RESUMO
Establishing modular binders as diagnostic detection agents represents a cost- and time-efficient alternative to the commonly used binders that are generated one molecule at a time. In contrast to these conventional approaches, a modular binder can be designed in silico from individual modules to, in principle, recognize any desired linear epitope without going through a selection and hit-validation process, given a set of preexisting, amino acid-specific modules. Designed armadillo repeat proteins (dArmRP) have been developed as modular binder scaffolds, and we report here the generation of highly specific dArmRP modules by yeast surface display selection, performed on a rationally designed dArmRP library. A selection strategy was developed to distinguish the binding difference resulting from a single amino acid mutation in the target peptide. Our reverse-competitor strategy introduced here employs the designated target as a competitor to increase the sensitivity when separating specific from cross-reactive binders that show similar affinities for the target peptide. With this switch in selection focus from affinity to specificity, we found that the enrichment during this specificity sort is indicative of the desired phenotype, regardless of the binder abundance. Hence, deep sequencing of the selection pools allows retrieval of phenotypic hits with only 0.1% abundance in the selectivity sort pool from the next-generation sequencing data alone. In a proof-of-principle study, a binder was created by replacing all corresponding wild-type modules with a newly selected module, yielding a binder with very high affinity for the designated target that has been successfully validated as a detection agent in western blot analysis.
Assuntos
Proteínas do Domínio Armadillo , Saccharomyces cerevisiae , Proteínas do Domínio Armadillo/genética , Proteínas do Domínio Armadillo/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Ligação Proteica , Peptídeos/metabolismo , Peptídeos/genética , Peptídeos/química , Epitopos/genética , Biblioteca de PeptídeosRESUMO
Long-read sequencing (LRS) technologies have provided extremely powerful tools to explore genomes. While in the early years these methods suffered technical limitations, they have recently made significant progress in terms of read length, throughput, and accuracy and bioinformatics tools have strongly improved. Here, we aim to review the current status of LRS technologies, the development of novel methods, and the impact on genomics research. We will explore the most impactful recent findings made possible by these technologies focusing on high-resolution sequencing of genomes and transcriptomes and the direct detection of DNA and RNA modifications. We will also discuss how LRS methods promise a more comprehensive understanding of human genetic variation, transcriptomics, and epigenetics for the coming years.
Assuntos
Genômica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Genômica/métodos , Análise de Sequência de DNA/métodos , Biologia Computacional , Perfilação da Expressão Gênica/métodosRESUMO
Approximately 5 to 15% of nonmedullary thyroid cancers (NMTC) present in a familial form (familial nonmedullary thyroid cancers [FNMTC]). The genetic basis of FNMTC remains largely unknown, representing a limitation for diagnostic and clinical management. Recently, germline mutations in DNA repair-related genes have been described in cases with thyroid cancer (TC), suggesting a role in FNMTC etiology. Here, two FNMTC families were studied, each with two members affected with TC. Ninety-four hereditary cancer predisposition genes were analyzed through next-generation sequencing, revealing two germline CHEK2 missense variants (c.962A > C, p.E321A and c.470T > C, p.I157T), which segregated with TC in each FNMTC family. p.E321A, located in the CHK2 protein kinase domain, is a rare variant, previously unreported in the literature. Conversely, p.I157T, located in CHK2 forkhead-associated domain, has been extensively described, having conflicting interpretations of pathogenicity. CHK2 proteins (WT and variants) were characterized using biophysical methods, molecular dynamics simulations, and immunohistochemistry. Overall, biophysical characterization of these CHK2 variants showed that they have compromised structural and conformational stability and impaired kinase activity, compared to the WT protein. CHK2 appears to aggregate into amyloid-like fibrils in vitro, which opens future perspectives toward positioning CHK2 in cancer pathophysiology. CHK2 variants exhibited higher propensity for this conformational change, also displaying higher expression in thyroid tumors. The present findings support the utility of complementary biophysical and in silico approaches toward understanding the impact of genetic variants in protein structure and function, improving the current knowledge on CHEK2 variants' role in FNMTC genetic basis, with prospective clinical translation.
