Towards Uncovering the Role of Incomplete Penetrance in Maculopathies through Sequencing of 105 Disease-Associated Genes.

Inherited macular dystrophies (iMDs) are a group of genetic disorders, which affect the central region of the retina. To investigate the genetic basis of iMDs, we used single-molecule Molecular Inversion Probes to sequence 105 maculopathy-associated genes in 1352 patients diagnosed with iMDs. Within this cohort, 39.8% of patients were considered genetically explained by 460 different variants in 49 distinct genes of which 73 were novel variants, with some affecting splicing. The top five most frequent causative genes were ABCA4 (37.2%), PRPH2 (6.7%), CDHR1 (6.1%), PROM1 (4.3%) and RP1L1 (3.1%). Interestingly, variants with incomplete penetrance were revealed in almost one-third of patients considered solved (28.1%), and therefore, a proportion of patients may not be explained solely by the variants reported. This includes eight previously reported variants with incomplete penetrance in addition to CDHR1:c.783G>A and CNGB3:c.1208G>A. Notably, segregation analysis was not routinely performed for variant phasing-a limitation, which may also impact the overall diagnostic yield. The relatively high proportion of probands without any putative causal variant (60.2%) highlights the need to explore variants with incomplete penetrance, the potential modifiers of disease and the genetic overlap between iMDs and age-related macular degeneration. Our results provide valuable insights into the genetic landscape of iMDs and warrant future exploration to determine the involvement of other maculopathy genes.

Aberrantly Expressed Hsa_circ_0060762 and CSE1L as Potential Peripheral Blood Biomarkers for ALS.

Amyotrophic lateral sclerosis (ALS) is a rapidly progressive adult-onset neurodegenerative disease that is often diagnosed with a delay due to initial non-specific symptoms. Therefore, reliable and easy-to-obtain biomarkers are an absolute necessity for earlier and more accurate diagnostics. Circular RNAs (circRNAs) have already been proposed as potential biomarkers for several neurodegenerative diseases. In this study, we further investigated the usefulness of circRNAs as potential biomarkers for ALS. We first performed a microarray analysis of circRNAs on peripheral blood mononuclear cells of a subset of ALS patients and controls. Among the differently expressed circRNA by microarray analysis, we selected only the ones with a host gene that harbors the highest level of conservation and genetic constraints. This selection was based on the hypothesis that genes under selective pressure and genetic constraints could have a major role in determining a trait or disease. Then we performed a linear regression between ALS cases and controls using each circRNA as a predictor variable. With a False Discovery Rate (FDR) threshold of 0.1, only six circRNAs passed the filtering and only one of them remained statistically significant after Bonferroni correction: hsa_circ_0060762 and its host gene CSE1L. Finally, we observed a significant difference in expression levels between larger sets of patients and healthy controls for both hsa_circ_0060762 and CSE1L. CSE1L is a member of the importin ß family and mediates inhibition of TDP-43 aggregation; the central pathogenicity in ALS and hsa_circ_0060762 has binding sites for several miRNAs that have been already proposed as biomarkers for ALS. In addition, receiver operating characteristics curve analysis showed diagnostic potential for CSE1L and hsa_circ_0060762. Hsa_circ_0060762 and CSE1L thus represent novel potential peripheral blood biomarkers and therapeutic targets for ALS.

Inflammation and Oxidative Stress Gene Variability in Retinal Detachment Patients with and without Proliferative Vitreoretinopathy.

This study investigated the association between certain genetic variations and the risk of developing proliferative vitreoretinopathy (PVR) after surgery. The study was conducted on 192 patients with primary rhegmatogenous retinal detachment (RRD) who underwent 3-port pars plana vitrectomy (PPV). The distribution of single nucleotide polymorphisms (SNPs) located in genes involved in inflammation and oxidative stress associated with PVR pathways were analyzed among patients with and without postoperative PVR grade C1 or higher. A total of 7 defined SNPs of 5 genes were selected for genotyping: rs4880 (SOD2); rs1001179 (CAT); rs1050450 (GPX1); rs1143623, rs16944, rs1071676 (IL1B); rs2910164 (MIR146A) using competitive allele-specific polymerase chain reaction. The association of SNPs with PVR risk was evaluated using logistic regression. Furthermore, the possible association of SNPs with postoperative clinical parameters was evaluated using non-parametric tests. The difference between two genotype frequencies between patients with or without PVR grade C1 or higher was found to be statistically significant: SOD2 rs4880 and IL1B rs1071676. Carriers of at least one polymorphic IL1B rs1071676 GG allele appeared to have better postoperative best-corrected visual acuity only in patients without PVR (p = 0.070). Our study suggests that certain genetic variations may play a role in the development of PVR after surgery. These findings may have important implications for identifying patients at higher risk for PVR and developing new treatments.

