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1.
PLoS Genet ; 16(8): e1008941, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32760060

RESUMO

Apolipoprotein B-containing lipoproteins (B-lps) are essential for the transport of hydrophobic dietary and endogenous lipids through the circulation in vertebrates. Zebrafish embryos produce large numbers of B-lps in the yolk syncytial layer (YSL) to move lipids from yolk to growing tissues. Disruptions in B-lp production perturb yolk morphology, readily allowing for visual identification of mutants with altered B-lp metabolism. Here we report the discovery of a missense mutation in microsomal triglyceride transfer protein (Mtp), a protein that is essential for B-lp production. This mutation of a conserved glycine residue to valine (zebrafish G863V, human G865V) reduces B-lp production and results in yolk opacity due to aberrant accumulation of cytoplasmic lipid droplets in the YSL. However, this phenotype is milder than that of the previously reported L475P stalactite (stl) mutation. MTP transfers lipids, including triglycerides and phospholipids, to apolipoprotein B in the ER for B-lp assembly. In vitro lipid transfer assays reveal that while both MTP mutations eliminate triglyceride transfer activity, the G863V mutant protein unexpectedly retains ~80% of phospholipid transfer activity. This residual phospholipid transfer activity of the G863V mttp mutant protein is sufficient to support the secretion of small B-lps, which prevents intestinal fat malabsorption and growth defects observed in the mttpstl/stl mutant zebrafish. Modeling based on the recent crystal structure of the heterodimeric human MTP complex suggests the G865V mutation may block triglyceride entry into the lipid-binding cavity. Together, these data argue that selective inhibition of MTP triglyceride transfer activity may be a feasible therapeutic approach to treat dyslipidemia and provide structural insight for drug design. These data also highlight the power of yolk transport studies to identify proteins critical for B-lp biology.


Assuntos
Proteínas de Transporte/genética , Lipídeos/genética , Lipoproteínas/genética , Triglicerídeos/genética , Animais , Fígado Gorduroso/genética , Fígado Gorduroso/patologia , Trato Gastrointestinal/metabolismo , Humanos , Imunoprecipitação , Gotículas Lipídicas/metabolismo , Lipoproteínas/metabolismo , Mutação de Sentido Incorreto/genética , Mutação Puntual/genética , Transporte Proteico/genética , Triglicerídeos/metabolismo , Peixe-Zebra/genética
2.
Nat Commun ; 11(1): 2169, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32358516

RESUMO

Cells possess an armamentarium of DNA repair pathways to counter DNA damage and prevent mutation. Here we use C. elegans whole genome sequencing to systematically quantify the contributions of these factors to mutational signatures. We analyse 2,717 genomes from wild-type and 53 DNA repair defective backgrounds, exposed to 11 genotoxins, including UV-B and ionizing radiation, alkylating compounds, aristolochic acid, aflatoxin B1, and cisplatin. Combined genotoxic exposure and DNA repair deficiency alters mutation rates or signatures in 41% of experiments, revealing how different DNA alterations induced by the same genotoxin are mended by separate repair pathways. Error-prone translesion synthesis causes the majority of genotoxin-induced base substitutions, but averts larger deletions. Nucleotide excision repair prevents up to 99% of point mutations, almost uniformly across the mutation spectrum. Our data show that mutational signatures are joint products of DNA damage and repair and suggest that multiple factors underlie signatures observed in cancer genomes.


Assuntos
Dano ao DNA/fisiologia , Reparo do DNA/fisiologia , Animais , Caenorhabditis elegans/genética , Dano ao DNA/genética , Reparo do DNA/genética , Genômica/métodos , Humanos , Mutação/genética , Mutação Puntual/genética
3.
J Vis Exp ; (159)2020 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-32449724

RESUMO

Custom designed endonucleases, such as RNA-guided Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9, enable efficient genome editing in mammalian cells. Here we describe detailed procedures to seamlessly genome edit the hepatocyte nuclear factor 4 alpha (HNF4α) locus as an example in human pluripotent stem cells. Combining a piggyBac-based donor plasmid and the CRISPR-Cas9 nickase mutant in a two-step genetic selection, we demonstrate correct and efficient targeting of the HNF4α locus.