Assuntos
Quinase do Ponto de Checagem 2 , Síndromes Neoplásicas Hereditárias , Câncer Papilífero da Tireoide , Neoplasias da Glândula Tireoide , Humanos , Quinase do Ponto de Checagem 2/química , Quinase do Ponto de Checagem 2/genética , Quinase do Ponto de Checagem 2/metabolismo , Predisposição Genética para Doença , Mutação em Linhagem Germinativa , Síndromes Neoplásicas Hereditárias/genética , Estudos Prospectivos , Câncer Papilífero da Tireoide/genética , Neoplasias da Glândula Tireoide/genética , Domínios Proteicos , Masculino , Feminino , Pessoa de Meia-IdadeRESUMO
RNA editing is a crucial post-transcriptional modification process in plant organellar RNA metabolism. rRNA removal-based total RNA-seq is one of the most common methods to study this event. However, the lack of commercial kits to remove rRNAs limits the usage of this method, especially for non-model plant species. DSN-seq is a transcriptome sequencing method utilizing duplex-specific nuclease (DSN) to degrade highly abundant cDNA species especially those from rRNAs while keeping the robustness of transcript levels of the majority of other mRNAs, and has not been applied to study RNA editing in plants before. In this study, we evaluated the capability of DSN-seq to reduce rRNA content and profile organellar RNA editing events in plants, as well we used commercial Ribo-off-seq and standard mRNA-seq as comparisons. Our results demonstrated that DSN-seq efficiently reduced rRNA content and enriched organellar transcriptomes in rice. With high sensitivity to RNA editing events, DSN-seq and Ribo-off-seq provided a more complete and accurate RNA editing profile of rice, which was further validated by Sanger sequencing. Furthermore, DSN-seq also demonstrated efficient organellar transcriptome enrichment and high sensitivity for profiling RNA editing events in Arabidopsis thaliana. Our study highlights the capability of rRNA removal-based total RNA-seq for profiling RNA editing events in plant organellar transcriptomes and also suggests DSN-seq as a widely accessible RNA editing profiling method for various plant species.
Assuntos
Edição de RNA , Transcriptoma , Transcriptoma/genética , Edição de RNA/genética , Organelas/genética , Organelas/metabolismo , RNA Ribossômico/genética , RNA Ribossômico/metabolismo , RNA de Plantas/genética , RNA de Plantas/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Análise de Sequência de RNA/métodosRESUMO
BACKGROUND: Standard ChIP-seq and RNA-seq processing pipelines typically disregard sequencing reads whose origin is ambiguous ("multimappers"). This usual practice has potentially important consequences for the functional interpretation of the data: genomic elements belonging to clusters composed of highly similar members are left unexplored. RESULTS: In particular, disregarding multimappers leads to the underrepresentation in epigenetic studies of recently active transposable elements, such as AluYa5, L1HS and SVAs. Furthermore, this common strategy also has implications for transcriptomic analysis: members of repetitive gene families, such the ones including major histocompatibility complex (MHC) class I and II genes, are under-quantified. CONCLUSION: Revealing inherent biases that permeate routine tasks such as functional enrichment analysis, our results underscore the urgency of broadly adopting multimapper-aware bioinformatic pipelines -currently restricted to specific contexts or communities- to ensure the reliability of genomic and transcriptomic studies.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Elementos de DNA Transponíveis/genética , Biologia Computacional/métodos , Perfilação da Expressão Gênica/métodos , Genômica/métodos , Análise de Sequência de RNA/métodosRESUMO
Hepatitis B, a persistent inflammatory liver condition, stands as a significant global health issue. In Romania, the prevalence of chronic hepatitis B virus (CHB) infection ranks among the highest in the European Union. The HLA genotype significantly impacts hepatitis B virus infection progression, indicating that certain HLA variants can affect the infection's outcome. The primary goal of the present work is to identify HLA alleles and specific amino acid residues linked to hepatitis B within the Romanian population. The study enrolled 247 patients with chronic hepatitis B; HLA typing was performed using next-generation sequencing. This study's main findings include the identification of certain HLA alleles, such as DQB1*06:03:01, DRB1*13:01:01, DQB1*06:02:01, DQA1*01:03:01, DRB5*01:01:01, and DRB1*15:01:01, which exhibit a significant protective effect against HBV. Additionally, the amino acid residue alanine at DQB1_38 is associated with a protective role, while valine presence may signal an increased risk of hepatitis B. The present findings are important in addressing the urgent need for improved methods of diagnosing and managing CHB, particularly when considering the disease's presence in diverse population groups and geographical regions.
