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1.
Artigo em Coreano | WPRIM (Pacífico Ocidental) | ID: wprim-786626

RESUMO

Addressing the increasing antibiotic resistance, including clarithromycin resistance, which affects Helicobacter pylori (H. pylori) eradication therapy, is a challenge for clinicians. Antibiotic resistance is the main reason for H. pylori eradication failure and the resistance rate for clarithromycin may drastically increase, up to 38.5%, due to 23S ribosomal RNA point mutations. Therefore, the standard triple regimen is no longer suitable as the first-line treatment in most regions. However, there is a growing interest in personalized care for patients. Increased eradication rates of tailored therapy based on antibiotic susceptibility have been reported using nucleic acid-based techniques for clarithromycin resistance with a focus on the first-line eradication therapy of H. pylori infection. Herein, we discuss the eradication therapy for H. pylori, with a diagnostic test and appropriate treatment for clarithromycin resistance.


Assuntos
Claritromicina , Testes Diagnósticos de Rotina , Resistência a Medicamentos , Resistência Microbiana a Medicamentos , Helicobacter pylori , Helicobacter , Humanos , Mutação Puntual , RNA Ribossômico 23S
2.
Artigo em Inglês | WPRIM (Pacífico Ocidental) | ID: wprim-766007

RESUMO

Although papillary thyroid carcinoma (PTC)–type nuclear changes are the most reliable morphological feature in the diagnosis of PTC, the nuclear assessment used to identify these changes is highly subjective. Here, we report a noninvasive encapsulated thyroid tumor with a papillary growth pattern measuring 23 mm at its largest diameter with a nuclear score of 2 in a 26-year-old man. After undergoing left lobectomy, the patient was diagnosed with an encapsulated PTC. However, a second opinion consultation suggested an alternative diagnosis of follicular adenoma with papillary hyperplasia. When providing a third opinion, we identified a low MIB-1 labeling index and a heterozygous point mutation in the KRAS gene but not the BRAF gene. We speculated that this case is an example of a novel borderline tumor with a papillary structure. Introduction of the new terminology “noninvasive encapsulated papillary RAS-like thyroid tumor (NEPRAS)” without the word “cancer” might relieve the psychological burden of patients in a way similar to the phrase “noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP).”


Assuntos
Adenoma , Adulto , Diagnóstico , Humanos , Hiperplasia , Variações Dependentes do Observador , Mutação Puntual , Encaminhamento e Consulta , Glândula Tireoide , Neoplasias da Glândula Tireoide
3.
Artigo em Inglês | WPRIM (Pacífico Ocidental) | ID: wprim-719506

RESUMO

PURPOSE: Hypoallergenic recombinant Der p 2 has been produced by various genetic manipulations, but mutation of a naturally polymorphic amino acid residue known to affect IgE binding has not been studied. This study aimed to determine the effect of a point mutation (S47W) of residue 47 of Der p 2 on its structure and immunoglobulin (Ig) E binding. Its ability to induce pro-inflammatory responses and to induce blocking IgG antibody was also determined. METHODS: S47 of recombinant Der p 2.0110, one of the predominant variants in Bangkok, was mutated to W (S47W). S47W secreted from Pichia pastoris was examined for secondary structure and for the formation of a hydrophobic cavity by 8-Anilino-1-naphthalenesulfonic acid (ANS) staining. Monoclonal and human IgE-antibody binding was determined by enzyme-linked immunosorbent assay. Allergen-induced degranulation by human epsilon receptor expressed-rat basophil was determined. Stimulation of the pro-inflammatory cytokine interleukin (IL)-8 release from human bronchial epithelial (BEAS2B) cells and inhibition of IgE binding to the wild type allergen by S47W-induced IgG were determined. RESULTS: S47W reduced secondary structure and failed to bind the hydrophobic ANS ligand as well as a monoclonal antibody known to be dependent on the nature of the side chain of residue 114 in an adjacent loop. It could also not stimulate IL-8 release from BEAS2B cells. IgE from house dust mite (HDM)-allergic Thais bound S47W with 100-fold weaker avidity, whereas IgE of HDM-allergic Australians did not. S47W still induced basophil degranulation, although requiring higher concentrations for some subjects. Anti-S47W antiserum-immunized mice blocked the binding of human IgE to wild type Der p 2. CONCLUSIONS: The mutant S47W had altered structure and reduced ability to stimulate pro-inflammatory responses and to bind IgE, but retained its ability to induce blocking antibodies. It thus represents a hypoallergen produced by a single mutation of a non-solvent-accessible amino acid.