Assuntos
Edição de Genes/métodos , Células-Tronco Pluripotentes/metabolismo , Mutação Puntual/genética , Sequência de Bases , Sistemas CRISPR-Cas/genética , Elementos de DNA Transponíveis/genética , Vetores Genéticos/genética , Genoma Humano , Humanos
4.
PLoS One ; 15(4): e0232167, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32353016

RESUMO

We examined seventy million well-characterized human mutations, and their impact on G+C-compositional dynamics, in order to understand the formation and maintenance of major genomic nucleotide sequence patterns. Among novel mutations, those that change a strong (S) base pair G:C/C:G to a weak (W) pair A:T/T:A occur at nearly twice the frequency of the opposite mutations. Such imbalance puts strong downward pressure on overall GC-content. However, along protracted paths to fixation, S→W mutations are much less likely to propagate than W→S mutations. The magnitude of relative propagation disadvantages for S→W mutations is inexplicable by any currently-accepted model. This fact forced us to re-examine the quantitative features of Biased Gene Conversion (BGC) theory. Revised parameters of BGC that, per average individual, convert 7-14 W base pairs into S pairs, would account for the S-content turnover differences between new and old mutations, and make BGC an instrumental force for nucleotide dynamics and evolution. BGC should thus be considered seriously in both theories and biomedical practice. In particular, BGC should be taken into account during allele imputations, where missing SNP alleles are computationally predicted based on the information about several neighboring alleles. Finally, we analyzed the effect of neighboring nucleotide context on the mutation frequencies, dynamics, and GC-composition turnover. For this purpose, we examined genomic regions having extremely biased nucleotide compositions (enriched for S-, W-, purine/pyrimidine strand asymmetry, or AC/GT-strand asymmetry). It was found that point mutations in these regions preferentially degrade the nucleotide inhomogeneities, decreasing the sequence biases. Degradation of sequence bias is highest for novel mutations, and considerably lower for older mutations (those widespread across populations). Besides BGC, there may be additional, still uncharacterized molecular mechanisms that either preserve genomic regions with biased nucleotide compositions from mutational degradation or fail to degrade such inhomogeneities in specific chromosomal regions.


Assuntos
Composição de Bases/genética , Conversão Gênica/genética , Genoma Humano/genética , Alelos , Evolução Molecular , Humanos , Taxa de Mutação , Mutação Puntual/genética , Polimorfismo de Nucleotídeo Único/genética
5.
J Gen Virol ; 101(5): 510-522, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32242791

RESUMO

Noroviruses are recognized as the major cause of non-bacterial gastroenteritis in humans. Molecular mechanisms driving norovirus evolution are the accumulation of point mutations and recombination. Recombination can create considerable changes in a viral genome, potentially eliciting a fitness cost, which must be compensated via the adaptive capacity of a recombinant virus. We previously described replicative fitness reduction of the first in vitro generated WU20-CW1 recombinant murine norovirus, RecMNV. In this follow-up study, RecMNV's capability of replicative fitness recuperation and genetic characteristics of RecMNV progenies at early and late stages of an adaptation experiment were evaluated. Replicative fitness regain of the recombinant was demonstrated via growth kinetics and plaque size differences between viral progenies prior to and post serial in vitro passaging. Point mutations at consensus and sub-consensus population levels of early and late viral progenies were characterized via next-generation sequencing and putatively associated to fitness changes. To investigate the effect of genomic changes separately and in combination in the context of a lab-generated inter-MNV infectious virus, mutations were introduced into a recombinant WU20-CW1 cDNA for subsequent DNA-based reverse genetics recovery. We thus associated fitness loss of RecMNV to a C7245T mutation and functional VP2 (ORF3) truncation and demonstrated individual and cumulative compensatory effects of one synonymous OFR2 and two non-synonymous ORF1 consensus-level mutations acquired during successive rounds of in vitro replication. Our data provide evidence of viral adaptation in a controlled environment via genetic drift after genetic shift induced a fitness cost of an infectious recombinant norovirus.


Assuntos
Norovirus/genética , Replicação Viral/genética , Animais , Infecções por Caliciviridae/virologia , Linhagem Celular , DNA Complementar/genética , Seguimentos , Deriva Genética , Genoma Viral/genética , Camundongos , Mutação Puntual/genética , Células RAW 264.7 , RNA Viral/genética
6.
Proc Natl Acad Sci U S A ; 117(14): 7745-7754, 2020 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-32198205