RESUMO
Multiple myeloma (MM) first-line treatment algorithms include immuno-chemotherapy (ICT) induction, high-dose chemotherapy (HDCT) and autologous stem cell transplant (ASCT) consolidation, followed by lenalidomide maintenance. After these initial therapies, most patients suffer a disease relapse and require subsequent treatment lines including ICT, additional HDCT and ASCT, or novel immunotherapies. The presence of somatic mutations in peripheral blood cells has been associated with adverse outcomes in a variety of hematological malignancies. Nonsense and frameshift mutations in the PPM1D gene, a frequent driver alteration in clonal hematopoiesis (CH), lead to the gain-of-function of Wip1 phosphatase, which may impair the p53-dependent G1 checkpoint and promote cell proliferation. Here, we determined the presence of PPM1D gene mutations in peripheral blood cells of 75 subsequent myeloma patients in remission after first or second HDCT/ASCT. The prevalence of truncating PPM1D gene mutations emerged at 1.3% after first HDCT/ASCT, and 7.3% after second HDCT/ASCT, with variant allele frequencies (VAF) of 0.01 to 0.05. Clinical outcomes were inferior in the PPM1D-mutated (PPM1Dmut) subset with median progression-free survival (PFS) of 15 vs. 37 months (p = 0.0002) and median overall survival (OS) of 36 vs. 156 months (p = 0.001) for the PPM1Dmut and PPM1Dwt population, respectively. Our data suggest that the occurrence of PPM1D gene mutations in peripheral blood cells correlates with inferior outcomes after ASCT in patients with multiple myeloma.
RESUMO
INTRODUCTION: Next generation sequencing technology has greatly reduced the cost and time required for sequencing a genome. An approach that is rapidly being adopted as an alternative method for CNV analysis is the low-pass whole genome sequencing (LP-WGS). Here, we evaluated the performance of LP-WGS to detect copy number variants (CNVs) in clinical cytogenetics. MATERIALS AND METHODS: DNA samples with known CNVs detected by chromosomal microarray analyses (CMA) were selected for comparison and used as positive controls; our panel included 44 DNA samples (12 prenatal and 32 postnatal), comprising a total of 55 chromosome imbalances. The selected cases were chosen to provide a wide range of clinically relevant CNVs, the vast majority being associated with intellectual disability or recognizable syndromes. The chromosome imbalances ranged in size from 75 kb to 90.3 Mb, including aneuploidies and two cases of mosaicism. RESULTS: All CNVs were successfully detected by LP-WGS, showing a high level of consistency and robust performance of the sequencing method. Notably, the size of chromosome imbalances detected by CMA and LP-WGS were compatible between the two different platforms, which indicates that the resolution and sensitivity of the LP-WGS approach are at least similar to those provided by CMA. DISCUSSION: Our data show the potential use of LP-WGS to detect CNVs in clinical diagnosis and confirm the method as an alternative for chromosome imbalances detection. The diagnostic effectiveness and feasibility of LP-WGS, in this technical validation study, were evidenced by a clinically representative dataset of CNVs that allowed a systematic assessment of the detection power and the accuracy of the sequencing approach. Further, since the software used in this study is commercially available, the method can easily be tested and implemented in a routine diagnostic setting.
Assuntos
Aneuploidia , Variações do Número de Cópias de DNA , Gravidez , Feminino , Humanos , Análise Custo-Benefício , Sequenciamento Completo do Genoma/métodos , DNARESUMO
The ongoing or anticipated therapeutic advances as well as previous experience in other malignancies, including acute myeloid leukaemia, have made molecular monitoring a potential interesting tool for predicting outcomes and demonstrating treatment efficacy in patients with myelodysplastic syndromes (MDS). The important genetic heterogeneity in MDS has made challenging the establishment of recommendations. In this context, high-throughput/next-generation sequencing (NGS) has emerged as an attractive tool, especially in patients with high-risk diseases. However, its implementation in clinical practice still suffers from a lack of standardization in terms of sensitivity, bioinformatics and result interpretation. Data from literature, mostly gleaned from retrospective cohorts, show NGS monitoring when used appropriately could help clinicians to guide therapy, detect early relapse and predict disease evolution. Translating these observations into personalized patient management requires a prospective evaluation in clinical research and remains a major challenge for the next years.
