Kristóf Árvai

Molecular Profiling of Parathyroid Hyperplasia, Adenoma and Carcinoma

The objective of the study was to examine proliferation and apoptosis associated gene expression in the whole sequence parathyroid lesions to reveal specific features of carcinoma. This study was based on surgically removed parathyroid tissues, gene expression analysis was performed both at gene and protein level. First, mRNA isolation was performed from deep-frozen tissue samples, and further apoptosis pathway-specific cDNA macroarray analysis was carried out. The results were validated with real-time PCR. Subsequently, protein expression was analyzed with immunhistochemistry on Tissue Micro Array multi-blocks derived from several paraffin-embedded samples. cDNA macroarrays revealed elevated expression of both pro-apoptotic (FAS receptor, TRAIL ligand, CASPASE8, and −4) and anti-apoptotic (cIAP1, APOLLON) genes in benign proliferative lesions compared to that in normal gland. TMA studies showed overexpression of KI67, P53, SURVIVIN and APOLLON protein and failure of expression of P27, BCL2, BAX, CHROMOGRANIN-A, SYNAPTOPHYSIN, CYCLIND1, FLIP, TRAIL, CK8, CK18, CK19 in parathyroid carcinoma was detected. These alterations in gene expression of the investigated products could be used in differentiation between beningn and malignant proliferative processes of the parathyroid gland. Authors conclude that a series of alterations in gene expression such as overexpression of APOLLON, P53, KI67 and suppression of P27, BCL2, BAX lead to uncontrolled cell proliferation, but still not leading to increased apoptotic activity in parathyroid carcinoma.

Fast and robust next-generation sequencing technique using ion torrent personal genome machine for the screening of neurofibromatosis type 1 (NF1) gene

Neurofibromatosis type 1 (NF1) gene exhibits one of the highest spontaneous mutation rates in the human genome. Identification of the NF1 mutation is challenging because the NF1 gene is very large and complex, lacking mutational “hot spots.” There is no clustering of mutations, there are several pseudogenes, and a wide spectrum of different types of mutation has been recognized. To date, NF1 mutations or deleted regions have been detected with a number of techniques. With the appearance of next-generation sequencing (NGS) machines, molecular biology is in a new revolutionary phase. Our aim was to work out a method to use the high-throughput NGS machine, Ion Torrent PGM, in diagnostic settings for neurofibromatosis type 1. In our examination, we could reveal 21 distinct variations in NF1 gene in seven patients. This is an absolutely new method for exploring the genetic background of neurofibromatosis type 1 exhibiting the extremely high throughput of NGS in a diagnostic setting.

Clinical Use of Next-Generation Sequencing in the Diagnosis of Wilson’s Disease

Objective. Wilsons disease is a disorder of copper metabolism which is fatal without treatment. The great number of disease-causing ATP7B gene mutations and the variable clinical presentation of WD may cause a real diagnostic challenge. The emergence of next-generation sequencing provides a time-saving, cost-effective method for full sequencing of the whole ATP7B gene compared to the traditional Sanger sequencing. This is the first report on the clinical use of NGS to examine ATP7B gene. Materials and Methods. We used Ion Torrent Personal Genome Machine in four heterozygous patients for the identification of the other mutations and also in two patients with no known mutation. One patient with acute on chronic liver failure was a candidate for acute liver transplantation. The results were validated by Sanger sequencing. Results. In each case, the diagnosis of Wilsons disease was confirmed by identifying the mutations in both alleles within 48 hours. One novel mutation (p.Ala1270Ile) was found beyond the eight other known ones. The rapid detection of the mutations made possible the prompt diagnosis of WD in a patient with acute liver failure. Conclusions. According to our results we found next-generation sequencing a very useful, reliable, time-saving, and cost-effective method for diagnosing Wilsons disease in selected cases.