RESUMO

Competence allows bacteria to internalize exogenous DNA fragments for the acquisition of new phenotypes such as antibiotic resistance or virulence traits. In most streptococci, competence is regulated by ComRS signaling, a system based on the mature ComS pheromone (XIP), which is internalized to activate the (R)RNPP-type ComR sensor by triggering dimerization and DNA binding. Cross-talk analyses demonstrated major differences of selectivity between ComRS systems and raised questions concerning the mechanism of pheromone-sensor recognition and coevolution. Here, we decipher the molecular determinants of selectivity of the closely related ComRS systems from Streptococcus thermophilus and Streptococcus vestibularis Despite high similarity, we show that the divergence in ComR-XIP interaction does not allow reciprocal activation. We perform the structural analysis of the ComRS system from S. vestibularis. Comparison with its ortholog from S. thermophilus reveals an activation mechanism based on a toggle switch involving the recruitment of a key loop by the XIP C terminus. Together with a broad mutational analysis, we identify essential residues directly involved in peptide binding. Notably, we generate a ComR mutant that displays a fully reversed selectivity toward the heterologous pheromone with only five point mutations, as well as other ComR variants featuring XIP bispecificity and/or neofunctionalization for hybrid XIP peptides. We also reveal that a single XIP mutation relaxes the strictness of ComR activation, suggesting fast adaptability of molecular communication phenotypes. Overall, this study is paving the way toward the rational design or directed evolution of artificial ComRS systems for a range of biotechnological and biomedical applications.


Assuntos
Feromônios/metabolismo , Transdução de Sinais , Streptococcus/metabolismo , Sequência de Aminoácidos , Luciferases/metabolismo , Modelos Moleculares , Mutação Puntual/genética , Estrutura Secundária de Proteína , Homologia Estrutural de Proteína
7.
Nat Commun ; 11(1): 1580, 2020 03 27.
Artigo em Inglês | MEDLINE | ID: mdl-32221286

RESUMO

ADAR RNA editing enzymes are high-affinity dsRNA-binding proteins that deaminate adenosines to inosines in pre-mRNA hairpins and also exert editing-independent effects. We generated a Drosophila AdarE374A mutant strain encoding a catalytically inactive Adar with CRISPR/Cas9. We demonstrate that Adar adenosine deamination activity is necessary for normal locomotion and prevents age-dependent neurodegeneration. The catalytically inactive protein, when expressed at a higher than physiological level, can rescue neurodegeneration in Adar mutants, suggesting also editing-independent effects. Furthermore, loss of Adar RNA editing activity leads to innate immune induction, indicating that Drosophila Adar, despite being the homolog of mammalian ADAR2, also has functions similar to mammalian ADAR1. The innate immune induction in fly Adar mutants is suppressed by silencing of Dicer-2, which has a RNA helicase domain similar to MDA5 that senses unedited dsRNAs in mammalian Adar1 mutants. Our work demonstrates that the single Adar enzyme in Drosophila unexpectedly has dual functions.


Assuntos
Adenosina Desaminase/genética , Encéfalo/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/imunologia , Imunidade Inata/genética , Edição de RNA/genética , Adenosina Desaminase/química , Monofosfato de Adenosina/metabolismo , Envelhecimento/patologia , Animais , Catálise , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Regulação da Expressão Gênica , Locomoção , Degeneração Neural/patologia , Mutação Puntual/genética , Domínios Proteicos , RNA Helicases/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ribonuclease III/metabolismo
8.
Nature ; 579(7800): 603-608, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32132710

RESUMO

Acetaldehyde is a highly reactive, DNA-damaging metabolite that is produced upon alcohol consumption1. Impaired detoxification of acetaldehyde is common in the Asian population, and is associated with alcohol-related cancers1,2. Cells are protected against acetaldehyde-induced damage by DNA crosslink repair, which when impaired causes Fanconi anaemia (FA), a disease resulting in failure to produce blood cells and a predisposition to cancer3,4. The combined inactivation of acetaldehyde detoxification and the FA pathway induces mutation, accelerates malignancies and causes the rapid attrition of blood stem cells5-7. However, the nature of the DNA damage induced by acetaldehyde and how this is repaired remains a key question. Here we generate acetaldehyde-induced DNA interstrand crosslinks and determine their repair mechanism in Xenopus egg extracts. We find that two replication-coupled pathways repair these lesions. The first is the FA pathway, which operates using excision-analogous to the mechanism used to repair the interstrand crosslinks caused by the chemotherapeutic agent cisplatin. However, the repair of acetaldehyde-induced crosslinks results in increased mutation frequency and an altered mutational spectrum compared with the repair of cisplatin-induced crosslinks. The second repair mechanism requires replication fork convergence, but does not involve DNA incisions-instead the acetaldehyde crosslink itself is broken. The Y-family DNA polymerase REV1 completes repair of the crosslink, culminating in a distinct mutational spectrum. These results define the repair pathways of DNA interstrand crosslinks caused by an endogenous and alcohol-derived metabolite, and identify an excision-independent mechanism.