RESUMO
BACKGROUND: Advancements in the field of precision medicine have prompted the European Society for Medical Oncology (ESMO) Precision Medicine Working Group to update the recommendations for the use of tumour next-generation sequencing (NGS) for patients with advanced cancers in routine practice. METHODS: The group discussed the clinical impact of tumour NGS in guiding treatment decision using the ESMO Scale for Clinical Actionability of molecular Targets (ESCAT) considering cost-effectiveness and accessibility. RESULTS: As for 2020 recommendations, ESMO recommends running tumour NGS in advanced non-squamous non-small-cell lung cancer, prostate cancer, colorectal cancer, cholangiocarcinoma, and ovarian cancer. Moreover, it is recommended to carry out tumour NGS in clinical research centres and under specific circumstances discussed with patients. In this updated report, the consensus within the group has led to an expansion of the recommendations to encompass patients with advanced breast cancer and rare tumours such as gastrointestinal stromal tumours, sarcoma, thyroid cancer, and cancer of unknown primary. Finally, ESMO recommends carrying out tumour NGS to detect tumour-agnostic alterations in patients with metastatic cancers where access to matched therapies is available. CONCLUSION: Tumour NGS is increasingly expanding its scope and application within oncology with the aim of enhancing the efficacy of precision medicine for patients with cancer.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala , Neoplasias , Medicina de Precisão , Humanos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Sequenciamento de Nucleotídeos em Larga Escala/normas , Medicina de Precisão/métodos , Medicina de Precisão/normas , Neoplasias/genética , Neoplasias/patologia , Neoplasias/terapia , Oncologia/métodos , Oncologia/normas , Europa (Continente)RESUMO
BACKGROUND: Genomic tumour profiling has a crucial role in the management of patients with solid cancers, as it helps selecting and prioritising therapeutic interventions based on prognostic and predictive biomarkers, as well as identifying markers of hereditary cancers. Harmonised approaches to interpret the results of genomic testing are needed to support physicians in their decision making, prevent inequalities in precision medicine and maximise patient benefit from available cancer management options. METHODS: The European Society for Medical Oncology (ESMO) Translational Research and Precision Medicine Working Group assembled a group of international experts to propose recommendations for preparing clinical genomic reports for solid cancers. These recommendations aim to foster best practices in integrating genomic testing within clinical settings. After review of available evidence, several rounds of surveys and focused discussions were conducted to reach consensus on the recommendation statements. Only consensus recommendations were reported. Recommendation statements were graded in two tiers based on their clinical importance: level A (required to maintain common standards in reporting) and level B (optional but necessary to achieve ideal practice). RESULTS: Genomics reports should present key information in a front page(s) followed by supplementary information in one or more appendices. Reports should be structured into sections: (i) patient and sample details; (ii) assay and data analysis characteristics; (iii) sample-specific assay performance and quality control; (iv) genomic alterations and their functional annotation; (v) clinical actionability assessment and matching to potential therapy indications; and (vi) summary of the main findings. Specific recommendations to prepare each of these sections are made. CONCLUSIONS: We present a set of recommendations aimed at structuring genomics reports to enhance physician comprehension of genomic profiling results for solid cancers. Communication between ordering physicians and professionals reporting genomic data is key to minimise uncertainties and to optimise the impact of genomic tests in patient care.
