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Introduction: X-linked Alport syndrome (XLAS) is caused by pathogenic variants in COL4A5 which lead to abnormalities of the glomerular basement membrane (GBM) structural and is characterized by progressive kidney disease, hearing loss, and ocular abnormalities. The aim of this study was to identify gene mutations in a Chinese family with XLAS by whole-exome sequencing (WES) and verified the pathogenicity of the mutation in vitro experiments. Case Presentation: A five-generation pedigree with a total of 49 family members originating from Hainan province of China was investigated in this study. The proband was a 23-year-old male who developed microscopic hematuria, proteinuria and end-stage kidney disease (ESKD) at age 17. WES identified a novel splicing mutation c.321+5G>A of COL4A5, which cause exon skip. Further co-segregation analysis confirmed that this mutation exists in relatives who had renal abnormalities using Sanger sequencing. According to American College of Medical Genetics and Genomics guidelines (ACMG), the mutation was determined to be of uncertain significance (VUS). In vitro splicing experiments have shown that the COL4A5 variant induces aberrant mRNA splicing and transcript deletion. Conclusion: We identified a novel intronic COL4A5 pathogenic mutation (c.321+5G>A) in a Chinese XLAS family and described the phenotypes of affected relatives. This study expands the mutation spectrum of COL4A5 gene in XLAS and demonstrates the importance of gene screening for AS.
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BACKGROUND: Krüppel-like factor 1 (KLF1), a crucial erythroid transcription factor, plays a significant role in various erythroid changes and haemolytic diseases. The rare erythrocyte Lutheran inhibitor (In(Lu)) blood group phenotype serves as an effective model for identifying KLF1 hypomorphic and loss-of-function variants. In this study, we aimed to analyse the genetic background of the In(Lu) phenotype in a population-based sample group by high-throughput technologies to find potentially clinically significant KLF1 variants. RESULTS: We included 62 samples with In(Lu) phenotype, screened from over 300,000 Chinese blood donors. Among them, 36 samples were sequenced using targeted Next Generation Sequencing (NGS), whereas 19 samples were sequenced using High Fidelity (HiFi) technology. In addition, seven samples were simply sequenced using Sanger sequencing. A total of 29 hypomorphic or loss-of-function variants of KLF1 were identified, 21 of which were newly discovered. All new variants discovered by targeted NGS or HiFi sequencing were validated through Sanger sequencing, and the obtained results were found to be consistent. The KLF1 haplotypes of all new variants were further confirmed using clone sequencing or HiFi sequencing. The lack of functional KLF1 variants detected in the four samples indicates the presence of additional regulatory mechanisms. In addition, some samples exhibited BCAM polymorphisms, which encodes antigens of the Lutheran (LU) blood group system. However, no BCAM mutations which leads to the absence of LU proteins were detected. CONCLUSIONS: High-throughput sequencing methods, particularly HiFi sequencing, were introduced for the first time into genetic analysis of the In(Lu) phenotype. Targeted NGS and HiFi sequencing demonstrated the accuracy of the results, providing additional advantages such as simultaneous analysis of other blood group genes and clarification of haplotypes. Using the In(Lu) phenotype, a powerful model for identifying hypomorphic or loss-of-function KLF1 variants, numerous novel variants have been detected, which have contributed to the comprehensive understanding of KLF1. These clinically significant KLF1 mutations can serve as a valuable reference for the diagnosis of related blood cell diseases.
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Antígenos de Grupos Sanguíneos , Fatores de Transcrição Kruppel-Like , Antígenos de Grupos Sanguíneos/genética , Sequenciamento de Nucleotídeos em Larga Escala , Sistema do Grupo Sanguíneo Lutheran/genética , Mutação , HumanosRESUMO
BACKGROUND: Preimplantation genetic testing (PGT), also referred to as preimplantation genetic diagnosis (PGD), is an advanced reproductive technology used during in vitro fertilization (IVF) cycles to identify genetic abnormalities in embryos prior to their implantation. PGT is used to screen embryos for chromosomal abnormalities, monogenic disorders, and structural rearrangements. DEVELOPMENT OF PGT: Over the past few decades, PGT has undergone tremendous development, resulting in three primary forms: PGT-A, PGT-M, and PGT-SR. PGT-A is utilized for screening embryos for aneuploidies, PGT-M is used to detect disorders caused by a single gene, and PGT-SR is used to detect chromosomal abnormalities caused by structural rearrangements in the genome. PURPOSE OF REVIEW: In this review, we thoroughly summarized and reviewed PGT and discussed its pros and cons down to the minutest aspects. Additionally, recent studies that highlight the advancements of PGT in the current era, including their future perspectives, were reviewed. CONCLUSIONS: This comprehensive review aims to provide new insights into the understanding of techniques used in PGT, thereby contributing to the field of reproductive genetics.
