Diamond-Blackfan anemia (DBA) is predominantly underlined by mutations in genes encoding ribosomal proteins (RP); however, its etiology remains unexplained in approximately 25 % of patients. We previously reported a novel heterozygous RPS7 mutation hg38 chr2:g.3,580,153G > T p.V134F in one female patient and two asymptomatic family members, in whom mild anemia and increased erythrocyte adenosine deaminase (eADA) activity were detected. We observed that altered erythrocyte metabolism and oxidative stress which may negatively affect the lifespan of erythrocytes distinguishes the patient from her asymptomatic family members. Pathogenicity of the RPS7 p.V134F mutation was extensively validated including molecular defects in protein translational activity and ribosomal stress activation in the cellular model of this variant.

• MeSH
• Diamondova-Blackfanova anemie * genetika   MeSH
• erytrocyty metabolismus   MeSH
• lidé MeSH
• missense mutace MeSH
• proteosyntéza MeSH
• ribozomální proteiny * genetika   MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• kazuistiky MeSH
• práce podpořená grantem MeSH

Diamond-Blackfan anemia (DBA) is a rare congenital erythroid aplasia, underlied by haploinsufficient mutations in genes coding for ribosomal proteins (RP) in approximately 70% of cases. DBA is frequently associated with somatic malformations, endocrine dysfunction and with an increased predisposition to cancer. Here we present clinical and genetic characteristics of 62 patients from 52 families enrolled in the Czech and Slovak DBA Registry. Whole exome sequencing (WES) and array comparative genomic hybridization (aCGH) were employed to identify causative mutations in newly diagnosed patients and in cases with previously unrecognized molecular pathology. RP mutation detection rate was 81% (50/62 patients). This included 8 novel point mutations and 4 large deletions encompassing some of the RP genes. Malignant or predisposing condition developed in 8/62 patients (13%): myelodysplastic syndrome in 3 patients; breast cancer in 2 patients; colorectal cancer plus ocular tumor, diffuse large B-cell lymphoma and multiple myeloma each in one case. These patients exclusively harbored RPL5, RPL11 or RPS19 mutations. Array CGH is beneficial for detection of novel mutations in DBA due to its capacity to detect larger chromosomal aberrations. Despite the importance of genotype-phenotype correlation in DBA, phenotypic differences among family members harboring an identical mutation were observed.

• MeSH
• Diamondova-Blackfanova anemie komplikace   epidemiologie   genetika   MeSH
• genetické asociační studie MeSH
• lidé MeSH
• mutace * MeSH
• nádory etiologie   MeSH
• registrace MeSH
• ribozomální proteiny genetika   MeSH
• rodina MeSH
• sekvenování exomu MeSH
• srovnávací genomová hybridizace MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Česká republika MeSH
• Slovenská republika MeSH

Costunolide, a natural sesquiterpene lactone, has multiple pharmacological activities such as neuroprotection or induction of apoptosis and eryptosis. However, the effects of costunolide on pro-survival factors and enzymes in human erythrocytes, e.g. glutathione and glucose-6-phosphate dehydrogenase (G6PDH) respectively, have not been studied yet. Our aim was to determine the mechanisms underlying costunolide-induced eryptosis and to reverse this process. Phosphatidylserine exposure was estimated from annexin-V-binding, cell volume from forward scatter in flow cytometry, and intracellular glutathione [GSH]i from high performance liquid chromatography. The oxidized status of intracellular glutathione and enzyme activities were measured by spectrophotometry. Treatment of erythrocytes with costunolide dose-dependently enhanced the percentage of annexin-V-binding cells, decreased the cell volume, depleted [GSH]i and completely inhibited G6PDH activity. The effects of costunolide on annexin-V-binding and cell volume were significantly reversed by pre-treatment of erythrocytes with the specific PKC-α inhibitor chelerythrine. The latter, however, had no effect on costunolide-induced GSH depletion. Costunolide induces eryptosis, depletes [GSH]i and inactivates G6PDH activity. Furthermore, our study reveals an inhibitory effect of chelerythrine on costunolide-induced eryptosis, indicating a relationship between costunolide and PKC-α. In addition, chelerythrine acts independently of the GSH depletion. Understanding the mechanisms of G6PDH inhibition accompanied by GSH depletion should be useful for development of anti-malarial therapeutic strategies or for synthetic lethality-based approaches to escalate oxidative stress in cancer cells for their sensitization to chemotherapy and radiotherapy.

