The importance of genomic profiling for differential diagnosis of pediatric lung disease patients with suspected ciliopathies

Uvod/Cilj Izmenjena funkcija aksonemalne strukture dovodi do ciliopatija (motornih i senzornih), koje su do sada povezane sa brojnim pedijatrijskim poremećajima, uključujući i respiratorne. Primarna cilijarna diskinezija (PCD) najčešća je ciliopatija, koja nastaje kao posledica poremećaja u motornim cilijama. Promenjena struktura i/ ili funkcija motornih cilija dovodi do neonatalnog respiratornog distresa, hroničnog vlažnog kašlja, simptoma nazalne sekrecije, bronhoektazija, hronične upale sinusa i uha, a 50% bolesnika ima i situs inversus. Ovi simptomi su prilično uobičajeni kod male dece i u drugim stanjima; stoga je uspostavljanje precizne dijagnoze otežano. Cilj ovog istraživanja je ukazivanje na značaj genomskog profilisanja bolesnika i dizajniranje strategije za genetičku analizu podataka kod bolesnika suspektnih na ciliopatije sa kliničkom slikom sličnom drugim bolestima pluća. Metode Sproveli smo bioinformatičku analizu podataka dobijenih metodom sekvenciranja nove generacije 21 bolesnika sa potvrđenom ili suspektnom dijagnozom PCD-a. Analizirano je 93 gena: 29 PCD gena, 45 gena asociranih sa pojedinačnim simptomima plućnih bolesti i 19 gena asociranih sa senzornim ciliopatijama. Rezultati Dizajnirani algoritam za genetičku analizu NAM je omogućio da potvrdimo kliničku i uspostavimo genetičku dijagnozu kod 17/21 (80,95%) bolesnika, među kojima je 11/21 (52,38%) PCD bolesnika. Kod 3/21 (14,28%) bolesnika detektovane su monoalelske varijante u PCD genima, kod 6/21 (28,57%) bolesnika detektovane su varijante u genima relevantnim za druga plućna oboljenja, dok je kod 1/21 (4,76%) bolesnika genetička osnovna bolesti ostala nerazjašnjena. Zaključak Dizajniranje strategije za lakše i brže uspostavljanje konačne dijagnoze ciliopatija je obavezno i uključuje i kliničku i genetičku potvrdu bolesti.Introduction/Objective Dysfunction of the axonemal structure leads to ciliopathies. Sensory and mo-tile ciliopathies have been associated with numerous pediatric diseases, including respiratory diseases. Primary ciliary dyskinesia (PCD) is ciliopathy linked to the dysfunction of motile cilia. Motile ciliary dys-function in childhood leads to chronic rhinosinusitis, persistent cough, neonatal respiratory distress, bronchiectasis, and situs inversus (SI) have 50% of patients. These symptoms are common among pediatric lung diseases, which additionally makes it difficult to establish the accurate diagnosis. The aim of the study was to point out the significance of genomic profiling for patients with suspected ciliopathies and to design a strategy for genomic analysis relevant for differential diagnosis of lung disease patients with suspected ciliopathies. Methods We conducted a bioinformatic analysis of data generated by New Generation Sequencing (NGS) approach of 21 patients with final or suspected diagnosis of PCD. It was analyzed 93 genes: 29 PCD genes, 45 genes related to individual symptoms of lung diseases, and 19 genes related to sensory ciliopathies. Results the algorithm we have designed, enabled us to establish the clinical and genetic diagnosis for 17/21 (80.95%) patients, among which 11/21 (52.38%) were PCD patients. In 3/21 (14.28%) patients we detected monoallelic variants in PCD disease-causing genes. In 6/21 (28.57%) patients, variants in genes for other pulmonary diseases were detected, and for one patient, genetic background of disease remained unclear. Conclusion an improved strategy for easier and faster establishment of final diagnosis of ciliopathies is mandatory and includes both, clinical and genetic confirmation of disease