Polymorphisms of CSF1 and TM7SF4 genes in a case of mild juvenile Paget's disease found using next-generation sequencing

Juvenile Paget’s disease (JPD) is a rare autosomal-recessive condition. It is diagnosed in young children and characterized by a generalized increase in bone turnover, bone pain, and skeletal deformity. Our patient was diagnosed after a pathological fracture when she was 11 years old. When we first examined her at the age of 30 she had bone pain and deformity in both the femur and tibia. Serum alkaline phosphatase (ALP) level, radiology, bone scintigraphy, and densitometry were monitored. Next generation sequencing (NGS) technology, namely semiconductor sequencing, was used to determine the genetic background of JPD. Seven target genes and regions were selected and analyzed after literature review (TM7SF4, SQSTM1, TNFRSF11A, TNFRSF11B, OPTN, CSF1, VCP). No clear pathogenic mutation was found, but we detected missense polymorphisms in CSF1 and TM7SF4 genes. After treatment with zoledronic acid, infusion bone pain and ALP level decreased. We can conclude that intravenous zoledronic acid therapy is effective and safe for suppressing bone turnover and improving symptoms in JPD, but the long-term effects on clinical outcomes are unclear. Our findings also suggest that NGS may help explore the pathogenesis and aid the diagnosis of JPD.

Next-generation sequencing of common osteogenesis imperfecta-related genes in clinical practice.

Next generation sequencing (NGS) is a rapidly developing area in genetics. Utilizing this technology in the management of disorders with complex genetic background and not recurrent mutation hot spots can be extremely useful. In this study, we applied NGS, namely semiconductor sequencing to determine the most significant osteogenesis imperfecta-related genetic variants in the clinical practice. We selected genes coding collagen type I alpha-1 and-2 (COL1A1, COL1A2) which are responsible for more than 90% of all cases. CRTAP and LEPRE1/P3H1 genes involved in the background of the recessive forms with relatively high frequency (type VII and VIII) represent less than 10% of the disease. In our six patients (1–41 years), we identified 23 different variants. We found a total of 14 single nucleotide variants (SNV) in COL1A1 and COL1A2, 5 in CRTAP and 4 in LEPRE1. Two novel and two already well-established pathogenic SNVs have been identified. Among the newly recognized mutations, one results in an amino acid change and one of them is a stop codon. We have shown that a new full-scale cost-effective NGS method can be developed and utilized to supplement diagnostic process of osteogenesis imperfecta with molecular genetic data in clinical practice.

Effects of imatinib and nilotinib on the whole transcriptome of cultured murine osteoblasts

Numerous clinical observations have confirmed that breakpoint cluster region-abelson fusion oncoprotein tyrosine kinase inhibitors used in leukemia treatment alter bone physiology in a complex manner. The aim of the present study was to analyze the whole transcriptome of cultured murine osteoblasts and determine the changes following treatment with imatinib and nilotinib using Sequencing by Oligonucleotide Ligation and Detection next generation RNA sequencing. This study also aimed to identify candidate signaling pathways and network regulators by multivariate Ingenuity Pathway Analysis. Based on the right-tailed Fishers exact test, significantly altered pathways including upstream regulators were defined for each drug. The correlation between these pathways and bone metabolism was also examined. The preliminary results suggest the two drugs have different mechanisms of action on osteoblasts, and imatinib was shown to have a greater effect on gene expression. Data also indicated the potential role of a number of genes and signaling cascades that may contribute to identifying novel targets for the treatment of metabolic bone diseases.

Next-generation Sequencing in the Clinical Decision Making in Hypertrophic Cardiomyopathy

Next generation sequencing (NGS) is becoming a valuable tool in clinical decisions. Here, we discuss the case of a family (2 parents with 3 children) with hypertrophic cardiomyopathy where we applied a NGS method developed by us to determine the genetic background of the disease. When the youngest sister underwent sudden cardiac arrest and successful reanimation, her genome was tested with this approach and two disease-causing heterozygous mutations in the MYBPC3 gene (p.R495Q and p.S593fs*11) were identified. After this, all of the family members were screened targeting these two mutations. The mother carried the frameshift mutation (p.S593fs*11) while the father’s genome contained the point mutation (p.R495Q). All the children were compound heterozygous. Information collected from our genetic testing panel helped to make the decision of implanting ICD that is associated potentially severe complications in children. This case further reinforces that a full-scale, cost-effective NGS method can be utilized to supplement diagnostic and therapeutic processes of hypertrophic cardiomyopathy in clinical practice.