RESUMO
BACKGROUND: The species composition of tree stands plays an important role in shaping the properties of forest soils. The aim of our research was to determine the influence on soil properties of the root systems of six species of trees which form forest stands in the temperate climatic zone. The research covered areas including six tree species - Scots pine (Pinus sylvestris L.), European larch (Larix deciduas Mill.), English oak (Quercus robur L.), English ash (Fraxinus excelsior L.), European beech (Fagus sylvatica L.) and European hornbeam (Carpinus betulus L.). In our study, we determined the characteristics of the roots and the amount of carbon excreted alongside their exudates. Enzymatic activity, and the composition and diversity of the fungi and bacteria, were also determined in addition to the basic physicochemical properties of the soil samples. RESULTS: A strong relationship between the root characteristics and soil properties, including the pH, basic cation content and phosphorus content, was confirmed. In addition, the enzymatic activity of phosphatase, ß-glucosidase, N-acetyl-ß-D-glucosaminidase and ß-D-cellobiosidase were positively correlated with the root characteristics. The study on soil bacteria across different tree species revealed Proteobacteria and Actinobacteriota to be the most abundant phylum. Fungal analysis showed Basidiomycota and Ascomycota as the dominant phyla. Ascomycota dominated in hornbeam and oak soils. Mortierellomycota was remarkably more present in pine soil. CONCLUSIONS: This analysis of root systems and soil properties confirmed the distinctness of ash stands, which were also more abundant in various microorganisms. It was also found that soils affected by different tree species were characterised by varied fungal and bacterial composition. The ash had particularly beneficial impact on soil microbiota.
Assuntos
Ascomicetos , Fagus , Pinus sylvestris , Quercus , Ecossistema , Árvores , Solo/química , Florestas , Exsudatos e Transudatos , Microbiologia do SoloRESUMO
BACKGROUND: Filarial worms are important vector-borne pathogens of a large range of animal hosts, including humans, and are responsible for numerous debilitating neglected tropical diseases such as, lymphatic filariasis caused by Wuchereria bancrofti and Brugia spp., as well as loiasis caused by Loa loa. Moreover, some emerging or difficult-to-eliminate filarioid pathogens are zoonotic using animals like canines as reservoir hosts, for example Dirofilaria sp. 'hongkongensis'. Diagnosis of filariasis through commonly available methods, like microscopy, can be challenging as microfilaremia may wane below the limit of detection. In contrast, conventional PCR methods are more sensitive and specific but may show limited ability to detect coinfections as well as emerging and/or novel pathogens. Use of deep-sequencing technologies obviate these challenges, providing sensitive detection of entire parasite communities, whilst also being better suited for the characterisation of rare or novel pathogens. Therefore, we developed a novel long-read metabarcoding assay for deep-sequencing the filarial nematode cytochrome c oxidase subunit I gene on Oxford Nanopore Technologies' (ONT) MinION™ sequencer. We assessed the overall performance of our assay using kappa statistics to compare it to commonly used diagnostic methods for filarial worm detection, such as conventional PCR (cPCR) with Sanger sequencing and the microscopy-based modified Knott's test (MKT). RESULTS: We confirmed our metabarcoding assay can characterise filarial parasites from a diverse range of genera, including, Breinlia, Brugia, Cercopithifilaria, Dipetalonema, Dirofilaria, Onchocerca, Setaria, Stephanofilaria and Wuchereria. We demonstrated proof-of-concept for this assay by using blood samples from Sri Lankan dogs, whereby we identified infections with the filarioids Acanthocheilonema reconditum, Brugia sp. Sri Lanka genotype and zoonotic Dirofilaria sp. 'hongkongensis'. When compared to traditionally used diagnostics, such as the MKT and cPCR with Sanger sequencing, we identified an additional filarioid species and over 15% more mono- and coinfections. CONCLUSIONS: Our developed metabarcoding assay may show broad applicability for the metabarcoding and diagnosis of the full spectrum of filarioids from a wide range of animal hosts, including mammals and vectors, whilst the utilisation of ONT' small and portable MinION™ means that such methods could be deployed for field use.
