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Methods in Molecular Biology (Clifton,... 2023Mitochondrial DNA (mtDNA) mutations are found in several human pathologies and are associated with aging. Deletion mutations in mtDNA result in the loss of essential...
Mitochondrial DNA (mtDNA) mutations are found in several human pathologies and are associated with aging. Deletion mutations in mtDNA result in the loss of essential genes for mitochondrial function. Over 250 deletion mutations have been reported and the common deletion is the most frequent mtDNA deletion linked to disease. This deletion removes 4977 base pairs of mtDNA. It has previously been shown that exposure to UVA radiation can promote the formation of the common deletion. Furthermore, aberrations in mtDNA replication and repair are associated with formation of the common deletion. However, molecular mechanisms describing the formation of this deletion are poorly characterized. This chapter describes a method to irradiate human skin fibroblasts with physiological doses of UVA and the subsequent detection of the common deletion by quantitative PCR analysis.
Topics: Humans; DNA, Mitochondrial; Sequence Deletion; Mitochondria; Aging; Mutation
PubMed: 36807799
DOI: 10.1007/978-1-0716-2922-2_20 -
Cold Spring Harbor Protocols Aug 2023This protocol continues a series of methods for the construction of an in-frame gene deletion in strain RN4220. To this end, we describe in this protocol an...
This protocol continues a series of methods for the construction of an in-frame gene deletion in strain RN4220. To this end, we describe in this protocol an allelic-exchange procedure for We have previously described how an allelic-exchange plasmid containing a desired gene deletion (in this case, pIMAY*-Δ) can be constructed and isolated from , then introduced into electrocompetent cells by electroporation. This plasmid contains a temperature-sensitive origin of replication, a counterselectable marker (* gene) and confers chloramphenicol resistance to As a specific example, we present the construction of strain RN4220*Δ from strain RN4220 carrying the pIMAY*-Δ plasmid. The protocol can be easily adapted for the construction of other gene deletions and/or allelic-exchange plasmids.
Topics: Staphylococcus aureus; Plasmids; Sequence Deletion; Gene Deletion
PubMed: 37117017
DOI: 10.1101/pdb.prot107948 -
Communications Biology Jun 2023CHD8 encodes chromodomain helicase DNA-binding protein 8 and its mutation is a highly penetrant risk factor for autism spectrum disorder (ASD). CHD8 serves as a key...
CHD8 encodes chromodomain helicase DNA-binding protein 8 and its mutation is a highly penetrant risk factor for autism spectrum disorder (ASD). CHD8 serves as a key transcriptional regulator on the basis of its chromatin-remodeling activity and thereby controls the proliferation and differentiation of neural progenitor cells. However, the function of CHD8 in postmitotic neurons and the adult brain has remained unclear. Here we show that Chd8 homozygous deletion in mouse postmitotic neurons results in downregulation of the expression of neuronal genes as well as alters the expression of activity-dependent genes induced by KCl-mediated neuronal depolarization. Furthermore, homozygous ablation of CHD8 in adult mice was associated with attenuation of activity-dependent transcriptional responses in the hippocampus to kainic acid-induced seizures. Our findings implicate CHD8 in transcriptional regulation in postmitotic neurons and the adult brain, and they suggest that disruption of this function might contribute to ASD pathogenesis associated with CHD8 haploinsufficiency.
Topics: Mice; Animals; Autistic Disorder; Autism Spectrum Disorder; Homozygote; Sequence Deletion; DNA-Binding Proteins; Neurons
PubMed: 37268684
DOI: 10.1038/s42003-023-04968-y -
Journal of Virology Sep 2023Porcine epidemic diarrhea virus (PEDV) leads to enormous economic losses for the pork industry. However, the commercial vaccines failed to fully protect against the...