Assuntos
Animais , Anticorpos Bloqueadores , Grupo com Ancestrais do Continente Asiático , Basófilos , Poeira , Ensaio de Imunoadsorção Enzimática , Humanos , Imunoglobulina E , Imunoglobulina G , Imunoglobulinas , Interleucina-8 , Interleucinas , Camundongos , Pichia , Mutação Puntual , Pyroglyphidae
4.
Artigo em Inglês | WPRIM (Pacífico Ocidental) | ID: wprim-719428

RESUMO

PURPOSE: With the emergence of next-generation sequencing (NGS) technology, profiling a wide range of genomic alterations has become a possibility resulting in improved implementation of targeted cancer therapy. In Asian populations, the prevalence and spectrum of clinically actionable genetic alterations has not yet been determined because of a lack of studies examining high-throughput cancer genomic data. MATERIALS AND METHODS: To address this issue, 1,071 tumor samples were collected from five major cancer institutes in Korea and analyzed using targeted NGS at a centralized laboratory. Samples were either fresh frozen or formalin-fixed, paraffin embedded (FFPE) and the quality and yield of extracted genomic DNA was assessed. In order to estimate the effect of sample condition on the quality of sequencing results, tissue preparation method, specimen type (resected or biopsied) and tissue storage time were compared. RESULTS: We detected 7,360 non-synonymous point mutations, 1,164 small insertions and deletions, 3,173 copy number alterations, and 462 structural variants. Fifty-four percent of tumors had one or more clinically relevant genetic mutation. The distribution of actionable variants was variable among different genes. Fresh frozen tissues, surgically resected specimens, and recently obtained specimens generated superior sequencing results over FFPE tissues, biopsied specimens, and tissues with long storage duration. CONCLUSION: In order to overcome, challenges involved in bringing NGS testing into routine clinical use, a centralized laboratory model was designed that could improve the NGS workflows, provide appropriate turnaround times and control costs with goal of enabling precision medicine.


Assuntos
Academias e Institutos , Grupo com Ancestrais do Continente Asiático , DNA , Humanos , Coreia (Geográfico) , Métodos , Parafina , Mutação Puntual , Medicina de Precisão , Prevalência
5.
Artigo em Chinês | WPRIM (Pacífico Ocidental) | ID: wprim-781310

RESUMO

OBJECTIVE@#To explore the serological and genotypic characteristics of a pedigree with B(A).06 subtype.@*METHODS@#Serological methods was used to identify the ABO phenotypes. Exons 6 and 7 of the ABO gene and flanking regions were subjected to direct sequencing and TA clonal sequencing in order to determine the genotype of individuals with inconsistent results for forward and reverse serological typing.@*RESULTS@#Among 12 individuals from 4 generations, 5 were identified with a AwB phenotype, along with a c.803C>G mutation in exon 7 of the B allele, which was named as B(A).06. The B(A).06/O.01.01 phenotype may be easily missed due to its weak anti-A antibody in the serum upon initial serological test.@*CONCLUSION@#A B(A).06 subtype family was identified. The serological phenotype of individuals carrying the B(A).06 allele may be affected by the opposite DNA strand.