Assuntos
Acetaldeído/química , Reagentes para Ligações Cruzadas/química , Dano ao DNA , Reparo do DNA , Replicação do DNA/fisiologia , DNA/química , Etanol/química , Anemia de Fanconi/metabolismo , Animais , Cisplatino/química , Cisplatino/farmacologia , Dano ao DNA/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , DNA Polimerase Dirigida por DNA/metabolismo , Etanol/farmacologia , Mutagênese/efeitos dos fármacos , Nucleotidiltransferases/metabolismo , Mutação Puntual/efeitos dos fármacos , Mutação Puntual/genética , Xenopus , Proteínas de Xenopus/metabolismo
9.
Cell ; 180(5): 819, 2020 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-32142671

RESUMO

Sickle cell disease (SCD) is caused by a point mutation in the ß-globin gene that creates hemoglobin S (HbS). Upon deoxygenation, HbS forms long polymers that distort the shape of red blood cells, causing hemolysis and vaso-occlusion. Voxelotor inhibits HbS polymerization, the root cause of SCD complications. To view this Bench to Bedside, open or download the PDF.


Assuntos
Anemia Falciforme/genética , Benzaldeídos/uso terapêutico , Hemoglobina Falciforme/antagonistas & inibidores , Pirazinas/uso terapêutico , Pirazóis/uso terapêutico , Globinas beta/genética , Anemia Falciforme/epidemiologia , Hemoglobina Falciforme/genética , Humanos , Mutação Puntual/genética , Polimerização/efeitos dos fármacos
10.
Infect Immun ; 88(5)2020 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-32094260

RESUMO

Antimicrobial peptides play an important role in host defense against Vibrio cholerae Generally, the V. cholerae O1 classical biotype is polymyxin B (PB) sensitive and El Tor is relatively resistant. Detection of classical biotype traits like the production of classical cholera toxin and PB sensitivity in El Tor strains has been reported in recent years, including in the devastating Yemen cholera outbreak during 2016-2018. To investigate the factor(s) responsible for the shift in the trend of sensitivity to PB, we studied the two-component system encoded by carRS, regulating the lipid A modification of El Tor vibrios, and found that only carR contains a single nucleotide polymorphism (SNP) in recently emerged PB-sensitive strains. We designated the two alleles present in PB-resistant and -sensitive strains carR r and carR s alleles, respectively, and replaced the carR s allele of a sensitive strain with the carR r allele, using an allelic-exchange approach. The sensitive strain then became resistant. The PB-resistant strain N16961 was made susceptible to PB in a similar fashion. Our in silico CarR protein models suggested that the D89N substitution in the more stable CarRs protein brings the two structural domains of CarR closer, constricting the DNA binding cleft. This probably reduces the expression of the carR-regulated almEFG operon, inducing PB susceptibility. Expression of almEFG in PB-sensitive strains was found to be downregulated under natural culturing conditions. In addition, the expression of carR and almEG decreased in all strains with increased concentrations of extracellular Ca2+ but increased with a rise in pH. The downregulation of almEFG in CarRs strains confirmed that the G265A mutation is responsible for the emergence of PB-sensitive El Tor strains.


Assuntos
Mutação Puntual/genética , Polimixina B/farmacologia , Transcrição Genética/genética , Vibrio cholerae O1/efeitos dos fármacos , Vibrio cholerae O1/genética , Alelos , Antibacterianos/farmacologia , Cálcio/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação para Baixo/genética , Escherichia coli/genética , Polimorfismo de Nucleotídeo Único/genética , Vibrio cholerae O1/metabolismo
11.
Arq Neuropsiquiatr ; 78(1): 34-38, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-32074192

