imagine (Institute of molecular genetics and genetic engineering)
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Genomic profiling of pediatric patients with liver glycogenosis: genotype-phenotype correlation and functional characterization of novel variants
Get PDFBolesti koje nastaju usled naslednog enzimskog poremećaja u sintezi ili razgradnji glikogena i koje primarno pogađaju jetru i bubrege, nazivaju se hepatične glikogenoze. Glikogenoza tip I (GSD tip I) je jedan od najčešćih oblika i zauzima posebno mesto u grupi ovih bolesti zbog prepoznatljivog kliničkog fenotipa koji jasno odražava složenu patofoziologiju poremećaja u metabolizmu glikogena. GSD tip Ia razvija se usled deficitarne aktivnosti enzima glukozo-6-fosfataze α (G6Paza-α ili G6PC), a GSD tip Ib nastaje kao posledica deficijencije transportera glukozo-6-fosfat translokaze (G6PT ili SLC37A4) koja se nalazi na membrani endoplazmatičnog retikuluma (ER). U jetri, bubrezima i mukozi creva, G6Paza-α i G6PT formiraju funkcionalni kompleks koji učestvuje u održavanju homeostaze glukoze u krvi između obroka. Ukoliko je ovaj proces narušen, dolazi do nakupljanja glikogena u jetri, bubrezima i intestinalnoj mukozi i razvoja hipoglikemije, hiperlaktatemije, hiperlipidemije, hiperurikemije, i hepatonefromegalije. Ove metaboličke abnormalnosti rezultiraju dugoročnim komplikacijama bubrežnih bolesti i hepatocelularnih adenoma/karcinoma, čiji su molekularni mehanizmi slabo istraženi. Kod pacijenata obolelih od GSD tip Ib dodatno se javlјaju neutropenija i disfunkcija neutrofila i makrofaga, pa zbog toga ovi pacijenti razvijaju rekurentne bakterijske infekcije, neretko praćene simptomima inflamatorne bolesti creva slične Kronovoj bolesti. Uzimajući u obzir patofiziološke posledice metaboličkog stresa kod pacijenata obolelih od GSD tip Ib, od velikog interesa su adaptivni odgovori i molekularni mehanizmi koje ćelije aktiviraju pod takvim uslovima. Poznato je da poremećaj homeostaze ER-a, nastalog kao posledica gubitka intraćelijske produkcije glukoze, dovodi do hronične aktivacije „odgovora nesavijenih proteina” (UPR) čime doprinosi patogenezi mnogih bolesti. Stres ER-a je impliciran u mnogim hroničnim bolestima, ali se vrlo malo zna o hroničnom stresu ER-a uzrokovanog nedostatkom G6PT. Osim GSD tip I, postoji još šest hepatičnih GSD (tipovi 0, III, IV, VI, IX i XI), kao i desetine tipova GSD koje pogađaju skeletne mišiće, srce i druge organe. Pošto su posledice bolesti ozbilјne i ireverzibilne, molekularno-genetičko testiranje je od presudne važnosti za precizno postavlјanje dijagnoze i što raniju primenu adekvatne terapije...Hepatic glycogen storage diseases (GSD) are inherited disorders of glycogen synthesis or degradation, which primarily affect the liver and kidneys. GSD type I is one of the most common types and takes a special place in the group of these diseases due to a recognizable clinical phenotype that clearly reflects the complex pathophysiology of disorders in glycogen metabolism. GSD type Ia and type Ib are due to a deficiency of an enzyme glucose-6-phosphatase α (G6Pase-α or G6PC) and endoplasmic reticulum (ER) glucose-6-phosphate translocase (G6PT or SLC37A4), respectively. G6Pase-α and G6PT form a functional complex on the ER membrane, that maintenance the blood glucose homeostasis between the meals, and its deficiency results in excessive accumulation of glycogen in the liver, kidney, and intestinal mucosa, leading to the hypoglycemia, hyperlactatemia, hyperlipidemia, hyperuricemia, and hepathonephromegaly. These metabolic abnormalities result in long-term complications of kidney diseases and hepatocellular adenomas/carcinoma, whose molecular mechanisms are poorly understood. Patients with GSD type Ib, also develop neutropenia and dysfunction of neutrophils and macrophages followed by recurrent bacterial infections, and symptoms of inflammatory bowel disease like Crohn’s disease. Considering the pathophysiological consequences of this metabolic stress, the adaptive responses and molecular markers which cells activate under such