Andrea Soltysova

Phenylalanine hydroxylase deficiency in the Slovak population: Genotype–phenotype correlations and genotype-based predictions of BH4-responsiveness

We investigated the mutation spectrum of the phenylalanine hydroxylase gene (PAH) in a cohort of patients from 135 Slovak PKU families. Mutational screening of the known coding region, including conventional intron splice sites, was performed using high-resolution melting analysis, with subsequent sequencing analysis of the samples showing deviated melting profiles compared to control samples. The PAH gene was also screened for deletions and duplications using MLPA analysis. Forty-eight different disease causing mutations were identified in our patient group, including 30 missense, 8 splicing, 7 nonsense, 2 large deletions and 1 small deletion with frameshift; giving a detection rate of 97.6%. The most prevalent mutation was the p.R408W, occurring in 47% of all alleles, which concurs with results from neighboring and other Slavic countries. Other frequent mutations were: p.R158Q (5.3%), IVS12+1G>A (5.3%), p.R252W (5.1%), p.R261Q (3.9%) and p.A403V (3.6%). We also identified three novel missense mutations: p.F233I, p.R270I, p.F331S and one novel variant: c.-30A>T in the proximal part of the PAH gene promoter. A spectrum of 84 different genotypes was observed and a genotype based predictions of BH4-responsiveness were assessed. Among all genotypes, 36 were predicted to be BH4-responsive represented by 51 PKU families. In addition, genotype-phenotype correlations were performed.

Sulphide signalling potentiates apoptosis through the up‐regulation of IP3 receptor types 1 and 2

To investigate an interaction between the calcium and sulphide signalling pathways, particularly effects of the slow H2S release donor morpholin‐4‐ium‐4‐methoxyphenyl‐(morpholino)‐phosphinodithioate (GYY4137) on the expression of inositol 1,4,5‐trisphosphate receptors (IP3R) with the possible impact on the apoptosis induction in HeLa cells.

A novel 3D mesenchymal stem cell model of the multiple myeloma bone marrow niche: biologic and clinical applications.

Specific niches within the tumor bone marrow (BM) microenvironment afford a sanctuary for multiple myeloma (MM) clones due to stromal cell-tumor cell interactions, which confer survival advantage and drug resistance. Defining the sequelae of tumor cell interactions within the MM niches on an individualized basis may provide the rationale for personalized therapies. To mimic the MM niche, we here describe a new 3D co-culture ex-vivo model in which primary MM patient BM cells are co-cultured with mesenchymal stem cells (MSC) in a hydrogel 3D system. In the 3D model, MSC with conserved phenotype (CD73+CD90+CD105+) formed compact clusters with active fibrous connections, and retained lineage differentiation capacity. Extracellular matrix molecules, integrins, and niche related molecules including N-cadherin and CXCL12 are expressed in 3D MSC model. Furthermore, activation of osteogenesis (MMP13, SPP1, ADAMTS4, and MGP genes) and osteoblastogenic differentiation was confirmed in 3D MSC model. Co-culture of patient-derived BM mononuclear cells with either autologous or allogeneic MSC in 3D model increased proliferation of MM cells, CXCR4 expression, and SP cells. We carried out immune profiling to show that distribution of immune cell subsets was similar in 3D and 2D MSC model systems. Importantly, resistance to novel agents (IMiDs, bortezomib, carfilzomib) and conventional agents (doxorubicin, dexamethasone, melphalan) was observed in 3D MSC system, reflective of clinical resistance. This 3D MSC model may therefore allow for studies of MM pathogenesis and drug resistance within the BM niche. Importantly, ongoing prospective trials are evaluating its utility to inform personalized targeted and immune therapy in MM.

