Multiplex PCR
Dotaz
Zobrazit nápovědu
PURPOSE: A new high-resolution next-generation sequencing (NGS)-based method was established to type closely related European type II Toxoplasma gondii strains. METHODS: T. gondii field isolates were collected from different parts of Europe and assessed by whole genome sequencing (WGS). In comparison to ME49 (a type II reference strain), highly polymorphic regions (HPRs) were identified, showing a considerable number of single nucleotide polymorphisms (SNPs). After confirmation by Sanger sequencing, 18 HPRs were used to design a primer panel for multiplex PCR to establish a multilocus Ion AmpliSeq typing method. Toxoplasma gondii isolates and T. gondii present in clinical samples were typed with the new method. The sensitivity of the method was tested with serially diluted reference DNA samples. RESULTS: Among type II specimens, the method could differentiate the same number of haplotypes as the reference standard, microsatellite (MS) typing. Passages of the same isolates and specimens originating from abortion outbreaks were identified as identical. In addition, seven different genotypes, two atypical and two recombinant specimens were clearly distinguished from each other by the method. Furthermore, almost all SNPs detected by the Ion AmpliSeq method corresponded to those expected based on WGS. By testing serially diluted DNA samples, the method exhibited a similar analytical sensitivity as MS typing. CONCLUSION: The new method can distinguish different T. gondii genotypes and detect intra-genotype variability among European type II T. gondii strains. Furthermore, with WGS data additional target regions can be added to the method to potentially increase typing resolution.
- MeSH
- genetická variace MeSH
- genotyp MeSH
- lidé MeSH
- multiplexová polymerázová řetězová reakce MeSH
- polymorfismus délky restrikčních fragmentů MeSH
- protozoální DNA genetika MeSH
- těhotenství MeSH
- Toxoplasma * genetika MeSH
- vysoce účinné nukleotidové sekvenování MeSH
- Check Tag
- lidé MeSH
- těhotenství MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
PURPOSE: Preimplantation genetic testing for monogenic disorders (PGT-M) allows early diagnosis in embryos conceived in vitro. PGT-M helps to prevent known genetic disorders in affected families and ensures that pathogenic variants in the male or female partner are not passed on to offspring. The trend in genetic testing of embryos is to provide a comprehensive platform that enables robust and reliable testing for the causal pathogenic variant(s), as well as chromosomal abnormalities that commonly occur in embryos. In this study, we describe PGT protocol that allows direct mutation testing, haplotyping, and aneuploidy screening. METHODS: Described PGT protocol called OneGene PGT allows direct mutation testing, haplotyping, and aneuploidy screening using next-generation sequencing (NGS). Whole genome amplification product is combined with multiplex PCR used for SNP enrichment. Dedicated bioinformatic tool enables mapping, genotype calling, and haplotyping of informative SNP markers. A commercial software was used for aneuploidy calling. RESULTS: OneGenePGT has been implemented for seven of the most common monogenic disorders, representing approximately 30% of all PGT-M indications at our IVF centre. The technique has been thoroughly validated, focusing on direct pathogenic variant testing, haplotype identification, and chromosome abnormality detection. Validation results show full concordance with Sanger sequencing and karyomapping, which were used as reference methods. CONCLUSION: OneGene PGT is a comprehensive, robust, and cost-effective method that can be established for any gene of interest. The technique is particularly suitable for common monogenic diseases, which can be performed based on a universal laboratory protocol without the need for set-up or pre-testing.
- MeSH
- aneuploidie MeSH
- blastocysta patologie MeSH
- genetické testování metody MeSH
- lidé MeSH
- mutace genetika MeSH
- preimplantační diagnóza * metody MeSH
- těhotenství MeSH
- vysoce účinné nukleotidové sekvenování metody MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- těhotenství MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
Standardy molekulárně-patologického testování se s pátým vydáním WHO klasifikace nádorů centrálního nervového systému razantně mění. Významnou změnou je zapojení molekulárně genetického testování a využívání komplexního přístupu v diagnostice. Některé typy nádorů pro své správné zařazení vyžadují vyšetření molekulárního profilu. Nejčastěji jde o vyšetření jednonukleotidových mutací, delecí a kodelecí, fúzí, případně metylací. Molekulární metody rozšiřují diagnostické spektrum založené na histopatologickém a imunohistochemickém zhodnocení. Využívají se molekulární metody, jako je PCR, Sangerovo sekvenování, I‐FISH, MLPA a/nebo NGS. Zcela zásadní je pro diagnostiku úzká spolupráce vyšetřujícího neuropatologa, molekulárního genetika a neuroonkologa, bez níž může dojít nesprávně užitými postupy k významnému poškození pacienta.
