Can Pharmacogenetic Variants in TPMT, MTHFR and SLCO1B1 Genes Be Used as Potential Markers of Outcome Prediction in Systemic Sclerosis Patients?

Systemic sclerosis (SSc) is a rare connective tissue disorder with highest morbidity and mortality among rheumatologic diseases. Disease progression is highly heterogeneous between patients, implying a strong need for individualization of therapy. 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. Association was found 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. 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 reactions.

Germline Variants in Cancer Predisposition Genes in Pediatric Patients with Central Nervous System Tumors.

Central nervous system (CNS) tumors comprise around 20% of childhood malignancies. Germline variants in cancer predisposition genes (CPGs) are found in approximately 10% of pediatric patients with CNS tumors. This study aimed to characterize variants in CPGs in pediatric patients with CNS tumors and correlate these findings with clinically relevant data. Genomic DNA was isolated from the peripheral blood of 51 pediatric patients and further analyzed by the next-generation sequencing approach. Bioinformatic analysis was done using an "in-house" gene list panel, which included 144 genes related to pediatric brain tumors, and the gene list panel Neoplasm (HP:0002664). Our study found that 27% of pediatric patients with CNS tumors have a germline variant in some of the known CPGs, like ALK, APC, CHEK2, ELP1, MLH1, MSH2, NF1, NF2 and TP53. This study represents the first comprehensive evaluation of germline variants in pediatric patients with CNS tumors in the Western Balkans region. Our results indicate the necessity of genomic research to reveal the genetic basis of pediatric CNS tumors, as well as to define targets for the application and development of innovative therapeutics that form the basis of the upcoming era of personalized medicine.

Analysis of the promoter regions of disease-causing genes in maturity-onset diabetes of the young patients.

Maturity-onset diabetes of the young (MODY) is a form of monogenic diabetes caused by the variants in MODY-related genes. In addition to coding variants, variants in the promoter region of MODY-related genes can cause the disease as well. In this study, we screened the promoter regions of the most common MODY-related genes GCK, HNF1A, HNF4A and HNF1B in our cohort of 29 MODY patients. We identified one genetic variant in the HNF1A gene, a 7 bp insertion c.-154-160insTGGGGGT, and three variants in the GCK gene, -282C>T; -194A>G; 402C>G appearing as set. Chloramphenicol acetyltransferase (CAT) assay was performed to test the effect of the 7 bp insertion and the variant set on the activity of the reporter gene in HepG2 and RIN-5F cell, respectively, where a decreasing trend was observed for both variants. In silico analysis and electrophoretic mobility shift assay showed that the 7 bp insertion did not create the binding site for new transcriptional factors, but gave rise to additional binding sites for the existing ones. Results from our study indicated that the 7 bp insertion in the HNF1A gene could be associated with the patient's diabetes. As for the GCK variant set, it is probably not associated with diabetes in patients, but it may modify the fasting glucose level by causing small elevation in variant set carriers. We have presented two promoter variants in MODY-related genes. Variant in the HNF1A gene is presumed to be disease-causing and the GCK promoter variant set could be a phenotype modifier.

Genetic predictors of celiac disease, lactose intolerance, and vitamin D function and presence of peptide morphins in urine of children with neurodevelopmental disorders.

Gastrointestinal 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>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.

Expanded national database collection and data coverage in the FINDbase worldwide database for clinically relevant genomic variation allele frequencies.

FINDbase (http://www.findbase.org) is a comprehensive data repository that records the prevalence of clinically relevant genomic variants in various populations worldwide, such as pathogenic variants leading mostly to monogenic disorders and pharmacogenomics biomarkers. The database also records the incidence of rare genetic diseases in various populations, all in well-distinct data modules. Here, we report extensive data content updates in all data modules, with direct implications to clinical pharmacogenomics. Also, we report significant new developments in FINDbase, namely (i) the release of a new version of the ETHNOS software that catalyzes development curation of national/ethnic genetic databases, (ii) the migration of all FINDbase data content into 90 distinct national/ethnic mutation databases, all built around Microsoft's PivotViewer (http://www.getpivot.com) software (iii) new data visualization tools and (iv) the interrelation of FINDbase with DruGeVar database with direct implications in clinical pharmacogenomics. The abovementioned updates further enhance the impact of FINDbase, as a key resource for Genomic Medicine applications.