conditions are of great interest. It is known that disturbance of ER homeostasis, resulting from a loss of intracellular glucose production, leads to the chronic activation of the unfolded protein response (UPR), which contributes to the pathogenesis of many diseases. ER stress is implicated in many chronic diseases, but little is known how the UPR corresponds to the chronic ER stress caused by a deficiency of G6PT. Besides GSD type I, there are six additional hepatic GSD types (0, III, IV, VI, IX and XI), as well as dozens of GSD forms that affect skeletal muscles, heart and other organs. Since the consequences of the disease are serious and irreversible, molecular-genetic testing is crucial for precise diagnosis and applying the appropriate therapy as early as possible..
Maturity onset diabetes of the young (mody): the importance of combined ngs and mlpa genetic testing
No full textMaturity onset diabetes of the young (MODY) is a rare form of diabetes characterized by
an onset of hyperglycaemia before 25 years of age, autosomal dominant inheritance and
in some cases insulin independence. MODY is caused by changes in one of the genes
important for function, regulation and development of pancreatic β-cells, glucose sensing or
interaction with insulin. Thus far, variants of 13 genes, most frequently HNF1A, GCK, HNF4A
and HNF1B, have been associated with 13 different MODY subtypes. Being clinically and
genetically heterogeneous, MODY is often misdiagnosed as type 1 or type 2 diabetes, leading
to inadequate therapy.
The aim of this study was to genetically characterize clinically suspected MODY patients,
evaluate the relative frequency of MODY subtypes and to determine the type of variants in
MODY genes of Serbian pediatric patients.
Twenty-nine unrelated pediatric patients were analyzed using TruSight One panel for nextgeneration
sequencing (NGS) and multiplex ligation-dependent probe amplification (MLPA)
assay. Usage of these two genetic tests enabled detection of both single nucleotide variants
and large deletions, characteristic for some MODY subtypes.
Variants in MODY genes were identified in 22 out of 29 patients (75.9%). Most of these
variants were located in the GCK gene, followed by variants in HNF1B. The rest of the variants
were found in the NEUROD1 and HNF1A genes. We identified one novel variant in the GCK
gene: c.596T>C, p.Val199Ala. The applied genetic tests excluded the suspected diagnosis of
MODY in two patients and revealed variants in other genes possibly associated with the
patient’s clinical phenotype.
The combined NGS and MLPA-based genetic tests used in this study present a comprehensive
approach for genetic characterization of patients with suspected MODY diabetes and provide
a successful differential diagnosis of MODY subtypes, important for the right therapy,
prognosis, and genetic counseling.Abstracts of the 6th CONGRESS OF THE SERBIAN GENETIC SOCIETY Vrnjačka Banja; October 13 to 17, 2019
Some are good, some are bad: an overview of available nuclear microsatellites in fagus species, and their utility in f. Sylvatica from the Central Balkans (Serbia)
No full textSelectively neutral nuclear microsatellites (SSRs) are among the most commonly
used molecular markers for genotyping in plant and animal species. They are also
used in studies focusing on adaptation along with adaptive markers because available
methods for identification of loci related to adaptation are sensitive to evolutionary
forces that can mimic selection, such as population structure. In studies of this kind, the
usage of selectively neutral molecular markers may provide insights into among- and
within-population genetic structure, and thus, may be used for the distinction of effects of
these phenomena from the effects of selection. Nuclear microsatellites have been
developed for Fagus sp., and have been used in F. sylvatica, an ecologically and
economically most important European Fagus species. We provide a comprehensive
overview of available Fagus sp. SSRs (185 loci), carry out meta-analysis of loci used in F.
sylvatica (62 loci employed in 62 surveys), and validate a set of 16 loci in 45 individuals of
this species from the central Balkans (Serbia). Erroneous usage of marker’s names/authors
is rather frequent, and loci successfully used in a number of studies are characterized by
other authors by high prevalence of null alleles and even multilocus amplification
products. Frequent occurrence of null alleles at FS4-46, used in 26 surveys to date, most
likely indicates a failure to record multiple alleles at this locus. Twelve loci are reliable/
informative in F. sylvatica from the Balkans (5-18 alleles/locus, HE ranging from 0.523 to
0.850), while four loci are characterized by high prevalence of null alleles (sfc0161 and
sfc1063) and multilocus amplification products (FS4-46 and Fagsyl_007038). Our findings
are important for future population genetic analyses and studies on adaptation of F.
sylvatica to its environment, because the latter rely on both selectively neutral and
markers under selection.Abstracts of the 6th CONGRESS OF THE SERBIAN GENETIC SOCIETY Vrnjačka Banja; October 13 to 17, 2019
Transformation protocol optimization for Bacillus subtilis, Bacillus licheniformis and Bacillus thuringiensis strains
No full textMany members of the genus Bacillus have the ability to produce a large number
of different enzymes, antimicrobial compounds and other secondary metabolites,
making them very important for many industries, agriculture and medicine. The
natural competence of these strains is the physiological and genetic property
expressed under certain conditions or constitutively. However, the incompetent
Bacillus strains can be brought into the state of competence by the treatment with
chemical or physical agents that enable their transformation with exogenous DNA.
For Bacillus spp. transformation pA13 and pAZIL plasmids were used. Transformation
of naturally competent Bacillus strains was more efficient if performed at the stage of
growth when cells are naturally competent. From four different methods examined
on the strain Bacillus subtilis 168 we determined that for successful transformation
the addition of manganese sulphate in the growth medium was essential. Bacillus
licheniformis 30.3 was successfully transformed when medium contained maleic
acid as buffer and succinate for cell regeneration. The protoplasts regeneration on
succinate was a key step in the production of B. licheniformis 30.3 transformants.
Bacillus thuringiensis HD1 was successfully transformed by the method which used
Tris-induced competence, and longer incubation in the medium of competence
was very significant for the transformation efficiency. All the transformants were
obtained only with pA13, a rolling circle replication plasmid. In conclusion, efficiency
of Bacillus spp. transformation is species specific and depends on the characteristics
of the plasmids used.Abstracts of the 6th CONGRESS OF THE SERBIAN GENETIC SOCIETY Vrnjačka Banja; October 13 to 17, 2019
Pharmacogenomic profiling of serbian patients with pediatric acute lymphoblastic leukemia
No full textmics is one of the cornerstones of personalized medicine. Treatment of
pediatric acute lymphoblastic leukemia (ALL) based upon the incorporation of the principles
of pharmacogenomics significantly improves the outcome of the disease. Standard treatment
options for pediatric ALL encompass cytotoxic agents grouped into so called therapeutic
phases, namely remission induction, consolidation, intensification and maintenance phase.
We performed PCR-based and Sanger sequencing analyses, and next-generation sequencing in
retrospective study to investigate variants in genes important for metabolizing corticosteroid
drugs used in the induction phase and 6-mercaptopurine and methotrexate drugs used in
the maintenance phase in 120 Serbian pediatric ALL patients and 100 healthy controls.
Variants in NR3C1, GSTP11 and ABCB1 genes have been associated with the response to
glucocorticoid treatment in children with ALL. Genetic variants in TPMT (both coding
and promoter regions), ITPA, ABCC4 and ABCB1 genes have been shown as predictors
of 6-mercaptopurine induced toxicity. Also, we have identified genetic variants in DHFR
(promoter region), TYMS, MTHFR and SLC19A1 genes related to side effects of methotrexate
use. Association study revealed potential new pharmacogenetics markers that have to be
validated in prospective studies as well as in different populations.
True understanding of processes leading to disease development and mechanisms of
treatment efficacy and toxicity, as well as gaining new knowledge from big data obtained
in large omics studies and validation studies from various populations, could be the way
to implement personalized treatment to each patient. Research efforts have to be focused
on data analysis and designing prediction model using machine learning algorithms.
Bioinformatics tools and implementation of artificial intelligence are expected to open the
door wide for personalized medicine in clinical practice of childhood ALL.Abstracts of the 6th CONGRESS OF THE SERBIAN GENETIC SOCIETY Vrnjačka Banja; October 13 to 17, 2019
Gene expression and biochemical parameters during germination of maize seeds under the low temperature condition
No full textMaize is one of the most important crops worldwide. During several past decades severe
yield losses due to climatic changes have been observed. Avoiding period of high
temperatures during the summer, which could have negative effect on flowering and
grain filling, could be achieved by earlier sowing. The main concern is how the
temperature lower than optimal could affect early phases of maize growing. The object of
this research was to test the changes in biochemical parameters and gene expression
related to low temperature effect during the imbibition phase in two maize lines,
contrasting in the level of tolerance according to breeder’s experience. Experiment was
set at 8°C as treatment and 22°C as control temperature. Both genotypes were
exposed to cold stress for 24h. Germination test performed under treatment/control
showed better results for the tolerant genotype. Cell conductivity was significantly
higher under cold treatment in the sensitive genotype compared to control conditions.
Lipid peroxidation was elevated under the low temperature in both genotypes, but it was
more prominent in the sensitive line. Also, expression of some genes proven to be
involved in plant response to abiotic stress was tested. Expression of GID1, gibberellin
receptor, was three fold higher in the sensitive line. The role of this gene in cold tolerance
is not yet completely elucidated. FAD2 (fatty acid desaturase 2) gene was not activated by
low temperature, while expression of FAD6 (fatty acid desaturase 6) was much higher in
the sensitive line which is in accordance to literature data for some plant species. Results
of these analyses confirmed previous presumption about the level of tolerance in tested
material which is important for breeding programs and gave some directions for
further more profound research in defining pathways involved in maize plant response
to low temperatures in germination phase.Abstracts of the 6th CONGRESS OF THE SERBIAN GENETIC SOCIETY Vrnjačka Banja; October 13 to 17, 2019
Expression of genes associated with inflammation and fibrosis and candidate genes for cell therapy optimization in patients with systemic sclerosis
No full textSistemska skleroza (SSc) je retka, heterogena, multisistemska, autoimuna bolest nepoznate etiologije sa varijabilnim kliničkim manifestacijama i nepredvidivim i često letalnim ishodom. Osnovne karakteristike ove bolesti su poremećaji u imunom sistemu i inflamacija, vaskulopatija, preterana produkcija komponenti vanćelijskog matriksa i progresivna fibroza kože i unutrašnjih organa. Pronalaženje novih biomarkera, koji bi omogućili bolje razumevanje patogeneze same bolesti, bilo bi od presudne važnosti za ranije uspostavljanje dijagnoze, bolju klasifikaciju pacijenata i primenu adekvatnije terapije. Stoga je u okviru ove teze ispitana asocijacija odabranih genetičkih varijanti u genima IRAK1 i miRNK-146a sa predispozicijom za razvoj SSc. Takođe, u cilju otkrivanja potencijalno novih i pouzdanih biomarkera praćena je ekspresija