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Testes Genéticos , Diagnóstico Pré-Implantação , Gravidez , Feminino , Humanos , Testes Genéticos/métodos , Diagnóstico Pré-Implantação/métodos , Implantação do Embrião , Fertilização in vitro , AneuploidiaRESUMO
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes multi-organ damage, which includes hepatic dysfunction, as observed in over 50% of COVID-19 patients. Angiotensin I converting enzyme (peptidyl-dipeptidase A) 2 (ACE2) is the primary receptor for SARS-CoV-2 entry into host cells, and studies have shown the presence of intracellular virus particles in human hepatocytes that express ACE2, but at extremely low levels. Consequently, we asked if hepatocytes might express receptors other than ACE2 capable of promoting the entry of SARS-CoV-2 into cells. To address this question, we performed a genome-wide CRISPR-Cas9 activation library screening and found that Asialoglycoprotein receptor 1 (ASGR1) promoted SARS-CoV-2 pseudovirus infection of HeLa cells. In Huh-7 cells, simultaneous knockout of ACE2 and ASGR1 prevented SARS-CoV-2 pseudovirus infection. In the immortalized THLE-2 hepatocyte cell line and primary hepatic parenchymal cells, both of which barely expressed ACE2, SARS-CoV-2 pseudovirus could successfully establish an infection. However, after treatment with ASGR1 antibody or siRNA targeting ASGR1, the infection rate significantly dropped, suggesting that SARS-CoV-2 pseudovirus infects hepatic parenchymal cells mainly through an ASGR1-dependent mechanism. We confirmed that ASGR1 could interact with Spike protein, which depends on receptor binding domain (RBD) and N-terminal domain (NTD). Finally, we also used Immunohistochemistry and electron microscopy to verify that SARS-CoV-2 could infect primary hepatic parenchymal cells. After inhibiting ASGR1 in primary hepatic parenchymal cells by siRNA, the infection efficiency of the live virus decreased significantly. Collectively, these findings indicate that ASGR1 is a candidate receptor for SARS-CoV-2 that promotes infection of hepatic parenchymal cells.
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COVID-19 , Humanos , COVID-19/genética , SARS-CoV-2/fisiologia , Receptor de Asialoglicoproteína/genética , Células HeLa , Enzima de Conversão de Angiotensina 2/genética , Enzima de Conversão de Angiotensina 2/química , Hepatócitos , RNA Interferente PequenoRESUMO
Several molecular biology methods are available for high-throughput blood typing. In this study, we aimed to build a high-throughput blood-group genetic screening system for high-frequency blood-group antigen-negative rare-blood groups in donors and patients. The amplification primers for all blood-type gene fragments involving the selected alleles were designed for detection. Single-base extend primers were also designed based on specific loci. DNA fragments were detected by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MS) for the last nucleotide identification of amplification products in the extend step. The accuracy was verified by known samples. Thirty-six random samples were detected by serological tests and sequencing to verify the system stability. After verification, according to the collected known rare-blood-type samples, all the alleles designed to be detected matched with the validated single-nucleotide polymorphisms. The verification tests showed that all genotyping results of the random samples were in accordance with the findings of serotyping and sequencing. Then, 1258 random donor samples were screened by the built typing system after the verification. Three Fy(a-) and four s- were screened out in 1258 random blood samples. The multiple polymerase chain reaction-based MS detection system can be used in rare-blood-type screening with good accuracy and stability.
