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Leejun C Wong - One of the best experts on this subject based on the ideXlab platform.
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Sequence Homology at the Breakpoint and Clinical Phenotype of Mitochondrial DNA Deletion Syndromes
2013Co-Authors: Bekim Sadikovic, Jing Wang, Ayman El-hattab, Megan L, Ganka Douglas, Ellen K. Brundage, William J. Craigen, Eric S. Schmitt, Leejun C WongAbstract:Mitochondrial DNA (mtDNA) Deletions are a common cause of mitochondrial disorders. Large mtDNA Deletions can lead to a broad spectrum of clinical features with different age of onset, ranging from mild mitochondrial myopathies (MM), progressive external ophthalmoplegia (PEO), and Kearns-Sayre syndrome (KSS), to severe Pearson syndrome. The aim of this study is to investigate the molecular signatures surrounding the Deletion breakpoints and their association with the clinical phenotype and age at onset. MtDNA Deletions in 67 patients were characterized using array comparative genomic hybridization (aCGH) followed by PCR-sequencing of the Deletion junctions. Sequence homology including both perfect and imperfect short repeats flanking the Deletion regions were analyzed and correlated with clinical features and patients’ age group. In all age groups, there was a significant increase in sequence homology flanking the Deletion compared to mtDNA background. The youngest patient group (,6 years old) showed a diffused pattern of Deletion distribution in size and locations, with a significantly lower sequence homology flanking the Deletion, and the highest percentage of Deletion mutant heteroplasmy. The older age groups showed rather discrete pattern of Deletions with 44 % of all patients over 6 years old carrying the most common 5 kb mtDNA Deletion, which was found mostly in muscle specimens (22/41). Only 15% (3/20) of the young patients (,6 years old) carry the 5 kb common Deletion, which is usually present in blood rather than muscle. This group of patients predominantly (16 out of 17) exhibit multisystem disorder and/or Pearson syndrome, whil
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Sequence homology at the breakpoint and clinical phenotype of mitochondrial DNA Deletion syndromes.
PloS one, 2010Co-Authors: Bekim Sadikovic, Jing Wang, Ayman El-hattab, Ganka Douglas, Ellen K. Brundage, William J. Craigen, Eric S. Schmitt, Megan Landsverk, Leejun C WongAbstract:Mitochondrial DNA (mtDNA) Deletions are a common cause of mitochondrial disorders. Large mtDNA Deletions can lead to a broad spectrum of clinical features with different age of onset, ranging from mild mitochondrial myopathies (MM), progressive external ophthalmoplegia (PEO), and Kearns-Sayre syndrome (KSS), to severe Pearson syndrome. The aim of this study is to investigate the molecular signatures surrounding the Deletion breakpoints and their association with the clinical phenotype and age at onset. MtDNA Deletions in 67 patients were characterized using array comparative genomic hybridization (aCGH) followed by PCR-sequencing of the Deletion junctions. Sequence homology including both perfect and imperfect short repeats flanking the Deletion regions were analyzed and correlated with clinical features and patients' age group. In all age groups, there was a significant increase in sequence homology flanking the Deletion compared to mtDNA background. The youngest patient group (
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sequence homology at the breakpoint and clinical phenotype of mitochondrial dna Deletion syndromes
