Genomics as a basis for precision medicine

Summary. Precision medicine, also known as genome-based medicine and personalized medicine, uses knowledge of the molecular basis of a disease in order to individualize treatment for each patient. The development of novel, powerful, high-throughput technologies has enabled better insight into the genomic, epigenomic, transcriptomic and proteomic landscape of many diseases, resulting in the application of personalized medicine approaches in healthcare. Research in the field of biomedicine in Serbia has followed the modern trends and has made a great contribution to the implementation of genomics in Serbian clinical practice. This is a review of the state of the art of scientific achievements and their application, which have paved the way for personalized medicine in Serbia

Nasleđe srpske hemije u Galeriji nauke i tehnike SANU

У септембру 2013. године навршило се 160 година од тренутка када је 1853. године кнез Александар Карађорђевић одобрио ново устројство београдског Лицеја, једине високошколске установе у ондашњој Србији. Документом „Устројеније Књажеско-Србског Лицеја“ предвиђено је увођење нових предмета „Химија“ и „Технологија“ у наставни план Лицеја. За потребе ових предмета Устројенијем су предвиђени „Химическа Лабораторија“ и „Технологически кабинет“. На место професора за ове предмете изабран је 26-годишњи Михаило Рашковић (1827–1872). Не занемарујући значај дела Павла Илића (1807–1871), државног апотекара и касније државног хемичара, чијим радом је заправо започела хемијска струка код нас, са оснивањем лицејске хемијске лабораторије дошао је нови период развоја хемијске струке и наставе хемије у Србији. Захваљујући прегалаштву Михаила Рашковића у обезбеђивању добрих услова за лабораторијски рад, постављен је темељ и за почетак хемијске науке код нас. То ће се догодити почетком 70-их година 19. века, пре свега у делу Симе Лозанића (1847–1935). Са намером да се обележи 160. годишњица отварања хемијске лабораторије на Лицеју, приређена је изложба Лабораторија великана–наслеђе српске хемије у Галерији науке и технике Српске академије наука и уметности. Овај рад представља кратак извештај о изложби

Mutations in the PAH gene: A Tool for population genetics study

Fenilketonurija je urođena metabolička bolest prouzrokovana mutacijama u genu za fenilalanin hidroksilazu (PAH). U srpskoj populaciji je identifikovano 19 različitih PAH mutacija. PAH mutacije korišćene su kao molekularni markeri za populaciono-genetičko istraživanje. Niska vrednost homozigotnosti PAH gena (0,10) ukazuje na heterogenost fenilketonurije u Srbiji i odražava brojne migracije u regionu jugoistočne Evrope. U skladu sa tim, osmišljena je strategija molekularne dijagnostike fenilketonurije za Srbiju. U cilju rasvetljavanja porekla najčešće mutacije koja uzrokuje fenilketonuriju u Srbiji, L48S, urađena je haplotipska analiza PCR-RFLP metodom. Naši rezultati sugerišu da je L48S mutacija poreklom iz više populacija sa različitim genetičkim karakteristikama. .Phenylketonuria (PKU), an inborn error of metabolism, is caused by mutations in the phenylalanine hydroxylase (PAH) gene. In the Serbian population, 19 different PAH mutations have been identified. We used PAH mutations as molecular markers for population genetics study. The low homozygosity value of the PAH gene (0.10) indicates that PKU in Serbia is heterogeneous, reflecting numerous migrations throughout Southeast Europe. The strategy for molecular diagnostics of PKU was designed accordingly. To elucidate the origin of the most common (L48S) PKU mutation in Serbia, we performed haplotype analysis by PCR-RFLP. Our results suggest that the L48S mutation was imported into Serbia from populations with different genetic backgrounds

Genetic characterization of GSD I in Serbian population revealed unexpectedly high incidence of GSD Ib and 3 novel SLC37A4 variants