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Antígenos de Grupos Sanguíneos , Humanos , Antígenos de Grupos Sanguíneos/genética , Genótipo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Alelos , Polimorfismo de Nucleotídeo Único , Primers do DNA/genéticaRESUMO
In this work, a promising treatment strategy for triggering robust antitumor immune responses in transarterial chemoembolization of hepatocellular carcinoma (HCC) is presented. The zeolitic imidazolate framework nanoparticles loaded with hypoxia-activated prodrug tirapazamine and immune adjuvant resiquimod facilitated in situ generation of nanovaccine via a facile approach. The nanovaccine can strengthen the ability of killing the liver cancer cells under hypoxic environment, while was capable of improving immunogenic tumor microenvironment and triggering strong antitumor immune responses by increasing the primary and distant intratumoral infiltration of immune cells such as cytotoxic T cells. Moreover, a porous microcarrier, approved by FDA as pharmaceutical excipient, was designed to achieve safe and effective delivery of the nanovaccine via transarterial therapy in rabbit orthotopic VX2 liver cancer model. The microcarrier exhibited the characteristics of excellent drug loading and occlusion of peripheral artery. The collaborative delivery of the microcarrier and nanovaccine demonstrated an exciting inhibitory effect on solid tumors and tumor metastases, which provided a great potential as novel combination therapy for HCC interventional therapy.
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Carcinoma Hepatocelular , Quimioembolização Terapêutica , Neoplasias Hepáticas , Animais , Coelhos , Carcinoma Hepatocelular/tratamento farmacológico , Neoplasias Hepáticas/patologia , Nanovacinas , Hipóxia/tratamento farmacológico , Microambiente TumoralRESUMO
Explicitly sharing individual level data in genomics studies has many merits comparing to sharing summary statistics, including more strict QCs, common statistical analyses, relative identification and improved statistical power in GWAS, but it is hampered by privacy or ethical constraints. In this study, we developed encG-reg, a regression approach that can detect relatives of various degrees based on encrypted genomic data, which is immune of ethical constraints. The encryption properties of encG-reg are based on the random matrix theory by masking the original genotypic matrix without sacrificing precision of individual-level genotype data. We established a connection between the dimension of a random matrix, which masked genotype matrices, and the required precision of a study for encrypted genotype data. encG-reg has false positive and false negative rates equivalent to sharing original individual level data, and is computationally efficient when searching relatives. We split the UK Biobank into their respective centers, and then encrypted the genotype data. We observed that the relatives estimated using encG-reg was equivalently accurate with the estimation by KING, which is a widely used software but requires original genotype data. In a more complex application, we launched a finely devised multi-center collaboration across 5 research institutes in China, covering 9 cohorts of 54,092 GWAS samples. encG-reg again identified true relatives existing across the cohorts with even different ethnic backgrounds and genotypic qualities. Our study clearly demonstrates that encrypted genomic data can be used for data sharing without loss of information or data sharing barrier.
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Estudo de Associação Genômica Ampla , Privacidade , Humanos , Estudo de Associação Genômica Ampla/métodos , Genótipo , Software , GenômicaRESUMO
Hereditary spherocytosis (HS), the most common inherited hemolytic anemia disorder, is characterized by osmotically fragile microspherocytic red cells with a reduced surface area on the peripheral blood smear. Pathogenic variants in five erythrocyte membrane structure-related genes ANK1 (Spherocytosis, type 1; MIM#182900), SPTB (Spherocytosis, type 2; MIM#616649), SPTA1 (Spherocytosis, type 3; MIM#270970), SLC4A1 (Spherocytosis, type 4; MIM#612653) and EPB42 (Spherocytosis, type 5; MIM#612690) have been confirmed to be related to HS. There have been many studies on the pathogenic variants and mechanisms of HS, however, studies on how to manage the transmission of HS to the next-generation have not been reported. In this study, we recruited a patient with HS. Targeted next-generation sequencing with a panel of 208 genes related to blood system diseases detected a novel heterozygous variant in the SPTB: c.300+2dup in the proband. Sanger sequencing of variant alleles and haplotype linkage analysis of single nucleotide polymorphism (SNP) based on next-generation sequencing were performed simultaneously. Five embryos were identified with one heterozygous and four not carrying the SPTB variant. Single-cell amplification and whole genome sequencing showed that three embryos had varying degrees of trisomy mosaicism. One of two normal embryos was transferred to the proband. Ultimately, a healthy boy was born, confirmed by noninvasive prenatal testing for monogenic conditions (NIPT-M) to be disease-free. This confirmed our successful application of PGT in preventing transmission of the pathogenic variant allele in the HS family.
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Introduction: The overdiagnosing of papillary thyroid carcinoma (PTC) in China necessitates the development of an evidence-based diagnosis and prognosis strategy in line with precision medicine. A landscape of PTC in Chinese cohorts is needed to provide comprehensiveness. Methods: 6 paired PTC samples were employed for whole-exome sequencing, RNA sequencing, and data-dependent acquisition mass spectrum analysis. Weighted gene co-expression network analysis and protein-protein interactions networks were used to screen for hub genes. Moreover, we verified the hub genes' diagnostic and prognostic potential using online databases. Logistic regression was employed to construct a diagnostic model, and we evaluated its efficacy and specificity based on TCGA-THCA and GEO datasets. Results: The basic multiomics landscape of PTC among local patients were drawn. The similarities and differences were compared between the Chinese cohort and TCGA-THCA cohorts, including the identification of PNPLA5 as a driver gene in addition to BRAF mutation. Besides, we found 572 differentially expressed genes and 79 differentially expressed proteins. Through integrative analysis, we identified 17 hub genes for prognosis and diagnosis of PTC. Four of these genes, ABR, AHNAK2, GPX1, and TPO, were used to construct a diagnostic model with high accuracy, explicitly targeting PTC (AUC=0.969/0.959 in training/test sets). Discussion: Multiomics analysis of the Chinese cohort demonstrated significant distinctions compared to TCGA-THCA cohorts, highlighting the unique genetic characteristics of Chinese individuals with PTC. The novel biomarkers, holding potential for diagnosis and prognosis of PTC, were identified. Furthermore, these biomarkers provide a valuable tool for precise medicine, especially for immunotherapeutic or nanomedicine based cancer therapy.
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Reactivation of fetal hemoglobin (HbF) is a critical goal for the treatment of patients with hemoglobinopathies. ß-globin disorders can trigger stress erythropoiesis in red blood cells (RBCs). Cell-intrinsic erythroid stress signals promote erythroid precursors to express high levels of fetal hemoglobin, which is also known as γ-globin. However, the molecular mechanism underlying γ-globin production during cell-intrinsic erythroid stress remains to be elucidated. Here, we utilized CRISPR-Cas9 to model a stressed state caused by reduced levels of adult ß-globin in HUDEP2 human erythroid progenitor cells. We found that a decrease in ß-globin expression correlates with the upregulation of γ-globin expression. We also identified transcription factor high-mobility group A1 (HMGA1; formerly HMG-I/Y) as a potential γ-globin regulator that responds to reduced ß-globin levels. Upon erythroid stress, there is a downregulation of HMGA1, which normally binds -626 to -610 base pairs upstream from the STAT3 promoter, to downregulate STAT3 expression. STAT3 is a known γ-globin repressor, so the downregulation of HMGA1 ultimately upregulates γ-globin expression. SIGNIFICANCE: This study demonstrated HMGA1 as a potential regulator in the poorly understood phenomenon of stress-induced globin compensation, and after further validation these results might inform new strategies to treat patients with sickle cell disease and ß-thalassemia.
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Globinas beta , gama-Globinas , Adulto , Humanos , Hemoglobina Fetal/metabolismo , gama-Globinas/genética , Proteína HMGA1a , Proteômica , Fatores de TranscriçãoRESUMO
The membrane frizzled-related protein (Mfrp) and C1-tumor necrosis factor related protein 5 (Ctrp5) genes are transcribed as a bicistronic unit and dysregulation of either gene is associated with retinal degeneration in the retinal pigment epithelium (RPE) cells. However, the mechanisms that regulate the expression of the bicistronic transcript remain controversial. Here, we identified a microRNA-based negative feedback loop that helps maintain a normal expression level of the bicistronic Mfrp and Ctrp5 transcript. Specifically, miR-149-3p, a conserved microRNA, binds to the 3'UTR of the Mfrp gene. In MFRP-deficient rd6 mice, the miR-149-3p levels were compromised compared with those in WT mice, resulting in an increase in the bicistronic transcript. We also report a capsid-modified rAAVDJ-3M vector that is capable of robustly and specifically transducing RPE cells following subretinal delivery. Compared with the parental vector, the modified vector elicited similar levels of serum anti-rAAV antibodies, but recruited fewer microglial infiltrations. Most significantly, we also demonstrate that simultaneous overexpressing of MFRP and knockdown of the bicistronic transcript was more effective in rescuing vision than MFRP overexpression alone. Our findings offer new insights into the function of MFRP and provide a promising therapeutic strategy for the treatment of MFRP-associated ocular diseases.