PLOS ONE, 2010Co-Authors: Bekim Sadikovic, Jing Wang, Ganka Douglas, Ellen K. Brundage, William J. Craigen, Eric S. Schmitt, Megan Landsverk, Ayman W Elhattab, Leejun C WongAbstract:Mitochondrial DNA (mtDNA) Deletions are a common cause of mitochondrial disorders. Large mtDNA Deletions can lead to a broad spectrum of clinical features with different age of onset, ranging from mild mitochondrial myopathies (MM), progressive external ophthalmoplegia (PEO), and Kearns-Sayre syndrome (KSS), to severe Pearson syndrome. The aim of this study is to investigate the molecular signatures surrounding the Deletion breakpoints and their association with the clinical phenotype and age at onset. MtDNA Deletions in 67 patients were characterized using array comparative genomic hybridization (aCGH) followed by PCR-sequencing of the Deletion junctions. Sequence homology including both perfect and imperfect short repeats flanking the Deletion regions were analyzed and correlated with clinical features and patients' age group. In all age groups, there was a significant increase in sequence homology flanking the Deletion compared to mtDNA background. The youngest patient group (<6 years old) showed a diffused pattern of Deletion distribution in size and locations, with a significantly lower sequence homology flanking the Deletion, and the highest percentage of Deletion mutant heteroplasmy. The older age groups showed rather discrete pattern of Deletions with 44% of all patients over 6 years old carrying the most common 5 kb mtDNA Deletion, which was found mostly in muscle specimens (22/41). Only 15% (3/20) of the young patients (<6 years old) carry the 5 kb common Deletion, which is usually present in blood rather than muscle. This group of patients predominantly (16 out of 17) exhibit multisystem disorder and/or Pearson syndrome, while older patients had predominantly neuromuscular manifestations including KSS, PEO, and MM. In conclusion, sequence homology at the Deletion flanking regions is a consistent feature of mtDNA Deletions. Decreased levels of sequence homology and increased levels of Deletion mutant heteroplasmy appear to correlate with earlier onset and more severe disease with multisystem involvement.
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Application of dual-genome oligonucleotidearray-based comparative genomic hybridization to the molecular diagnosis of mitochondrial DNA Deletion and depletion syndromes
Genetics in Medicine, 2009Co-Authors: A. C. Chinault, Ellen K. Brundage, Chad A Shaw, Lin-ya Tang, Leejun C WongAbstract:Purpose: Mitochondrial disorders constitute a group of clinically and genetically heterogeneous diseases for which molecular diagnosis has been a challenge. The current procedures for diagnosis of mitochondrial DNA Deletion and depletion syndromes based on Southern analysis and quantitative polymerase chain reaction are particularly inefficient for determining important parameters of Deletion endpoints and percent heteroplasmy. We have developed an improved approach for routine analyses of these disorders in a clinical laboratory. Methods: A custom-designed oligonucleotide array-based comparative genomic hybridization platform was developed to provide both tiled coverage of the entire 16.6-kb mitochondrial genome and high-density coverage of nuclear genes involved in mitochondrial biogenesis and function, for quick evaluation of mitochondrial DNA Deletion and depletion. Results: For initial validation, the performance of this array was characterized in 20 samples with known mitochondrial DNA Deletions and 12 with apparent depletions. All previously known Deletions were clearly detected and the break points were correctly identified by the oligonucleotide array-based comparative genomic hybridization, within the limits of resolution of the array. The extent of mitochondrial DNA depletion and the percentage of Deletion heteroplasmy were estimated using an automated computational approach that gave results comparable to previous methods. Conclusions from subsequent application of this approach with >300 new clinical samples have been in 100% concordance with those from standard methods. Finally, for one sample, we were able to identify an intragenic Deletion in a nuclear gene that was responsible for the observed mitochondrial DNA depletion. Conclusion: We conclude that this custom array is capable of reliably detecting mitochondrial DNA Deletion with elucidation of the Deletion break points and the percentage of heteroplasmy. In addition, simultaneous detection of the copy number changes in both nuclear and mitochondrial genomes makes this dual genome array of tremendous value in the diagnoses of mitochondrial DNA depletion syndromes.