RESUMO

OBJECTIVE: Brain tumors are one of the most common causes of cancer-related deaths around the world. Angiogenesis is critical in high-grade malignant gliomas, such as glioblastoma multiforme. The aim of this study is to comparatively analyze the angiogenesis-related genes, namely VEGFA, VEGFB, KDR, CXCL8, CXCR1 and CXCR2 in LGG vs. GBM to identify molecular distinctions using datasets available on The Cancer Genome Atlas (TCGA). METHODS: DNA sequencing and mRNA expression data for 514 brain lower grade glioma (LGG) and 592 glioblastoma multiforme (GBM) patients were acquired from The Cancer Genome Atlas (TCGA), and the genetic alterations and expression levels of the selected genes were analyzed. RESULTS: We identified six distinct KDR mutations in the LGG patients and 18 distinct KDR mutations in the GBM patients, including missense and nonsense mutations, frame shift deletion and altered splice region. Furthermore, VEGFA and CXCL8 were significantly overexpressed within GBM patients. CONCLUSIONS: VEGFA and CXCL8 are important factors for angiogenesis, which are suggested to have significant roles during tumorigenesis. Our results provide further evidence that VEGFA and CXCL8 could induce angiogenesis and promote LGG to progress into GBM. These findings could be useful in developing novel targeted therapeutics approaches in the future.


Assuntos
Neoplasias Encefálicas/genética , Carcinogênese/genética , Glioblastoma/genética , Glioma/genética , Neovascularização Patológica/genética , Expressão Gênica , Glioblastoma/patologia , Glioma/patologia , Humanos , Interleucina-8/análise , Mutação Puntual/genética , Receptores de Interleucina-8A/análise , Receptores de Interleucina-8B/análise , Valores de Referência , Fator A de Crescimento do Endotélio Vascular/análise , Fator B de Crescimento do Endotélio Vascular/análise , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/análise
12.
Invest Ophthalmol Vis Sci ; 61(2): 9, 2020 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-32049341

RESUMO

Purpose: Variant B precursor cysteine protease inhibitor cystatin C, a known recessive risk factor for developing exudative age-related macular degeneration (AMD), presents altered intracellular trafficking and reduced secretion from retinal pigment epithelial (RPE) cells. Because cystatin C inhibits multiple extracellular matrix (ECM)-degrading cathepsins, this study evaluated the role of this mutation in inducing ECM-related functional changes in RPE cellular behavior. Methods: Induced pluripotent stem cells gene-edited bi-allelically by CRISPR/Cas9 to express the AMD-linked cystatin C variant were differentiated to RPE cells and assayed for their ability to degrade fluorescently labeled ECM proteins. Cellular migration and adhesion on multiple ECM proteins, differences in transepithelial resistance and polarized protein secretion were tested. Vessel formation induced by gene edited cells-conditioned media was quantified using primary human dermal microvascular epithelial cells. Results: Variant B cystatin C-expressing induced pluripotent stem cells-derived RPE cells displayed a significantly higher rate of laminin and fibronectin degradation 3 days after seeding on fluorescently labeled ECM (P < 0.05). Migration on matrigel, collagen IV and fibronectin was significantly faster for edited cells compared with wild-type (WT) cells. Both edited and WT cells displayed polarized secretion of cystatin C, but transepithelial resistance was lower in gene-edited cells after 6 weeks culture, with significantly lower expression of tight junction protein claudin-3. Media conditioned by gene-edited cells stimulated formation of significantly longer microvascular tubes (P < 0.05) compared with WT-conditioned media. Conclusions: Reduced levels of cystatin C lead to changes in the RPE ability to degrade, adhere, and migrate supporting increased invasiveness and angiogenesis relevant for AMD pathology.


Assuntos
Cistatina C/fisiologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Degeneração Macular/patologia , Epitélio Pigmentado da Retina/citologia , Movimento Celular/fisiologia , Células Cultivadas , Cistatina C/genética , Cistatina C/metabolismo , Fibronectinas/metabolismo , Edição de Genes , Humanos , Laminina/metabolismo , Mutação Puntual/genética
13.
PLoS One ; 15(2): e0228925, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32053675

