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Nucleic Acids Research Oct 2016The accumulation of somatic mitochondrial DNA (mtDNA) mutations contributes to the pathogenesis of human disease. Currently, mitochondrial mutations are largely...
The accumulation of somatic mitochondrial DNA (mtDNA) mutations contributes to the pathogenesis of human disease. Currently, mitochondrial mutations are largely considered results of inaccurate processing of its heavily damaged genome. However, mainly from a lack of methods to monitor mtDNA mutations with sufficient sensitivity and accuracy, a link between mtDNA damage and mutation has not been established. To test the hypothesis that mtDNA-damaging agents induce mtDNA mutations, we exposed MutaMouse mice to benzo[a]pyrene (B[a]P) or N-ethyl-N-nitrosourea (ENU), daily for 28 consecutive days, and quantified mtDNA point and deletion mutations in bone marrow and liver using our newly developed Digital Random Mutation Capture (dRMC) and Digital Deletion Detection (3D) assays. Surprisingly, our results demonstrate mutagen treatment did not increase mitochondrial point or deletion mutation frequencies, despite evidence both compounds increase nuclear DNA mutations and demonstrated B[a]P adduct formation in mtDNA. These findings contradict models of mtDNA mutagenesis that assert the elevated rate of mtDNA mutation stems from damage sensitivity and abridged repair capacity. Rather, our results demonstrate induced mtDNA damage does not readily convert into mutation. These findings suggest robust mitochondrial damage responses repress induced mutations after mutagen exposure.
Topics: Animals; Benzo(a)pyrene; Bone Marrow; Cell Nucleus; DNA Adducts; DNA, Mitochondrial; Ethylnitrosourea; Liver; Male; Mice; Mutagenesis; Mutagens; Point Mutation; Sequence Deletion
PubMed: 27550180
DOI: 10.1093/nar/gkw716 -
PloS One 2016The aim of this study is to identify the molecular basis of autosomal recessive congenital cataracts (arCC) in a large consanguineous pedigree.
PURPOSE
The aim of this study is to identify the molecular basis of autosomal recessive congenital cataracts (arCC) in a large consanguineous pedigree.
METHODS
All participating individuals underwent a detailed ophthalmic examination. Each patient's medical history, particularly of cataracts and other ocular abnormalities, was compiled from available medical records and interviews with family elders. Blood samples were donated by all participating family members and used to extract genomic DNA. Genetic analysis was performed to rule out linkage to known arCC loci and genes. Whole-exome sequencing libraries were prepared and paired-end sequenced. A large deletion was found that segregated with arCC in the family, and chromosome walking was conducted to estimate the proximal and distal boundaries of the deletion mutation.
RESULTS
Exclusion and linkage analysis suggested linkage to a region of chromosome 6p24 harboring GCNT2 (glucosaminyl (N-acetyl) transferase 2) with a two-point logarithm of odds score of 5.78. PCR amplifications of the coding exons of GCNT2 failed in individuals with arCC, and whole-exome data analysis revealed a large deletion on chromosome 6p in the region harboring GCNT2. Chromosomal walking using multiple primer pairs delineated the extent of the deletion to approximately 190 kb. Interestingly, a failure to amplify a junctional fragment of the deletion break strongly suggests an insertion in addition to the large deletion.
CONCLUSION
Here, we report a novel insertion/deletion mutation at the GCNT2 locus that is responsible for congenital cataracts in a large consanguineous family.
Topics: Animals; Cataract; Child; Child, Preschool; Consanguinity; Female; Genetic Linkage; Genetic Loci; Humans; Infant; Male; Mice; Microsatellite Repeats; N-Acetylglucosaminyltransferases; N-Acetylhexosaminyltransferases; Pedigree; Sequence Deletion
PubMed: 27936067
DOI: 10.1371/journal.pone.0167562 -
Clinical Biochemistry May 2010Lack of sequencing validation and complexity of deletion testing hinder genetic diagnosis of SDH-associated paraganglioma/pheochromocytoma.
BACKGROUND
Lack of sequencing validation and complexity of deletion testing hinder genetic diagnosis of SDH-associated paraganglioma/pheochromocytoma.
METHODS
We developed sequencing assays and multiplex ligation-dependent probe amplification (MLPA) deletion detection for SDHB, SDHC and SDHD. Clinical performance was validated on 141 blinded samples, previously tested at NIH.
RESULTS
Sequencing and deletion detection were highly reproducible and agreed with previous NIH results in 99.3% and 100%, respectively.
CONCLUSIONS
DNA sequencing combined with MLPA allows reliable and simplified genotyping of SDHB, SDHC and SDHD.