Leber Hereditary Optic Neuropathy in a Family of Carriers of MT-ND5 m.13042G>T (A236S) Novel Variant.

BACKGROUND: A Slovenian three-generation family with 3 individuals with bilateral optic neuropathy and 2 unaffected relatives with a novel homoplasmic missense variant m.13042G > T (A236S) in the ND5 gene is described. A detailed phenotype at initial diagnosis and a follow-up of bilateral optic neuropathy progression is presented for 2 affected individuals. METHODS: A detailed phenotype analysis with clinical examination in the early and chronic phase with electrophysiology and OCT segmentation is presented. Genotype analysis with full mitochondrial genome sequencing was performed. RESULTS: Two affected male individuals (maternal cousins) had a profound visual loss at an early age (11 and 20 years) with no recovery. The maternal grandmother exhibited bilateral optic atrophy with a history of visual loss at the age 58 years. The visual loss of both affected male individuals was characterized by centrocecal scotoma, abnormal color vision, abnormal PERG N95, and VEP. Later with disease progression, retinal nerve fiber layer thinning was observed on OCT. We observed no other extraocular clinical features. Mitochondrial sequencing identified a homoplasmic novel variant m.13042G > T (A236S) in the MT-ND5 gene, belonging to a haplogroup K1a. CONCLUSIONS: Novel homoplasmic variant m.13042G > T (A236S) in the ND5 gene in our family was associated with Leber hereditary optic neuropathy-like phenotype. However, predicting the pathogenicity of a novel ultra-rare missense variant in the mitochondrial ND5 gene is challenging. Genetic counseling should consider genotypic and phenotypic heterogeneity, incomplete penetrance, haplogroup type, and tissue-specific thresholds.

The Potential Connection between Molecular Changes and Biomarkers Related to ALS and the Development and Regeneration of CNS.

Neurodegenerative diseases are one of the greatest medical burdens of the modern age, being mostly incurable and with limited prognostic and diagnostic tools. Amyotrophic lateral sclerosis (ALS) is a fatal, progressive neurodegenerative disease characterized by the loss of motoneurons, with a complex etiology, combining genetic, epigenetic, and environmental causes. The neuroprotective therapeutic approaches are very limited, while the diagnostics rely on clinical examination and the exclusion of other diseases. The recent advancement in the discovery of molecular pathways and gene mutations involved in ALS has deepened the understanding of the disease pathology and opened the possibility for new treatments and diagnostic procedures. Recently, 15 risk loci with distinct genetic architectures and neuron-specific biology were identified as linked to ALS through common and rare variant association analyses. Interestingly, the quantity of related proteins to these genes has been found to change during early postnatal development in mammalian spinal cord tissue (opossum Monodelphis domestica) at the particular time when neuroregeneration stops being possible. Here, we discuss the possibility that the ALS-related genes/proteins could be connected to neuroregeneration and development. Moreover, since the regulation of gene expression in developmental checkpoints is frequently regulated by non-coding RNAs, we propose that studying the changes in the composition and quantity of non-coding RNA molecules, both in ALS patients and in the developing central nervous (CNS) system of the opossum at the time when neuroregeneration ceases, could reveal potential biomarkers useful in ALS prognosis and diagnosis.

Genetic Variability of Inflammation and Oxidative Stress Genes Affects Onset, Progression of the Disease and Survival of Patients with Amyotrophic Lateral Sclerosis.

