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Infection and Immunity Sep 2014The collagen adhesin Acm was the first virulence determinant reported to be important for the pathogenesis of Enterococcus faecium in a rat infective endocarditis model....
The collagen adhesin Acm was the first virulence determinant reported to be important for the pathogenesis of Enterococcus faecium in a rat infective endocarditis model. We had previously reported that there was a slight growth delay associated with acm allelic replacement (cat) mutant strain TX6051 used in that study. Recently, we generated a nonpolar markerless acm deletion mutant and did not observe a delay in growth. We therefore performed comparative genome sequence analysis of wild-type strain TX82 and TX6051 and found a single mutation, a nonsense mutation in the ccpA gene of TX6051. After correcting this mutation, the growth defect of TX6051 was abolished, implicating a role for CcpA in the growth of E. faecium. To confirm this, we created a ccpA deletion mutant of TX82, which also exhibited a slight delay in growth. Furthermore, the ccpA deletion mutant was attenuated (P = 0.0024) in a mixed-inoculum (TX82 plus TX82 ΔccpA) rat endocarditis model and also in an in vitro competitive growth assay; a ccpA-complemented strain showed neither reduced growth nor reduced virulence. We also found attenuation in the endocarditis model with the new acm deletion mutant although not as great as that previously observed with TX6051 carrying the ccpA mutation. Taken together, our data confirm the role of Acm in the pathogenesis of endocarditis. We also show that CcpA affects the growth of E. faecium, that an intact ccpA gene is important for full virulence, and that a ccpA mutation was partly responsible for the highly attenuated phenotype of TX6051.
Topics: Adhesins, Bacterial; Amino Acid Sequence; Animals; Bacterial Adhesion; Bacterial Proteins; Biofilms; Endocarditis, Bacterial; Enterococcus faecium; Molecular Sequence Data; Rats; Sequence Deletion; Virulence
PubMed: 24914215
DOI: 10.1128/IAI.01911-14 -
Journal of Veterinary Internal Medicine 2014Pituitary dwarfism in German Shepherd Dogs is associated with autosomal recessive inheritance and a mutation in LHX3, resulting in combined pituitary hormone deficiency....
BACKGROUND
Pituitary dwarfism in German Shepherd Dogs is associated with autosomal recessive inheritance and a mutation in LHX3, resulting in combined pituitary hormone deficiency. Congenital dwarfism also is encountered in breeds related to German Shepherd Dogs, such as Saarloos and Czechoslovakian wolfdogs.
OBJECTIVES
To investigate whether Saarloos and Czechoslovakian wolfdog dwarfs have the same LHX3 mutation as do Germans Shepherd Dog dwarfs. A specific aim was to determine the carrier frequency among Saarloos and Czechoslovakian wolfdogs used for breeding.
ANIMALS
Two client-owned Saarloos wolfdogs and 4 client-owned Czechoslovakian wolfdogs with pituitary dwarfism, 239 clinically healthy client-owned Saarloos wolfdogs, and 200 client-owned clinically healthy Czechoslovakian wolfdogs.
METHODS
Genomic DNA was amplified using polymerase chain reaction (PCR). In the Saarloos and Czechoslovakian wolfdog dwarfs, PCR products were analyzed by sequencing. DNA fragment length analysis was performed on the samples from the clinically healthy dogs.
RESULTS
Saarloos and Czechoslovakian wolfdog dwarfs have the same 7 bp deletion in intron 5 of LHX3 as do German Shepherd Dog dwarfs. The frequency of carriers of this mutation among clinically healthy Saarloos and Czechoslovakian wolfdogs used for breeding was 31% and 21%, respectively.
CONCLUSIONS AND CLINICAL IMPORTANCE
An LHX3 mutation is associated with pituitary dwarfism in Saarloos and Czechoslovakian wolfdogs. The rather high frequency of carriers of the mutated gene in the 2 breeds emphasizes the need for screening before breeding. If all breeding animals were genetically tested for the presence of the LHX3 mutation and a correct breeding policy would be implemented, this disease could be eradicated completely.