Assuntos
Coinfecção , Filariose , Filarioidea , Humanos , Animais , Cães , Filarioidea/genética , Filariose/diagnóstico , Filariose/veterinária , Filariose/parasitologia , Brugia/genética , Wuchereria bancrofti/genética , MamíferosRESUMO
Since variants of uncertain significance (VUS) reported in genetic testing cannot be acted upon clinically, this classification may delay or prohibit precise diagnosis and genetic counseling in adult genetic disorders patients. Large-scale analyses about qualitatively distinct lines of evidence used for VUS can make them re-classification more accurately. We analyzed 458 Chinese adult patients WES data, within 15 pathogenic evidence PS1, PS2, PM1, PM6 and PP4 were not used for VUS pathogenic classification, meanwhile the PP3, BP4, PP2 were used much more frequently. The PM2_Supporting was used most widely for all reported variants. There were also 31 null variants (nonsense, frameshift, canonical ±1 or 2 splice sites) which were probably the disease-causing variants of the patients were classified as VUS. By analyzed the evidence used for all VUS we recommend that appropriate genetic counseling, reliable releasing of in-house data, allele frequency comparison between case and control, expanded verification in patient family, co-segregation analysis and functional assays were urgent need to gather more evidence to reclassify VUS. We also found adult patients with nervous system disease were reported the most phenotype-associated VUS and the lower the phenotypic specificity, the more reported VUS. This result emphasized the importance of pretest genetic counseling which would make less reporting of VUS. Our result revealed the characteristics of the pathogenic classification evidence used for VUS in adult genetic disorders patients for the first time, recommend a rules-based process to evaluate the pathogenicity of VUS which could provide a strong basis for accurately evaluating the pathogenicity and clinical grade information of VUS. Meanwhile, we further expanded the genetic spectrum and improve the diagnostic rate of adult genetic disorders.
Assuntos
Aconselhamento Genético , Doenças Genéticas Inatas , Testes Genéticos , Humanos , Adulto , Testes Genéticos/métodos , Doenças Genéticas Inatas/genética , Doenças Genéticas Inatas/classificação , Doenças Genéticas Inatas/diagnóstico , Variação Genética , Fenótipo , Frequência do Gene , Feminino , Sequenciamento do Exoma , Masculino , Predisposição Genética para DoençaRESUMO
Malaria cases are frequently recorded in the Ethiopian highlands even at altitudes above 2000 m. The epidemiology of malaria in the Ethiopian highlands, and, in particular, the role of importation by human migration from the highly endemic lowlands is not well understood. We sequenced 187 Plasmodium falciparum samples from two sites in the Ethiopian highlands, Gondar (n = 159) and Ziway (n = 28), using a multiplexed droplet digital PCR (ddPCR)-based amplicon sequencing method targeting 35 microhaplotypes and drug resistance loci. Here, we characterize the parasite population structure and genetic relatedness. We identify moderate parasite diversity (mean HE : 0.54) and low infection complexity (74.9% monoclonal). A significant percentage of infections share microhaplotypes, even across transmission seasons and sites, indicating persistent local transmission. We identify multiple clusters of clonal or near-clonal infections, highlighting high genetic relatedness. Only 6.3% of individuals diagnosed with P. falciparum reported recent travel. Yet, in clonal or near-clonal clusters, infections of travellers were frequently observed first in time, suggesting that parasites may have been imported and then transmitted locally. 31.1% of infections are pfhrp2-deleted and 84.4% pfhrp3-deleted, and 28.7% have pfhrp2/3 double deletions. Parasites with pfhrp2/3 deletions and wild-type parasites are genetically distinct. Mutations associated with resistance to sulphadoxine-pyrimethamine or suggested to reduce sensitivity to lumefantrine are observed at near-fixation. In conclusion, genomic data corroborate local transmission and the importance of intensified control in the Ethiopian highlands.
Assuntos
Malária Falciparum , Malária , Parasitos , Animais , Humanos , Plasmodium falciparum/genética , Proteínas de Protozoários/genética , Antígenos de Protozoários/genética , Etiópia/epidemiologia , Deleção de Genes , Malária Falciparum/genética , Malária/genéticaRESUMO
AIMS: Asians have a high prevalence of young-onset diabetes, but the pattern of monogenic diabetes is unknown. We aimed to determine the prevalence of monogenic diabetes in Chinese patients with young-onset diabetes and compare the clinical characteristics and outcome between patients with and without monogenic diabetes. MATERIALS AND METHODS: We sequenced a targeted panel of 33 genes related to monogenic diabetes in 1021 Chinese patients with non-type 1 diabetes diagnosed at age ≤40 years. Incident complications including cardiovascular disease (CVD), end-stage kidney disease (ESKD) and all-cause death were captured since enrolment (1995-2012) until 2019. RESULTS: In this cohort (mean ± SD age at diagnosis: 33.0 ± 6.0 years, median[IQR] diabetes duration 7.0[1.0-15.0] years at baseline, 44.9% men), 22(2.2%, 95% confidence interval[CI] 1.4%-3.2%) had monogenic diabetes. Pathogenic (P) or likely pathogenic (LP) variants were detected in GCK (n = 6), HNF1A (n = 9), HNF4A (n = 1), PLIN1 (n = 1) and PPARG (n = 2), together with copy number variations in HNF1B (n = 3). Over a median follow-up of 17.1 years, 5(22.7%) patients with monogenic diabetes (incidence rate 12.3[95% CI 5.1-29.4] per 1000 person-years) versus 254(25.4%) without monogenic diabetes (incidence rate 16.7[95% CI 14.8-18.9] per 1000 person-years) developed the composite outcome of CVD, ESKD and/or death (p = 0.490). The multivariable Cox model did not show any difference in hazards for composite events between groups. CONCLUSIONS: In Chinese with young-onset non-type 1 diabetes, at least 2% of cases were contributed by monogenic diabetes, over 80% of which were accounted for by P/LP variants in common MODY genes. The incidence of diabetes complications was similar between patients with and without monogenic diabetes.