Porcine epidemic diarrhea virus (PEDV) leads to enormous economic losses for the pork industry. However, the commercial vaccines failed to fully protect against the epidemic strains. Previously, the rCH/SX/2016-S strain with the entire E protein and the rCH/SX/2015 strain with the deletion of 7-amino-acid (7-aa) at positions 23-29 in E protein were constructed and rescued. The pathogenicity assay indicated that rCH/SX/2015 is an attenuated strain, but rCH/SX/2016-S belongs to the virulent strains. Then, the recombination PEDV (rPEDV-E)strain with a 7-aa deletion in the E protein was generated, using the highly virulent rCH/SX/2016-S strain (rPEDV-E) as the backbone. Compared with the rPEDV-E strain, the release and infectivity of the rPEDV-E strain were significantly reduced , but stronger interferon (IFN) responses were triggered both and . The pathogenicity assay showed that the parental strain resulted in severe diarrhea (100%) and death (100%) in all piglets. Compared with the parental strain group, rPEDV-E caused lower mortality (33%) and diminished fecal PEDV RNA shedding. At 21 days, all surviving pigs were challenged orally with rPEDV-E. No pigs died in the two groups. Compared with the mock group, significantly delayed and milder diarrhea and reduced fecal PEDV RNA shedding were detected in the rPEDV-E group. In conclusion, the deletion of a 7-aa fragment in the E protein (E) attenuated PEDV but retained its immunogenicity, which can offer new ideas for the design of live attenuated vaccines and provide new insights into the attenuated mechanism of PEDV. IMPORTANCE Porcine epidemic diarrhea virus (PEDV) causes high mortality in neonatal piglets and remains a large challenge to the pork industry. Unfortunately, no safe and effective vaccines are available yet. The pathogenesis and molecular basis of the attenuation of PEDV remain unclear, which seriously hinders the development of PEDV vaccines. This study found that the rPEDV carrying E mutation in the E protein induced significantly higher IFN responses than the parental virus, partially attenuated, and remained immunogenic in piglets. For the first time, PEDV E was verified as an IFN antagonist in the infection context and identified as a virulence factor of PEDV. Our data also suggested that E mutation can be a good target for the development of live attenuated vaccines for PEDV and also provide new perspectives for the attenuated mechanism of PEDV.
Topics: Animals; Coronavirus Infections; Interferons; Porcine epidemic diarrhea virus; RNA; Swine; Swine Diseases; Vaccines, Attenuated; Sequence Deletion; Viral Envelope Proteins
PubMed: 37681956
DOI: 10.1128/jvi.00847-23 -
Analytical Biochemistry Mar 2021Deletion mutation has been proved as the important factor for occurrence and development of disease, especially those with cancer. With the popularity of precision...
Deletion mutation has been proved as the important factor for occurrence and development of disease, especially those with cancer. With the popularity of precision medicine, the individual cancer therapeutic strategy has highlighted the requirement to develop a straightforward and competent strategy for deletion mutation determination. Hence, the present study is dedicated to develop a one-step assay to identify deletion mutation with sequence specificity for clinical practice. Taking advantage of loop-mediated isothermal amplification, an ultrasensitive and rapid deletion mutation determination method is established, which allow as low as 30 copies or 0.1% target variants under strong interferential background can be accurately distinguished in 30 min dispensing with professional operation and complex data interpretation. As a demonstration, the epidermal growth factor receptor p.E746-A750del, a crucial factor for the susceptibility of tyrosine kinase inhibitor in non-small-cell lung cancer treatment, has been accurately identified by this method with both cell lines and real clinical samples. By tailor-made primer set, this method can be extended for other deletion mutants, making it a molecular diagnostic tool and could be readily adapted for cancer diagnosis, therapy and prognosis in point of care diagnostic test scenario.
Topics: Biomarkers, Tumor; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; DNA Mutational Analysis; DNA Primers; ErbB Receptors; Humans; Lung Neoplasms; Molecular Diagnostic Techniques; Nucleic Acid Amplification Techniques; Sensitivity and Specificity; Sequence Deletion
PubMed: 33352189
DOI: 10.1016/j.ab.2020.114087 -
Current Protocols in Chemical Biology Sep 2019Recombineering inserts PCR products into DNA using homologous recombination. A pair of short homology arms (50 base pairs) on the ends of a PCR cassette target the...
Recombineering inserts PCR products into DNA using homologous recombination. A pair of short homology arms (50 base pairs) on the ends of a PCR cassette target the cassette to its intended location. These homology arms can be easily introduced as 5' primer overhangs during the PCR reaction. The flexibility to choose almost any pair of homology arms enables the precise modification of virtually any DNA for purposes of sequence deletion, replacement, insertion, or point mutation. Recombineering often offers significant advantages relative to previous homologous recombination methods that require the construction of cassettes with large homology arms, and relative to traditional cloning methods that become intractable for large plasmids or DNA sequences. However, the tremendous number of variables, options, and pitfalls that can be encountered when designing and performing a recombineering protocol for the first time introduce barriers that can make recombineering a challenging technique for new users to adopt. This article focuses on three recombineering protocols we have found to be particularly robust, providing a detailed guide for choosing the simplest recombineering method for a given application and for performing and troubleshooting experiments. © 2019 by John Wiley & Sons, Inc.