Assuntos
Sistema ABO de Grupos Sanguíneos , Genética , Alelos , Genótipo , Humanos , Linhagem , Fenótipo , Mutação Puntual
6.
Artigo em Inglês | WPRIM (Pacífico Ocidental) | ID: wprim-761887

RESUMO

Androgen receptor (AR) is a steroid receptor transcriptional factor for testosterone and dihydrotestosterone consisting of four main domains, the N-terminal domain, DNA-binding domain, hinge region, and ligand-binding domain. AR plays pivotal roles in prostate cancer, especially castration-resistant prostate cancer (CRPC). Androgen deprivation therapy can suppress hormone-naïve prostate cancer, but prostate cancer changes AR and adapts to survive under castration levels of androgen. These mechanisms include AR point mutations, AR overexpression, changes of androgen biosynthesis, constitutively active AR splice variants without ligand binding, and changes of androgen cofactors. Studies of AR in CRPC revealed that AR was still active in CRPC, and it remains as a potential target to treat CRPC. Enzalutamide is a second-generation antiandrogen effective in patients with CRPC before and after taxane-based chemotherapy. However, CRPC is still incurable and can develop drug resistance. Understanding the mechanisms of this resistance can enable new-generation therapies for CRPC. Several promising new AR-targeted therapies have been developed. Apalutamide is a new Food and Drug Administration-approved androgen agonist binding to the ligand-binding domain, and clinical trials of other new AR-targeted agents binding to the ligand-binding domain or N-terminal domain are underway. This review focuses on the functions of AR in prostate cancer and the development of CRPC and promising new agents against CRPC.


Assuntos
Antagonistas de Androgênios , Castração , Di-Hidrotestosterona , Resistência a Medicamentos , Tratamento Farmacológico , Humanos , Mutação Puntual , Próstata , Neoplasias da Próstata , Receptores Androgênicos , Receptores de Esteroides , Testosterona
7.
Artigo em Coreano | WPRIM (Pacífico Ocidental) | ID: wprim-764093

RESUMO

Thyroid nodules are the most common endocrine tumor. Ultrasonography and fine-needle aspiration (FNA) are currently accurate diagnostic tools for evaluating thyroid nodules. However, 10–30% of FNA specimens are cytologically indeterminate. Making an accurate diagnosis between benign and malignant nodules is important so that patients with malignant nodule receive proper treatment and patients with benign nodule can avoid unnecessary treatment. Several genetic changes such as point mutations of the BRAF or RAS and rearrangements of the RET/PTC1, RET/PTC3, PAX8/PPARY are used to adjust to indeterminate FNA. Such a mutational analysis has an excellent positive predictive value (PPV), but there is a weakness in the low negative predictive value (NPV). Gene-expression classifier (GEC) has been found helpful in identify nodules that are benign rather than malignant. GEC has an excellent NPV, but there is a weakness of low PPV. Multiplatform mutational and miRNA test (MPT) and next-generation sequencing assay (NGS) are being studied to compensate for these weaknesses. Molecular tests appear to be a good solution for improving the accuracy of indeterminate FNA cytology specimens and support the clinical management decisions in patients with indeterminate cytologic nodules, but further prospective multicenter trials are required for validation of reported findings and need evaluation of cost-effectiveness. This paper will review recently available molecular diagnostic tools of thyroid nodule.


Assuntos
Biópsia por Agulha Fina , Diagnóstico , Humanos , MicroRNAs , Estudos Multicêntricos como Assunto , Patologia Molecular , Mutação Puntual , Estudos Prospectivos , Glândula Tireoide , Neoplasias da Glândula Tireoide , Nódulo da Glândula Tireoide , Ultrassonografia
8.
Artigo em Inglês | WPRIM (Pacífico Ocidental) | ID: wprim-760199

RESUMO

Alport syndrome (ATS) is an inherited glomerular disease caused by mutations in one of the type IV collagen novel chains (α3, α4, and α5). ATS is characterized by persistent microscopic hematuria that starts during infancy, eventually leading to either progressive nephritis or end-stage renal disease. There are 3 known genetic forms of ATS, namely X-linked ATS, autosomal recessive ATS, and autosomal dominant ATS. About 80% of patients with ATS have X-linked ATS, which is caused by mutations in the type IV collagen α5 chain gene, COL4A5. Although an 80% mutation detection rate is observed in men with X-linked ATS, some difficulties do exist in the genetic diagnosis of ATS. Most mutations are point mutations without hotspots in the COL4A3, COL4A4, and COL4A5 genes. Further, there are insufficient data on the detection of COL4A3 and COL4A4 mutations for their comparison between patients with autosomal recessive or dominant ATS. Therefore, diagnosis of ATS in female patients with no apparent family history can be challenging. Therefore, in this study, we used whole-exome sequencing (WES) to identify mutations in type IV collagen in 2 girls with glomerular basement membrane structural changes suspected to be associated with ATS; these patients had no relevant family history. Our results revealed de novo c.4688G>A (p.Arg1563Gln) and c.2714G>A (p.Gly905Asp) mutations in COL4A5. Therefore, we suggest that WES is an effective approach to obtain genetic information in ATS, particularly in female patients without a relevant family history, to detect unexpected DNA variations.