Topics: Adrenal Gland Neoplasms; DNA Mutational Analysis; Membrane Proteins; Nucleic Acid Amplification Techniques; Paraganglioma; Pheochromocytoma; Polymerase Chain Reaction; Sequence Deletion; Succinate Dehydrogenase
PubMed: 20153743
DOI: 10.1016/j.clinbiochem.2010.01.016 -
PLoS Genetics Mar 2019The simplicity and the versatility of clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR-Cas) systems have enabled the genetic...
The simplicity and the versatility of clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR-Cas) systems have enabled the genetic modification of virtually every organism and offer immense therapeutic potential for the treatment of human disease. Although these systems may function efficiently within eukaryotic cells, there remain concerns about the accuracy of Cas endonuclease effectors and their use for precise gene editing. Recently, two independent reports investigating the editing accuracy of the CRISPR-Cas9 system were published by separate groups at the Wellcome Sanger Institute; our study-Iyer and colleagues [1]-defined the landscape of off-target mutations, whereas the other by Kosicki and colleagues [2] detailed the existence of on-target, potentially deleterious deletions. Although both studies found evidence of large on-target CRISPR-induced deletions, they reached seemingly very different conclusions.
Topics: Animals; CRISPR-Cas Systems; Cell Cycle; Cell Division; Gene Editing; Genetic Therapy; Genome; Genomics; Genotype; Humans; Mammals; Mutation Rate; Sequence Deletion; Zygote
PubMed: 30870431
DOI: 10.1371/journal.pgen.1007994 -
PloS One 2015Despite that Vibrio spp. have a significant impact on the health of humans and aquatic animals, the molecular basis of their pathogenesis is little known, mainly due to...
Developing Universal Genetic Tools for Rapid and Efficient Deletion Mutation in Vibrio Species Based on Suicide T-Vectors Carrying a Novel Counterselectable Marker, vmi480.
Despite that Vibrio spp. have a significant impact on the health of humans and aquatic animals, the molecular basis of their pathogenesis is little known, mainly due to the limited genetic tools for the functional research of genes in Vibrio. In some cases, deletion of target DNAs in Vibrio can be achieved through the use of suicide vectors. However, these strategies are time-consuming and lack universality, and the widely used counterselectable gene sacB does not work well in Vibrio cells. In this study, we developed universal genetic tools for rapid and efficient deletion mutations in Vibrio species based on suicide T-Vectors carrying a novel counterselectable marker, vmi480. We explored two uncharacterized genes, vmi480 and vmi470, in a genomic island from Vibrio mimicus VM573 and confirmed that vmi480 and vmi470 constitute a two-component toxin-antitoxin system through deletion and expression of vmi480 and vmi470. The product of vmi480 exhibited strong toxicity to Escherichia coli cells. Based on vmi480 and the PBAD or PTAC promoter system, we constructed two suicide T-vectors, pLP11 and pLP12, and each of these vectors contained a multiple cloning region with two AhdI sites. Both vectors linearized by AhdI digestion could be stored and directly ligated with purified PCR products without a digestion step. By using pLP11 and pLP12 coupled with a highly efficient conjugation system provided by E. coli β2163, six genes from four representative Vibrio species were easily deleted. By using the counterselective marker vmi480, we obtained 3-12 positive colonies (deletion mutants) among no more than 20 colonies randomly selected on counterselection plates. The strategy does not require the digestion of PCR products and suicide vectors every time, and it avoids large-scale screening colonies on counterselective plates. These results demonstrate that we successfully developed universal genetic tools for rapid and efficient gene deletion in Vibrio species.
Topics: Bacterial Toxins; Escherichia coli; Genetic Vectors; Humans; Plasmids; Sequence Deletion; Vibrio
PubMed: 26641275
DOI: 10.1371/journal.pone.0144465 -
Experimental & Molecular Medicine Oct 2008Phenylketonuria (PKU; MIM 261600) is an autosomal recessive metabolic disorder caused by a deficiency of phenylalanine hydroxylase (PAH; EC 1.14.16.1). Point mutations...