Topics: DNA; Escherichia coli; Gene Deletion; Genetic Engineering; Mutagenesis, Insertional; Plasmids; Point Mutation; Polymerase Chain Reaction; Research Design
PubMed: 31483098
DOI: 10.1002/cpch.70 -
Journal of Neuropathology and... Apr 2021Numerous recent studies have demonstrated that the vast majority of IDH-wildtype astrocytomas with WHO grade II/III histology have clinical outcomes equivalent to...
Numerous recent studies have demonstrated that the vast majority of IDH-wildtype astrocytomas with WHO grade II/III histology have clinical outcomes equivalent to IDH-wildtype glioblastomas. This has called into question the existence of an IDH-wildtype lower-grade astrocytoma (LGA) category, and the cIMPACT-NOW study group has suggested 3 molecular features which, if present, warrant upgrading IDH-wildtype LGA to glioblastoma: EGFR amplification, 7+/10-, and TERT promoter mutation. Herein, we evaluate the clinical, histologic, and molecular features of IDH-wildtype low-grade astrocytomas, defined here as infiltrative adult astrocytoma lacking histologic features of glioblastoma (microvascular proliferation and/or necrosis), IDH1/2 mutation, and all 3 of the cIMPACT-NOW update 3 factors. Compared with their counterparts with cIMPACT-NOW features of glioblastoma (LGA-C+; n = 108), IDH-wildtype LGAs lacking these features (LGA-C0; n = 36) occur in significantly younger patients, are more frequently WHO grade II, have less total copy number variation distributed across the entire genome, less frequent homozygous deletion of CDKN2A, less frequent PTEN and PIK3CA alterations, and more frequent NF1 alterations. These results suggest that although rare, a "true" IDH-wildtype LGA category does exist, and has distinct clinical and molecular features consistent with relatively beneficial clinical outcomes in these patients.
Topics: Astrocytoma; DNA Copy Number Variations; Glioblastoma; Homozygote; Humans; Necrosis; Sequence Deletion; Vascular Diseases
PubMed: 33829259
DOI: 10.1093/jnen/nlab023 -
Journal of Neuropathology and... Sep 2023Homozygous deletion of CDKN2A/B is currently considered a molecular signature for grade 4 in IDH-mutant astrocytomas, irrespective of tumor histomorphology. The 2021 WHO...
Homozygous deletion of CDKN2A/B is currently considered a molecular signature for grade 4 in IDH-mutant astrocytomas, irrespective of tumor histomorphology. The 2021 WHO Classification of CNS Tumors does not currently include grading recommendations for histologically lower-grade (grade 2-3) IDH-mutant astrocytoma with CDKN2A mutation or other CDKN2A alterations, and little is currently known about the prognostic implications of these alternative CDKN2A inactivating mechanisms. To address this, we evaluated a cohort of institutional and publicly available IDH-mutant astrocytomas, 15 with pathogenic mutations in CDKN2A, 47 with homozygous CDKN2A deletion, and 401 with retained/wildtype CDKN2A. The IDH-mutant astrocytomas with mutant and deleted CDKN2A had significantly higher overall copy number variation compared to those with retained/wildtype CDKN2A, consistent with more aggressive behavior. Astrocytoma patients with CDKN2A mutation had significantly worse progression-free (p = 0.0025) and overall survival (p < 0.0001) compared to grade-matched patients with wildtype CDKN2A, but statistically equivalent progression-free survival and overall survival outcomes to patients with CDKN2A deletion. No significant survival difference was identified between CDKN2A mutant cases with or without loss of the second allele. These findings suggest that CDKN2A mutation has a detrimental effect on survival in otherwise lower-grade IDH-mutant astrocytomas, similar to homozygous CDKN2A deletion, and should be considered for future grading schemes.