Assuntos
Idade de Início , Humanos , Masculino , Feminino , Seguimentos , Hong Kong/epidemiologia , Adulto , Estudos Prospectivos , Prognóstico , Povo Asiático/genética , Adulto Jovem , Diabetes Mellitus/genética , Diabetes Mellitus/epidemiologia , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/epidemiologia , Prevalência , Adolescente , Incidência , População do Leste AsiáticoRESUMO
BACKGROUND: Next-generation sequencing (NGS) is essential for lung cancer treatment. It is important to collect sufficient tissue specimens, but sometimes we cannot obtain large enough samples for NGS analysis. We investigated the yield of NGS analysis by frozen cytology pellets using an Oncomine Comprehensive Assay or Oncomine Precision Assay. METHODS: We retrospectively enrolled patients with lung cancer who underwent bronchoscopy at Kobe University Hospital and were enrolled in the Lung Cancer Genomic Screening Project for Individualized Medicine. We investigated the amount of extracted DNA and RNA and determined the NGS success rates. We also compared the amount of DNA and RNA by bronchoscopy methods. To create the frozen cytology pellets, we first effectively collected the cells and then quickly centrifuged and cryopreserved them. RESULTS: A total of 132 patients were enrolled in this study between May 2016 and December 2022; of them, 75 were subjected to frozen cytology pellet examinations and 57 were subjected to frozen tissue examinations. The amount of DNA and RNA obtained by frozen cytology pellets was nearly equivalent to frozen tissues. Frozen cytology pellets collected by endobronchial ultrasound-guided transbronchial needle aspiration yielded significantly more DNA than those collected by transbronchial biopsy methods. (P < 0.01) In RNA content, cytology pellets were not inferior to frozen tissue. The success rate of NGS analysis with frozen cytology pellet specimens was comparable to the success rate of NGS analysis with frozen tissue specimens. CONCLUSIONS: Our study showed that frozen cytology pellets may have equivalent diagnostic value to frozen tissue for NGS analyses. Bronchial cytology specimens are usually used only for cytology, but NGS analysis is possible if enough cells are collected to create pellet specimens. In particular, the frozen cytology pellets obtained by endobronchial ultrasound-guided transbronchial needle aspiration yielded sufficient amounts of DNA. TRIAL REGISTRATION: This was registered with the University Medical Hospital Information Network in Japan (UMINCTR registration no. UMIN000052050).
