imagine (Institute of molecular genetics and genetic engineering)
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Seeking an optimal variant calling pipeline for medical genetics
Get PDFAccurate and comprehensive variant discovery is extremely important for rare disease
diagnostics using next-generation sequencing (NGS) methods. Over the recent years, a
plethora of methods have been developed for short variant calling from NGS data, and the
most recent tools extensively use machine learning algorithms for both variant discovery
and filtering. In our study, we took an effort to systematically evaluate the performance of
different pipelines for short variant calling in the human genome.
To perform such a systematic comparison, we collected a large dataset of both “gold
standard” (provided by the Genome In A Bottle (GIAB) consortium) and in-house wholeexome
sequencing (WES) and whole-genome sequencing (WGS) datasets. (a total of 20
different datasets was used). We tested all combinations of 4 popular short read aligners
(BWA, Bowtie2, Isaac, and Novoalign) and 9 novel and well-established variant calling
and filtering methods (Freebayes, Clair3, DeepVariant, Genome Analysis ToolKit (GATK),
Octopus, Strelka2). We also used several different tools for preprocessing of short reads.
Our analysis showed negligible effects of adapter trimming on the accuracy of short
variant calling. Among read aligners, Bowtie2 performed significantly worse than other
tools, suggesting it should not be used for medical variant calling. For pipelines based
on BWA, Isaac, and Novoalign, the accuracy of variant discovery mostly depended on
the variant caller and not the read aligner. DeepVariant consistently showed the best
performance and the greatest robustness compared to all other tested variant callers. We
have also compared the consistency of variant calls in GIAB and non-GIAB samples. With
few important caveats, best-performing tools have shown little evidence of overfitting.
Taken together, our study showed that modern strategies for NGS data analysis allow for
high accuracy of genetic variant discovery within coding regions of the human genome.
However, there is still a need for development of new library preparation and variant
calling methods to enhance variant discovery in the challenging regions of the human
genome.Book of abstract: 4th Belgrade Bioinformatics Conference, June 19-23, 202
Supplementary material for: Spasovski, V.; Srzentić Dražilov, S.; Nikčević, G.; Baščarević, Z.; Stojiljković, M.; Pavlović, S.; Spasovski, D. Molecular Biomarkers in Perthes Disease: A Review. Diagnostics 2023, 13 (3), 471. https://doi.org/10.3390/diagnostics13030471.
Get PDFBackground: 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 material for: [https://doi.org/10.3390/diagnostics13030471]Related to the published version: [https://imagine.imgge.bg.ac.rs/handle/123456789/1853
Discovery of novel HDAC inhibitors for therapy of triple-negative breast cancer – preclinical study
Get PDFTriple-negative breast cancer (TNBC) is an aggressive form of breast cancer that has poor survival rates due to the
absence of specifi c molecular markers such as estrogen receptor (ER), progesterone receptor (PR), and human
epidermal growth factor receptor 2 (HER2). In the era of precision oncology, it is recognized that an imbalance in posttransla
tional modifi cations of histones, such as histone lysine acetylation and deacetylation, is closely linked to tumor
initiation and progression. Two groups of enzymes control the reversible nature of histone post-translational acetylation:
histone acetyltransferases (HATs) and histone deacetylases (HDACs). Isoform-specifi c targeting of HDACs is considered
a rational strategy to develop safe anticancer therapeutics compared to non-selective HDAC inhibitors. However, nonselec
tive HDAC inhibitors have been more extensively studied in clinical trials. This work presents the design and
discovery of potent HDAC inhibitors that selectively target HDAC6 isozyme, using 1-benzhydryl piperazine as a surface
recognition group with diff erent hydrocarbon linkers. Through in vitro screening, two HDAC6-selective inhibitors with
nanomolar IC50 values and two non-selective HDAC inhibitors were identifi ed. Structure-based molecular modelling
was utilized to investigate the impact of linker chemistry on the potency of synthesized inhibitors against HDAC6. The
anti-cancer, anti-migratory, and anti-invasive activities of these compounds were evaluated using breast cancer cell
lines (MDA-MB-231 and MCF-7). Experiments on a zebrafi sh MDA-MB-231 xenograft model demonstrated that a novel
non-selective HDAC inhibitor (compound 8b) with a seven-carbon-atom linker exhibited potent eff ects against tumor
growth, metastasis, and angiogenesis at low micromolar concentrations
Sugars and their substitutes increase pathogenicity of Pseudomonas aeruginosa
Get PDFIntroduction: Differentsugars are commonly used in the diet, but little is understood about the various
effects of human health that they can affect. Hence, the impact of sugars and their substitutes used in
diet on the development of virulence in Pseudomonas aeruginosa PAO1 was investigated. Sugars (fructose, demerara, coconut sugar, and cane sugar) and sugar substitutes (erythritol and stevia) were selected. The genes from three P. aeruginosa QS networks (las - lasI, lasR; rhl - rhlI, rhlR; PQS - pqsA, mvfR)
were used for RT-qPCR analysis in order to investigate whether the expression of these genes changes.
