Rearrangements of T- and B-cell receptor (TCR and BCR) genes are useful markers for clonality assessment as well as for minimal residual disease (MRD) monitoring during the treatment of haematological malignancies. Currently, rearrangements of three out of four TCR and all BCR loci are used for this purpose. The fourth TCR gene, TRA, has not been used so far due to the lack of a method for its rearrangement detection in genomic DNA. Here we propose the first high-throughput sequencing based method for the identification of clonal TRA gene rearrangements at the DNA level. The method is based on target amplification of the rearranged TRA locus using an advanced multiplex polymerase chain reaction system and high-throughput sequencing, and has been tested on DNA samples from peripheral blood of healthy donors. Combinations of all functional V- and J-segments were detected, indicating the high sensitivity of the method. Additionally, we identified clonal TRA rearrangements in 57 out of 112 tested DNA samples of patients with various T-lineage lymphoproliferative disorders. The method fills the existing gap in utilizing the TRA gene for a wide range of studies, including clonality assessment, MRD monitoring and clonal evolution analysis in different lymphoid malignancies.
- MeSH
- DNA nádorová genetika MeSH
- genová přestavba - alfa řetězec receptoru antigenů T-buněk * MeSH
- hematologické nádory genetika MeSH
- lidé MeSH
- lymfoproliferativní nemoci genetika MeSH
- multiplexová polymerázová řetězová reakce * MeSH
- vysoce účinné nukleotidové sekvenování * MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Publikační typ
- abstrakt z konference MeSH
- Publikační typ
- abstrakt z konference MeSH
Trace analysis of microorganisms in real biological samples needs very sensitive methods for their detection. Most procedures for detecting and quantifying pathogens require a sample preparation step including concentrating microorganisms from large sample volumes with high and reproducible efficiency. Electromigration techniques have great potential to include the preconcentration, separation, and detection of whole cells and therefore they can rapidly indicate the presence of pathogens. The preconcentration and separation of microorganisms from real suspensions utilising a combination of filtration and capillary isoelectric focusing was developed and the possibility for its application to real samples was verified. For our experiments, spores of Monilinia species and of Penicillium expansum were selected as model bioparticles, as they cause major losses in agrosystems. The isoelectric points of the spores of M. laxa, M. fructigena, M. fruticola, and P. expansum were determined and the method was verified using real samples taken directly from infected apples. The coupling of a filtration cartridge with a separation capillary can improve the detection limit of isoelectric focusing with UV detection by at least 4 orders of magnitude. Spores of M. fructigena and of M. laxa in numbers of hundreds of particles per milliliter were detected on a visually noninfected apple surface which was cross-contaminated during handling and storage. The efficiency of preconcentration and a preliminary identification was verified by the phenotyping technique after cultivation of the spores sampled from the apple surface.
- MeSH
- Ascomycota izolace a purifikace MeSH
- design vybavení MeSH
- elektroforéza kapilární přístrojové vybavení metody MeSH
- filtrace přístrojové vybavení metody MeSH
- isoelektrická fokusace přístrojové vybavení metody MeSH
- Malus mikrobiologie MeSH
- Penicillium izolace a purifikace MeSH
- senzitivita a specificita MeSH
- spory hub izolace a purifikace MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
- práce podpořená grantem MeSH
Limb girdle muscular dystrophy type 2A (LGMD2A) is caused by single or small nucleotide changes widespread along the CAPN3 gene, which encodes the muscle-specific proteolytic enzyme calpain-3. About 356 unique allelic variants of CAPN3 have been identified to date. We performed analysis of the CAPN3 gene in LGMD2A patients at both the mRNA level using reverse transcription-PCR, and at the DNA level using PCR and denaturing high performance liquid chromatography. In four patients, we detected homozygous occurrence of a missense mutation or an in-frame deletion at the mRNA level although the DNA was heterozygous for this mutation in conjunction with a frame-shift mutation. The relationship observed in 12 patients between the quantity of CAPN3 mRNA, determined using real-time PCR, and the genotype leads us to propose that CAPN3 mRNAs which contain frame-shift mutations are degraded by nonsense-mediated mRNA decay. Our results illustrate the importance of DNA analysis for reliable establishment of mutation status, and provide a new insight into the process of mRNA decay in cells of LGMD2A patients.