Assuntos
Neoplasias Pulmonares , Humanos , Estudos Retrospectivos , Neoplasias Pulmonares/patologia , Aspiração por Agulha Fina Guiada por Ultrassom Endoscópico/métodos , Broncoscopia/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , DNA , RNA , Linfonodos/patologiaRESUMO
BACKGROUND: Esophageal cancer (EC) is a deadly disease with limited therapeutic options. Although circulating tumor DNA (ctDNA) could be a promising tool in this regard, the availiable evidence is limited. We performed a systematic review and meta-analysis to summarize the clinical applicability of the next-generation sequencing (NGS) and droplet digital polymerase chain reaction (ddPCR) technology on the ctDNA detection of the EC and listed the current challenges. METHODS: We systematically searched MEDLINE (via PubMed), Embase (via OVID), ISI Web of Science database and Cochrane Library from January, 2000 to April, 2023. Progression-free survival (PFS) and overall survival (OS) were set as primary outcome endpoints. Pathologic response was evaluated by tumor regression grade (TRG), according to the eighth edition of the American Joint Committee on Cancer (AJCC). Major pathologic regression (MPR) was defined as TRG 1 and 2. The MPR was set as secondary endpoint. Hazard rate (HR) and associated 95% CI were used as the effect indicators the association between ctDNA and prognosis of EC. MPR rates were also calculated. Fixed-effect model (Inverse Variance) or random-effect model (Mantel-Haenszel method) was performed depending on the statistically heterogeneity. RESULTS: Twenty-two studies, containing 1144 patients with EC, were included in this meta-analysis. The results showed that OS (HR = 3.87; 95% CI, 2.86-5.23) and PFS (HR = 4.28; 95% CI, 3.34-5.48) were shorter in ctDNA-positive patients. In the neoadjuvant therapy, the sensitivity analysis showed the clarified HR of ctDNA-positive was 1.13(95% CI, 1.01-1.28). We also found that TP53, NOTCH1, CCND1 and CNKN2A are the most frequent mutation genes. CONCLUSIONS: Positive ctDNA is associated with poor prognosis, which demonstrated clinical value of ctDNA. Longitudinal ctDNA monitoring showed potential prognostic value in the neoadjuvant therapy. In an era of precision medicine, ctDNA could be a promising tool to individualize treatment planning and to improve outcomes in EC. PROSPERO REGISTRATION NUMBER: CRD42023412465.
Assuntos
DNA Tumoral Circulante , Neoplasias Esofágicas , Humanos , DNA Tumoral Circulante/genética , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/terapia , Bases de Dados Factuais , Biblioteca Gênica , Genes cdcRESUMO
Typical (or classic) Rett syndrome (RTT) is an X-linked neurodevelopmental disorder characterized by a period of regression, partial or complete loss of purposeful hand movements, and acquired speech, impaired gait, and stereotyped hand movements. In over 95% of typical RTT, a pathogenic variant is found in the methyl-CPG binding protein 2 gene (MECP2). Here, we describe a young woman with clinically diagnosed typical RTT syndrome who lacked a genetic diagnosis despite 20 years of investigation and multiple rounds of sequencing the MECP2 gene. Recently, additional genetic testing using next-generation sequencing was completed, which revealed a partial insertion of the BCL11A gene within exon 4 of MECP2, resulting in a small deletion in MECP2, causing likely disruption of MeCP2 function due to a frameshift. This case demonstrates the ever-changing limitations of genetic testing, as well as the importance of continual pursuit of a diagnosis as technologies improve and are more widely utilized.
RESUMO
In this pilot study, we aimed to evaluate the feasibility of whole genome sequencing (WGS) as a first-tier diagnostic test for infants hospitalized in neonatal intensive care units in the Brazilian healthcare system. The cohort presented here results from a joint collaboration between private and public hospitals in Brazil considering the initiative of a clinical laboratory to provide timely diagnosis for critically ill infants. We performed trio (proband and parents) WGS in 21 infants suspected of a genetic disease with an urgent need for diagnosis to guide medical care. Overall, the primary indication for genetic testing was dysmorphic syndromes (n = 14, 67%) followed by inborn errors of metabolism (n = 6, 29%) and skeletal dysplasias (n = 1, 5%). The diagnostic yield in our cohort was 57% (12/21) based on cases that received a definitive or likely definitive diagnostic result from WGS analysis. A total of 16 pathogenic/likely pathogenic variants and 10 variants of unknown significance were detected, and in most cases inherited from an unaffected parent. In addition, the reported variants were of different types, but mainly missense (58%) and associated with autosomal diseases (19/26); only three were associated with X-linked diseases, detected in hemizygosity in the proband an inherited from an unaffected mother. Notably, we identified 10 novel variants, absent from public genomic databases, in our cohort. Considering the entire diagnostic process, the average turnaround time from enrollment to medical report in our study was 53 days. Our findings demonstrate the remarkable utility of WGS as a diagnostic tool, elevating the potential of transformative impact since it outperforms conventional genetic tests. Here, we address the main challenges associated with implementing WGS in the medical care system in Brazil, as well as discuss the potential benefits and limitations of WGS as a diagnostic tool in the neonatal care setting.