• MeSH
• apoptóza účinky léků   MeSH
• benzofenantridiny farmakologie   MeSH
• eryptóza účinky léků   genetika   MeSH
• erytrocyty účinky léků   patologie   MeSH
• glukosa-6-fosfátdehydrogenasa antagonisté a inhibitory   genetika   MeSH
• glutathion genetika   MeSH
• inhibitory enzymů farmakologie   MeSH
• lidé MeSH
• oxidační stres účinky léků   MeSH
• proteinkinasa C-alfa antagonisté a inhibitory   genetika   MeSH
• reaktivní formy kyslíku MeSH
• seskviterpeny farmakologie   MeSH
• vápník metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Inflammatory and oncogenic signaling, both known to challenge genome stability, are key drivers of BCR-ABL-positive chronic myeloid leukemia (CML) and JAK2 V617F-positive chronic myeloproliferative neoplasms (MPNs). Despite similarities in chronic inflammation and oncogene signaling, major differences in disease course exist. Although BCR-ABL has robust transformation potential, JAK2 V617F-positive polycythemia vera (PV) is characterized by a long and stable latent phase. These differences reflect increased genomic instability of BCR-ABL-positive CML, compared to genome-stable PV with rare cytogenetic abnormalities. Recent studies have implicated BCR-ABL in the development of a "mutator" phenotype fueled by high oxidative damage, deficiencies of DNA repair, and defective ATR-Chk1-dependent genome surveillance, providing a fertile ground for variants compromising the ATM-Chk2-p53 axis protecting chronic phase CML from blast crisis. Conversely, PV cells possess multiple JAK2 V617F-dependent protective mechanisms, which ameliorate replication stress, inflammation-mediated oxidative stress and stress-activated protein kinase signaling, all through up-regulation of RECQL5 helicase, reactive oxygen species buffering system, and DUSP1 actions. These attenuators of genome instability then protect myeloproliferative progenitors from DNA damage and create a barrier preventing cellular stress-associated myelofibrosis. Therefore, a better understanding of BCR-ABL and JAK2 V617F roles in the DNA damage response and disease pathophysiology can help to identify potential dependencies exploitable for therapeutic interventions.

• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

Erytrocytární enzymopatie je genetické onemocnění vyznačující se vrozenými kvantitativními či kvalitativními abnormalitami erytrocytárních enzymů, které jsou jednou z příčin vzniku vrozené nesférocytární hemolytické anémie. V případě zralých erytrocytů, které neobsahují jádro, mitochondrie ani endoplazmatické retikulum, se enzymopatie týkají glykolýzy a pentózafosfátového cyklu, popř. metabolismu glutathionu a nukleotidů. Mezi nejrozšířenější abnormality patří deficit pyruvátkinázy a glukóza-6-fosfátdehydrogenázy, které jsou vzácnou příčinou vzniku hemolytické anémie v české i slovenské populaci. Enzymopatie jsou charakterizovány normo-/lehce makrocytární normochromní anémií s obecnými znaky hemolýzy. Při negativní rodinné anamnéze, je diagnostika enzymopatií vzhledem k minimálním charakteristickým abnormalitám v morfologii erytrocytů komplikovaná (proto byly dříve označovány jako vrozené nesférocytární hemolytické anémie). Základní diferenciální diagnostika nedokáže rozlišit jednotlivé enzymové defekty. Přesná diagnóza může být určena až na základě snížené specifické aktivity daného enzymu a potvrzena dále na molekulárně-genetické úrovni. Deficit glukóza-6-fosfátdehydrogenázy (G6PD) a pyruvátkinázy (PK) byl v české a slovenské populaci popsán již v 80. letech. Ucelený přehled o výskytu tohoto onemocnění v české a slovenské populaci, zvláště pak o jeho vzácnějších formách, doposud chybí. Od roku 2013 se naše laboratoř zabývá metodou přímého stanovení aktivity enzymů s následným genetickým vyšetřením u pacientů se suspektní enzymopatií. V současné době jsme jedinou specializovanou laboratoří zabývající se touto diagnostikou v České republice, resp. ve střední Evropě. Doposud byl enzymový deficit diagnostikován u dvaceti čtyř pacientů. Vedle nejčastějších deficitů G6PD a PK (9 a 12 případů), byly identifikovány 2 rodiny s deficitem glukózafosfátizomerázy (GPI) a 1 rodina s velmi vzácným deficitem hexokinázy (HK). Deficit GPI a HK jsme v české a slovenské populaci diagnostikovali poprvé. Celkově bylo dosud identifikováno dvacet dva kauzálních mutací, z nichž pět bylo v literatuře popsáno poprvé – G6PD p.(Phe216Tyr), PK p.(Arg518Leufs*12), p.(Asp293Val), GPI p.(Ser160Pro) a p.(Arg472Cys).

Hereditary red blood cell (RBC) enzymopathies are genetic disorders affecting genes encoding red blood cell enzymes. They cause a specific type of anaemia designated hereditary nonspherocytic haemolytic anaemia. Enzymopathies affect cellular metabolism, which in the RBC mainly consists of anaerobic glycolysis, the hexose monophosphate shunt, glutathione metabolism and nucleotide metabolism. Enzymopathies are commonly associated with normocytic/slightly macrocytic normochromic haemolytic anaemia. In general, RBC enzymopathies are not characterized by specific abnormalities in RBC morphology. Routinely used haematology methods are unable to pinpoint a specific enzyme defect. Definitive diagnosis is based on the detection of reduced specific enzyme activity and molecular characterization of the defect at DNA level. This work represents advanced and up-to-date information regarding RBC enzyme deficiencies in the Czech and Slovak populations dating from the 1980s, when mutations in Czech and Slovak subjects resulting in glucose-6-phosphate dehydrogenase (G6PD) and pyruvate kinase (PK) deficiencies were first described. Both deficiencies represent the most common erythroenzymopathies worldwide. Since 2013, our laboratory has focused on the introduction of direct enzyme assays supplemented by genetic testing in patients with haemolytic anaemia and suspected erythroenzymopathy. To date, several different enzyme defects have been diagnosed in twenty-four patients. Apart from G6PD and PK deficiencies (9 and 12 cases), 2 families with glucose phosphate isomerase (GPI) defect and 1 family with the very rare hexokinase (HK) deficiency have been identified. The two latter deficiencies were diagnosed in the Czech and Slovak populations for the first time. Among the 22 identified mutations, were novel and have not been previously reported in literature: 5, namely G6PD p.(Phe216Tyr), PK p.(Arg518Leufs*12), p.(Asp293Val) and GPI p.(Ser160Pro), p.(Arg472Cys).