Topics: Humans; Prognosis; Brain Neoplasms; Homozygote; DNA Copy Number Variations; Sequence Deletion; Isocitrate Dehydrogenase; Astrocytoma; Mutation; Cyclin-Dependent Kinase Inhibitor p16
PubMed: 37550258
DOI: 10.1093/jnen/nlad063 -
Movement Disorders : Official Journal... Jul 2021Mutations in PRKN are the most common cause of autosomal recessive juvenile parkinsonism. The objective of this study was to investigate the association between genotype...
BACKGROUND
Mutations in PRKN are the most common cause of autosomal recessive juvenile parkinsonism. The objective of this study was to investigate the association between genotype and pathology in patients with PRKN mutations.
METHODS
We performed a sequence and copy number variation analysis of PRKN, mRNA transcripts, Parkin protein expression, and neuropathology in 8 autopsied patients.
RESULTS
All the patients harbored biallelic PRKN mutations. Two patients were homozygous and heterozygous, respectively, for the missense mutation p.C431F. Seven patients had exon rearrangements, including 2 patients from a single family who harbored a homozygous deletion of exon 4, and 3 patients who carried a homozygous duplication of exons 6-7, a homozygous duplication of exons 10-11, and a heterozygous duplication of exons 2-4. In the other 2 patients, we found a compound heterozygous duplication of exon 2, deletion of exon 3, and a heterozygous duplication of exon 2. However, sequencing of cDNA prepared from mRNA revealed 2 different transcripts derived from triplication of exon 2 and deletion of exons 2-3 and from duplication of exons 2-4 and deletion of exons 3-4. Western blotting and immunohistochemistry revealed faint or no expression of Parkin in their brains. In the substantia nigra pars compacta, a subfield-specific pattern of neuronal loss and mild gliosis were evident. Lewy bodies were found in 3 patients. Peripheral sensory neuronopathy was a feature.
CONCLUSIONS
Genomic and mRNA analysis is needed to identify the PRKN mutations. Variable mutations may result in no or little production of mature Parkin and the histopathologic features may be similar. © 2021 International Parkinson and Movement Disorder Society.
Topics: DNA Copy Number Variations; Homozygote; Humans; Mutation; Sequence Deletion; Ubiquitin-Protein Ligases
PubMed: 33570211
DOI: 10.1002/mds.28521 -
Reproductive Sciences (Thousand Oaks,... Feb 2022Acephalic spermatozoa syndrome (ASS) is a severe form of teratozoospermia, previous studies have shown that SUN5 mutations are the major cause of acephalic spermatozoa...
Acephalic spermatozoa syndrome (ASS) is a severe form of teratozoospermia, previous studies have shown that SUN5 mutations are the major cause of acephalic spermatozoa syndrome. This study is to identify the pathogenic mutations in SUN5 leading to ASS. PCR and Sanger sequence were performed to define the breakpoints and mutations in SUN5. Whole genome sequencing (WGS) was performed to detect heterozygous deletion. Western blotting and immunofluorescence analysis detected the expression level and localization of SUN5. Furthermore, the pathogenicity of the mutant SUN5 was predicted in silico and was verified by the experiments in vitro. We identified one novel homozygous missense mutation (c.775G>A; p.G259S) and one compound heterozygous including one reported missense mutation (c.1043A>T; p.N348I) and a large deletion that contains partial EFCAB8 ( NM_001143967 .1) and BPIFB2 ( NM_025227 ) and complete SUN5 ( NM_080675 ), and one recurrent homozygous splice-site mutation (c.340G>A; p.G114R) in SUN5 in three patients with ASS. Our results showed that SUN5 could not be detected in the patients' spermatozoa and the exogenous expression level of the mutant protein was decreased in transfected HEK-293T cells. This study expands the mutational spectrum of SUN5. We recommended a clinical diagnostic strategy for SUN5 genomic deletion to screen heterozygous deletions and indicated that the diagnostic value of screening for SUN5 mutations and deletions in infertile men with ASS.
Topics: Adult; Blotting, Western; Fluorescent Antibody Technique; HEK293 Cells; Humans; Infertility, Male; Male; Membrane Proteins; Mutation, Missense; Pedigree; Sequence Deletion; Spermatozoa; Syndrome; Teratozoospermia; Whole Genome Sequencing
PubMed: 34159570
DOI: 10.1007/s43032-021-00665-5