Glycogen storage disease (GSD) type I is inborn metabolic disease characterized by accumulation of glycogen in multiple organs. We analyzed 38 patients with clinical suspicion of GSD I using Sanger and next-generation sequencing (NGS). We identified 28 GSD Ib and 5 GSD Ia patients. In 5 patients, GSD III, VI, IX, cholesteryl-ester storage disease and Shwachman-Diamond syndrome diagnoses were set using NGS. Incidences for GSD Ia and GSD Ib were estimated at 1:172746 and 1:60461 live-births, respectively. Two variants were identified in G6PC gene: c.247C gt T (p.Arg83Cys) and c.518T gt C (p.Leu173Pro). In SLC37A4 gene, 6 variants were detected. Three previously reported variants c.81T gt A (p.Asn27Lys), c.162C gt A (p.Ser54Arg) and c.1042_1043delCT (p.Leu348Valfs*53) accounted for 87% of all analyzed alleles. Computational, transcription studies and/or clinical presentation in patients confirmed pathogenic effect of 3 novel variants: c.248G gt A (p.Gly83Glu), c.404G gt A (p.Gly135Asp) and c.785G gt A (p.Ser263Glyfs*33 or p.Gly262Asp). In the cohort, hepatomegaly, hypoglycemia and failure to thrive were the most frequent presenting signs of GSD Ia, while hepatomegaly and recurrent bacterial infections were clinical hallmarks of GSD Ib. All GSD Ib patients developed neutropenia while 20.6% developed inflammatory bowel disease. Our study revealed the highest worldwide incidence of GSD Ib. Furthermore, description of 3 novel variants will facilitate medical genetic practice.This is the peer reviewed version of the paper: Skakic, A., Djordjevic, M., Sarajlija, A., Klaassen, K., Tosic, N., Kecman, B., Ugrin, M., Spasovski, V., Pavlovic, S., & Stojiljkovic, M. (2018). Genetic characterization of GSD I in Serbian population revealed unexpectedly high incidence of GSD Ib and 3 novel SLC37A4 variants. Clinical Genetics, 93(2), 350–355. [https://doi.org/10.1111/cge.13093

Chemometric and ICP-OES analyses of Forsythia europaea Degen & Bald. and its extracts

Forsythia represents a group of plants originating mainly from China and Japan, but one species is endemic and grows in the Balkans (Balkan forsythia, Forsythia europaea Degen & Bald.). Our previous studies on polyphenols in investigated extracts of Balkan forsythia showed that this plant is a good source of polyphenols. Analysis of the various extracts of Balkan forsythia (Forsythia europaea Degen & Bald.) by the application of ICP-OES method showed that they are rich in different macro and microelements. The abundance order of macroelements is K>Ca>P>Mg>Na in all extracts. Among the transition metals iron, manganese, zinc and copper are particularly important, and the order of abundance is Zn>Fe>Cu>Mn. Heavy metals which are the most frequent contaminants of food are lead, cadmium and arsenic, and the determination of their contents is of special importance on the safe use of plant species. The determination shows that aqueous extracts contain the highest quantity of elements, which is especially important. The contents of toxic elements are significantly lower than the permitted values. Statistical methods (Principal Component Analysis (PCA) and Agglomerative Hierarchical Clustering (AHC)) are useful tools for the grouping of samples and determining relations between investigated elements. This analysis shows that when higher quantities of Cr and Ba are present, the lower quantities of V are present, and vice versa. Based on our studies on polyphenols and minerals, we can expect the anti-inflammatory effects of extracts of Balkan forsythia

Molecular diagnosis of Fabry disease in patients with chronic renal failure of unknown etiology

Background/Objectives: Fabry disease (FD) is a rare X-linked disorder caused by variants in the GLA gene leading to the deficiency of lysosomal α-galactosidase-A and progressive accumulation of globotriaosylceramide affecting the heart, nervous system, and kidneys. FD has overlapping phenotypes and often remains undiagnosed. Therefore, the precise molecular-genetic diagnosis and the earliest possible treatment are essential to avoid significant disease progression. Methods: We analyzed 95 (34 female and 61 male) hemodialysis patients with clinical suspicion of FD using Sanger sequencing of all coding exons (7) and flanking intron regions of the GLA gene, and measured the relative expression of the GLA gene in available samples. Results: The genetic analysis revealed 3 patients with a missense variant (p.Asp313Tyr), and 10 patients with combinations of non-coding variants, described as complex intronic haplotypes (CIHs). CIH1 (c.-10C>T, c.370-81_370-77delCAGCC, c.640-16A>G, c.1000-22C>T), the most frequent haplotype, was detected in 7 (7.4%) patients. Lyso-Gb3 biomarker levels were within the normal range in each tested patient. However, RT-qPCR analysis revealed decreased relative expression of GLA gene in PBMC of 2 female patients with CIH1 and one female patient carrying only c.-10C>T variant by 9,1%, 7,4%, 46,3%, respectively, pointing out that further analyses are needed to confirm/exclude FD in these patients. Conclusion: Because the effects of CIHs are not yet fully understood, our work highlights the importance of analyzing intronic regions of the GLA gene as genetic modifiers and the need to include expression analysis in the diagnostic algorithm.Abstracts from the 55th European Society of Human Genetics (ESHG) Conferenc