Assuntos
Criança , Colágeno Tipo IV , Diagnóstico , DNA , Exoma , Feminino , Membrana Basal Glomerular , Hematúria , Humanos , Falência Renal Crônica , Coreia (Geográfico) , Masculino , Nefrite , Nefrite Hereditária , Mutação Puntual
9.
Artigo em Chinês | WPRIM (Pacífico Ocidental) | ID: wprim-775836

RESUMO

OBJECTIVE@#To explore the characteristics of PAH gene variants among 113 phenylketonuria patients from Henan Province.@*METHODS@#The 13 exons of the PAH gene were subjected to PCR amplification and direct sequencing. Large fragment deletion and duplication of the PAH gene were detected with a multiple ligation-dependent probe amplification (MLPA) assay.@*RESULTS@#In total 195 point variants and 3 large fragment deletions were detected among the 226 alleles, with the detection rates being 86.28% and 1.33%, respectively. Variants of p.Arg243Gln (18.14%), p.Arg111X (6.19%), p.Arg53His (5.31%), EX6-96A>G (5.31%), p.Tyr356X (4.87%) and p.Val399Val (4.42%) were relatively common. Most of the variants were located in exons 7, 11, 3 and 6. Missense variations were most common. Four novel variations were detected, which included c.1016C>A (p.Ser339Tyr), c.1000T>C (p.Cys334Arg), c.1110G>T (p.Glu370Asp), and IVS6+1G>T.@*CONCLUSION@#The PAH gene variations in Henan Province have featured extensive allelic heterogeneity and variety.


Assuntos
China , Éxons , Humanos , Mutação de Sentido Incorreto , Fenilalanina Hidroxilase , Genética , Fenilcetonúrias , Genética , Mutação Puntual , Deleção de Sequência
10.
Gut and Liver ; : 641-647, 2018.
Artigo em Inglês | WPRIM (Pacífico Ocidental) | ID: wprim-718123

RESUMO

BACKGROUND/AIMS: Helicobacter pylori eradication rates are decreasing because of increases in clarithromycin resistance. Thus, finding an easy and accurate method of detecting clarithromycin resistance is important. METHODS: We evaluated 70 H. pylori isolates from Korean patients. Dual-labeled peptide nucleic acid (PNA) probes were designed to detect resistance associated with point mutations in 23S ribosomal ribonucleic acid gene domain V (A2142G, A2143G, and T2182C). Data were analyzed by probe-based fluorescence melting curve analysis based on probe-target dissociation temperatures and compared with Sanger sequencing. RESULTS: Among 70 H. pylori isolates, 0, 16, and 58 isolates contained A2142G, A2143G, and T2182C mutations, respectively. PNA probe-based analysis exhibited 100.0% positive predictive values for A2142G and A2143G and a 98.3% positive predictive value for T2182C. PNA probe-based analysis results correlated with 98.6% of Sanger sequencing results (κ-value=0.990; standard error, 0.010). CONCLUSIONS: H. pylori clarithromycin resistance can be easily and accurately assessed by dual-labeled PNA probe-based melting curve analysis if probes are used based on the appropriate resistance-related mutations. This method is fast, simple, accurate, and adaptable for clinical samples. It may help clinicians choose a precise eradication regimen.