Topics: Animals; Dog Diseases; Dogs; Dwarfism, Pituitary; Female; Genetic Association Studies; Growth Hormone; Heterozygote; Insulin-Like Growth Factor I; LIM-Homeodomain Proteins; Male; Sequence Deletion; Transcription Factors
PubMed: 25273400
DOI: 10.1111/jvim.12448 -
Animal Genetics Oct 2023Cerebellar hypoplasia is a heterogeneous neurological condition in which the cerebellum is smaller than usual or not completely developed. The condition can have genetic...
Cerebellar hypoplasia is a heterogeneous neurological condition in which the cerebellum is smaller than usual or not completely developed. The condition can have genetic origins, with Mendelian-effect mutations described in several mammalian species. Here, we describe a genetic investigation of cerebellar hypoplasia in White Swiss Shepherd dogs, where two affected puppies were identified from a litter with a recent common ancestor on both sides of their pedigree. Whole genome sequencing was conducted for 10 dogs in this family, and filtering of these data based on a recessive transmission hypothesis highlighted five protein-altering candidate variants - including a frameshift-deletion of the Reelin (RELN) gene (p.Val947*). Given the status of RELN as a gene responsible for cerebellar hypoplasia in humans, sheep and mice, these data strongly suggest the loss-of-function variant as underlying these effects. This variant has not been found in other dog breeds nor in a cohort of European White Swiss Shepherds, suggesting a recent mutation event. This finding will support the genotyping of a more diverse sample of dogs, and should aid future management of the harmful allele through optimised mating schemes.
Topics: Animals; Dogs; Humans; Cerebellum; Dog Diseases; Frameshift Mutation; Mammals; Mutation; Sequence Deletion; Switzerland; Reelin Protein
PubMed: 37334487
DOI: 10.1111/age.13336 -
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 -
Current Opinion in Genetics &... Dec 2016Over the past decade, there has been both great interest and confusion about whether recent demographic events-notably the Out-of-Africa-bottleneck and recent population... (Review)
Review
Over the past decade, there has been both great interest and confusion about whether recent demographic events-notably the Out-of-Africa-bottleneck and recent population growth-have led to differences in mutation load among human populations. The confusion can be traced to the use of different summary statistics to measure load, which lead to apparently conflicting results. We argue, however, that when statistics more directly related to load are used, the results of different studies and data sets consistently reveal little or no difference in the load of non-synonymous mutations among human populations. Theory helps to understand why no such differences are seen, as well as to predict in what settings they are to be expected. In particular, as predicted by modeling, there is evidence for changes in the load of recessive loss of function mutations in founder and inbred human populations. Also as predicted, eastern subspecies of gorilla, Neanderthals and Denisovans, who are thought to have undergone reductions in population sizes that exceed the human Out-of-Africa bottleneck in duration and severity, show evidence for increased load of non-synonymous mutations (relative to western subspecies of gorillas and modern humans, respectively). A coherent picture is thus starting to emerge about the effects of demographic history on the mutation load in populations of humans and close evolutionary relatives.
Topics: Africa; Animals; Evolution, Molecular; Genetic Variation; Genetics, Population; Gorilla gorilla; Humans; Neanderthals; Sequence Deletion
PubMed: 27744216
DOI: 10.1016/j.gde.2016.09.006 -
Journal of the American Heart... Sep 2021Background Vascular endothelial cell proliferation, migration, and network formation are key proangiogenic processes involving the prototypic immediate early gene...