Deregulation of energetic metabolism in the clear cell renal cell carcinoma: A multiple pathway analysis based on microarray profiling

Clear cell renal cell carcinoma (ccRCC) is the most frequent type of kidney cancer. In order to better understand the biology of ccRCC, we accomplished the gene profiling of fresh tissue specimens from 11 patients with the renal tumors (9 ccRCCs, 1 oncocytoma and 1 renal B-lymphoma), in which the tumor-related data were compared to the paired healthy kidney tissues from the same patients. All ccRCCs exhibited a considerably elevated transcription of the gene coding for carbonic anhydrase IX (CAIX). Moreover, the ccRCC tumors consistently displayed increased expression of genes encoding the glycolytic pathway enzymes, e.g. hexokinase II (HK2) and lactate dehydrogenase A (LDHA) and a decreased expression of genes for the mitochondrial electron transport chain components, indicating an overall reprogramming of the energetic metabolism in this tumor type. This appears to be accompanied by altered expression of the genes of the pH regulating machinery, including ion and lactate transporters. Immunohistochemical staining of tumor tissue sections confirmed the increased expression of CAIX, HK2 and LDHA in ccRCC, validating the microarray data and supporting their potential as the energetic metabolism-related biomarkers of the ccRCC.

Triptolide induces apoptosis through the SERCA 3 upregulation in PC12 cells.

Diterpenoid triepoxide - Triptolide (TTL) - increased protein levels of the noradrenaline transporter in three pheochromocytoma cell lines. This transporter is involved in the apoptosis induction through the inhibition of a transcription factor NF-kappa B. Nevertheless, calcium release from the endoplasmic reticulum can also induce inner mitochondrial pathway of apoptosis in variety of cells. Therefore, the aim of this work was to evaluate an involvement of calcium and, more specifically, the intracellular calcium transport systems in the apoptosis induction in pheochrocytoma cell line PC12. We observed significantly increased amount of reticular calcium in TTL-treated cells compared to control, untreated cells. Surprisingly, gene expression of the IP3 receptors was not changed after the TTL treatment, but ryanodine receptor of the type 2 (RyR2) was downregulated and sarco/endoplasmic reticulum calcium ATPase type 3 (SERCA 3) was upregulated in TTL- treated cells, compared to untreated controls. SERCA 3 blocking with the specific blocker thapsigargin prevented increase in apoptosis observed by the TTL treatment. Decrease in the ATP production by a replacement of glucose in the cultivation medium for its nonutilizable analog 2-deoxyglucose also prevented induction of the apoptosis in TTL-treated PC12 cells. Thus, these results suggest that upregulation of the SERCA 3 is ultimately involved in the TTL-induced apoptosis in PC12 cells.

Augmenting Clinical Interpretability of Thiopurine Methyltransferase Laboratory Evaluation

Objective: Individuals with decreased thiopurine methyltransferase (TPMT) activity are at risk of adverse effects of thiopurine administration whereas its increased activity may inactivate drugs faster. We evaluated genotype-phenotype correlations in patients with suspected hematological malignancies and inflammatory bowel disease from our region based on findings of nonlinear TPMT enzyme kinetics previously unreported. Patients and Methods: The study group comprised 267 individuals. They were screened for the most common variants of low TPMT activity. TPMT activity was measured in erythrocytes using the HPLC rate-blanked method. Results: Thirty-three patients (12.4%) were heterozygous (26 were TPMT*1/*3A, 5 TPMT*1/*2, 2 TPMT *1/*3C) and 1 was a compound heterozygote (*2/*3A). Normal and low normal TPMT activities substantially overlapped in wild-type and heterozygous individuals, whereas high activities were found in 29 wild-type genotyped patients. Extreme and life-threatening toxicity was observed in the compound heterozygote patient. Conclusion: Activity measurement performed at diagnosis provides clinicians with information on immediate pharmacokinetic-related adverse events and/or hypermetabolism, and genotyping may indicate the rate of pharmacodynamic thioguanine nucleotide accumulation due to slower overall thiopurine metabolism.