Novel genetic risk variants for pediatric celiac disease.

BACKGROUND: Celiac disease is a complex chronic immune-mediated disorder of the small intestine. Today, the pathobiology of the disease is unclear, perplexing differential diagnosis, patient stratification, and decision-making in the clinic. METHODS: Herein, we adopted a next-generation sequencing approach in a celiac disease trio of Greek descent to identify all genomic variants with the potential of celiac disease predisposition. RESULTS: Analysis revealed six genomic variants of prime interest: SLC9A4 c.1919G>A, KIAA1109 c.2933T>C and c.4268_4269delCCinsTA, HoxB6 c.668C>A, HoxD12 c.418G>A, and NCK2 c.745_746delAAinsG, from which NCK2 c.745_746delAAinsG is novel. Data validation in pediatric celiac disease patients of Greek (n = 109) and Serbian (n = 73) descent and their healthy counterparts (n = 111 and n = 32, respectively) indicated that HoxD12 c.418G>A is more prevalent in celiac disease patients in the Serbian population (P < 0.01), while NCK2 c.745_746delAAinsG is less prevalent in celiac disease patients rather than healthy individuals of Greek descent (P = 0.03). SLC9A4 c.1919G>A and KIAA1109 c.2933T>C and c.4268_4269delCCinsTA were more abundant in patients; nevertheless, they failed to show statistical significance. CONCLUSIONS: The next-generation sequencing-based family genomics approach described herein may serve as a paradigm towards the identification of novel functional variants with the aim of understanding complex disease pathobiology.

Functional Analysis of an (A)γ-Globin Gene Promoter Variant (HBG1: g.-225_-222delAGCA) Underlines Its Role in Increasing Fetal Hemoglobin Levels Under Erythropoietic Stress.

Hereditary persistence of fetal hemoglobin (HPFH) is a condition characterized by persistent γ-globin gene expression and synthesis of high levels of fetal hemoglobin (Hb F; α2γ2) during adult life. It is usually caused by promoter variants or large deletions affecting the human fetal globin (HBG1 and HBG2) genes. Some of these HPFH-causing variants, such as HBG2: g.-158 C > T, exert their effect only under conditions of erythropoietic stress, typical for ß-thalassemia (ß-thal) patients. Namely, the presence of HBG2: g.-158 C > T favors a higher Hb F response, while it has little effect in healthy individuals. We analyzed a previously reported deletion residing in the promoter region of the HBG1 gene (HBG1: g.-225_-222delAGCA), both in normal conditions and under conditions of erythropoietic stress. Our results indicate that this deletion is responsible for decreased HBG1 gene expression. Specifically, this deletion was shown to result in drastically reduced reporter gene expression in K562 cells, compared to the wild-type sequence but only under conditions of erythropoietic stress, mimicked by introduction of erythropoietin (EPO) into the cell culture. Also, electrophoretic mobility shift analysis showed that the HBG1: g.-225_-222delAGCA deletion creates additional transcriptional factors' binding sites, which, we propose, bind a transcriptional repressor, thus decreasing the HBG1 gene promoter activity. These results are consistent with in silico analysis, which indicated that this deletion creates a binding site for GATA1, known to be a repressor of the γ-globin gene expression. These data confirm the regulatory role of the HBG1: g.-225_-222 region that exerts its effect under conditions of erythropoietic stress characteristic for ß-thal patients.

6th Golden Helix Pharmacogenomics Day: pharmacogenomics and individualized therapy.