Az onkohematológiai betegségek kezelésében használt tirozinkináz-gátló imatinib és nilotinib csonthatásainak irodalmi áttekintése és a saját kutatási eredmények bemutatása

Absztrakt A tirozinkinaz-gatlok bizonyos onkohematologiai betegsegek kezeleseben elterjedten hasznalt gyogyszerek. Tobb klinikai tanulmany igazolta, hogy a BCR-ABL specifikus tirozinkinaz-gatlok alkalmazasa komplex es meg nem egyertelműen azonositott modon valtoztatja meg a csontszovet elettani folyamatait. Mivel a kezelesek egyre tobb beteget erintenek, illetve hosszu evtizedekig vagy akar elethosszig is tarthatnak, indokolt ezen mechanizmusok molekularis hatterenek reszletesebb megismerese. A szerzők osszefoglaljak az imatinibbel es a nilotinibbel vegzett, csontanyagcserehez kapcsolodo alapkutatasi eredmenyeket, human klinikai megfigyeleseket, kiegeszitve in vitro osteoblast-sejtkulturakon vegzett sajat kiserleteik eredmenyeivel. Az osszefoglalt kutatasi eredmenyek alapjan az imatinib es a nilotinib csontsejtekre gyakorolt hatasa fugg az alkalmazott hatoanyag-koncentraciotol, a sejtek erettsegi allapotatol, illetve az altaluk kotott receptor-tirozinkinaz utvonalak megoszlasi aranyatol. Jelen kozlemenyben elsőkent keszitettek a hazai szakirodalomban hianypotlo, atfogo irodalmi attekintest a tirozinkinaz-gatlok csontanyagcseret befolyasolo hatasaival kapcsolatban es vegeztek teljes transzkriptom-analizist osteoblastokon a sejtszintű hatasmechanizmus jobb megerteset szolgalva. Orv. Hetil., 2016, 157(36), 1429–1437. | Abstract Tyrosine kinase inhibitors are widely used for treatment of certain oncohematological diseases. Several clinical studies have confirmed that specific BCR-ABL tyrosine kinase inhibitors alter the physiological process of bone tissue in a complex and unclearly identified manner. Since these treatments are being given to more and more patients, and the therapy takes decades or lasts even lifelong, it is justifiable to obtain more detailed knowledge of the molecular background of these mechanisms. In this article the authors summarize preliminary research results and human clinical observations on imatinib and nilotinib which are related to bone metabolism, and present the results of their own experiments in in vitro osteoblast cultures. Based on the presented results, the effects of imatinib and nilotinib on bone cells depend on the concentration of imatinib and nilotinib, the maturation stage of the cells and the distribution ratio of receptor tyrosine kinase signaling pathways. In this study the authors firstly prepared a stop-gap, comprehensive review in the Hungarian literature, regarding the effects of tyrosine kinase inhibitors on bone metabolism. In addition they firstly performed whole transcriptome analysis on osteoblasts in order to obtain a better understanding of the cellular molecular mechanisms. Orv. Hetil., 2016, 157(36), 1429–1437.Tyrosine kinase inhibitors are widely used for treatment of certain oncohematological diseases. Several clinical studies have confirmed that specific BCR-ABL tyrosine kinase inhibitors alter the physiological process of bone tissue in a complex and unclearly identified manner. Since these treatments are being given to more and more patients, and the therapy takes decades or lasts even lifelong, it is justifiable to obtain more detailed knowledge of the molecular background of these mechanisms. In this article the authors summarize preliminary research results and human clinical observations on imatinib and nilotinib which are related to bone metabolism, and present the results of their own experiments in in vitro osteoblast cultures. Based on the presented results, the effects of imatinib and nilotinib on bone cells depend on the concentration of imatinib and nilotinib, the maturation stage of the cells and the distribution ratio of receptor tyrosine kinase signaling pathways. In this study the authors firstly prepared a stop-gap, comprehensive review in the Hungarian literature, regarding the effects of tyrosine kinase inhibitors on bone metabolism. In addition they firstly performed whole transcriptome analysis on osteoblasts in order to obtain a better understanding of the cellular molecular mechanisms. Orv. Hetil., 2016, 157(36), 1429-1437.