RESUMO

Adenocarcinoma is the most common type of non-small cell lung cancer. Some causative genomic alterations in epidermal growth factor receptor (EGFR), including deletions in exon 19 (E19 dels) and a point mutation in E21, are known to have favourable prognoses due to sensitivity to tyrosine kinase inhibitors; however, the prognoses of other uncommon mutations are unclear. This study analysed the clinical significance of EGFR mutation types in lung adenocarcinoma. We retrospectively reviewed 1,020 subjects (mean age: 66.8 years, female: 41.7%) who were diagnosed with advanced lung adenocarcinoma, had EGFR mutation data, and did not undergo surgery from five medical institutes between 2010 and 2016. Subjects were classified according to EGFR mutation status, particularly for exon-specific mutations. EGFR positivity was defined as the presence of mutation and EGFR negativity was defined as wild-type EGFR. EGFR positivity was 38.0%, with the incidence of mutations in E18, E19, E20, and E21 was 3.6%, 51.0%, 3.4%, and 42.0%, respectively. The EGFR positive group survived significantly longer than the negative group (p<0.001), and there was a significant difference in survival among the four EGFR mutation sites (p = 0.003); E19 dels were the only significant factor that lowered mortality (HR: 0.678, p = 0.002), while an E21 mutation was the prognostic factor associated with the most increased mortality (HR: 1.365, p = 0.015). Amongst EGFR positive subjects, the proportion of E19 dels in TKI-responders was significantly higher and that of E21 mutations significantly lower, compared with non-responders. In TKI treatment, mutations in E18 and E20 were not worse factors than the E21 L858R mutation. In conclusion, the presence of EGFR mutations in advanced lung adenocarcinoma can predict a good prognosis; E19 dels prospect to have a better prognosis than other mutations, while an E21 mutation is expected to increase mortality.


Assuntos
Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/mortalidade , Adenocarcinoma de Pulmão/metabolismo , Idoso , Grupo com Ancestrais do Continente Asiático , Carcinoma Pulmonar de Células não Pequenas/patologia , Receptores ErbB/genética , Receptores ErbB/metabolismo , Éxons/genética , Feminino , Humanos , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Mutação/genética , Mutação Puntual/genética , Prognóstico , República da Coreia , Estudos Retrospectivos , Deleção de Sequência/genética
14.
Mol Genet Genomics ; 295(2): 505-514, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31897801

RESUMO

α-thalassemia is an inherited blood disorder commonly caused by deletions or point mutations involving one or both α-globin genes. Recent studies shed new light on the critical role of upstream enhancers multi-species conserved sequences (MCSs) in the ordered regulation of α-globin gene expression. Herein, we reported two unrelated probands with deletions in α-globin genes and MCSs, respectively. The proband from Family A is a compound heterozygote carrying a known α+ mutation (-α3.7) and a novel 60.2 kb deletion causing the absence of both α-globin genes. The proband from Family B, on the other hand, is a compound heterozygote with a known α0 mutation (--SEA) and a novel deletion involving only upstream regulatory elements MCS-R1, R2 and R3, while the α-globin genes remain intact. Notably, both these two patients suffered varied extent of anemia, indicating that the loss of enhancer elements could equally lead to reduced synthesis of α-globin. Upon these observations, we then confirmed the exact breakpoints of these two novel deletions using a targeted next-generation sequencing (NGS) previously established by our group, which may enable further elucidation of the rearrangement mechanisms on these deletions and functional dissection of MCSs. Taken together, our study reports a reliable NGS-based molecular screening approach for accurate identification of copy number variations (CNVs) in the α-globin cluster and the genetic diagnosis of these two probands may help to extend the spectrum of α-thalassemia mutations in Chinese population.


Assuntos
Elementos Alu/genética , Anemia/genética , alfa-Globinas/genética , Talassemia alfa/genética , Adulto , Anemia/sangue , Anemia/patologia , Variações do Número de Cópias de DNA/genética , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Pessoa de Meia-Idade , Família Multigênica/genética , Linhagem , Mutação Puntual/genética , Deleção de Sequência/genética , Talassemia alfa/sangue , Talassemia alfa/patologia
15.
BMC Infect Dis ; 20(1): 19, 2020 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-31910878