Assuntos
Claritromicina , Fluorescência , Congelamento , Helicobacter pylori , Helicobacter , Humanos , Métodos , Ácidos Nucleicos Peptídicos , Mutação Puntual , RNA
11.
Cancer Research and Treatment ; : 1294-1303, 2018.
Artigo em Inglês | WPRIM (Pacífico Ocidental) | ID: wprim-717739

RESUMO

PURPOSE: The main objective of this study was to investigate the relationship among the clinical characteristics and the frequency of T790M mutation in advanced epidermal growth factor receptor (EGFR)–mutant lung adenocarcinoma patients with acquired resistance after firstline EGFR–tyrosine kinase inhibitor (TKI) treatment. MATERIALS AND METHODS: We enrolled EGFR-mutant stage IIIB-IV lung adenocarcinoma patients, who had progressed to prior EGFR-TKI therapy, and evaluated their rebiopsy EGFR mutation status. RESULTS: A total of 205 patients were enrolled for analysis. The overall T790M mutation rate of rebiopsy was 46.3%. The T790M mutation rates among patients with exon 19 deletion mutation, exon 21 L858R point mutation, and other mutations were 55.0%, 37.3%, and 27.3%, respectively. Baseline exon 19 deletion was associated with a significantly higher frequency of T790M mutation (adjusted odds ratio, 2.14; 95% confidence interval [CI], 1.20 to 3.83; p=0.010). In the exon 19 deletion subgroup, there was a greater prevalence of T790M mutation than other exon 19 deletion subtypes in patients with the Del E746-A750 mutation (61.6% vs. 40.6%; odds ratio, 2.35; 95% CI, 1.01 to 5.49; p=0.049). The progression-free survival (PFS) of first-line TKI treatment > 11 months was also associated with a higher T790M mutation rate (54.1% vs. 39.3%; adjusted odds ratio, 1.82; 95% CI, 1.02 to 3.25; p=0.044). Patients who underwent rebiopsy at metastatic sites had more chance to harbor T790M mutation (52.6% vs. 33.8%; adjusted odds ratio, 1.97; 95% CI, 1.06 to 3.67; p=0.032). CONCLUSION: PFS of first-line EGFR-TKI, rebiopsy site, EGFR exon 19 deletion and its subtype Del E746-A750 mutation are associated with the frequency of T790M mutation.


Assuntos
Adenocarcinoma , Intervalo Livre de Doença , Fator de Crescimento Epidérmico , Éxons , Humanos , Neoplasias Pulmonares , Pulmão , Taxa de Mutação , Razão de Chances , Fosfotransferases , Mutação Puntual , Prevalência , Receptores ErbB , Deleção de Sequência
12.
Cancer Research and Treatment ; : 1452-1457, 2018.
Artigo em Inglês | WPRIM (Pacífico Ocidental) | ID: wprim-717509

RESUMO

Microcystic stromal tumor (MCST) is a rare subtype of sex cord-stromal neoplasm. Tumors from all 31 previously reported cases were located in the ovary. Herein, we present a unique case of a right-side testicular tumor in a 33-year-old Chinese male. The tumor is composed of predominantly lobulated cellular nodules separated by hyalinized fibrous stroma and they expressed CD10, β-catenin (nuclear), and cyclin D1. Molecular analysis identified a point mutation (c.110C>G) in exon 3 of CTNNB1. The histopathological features, immunohistochemistry profiles, and molecular analysis of this tumor were consistent with MCST of the ovary. Therefore, a diagnosis of MCST of the right testicle was determined. To the best of our knowledge, this is the first case of MCST occurring in the testicles. The study may provide new insights to the tumor biology of MCST and a better understanding of this rare entity.


Assuntos
Adulto , Grupo com Ancestrais do Continente Asiático , Biologia , Ciclina D1 , Diagnóstico , Éxons , Feminino , Humanos , Hialina , Imuno-Histoquímica , Masculino , Ovário , Mutação Puntual , Testículo
14.
Artigo em Inglês | WPRIM (Pacífico Ocidental) | ID: wprim-715205

RESUMO

Point mutations in the human cardiac homeobox gene NKX2.5 are associated with familial atrial septal defect (ASD), atrioventricular (AV) conduction disturbance, as well as sudden cardiac death. To date, more than 60 NKX2.5 mutations have been documented, but there are no reports in Korea. We are reporting the first Korean family with ASD and AV block associated with a novel mutation in the NKX2.5 coding region. A 9-year-old boy presented with a slow and irregular pulse, and was diagnosed with secundum ASD and first degree AV block. The boy's father, who had a history of ASD correction surgery, presented with second degree AV block and atrial fibrillation. The boy's brother was also found to have secundum ASD and first degree AV block. There were two sudden deaths in the family. Genetic testing revealed a novel mutation of NKX2.5 in all affected members of the family.