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Simultaneous detection of mitochondrial DNA depletion and single-exon Deletion in the deoxyguanosine gene using array-based comparative genomic hybridisation
Archives of disease in childhood, 2009Co-Authors: Ni-chung Lee, D. Dimmock, Wuh-liang Hwu, L. Y. Tang, Wei Chen Huang, A. C. Chinault, Leejun C WongAbstract:Intragenic exonic Deletions, which cannot be detected by direct DNA sequencing, are a common cause of Mendelian disease. Array-based comparative genomic hybridisation (aCGH) is now widely used for the clinical diagnosis of large chromosomal Deletions, but not small Deletions or analysis of the mitochondrial genome. An oligonucleotide-based microarray that provides high-density coverage of the entire mitochondrial genome and nuclear genes related to mitochondrial disorders has been developed. In this report, the case of an infant referred with tyrosinaemia on newborn screening who developed liver failure is presented. DNA sequencing revealed a heterozygous missense mutation (c.679G>A, p.E227K) in the deoxyguanosine gene (DGUOK). Oligonucleotide aCGH allowed simultaneous detection of an intragenic heterozygous Deletion of exon 4 of DGUOK and mitochondrial DNA depletion in blood and liver. Screening of the parents' DNA samples indicated that the patient was compound heterozygous for these mutations. An older sibling who had died from liver failure was then retrospectively diagnosed with the same mutations. This report shows the clinical utility of this oligoarray in the detection of changes in DNA copy number in both the mitochondrial and nuclear genomes, thus greatly improving the molecular diagnosis of mitochondrial disorders caused by nuclear genes involved in mitochondrial DNA biosynthesis.
Jacky Chung - One of the best experts on this subject based on the ideXlab platform.
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characterization of genomic Deletion efficiency mediated by clustered regularly interspaced palindromic repeats crispr cas9 nuclease system in mammalian cells
Journal of Biological Chemistry, 2014Co-Authors: Matthew C Canver, Daniel E Bauer, Abhishek Dass, Yvette Y Yien, Jacky Chung, Takeshi Masuda, Takahiro Maeda, Barry H PawAbstract:The clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 nuclease system has provided a powerful tool for genome engineering. Double strand breaks may trigger nonhomologous end joining repair, leading to frameshift mutations, or homology-directed repair using an extrachromosomal template. Alternatively, genomic Deletions may be produced by a pair of double strand breaks. The efficiency of CRISPR/Cas9-mediated genomic Deletions has not been systematically explored. Here, we present a methodology for the production of Deletions in mammalian cells, ranging from 1.3 kb to greater than 1 Mb. We observed a high frequency of intended genomic Deletions. Nondeleted alleles are nonetheless often edited with inversions or small insertion/Deletions produced at CRISPR recognition sites. Deleted alleles also typically include small insertion/Deletions at predicted Deletion junctions. We retrieved cells with biallelic Deletion at a frequency exceeding that of probabilistic expectation. We demonstrate an inverse relationship between Deletion frequency and Deletion size. This work suggests that CRISPR/Cas9 is a robust system to produce a spectrum of genomic Deletions to allow investigation of genes and genetic elements.
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characterization of genomic Deletion efficiency mediated by clustered regularly interspaced short palindromic repeats crispr cas9 nuclease system in mammalian cells
Journal of Biological Chemistry, 2014Co-Authors: Matthew C Canver, Daniel E Bauer, Abhishek Dass, Yvette Y Yien, Jacky Chung, Takeshi Masuda, Takahiro Maeda, Stuart H OrkinAbstract:Abstract The clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 nuclease system has provided a powerful tool for genome engineering. Double strand breaks may trigger nonhomologous end joining repair, leading to frameshift mutations, or homology-directed repair using an extrachromosomal template. Alternatively, genomic Deletions may be produced by a pair of double strand breaks. The efficiency of CRISPR/Cas9-mediated genomic Deletions has not been systematically explored. Here, we present a methodology for the production of Deletions in mammalian cells, ranging from 1.3 kb to greater than 1 Mb. We observed a high frequency of intended genomic Deletions. Nondeleted alleles are nonetheless often edited with inversions or small insertion/Deletions produced at CRISPR recognition sites. Deleted alleles also typically include small insertion/Deletions at predicted Deletion junctions. We retrieved cells with biallelic Deletion at a frequency exceeding that of probabilistic expectation. We demonstrate an inverse relationship between Deletion frequency and Deletion size. This work suggests that CRISPR/Cas9 is a robust system to produce a spectrum of genomic Deletions to allow investigation of genes and genetic elements.