The Golden Helix Pharmacogenomics Days are international scientific meetings aiming to educate healthcare professionals and biomedical scientists about pharmacogenomics and personalized medicine. In this meeting report, we provide an overview of the scientific lectures and the topics discussed during the 6th Golden Helix Pharmacogenomics Day that was held in Belgrade, Serbia last June 5, 2012. The scientific program included lectures by the local and international speakers from Europe and the United States.

Functional analysis of a novel KLF1 gene promoter variation associated with hereditary persistence of fetal hemoglobin.

Hereditary persistence of fetal hemoglobin (HPFH) is a rare hereditary condition resulting in elevated levels of fetal hemoglobin (HbF) in adults. Typical HPFH is associated with promoter mutations or large deletions affecting the human fetal globin (HBG1 and HBG2) genes, while genetic defects in other genes involved in human erythropoiesis, e.g. KLF1, also result in atypical HPFH. Here, we report the first KLF1 gene promoter mutation (KLF1:g.-148G > A) that is associated with increased HbF level. This mutation was shown to result in drastically reduced CAT reporter gene expression in K562 cells, compared to the wild-type sequence (p = 0.009) and also in reduced KLF1 gene expression in vivo. Furthermore, consistent with in silico analysis, electrophoretic mobility shift analysis showed that the KLF1:g.-148G > A mutation resides in a Sp1 binding site and further that this mutation leads to the ablation of Sp1 binding in vitro. These data suggest that the KLF1:g-148G > A mutation could play a role in increasing HbF levels in adults and further underlines the role of KLF1 as one of the key transcription factors involved in human fetal globin gene switching.

The Sixth European Society of Pharmacogenomics and Personalised Therapy Congress.

On 8-9 November 2022, the European Society of Pharmacogenomics and Personalised Therapy organized its sixth biennial congress, in Belgrade, Serbia (congress website: www.sspt.rs). The congress aimed to address the current status and future perspectives of pharmacogenomics, share latest knowledge in the field of precision medicine and showcase the implementation of clinical applications in pharmacogenomics/pharmacogenetics. The 2 day congress consisted of 17 lectures given by key-opinion leaders and included a poster session plus discussions. The meeting was a great success by generating an informal environment and enabling the exchange of information between 162 participants from 16 different countries.

Diagnostic and Therapeutic Implications of Long Non-Coding RNAs in Leukemia.

Leukemia is a heterogenous group of hematological malignancies categorized in four main types (acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML) and chronic lymphocytic leukemia (CLL). Several cytogenetic and molecular markers have become a part of routine analysis for leukemia patients. These markers have been used in diagnosis, risk-stratification and targeted therapy application. Recent studies have indicated that numerous regulatory RNAs, such as long non-coding RNAs (lncRNAs), have a role in tumor initiation and progression. When it comes to leukemia, data for lncRNA involvement in its etiology, progression, diagnosis, treatment and prognosis is limited. The aim of this review is to summarize research data on lncRNAs in different types of leukemia, on their expression pattern, their role in leukemic transformation and disease progression. The usefulness of this information in the clinical setting, i.e., for diagnostic and prognostic purposes, will be emphasized. Finally, how particular lncRNAs could be used as potential targets for the application of targeted therapy will be considered.

Genome-Wide Association Study of COVID-19 Outcomes Reveals Novel Host Genetic Risk Loci in the Serbian Population.