The standards of molecular-pathological testing are transforming with the fifth edition of the WHO classification of central nervous system tumors. A significant change involves incorporating molecular genetic testing and adopting a comprehensive diagnostic approach. Certain tumors require examining the molecular profile for accurate classification, typically analyzing single nucleotide variants, deletions, codeletions, fusions, or methylation. Molecular methods extend the diagnostic spectrum beyond histopathological and immunohistochemical assessments, using techniques like PCR, Sanger sequencing, I-FISH, MLPA, and/or NGS. Collaboration among neuropathologists, molecular geneticists, and neurooncologists is crucial for accurate diagnosis and preventing potential harm to patients.
- MeSH
- diagnostické techniky molekulární metody MeSH
- hybridizace in situ fluorescenční MeSH
- imunohistochemie MeSH
- lidé MeSH
- metylace DNA MeSH
- multiplexová polymerázová řetězová reakce MeSH
- mutace genetika MeSH
- nádory centrálního nervového systému * diagnóza genetika patologie MeSH
- polymerázová řetězová reakce MeSH
- sekvenční analýza DNA MeSH
- vysoce účinné nukleotidové sekvenování MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- přehledy MeSH
- MeSH
- antibakteriální látky aplikace a dávkování terapeutické užití MeSH
- Borrelia burgdorferi komplex MeSH
- imunoanalýza MeSH
- lidé MeSH
- lymeská nemoc * diagnóza epidemiologie farmakoterapie MeSH
- lymská neuroborelióza diagnóza farmakoterapie MeSH
- polymerázová řetězová reakce MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- přehledy MeSH
BACKGROUND: The BCR::ABL1 is a hallmark of chronic myeloid leukemia (CML) and is also found in acute lymphoblastic leukemia (ALL). Most genomic breaks on the BCR side occur in two regions - Major and minor - leading to p210 and p190 fusion proteins, respectively. METHODS: By multiplex long-distance PCR or next-generation sequencing technology we characterized the BCR::ABL1 genomic fusion in 971 patients (adults and children, with CML and ALL: pediatric ALL: n = 353; pediatric CML: n = 197; adult ALL: n = 166; adult CML: n = 255 patients) and designed "Break-App" web tool to allow visualization and various analyses of the breakpoints. Pearson's Chi-Squared test, Kolmogorov-Smirnov test and logistic regression were used for statistical analyses. RESULTS: Detailed analysis showed a non-random distribution of breaks in both BCR regions, whereas ABL1 breaks were distributed more evenly. However, we found a significant difference in the distribution of breaks between CML and ALL. We found no association of breakpoints with any type of interspersed repeats or DNA motifs. With a few exceptions, the primary structure of the fusions suggests non-homologous end joining being responsible for the BCR and ABL1 gene fusions. Analysis of reciprocal ABL1::BCR fusions in 453 patients showed mostly balanced translocations without major deletions or duplications. CONCLUSIONS: Taken together, our data suggest that physical colocalization and chromatin accessibility, which change with the developmental stage of the cell (hence the difference between ALL and CML), are more critical factors influencing breakpoint localization than presence of specific DNA motifs.
- MeSH
- akutní lymfatická leukemie * genetika patologie MeSH
- bcr-abl fúzní proteiny * genetika MeSH
- body zlomu chromozomu * MeSH
- chronická myeloidní leukemie * genetika patologie MeSH
- dítě MeSH
- dospělí MeSH
- lidé MeSH
- vysoce účinné nukleotidové sekvenování MeSH
- Check Tag
- dítě MeSH
- dospělí MeSH
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- dopisy MeSH
BACKGROUND: A proportion of head and neck carcinomas (HNSCCs) are induced by high-risk human papillomaviruses (HPVs) and are associated with better patient outcomes compared to patients with HNSCCs related to tobacco and alcohol abuse. In the microenvironment of solid tumors, including HNSCCs, oxygen levels are often reduced, and a hypoxic state is induced. This can lead to a poor treatment response and a worse patient prognosis. One of the hypoxia-responsive genes is aspartate-β-hydroxylase (ASPH), whose activity promotes the growth, invasiveness, and metastasis of many types of solid tumors. METHODS: In our study, HNSCC samples were analyzed for the expression of ASPH and selected endogenous hypoxia markers by real-time PCR and/or multiplex fluorescence immunohistochemistry. RESULTS: Except for the EPAS1 gene, which had higher mRNA expression in the HPV-negative group of HNSCC (p < 0.05), we found no other differences in the expression of the tested genes that were related to HPV status. On the contrary, a statistically significantly higher number of cells producing ASPH (p < 0.0001), HIF1A (p < 0.0001), GLUT1 (p < 0.0001), and MMP13 (p < 0.05) proteins were detected in the HPV-positive tumor group than in the HPV-negative sample group. All the evaluated markers, except for MMP9/13, were more abundant in the tumor parenchyma than in the tumor stroma. The Cox proportional hazard models showed that increased numbers of cells with GLUT1 and HIF1A protein expression were positive prognostic markers for overall and disease-specific survival in patients independent of HPV tumor status. CONCLUSION: The study examined HNSCC samples and found that elevated ASPH and hypoxia marker proteins, typically associated with poor prognosis, may actually indicate active HPV infection, the strongest prognostic factor in HNSCC patients. In cases where HPV status is uncertain, increased expression of HIF1A and GLUT1 can serve as positive prognostic factors.
