Molecular mechanisms of pathogenesis of myeloproliferative neoplasms: deregulation of genes involved in cell proliferation and apoptosis

Mijeloproliferativne neoplazije (MPN) su hronični hematološki maligniteti koji se odlikuju autonomnom proliferacijom opredeljenih progenitora hematopoeze i aberantnom aktivacijom tirozin kinaznih signalnih puteva u kombinaciji sa snažnim odgovorom na citokine i faktore rasta. Tri bolesti predstavljaju MPN u užem smislu: policitemija vera (PV), esencijalna trombocitemija (ET) i mijelofibroza (MF). Jedna od komplikacija ovih oboljenja je njihova kasna evolucija u akutnu mijeloidnu leukemiju (AML). Važno obeležje ovih bolesti ja prisustvo „missense“ JAK2-V617F mutacije u sve tri bolesti, a procenat zastupljenosti mutacije po bolestima je različit. TakoĎe pokazano je da kod ovih pacijenata postoji tzv. efekat “doze gena”, odnosno da različit nivo V617F alela utiče kliničku sliku bolesti. JAK2-V617F mutacija dogaĎa se u 80% slučajeva na specifičnom haplotipu koji je nazvan 46/1 haplotip. Na koji način ovaj niz SNP-ova, koji se nalaze u JAK2 genu, predisponira nastajanje mutacije kao i njen uticaj na fenotip MPN, još nije utvrĎeno. Jedan od mogućih mehanizama je uticaj ovog haplotipa na transkripciju. Posebnu pažnju u okviru 46/1 haplotipa je privukao SNP rs12343867, koji u potpunosti asocira sa MPN. Proces apoptoze je deregulisan u hematološkim malignitetima, što dovodi do rezistencije malignih ćelija na signale smrti i obezbeĎuje im duži život u odnosu na normalne ćelije. Proces apoptoze nije detaljno izučen kod MPN, mada se zna da je direktno pogoĎen JAK2-V617F mutacijom. Naime, glavni signalni put preko STAT5 (Signal Transducers and Activators of Transcription) proteina direktno aktivira anti- apoptotski BCL2-xL protein, čime se smanjuje apoptoza. Deregulacija ostalih apoptotskih puteva u MPN nije u potpunosti rasvetljenaMyeloproliferative neoplasms (MPN) are chronic hematological malignancies that are characterized by autonomous proliferation of committed hematopoietic progenitors and aberrant activation of tyrosine kinase signaling pathways, in combination with a strong response to cytokines and growth factors. Three major entities constitute MPN: polycythaemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). One of the complications of these diseases is their late evolution into acute myeloid leukemia. Important feature of these diseases is the presence of missense mutation JAK2- V617F and its variable representation among MPN entities. It is also shown that there is socalled effect of "gene dosage" in these patients, meaning that a different level of V617F alleles influences the clinical picture of the disorders. JAK2-V617F mutation occurs in 80% of cases on a specific haplotype, called 46/1 haplotype. Exact mechanism of action of this set of SNPs, that are located within the JAK2 gene, has not been determined yet. One of the possible mechanisms could be that it effects transcription. Among eight SNPs, included in this haplotype, SNP rs12343867 has drawn special attention because of its strong association with the MPN. The process of apoptosis is deregulated in hematological malignancies, leading to resistance of cancer cells to death signals, thus providing them a longer life span compared to normal cells. The process of apoptosis has not been extensively studied in MPN, although it is known that it is directly affected by the JAK2-V617F mutation. Specifically, the main signaling pathway through STAT5 protein directly activates anti-apoptotic BCL2- xL protein, thereby reducing apoptosis. Deregulation of other apoptotic pathways in MPN is not fully understoo

Molecular markers in systemic sclerosis: candidate genes and therapeutic modalities

Sistemska skleroza je izuzetno složeno i heterogeno oboljenje u pogledu kliničke prezentacije, progresije bolesti, zahvaćenosti organa i ishodu. Na individualnom nivou, ova složenost predstavlja izazov u predviđanju progresije bolesti i lečenju. U ovom radu će biti prikazana dosadašnja saznanja o molekularnim markerima u sistemskoj sklerozi, signalnim putevima koji su uključeni u patogenezu, kao i njihova uloga u kliničkoj prezentaciji bolesti. Biće predstavljeni novi terapijski pristupi u lečenju, sa posebnim osvrtom na biološku terapiju zasnovanu na genima kandidatima, kao i mogućnosti koje regenerativna medicina nudi u lečenju ove autoimune sistemske bolesti.Systemic sclerosis is an extremely complex and heterogeneous disease in terms of clinical presentation, disease progression, organ involvement, and outcome. At the individual level, this complexity presents a challenge in predicting disease progression and treatment. This paper will present the current knowledge about molecular markers in systemic sclerosis, signaling pathways involved in pathogenesis, as well as their role in the clinical presentation of the disease. New therapeutic approaches in treatment will be presented, with special emphasis on biological therapy based on candidate genes, as well as the possibilities that regenerative medicine offers in the treatment of this autoimmune systemic disease

Intra-articular injection of autologous adipose-derived mesenchymal stem cells in the treatment of knee osteoarthritis

BackgroundOsteoarthritis (OA) is a chronic degenerative joint disease and is considered to be the fourth leading cause of disability and the second cause of inability to work in men. Recently, adipose-derived mesenchymal stem cells (AD-MSCs) came into focus for regenerative medicine as a promising tool for the treatment of OA. The administration of stem cells into impaired joints results in pain relief and improves quality of life, accompanied by restoration of hyaline articular cartilage. MethodsIn the present study, nine patients (including two patients with bilateral symptoms) diagnosed with osteoarthritis (International Knee Documentation grade B in 5 and grade D in six knees) were treated using a single injection of AD-MSCs at a concentration of 0.5-1.0 x 10(7) cells and were followed up for 18months. During follow-up, all the cases were evaluated clinically by Knee Society score (KSS), Hospital for Special Surgery knee score (HSS-KS), Tegner-Lysholm (T-L) score and visual analogue scale (VAS) of pain, as well as by plain radiography and by magnetic resonance imaging visualization with 2D Magnetic Resonance Observation of Cartilage Repair Tissue (MOCART) score assessment. ResultsSignificant improvement of all four clinical scores was observed within the first 6months (KSS for 41.4 points, HSS-KS for 33.9 points, T-L score for 44.8 points, VAS of pain from 54.5 to 9.3) and improvement persisted throughout the rest of the follow-up. MOCART score showed significant cartilage restoration (from 43 7.2 to 63 +/- 17.1), whereas radiography showed neither improvement, nor further joint degeneration. ConclusionsThe results obtained in the present study provide good basis for prospective randomized controlled clinical trials with respect to the use of AD-MSCs in the treatment of osteoarthritis.This is the peer reviewed version of the paper : Spasovski, D., Spasovski, V., Baščarević, Z., Stojiljković, M., Vreća, M., Anđelković, M., & Pavlović, S. (2018). Intra-articular injection of autologous adipose-derived mesenchymal stem cells in the treatment of knee osteoarthritis. The Journal of Gene Medicine, 20(1), e3002. [https://doi.org/10.1002/jgm.3002

Novel Patched 1 mutations in patients with nevoid basal cell carcinoma syndrome - case report

Nevoid basal cell carcinoma syndrome (Gorlin syndrome) is a rare autosomal dominant disorder characterized by numerous basal cell carcinomas, keratocystic odontogenic tumors of the jaws, and diverse developmental defects. This disorder is associated with mutations in tumor suppressor gene Patched 1 (PTCH1). We present two patients with Gorlin syndrome, one sporadic and one familial. Clinical examination, radiological and CT imaging, and mutation screening of PTCH1 gene were performed. Family members, as well as eleven healthy controls were included in the study. Both patients fulfilled the specific criteria for diagnosis of Gorlin syndrome. Molecular analysis of the first patient showed a novel frameshift mutation in exon 6 of PTCH1gene (c.903delT). Additionally, a somatic frameshift mutation in exon 21 (c.3524delT) along with germline mutation in exon 6 was detected in tumor-derived tissue sample of this patient. Analysis of the second patient, as well as two affected family members, revealed a novel nonsense germline mutation in exon 8 (c.1148 C gt A)