Slow sulfide donor GYY4137 differentiates NG108-15 neuronal cells through different intracellular transporters than dbcAMP

Cellular differentiation is the process, by which a cell changes from one cell type to another, preferentially to the more specialized one. Calcium fluxes play an important role in this action. Differentiated NG108-15 or PC12 cells serve as models for studying neuronal pathways. NG108-15 cell line is a reliable model of cholinergic neuronal cells. These cells differentiate to a neuronal phenotype due to the dibutyryl cAMP (dbcAMP) treatment. We have shown that a slow sulfide donor - GYY4137 - can also act as a differentiating factor in NG108-15 cell line. Calcium is an unavoidable ion required in NG108-15 cell differentiation by both, dbcAMP and GYY4137, since cultivation in EGTA completely prevented differentiation of these cells. In this work we focused primarily on the role of reticular calcium in the process of NG108-15 cell differentiation. We have found that dbcAMP and also GYY4137 decreased reticular calcium concentration by different mechanisms. GYY4137 caused a rapid decrease in type 2 sarco/endoplasmic calcium ATPase (SERCA2) mRNA and protein, which results in lower calcium levels in the endoplasmic reticulum compared to the control, untreated group. The dbcAMP revealed rapid increase in expression of the type 3 IP3 receptor, which participates in a calcium clearance from the endoplasmic reticulum. These results point to the important role of reticular calcium in a NG108-15 cell differentiation.

Novel SCN1A variants in Dravet syndrome and evaluating a wide approach of patient selection.

Voltage-gated sodium channels are essential for generation and propagation of the action potential mainly in nerve and muscle cells. Causative variants in SCN1A gene which codes the main, pore-forming subunit of the channel expressed in central nervous system are associated predominantly with Dravet syndrome (DS), as well as with generalized epilepsy with febrile seizures plus (GEFS+) making it one of the most significant epilepsy gene. Our goal was to determine whether SCN1A screening is relevant in patients with a broad range of epileptic syndromes. 52 patients diagnosed with DS, generalized epilepsy with GEFS+ or similar types of epileptic syndromes were included. Sequencing of the protein coding parts of the gene complemented with MLPA analysis was carried out. One already described nonsense variant, four novel protein truncating variants and a deletion encompassing the whole SCN1A gene were revealed, all in heterozygous state. All identified variants were found in DS patients with 85.7% sensitivity, thus supporting the role of profound SCN1A gene variants in etiology of DS phenotype. No causative variants were identified in any of non-DS epileptic patients in our cohort, suggesting a minor, but not irrelevant role for SCN1A in patients with other types of childhood epilepsy.

Comprehensive genetic study of cystic fibrosis in Slovak patients in 25 years of genetic diagnostics

Cystic fibrosis (CF) has one of the longest histories in hereditary disease molecular diagnostics. However, identification of causative mutations in the CFTR gene is complicated by over 2000 currently identified mutations; with more still being discovered. Knowledge of mutation spectrum may improve effective routine diagnostics and is obligatory in mutation‐specific treatment.

Endogenous H 2 S producing enzymes are involved in apoptosis induction in clear cell renal cell carcinoma

BackgroundKnowledge about the expression and thus a role of enzymes that produce endogenous H2S - cystathionine-β-synthase, cystathionine γ-lyase and mercaptopyruvate sulfurtransferase - in renal tumors is still controversial. In this study we aimed to determine the expression of these enzymes relatively to the expression in unaffected part of kidney from the same patient and to found relation of these changes to apoptosis. To evaluate patient’s samples, microarray and immunohistochemistry was used.MethodsTo determine the physiological importance, we used RCC4 stable cell line derived from clear cell renal cell carcinoma, where apoptosis induction by a mixture of five chemotherapeutics with/without silencing of H2S-producing enzymes was detected. Immunofluorescence was used to determine each enzyme in the cells.ResultsIn clear cell renal cell carcinomas, expression of H2S-producing enzymes was mostly decreased compared to a part of kidney that was distal from the tumor. To evaluate a potential role of H2S-producing enzymes in the apoptosis induction, we used RCC4 stable cell line. We have found that silencing of cystathionine-β-synthase and cystathionine γ-lyase prevented induction of apoptosis. Immunofluorescence staining clearly showed that these enzymes were upregulated during apoptosis in RCC4 cells.ConclusionBased on these results we concluded that in clear cell renal cell carcinoma, reduced expression of the H2S-producing enzymes, mainly cystathionine γ-lyase, might contribute to a resistance to the induction of apoptosis. Increased production of the endogenous H2S, or donation from the external sources might be of a therapeutic importance in these tumors.