- Publikační typ
- časopisecké články MeSH
AIMS: This retrospective non-randomised study aims to identify new and rare fusion partners with USP6 in the setting of nodular fasciitis. It has been proven, that nodular fasciitis can harbour different variants of USP6 fusions, which can be used in routine diagnostics and even determine the biological behaviour of the process. METHODS: A total of 19 cases of nodular fasciitis examined between 2011 and 2022 at Motol University Hospital in Prague were included into this study. Next to the histopathological evaluation, all cases were assessed using immunohistochemistry, RT-PCR and Anchored multiplex RNA methods. Patient's main demographic characteristics and corresponding clinical data were also analysed. RESULTS: This study presents one novel (KIF1A) and five rare examples (TMP4, SPARC, EIF5A, MIR22HG, COL1A2) of fusion partners with USP6 among 19 cases of nodular fasciitis. CONCLUSION: Identification of USP6 fusion partners in nodular fasciitis helps to understand the biology of such lesions. Moreover, it can be useful in routine histopathological practice of soft-tissues diagnostics, especially in preventing possible misdiagnosis of malignancy.
- MeSH
- dospělí MeSH
- fasciitida * genetika patologie MeSH
- fúzní onkogenní proteiny genetika MeSH
- genová přestavba * MeSH
- imunohistochemie MeSH
- kineziny genetika MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladý dospělý MeSH
- retrospektivní studie MeSH
- senioři MeSH
- thiolesterasa ubikvitinu * genetika MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladý dospělý MeSH
- mužské pohlaví MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
The current study assessed the performance of the fully automated RT-PCR-based IdyllaTM GeneFusion Assay, which simultaneously covers the advanced non-small cell lung carcinoma (aNSCLC) actionable ALK, ROS1, RET, and MET exon 14 rearrangements, in a routine clinical setting involving 12 European clinical centers. The IdyllaTM GeneFusion Assay detects fusions using fusion-specific as well as expression imbalance detection, the latter enabling detection of uncommon fusions not covered by fusion-specific assays. In total, 326 archival aNSCLC formalin-fixed paraffin-embedded (FFPE) samples were included of which 44% were resected specimen, 46% tissue biopsies, and 9% cytological specimen. With a total of 179 biomarker-positive cases (i.e., 85 ALK, 33 ROS1, 20 RET fusions and 41 MET exon 14 skipping), this is one of the largest fusion-positive datasets ever tested. The results of the IdyllaTM GeneFusion Assay were compared with earlier results of routine reference technologies including fluorescence in situ hybridization, immunohistochemistry, reverse-transcription polymerase chain reaction, and next-generation sequencing, establishing a high sensitivity/specificity of 96.1%/99.6% for ALK, 96.7%/99.0% for ROS1, 100%/99.3% for RET fusion, and 92.5%/99.6% for MET exon 14 skipping, and a low failure rate (0.9%). The IdyllaTM GeneFusion Assay was found to be a reliable, sensitive, and specific tool for routine detection of ALK, ROS1, RET fusions and MET exon 14 skipping. Given its short turnaround time of about 3 h, it is a time-efficient upfront screening tool in FFPE samples, supporting rapid clinical decision making. Moreover, expression-imbalance-based detection of potentially novel fusions may be easily verified with other routine technologies without delaying treatment initiation.
- MeSH
- anaplastická lymfomová kináza * genetika MeSH
- exony * genetika MeSH
- fúzní onkogenní proteiny * genetika MeSH
- genová přestavba MeSH
- hybridizace in situ fluorescenční metody MeSH
- lidé MeSH
- multiplexová polymerázová řetězová reakce MeSH
- nádorové biomarkery genetika analýza MeSH
- nádory plic * genetika patologie MeSH
- nemalobuněčný karcinom plic * genetika patologie MeSH
- protoonkogenní proteiny c-met * genetika MeSH
- protoonkogenní proteiny c-ret * genetika MeSH
- protoonkogenní proteiny * genetika MeSH
- tyrosinkinasy * genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
- multicentrická studie MeSH