odabranih učesnika signalnih puteva JAK2/STAT3 i NF-kB, uključenih u proces inflamacije na sistemskom i proces fibroze na lokalnom nivou. Upotreba ćelijske terapije u lečenju oštećenja malih zglobova kod ovih pacijenata jedan je od potencijalno novih terapeutskih pristupa. Uprkos rastućem znanju i upotrebi mezenhimskih matičnih ćelija izolovanih iz adopoznog tkiva (aMMĆ) u ćelijskoj terapiji, veoma malo se zna kako gajenje ovih ćelija u laboratorijskim uslovima utiče na njihovu sposobnost samoobnove i potencijal za diferencijaciju u hondrocite. U cilju ispitivanja uticaja dužine gajenja ćelija na njihovu sposobnost samoobnove i potencijala za hondrogenu diferencijaciju, praćena je ekspresija gena markera matičnosti i hondrogene diferencijacije tokom in vitro kultivacije ovih ćelija.Studija asocijacije odabranih genetičkih varijanti, IRAK1 rs3027898 C>A i miRNK-146a rs2910164 G>C izvedena je na grupi od 102 SSc pacijenata i 66 zdravih ispitanika, direktnim sekvenciranjem po Sangeru. Relativna kvantifikaciona analiza ekspresije gena IL6, JAK2, STAT3, TLR7, TLR9, IRAK1 i miRNK-146a u mononuklearnim ćelijama periferne krvi (MNĆPK), izvršena je na podgrupi od 50 SSc pacijenata i 13 zdravih ispitanika, dok je ekspresiona analiza odabranih gena na lokalnom nivou izvedena na uzorcima tkiva kože, sa i bez fibrotičnih promena, poreklom iz 5 SSc pacijenata...Systemic sclerosis (SSc) is a rare heterogeneous, multisystem, autoimmune disease with unknown etiology, characterized with variable clinical manifestations, and unpredictable and often lethal outcome. The basic mechanism of the disease appears to involve aberrant immune activation and inflammation, vasculopathy, overproduction of extracellular matrix proteins and progressive fibrosis of skin and internal organs. The identification of novel biomarkers will not only enable a better understanding of the disease pathogenesis, but also contribute to earlier establishment of diagnosis, better classification of patients, and the application of more appropriate therapy. Therefore, within this thesis, it was examined the association of selected genetic variants in the IRAK1 and miRNA-146a genes with the predisposition for development of SSc. In order to discover potentially novel and reliable biomarkers, the expression of selected participants of JAK2/STAT3 and NF-kB signal pathways, involved in the process of inflammation at the systemic and process of fibrosis at the local level, was examined. Stem cell therapy, is one of the potential new therapeutic approaches in the treatment of small-joint damage in SSc patients. Despite growing knowledge and utilization of adipose tissue-derived mesenchymal stem cell (aMSC) in therapy, very little known about how prolonged cultivation of these cells in laboratory conditions affects their ability for self-renewal and their potential for differentiation into functional chondrocytes. In order to investigate the influence of the long-term ex vivo cultivation on their potency and potential for chondrogenic diferrentiation, the expression of genes, markers of stemness, as well as markers for chondrogenic differentiation, during prolonged in vitro aMSC cultivation, was investigated.The association study between selected genetic variants, IRAK1 rs3027898 C> A and miRNA-146a rs2910164 G> C, and susceptibility to SSc was performed on a group of 102 SSc patients and 66 healthy subjects, by Sanger sequencing. Relative quantification analysis of the IL6, JAK2, STAT3, TLR7, TLR9, IRAK1 and miRNA-146a gene expressions in peripheral blood mononuclear cells, were performed on a subgroup of 50 SSc patients and13 healthy subjects, while the expression analyzes of selected genes at the local level were performed on skin tissue samples, with and without fibrotic changes, originating from 5 SSc patients..
