duchenne and Becker Muscular Dystrophy in the Personalized Medicine Era

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