• Klíčová slova
• erytrocytární enzymopatie,
• MeSH
• enzymy metabolismus   MeSH
• erytrocyty metabolismus   MeSH
• glykolýza fyziologie   MeSH
• hemolytická nesférocytická kongenitální anemie genetika   MeSH
• hemolytické anemie * diagnóza   terapie   MeSH
• lidé MeSH
• pyruvátkinasa genetika   nedostatek   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• práce podpořená grantem MeSH
• souhrny MeSH

Glucose-6-phosphate isomerase (GPI) deficiency, a genetic disorder responsible for chronic nonspherocytic hemolytic anemia, is the second most common red blood cell glycolytic enzymopathy. We report three patients from two unrelated families of Czech and Slovak origin with macrocytic hemolytic anemia due to GPI deficiency. The first patient had 15% of residual GPI activity resulting from two new heterozygous missense mutations c.478T>C and c.1414C>T leading to substitutions p.(Ser160Pro) and p.(Arg472Cys). Two other patients (siblings) inherited the same c.1414C>T p.(Arg472Cys) mutation in a homozygous constitution and lost approximately 89% of their GPI activity. Erythroid hyperplasia with dysplastic features was observed in the bone marrow of all three patients. Low hepcidin/ferritin ratio and elevated soluble transferrin receptor detected in our GPI-deficient patients suggest disturbed balance between erythropoiesis and iron metabolism contributing to iron overload.

• MeSH
• alely MeSH
• biologické markery MeSH
• biopsie MeSH
• dítě MeSH
• erytrocytární znaky MeSH
• erytroidní buňky metabolismus   MeSH
• erytropoéza genetika   MeSH
• genotyp MeSH
• glukosa-6-fosfátisomerasa chemie   genetika   MeSH
• hemolytická nesférocytická kongenitální anemie krev   diagnóza   genetika   MeSH
• hepcidiny krev   MeSH
• konformace proteinů MeSH
• kostní dřeň patologie   MeSH
• lidé MeSH
• molekulární modely MeSH
• mutace * MeSH
• regulace genové exprese MeSH
• sekvenční analýza DNA MeSH
• substituce aminokyselin * MeSH
• vztahy mezi strukturou a aktivitou MeSH
• železo metabolismus   MeSH
• Check Tag
• dítě MeSH
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

We report two brothers with mild intellectual deficiency, exercise intolerance, rhabdomyolysis, seizures and no hemolysis. Phosphoglycerate kinase (PGK) activity was strongly decreased in their red blood cells. Subsequent molecular analysis of PGK1 revealed hemizygosity for a novel mutation c.756 + 3A > G, in intron 7. Analysis of the effect of this mutation on pre-mRNA processing demonstrated markedly decreased levels of normal PGK1 mRNA. In addition, the c.756 + 3A > G change resulted in abnormally spliced transcripts. If translated, these transcripts mostly encode for C-terminally truncated proteins. The consequences of the c.756 + 3A > G mutation is discussed, as well as the genotype-to-phenotype correlation with regard to previously described mutations (PGK Fukuroi and PGK Antwerp), which also result in C-terminal truncated proteins.

• MeSH
• fenotyp MeSH
• fosfoglycerátkinasa nedostatek   genetika   ultrastruktura   MeSH
• genetické nemoci vázané na chromozom X komplikace   diagnóza   genetika   MeSH
• genotyp MeSH
• hemolýza MeSH
• kosterní svaly patologie   MeSH
• lidé MeSH
• mentální retardace komplikace   MeSH
• mladiství MeSH
• mutace MeSH
• myoglobinurie komplikace   MeSH
• sourozenci MeSH
• vrozené poruchy metabolismu komplikace   diagnóza   genetika   MeSH
• záchvaty komplikace   MeSH
• Check Tag
• lidé MeSH
• mladiství MeSH
• mužské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• kazuistiky MeSH
• práce podpořená grantem MeSH