Genetic predictors of celiac disease, lactose intolerance, and vitamin D function and presence of peptide morphins in urine of children with neurodevelopmental disorders
No full textGastrointestinal disturbances, nutritional deficiencies, and food intolerances are frequently observed in children with neurodevelopmental disorders (NDD). To reveal possible association of celiac disease risk variants (HLA-DQ), lactose intolerance associated variant (LCT-13910C gt T) as well as variant associated with vitamin D function (VDR FokI) with NDD, polymerase chain reaction-based methodology was used. Additionally, intestinal peptide permeability was estimated in NDD patients and healthy children by measuring the level of peptides in urine using high-performance liquid chromatography. Levels of opioid peptides, casomorphin 8, and gluten exorphin C were significantly elevated in urine samples of NDD patients (P = 0.004 and P = 0.005, respectively), but no association of genetic risk variants for celiac disease and lactose intolerance with NDD was found. Our results indicate that increased intestinal peptide permeability observed in analyzed NDD patients is not associated with genetic predictors of celiac disease or lactose intolerance. We have also found that FF genotype of VDR FokI and lower serum levels of vitamin D (25-OH) showed association with childhood autism (CHA), a subgroup of NDD. We hypothesize that vitamin D might be important for the development of CHA
The importance of combined NGS and MLPA genetic tests for differential diagnosis of maturity onset diabetes of the young
Get PDFIntroduction: Maturity onset diabetes of the young (MODY) is a rare form of monogenic diabetes. Being clinically and genetically heterogeneous, it is often misdiagnosed as type 1 or type 2 diabetes, leading to inappropriate therapy. MODY is caused by a single gene mutation. Thirteen genes, defining 13 subtypes, have been identified to cause MODY. A correct diagnosis is important for the right therapy, prognosis, and genetic counselling. Material and methods: Twenty-nine unrelated paediatric patients clinically suspected of having MODY diabetes were analysed using TruSight One panel for next-generation sequencing (NGS) and multiplex ligation-dependent probe amplification (MLPA) assay. Results: In this study we identified variants in MODY genes in 22 out of 29 patients (75.9%). Using two genetic tests, NGS and MLPA, we detected both single nucleotide variants and large deletions in patients. Most of the patients harboured a variant in the GCK gene (11/22), followed by HNF1B (5/22). The rest of the variants were found in the NEUROD1 and HNF1A genes. We identified one novel variant in the GCK gene: c.596T gt C, p.Val199Ala. The applied genetic tests excluded the suspected diagnosis of MODY in two patients and revealed variants in other genes possibly associated with the patient's clinical phenotype. Conclusions: In our group of MODY patients most variants were found in the GCK gene, followed by variants in HNF1B, NEUROD1, and HNF1A genes. The combined NGS and MLPA-based genetic tests presented a comprehensive approach for analysing patients with suspected MODY diabetes and provided a successful differential diagnosis of MODY subtypes
Binding activity to intestinal cells and transient colonization in mice of two Lactobacillus paracasei subsp. paracasei strains with high aggregation potential
No full textSurface properties like hydrophobicity, aggregation ability, adhesion to mucosal surfaces and epithelial cells and transit time are key features for the characterization of probiotic strains. In this study, we used two Lactobacillus paracasei subsp. paracasei strains (BGNJ1-64 and BGSJ2-8) strains which were previously described with very strong aggregation capacity. The aggregation promoting factor (AggLb) expressed in these strains showed high level of binding to collagen and fibronectin, components of extracellular matrix. The working hypothesis was that strains able to aggregate have an advantage to resist in intestinal tract. So, we assessed whether these strains and their derivatives (without aggLb gene) are able to bind or not to intestinal components and we compared the transit time of each strains in mice. In that purpose parental strains (BGNJ1-64 and BGSJ2-8) and their aggregation negative derivatives (BGNJ1-641 and BGSJ2-83) were marked with double antibiotic resistance in order to be tracked in in vivo experiments in mice. Comparative analysis of binding ability of WT and aggregation negative strains to different human intestinal cell lines and mucin revealed no significant difference among them, excluding involvement of AggLb in interaction with surface of intestinal cells and mucin. In vivo experiments showed that surviving and transit time of marked strains in mice did not drastically depend on the presence of the AggLb aggregation factor