Gene Mutation Profiles in Primary Diffuse Large B Cell Lymphoma of Central Nervous System: Next Generation Sequencing Analyses

The existence of a potential primary central nervous system lymphoma-specific genomic signature that differs from the systemic form of diffuse large B cell lymphoma (DLBCL) has been suggested, but is still controversial. We investigated 19 patients with primary DLBCL of central nervous system (DLBCL CNS) using the TruSeq Amplicon Cancer Panel (TSACP) for 48 cancer-related genes. Next generation sequencing (NGS) analyses have revealed that over 80% of potentially protein-changing mutations were located in eight genes (CTNNB1, PIK3CA, PTEN, ATM, KRAS, PTPN11, TP53 and JAK3), pointing to the potential role of these genes in lymphomagenesis. TP53 was the only gene harboring mutations in all 19 patients. In addition, the presence of mutated TP53 and ATM genes correlated with a higher total number of mutations in other analyzed genes. Furthermore, the presence of mutated ATM correlated with poorer event-free survival (EFS) (p = 0.036). The presence of the mutated SMO gene correlated with earlier disease relapse (p = 0.023), inferior event-free survival (p = 0.011) and overall survival (OS) (p = 0.017), while mutations in the PTEN gene were associated with inferior OS (p = 0.048). Our findings suggest that the TP53 and ATM genes could be involved in the molecular pathophysiology of primary DLBCL CNS, whereas mutations in the PTEN and SMO genes could affect survival regardless of the initial treatment approach

Expression Pattern and Prognostic Significance of the Long Non-Coding RNA Metastasis-Associated Lung Adenocarcinoma Transcript 1 in Chronic Lymphocytic Leukemia

Dysregulated expression of the long non-coding RNA MALAT1 has been implicated in the pathogenesis and progression of a variety of cancers, including hematological malignancies, but it has been poorly investigated in chronic lymphocytic leukemia (CLL). In this study, the expression of MALAT1 was measured using a quantitative reverse-transcriptase polymerase chain reaction in the peripheral blood mononuclear cells of 114 unselected, newly diagnosed CLL patients in order to analyze its association with clinical, laboratory, and molecular patients’ characteristics at diagnosis, as well as its prognostic relevance. MALAT1 was found to be upregulated in CLL patients in comparison to healthy controls, and expression levels were not related to age, leukocyte, lymphocyte and platelet count, serum β2-microglobulin, and IGHV somatic hypermutational status. On the other hand, high MALAT1 expression was associated with several favorable prognostic markers (high hemoglobin, low serum lactate dehydrogenase, earlier clinical stages, CD38-negative status), but also with unfavorable cytogenetics. Furthermore, an association between high MALAT1 levels and longer time to first treatment and overall survival in IGHV-unmutated CLL subtype was observed. In summary, our results imply that high MALAT1 expression at diagnosis may be a predictor of better prognosis and point to MALAT1 expression profiling as a candidate biomarker potentially useful in clinical practice