Assuntos
Fibrilação Atrial , Bloqueio Atrioventricular , Criança , Codificação Clínica , Morte Súbita , Morte Súbita Cardíaca , Pai , Genes Homeobox , Testes Genéticos , Comunicação Interatrial , Humanos , Coreia (Geográfico) , Masculino , Mutação Puntual , Irmãos
15.
Infection and Chemotherapy ; : 357-361, 2018.
Artigo em Inglês | WPRIM (Pacífico Ocidental) | ID: wprim-722309

RESUMO

While carbapenems are the drug of choice to treat extended-spectrum-β-lactamase (ESBL)-producing strains, some alternative carbapenem-sparing regimens are suggested for antibiotic stewardship. We experienced a case of ciprofloxacin treatment failure for acute pyelonephritis caused by an apparently susceptible Escherichia coli. A 71-year-old woman presented the emergency department with fever for 7 days and bilateral flank pain for 2 days. The laboratory results and abdominopelvic computed tomography finding were compatible with acute pyelonephritis. During 3-day ciprofloxacin therapy, the patient remained febrile with persistent bacteremia. After the change in antibiotics to ertapenem, the patient’s clinical course started to improve. ESBL-producing E. coli isolates were identified in all three consecutive blood samples. Pulsed-field gel electrophoresis (PFGE) patterns, serotypes, and sequence types showed the three isolates were derived from the identical strain. The isolates produced CTX-M-14 type ESBL belonging to the ST69 clonal group. Despite in vitro susceptibility, the failure was attributed to a gyrA point mutation encoding Ser83Leu within quinolone resistance-determining regions. This case suggests that ciprofloxacin should be used cautiously in the treatment of serious infections caused by ciprofloxacin-susceptible, ESBL-producing E. coli, even in acute pyelonephritis because in-vitro susceptibility tests could fail to detect certain genetic mutations.


Assuntos
Idoso , Antibacterianos , Bacteriemia , Carbapenêmicos , Ciprofloxacino , Eletroforese em Gel de Campo Pulsado , Serviço Hospitalar de Emergência , Escherichia coli , Escherichia , Feminino , Febre , Dor no Flanco , Humanos , Técnicas In Vitro , Mutação Puntual , Pielonefrite , Sorogrupo , Falha de Tratamento
16.
Infection and Chemotherapy ; : 357-361, 2018.
Artigo em Inglês | WPRIM (Pacífico Ocidental) | ID: wprim-721804

RESUMO

While carbapenems are the drug of choice to treat extended-spectrum-β-lactamase (ESBL)-producing strains, some alternative carbapenem-sparing regimens are suggested for antibiotic stewardship. We experienced a case of ciprofloxacin treatment failure for acute pyelonephritis caused by an apparently susceptible Escherichia coli. A 71-year-old woman presented the emergency department with fever for 7 days and bilateral flank pain for 2 days. The laboratory results and abdominopelvic computed tomography finding were compatible with acute pyelonephritis. During 3-day ciprofloxacin therapy, the patient remained febrile with persistent bacteremia. After the change in antibiotics to ertapenem, the patient’s clinical course started to improve. ESBL-producing E. coli isolates were identified in all three consecutive blood samples. Pulsed-field gel electrophoresis (PFGE) patterns, serotypes, and sequence types showed the three isolates were derived from the identical strain. The isolates produced CTX-M-14 type ESBL belonging to the ST69 clonal group. Despite in vitro susceptibility, the failure was attributed to a gyrA point mutation encoding Ser83Leu within quinolone resistance-determining regions. This case suggests that ciprofloxacin should be used cautiously in the treatment of serious infections caused by ciprofloxacin-susceptible, ESBL-producing E. coli, even in acute pyelonephritis because in-vitro susceptibility tests could fail to detect certain genetic mutations.