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Drosophila is a crucial biological experimental teaching material extensively utilized in experimental teaching. In this experimental teaching, each student typically needs to manually identify hundreds of fruit flies and record multiple of each fly. This task involves substantial workload, and the classification standards can be inconsistent. To address this issue, we introduce a deep convolutional neural network that classifies the traits of every fruit fly, using a two-stage consisting of an object detector and a trait classifier. We propose a keypoint-assisted classification model with tailored training session for the trait classification task and significantly enhanced the model interpretability. Additionally, we've enhanced the RandAugment method to better fit the features of our task. The model is trained with progressive learning and adaptive regularization under limited computational resources. The final classification model, which utilizes MobileNetV3 as backbone, achieves an accuracy of 97.5%, 97.5% and 98% for the eyes, wings, gender tasks, respectively. After optimization, the model is highly lightweight, classifying 600 fruit fly traits from raw images in 10 seconds and having a size less than 5 MB. It can be easily deployed on any android device. The development of this system is conducive to promoting the experimental teaching, such as verifying genetic laws with Drosophila as the research object. It can also be used for scientific research involving a large number of Drosophila classifications, statistics and analyses.
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Drosophila , Redes Neurais de Computação , Animais , Drosophila/genética , Computadores , TecnologiaRESUMO
KNL1 (kinetochore scaffold 1) has attracted much attention as one of the assembly elements of the outer kinetochore, and the functions of its different domains have been gradually revealed, most of which are associated with cancers, but few links have been made between KNL1 and male fertility. Here, we first linked KNL1 to male reproductive health and the loss-function of KNL1 resulted in oligospermia and asthenospermia in mice (an 86.5% decrease in total sperm number and an 82.4% increase in static sperm number, respectively) through CASA (computer-aided sperm analysis). Moreover, we introduced an ingenious method to pinpoint the abnormal stage in the spermatogenic cycle using flow cytometry combined with immunofluorescence. Results showed that 49.5% haploid sperm was reduced and 53.2% diploid sperm was increased after the function of KNL1 was lost. Spermatocytes arrest was identified at the meiotic prophase I of spermatogenesis, which was induced by the abnormal assembly and separation of the spindle. In conclusion, we established an association between KNL1 and male fertility, providing a guide for future genetic counseling regarding oligospermia and asthenospermia, and a powerful method for further exploring spermatogenic dysfunction by utilizing flow cytometry and immunofluorescence.
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Astenozoospermia , Proteínas Associadas aos Microtúbulos , Oligospermia , Animais , Masculino , Camundongos , Citometria de Fluxo , Imunofluorescência , Meiose , Sêmen , Proteínas Associadas aos Microtúbulos/genéticaRESUMO
During the period of 2018-2020, we first combined reported low-pass whole genome sequencing and NGS-based STR tests for miscarriage samples analysis. Compared with G-banding karyotyping, the system increased the detection rate of chromosomal abnormalities in miscarriage samples to 56.4% in 500 unexplained recurrent spontaneous abortions. In this study, a total of 386 STR loci were developed on twenty-two autosomes and two sex chromosomes (X and Y chromosomes), which can help to distinguish triploidy, uniparental diploidy and maternal cell contamination and can trace the parental origin of erroneous chromosomes. It is not possible to accomplish this with existing methods of detection in miscarriage samples. Among the tested aneuploid errors, the most frequently detected error was trisomy (33.4% in total and 59.9% in the error chromosome group). In the trisomy samples, 94.7% extra chromosomes were of maternal origin and 5.31% were of paternal origin. This novel system improves the genetic analysis method of miscarriage samples and provides more reference information for clinical pregnancy guidance.