Phenylketonuria (PKU; MIM 261600) is an autosomal recessive metabolic disorder caused by a deficiency of phenylalanine hydroxylase (PAH; EC 1.14.16.1). Point mutations in the PAH gene are known to cause PKU in various ethnic groups, and large deletions or duplications account for up to 3% of the PAH mutations in some ethnic groups. However, a previous study could not identify approximately 14% of the mutant alleles by sequence analysis in Korean patients with PKU, which suggests that large deletions or duplication might be frequent causes of PKU in Koreans. To test this hypothesis, we performed multiplex ligation-dependent probe amplification (MLPA) for the identification of uncharacterized mutant alleles after PAH sequence analysis of 33 unrelated Korean patients with PKU. Bi-directional sequencing of the PAH exons and flanking intronic regions revealed 27 different mutations, including four novel mutations (two missense and two deletion mutations), comprising 57/66 (86%) mutant alleles. MLPA identified a large deletion that encompassed exons 5 and 6 in four patients, another large deletion that extended from exon 4 to exon 7 in one patient, and a duplication of exon 4 in one patient. Chromosomal walking characterized the deletion breakpoint of the most common large deletion that involved exons 5 and 6 (c.456_706+138del). The present study shows that the allelic frequency of exon deletion or duplication is 9% (6/66) in Korean PKU patients, which suggests that these mutations may be frequent causes of PKU in Korean subjects.
Topics: Asian People; Binding Sites; DNA Mutational Analysis; Exons; Humans; Korea; Models, Molecular; Phenylalanine Hydroxylase; Phenylketonurias; Protein Structure, Tertiary; Sequence Deletion
PubMed: 18985011
DOI: 10.3858/emm.2008.40.5.533 -
Journal of Biomedicine & Biotechnology 2011The advent of cloning herpesviral genomes as bacterial artificial chromosomes (BACs) has made herpesviruses accessible to bacterial genetics and has thus revolutionised... (Review)
Review
The advent of cloning herpesviral genomes as bacterial artificial chromosomes (BACs) has made herpesviruses accessible to bacterial genetics and has thus revolutionised their mutagenesis. This opened all possibilities of reverse genetics to ask scientific questions by introducing precisely accurate mutations into the viral genome for testing their influence on the phenotype under study or to create phenotypes of interest. Here, we report on our experience with using BAC technology for a designed modulation of viral antigenicity and immunogenicity with focus on the CD8 T-cell response. One approach is replacing an intrinsic antigenic peptide in a viral carrier protein with a foreign antigenic sequence, a strategy that we have termed "orthotopic peptide swap". Another approach is the functional deletion of an antigenic peptide by point mutation of its C-terminal MHC class-I anchor residue. We discuss the concepts and summarize recently published major scientific results obtained with immunological mutants of murine cytomegalovirus.
Topics: Animals; Antigens, Viral; CD8-Positive T-Lymphocytes; Cytomegalovirus; Epitopes, T-Lymphocyte; Humans; Mutagenesis, Insertional; Sequence Deletion
PubMed: 21253509
DOI: 10.1155/2011/812742 -
BMC Genomics Jul 2015Small insertions and deletions (InDels) constitute the second most abundant class of genetic variants and have been found to be associated with many traits and diseases....
BACKGROUND
Small insertions and deletions (InDels) constitute the second most abundant class of genetic variants and have been found to be associated with many traits and diseases. The present study reports on the detection and characterisation of about 883 K high quality InDels from the whole-genome analysis of several modern layer chicken lines from diverse breeds.
RESULTS
To reduce the error rates seen in InDel detection, this study used the consensus set from two InDel-calling packages: SAMtools and Dindel, as well as stringent post-filtering criteria. By analysing sequence data from 163 chickens from 11 commercial and 5 experimental layer lines, this study detected about 883 K high quality consensus InDels with 93% validation rate and an average density of 0.78 InDels/kb over the genome. Certain chromosomes, viz, GGAZ, 16, 22 and 25 showed very low densities of InDels whereas the highest rate was observed on GGA6. In spite of the higher recombination rates on microchromosomes, the InDel density on these chromosomes was generally lower relative to macrochromosomes possibly due to their higher gene density. About 43-87% of the InDels were found to be fixed within each line. The majority of detected InDels (86%) were 1-5 bases and about 63% were non-repetitive in nature while the rest were tandem repeats of various motif types. Functional annotation identified 613 frameshift, 465 non-frameshift and 10 stop-gain/loss InDels. Apart from the frameshift and stopgain/loss InDels that are expected to affect the translation of protein sequences and their biological activity, 33% of the non-frameshift were predicted as evolutionary intolerant with potential impact on protein functions. Moreover, about 2.5% of the InDels coincided with the most-conserved elements previously mapped on the chicken genome and are likely to define functional elements. InDels potentially affecting protein function were found to be enriched for certain gene-classes e.g. those associated with cell proliferation, chromosome and Golgi organization, spermatogenesis, and muscle contraction.