Immunoglobulin heavy chain gene rearrangements in patients with Gaucher disease

Uvod: Nekoliko studija u literaturi navode dokaze o povećanoj incidenci hematoloških komplikacija u bolesnika sa Gošeovom bolešću, uključujući monoklonsku i poliklonsku gamapatiju i hematološke malignitete, a posebno multipli mijelom. Metode: Određivana je serumska koncentracija imunoglobulina kao i rearanžman gena za teški lanac imunoglobulina - IGH, PCR analizom. Klonalni PCR produkti su direktno sekvencirani i analizirani koristeći adekvatne alate i baze podataka. Monoklonski proteini seruma su detektovani i identifikovani metodom elektroforeze. Rezultati: Među 27 bolesnika, klonalni IGH rearanžman je otkriven kod osmoro, od kojih je petoro imalo i monot klonski protein u serumu. Hipergamaglobulinemija je otkrivena u 9/27 bolesnika. Podaci o praćenju za 17 bolesnika su pokazali da je klonalni rearanžman ostao isti u četiri bolesnika, dok je u jednog bolesnika iščezao tokom perioda praćenja. Preostalih 12/17 bolesnika nisu imali klonalni IGH rearanžman niti su ga stekli nakon perioda praćenja. Zaključak: Iako klonska ekspanzija može da nastane relativno rano u toku Gošeove bolesti, barem sudeći prema rearanžmanu IGH gena, detektovani klonovi mogu biti tranzitorni. Pažljivo kliničko praćenje ovih bolesnika je obavezno, uključujući i nadzor nad limfoidnim neoplazmama, posebno multiplim mijelomom.Background: Several studies support the evidence of increased incidence of hematological complications in Gaucher disease including monoclonal and polyclonal gammopathies and blood malignancies, especially multiple myeloma. Methods: Serum concentrations of immunoglobulins and PCR analysis of the IGH gene rearrangements were performed. The clonal PCR products were directly sequenced and analyzed with the appropriate database and tools. Serum monoclonal proteins were detected and identified by electrophoresis. Results: Among 27 Gaucher patients, clonal IGH rearrangement was discovered in eight, with 5/8 having also serum monoclonal protein. Elevated immunoglobulins were detected in 9/27 patients. Follow-up data for 17 patients showed that the clonal rearrangement remained the same in four of them, however, in one patient it disappeared after the follow-up period. The remaining 12/17 patients were without previous IGH clonal rearrangement and remained so after the follow-up. Conclusions: Although clonal expansion may occur relatively early in the disease course, at least judging by the IGH gene rearrangements in Gaucher patients, the detected clones may be transient. A careful clinical follow-up in these patients is mandatory, including monitoring for lymphoid neoplasms, especially multiple myeloma

Wilms tumor (wt)1 gene expression in children with acute leukemia in Serbia

Acute leukemias constitute the most common malignancy in childhood, accounting for 25-35% of all cancer in children. They are divided into acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). Genetic susceptibility is known to play a major role in childhood leukemias. Wilms tumor (WT)1 is a zinc finger transcription factor involved in regulating the process of cell differentiation; it has been implicated in a wide range of human neoplasms. WT1 overexpression in the bone marrow at diagnosis is reported to be an independent negative prognostic factor in adults and children with AML. The aim of the present investigation was to determine the expression of WT1 in the bone marrow of children with AML and ALL in Serbia and its possible impact on patient survival. We determined bone marrow WT1 expression levels by reverse -transcription polymerase chain reaction (RT-PCR) at diagnosis in 20 children with AML and 20 children with ALL (16 B-ALL and 4 T-ALL), as well as 15 age- and sex-matched controls who were evaluated for immune thrombocytopenic purpura (ITP). For children with AML, follow-up samples were also analyzed one month after treatment initiation and at variable later timepoints of control punctures. The results were normalized based on WT1 expression in controls. We found that children with AML had significantly higher WT1 expression at diagnosis (median SD: 139.42 244.03) than those with ALL (1.18 54.37; Mann -Whitney U=82; p lt 0.01) and ITP (0.76 1.01; U=32; p lt 0.01). Patients with T-ALL had higher WT1 expression than those with B-ALL, though significance was not reached due to subgroup size; differences between AML subgroups according to the French-American-British (FAB) classification were also below the level of significance, though a tendency toward higher values in M3 and M4 leukemias was notable. There was also a tendency toward higher values in 14 children with AML who were still alive after a median follow-up of 1.5 years (181.42 192.52) than in 6 who succumbed to the disease (104.29 354.87). All children with AML who had WT1 expression 1 month after diagnosis below the fourth quartile (10 of 10) were still alive, while only 2 of 5 with 1 -month WT1 expression in the fourth quartile survived (Fisher's exact test: p=0.0952). Taken together, our results support a role for WT1 in the diagnostic workup in children with acute leukemia, although it needs to be considered in view of a complex and indvidualized context

Structural study of Pt(II) and Pd(II) complexes with quinoline-2-carboxaldehyde thiosemicarbazone

Two square-planar complexes, [PtLCl] (1) and [PdLCl] (2), were synthesized with quinoline-2-carboxaldehyde thiosemicarbazone ligand (HL), and characterized by IR and NMR spectroscopy and single crystal X-ray diffraction analysis. In both complexes, L- is coordinated tridentately via the same donor atom set, while the fourth coordination site is occupied by a chloride ion. However, the complexes are not isostructural due to different types of non-covalent intermolecular interactions. These interactions were analyzed using Hirshfeld surfaces and two-dimensional fingerprint plots