Host genetics, an important contributor to the COVID-19 clinical susceptibility and severity, currently is the focus of multiple genome-wide association studies (GWAS) in populations affected by the pandemic. This is the first study from Serbia that performed a GWAS of COVID-19 outcomes to identify genetic risk markers of disease severity. A group of 128 hospitalized COVID-19 patients from the Serbian population was enrolled in the study. We conducted a GWAS comparing (1) patients with pneumonia (n = 80) against patients without pneumonia (n = 48), and (2) severe (n = 34) against mild disease (n = 48) patients, using a genotyping array followed by imputation of missing genotypes. We have detected a significant signal associated with COVID-19 related pneumonia at locus 13q21.33, with a peak residing upstream of the gene KLHL1 (p = 1.91 × 10-8). Our study also replicated a previously reported COVID-19 risk locus at 3p21.31, identifying lead variants in SACM1L and LZTFL1 genes suggestively associated with pneumonia (p = 7.54 × 10-6) and severe COVID-19 (p = 6.88 × 10-7), respectively. Suggestive association with COVID-19 pneumonia has also been observed at chromosomes 5p15.33 (IRX, NDUFS6, MRPL36, p = 2.81 × 10-6), 5q11.2 (ESM1, p = 6.59 × 10-6), and 9p23 (TYRP1, LURAP1L, p = 8.69 × 10-6). The genes located in or near the risk loci are expressed in neural or lung tissues, and have been previously associated with respiratory diseases such as asthma and COVID-19 or reported as differentially expressed in COVID-19 gene expression profiling studies. Our results revealed novel risk loci for pneumonia and severe COVID-19 disease which could contribute to a better understanding of the COVID-19 host genetics in different populations.

Machine Learning Modeling from Omics Data as Prospective Tool for Improvement of Inflammatory Bowel Disease Diagnosis and Clinical Classifications.

Research of inflammatory bowel disease (IBD) has identified numerous molecular players involved in the disease development. Even so, the understanding of IBD is incomplete, while disease treatment is still far from the precision medicine. Reliable diagnostic and prognostic biomarkers in IBD are limited which may reduce efficient therapeutic outcomes. High-throughput technologies and artificial intelligence emerged as powerful tools in search of unrevealed molecular patterns that could give important insights into IBD pathogenesis and help to address unmet clinical needs. Machine learning, a subtype of artificial intelligence, uses complex mathematical algorithms to learn from existing data in order to predict future outcomes. The scientific community has been increasingly employing machine learning for the prediction of IBD outcomes from comprehensive patient data-clinical records, genomic, transcriptomic, proteomic, metagenomic, and other IBD relevant omics data. This review aims to present fundamental principles behind machine learning modeling and its current application in IBD research with the focus on studies that explored genomic and transcriptomic data. We described different strategies used for dealing with omics data and outlined the best-performing methods. Before being translated into clinical settings, the developed machine learning models should be tested in independent prospective studies as well as randomized controlled trials.

Expression Profiles of Long Non-Coding RNA GAS5 and MicroRNA-222 in Younger AML Patients.

Acute myeloid leukemia (AML) is a heterogeneous malignant disease both on clinical and genetic levels. AML has poor prognosis and, therefore, there is a constant need to find new prognostic markers, as well as markers that can be used as targets for innovative therapeutics. Recently, the search for new biomarkers has turned researchers' attention towards non-coding RNAs, especially long non-coding RNAs (lncRNAs) and micro RNAs (miRNAs). We investigated the expression level of growth arrest-specific transcript 5 (GAS5) lncRNA in 94 younger AML patients, and also the expression level of miR-222 in a cohort of 39 AML patients with normal karyotype (AML-NK), in order to examine their prognostic potential. Our results showed that GAS5 expression level in AML patients was lower compared to healthy controls. Lower GAS5 expression on diagnosis was related to an adverse prognosis. 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. This is the first study focused on examining the GAS5 and miR-222 expression pattern in AML patients. Its initial findings indicate the need for further investigation of these two non-coding RNAs, their potential roles in leukemogenesis, and the prognosis of AML patients.

The first insight into the genetic structure of the population of modern Serbia.

The complete understanding of the genomic contribution to complex traits, diseases, and response to treatments, as well as genomic medicine application to the well-being of all humans will be achieved through the global variome that encompasses fine-scale genetic diversity. Despite significant efforts in recent years, uneven representation still characterizes genomic resources and among the underrepresented European populations are the Western Balkans including the Serbian population. Our research addresses this gap and presents the first ever targeted sequencing dataset of variants in clinically relevant genes. By measuring population differentiation and applying the Principal Component and Admixture analysis we demonstrated that the Serbian population differs little from other European populations, yet we identified several novel and more frequent variants that appear as its unique genetic determinants. We explored thoroughly the functional impact of frequent variants and its correlation with the health burden of the population of Serbia based on a sample of 144 individuals. Our variants catalogue improves the understanding of genetics of modern Serbia, contributes to research on ancestry, and aids in improvements of well-being and health equity. In addition, this resource may also be applicable in neighboring regions and valuable in worldwide functional analyses of genetic variants in individuals of European descent.

Association of Vitamin D, Zinc and Selenium Related Genetic Variants With COVID-19 Disease Severity.

Background: COVID-19 pandemic has proved to be an unrelenting health threat for more than a year now. The emerging amount of data indicates that vitamin D, zinc and selenium could be important for clinical presentation of COVID-19. Here, we investigated association of genetic variants related to the altered level and bioavailability of vitamin D, zinc and selenium with clinical severity of COVID-19. Methods: We analyzed variants in genes significant for the status of vitamin D (DHCR7/NADSYN1 rs12785878, GC rs2282679, CYP2R1 rs10741657, and VDR rs2228570), zinc (PPCDC rs2120019) and selenium (DMGDH rs17823744) in 120 Serbian adult and pediatric COVID-19 patients using allelic discrimination. Furthermore, we carried out comparative population genetic analysis among European and other worldwide populations to investigate variation in allelic frequencies of selected variants. Results: Study showed that DHCR7/NADSYN rs12785878 and CYP2R1 rs10741657 variants were associated with severe COVID-19 in adults (p = 0.03, p = 0.017, respectively); carriers of DHCR7/NADSYN TG+GG and CYP2R1 GG genotypes had 0.21 and 5.9 the odds for developing severe disease, OR 0.21 (0.05-0.9) and OR 5.9 (1.4-25.2), respectively. There were no associations between selected genetic variants and disease severity in pediatric patients. Comparative population genetic analysis revealed that Serbian population had the lowest frequency of CYP2R1 rs10741657 G allele compared to other non-Finish Europeans (0.58 compared to 0.69 and 0.66 in Spanish and Italian population, respectively), suggesting that other populations should also investigate the relationship of CYP2R1 variant and the COVID-19 disease course. Conclusion: The results of the study indicated that vitamin D related genetic variants were implicated in severe COVID-19 in adults. This could direct prevention strategies based on population specific nutrigenetic profiles.

The FKBP5 genotype and childhood trauma effects on FKBP5 DNA methylation in patients with psychosis, their unaffected siblings, and healthy controls.

Hypothalamic-pituitary-adrenal (HPA) axis activity mediates the relationship between childhood trauma (CT) and psychosis. The FKBP5 gene, one of the key regulators of HPA axis activity after stress exposure, has been found associated with psychosis. Allele-specific and CT related FKBP5 demethylation in intron 7 was revealed in different psychiatric disorders. However, no studies have investigated FKBP5 methylation in subjects with different genetic liability for psychosis. A total of 144 participants were included in the study: 48 patients with psychotic disorders, 50 unaffected siblings, and 46 healthy controls. CT was assessed by Childhood Trauma Questionnaire. The FKBP5 rs1360780 was genotyped and FKBP5 methylation analyses were performed using bisulfite conversion followed by Sanger sequencing at three CpG sites in intron 7. Mixed linear model was used to assess group differences depending on rs1360780 T allele and CT. Results showed a significant T allele-dependent decrease of FKBP5 methylation in patients compared to unaffected siblings and controls. Effect of interaction between T allele and CT exposure on FKBP5 demethylation was found in controls. No effect of both risk factors (T allele and CT) on FKBP5 methylation level was found in unaffected siblings. We confirmed previous evidence of the association between the FKBP5 rs1360780 T allele, CT, and decreased FKBP5 methylation in intron 7. Allele-specific FKBP5 demethylation found in patients could shed a light on altered HPA axis activity in a subgroup of patients related to stress-induced psychosis. FKBP5 methylation and potential protective mechanisms in unaffected siblings after trauma exposure require further investigation.