Assuntos
Idoso , Antibacterianos , Bacteriemia , Carbapenêmicos , Ciprofloxacino , Eletroforese em Gel de Campo Pulsado , Serviço Hospitalar de Emergência , Escherichia coli , Escherichia , Feminino , Febre , Dor no Flanco , Humanos , Técnicas In Vitro , Mutação Puntual , Pielonefrite , Sorogrupo , Falha de Tratamento
17.
Artigo em Chinês | WPRIM (Pacífico Ocidental) | ID: wprim-689603

RESUMO

Three boys aged 7-13 months visited the hospital due to unusual facies (prominent forehead, hypertelorism, or long mandible), motor developmental delay, and mental retardation. As for body length and head circumference, only one patient had a head circumference of >2 SD. Two patients had an advanced bone age, one had electroencephalographic abnormalities, and 3 had enlarged ventricles on head CT. The whole-genome microarray analysis showed the deletion of a copy with a size of 1.75 Mb in the chromosomal region 5q35.2 in one patient, which contained the NSD1 gene. Quantitative real-time PCR was performed for the validation of the region with copy number variation, and the results showed that the copy number of the NSD1 gene in this patient was reduced by half. High-throughput sequencing identified two heterozygous mutations, c.1157T>G and c.1177G>T, in the NSD1 gene in two patients. c.1157T>G mutations had not been reported before, but the bioinformatics analysis showed that this mutation had pathogenicity. All three boys were diagnosed with Sotos syndrome. Sotos syndrome is a congenital overgrowth syndrome with autosomal dominant inheritance; 70%-90% of patients have NSD1 gene mutations, and about 10% of patients have depletion in the 5q35 region (containing the NSD1 gene).


Assuntos
Sequência de Aminoácidos , Sequência de Bases , Variações do Número de Cópias de DNA , Humanos , Lactente , Peptídeos e Proteínas de Sinalização Intracelular , Genética , Masculino , Proteínas Nucleares , Genética , Fenótipo , Mutação Puntual , Deleção de Sequência , Síndrome de Sotos , Genética
18.
Artigo em Inglês | WPRIM (Pacífico Ocidental) | ID: wprim-727952

RESUMO

Familial Parkinson's disease (PD) has been linked to point mutations and duplication of the α-synuclein (α-syn) gene. Mutant α-syn expression increases the vulnerability of neurons to exogenous insults. In this study, we developed a new PD model in the transgenic mice expressing mutant hemizygous (hemi) or homozygous (homo) A53T α-synuclein (α-syn Tg) and their wildtype (WT) littermates by treatment with sub-toxic (10 mg/kg, i.p., daily for 5 days) or toxic (30 mg/kg, i.p., daily for 5 days) dose of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Tyrosine hydroxylase and Bcl-2 levels were reduced in the α-syn Tg but not WT mice by sub-toxic MPTP injection. In the adhesive removal test, time to remove paper was significantly increased only in the homo α-syn Tg mice. In the challenging beam test, the hemi and homo α-syn Tg mice spent significantly longer time to traverse as compared to that of WT group. In order to find out responsible proteins related with vulnerability of mutant α-syn expressed neurons, DJ-1 and ubiquitin enzyme expressions were examined. In the SN, DJ-1 and ubiquitin conjugating enzyme, UBE2N, levels were significantly decreased in the α-syn Tg mice. Moreover, A53T α-syn overexpression decreased DJ-1 expression in SH-SY5Y cells. These findings suggest that the vulnerability to oxidative injury such as MPTP of A53T α-syn mice can be explained by downregulation of DJ-1.