In this work, the focusis on the expression of genesinvolved in QS and the ability to form biofilms(a type
of structured community of microorganisms that is attached to the surface and connected by an exopolysaccharide matrix), as well as determining minimal inhibitory concentration of antibiotics in presence of tested compounds.
Methods: Microdiltuion assay, Antibiofilm assay, RT- qPCR
Results: In the presence of tested sugars and theirsubstitutes, the minimum inhibitory concentration of
commercial antibiotics increased, as well as the percentages of biofilm formation (for instance, the percentage of biofilm formation is 171% in the presence of coconut sugar). Furthermore, exposure of P.
aeruginosa to tested compounds caused the greatest increase in expression of virulence associated with
the lasI and pvdF genes.
Conclusion: More awareness and research is needed to highlight the effectssugars can have on P. aeruginosa and to propose new strategies to reduce this negative aspect
Influence of intestinal microbiota composition on immunomodulatory properties and immunotherapeutic potential of dendritic cells and myeloid derived suppressor cells
Get PDFTransfer mijeloidnih ćelija imunogenih ili supresivnih svojstava, ima velikipotencijal u lečenju malignih tumora ali i autoimunskih bolesti. Međutim, nije do krajaispitana veza funkcijskog potencijala mijeloidnih ćelija i sastava mikrobiote creva, kaojednog od ključnih regulatora imunskog sistema. U ovoj tezi je po prvi put ispitivana vezaizmeđu sastava mikrobiote creva i funkcijskog potencijala mijeloidnih ćelija, i to humanihdendritskih ćelija (DC) i supresorskih ćelija mijeloidnog porekla (MDSC) in vitro, kao ipotencijal MDSC pacova da suprimiraju autoimunski proces u modelu eksperimentalnogautoimunskog encefalomijelitisa (EAE). Rezultati su pokazali da DC dobijene od zdravihdonora koji su imali veći diverzitet mikrobiote creva i veću zastupljenost bakterija kojeproizvode masne kiseline kratkog lanca (SCFA) poseduju slabiji imunogeni potencijal uodnosu na DC poreklom iz donora koji su imali manji diverzitet mikrobiote i većuzastupljenost rodova Bifidobacterium i Collinsella. U modelu humanih MDSC je pokazanoda poliamini mikrobiote potenciraju imunosupresivna svojstva MDSC posredstvomintestinalnih epitelnih ćelija. Takođe, transfer MDSC aktiviranih prostaglandinom E2(MDSC-PGE2) u ţivotinje sa indukovanim EAE, ublaţava simptome EAE nakon migracijeu limfni sistem creva. Fenomen ublaţavanja simptoma EAE nakon transfera MDSC-PGE2je bio povezan sa smanjenom infiltracijom proinflamatornih i povećanom infiltracijomregulatornih ćelija u CNS i slezinu, očuvanjem integriteta intestinalne barijere, diverzitetamikrobiote creva i povećanjem zastupljenosti bakterija koje ispoljavaju imunoregulatorneosobine, poliamina i SCFA u fecesu. Ovi rezultati ukazuju na blisku povezanost mikro-biote creva i imunogenosti/supresivnosti mijeloidnih ćelija, što moţe biti iskorišćeno urazvoju novih efikasnijih terapija za maligne tumore i autoimunske bolesti.The transfer of myeloid cells with immunogenic or suppressive properties holdsgreat potential for the treatment of malignant tumors or autoimmune diseases,respectively. However, the relationship between the functional potential of myeloid cellsand the composition of the gut microbiota, one of the most important regulators of theimmune system, has not yet been elucidated. In this thesis, we investigated for the firsttime the relationship between the gut microbiota composition and the functional potentialof myeloid cells, in human dendritic cell (DC) and myeloid derived suppressor cells(MDSC) differentiated in vitro, and the potential of rat MDSCs to suppress theautoimmune response in experimental autoimmune encephalomyelitis (EAE) model invivo. The results showed that DCs from healthy donors with higher gut microbiotadiversity and higher abundance of short-chain fatty acid (SCFA)-producing bacteria havelower immunogenic potential, in contrast to DCs from donors with lower gut microbiotadiversity and higher abundance of Bifidobacterium and Collinsella genera. In the humanMDSC model, polyamines produced by the microbiota were shown to enhance theimmunosuppressive properties of MDSCs by intestinal epithelial cells. Also, transfer ofanimal MDSCs activated with prostaglandin E2 (MDSC-PGE2) into animals with inducedEAE alleviates EAE symptoms after migration to the intestinal lymphatic system. Thephenomenon of alleviation of EAE symptoms after MDSC-PGE2 transfer was associatedwith decreased infiltration of pro-inflammatory and increased infiltration of regulatorycells in the CNS and spleen, maintenance of intestinal barrier integrity, diversity of theintestinal microbiota, and an increase in the abundance of immunoregulatory bacteria,polyamines, and SCFA in the feces. These results suggest a strong association between thegut microbiota and the immunogenicity/suppressiveness of myeloid cells that can beexploited in the development of new, more effective therapies for malignancies andautoimmune diseases
Can pharmacogenetic variants in TPMT, MTHFR and SLCO1B1 genes be used as potential markers of outcome prediction in systemic sclerosis patients?
Get PDFIntroduction: Systemic sclerosis(SSc) is a rare autoimmune disorder that affects connective tissues and
hasthe highest morbidity and mortality among rheumatologic diseases. Clinical presentations as well as
disease progression are highly heterogeneous between patients, implying a strong need for individualization of therapy.
Methods: Four pharmacogenetic variants, namely TPMT rs1800460, TPMT rs1142345, MTHFR rs1801133
and SLCO1B1 rs4149056 were tested for association with severe disease outcomes in 102 patients with
SSc from Serbia treated either with immunosuppressants azathioprine (AZA) and methotrexate (MTX) or
with other types of medications. Genotyping was performed using PCR-RFLP and direct Sanger sequencing. R software was used for statistical analysis and development of polygenic risk score (PRS)
model.
Results: Association wasfound between MTHFR rs1801133 and higher risk for elevated systolic pressure
in all patients except those prescribed with MTX, and higher risk for kidney insufficiency in patients prescribed with other types of drugs. In patients treated with MTX, variant SLCO1B1 rs4149056 was protective against kidney insufficiency. For patients receiving MTX a trend was shown for having a higher PRS
rank and elevated systolic pressure.
Conclusion: Our results open a door wide for more extensive research on pharmacogenomics markers
in patients with SSc. Altogether, pharmacogenomics markers could predict the outcome of patients with
SSc and help in prevention of adverse drug reaction
Expression profiles of long non-coding RNA gas5 and microrna-222 in younger AML patients
Get PDFIntroduction: Acute myeloid leukemia (AML) is a heterogeneous malignant disease, that accounts for
80% of all acute leukemias in adults. Imprecise risk stratification and lack of personalized treatment creates a constant need to find new prognostic markers and targets for innovative therapeutics. Recently,
thissearch has pointed towards non-coding RNAs(ncRNA). Numerousstudies have shown dysregulation
of lncRNA GAS5 in cancers, but it was poorly investigated in AML. Since GAS5 actslike a molecularsponge
for miR-222, co-expression profiles of these non-coding RNAs could be novel prognostic markersin AML.
Methods: GAS5 expression levels were analysed in 94 AML patients and 14 healthy controls using RealTime PCR and miR-222 expression levels were analysed in a subgroup of 39 patients with normal karyotype (AML-NK). ROC curve analyses were used to find a cut-off value between GAS5high and GAS5low,
while the median value was used for distinguishing between miR-222high and miR-222low.
Results: We showed that GAS5 expression in AML patients was lower compared to healthy controls.
Lower GAS5 expression on diagnosis was related to an adverse prognosis. The disease-free survival and
the overallsurvival were lower in the GAS5low group butsurvival analysisfailed to confirm thisfinding. In
the AML-NK group patients had higher expression of miR-222 compared to healthy controls. A synergistic
effect of GAS5low/miR-222high status on disease prognosis was not established.
Conclusion:Our findingsindicate the potential prognostic significance of GAS5 expression and the need
for further investigation of these two non-coding RNAs and their potential roles in leukemogenesis
Generation of induced pluripotent stem cells derived from patients with 22q11.2 deletion syndrome as a tool for studying neurodevelopmental disorders
Get PDFIntroduction: Neurodevelopmental disorders (NDDs), such as autism spectrum disorders (ASD), intellectual disability (ID),schizophrenia, and bipolar disorder, are caused by the alterationsin early brain development. They affect approximately 4% of the European population and represent a high
socio-economic impact and financial burden. Treatments of NDDs are focused on symptoms since molecular mechanisms underlying NDDs are still unknown. One of the syndromes with a high risk for NDDs
is 22q11.2 Deletion Syndrome (22q11.2DS) caused by microdeletion 22q11.2. 22q11.2 microdeletion is
the most common microdeletion in humans; it is one of the strongest known risk factorsfor development
of psychiatric illness and the highest known genetic risk for schizophrenia (approximately, 25% of patients with 22q11.2DS develop schizophrenia compared to 1% in the general population).
Methods: Genomic and clinical findings in 35 patients with 22q11.2DS were analyzed and peripheral
blood mononuclear cells of patients with 22q11.2DS and healthy controls were reprogrammed.
Results: The majority of patients have 3 Mb deletion and nine of them have inherited 22q11.2 microdeletion from parents. Twenty-one different clinical presentations are revealed in the cohort with developmental delay detected in about 50% of patients. iPSCs were generated from four patients with
22q11.2 microdeletion and five healthy controls.
Conclusion: Cohort of patients with 22q11.2DS isform and iPSCs were generated which enable research
of molecular mechanisms underlying NDDs
Microbial degradation of bis (2-hydroxyethyl) terephthalate
Get PDFProizvodnja plastike i zamena staklene i keramičke ambalaže plastičnim materijalima
doveli su do nagomilavanja plastičnog otpada. Neophodno je naći povoljan sistem za
degradaciju plastičnog otpada, bez nastanka toksičnih produkata ili dodatnog zagađenja
životne sredine. Polietilen-tereftalat (PET) je jedan od najčešće proizvedenih plastičnih
polimera. Proizvodnja PET-a započinje esterifikacijom tereftalne kiseline i etilen glikola,
pri čemu nastaje bis-(2-hidroksietil)-tereftalat (BHET), koji se dalje polikondenzuje do
polimera. U poslednje vreme, BHET se često koristi kao model jedinjenje za identifikovanje
novih biokatalizatora za degradaciju PET-a [1,2].
Cilj ovog rada bio je ispitivanje mehanizma degradacije BHET-a pomoću
mikroorganizama.
U preliminarnom testu na čvrstim podlogama, kapacitet za degradaciju BHET-a je
testiran kod stotinak mikroorganizama, nakon čega su odabrani najefikasniji sojevi, koji su
identifikovani sekvenciranjem gena za 16s rRNK. Dalje, ispitivana je degradacija u tečnoj
podlozi gde je BHET bio glavni izvor ugljenika. Eksperiment je trajao 7 dana, a degradacija
je praćena nakon drugog, petog i sedmog dana upotrebom tečne hromatografije (HPLC).
Kao najefikasniji sojevi pokazali su se pripadnici roda Pseudomonas. Oni su u potpunosti
transformisali BHET do različitih intermedijera.
Rezultati su pokazali da ispitivani sojevi mogu da transformišu BHET, korišćenjem
najmanje dva različita puta, pa će se naredni eksperimenti usmeriti na identifikaciju
intermedijera degradacije. Takođe, radi optimizacije degradacije, ispitivaće se simbiotsko i
sinergističko dejstvo različitih konzorcijuma, kako bi se obezbedila potpuna degradacija
ovog model jedinjenja.KNJIGA IZVODA: 9. simpozijum Hemija i zaštita životne sredine Kladovo, 4-7. jun 2023. BOOK OF ABSTRACTS : 9th Symposium Chemistry and Environmental Protection Kladovo, 4-7th June 202
CHARACTERIZATION OF 16 NOVEL GENETIC VARIANTS IN GENES RELATED TO CHILDHOOD EPILEPSIES
Get PDFBackground: Childhood epilepsies are
caused by heterogeneous underlying disorders where
approximately 40% can be attributed to genetic
factors. Application of next-generation sequencing
(NGS) has revolutionized diagnostics and therefore
has enabled the identification of disease-causing
genes and variants in childhood epilepsies.
Materials and Methods: Patients who presented
with epilepsy of unknown etiology in childhood,
with suspicion of a genetic cause were included
in this study. In total, 55 patients from unrelated
non-consanguineous families were included and analyzed
by NGS either using clinical-exome sequencing
(MiSeq, Illumina) or whole-exome sequencing
(DNBSEQ-G400, MGI). Variants were prioritized
using Variant Interpreter and VarSome and classified
according to the ACMG recommendations.
Results: Using CES we analyzed 38 patients,
and for 22 of them a diagnosis was established.
Using WES we analyzed 17 patients with childhood epilepsy, which led to the identification of
disease-causing genes in 11 patients. The diagnostic
success rate for CES was 55.3% (21/38) and the
diagnostic rate for WES was 64.7% (11/17), with
the overall diagnostic rate being 58.2% (32/55). For
these patients, we detected pathogenic, likely pathogenic
variants or VUS in 24 epilepsy genes that
correlate well to the observed phenotype. Sixteen
novel genetic variants were identified and characterized
using various in silico algorithms.
Conclusion: This is the first study reporting
the molecular-genetic basis of childhood epilepsy
in Serbia. The prompt establishment of a specific
diagnosis is essential in order to make available the
prognosis, optimize therapy, and enable counseling
on recurrence risk in future pregnancies.ABSTRACT BOOK: “Genetic Diseases from Diagnostics to Prevention and Therapy” October 05-14th Balkan Congress of Human Genetics & 9th Rare Disease SEE Meeting 2023; Skopje, October 05-07, 202