CONCLUSIONS
The large catalogue of InDels presented in this study along with their associated information such as functional annotation, estimated allele frequency, etc. are expected to serve as a rich resource for application in future research and breeding in the chicken.
Topics: Amino Acid Sequence; Animals; Chickens; Genome; INDEL Mutation; Polymorphism, Single Nucleotide; Sequence Deletion
PubMed: 26227840
DOI: 10.1186/s12864-015-1711-1 -
PloS One 2018All-trans retinoic acid (ATRA) and arsenic trioxide (ATO) are essential for acute promyelocytic leukemia (APL) treatment. It has been reported that mutations in PML-RARA...
All-trans retinoic acid (ATRA) and arsenic trioxide (ATO) are essential for acute promyelocytic leukemia (APL) treatment. It has been reported that mutations in PML-RARA confer resistance to ATRA and ATO, and are associated with poor prognosis. Although most PML-RARA mutations were point mutations, we identified a novel seven amino acid deletion mutation (p.K227_T233del) in the RARA region of PML-RARA in a refractory APL patient. Here, we analyzed the evolution of the mutated clone and demonstrated the resistance of the mutated clone to retinoic acid (RA). Mutation analysis of PML-RARA was performed using samples from a chemotherapy- and ATRA-resistant APL patient, and the frequencies of mutated PML-RARA transcript were analyzed by targeted deep sequencing. To clarify the biological significance of the identified PML-RARA mutations, we analyzed the ATRA-induced differentiation and PML nuclear body formation in mutant PML-RARA-transduced HL-60 cells. At molecular relapse, the p.K227_T233del deletion and the p.R217S point-mutation in the RARA region of PML-RARA were identified, and their frequencies increased after re-induction therapy with another type of retinoiec acid (RA), tamibarotene. In deletion PML-RARA-transduced cells, the CD11b expression levels and NBT reducing ability were significantly decreased compared with control cells and the formation of PML nuclear bodies was rarely observed after RA treatment. These results indicate that this deletion mutation was closely associated with the disease progression during RA treatment.
Topics: CD11b Antigen; Cell Line, Tumor; Disease Progression; Down-Regulation; Drug Resistance, Neoplasm; Female; Gene Expression Regulation, Neoplastic; HL-60 Cells; Humans; Leukemia, Promyelocytic, Acute; Mutation; Oncogene Proteins, Fusion; Point Mutation; Sequence Deletion; Tretinoin
PubMed: 30289902
DOI: 10.1371/journal.pone.0204850 -
Bosnian Journal of Basic Medical... Feb 2020Presbycusis, or age-related hearing loss, is a prevalent disease that severely affects the physical and mental health of the elderly. Oxidative stress and mitochondrial...
Presbycusis, or age-related hearing loss, is a prevalent disease that severely affects the physical and mental health of the elderly. Oxidative stress and mitochondrial (mt)DNA deletion mutation are considered as major factors in the pathophysiology of age-related hearing loss. The 4977-bp deletion in human mtDNA (common deletion, corresponding to the 4834-bp mtDNA deletion in rats) is suggested to be closely associated with the pathogenesis of age-related hearing loss. Superoxide dismutase 2 (SOD2), an isoform of SOD that is exclusively expressed in the intracellular mitochondrial matrix, plays a crucial role in oxidative resistance against mitochondrial superoxide. Previous research has shown that methylation of the promoter region of the SOD2 gene decreased the expression of SOD2 in marginal cells (MCs) extracted from the inner ear of rats subjected to D-galactose-induced mtDNA4834 deletion. However, the relationship between SOD2 methylation and mtDNA4834 deletion under oxidative stress remains to be elucidated. Herein, an oxidative damage model was established in the extracted MCs using hydrogen peroxide (H2O2), which increased the methylation level of SOD2 and the copy number of mtDNA4834 mutation in MCs. Decreasing the methylation level of SOD2 using 5-azacytidine, a DNA methylation inhibitor, reduced oxidative stress and the copy number of mtDNA4834 mutation and inhibited H2O2-induced apoptosis. The present work demonstrates that decreasing the methylation of SOD2 suppresses the mtDNA4834 deletion in MCs under oxidative stress and provides potential insights to the intervention therapy of aging-related hearing loss.
Topics: Animals; Cell Culture Techniques; DNA, Mitochondrial; Disease Models, Animal; Ear, Inner; Hydrogen Peroxide; Methylation; Oxidative Stress; Presbycusis; Rats; Rats, Wistar; Sequence Deletion; Superoxide Dismutase
PubMed: 31465718
DOI: 10.17305/bjbms.2019.4353