Takahiro Maeda - One of the best experts on this subject based on the ideXlab platform.
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characterization of genomic Deletion efficiency mediated by clustered regularly interspaced palindromic repeats crispr cas9 nuclease system in mammalian cells
Journal of Biological Chemistry, 2014Co-Authors: Matthew C Canver, Daniel E Bauer, Abhishek Dass, Yvette Y Yien, Jacky Chung, Takeshi Masuda, Takahiro Maeda, Barry H PawAbstract:The clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 nuclease system has provided a powerful tool for genome engineering. Double strand breaks may trigger nonhomologous end joining repair, leading to frameshift mutations, or homology-directed repair using an extrachromosomal template. Alternatively, genomic Deletions may be produced by a pair of double strand breaks. The efficiency of CRISPR/Cas9-mediated genomic Deletions has not been systematically explored. Here, we present a methodology for the production of Deletions in mammalian cells, ranging from 1.3 kb to greater than 1 Mb. We observed a high frequency of intended genomic Deletions. Nondeleted alleles are nonetheless often edited with inversions or small insertion/Deletions produced at CRISPR recognition sites. Deleted alleles also typically include small insertion/Deletions at predicted Deletion junctions. We retrieved cells with biallelic Deletion at a frequency exceeding that of probabilistic expectation. We demonstrate an inverse relationship between Deletion frequency and Deletion size. This work suggests that CRISPR/Cas9 is a robust system to produce a spectrum of genomic Deletions to allow investigation of genes and genetic elements.
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characterization of genomic Deletion efficiency mediated by clustered regularly interspaced short palindromic repeats crispr cas9 nuclease system in mammalian cells
Journal of Biological Chemistry, 2014Co-Authors: Matthew C Canver, Daniel E Bauer, Abhishek Dass, Yvette Y Yien, Jacky Chung, Takeshi Masuda, Takahiro Maeda, Stuart H OrkinAbstract:Abstract The clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 nuclease system has provided a powerful tool for genome engineering. Double strand breaks may trigger nonhomologous end joining repair, leading to frameshift mutations, or homology-directed repair using an extrachromosomal template. Alternatively, genomic Deletions may be produced by a pair of double strand breaks. The efficiency of CRISPR/Cas9-mediated genomic Deletions has not been systematically explored. Here, we present a methodology for the production of Deletions in mammalian cells, ranging from 1.3 kb to greater than 1 Mb. We observed a high frequency of intended genomic Deletions. Nondeleted alleles are nonetheless often edited with inversions or small insertion/Deletions produced at CRISPR recognition sites. Deleted alleles also typically include small insertion/Deletions at predicted Deletion junctions. We retrieved cells with biallelic Deletion at a frequency exceeding that of probabilistic expectation. We demonstrate an inverse relationship between Deletion frequency and Deletion size. This work suggests that CRISPR/Cas9 is a robust system to produce a spectrum of genomic Deletions to allow investigation of genes and genetic elements.
Matthew C Canver - One of the best experts on this subject based on the ideXlab platform.