Influence of clonal b cell proliferation and cytokine gene polymorphisms on disease severity and occurence of monoclonal gammopathy in patients with Gaucher disease

Gošeova bolest je oboljenje lizozoma kod kojeg je korelacija između genotipa i fenotipske ekspresije bolesti slaba te se samim tim ni težina bolesti ne može pouzdano predvideti. Citopatološke promene u ovoj bolesti nisu izazvane samo nakupljanjem nerazgrađenog glikolipida, već i aktivacijom makrofaga, a koncentracije različitih citokina, važnih za proces inflamacije, su povišene u serumu ovih bolesnika. Takođe, nekoliko studija u literaturi navode dokaze o povećanoj incidenciji hematoloških komplikacija u bolesnika sa Gošeovom bolešću, uključujući monoklonsku i poliklonsku gamapatiju i hematološke malignitete, a posebno multipli mijelom. U kohorti naših bolesnika u Srbiji je ranije već opisana visoka incidencija rearanžmana gena za teški lanac imunoglobulina, što sugeriše da se aktivacija imunskog sistema može javiti relativno rano tokom bolesti. Predmet rada istraživanja ove studije je bila analiza uticaja klonskog rearanžmana gena za teški lanac imunoglobulina i/ili prisustva pojedinih alela gena koji kodiraju molekule važne u inflamaciji (TNFα, IL-10 i IL-6) na težinu kliničke slike obolelih od Gošeove bolesti. Materijal i metod: U ovu kohortnu studiju su bili uključeni pacijenti oboleli od Gošeove bolesti u Srbiji koji su u periodu od 2008 – 2016. godine lečeni na Klinici za hematologiju i Klinici za endokrinologiju, dijabetes i bolesti metabolizma Kliničkog centra Srbije, u Beogradu, kao i Klinici za hematologiju Kliničkog centra u Nišu. Kriterijumi za isključenje iz studije su bili: odbijanje pacijenta da učestvuje u studiji, smrt pacijenta ili emigracija u druge države. Kontrolna grupa za poređenje učestalosti ispitivanih polimorfnih genotipova za citokinske gene je bila selektovana iz baze DNK uzoraka Laboratorije za imunologiju Instituta za Mikrobiologiju i imunologiju. Svaki od ispitanika je detaljno obavešten o cilju studije i potpisao je dobrovoljan pristanak za učestvovanje u njoj. Iz uzoraka pune venske krvi je vršena izolacija DNK, a detekcija i analiza polimorfizama citokinskih gena TNF, IL10 i IL6 je vršena metodom Real-time PCR, u Laboratoriji za imunologiju Instituta za mikrobiologiju i imunologiju Medicinskog fakulteta Univerziteta u Beogradu...Gaucher disease is a lysosomal storage disease showing weak genotype-phenotype correlation, with poorly predictable disease severity. Cytopathological alterations are caused not only by the accumulation of the undegraded glycolipid substrate, but also by macrophage activation, displaying serum elevation of various cytokines with important influence to inflammation. Several studies in the literature showed increased incidence of hematological complications in Gaucher patients, including monoclonal and polyclonal gammopathies as well as hematological malignancies, especially multiple myeloma. We had already described a high incidence of immunoglobulin heavy chain gene rearrangements in a cohort of Serbian patients, suggesting that an early occurrence of immune system activation is possible early in the disease course. The object of this study was the analysis of clonal rearrangements of IGH genes and frequencies of alleles of cytokine genes important for inflammation (TNFα, IL-10 and IL-6) as well as their influence on disease severity in Gaucher patients. Material and methods: the cohort study included patients with Gaucher disease treated in Serbia since 2008 – 2016, at the Clinic of hematology and Clinic for endocrinology, diabetes and metabolic diseases of Clinical center of Serbia in Belgrade, as well as Clinic of hematology, Clinical Center of Niš. Exclusion criteria were: patient refusal to participate in the study, death of a patient and / or emmigration of a patient. Control group for frequency analysis of polymorphic genotypes of cytokine genes was selected out of the database of DNA samples acquired from Laboratory of immunology, Institute of microbiology and immunology. Every subject was thoroughly informed of the aim of the study and signed the informed consent in order to participate in it voluntarily. Venous blood samples were taken with subsequent DNA isolation permitting detection and analysis of cytokine gene polymorphisms (TNF, IL10 and IL6) using the method of Real-time PCR. This was performed in the Laboratory of immunology, Institute of microbiology and immunology, School of medicine, University of Belgrade..