Background Vascular endothelial cell proliferation, migration, and network formation are key proangiogenic processes involving the prototypic immediate early gene product, Egr-1 (early growth response-1). Egr-1 undergoes phosphorylation at a conserved Ser26 but its function is completely unknown in endothelial cells or any other cell type. Methods and Results A CRISPR/Cas9 strategy was used to introduce a homozygous Ser26>Ala mutation into endogenous Egr-1 in human microvascular endothelial cells. In the course of generating mutant cells, we produced cells with homozygous deletion in caused by frameshift and premature termination. We found that Ser26 mutation in Egr-1, or Egr-1 deletion, perturbed endothelial cell proliferation in models of cell counting or real-time growth using the xCELLigence System. We found that Ser26 mutation or Egr-1 deletion ameliorated endothelial cell migration toward VEGF-A (vascular endothelial growth factor-A) in a dual-chamber model. On solubilized basement membrane preparations, Ser26 mutation or Egr-1 deletion prevented endothelial network (or tubule) formation, an in vitro model of angiogenesis. Flow cytometry further revealed that Ser26 mutation or Egr-1 deletion elevated early and late apoptosis. Finally, we demonstrated that Ser26 mutation or Egr-1 deletion increased VE-cadherin (vascular endothelial cadherin) expression, a regulator of endothelial adhesion and signaling, permeability, and angiogenesis. Conclusions These findings not only indicate that Egr-1 is essential for endothelial cell proliferation, migration, and network formation, but also show that point mutation in Ser26 is sufficient to impair each of these processes and trigger apoptosis as effectively as the absence of Egr-1. This highlights the importance of Ser26 in Egr-1 for a range of proangiogenic processes.
Topics: Cell Proliferation; Endothelial Cells; Homozygote; Humans; Sequence Deletion; Serine; Vascular Endothelial Growth Factor A
PubMed: 34476983
DOI: 10.1161/JAHA.120.020521 -
Aging Cell Dec 2015Sarcopenia, the age-induced loss of skeletal muscle mass and function, results from the contributions of both fiber atrophy and loss of myofibers. We have previously...
Sarcopenia, the age-induced loss of skeletal muscle mass and function, results from the contributions of both fiber atrophy and loss of myofibers. We have previously characterized sarcopenia in FBN rats, documenting age-dependent declines in muscle mass and fiber number along with increased fiber atrophy and fibrosis in vastus lateralis and rectus femoris muscles. Concomitant with these sarcopenic changes is an increased abundance of mitochondrial DNA deletion mutations and electron transport chain (ETC) abnormalities. In this study, we used immunohistological and histochemical approaches to define cell death pathways involved in sarcopenia. Activation of muscle cell death pathways was age-dependent with most apoptotic and necrotic muscle fibers exhibiting ETC abnormalities. Although activation of apoptosis was a prominent feature of electron transport abnormal muscle fibers, necrosis was predominant in atrophic and broken ETC-abnormal fibers. These data suggest that mitochondrial dysfunction is a major contributor to the activation of cell death processes in aged muscle fibers. The link between ETC abnormalities, apoptosis, fiber atrophy, and necrosis supports the hypothesis that mitochondrial DNA deletion mutations are causal in myofiber loss. These studies suggest a progression of events beginning with the generation and accumulation of a mtDNA deletion mutation, the concomitant development of ETC abnormalities, a subsequent triggering of apoptotic and, ultimately, necrotic events resulting in muscle fiber atrophy, breakage, and fiber loss.
Topics: Aging; Animals; Apoptosis; Biomarkers; DNA, Mitochondrial; Electron Transport; Male; Mitochondria; Muscle Fibers, Skeletal; Necrosis; Quadriceps Muscle; Rats; Rats, Inbred F344; Sarcopenia; Sequence Deletion
PubMed: 26365892
DOI: 10.1111/acel.12399 -
Scientific Reports Aug 2023Mutations in whiB7 have been associated with both hypersusceptibility and resistance to various antibiotics in Mycobacterium tuberculosis (Mtb). Unlocking the secrets of...
Mutations in whiB7 have been associated with both hypersusceptibility and resistance to various antibiotics in Mycobacterium tuberculosis (Mtb). Unlocking the secrets of antibiotic resistance in the bacterium, we examined mutations in the coding sequences of whiB7 of over 40,000 diverse Mtb isolates. Our results unveil the dominant c.191delG (Gly64delG) mutation, present in all members of the lineage L1.2.2 and its impact on WhiB7's conserved GVWGG-motif, causing conformational changes and deletion of the C-terminal AT-hook. Excitingly, we discovered six unique mutations associated with partial or total deletion of the AT-hook, specific to certain sublineages. Our findings suggest the selective pressures driving these mutations, underlining the potential of genomics to advance our understanding of Mtb's antibiotic resistance. As tuberculosis remains a global health threat, our study offers valuable insights into the diverse nature and functional consequences of whiB7 mutations, paving the way for the development of novel therapeutic interventions.