PB1917: EXPRESSION OF THE LONG NON-CODING RNA MALAT1 IN CHRONIC LYMPHOCYTIC LEUKEMIA

Background: The long non-coding RNA (lncRNA) MALAT1 (metastasis-associated lung adenocarcinoma transcript 1) dysregulated expression and prognostic significance have been reported in a variety of cancers, including hematological malignancies, but have been poorly investigated in chronic lymphocytic leukemia (CLL). Acting through regulation of gene expression at transcriptional and post-transcriptional level, lncRNA MALAT1 is involved in many cellular processes such as proliferation, apoptosis, migration and drug resistance. However, its role as either an oncogene or a tumor-supressor is still controversial, and clearly tumor type-dependent. Aims: To analyze the expression pattern of lncRNA MALAT1 in CLL, and evaluate its prognostic relevance. Methods: This study enrolled 114 unselected CLL patients (pts) and 20 healthy controls (hcs). Clinical and laboratory characteristics of pts were determined at diagnosis, while genetic analyses were performed during the period prior to first treatment. The expression of MALAT1 was analyzed in peripheral blood mononuclear cells by RQ-PCR, using TaqMan chemistry and GAPDH as endogenous control; relative quantification was made by comparative ddCt method, using hcs as calibrator. Results: CLL cohort consisted of 81 males and 33 females (male/female=2.45), with median age at diagnosis of 59 years (range 33-80). Hcs group consisted of 15 males and 5 females (male/female=3), with median age at diagnosis of 71 years (range 65-85). Distribution of Binet stages (112/114 pts) was as follows: A-46.4%, B-39.3%, C-14.3%. Del13q, normal karyotype, trisomy12, del11q and del17p were detected by FISH in 33%, 35%, 9.3%, 10.3% and 12.4% of pts, respectively (97/114 pts). CD38 status (85/114 pts) was negative in 70.6% and positive in 29.4% of pts. Regarding IGHV mutational status (114 pts), 41.2% of pts were mutated, and 58.8% unmutated. Median follow-up was 72 months (range 1-360). LncRNA MALAT1 was overexpressed in CLL pts compared to hcs (p<0.001). Median value of MALAT1 expression was used to divide the cohort into MALAT1low and MALAT1high groups, and association with clinical and biological features at diagnosis was assessed. In both pts and hcs MALAT1 expression was not associated with age but, unlike hcs, MALAT1high status was significantly associated with male sex in CLL (p=0.003). Regarding laboratory parameters, MALAT1 expression showed no correlation with leukocyte, lymphocyte and platelet counts, and serum β2-microglobulin, but exerted a positive correlation with hemoglobin level (r=0.315, p=0.003) and a negative correlation with lactate dehydrogenase (LDH) level (r=-0.303, p=0.004). MALAT1 expression was higher in Binet A and B pts vs. Binet C pts (p=0.037). There was also a trend toward higher MALAT1 expression in pts with favorable (del13q) and intermediate (normal karyotype, trisomy12) cytogenetics in comparison to pts with unfavorable (del11q and del17p) cytogenetics (p=0.059). In addition, MALAT1high status was associated with CD38-negative status (p=0.017), but not with IGHV mutational status. Finally, while the association of MALAT1 expression with the time to first treatment was not detected, longer median overall survival (OS) in MALAT1high vs. MALAT1low group was observed (142 vs. 82 months, log rank p=0.032). Summary/Conclusion: LncRNA MALAT1 is up-regulated in CLL. However, high MALAT1 expression is associated with several favorable prognostic markers (high hemoglobin, low LDH, early clinical stages, negative CD38 status), as well as longer OS. The exact mechanisms of MALAT1 function in CLL pathogenesis and/or progression remain to be determined

Prognostic significance of the long non-coding rna malat1 expression in chronic lymphocytic leukemia

Introduction: The long non-coding RNA (lncRNA) MALAT1 (metastasis-associated lung adenocarcinoma transcript 1) dysregulated expression has been reported in a variety of cancers, but has been poorly investigated in chronic lymphocytic leukemia (CLL). The aim of thisstudy wasto investigate the expression pattern of lncRNA MALAT1 in CLL, and evaluate its prognostic significance. Methods: MALAT1 expression was analyzed in peripheral blood mononuclear cells of 114 newly-diagnosed CLL patients and 20 healthy controls by qRT-PCR, and association with clinical and biological features at diagnosis was assessed. Results: MALAT1 was overexpressed in CLL compared to controlsamples(p<0.001). MALAT1 expression was higher in male patients (p=0.003). It showed no correlation with age, leukocyte, lymphocyte and platelet count, and serum β2-microglobulin, but exerted a positive correlation with hemoglobin level (r=0.315, p=0.003) and a negative correlation with lactate dehydrogenase level (r=-0.303, p=0.004). MALAT1 expression was higher in Binet A and B patients vs. Binet C patients (p=0.037). There was also a trend toward higher MALAT1 expression in patients with favorable (del13q) and intermediate (normal karyotype, trisomy12) cytogeneticsin comparison to patients with unfavorable (del11q and del17p) cytogenetics (p=0.059). In addition, high MALAT1 levels were associated with CD38-negative status (p=0.017), but not with IGHV mutational status. While there was no association with the time to first treatment, longer median overall survival in MALAT1 high- vs. MALAT1 low-expressing cases was observed (142 vs. 82 months, log rank p=0.032). Conclusion: LncRNA MALAT1 is up-regulated in CLL. High MALAT1 expression at diagnosis may be associated with better prognosis