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Recessive dystrophic epidermolysis bullosa (RDEB) is a rare and severe hereditary skin disease, caused by mutations in the COL7A1. However, whether non-invasive prenatal testing (NIPT) can be used for this monogenic genodermatosis remains unknown. Accordingly, we conducted a study in which one couple at high risk of having a fetus with RDEB were recruited and tested by haplotyping-based NIPT. Next-generation sequencing-based multi-gene panel testing was carried out in this couple and their first child as proband who was affected with RDEB. We deduced parental haplotypes via single nucleotide polymorphism (SNP)-based haplotype linkage analysis. Then the maternal plasma cell-free DNA was also sequenced to determine the fetal haplotypes using a parental haplotype-assisted hidden Markov model (HMM) analysis. Results show that the fetus was only a heterozygous mutation carrier in COL7A1 and the identical results were obtained after birth. These results demonstrate that haplotyping-based NIPT is a feasible method for NIPT of RDEB.
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Epidermólise Bolhosa Distrófica , Gravidez , Criança , Feminino , Humanos , Epidermólise Bolhosa Distrófica/diagnóstico , Epidermólise Bolhosa Distrófica/genética , Haplótipos , Mutação , Colágeno/genética , Genes Recessivos , Sequenciamento de Nucleotídeos em Larga Escala , Diagnóstico Pré-Natal , Colágeno Tipo VII/genéticaRESUMO
In recent years, the development of bispecific antibodies (bsAbs) has become a major trend in the biopharmaceutical industry. By simultaneously engaging two molecular targets, bsAbs have exhibited unique mechanisms of action that could lead to clinical benefits unattainable by conventional monoclonal antibodies. The type of structure used to construct a bsAb directly influences the distance, angle, degree of freedom, and affinity between the two antibody binding sites and the interaction between the two antigens or the cells where the antigens are located, which have been bound by the antibody. Consequently, the structure of the bsAb is one of the most vital factors affecting its function. Herein, we reported for the first time a novel basic module bsAb format, VFV (Variable domain-Fab-Variable domain). And then, the feasibility of the VFV format was demonstrated by constructing a series of engager-like basic module bsAbs. Next, a series of VFV bsAbs containing Fc (VFV-Ig), Fab (VFV-Fab), or Hinge (VFV-Hinge) were developed based on Hxb module, and all of them had adequate purity and activity. Finally, a T cell engager bsAb with the potential to overcome on-target off-tumor activity was constructed according to the structural characteristics of VFV, which validated that the VFV module can be used as a new brick for the construction of various bsAbs. In a word, the successful construction of this bsAb format for the first time not only enriches the arsenal of the bsAb format, but also provides inspiration for the construction of new bsAbs. Nevertheless, we are fully aware that as a proof-of-concept study, this paper has many shortcomings, and there is still a lot of work to be done to determine whether VFV can serve as a platform for drug development.
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Anticorpos Biespecíficos , Neoplasias , Humanos , Anticorpos Monoclonais , Sítios de Ligação de Anticorpos , Linfócitos TRESUMO
The detection of mutations in KRAS, NRAS, BRAF, and PIK3CA has become essential in managing the treatment of metastatic colorectal cancer (CRC) with the approval of new targeted therapies. We developed novel multiplex drop-off digital PCR (MDO-dPCR) assays by combining amplitude-/ratio-based multiplexing with drop-off/double drop-off strategies that allow for the detection of at least the 69 most frequent hotspot mutations in all four genes with only three reactions. The analytical performance of the assays was assessed using synthetic oligonucleotides, which were further validated on plasma cell-free DNA samples from a large cohort of CRC patients and compared with next-generation sequencing data. The MDO-dPCR assays showed a high sensitivity with a limit of detection ranging from 0.084% to 0.182% in mutant allelic frequency. The screening of plasma cell-free DNAs from 106 CRC patients identified mutations in 42.45% of them, with a sensitivity of 95.24%, a specificity of 98.53%, and an accuracy of 96.98% for mutation detection, and a strong correlation of measured mutant allelic frequencies compared with next-generation sequencing results. The high sensitivity and comprehensive mutation coverage of the MDO-dPCR assays make them suitable for rapid and cost-effective detection of KRAS, NRAS, BRAF, and PIK3CA mutations in the plasma of CRC patients, and could be useful in early response assessment and longitudinal disease monitoring.