RESUMO

BACKGROUND: Pyrazinamide still may be a useful drug for treatment of rifampin-resistant (RR-TB) or multidrug-resistant tuberculosis (MDR-TB) in China while awaiting scale up of new drugs and regimens including bedaquiline and linezolid. The level of pyrazinamide resistance among MDR-TB patients in China is not well established. Therefore, we assessed pyrazinamide resistance in a representative sample and explored determinants and patterns of pncA mutations. METHODS: MDR-TB isolates from the 2007 national drug resistance survey of China were sub-cultured and examined for pyrazinamide susceptibility by BACTEC MGIT 960 method. pncA mutations were identified by sequencing. Characteristics associated with pyrazinamide resistance were analyzed using univariable and multivariable log-binominal regression. RESULTS: Of 401 MDR-TB isolates, 324 were successfully sub-cultured and underwent drug susceptibility testing. Pyrazinamide resistance was prevalent in 40.7% of samples, similarly among new and previously treated MDR-TB patients. Pyrazinamide resistance in MDR-TB patients was associated with lower age (adjusted OR 0.54; 95% CI, 0.34-0.87 for those aged ≧60 years compared to < 40 years). Pyrazinamide resistance was not associated with gender, residential area, previous treatment history and Beijing genotype. Of 132 patients with pyrazinamide resistant MDR-TB, 97 (73.5%) had a mutation in the pncA gene; with 61 different point mutations causing amino acid change, and 11 frameshifts in the pncA gene. The mutations were scattered throughout the whole pncA gene and no hot spot region was identified. CONCLUSIONS: Pyrazinamide resistance among MDR-TB patients in China is common, although less so in elderly patients. Therefore, pyrazinamide should only be used for treatment of RR/MDR-TB in China if susceptibility is confirmed. Molecular testing for detection of pyrazinamide resistance only based on pncA mutations has certain value for the rapid detection of pyrazinamide resistance in MDR-TB strains but other gene mutations conferring to pyrazinamide resistance still need to be explored to increase its predictive ability .


Assuntos
Antituberculosos/uso terapêutico , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/genética , Pirazinamida/uso terapêutico , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico , Tuberculose Resistente a Múltiplos Medicamentos/epidemiologia , Adulto , Fatores Etários , Amidoidrolases/genética , Antituberculosos/efeitos adversos , Sequência de Bases/genética , China/epidemiologia , Diarilquinolinas/uso terapêutico , Genes Bacterianos/genética , Genótipo , Humanos , Linezolida/uso terapêutico , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Mycobacterium tuberculosis/isolamento & purificação , Mutação Puntual/genética , Polimorfismo de Nucleotídeo Único/genética , Prevalência , Pirazinamida/efeitos adversos , Rifampina/efeitos adversos , Rifampina/uso terapêutico , Fatores de Risco , Tuberculose Resistente a Múltiplos Medicamentos/diagnóstico
16.
BMC Med Genet ; 21(1): 13, 2020 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-31937257

RESUMO

BACKGROUND: Mutations of the WFS1 gene are responsible for most cases of Wolfram syndrome (WS), a rare, recessively inherited neurodegenerative disorder characterized by juvenile-onset non-autoimmune diabetes mellitus and optic atrophy. Variants of WFS1 are also associated with non-syndromic hearing loss and type-2 diabetes mellitus (T2DM). Our study adds to literature significant associations between WS and T2DM. CASE PRESENTATION: In this study, we analyzed the clinical and genetic data of two families with high prevalence of WS and T2DM. Genetic linkage analysis and DNA sequencing were exploited to identify pathogenic variants. One novel pathogenic variant (c.2243-2244insC) and one known pathogenic (c.1232_1233delCT) (frameshift) variant were identified in exon eight of WFS1 gene. CONCLUSIONS: The mutational and phenotypic spectrum of WS is broadened by our report of novel WFS1 mutation. Our results reveal the value of molecular analysis of WFS1 in the improvement of clinical diagnostics for WS. This study also confirms the role of WFS1 in T2DM.


Assuntos
Diabetes Mellitus Tipo 2/genética , Testes Genéticos , Proteínas de Membrana/genética , Síndrome de Wolfram/genética , Adulto , Criança , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/patologia , Éxons/genética , Feminino , Mutação da Fase de Leitura/genética , Ligação Genética , Predisposição Genética para Doença , Perda Auditiva/complicações , Perda Auditiva/genética , Perda Auditiva/patologia , Humanos , Irã (Geográfico)/epidemiologia , Masculino , Atrofia Óptica/complicações , Atrofia Óptica/genética , Atrofia Óptica/patologia , Linhagem , Fenótipo , Mutação Puntual/genética , Síndrome de Wolfram/complicações , Síndrome de Wolfram/patologia , Adulto Jovem
17.
PLoS One ; 15(1): e0227621, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31923916

RESUMO

Performing a complete deep mutational scan with all single point mutations may not be practical, and may not even be required, especially if predictive computational models can be developed. Computational models are however naive to cellular response in the myriads of assay-conditions. In a realistic paradigm of assay context-aware predictive hybrid models that combine minimal experimental data from deep mutational scans with structure, sequence information and computational models, we define and evaluate different strategies for choosing this minimal set. We evaluated the trivial strategy of a systematic reduction in the number of mutational studies from 85% to 15%, along with several others about the choice of the types of mutations such as random versus site-directed with the same 15% data completeness. Interestingly, the predictive capabilities by training on a random set of mutations and using a systematic substitution of all amino acids to alanine, asparagine and histidine (ANH) were comparable. Another strategy we explored, augmenting the training data with measurements of the same mutants at multiple assay conditions, did not improve the prediction quality. For the six proteins we analyzed, the bin-wise error in prediction is optimal when 50-100 mutations per bin are used in training the computational model, suggesting that good prediction quality may be achieved with a library of 500-1000 mutations.