Topics: Mycobacterium tuberculosis; AT-Hook Motifs; Anti-Bacterial Agents; Exons; Sequence Deletion
PubMed: 37587174
DOI: 10.1038/s41598-023-40152-2 -
Cold Spring Harbor Molecular Case... Oct 2022Neonatal Marfan syndrome (nMFS) is a rare and severe form of Marfan syndrome (MFS) with a poor prognosis, that presents with a highly variable phenotype, particularly...
Neonatal Marfan syndrome (nMFS) is a rare and severe form of Marfan syndrome (MFS) with a poor prognosis, that presents with a highly variable phenotype, particularly regarding skeletal, ocular, and cardiovascular manifestations. Mutations in the fibrillin-1 () gene are known as the principal cause of MFS and MFS-related syndromes. Here, we report on a full-term female neonate with postnatal characteristics suggestive of nMFS, including severe cardiovascular disease resulting in cardiorespiratory failure and death by 4 mo of age. We identified a novel large genomic in-frame deletion of exons 42-45, c.(5065 + 1_5066 - 1)_(5545 + 1_5546 - 1)del. Large in-frame deletions between exons 24 and 53 have been associated with severe MFS. The deletion in our patient differs from the region associated with the majority of nMFS cases, exons 24-32.
Topics: Female; Humans; Exons; Fibrillin-1; Marfan Syndrome; Mutation; Phenotype; Sequence Deletion
PubMed: 36307213
DOI: 10.1101/mcs.a006213 -
Cancer Research and Treatment Oct 2016, the most frequently mutated gene in breast cancer, is more frequently altered in HER2-enriched and basal-like breast cancer. However, no studies have clarified the...
PURPOSE
, the most frequently mutated gene in breast cancer, is more frequently altered in HER2-enriched and basal-like breast cancer. However, no studies have clarified the role of status as a prognostic and predictive marker of triple-negative breast cancer (TNBC).
MATERIALS AND METHODS
We performed p53 immunohistochemistry (IHC), nCounter mRNA expression assay, and DNA sequencing to determine the relationship between alteration and clinical outcomes of TNBC patients.
RESULTS
Seventy-seven of 174 TNBC patients were found to harbor a mutation. Patients with missense mutations showed high protein expression in contrast to patients with deletion mutations (positivity of IHC: wild type vs. missense vs. deletion mutation, 53.6% vs. 89.8% vs. 25.0%, respectively; p < 0.001). mRNA expression was influenced by mutation status (mRNA expression [median]: wild type vs. missense vs. deletion mutation, 207.36± 132.73 vs. 339.61±143.21 vs. 99.53±99.57, respectively; p < 0.001). According to survival analysis, neither class of mutation nor protein or mRNA expression status had any impact on patient prognosis. In subgroup analysis, low mRNA expression was associated with poor prognosis in patients with a missense mutation (5-year distant recurrence-free survival [5Y DRFS]: low vs. high, 50.0% vs. 87.8%; p=0.009), while high mRNA expression with a deletion mutation indicated poor prognosis (5Y DRFS: low vs. high, 91.7% vs. 75.0%; p=0.316).
CONCLUSION
Association between mutation and expression indicates a potential prognostic marker of TNBC; hence both DNA sequencing and mRNA expression analysis may be required to predict the prognosis of TNBC patients.
Topics: Adult; Aged; Biomarkers, Tumor; Disease-Free Survival; Female; Gene Expression Regulation, Neoplastic; High-Throughput Nucleotide Sequencing; Humans; Middle Aged; Mutation, Missense; Neoplasm Staging; Prognosis; RNA, Messenger; Sequence Deletion; Triple Negative Breast Neoplasms; Tumor Suppressor Protein p53
PubMed: 26910472
DOI: 10.4143/crt.2015.430