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characterization of genomic Deletion efficiency mediated by clustered regularly interspaced palindromic repeats crispr cas9 nuclease system in mammalian cells
Journal of Biological Chemistry, 2014Co-Authors: Matthew C Canver, Daniel E Bauer, Abhishek Dass, Yvette Y Yien, Jacky Chung, Takeshi Masuda, Takahiro Maeda, Barry H PawAbstract:The clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 nuclease system has provided a powerful tool for genome engineering. Double strand breaks may trigger nonhomologous end joining repair, leading to frameshift mutations, or homology-directed repair using an extrachromosomal template. Alternatively, genomic Deletions may be produced by a pair of double strand breaks. The efficiency of CRISPR/Cas9-mediated genomic Deletions has not been systematically explored. Here, we present a methodology for the production of Deletions in mammalian cells, ranging from 1.3 kb to greater than 1 Mb. We observed a high frequency of intended genomic Deletions. Nondeleted alleles are nonetheless often edited with inversions or small insertion/Deletions produced at CRISPR recognition sites. Deleted alleles also typically include small insertion/Deletions at predicted Deletion junctions. We retrieved cells with biallelic Deletion at a frequency exceeding that of probabilistic expectation. We demonstrate an inverse relationship between Deletion frequency and Deletion size. This work suggests that CRISPR/Cas9 is a robust system to produce a spectrum of genomic Deletions to allow investigation of genes and genetic elements.
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characterization of genomic Deletion efficiency mediated by clustered regularly interspaced short palindromic repeats crispr cas9 nuclease system in mammalian cells
Journal of Biological Chemistry, 2014Co-Authors: Matthew C Canver, Daniel E Bauer, Abhishek Dass, Yvette Y Yien, Jacky Chung, Takeshi Masuda, Takahiro Maeda, Stuart H OrkinAbstract:Abstract The clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 nuclease system has provided a powerful tool for genome engineering. Double strand breaks may trigger nonhomologous end joining repair, leading to frameshift mutations, or homology-directed repair using an extrachromosomal template. Alternatively, genomic Deletions may be produced by a pair of double strand breaks. The efficiency of CRISPR/Cas9-mediated genomic Deletions has not been systematically explored. Here, we present a methodology for the production of Deletions in mammalian cells, ranging from 1.3 kb to greater than 1 Mb. We observed a high frequency of intended genomic Deletions. Nondeleted alleles are nonetheless often edited with inversions or small insertion/Deletions produced at CRISPR recognition sites. Deleted alleles also typically include small insertion/Deletions at predicted Deletion junctions. We retrieved cells with biallelic Deletion at a frequency exceeding that of probabilistic expectation. We demonstrate an inverse relationship between Deletion frequency and Deletion size. This work suggests that CRISPR/Cas9 is a robust system to produce a spectrum of genomic Deletions to allow investigation of genes and genetic elements.
Stuart H Orkin - One of the best experts on this subject based on the ideXlab platform.
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characterization of genomic Deletion efficiency mediated by clustered regularly interspaced short palindromic repeats crispr cas9 nuclease system in mammalian cells
Journal of Biological Chemistry, 2014Co-Authors: Matthew C Canver, Daniel E Bauer, Abhishek Dass, Yvette Y Yien, Jacky Chung, Takeshi Masuda, Takahiro Maeda, Stuart H OrkinAbstract:Abstract The clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 nuclease system has provided a powerful tool for genome engineering. Double strand breaks may trigger nonhomologous end joining repair, leading to frameshift mutations, or homology-directed repair using an extrachromosomal template. Alternatively, genomic Deletions may be produced by a pair of double strand breaks. The efficiency of CRISPR/Cas9-mediated genomic Deletions has not been systematically explored. Here, we present a methodology for the production of Deletions in mammalian cells, ranging from 1.3 kb to greater than 1 Mb. We observed a high frequency of intended genomic Deletions. Nondeleted alleles are nonetheless often edited with inversions or small insertion/Deletions produced at CRISPR recognition sites. Deleted alleles also typically include small insertion/Deletions at predicted Deletion junctions. We retrieved cells with biallelic Deletion at a frequency exceeding that of probabilistic expectation. We demonstrate an inverse relationship between Deletion frequency and Deletion size. This work suggests that CRISPR/Cas9 is a robust system to produce a spectrum of genomic Deletions to allow investigation of genes and genetic elements.