Wastewater samples from a Swedish chemi-thermo-mechanical pulp (CTMP) mill collected at different purification stages in a wastewater treatment plant (WWTP) were analyzed with an amperometric enzyme-based biosensor array in a flow-injection system. In order to resolve the complex composition of the wastewater, the array consists of several sensing elements which yield a multidimensional response. We used principal component analysis (PCA) to decompose the array's responses, and found that wastewater with different degrees of pollution can be differentiated. With the help of partial least squares regression (PLS-R), we could link the sensor responses to the Microtox® toxicity parameter, as well as to global organic pollution parameters (COD, BOD, and TOC). From investigating the influences of individual sensors in the array, it was found that the best models were in most cases obtained when all sensors in the array were included in the PLS-R model. We find that fast simultaneous determination of several global environmental parameters characterizing wastewaters is possible with this kind of biosensor array, in particular because of the link between the sensor responses and the biological effect onto the ecosystem into which the wastewater would be released. In conjunction with multivariate data analysis tools, there is strong potential to reduce the total time until a result is yielded from days to a few minutes.

• MeSH
• biosenzitivní techniky * MeSH
• chemické látky znečišťující vodu chemie   izolace a purifikace   MeSH
• monitorování životního prostředí * MeSH
• odpadní voda chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

In mature erythrocytes, glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH) yield NADPH, a crucial cofactor of the enzyme glutathione reductase (GR) converting glutathione disulfide (GSSG) into its reduced state (GSH). GSH is essential for detoxification processes in and survival of erythrocytes. We explored whether the anti-inflammatory compounds Bay 11-7082, parthenolide and dimethyl fumarate (DMF) were able to completely deplete a common target (GSH), and to impair the function of upstream enzymes of GSH recycling and replenishment. Treatment of erythrocytes with Bay 11-7082, parthenolide or DMF led to concentration-dependent eryptosis resulting from complete depletion of GSH. GSH depletion was due to strong inhibition of G6PDH activity. Bay 11-7082 and DMF, but not parthenolide, were able to inhibit the GR activity. This approach "Inhibitors, Detection of their common target that is completely depleted or inactivated when pharmacologically relevant concentrations of each single inhibitor are applied, Subsequent functional analysis of upstream enzymes for this target" (IDS), can be applied to a broad range of inhibitors and cell types according to the selected target. The specific G6PDH inhibitory effect of these compounds may be exploited for the treatment of human diseases with high NADPH and GSH consumption rates, including malaria, trypanosomiasis, cancer or obesity.

• MeSH
• dimethyl fumarát farmakologie   MeSH
• eryptóza účinky léků   MeSH
• erytrocyty enzymologie   MeSH
• glukosa-6-fosfátdehydrogenasa * antagonisté a inhibitory   metabolismus   MeSH
• lidé MeSH
• nitrily farmakologie   MeSH
• seskviterpeny farmakologie   MeSH
• sulfony farmakologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Hexokinase (HK) is a key enzyme of glycolysis, the only metabolic pathway able to provide the red blood cell with ATP. HK deficiency is a very rare hereditary disorder with severe chronic nonspherocytic hemolytic anemia (HNSHA) as a major clinical feature. To date, only 24 patients with HK deficiency have been identified. Here, we report the molecular analysis of six new cases of HK deficiency. A total of six different mutations were detected in HK1, four of them described here for the first time: c.2599C>T p.(His867Tyr), c.1799C>T p.(Thr600Met), c.873-2A>G and c.493-1G>A. The pathogenic nature of the identified missense mutations was confirmed by biochemical and 3-dimensional structural analysis. The effects of the novel splice site mutation c.873-2A>G were studied at the level of pre-mRNA processing, and confirmed at the protein level. All together, these results provide a better insight into the pathogenesis of this rare red cell disorder, and contribute to a better understanding of the genotype-phenotype correlation in HK deficiency.

• MeSH
• dítě MeSH
• erytrocyty enzymologie   MeSH
• genetické asociační studie MeSH
• hexokinasa nedostatek   genetika   MeSH
• kojenec MeSH
• lidé MeSH
• missense mutace MeSH
• místa sestřihu RNA genetika   MeSH
• mladý dospělý MeSH
• mutace MeSH
• předškolní dítě MeSH
• Check Tag
• dítě MeSH
• kojenec MeSH
• lidé MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• předškolní dítě MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH