Genetic Association Studies on Prostate Cancer

The modern research on molecular basis of prostate cancer (PCa) development includes studies aiming to identify potential genetic markers which could be used in diagnostics and/or monitoring of PCa. Genome-wide association studies (GWASs) have identified over 75 variants associated with PCa risk. One of the major PCa-related regions identified through GWASs is found to be a segment of 8q24. Other important PCa-susceptibility regions are 17q12, 17q24, 10q11, and 19q13. Candidate-gene based approach has also provided evidence of association between PCa risk and genetic variants located in functionally significant genes (both protein-coding and noncoding RNA genes) involved in normal prostatic cell growth, malignant transformation, or in the development of metastases. Nevertheless, the success of these studies is questionable, since numerous candidates for PCa-susceptibility variants were identified, but these results failed to replicate. The main aim of both types of genetic association studies on PCa is the identification of potential PCa genetic markers which could be used for constructing reliable algorithms for evaluating the risk for PCa development and/or PCa progression

Efekat epistatičkih interakcija između varijanti u genima za mikrornk i genima za proteine utišavajućeg kompleksa na rizik za razvoj i progresiju karcinoma prostate

Dobrijević Z., J. Karanović, D. Savić-Pavićević, G. Brajušković (2023). The effect of epistatic interactions between genetic variants located in microRNA and silencing complex genes on prostate cancer progression risk.- Genetika, Vol 55, No.1, 263-275. Previous studies conducted in Asian and European populations have provided evidence of the association between microRNA-related genetic variants and prostate cancer (PCa) risk and/or progression. Nevertheless, the results obtained in these studies are inconsistent, which could be explained by the limitations of single-locus main effect evaluations to detect joint effects of multiple genetic variants, reflected in statistical epistases. Therefore, we conducted the analysis of potential epistatic interactions between variants located in microRNA genes and in genes encoding the components of RNA-induced silencing complex (RISC) in relation with PCa risk/aggressiveness. Raw data on genotyping results from our previous studies involving four microRNA polymorphisms and five variants in RISC genes were subjected to the exclusion of samples based on missing data criterion, followed by the re-evaluation of Hardy-Weinberg equilibrium. Afterwards, these genotyping results were included in the Multifactor dimensionality reduction (MDR) analysis. Permutation testing was conducted in order to assess statistical significance of the best models from MDR tests. MDR tests on the risk of developing PCa yielded statistically insignificant results. Nevertheless, the MDR results for comparison of PCa patients with high and low cancer progression risk were statistically significant for the analysis that included rs11614913, with the 3-locus best model comprising this genetic variant, rs7813 and rs784567. We conclude that statistical epistasis between rs11614913 in hsa-miR-196a2, rs7813 in GEMIN4 and rs784567 in TARBP2 shows association with the invasiveness of PCa.Ranije studije u evropskim i azijskim populacijama ukazale su na značajnu asocijaciju genetičkih varijanti sa efektom na funkciju mikroRNK sa rizikom za razvoj i/ili progresiju karcinoma prostate (KP). Međutim, rezultati navedenih studija su nepodudarni, za šta je jedan od mogućih razloga nemogućnost pristupa baziranih na proceni pojedinačnih efekata genetičkih varijanti da detektuju značajne zajedničke efekte više varijanti, a koji se reflektuju u statističke epistaze. Iz tog razloga, cilj naše studije bila je analiza potencijalnih epistatičkih interakcija između varijanti lociranih u genima za mikroRNK molekule i u genima za komponente utišavajućeg kompleksa indukovanog sa RNK (RISC) kao faktora rizika za razvoj i/ili progresiju KP. Rezultati genotipizacije dobijeni tokom sprovođenja naših ranijih studija, a koji uključuju podatke za četiri varijante u genima za mikroRNK i pet u genima za komponente RICS, podvrgnuti su inicijalnoj obradi podataka u smislu isključivanja uzoraka sa nedostajućim rezultatima, nakon čega je procenjeno odstupanje od Hardi-Vajnbergove ravnoteže. Rezultati su zatim analizirani metodom redukcije dimenzionalnosti višestrukih faktora (Multifactor dimensionality reduction - MDR analysis). Permutacioni testovi su sprovedeni sa ciljem procene statističke značajnosti najboljih modela iz MDR analize. Dobijeni rezultati MDR testova koji su se odnosili na rizik za razvoj KP nisu bili statistički značajni. S druge strane, MDR rezultati koji se odnose na rizik za progresiju KP bili su značajni za model koji uključuje tri lokusa: rs11614913, rs7813 and rs784567. Stoga, zaključci naše studije ukazuju na značaj epistatičkih interakcija između varijanti rs11614913 u hsa-miR-196a2, rs7813 u GEMIN4 i rs784567 u TARBP2 kao faktora koji ispoljavaju efekat na invazivnost KP

Asocijacija varijanti u genima PRMT6, PEX10 I SOX5 sa idiopatskim muškim sterilitetom: dokazi iz populacije Severne Makedonije i ažurirana meta-analiza

PRMT6, PEX10 and SOX5 genetic variants were identified as male infertility-associated loci in a genome-wide association study and further validated in various populations. Still, the results of previous case-control studies varied, which could be due to differences in participants’ ethnic backgrounds. The main purpose of the present study was to evaluate the supposed association of these variants with idiopathic male infertility in North Macedonian population. Furthermore, we aimed to conduct the systematic quantitative data synthesis which includes the results of previous studies on the same issue in other European and non-European populations. A total of 137 men from North Macedonia diagnosed with idiopathic infertility and 130 age-matched fertile controls were included in the present case-control study. PCR-RFLP method was used for genotyping. Meta-analysis was performed by OpenMeta-analyst statistical software. Variants rs10842262 in SOX5, rs2477686 in PEX10 and rs12097821 in PRMT6 showed the lack of statistically significant differences in genotype distributions between men diagnosed with idiopathic infertility and the control group. Still, rs10842262 allele G frequency was significantly increased in men with poor sperm concentration (P= 0.024, OR = 2.10, 95%CI 1.08-4.06). Meta-analysis further showed the association of rs10842262 and rs12097821 with the risk of idiopathic male infertility. Our results obtained in North Macedonian population supported the previous reports on the involvement of rs10842262 in the genetic basis of male infertility. The meta-analysis confirmed the association of rs10842262 and rs12097821 with male infertility occurrence. Still, additional studies are needed to support the present findings.Asocijacija varijanti u genima PRMT6, PEX10 i SOX5 sa muškim sterilitetom identifikovana je u studiji genetičke asocijacije na čitavom genomu i kasnije analizirana u studijama slučajeva i kontrola u različitim populacijama. Rezultati prethodnih studija su pokazali značajnu varijabilnost, što može biti posledica razlika u etničkom poreklu studijskih grupa. Osnovni cilj ovog istraživanja je analiza asocijacije navedenih genetičkih varijanti sa rizikom za pojavu idiopatskog muškog steriliteta u populaciji Severne Makedonije. Takođe, naš cilj je bio i sprovođenje sistematske kvantitativne sinteze podataka iz studija sa istom ili sličnom temom istraživanja sprovedenim u drugim evropskim i neevropskim populacijama. Ukupno 137 muškaraca sa idiopatskim sterilitetom iz Severne Makedonije i 130 fertilnih kontrola slične starosti uključeno je u studiju slučajeva i kontrola. Genotipizacaija je vršena PCR-RFLP metodom, dok je za meta-analizu korišćen statistički softver OpenMeta-analyst. Za varijante rs10842262 u SOX5, rs2477686 u PEX10 i rs12097821 u PRMT6 nije utvrđena statistički značajna razlika u distribucijama genotipova između grupe ispitanika sa idiopatskim sterilitetom i kontrolne grupe. Međutim, učestalost alela G varijante rs10842262 bila je značajno povećana kod muškaraca sa niskom koncentracijom spermatozoida (P= 0.024, OR = 2.10, 95%CI 1.08– 4.06). Meta-analizom pokazana je asocijacija rs10842262, ali i rs12097821, sa rizikom za razvoj idiopatskog muškog steriliteta. Naši rezultati ustanovljeni u populaciji Severne Makedonije idu u prilog prethodnim navodima o učešću rs10842262 u genetičkoj osnovi muškog steriliteta. Ipak, dodatne studije su neophodne kako bi potvrdile značaj rezultata ovog istraživanja

Two main skeletal muscle molecular phenotypes of mouse dm1 models: a comparative transcriptomic analysis

Introduction:Myotonic dystrophy type 1 (DM1) is a rare, incurable multisystemic disease, with the main symptoms being skeletal muscle weakness, atrophy, and myotonia. It is caused by CTG expansion in the 3' UTR of the DMPK gene whose RNA acquires toxic functions and sequesters MBNL proteins, resulting in globally altered RNA metabolism. To better understand the DM1 transcriptome, we systematically analyzed gene expression in the skeletal muscles of various mouse DM1 models. Methods:We retrieved 13 publicly available RNA-seq datasets from mouse models expressing expanded CTG repeats (HSALR, CTG480KI, TREDT960I) and Mbnl knockout models (SKO, DKO, TKO). Our bioinformatic pipeline with unified parameters consisted of preprocessing, differential expression (DESeq2), gene network analysis (WGCNA), comparison of gene network interactions with the STRING database, and network node enrichment analysis (Cytoscape). Results: In models expressing CTG repeats, the average number of up-regulated genes was 787, compared to 676 in Mbnl knockout models, while there was 642 and 380 down-regulated genes, respectively (log2FC>1, padj>0.05). Both model groups had network modules whose nodes were enriched for muscle and secretory functions (FDR<0.05). There were modules related to immune response, lipid transfer, and insulin in models expressing repeats and modules related to immunoglobulins and extracellular matrix in knockout models. Conclusion: Gene expression patterns separated Mbnl knockouts from models expressing CTG repeats that had a greater number of smaller functionally distinct network modules. Our results revealed novel pathway changes in DM1 skeletal muscles, among which immunological and secretory are particularly interesting as molecular targets for further investigation

Assessment of possible association between rs378854 and prostate cancer risk in the Serbian population

Prostate cancer (PCa) is the second most commonly diagnosed cancer among men worldwide. Despite its high incidence rate, the molecular basis of PCa onset and its progression remains little understood. Genome-wide association studies (GWAS) have greatly contributed to the identification of single nucleotide polymorphisms (SNP) associated with PCa risk. Several GWAS identified 8q24 as one of the most significant PCa-associated regions. The aim of this study was to evaluate the association of SNP rs378854 at 8q24 with PCa risk in the Serbian population. The study population included 261 individuals diagnosed with PCa, 257 individuals diagnosed with benign prostatic hyperplasia (BPH) and 106 healthy controls. Data quality analysis yielded results showing deviations from Hardy-Weinberg equilibrium in groups of PCa patients and BPH patients as well as in the control group. There was no significant association between alleles and genotypes of the genetic variant rs378854 and PCa risk in the Serbian population. [Projekat Ministarstva nauke Republike Srbije, br. 173016

Identification of potentally causal variants for myasthenia gravis: a bioinformatics-driven fine-mapping approach combined with genetic association study

Introduction: Genome-wide association studies (GWAS) identify genomic loci that contain genetic determinants of complex diseases. Subsequent functional genomic approaches, such as bioinformatic finemapping and transcriptome-wide association studies (TWAS), can reveal potentially causal single nucleotide variants (SNVs) that can be tested on patient samples. We applied this approach to study causal SNVs for acetylcholine receptor (AChR) seropositive myasthenia gravis (MG). We focused on CHRNA1 and CHRNB1 loci, coding AChR subunits, and CTLA-4 locus, coding protein transmitting an inhibitory signal to T cells. Methods: CHRNA1 was fine-mapped by PAINTOR using data from GWAS summary statistics, 1000 genome and RegulomeDB. Alongside, rs4151121 identified by TWAS in CHRNB1, and rs231735 and rs231770 identified by fine-mapping in CTLA-4 were studied. SNVs were genotyped using allele discrimination assays in 447 Serbian AChR-MG patients (183 early-onset and 264 late-onset) and 447 sex- and age-matched controls. Results: CHRNA1 rs35274388 was fine-mapped as a potentially causal variant (PIP2=92%) exhibiting transcription factor binding and chromatin accessibility peaks. CHRNA1 rs35274388 minor allele A and CHRNAB1 rs4151121 minor allele G increased the risk for late-onset MG (OR=1.669, 95% CI=1.05-2.638, p=0.027, p10e6 permutation=0.031 and OR=1.322, 95% CI=1.063-1.644, p10e6 permutation=0.014, respectively). On the other hand, CTLA-4 rs231735 recessive genotype TT decreased, while rs231735- rs231770 haplotype GC increased the susceptibility to early-onset MG (OR=0.548, 95% CI=0.339-0.888, p=0.014, p10e6 permutation=0.014 and OR=1.360, p=0.027, p10e6 permutation=0.027, respectively). Conclusion: CHRNA1 rs35274388 and CHRNAB1 rs4151121 loci could be causal genetic factors for lateonset MG while CTLA-4 rs231735 and rs231770 could be causal genetic factors for early-onset MG in Serbian population

One year of newborn screening for spinal muscular atrophy – results of a Serbian pilot project

Spinal muscular atrophy (SMA) is the most common genetic cause of death in childhood. Innovative therapies show the greatest benefit only when administered in the presymptomatic period, making newborn screening an ethical and medical priority in many countries. In 2022 Centre for Human Molecular Genetics initiated a feasibility study of the newborn screening for SMA in close collaboration with the University Children's Hospital Tirsova, Association SMA Serbia and with financial support from Novartis Gene Therapies, Roche and Biogen/Medis Pharma aiming to screen up to 8000 babies from the Obstetrics and Gynaecology Clinic Narodni Front during one year. A total of 6950 newborns were tested and SMA was confirmed in two unrelated newborns from families with no history of SMA. A 16-month old sibling of the first baby was also tested, even though he was completely asymptomatic, and SMA was also confirmed. Average time between birth and the first screen-positive result was 5 days, and 8 days between birth and final confirmation of diagnosis. All three children received modifying therapies in less than 10 days from final diagnosis. So far, no false-negatives have been reported among the newborns who tested negative in the screening. As pioneers and leaders in this field, we created synchronised work at different levels of healthcare system, established screening and diagnostic algorithms and follow-up protocols. We are currently involved in scaling up screening to include an additional maternity hospital and preparing the ground for the implementation of the newborn screening for SMA as the official national screening program.BOOK OF ABSTRACTS: 8th CONGRESS OF SERBIAN NEUROSCIENCE SOCIETY with international participation 31 May – 2 June 2023. Belgrade, Serbi

Repeat Interruptions Modify Age at Onset in Myotonic Dystrophy Type 1 by Stabilizing DMPK Expansions in Somatic Cells

CTG expansions in DMPK gene, causing myotonic dystrophy type 1 (DM1), are characterized by pronounced somatic instability. A large proportion of variability of somatic instability is explained by expansion size and patient’s age at sampling, while individual-specific differences are attributed to additional factors. The age at onset is extremely variable in DM1, and inversely correlates with the expansion size and individual-specific differences in somatic instability. Three to five percent of DM1 patients carry repeat interruptions and some appear with later age at onset than expected for corresponding expansion size. Herein, we characterized somatic instability of interrupted DMPK expansions and the effect on age at onset in our previously described patients. Repeat-primed PCR showed stable structures of different types and patterns of repeat interruptions in blood cells over time and buccal cells. Single-molecule small-pool PCR quantification of somatic instability and mathematical modeling showed that interrupted expansions were characterized by lower level of somatic instability accompanied by slower progression over time. Mathematical modeling demonstrated that individual-specific differences in somatic instability had greater influence on age at onset in patients with interrupted expansions. Therefore, repeat interruptions have clinical importance for disease course in DM1 patients due to stabilizing effect on DMPK expansions in somatic cells

SMN1 copy number as a modifying factor of survival in Serbian patients with sporadic amyotrophic lateral sclerosis

Introduction/Objective. Amyotrophic lateral sclerosis (ALS) is a devastating motor neuron disease. The majority of cases are apparently sporadic ALS (SALS) with variants in susceptibility genes or sometimes in high-risk ALS genes. Two ALS susceptibility genes are SMN1, whose functional loss causes spinal muscular atrophy (SMA), and a nearly identical SMN2 gene, which modulates SMA severity. In this study we examined the association of copy number variations (CNVs) of SMN1 and SMN2 genes and two additional genes, SERF1 and NAIP, residing in the same genomic region (i.e. 5q13.2 segmental duplication), with SALS in patients from Serbia. Methods. Multiplex ligation-dependent probe amplification was used to determine CNVs of each gene in a clinically well-characterised group of 153 Serbian SALS patients and 153 controls. Results. Individual association between SMN1, SMN2, SERF1 or NAIP CNVs and SALS susceptibility or survival was not found. Survival curves based on the multivariable Cox regression analysis showed that three SMN1 copies, lower ALS Functional Rating Scale Revised (ALSFRS-R) score at the time of diagnosis, faster decline of the ALSFRS-R score over time, and shorter diagnostic delay result in shorter survival of Serbian SALS patients. Conclusion. Clinical variables might be complemented with the SMN1 copy number to improve prediction of survival in Serbian SALS patients

Effect of enriched environment on serotonin and RNA editing of serotonin 2C receptor is specific for brain regions and mouse strains

Serotonin neurotransmission is sensitive to environmental stimuli. Serotonin receptor 2C (HTR2C) undergoes dynamic A-to-I editing that fine-tunes cell response to serotonin and is altered in depressed suicide victims and by pharmacological treatments. We aimed to explore a mediating role of Htr2c mRNA editing in response to enriched environment and factors involved in this response. Three-week-old BALB/c and C57BL/6 male mice were housed in enriched and standard conditions for four weeks. Htr2c mRNA editing pattern and expression, serotonin level and expression of Adar and Adarb1 mRNAs (coding enzymes catalyzing A-to-I editing) and Snord115 RNA (regulating Htr2c mRNA alternative splicing and editing) were measured in prefrontal cortex (PFC) and hippocampus (HC), brain regions implicated in suicidal behavior. BALB/c mice, a “stress-sensitive” strain due to genetically determined lower serotonin level, responded to enriched conditions by adapting the Htr2c editing pattern to a slight serotonin decrease in PFC and a significant increase in HC. C57BL/6 mice, a “stress-resilient” strain, responded to enriched environment by increasing the serotonin level and changing Adar and Adarb1 mRNAs expression in HC, and without changes in PFC. Our findings suggest that the enriched environment effect on the serotonin level and a mediating role of Htr2c mRNA editing in PFC depend on the genetic background and its interactions with the environment. On the other hand, changes in HC are primarily driven by enriched environment. Our results imply usefulness of enriched environment paradigm for understanding interactions of genetic and environmental factors underlying suicidal behavior, which might improve psychological treatments.BOOK OF ABSTRACTS: 8th CONGRESS OF SERBIAN NEUROSCIENCE SOCIETY with international participation 31 May – 2 June 2023. Belgrade, Serbi