The role of FasR/FasL system in pathogenesis of myeloprolyferative neoplasms

Mijeloproliferativne neoplazije (MPN) su hematološki maligniteti koji se karakterišu nekontrolisanom ćelijskom proliferacijom i poremećajem u procesu apoptoze. Sistem FasR/FasL je uključen u kontrolu apoptoze u različitim tipovima ćelija. U ovom radu je izučavana uloga sistema FasR/FasL u patogenezi mijeloproliferativnih neoplazija. Upoređena je ekspresija FasR i FasL između pacijenata sa MPN (24) i zdravih kontrola korišćenjem metode 'real-time' PCR. Detektovana je povećana ekspresija FasR kod pacijenata sa MPN. Nije utvrđena razlika u ekspresiji FasL. Mutacija B617F u JAK2 genu, karakteristična za MPN, je nađena kod 13 od 24 pacijenta. Pokazano je da ekspresija FasR i FasL nije povezana sa prisustvom B617F JAK2 mutacije.Myeloproliferative neoplasms (MPN) are hematological malignancies characterized by uncontrolled cell proliferation and impaired apoptosis. The FasR/FasL system is involved in the control of apoptosis in different cell types. Here we have investigated the role of FasR/FasL in the pathogenesis of MPNs. We compared FasR/FasL expression between MPN patients (24) and healthy individuals using the real-time PCR assay. We found an increase of FasR expression in MPN patients. No difference was detected in FasL expression. Mutation V617F in the JAK2 gene, a hallmark of MPN, was detected in 13/24 patients. We found that neither FasR nor FasL expression were related to the presence of JAK2 V617F mutation

Impact of alterations in X-linked IRAK1gene and miR-146a on susceptibility and clinical manifestations in patients with systemic sclerosis

Systemic sclerosis (SSc) is a heterogeneous multisystem autoimmune disease with unknown etiology. Numerous studies have indicated that the disease heterogeneity implies various genetic abnormalities. Considering that SSc is characterized by a strong sex bias and that the position of IRAK1 gene is on the X chromosome, we assume that variations in IRAK1 gene could explain female predominance of SSc. It was previously described that miR-146a has a role in 'fine-tuning' regulation of the TLR/NF-kB signaling pathway through down-regulation of IRAK1 gene. The aim of the present study was to analyze both variants and expression level of IRAK1 and miR-146a genes in terms of susceptibility to SSc and clinical presentation of SSc patients. We analyzed variants IRAK1 rs3027898 C gt A and miR-146a rs2910164 C gt G in 102 SSc patients and 66 healthy subjects. Genotyping was performed by Sanger sequencing. Expression level of IRAK1 mRNA and miR-146a in PBMCs was performed in subset of 50 patients and 13 healthy controls by RT-qPCR. Our results showed that there was no association between IRAK1 rs3027898 and the risk of SSc in women. However, the analysis of genotype distribution of the mir-146a rs2910164 C gt G variant indicated that CC genotype shows strong association with lung fibrosis and active form of the disease. When expression level of IRAK1 gene was analyzed, we detected significant downregulation of IRAK1 mRNA in SSc patients compared to controls, as well as in male compared to female patients, in patients with ACAS autoantibodies and in patients with severe skin involvement. Regarding the expression level of miR-146a, we have found significantly reduced expression in SSc patients, in patients with skin involvement and in male SSc patients. The results from this study indicate that expression of IRAK1 gene could explain phenotypic heterogeneity of SSc and may be involved in the pathogenesis of SSc due to its differential expression in certain subgroups. Our results also suggested that miR-146a rs2910164 CC genotype may be predisposing factor for development lung fibrosis and more progressive form of SSc. Results from relative expression analysis of miR-146a demonstrated that changes in the level of this miRNA may have an impact on development and clinical course of SSc.This is the peer reviewed version of the paper: Vreca, M., Andjelkovic, M., Tosic, N., Zekovic, A., Damjanov, N., Pavlovic, S., & Spasovski, V. (2018). Impact of alterations in X-linked IRAK1gene and miR-146a on susceptibility and clinical manifestations in patients with systemic sclerosis. Immunology Letters, 204, 1–8. [https://doi.org/10.1016/j.imlet.2018.10.002