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Neoplasias Colorretais , Proteínas Proto-Oncogênicas B-raf , Humanos , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Mutação , Reação em Cadeia da Polimerase Multiplex , Classe I de Fosfatidilinositol 3-Quinases/genética , Proteínas de Membrana/genética , GTP Fosfo-Hidrolases/genéticaRESUMO
The CRISPR-Cas system can treat autosomal dominant diseases by nonhomologous end joining (NHEJ) gene disruption of mutant alleles. However, many single-nucleotide mutations cannot be discriminated from wild-type alleles by current CRISPR-Cas systems. Here, we functionally screened six Cas12j nucleases and determined Cas12j-8 as an ideal genome editor with a hypercompact size. Cas12j-8 displayed comparable activity to AsCas12a and Un1Cas12f1. Cas12j-8 is a highly specific nuclease sensitive to single-nucleotide mismatches in the protospacer adjacent motif (PAM)-proximal region. We experimentally proved that Cas12j-8 enabled allele-specific disruption of genes with a single-nucleotide polymorphism (SNP). Cas12j-8 recognizes a simple TTN PAM that provides for high target site density. In silico analysis reveals that Cas12j-8 enables allele-specific disruption of 25,931 clinically relevant variants in the ClinVar database, and 485,130,147 SNPs in the dbSNP database. Therefore, Cas12j-8 would be particularly suitable for therapeutic applications.
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Sistemas CRISPR-Cas , Edição de Genes , Alelos , Endonucleases/metabolismo , NucleotídeosRESUMO
The aim of this study was to assess the narrow-sense validity of polygenic risk score (PRS) for prostate cancer (PCa) in a Chinese prostate biopsy cohort. We performed an observational prospective study with 2640 men who underwent prostate biopsy. Germline DNA samples were genotyped and PRS was calculated for each subject using 17 PCa risk-associated genetic variants. Additional GWAS data of the ChinaPCa dataset was also used to compliment the evaluation process. The mean PRS was 1.02 in patients with negative biopsy results, which met the baseline benchmark. The mean PRS was significantly higher in the PCa cases (1.32 vs. 1.02, p = 5.56 × 10-17 ). Significant dose-response associations between PRS values and odds ratios for PCa were observed. However, the raw calibration slope was 0.524 and the average bias score between the observed risk and uncorrected PRS value was 0.307 in the entire biopsy cohort. After applying a correction factor derived from a training set, the corrected calibration slope improved to 1.002 in a testing set. Similar and satisfied results were also seen in the ChinaPCa dataset and two datasets combined, while the calibration results were inaccurate when the calibration process were performed mutually between two different study populations. In conclusion, assessing the narrow-sense validity of PRS is necessary prior to its clinical implementation for accurate individual risk assessment.
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Próstata , Neoplasias da Próstata , Humanos , Masculino , Biópsia , População do Leste Asiático , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Estudos Prospectivos , Próstata/patologia , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Medição de Risco/métodos , Fatores de RiscoRESUMO
X-linked myopia 26 (Myopia 26, MIM #301010), which is caused by the variants of ARR3 (MIM *301770), is characterized by female-limited early-onset high myopia (eo-HM). Clinical characteristics include a tigroid appearance in the fundus and a temporal crescent of the optic nerve head. At present, the limited literature on eo-HM caused by ARR3 mutations shows that its inheritance mode is complex, which brings certain difficulties to pre-pregnancy genetic counseling, pre-implantation genetic diagnosis, and prenatal diagnosis. Here, we investigated the genetic underpinning of a Chinese family with eo-HM. Whole exome sequencing of the proband revealed a novel frameshift mutation in ARR3 (NM_004312, exon10, c.666delC, p. Asn222LysfsTer22). Although the mode of inheritance of the eo-HM family fits the X-linked pattern of ARR3, the phenotypes of three patients deviate from the typical early-onset high myopia. Through X-chromosome inactivation experiments, the patient's different phenotypes can be precisely explained. In addition, this study not only enhanced the correlation between ARR3 and early-onset high myopia but also provided explanations for different phenotypes, which may inspire follow-up studies. Our results enrich the knowledge of the variant spectrum in ARR3 and provide critical information for preimplantation and prenatal genetic testing, diagnosis, and counseling.