Novel transcriptional regulatory element in the phenylalanine hydroxylase gene intron 8.

We present the first transcriptional regulatory element found in a PAH gene intron. The element is located in the PAH gene intron 8, acts as an enhancer specifically in the hepatoma cell line, and binds GATA-1 transcription factor. Herein the presented data could unlock a new area for the analysis of PAH gene expression and could contribute to refining genotype-phenotype correlation.

Thalassemia syndromes in Serbia: an update.

Thalassemia syndromes constitute a group of genetic disorders, widespread throughout the world. The present study contains data on thalassemia syndromes and their chromosomal environment obtained in Serbia over a period of 10 years. Ten different ß-thalassemia (ß-thal) mutations and two hemoglobin (Hb) variants were detected in 127 members of 68 families. Hb Lepore-Boston-Washington (Lepore-BW) (δ87Gln-ß-IVS-II-8), a thalassemic Hb variant, was shown to be the most common cause of thalassemia in Serbia. Haplotype analyses of the ß-globin gene clusters of healthy individuals as well as of individuals affected with ß-thal showed that haplotype I was the most frequent haplotype in the Serbian population, followed by haplotypes II and IX. Two novel haplotypes were detected. Haplotype analyses showed the association between certain haplotypes and the most common thalassemic mutations. Results presented in this paper will update the Serbian national mutation database and contribute to a better understanding of genographic history of South European and Balkan populations.

Functional prediction and comparative population analysis of variants in genes for proteases and innate immunity related to SARS-CoV-2 infection.

New coronavirus SARS-CoV-2 is capable to infect humans and cause a novel disease COVID-19. Aiming to understand a host genetic component of COVID-19, we focused on variants in genes encoding proteases and genes involved in innate immunity that could be important for susceptibility and resistance to SARS-CoV-2 infection. Analysis of sequence data of coding regions of FURIN, PLG, PRSS1, TMPRSS11a, MBL2 and OAS1 genes in 143 unrelated individuals from Serbian population identified 22 variants with potential functional effect. In silico analyses (PolyPhen-2, SIFT, MutPred2 and Swiss-Pdb Viewer) predicted that 10 variants could impact the structure and/or function of proteins. These protein-altering variants (p.Gly146Ser in FURIN; p.Arg261His and p.Ala494Val in PLG; p.Asn54Lys in PRSS1; p.Arg52Cys, p.Gly54Asp and p.Gly57Glu in MBL2; p.Arg47Gln, p.Ile99Val and p.Arg130His in OAS1) may have predictive value for inter-individual differences in the response to the SARS-CoV-2 infection. Next, we performed comparative population analysis for the same variants using extracted data from the 1000 Genomes project. Population genetic variability was assessed using delta MAF and Fst statistics. Our study pointed to 7 variants in PLG, TMPRSS11a, MBL2 and OAS1 genes with noticeable divergence in allelic frequencies between populations worldwide. Three of them, all in MBL2 gene, were predicted to be damaging, making them the most promising population-specific markers related to SARS-CoV-2 infection. Comparing allelic frequencies between Serbian and other populations, we found that the highest level of genetic divergence related to selected loci was observed with African, followed by East Asian, Central and South American and South Asian populations. When compared with European populations, the highest divergence was observed with Italian population. In conclusion, we identified 4 variants in genes encoding proteases (FURIN, PLG and PRSS1) and 6 in genes involved in the innate immunity (MBL2 and OAS1) that might be relevant for the host response to SARS-CoV-2 infection.