Assuntos
Biologia Computacional/métodos , Análise Mutacional de DNA/métodos , Mutagênese Sítio-Dirigida/métodos , Sequência de Aminoácidos , Animais , Simulação por Computador , Humanos , Modelos Teóricos , Mutação , Mutação Puntual/genética , Proteínas
18.
Nucleic Acids Res ; 48(2): 761-769, 2020 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-31777935

RESUMO

Identifying the molecular mechanisms that give rise to genetic variation is essential for the understanding of evolutionary processes. Previously, we have used adaptive laboratory evolution to enable biomass synthesis from CO2 in Escherichia coli. Genetic analysis of adapted clones from two independently evolving populations revealed distinct enrichment for insertion and deletion mutational events. Here, we follow these observations to show that mutations in the gene encoding for DNA topoisomerase I (topA) give rise to mutator phenotypes with characteristic mutational spectra. Using genetic assays and mutation accumulation lines, we find that point mutations in topA increase the rate of sequence deletion and duplication events. Interestingly, we observe that a single residue substitution (R168C) results in a high rate of head-to-tail (tandem) short sequence duplications, which are independent of existing sequence repeats. Finally, we show that the unique mutation spectrum of topA mutants enhances the emergence of antibiotic resistance in comparison to mismatch-repair (mutS) mutators, and leads to new resistance genotypes. Our findings highlight a potential link between the catalytic activity of topoisomerases and the fundamental question regarding the emergence of de novo tandem repeats, which are known modulators of bacterial evolution.


Assuntos
Dióxido de Carbono/metabolismo , DNA Topoisomerases Tipo I/genética , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Proteína MutS de Ligação de DNA com Erro de Pareamento/genética , Biomassa , Dióxido de Carbono/química , DNA Topoisomerases Tipo I/química , Farmacorresistência Bacteriana/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Evolução Molecular , Duplicação Gênica/genética , Genótipo , Proteína MutS de Ligação de DNA com Erro de Pareamento/química , Mutação , Mutação Puntual/genética
19.
Asian Pac J Cancer Prev ; 20(12): 3611-3615, 2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31870101

RESUMO

BACKGROUND: Janus Tyrosine Kinase-2 (JAK2 V617F), a novel point mutation affecting the MPD'S is a somatic gain-of-function mutation. It alters a highly conserved amino acid valine in the negative regulatory JH2 domain to phenylalanine predicted to dysregulate kinase activity. AIM: To evaluate the prevalence and clinical significance of JAK2 V617F mutation in various MPD's as well as in hematological malignancies. SUBJECTS AND METHODS: JAK2 mutation was assessed in 90 patients with myeloproliferative disorders and 47 leukemic patients. In addition, peripheral blood samples from 90 healthy donors were also collected as control. We used a highly sensitive Allele-Specific polymerase chain reaction (AS-PCR) for the detection and confirmed the mutation further by direct sequencing. RESULTS: Our results showed significant differences between various disorders with respect to either the proportion of positivity or that of mutant alleles. JAK2-V617F was detected in 67/90 MPD patients and 02/17 for AML,01/11 for ALL-L1,02/12 for ALL-L2 and 02/07 for CML and 90 healthy controls. CONCLUSION: From the above findings it is evident that the JAK2 V617F mutation is widespread not only in MPD's but also in hematological malignancies, which might as well lead to the new classification of MPD'S. Our data also suggest that different genetic events may lead to JAK-STAT pathway activation in different malignancies.


Assuntos
Neoplasias da Medula Óssea/genética , Predisposição Genética para Doença/genética , Neoplasias Hematológicas/genética , Janus Quinase 2/genética , Transtornos Mieloproliferativos/genética , Adolescente , Adulto , Substituição de Aminoácidos/genética , Criança , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mutação Puntual/genética , Adulto Jovem
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