Assuntos
1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina , Adesivos , Animais , Apoptose , Dopamina , Neurônios Dopaminérgicos , Regulação para Baixo , Hominidae , Humanos , Camundongos , Camundongos Transgênicos , Neurônios , Doença de Parkinson , Mutação Puntual , Sinucleínas , Tirosina 3-Mono-Oxigenase , Ubiquitina
19.
Protein & Cell ; (12): 601-611, 2017.
Artigo em Inglês | WPRIM (Pacífico Ocidental) | ID: wprim-756965

RESUMO

Targeted point mutagenesis through homologous recombination has been widely used in genetic studies and holds considerable promise for repairing disease-causing mutations in patients. However, problems such as mosaicism and low mutagenesis efficiency continue to pose challenges to clinical application of such approaches. Recently, a base editor (BE) system built on cytidine (C) deaminase and CRISPR/Cas9 technology was developed as an alternative method for targeted point mutagenesis in plant, yeast, and human cells. Base editors convert C in the deamination window to thymidine (T) efficiently, however, it remains unclear whether targeted base editing in mouse embryos is feasible. In this report, we generated a modified high-fidelity version of base editor 2 (HF2-BE2), and investigated its base editing efficacy in mouse embryos. We found that HF2-BE2 could convert C to T efficiently, with up to 100% biallelic mutation efficiency in mouse embryos. Unlike BE3, HF2-BE2 could convert C to T on both the target and non-target strand, expanding the editing scope of base editors. Surprisingly, we found HF2-BE2 could also deaminate C that was proximal to the gRNA-binding region. Taken together, our work demonstrates the feasibility of generating point mutations in mouse by base editing, and underscores the need to carefully optimize base editing systems in order to eliminate proximal-site deamination.


Assuntos
Desaminase APOBEC-1 , Genética , Metabolismo , Animais , Proteínas de Bactérias , Genética , Metabolismo , Sequência de Bases , Proteína 9 Associada à CRISPR , Sistemas CRISPR-Cas , Citidina , Genética , Metabolismo , Transferência Embrionária , Embrião de Mamíferos , Endonucleases , Genética , Metabolismo , Edição de Genes , Métodos , Células HEK293 , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Microinjeções , Plasmídeos , Química , Metabolismo , Mutação Puntual , RNA Guia , Genética , Metabolismo , Timidina , Genética , Metabolismo , Zigoto , Metabolismo , Transplante
20.
Protein & Cell ; (12): 811-822, 2017.
Artigo em Inglês | WPRIM (Pacífico Ocidental) | ID: wprim-756922

RESUMO

β-Thalassemia is a global health issue, caused by mutations in the HBB gene. Among these mutations, HBB -28 (A>G) mutations is one of the three most common mutations in China and Southeast Asia patients with β-thalassemia. Correcting this mutation in human embryos may prevent the disease being passed onto future generations and cure anemia. Here we report the first study using base editor (BE) system to correct disease mutant in human embryos. Firstly, we produced a 293T cell line with an exogenous HBB -28 (A>G) mutant fragment for gRNAs and targeting efficiency evaluation. Then we collected primary skin fibroblast cells from a β-thalassemia patient with HBB -28 (A>G) homozygous mutation. Data showed that base editor could precisely correct HBB -28 (A>G) mutation in the patient's primary cells. To model homozygous mutation disease embryos, we constructed nuclear transfer embryos by fusing the lymphocyte or skin fibroblast cells with enucleated in vitro matured (IVM) oocytes. Notably, the gene correction efficiency was over 23.0% in these embryos by base editor. Although these embryos were still mosaic, the percentage of repaired blastomeres was over 20.0%. In addition, we found that base editor variants, with narrowed deamination window, could promote G-to-A conversion at HBB -28 site precisely in human embryos. Collectively, this study demonstrated the feasibility of curing genetic disease in human somatic cells and embryos by base editor system.


Assuntos
Desaminase APOBEC-1 , Genética , Metabolismo , Sequência de Bases , Blastômeros , Biologia Celular , Metabolismo , Sistemas CRISPR-Cas , Embrião de Mamíferos , Metabolismo , Patologia , Feminino , Fibroblastos , Metabolismo , Patologia , Edição de Genes , Métodos , Expressão Gênica , Células HEK293 , Heterozigoto , Homozigoto , Humanos , Mutação Puntual , Cultura Primária de Células , Regiões Promotoras Genéticas , Análise de Sequência de DNA , Globinas beta , Genética , Metabolismo , Talassemia beta , Genética , Metabolismo , Patologia , Terapêutica
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