Molecular Biomarkers in Perthes Disease: A Review

Background: Perthes disease is a juvenile form of osteonecrosis of the femoral head that affects children under the age of 15. One hundred years after its discovery, some light has been shed on its etiology and the biological factors relevant to its etiology and disease severity. Methods: The aim of this study was to summarize the literature findings on the biological factors relevant to the pathogenesis of Perthes disease, their diagnostic and clinical significance, and their therapeutic potential. A special focus on candidate genes as susceptibility factors and factors relevant to clinical severity was made, where studies reporting clinical or preclinical results were considered as the inclusion criteria. PubMed databases were searched by two independent researchers. Sixty-eight articles were included in this review. Results on the factors relevant to vascular involvement and inflammatory molecules indicated as factors that contribute to impaired bone remodeling have been summarized. Moreover, several candidate genes relevant to an active phase of the disease have been suggested as possible biological therapeutic targets. Conclusions: Delineation of molecular biomarkers that underlie the pathophysiological process of Perthes disease can allow for the provision of earlier and more accurate diagnoses of the disease and more precise follow-ups and treatment in the early phases of the disease.Supplementary information:[https://hdl.handle.net/21.15107/rcub_imagine_2089

Crosstalk between Glycogen-Selective Autophagy, Autophagy and Apoptosis as a Road towards Modifier Gene Discovery and New Therapeutic Strategies for Glycogen Storage Diseases

Glycogen storage diseases (GSDs) are rare metabolic monogenic disorders characterized by an excessive accumulation of glycogen in the cell. However, monogenic disorders are not simple regarding genotype-phenotype correlation. Genes outside the major disease-causing locus could have modulatory effect on GSDs, and thus explain the genotype-phenotype inconsistencies observed in these patients. Nowadays, when the sequencing of all clinically relevant genes, whole human exomes, and even whole human genomes is fast, easily available and affordable, we have a scientific obligation to holistically analyze data and draw smarter connections between genotype and phenotype. Recently, the importance of glycogen-selective autophagy for the pathophysiology of disorders of glycogen metabolism have been described. Therefore, in this manuscript, we review the potential role of genes involved in glycogen-selective autophagy as modifiers of GSDs. Given the small number of genes associated with glycogen-selective autophagy, we also include genes, transcription factors, and non-coding RNAs involved in autophagy. A cross-link with apoptosis is addressed. All these genes could be analyzed in GSD patients with unusual discrepancies between genotype and phenotype in order to discover genetic variants potentially modifying their phenotype. The discovery of modifier genes related to glycogen-selective autophagy and autophagy will start a new chapter in understanding of GSDs and enable the usage of autophagy-inducing drugs for the treatment of this group of rare-disease patients

Variants in vdr and nramp1 genes as susceptibility factors for tuberculosis in the population of Serbia

Tuberculosis (TB) is granulomatous diseases caused by Mycobacterium tuberculosis (MTB). TB is a highly infectious disease that primarily affects the lungs. One-third of human population is infected with MTB, therefore it is of utmost significance to determine the factors that influence the individual susceptibility to the disease. Host genetic factors have been recognized as essential for susceptibility to TB, since only 5% to 10% of infected individuals develop the disease. A number of candidate genes has been intensively studied, the most of which were connected with the function of macrophages, thus participating in immune response. Here we examined the gene variants of VDR (FokI) and NRAMP1 (INT4, D543N, 3'UTR) genes in aim to make the correlation between these genetic factors and risk of TB in Serbian patients. This study included 110 TB patients and 67 healthy controls. Pulmonary TB was diagnosed by clinical symptoms, radiological evidence of TB and bacteriological criteria (Culture-positive/smear-positive). Genotyping was performed using PCR-RFLP method. Our findings revealed significant prevalence of ff genotype and variant allele f of the FokI VDR gene variant in patients compared to control group. Based on the our results the carriers of ff genotype are five times more at risk to tuberculosis than carriers of FF and Ff genotype in our population. The results of analyzed SNPs in NRAMP1 gene showed no statistically significant difference in distribution of the gene variants between patient and control groups. Therefore, we could conclude that the genotype ff of the VDR gene is factor that strongly contribute to susceptibility to TB in Serbian population