Hipertekstualnost kao oblik digitalizacije teorije / Hypertextuality as a Form of Digitalization of Theory

The introduction of the notion of hypertextuality (as the key concept of the new media) into the fields of visual arts, film, fashion, photography, postdramatic theatre, dance and music has resulted in the formation of constructivist or soft art theory. The basic method of art theory thus labelled is reading and the (hyper) text is the fundamental category founded on the concept of the sign and on the discursively encoded order of signs in textual form

Influence of clonal b cell proliferation and cytokine gene polymorphisms on disease severity and occurence of monoclonal gammopathy in patients with Gaucher disease

Gošeova bolest je oboljenje lizozoma kod kojeg je korelacija između genotipa i fenotipske ekspresije bolesti slaba te se samim tim ni težina bolesti ne može pouzdano predvideti. Citopatološke promene u ovoj bolesti nisu izazvane samo nakupljanjem nerazgrađenog glikolipida, već i aktivacijom makrofaga, a koncentracije različitih citokina, važnih za proces inflamacije, su povišene u serumu ovih bolesnika. Takođe, nekoliko studija u literaturi navode dokaze o povećanoj incidenciji hematoloških komplikacija u bolesnika sa Gošeovom bolešću, uključujući monoklonsku i poliklonsku gamapatiju i hematološke malignitete, a posebno multipli mijelom. U kohorti naših bolesnika u Srbiji je ranije već opisana visoka incidencija rearanžmana gena za teški lanac imunoglobulina, što sugeriše da se aktivacija imunskog sistema može javiti relativno rano tokom bolesti. Predmet rada istraživanja ove studije je bila analiza uticaja klonskog rearanžmana gena za teški lanac imunoglobulina i/ili prisustva pojedinih alela gena koji kodiraju molekule važne u inflamaciji (TNFα, IL-10 i IL-6) na težinu kliničke slike obolelih od Gošeove bolesti. Materijal i metod: U ovu kohortnu studiju su bili uključeni pacijenti oboleli od Gošeove bolesti u Srbiji koji su u periodu od 2008 – 2016. godine lečeni na Klinici za hematologiju i Klinici za endokrinologiju, dijabetes i bolesti metabolizma Kliničkog centra Srbije, u Beogradu, kao i Klinici za hematologiju Kliničkog centra u Nišu. Kriterijumi za isključenje iz studije su bili: odbijanje pacijenta da učestvuje u studiji, smrt pacijenta ili emigracija u druge države. Kontrolna grupa za poređenje učestalosti ispitivanih polimorfnih genotipova za citokinske gene je bila selektovana iz baze DNK uzoraka Laboratorije za imunologiju Instituta za Mikrobiologiju i imunologiju. Svaki od ispitanika je detaljno obavešten o cilju studije i potpisao je dobrovoljan pristanak za učestvovanje u njoj. Iz uzoraka pune venske krvi je vršena izolacija DNK, a detekcija i analiza polimorfizama citokinskih gena TNF, IL10 i IL6 je vršena metodom Real-time PCR, u Laboratoriji za imunologiju Instituta za mikrobiologiju i imunologiju Medicinskog fakulteta Univerziteta u Beogradu...Gaucher disease is a lysosomal storage disease showing weak genotype-phenotype correlation, with poorly predictable disease severity. Cytopathological alterations are caused not only by the accumulation of the undegraded glycolipid substrate, but also by macrophage activation, displaying serum elevation of various cytokines with important influence to inflammation. Several studies in the literature showed increased incidence of hematological complications in Gaucher patients, including monoclonal and polyclonal gammopathies as well as hematological malignancies, especially multiple myeloma. We had already described a high incidence of immunoglobulin heavy chain gene rearrangements in a cohort of Serbian patients, suggesting that an early occurrence of immune system activation is possible early in the disease course. The object of this study was the analysis of clonal rearrangements of IGH genes and frequencies of alleles of cytokine genes important for inflammation (TNFα, IL-10 and IL-6) as well as their influence on disease severity in Gaucher patients. Material and methods: the cohort study included patients with Gaucher disease treated in Serbia since 2008 – 2016, at the Clinic of hematology and Clinic for endocrinology, diabetes and metabolic diseases of Clinical center of Serbia in Belgrade, as well as Clinic of hematology, Clinical Center of Niš. Exclusion criteria were: patient refusal to participate in the study, death of a patient and / or emmigration of a patient. Control group for frequency analysis of polymorphic genotypes of cytokine genes was selected out of the database of DNA samples acquired from Laboratory of immunology, Institute of microbiology and immunology. Every subject was thoroughly informed of the aim of the study and signed the informed consent in order to participate in it voluntarily. Venous blood samples were taken with subsequent DNA isolation permitting detection and analysis of cytokine gene polymorphisms (TNF, IL10 and IL6) using the method of Real-time PCR. This was performed in the Laboratory of immunology, Institute of microbiology and immunology, School of medicine, University of Belgrade..