- MeSH
- delece genu MeSH
- dítě MeSH
- DNA biosyntéza genetika MeSH
- dospělí MeSH
- financování organizované MeSH
- genotyp MeSH
- introny genetika MeSH
- kalpain genetika MeSH
- kojenec MeSH
- lidé MeSH
- messenger RNA genetika MeSH
- mladiství MeSH
- nesmyslný kodon genetika MeSH
- pletencové svalové dystrofie genetika MeSH
- polymerázová řetězová reakce s reverzní transkripcí MeSH
- posunová mutace genetika MeSH
- předškolní dítě MeSH
- svalové proteiny genetika MeSH
- western blotting MeSH
- Check Tag
- dítě MeSH
- dospělí MeSH
- kojenec MeSH
- lidé MeSH
- mladiství MeSH
- mužské pohlaví MeSH
- předškolní dítě MeSH
- ženské pohlaví MeSH
Spinal muscular atrophy (SMA) is caused by homozygous deletion of the SMN1 gene in approximately 96% of cases. Four percent of SMA patients have a combination of the deletion or conversion on one allele and an intragenic mutation on the second one. We performed analysis of point mutations in a set of our patients with suspicion of SMA and without homozygous deletion of the SMN1 gene. A quantitative test determining SMN1 copy number (using real-time PCR and/or MLPA analysis) was performed in 301 patients and only 1 SMN1 copy was detected in 14 of them. When these 14 patients were screened for the presence of point mutations we identified 6 mutations, p.Y272C (in three patients) and p.T274I, p.I33IfsX6, and p.A188S (each in one case). The mutations p.I33IfsX6 and p.A188S were found in two SMAI patients and were not detected previously. Further, evaluation of the relationship between mutation type, copy number of the SMN2 gene and clinical findings was performed. Among our SMA patients with a SMN1 homozygous deletion, we found a family with two patients: the son with SMAII possesses 3 SMN2 copies and the nearly asymptomatic father has a homozygous deletion of SMN1 exon 7 and carries 4 SMN2 copies. Generally, our results illustrate that an increased SMN2 gene copy number is associated with a milder SMA phenotype.
- MeSH
- bodová mutace genetika MeSH
- delece genu MeSH
- DNA genetika MeSH
- dospělí MeSH
- exony genetika MeSH
- fenotyp MeSH
- financování organizované MeSH
- genová dávka MeSH
- homozygot MeSH
- lidé MeSH
- mladiství MeSH
- polymerázová řetězová reakce s reverzní transkripcí MeSH
- předškolní dítě MeSH
- protein přežití motorických neuronů 1 MeSH
- protein přežití motorických neuronů 2 MeSH
- protein vázající cAMP responzivní element genetika MeSH
- proteinový komplex SMN MeSH
- proteiny nervové tkáně genetika MeSH
- proteiny vázající RNA genetika MeSH
- spinální svalová atrofie genetika MeSH
- techniky amplifikace nukleových kyselin MeSH
- Check Tag
- dospělí MeSH
- lidé MeSH
- mladiství MeSH
- předškolní dítě MeSH
Limb-girdle muscular dystrophy type 2A (LGMD2A) is an autosomal-recessive disorder characterized by selective atrophy and progressive weakness of proximal girdle muscles. LGMD2A, the most prevalent form of LGMD, is caused by mutations in the CAPN3 gene that encodes the skeletal muscle-specific member of the calpain family, calpain-3 (p 94). We examined the histopathologic and molecular pathologic findings in 14 Czech LGMD2A patients. Analysis of the CAPN3 gene was performed at the mRNA level, using reverse transcription-polymerase chain reaction (RT-PCR) and sequencing, and/or DNA level, using PCR and denaturing high-performance liquid chromatography (DHPLC). Our results confirm that mutation 550 delA is the most frequent CAPN3 defect in Czech LGMD2A patients (9 alleles of 28). Furthermore, we established that, in a patient with the 550 delA/R490W genotype, mRNA carrying frameshift mutation 550 delA was not detected, probably due to its degradation by nonsense-mediated mRNA decay. In muscle biopsies of two LGMD2A patients, a neurogenic pattern simulating a neurogenic lesion was observed. Immunoblot analysis revealed the deficiency of p 94 in all genetically confirmed cases of LGMD2A, and secondary dysferlin deficiency was demonstrated on muscle membranes in 6 patients using immunofluorescence. Thus, we find a combination of DNA and mRNA mutational analysis to be useful in the diagnosis of LGMD2A. Moreover, our study expands the spectrum of calpainopathies to cases that simulate a neurogenic lesion in muscle biopsies, and the knowledge of possible secondary deficiencies of muscular proteins also contributes to a diagnosis of LGMD2A. Muscle Nerve, 2006.
- MeSH
- alely MeSH
- dítě MeSH
- dospělí MeSH
- financování organizované MeSH
- fluorescenční protilátková technika MeSH
- genotyp MeSH
- imunohistochemie MeSH
- izoenzymy metabolismus MeSH
- kalpain metabolismus MeSH
- kosterní svaly patologie MeSH
- lidé středního věku MeSH
- lidé MeSH
- membránové proteiny imunologie metabolismus MeSH
- messenger RNA biosyntéza genetika MeSH
- mladiství MeSH
- mutační analýza DNA MeSH
- NAD metabolismus MeSH
- pletencové svalové dystrofie genetika patologie MeSH
- polymerázová řetězová reakce s reverzní transkripcí MeSH
- předškolní dítě MeSH
- proteiny nervové tkáně metabolismus MeSH
- svalové proteiny imunologie metabolismus MeSH
- vysokoúčinná kapalinová chromatografie MeSH
- Check Tag
- dítě MeSH
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladiství MeSH
- mužské pohlaví MeSH
- předškolní dítě MeSH
- ženské pohlaví MeSH
- Geografické názvy
- Česká republika MeSH