Inflammation and oxidative stress are recognized as important contributors to amyotrophic lateral sclerosis (ALS) disease pathogenesis. Our aim was to evaluate the impact of selected single-nucleotide polymorphisms in genes involved in inflammation and oxidative stress on ALS susceptibility and modification. One-hundred-and-eighty-five ALS patients and 324 healthy controls were genotyped for nine polymorphisms in seven antioxidant and inflammatory genes using competitive allele-specific PCR. Logistic regression; nonparametric tests and survival analysis were used in the statistical analysis. Investigated polymorphisms were not associated with ALS susceptibility. Carriers of at least one polymorphic SOD2 rs4880 T or IL1B rs1071676 C allele more often had bulbar ALS onset (p = 0.036 and p = 0.039; respectively). IL1B rs1071676 was also associated with a higher rate of disease progression (p = 0.015). After adjustment for clinical parameters; carriers of two polymorphic IL1B rs1071676 C alleles had shorter survival (HR = 5.02; 95% CI = 1.92-13.16; p = 0.001); while carriers of at least one polymorphic CAT rs1001179 T allele had longer survival (HR = 0.68; 95% CI = 0.47-0.99; p = 0.046). Our data suggest that common genetic variants in the antioxidant and inflammatory pathways may modify ALS disease. Such genetic information could support the identification of patients that may be responsive to the immune or antioxidant system-based therapies.

The Role of Vitamin A in Retinal Diseases.

Vitamin A is an essential fat-soluble vitamin that occurs in various chemical forms. It is essential for several physiological processes. Either hyper- or hypovitaminosis can be harmful. One of the most important vitamin A functions is its involvement in visual phototransduction, where it serves as the crucial part of photopigment, the first molecule in the process of transforming photons of light into electrical signals. In this process, large quantities of vitamin A in the form of 11-cis-retinal are being isomerized to all-trans-retinal and then quickly recycled back to 11-cis-retinal. Complex machinery of transporters and enzymes is involved in this process (i.e., the visual cycle). Any fault in the machinery may not only reduce the efficiency of visual detection but also cause the accumulation of toxic chemicals in the retina. This review provides a comprehensive overview of diseases that are directly or indirectly connected with vitamin A pathways in the retina. It includes the pathophysiological background and clinical presentation of each disease and summarizes the already existing therapeutic and prospective interventions.

Resolving the dark matter of ABCA4 for 1054 Stargardt disease probands through integrated genomics and transcriptomics.

PURPOSE: Missing heritability in human diseases represents a major challenge, and this is particularly true for ABCA4-associated Stargardt disease (STGD1). We aimed to elucidate the genomic and transcriptomic variation in 1054 unsolved STGD and STGD-like probands. METHODS: Sequencing of the complete 128-kb ABCA4 gene was performed using single-molecule molecular inversion probes (smMIPs), based on a semiautomated and cost-effective method. Structural variants (SVs) were identified using relative read coverage analyses and putative splice defects were studied using in vitro assays. RESULTS: In 448 biallelic probands 14 known and 13 novel deep-intronic variants were found, resulting in pseudoexon (PE) insertions or exon elongations in 105 alleles. Intriguingly, intron 13 variants c.1938-621G>A and c.1938-514G>A resulted in dual PE insertions consisting of the same upstream, but different downstream PEs. The intron 44 variant c.6148-84A>T resulted in two PE insertions and flanking exon deletions. Eleven distinct large deletions were found, two of which contained small inverted segments. Uniparental isodisomy of chromosome 1 was identified in one proband. CONCLUSION: Deep sequencing of ABCA4 and midigene-based splice assays allowed the identification of SVs and causal deep-intronic variants in 25% of biallelic STGD1 cases, which represents a model study that can be applied to other inherited diseases.

Circulating RNAs as Potential Biomarkers in Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a complex multi-system neurodegenerative disorder with currently limited diagnostic and no therapeutic options. Despite the intense efforts no clinically applicable biomarkers for ALS are yet established. Most current research is thus focused, in particular, in identifying potential non-invasive circulating biomarkers for more rapid and accurate diagnosis and monitoring of the disease. In this review, we have focused on messenger RNA (mRNA), non-coding RNAs (lncRNAs), micro RNAs (miRNAs) and circular RNA (circRNAs) as potential biomarkers for ALS in peripheral blood serum, plasma and cells. The most promising miRNAs include miR-206, miR-133b, miR-27a, mi-338-3p, miR-183, miR-451, let-7 and miR-125b. To test clinical potential of this miRNA panel, a useful approach may be to perform such analysis on larger multi-center scale using similar experimental design. However, other types of RNAs (lncRNAs, circRNAs and mRNAs) that, together with miRNAs, represent RNA networks, have not been yet extensively studied in blood samples of patients with ALS. Additional research has to be done in order to find robust circulating biomarkers and therapeutic targets that will distinguish key RNA interactions in specific ALS-types to facilitate diagnosis, predict progression and design therapy.

SINGLE NUCLEOTIDE POLYMORPHISMS IN RETINAL DETACHMENT PATIENTS WITH AND WITHOUT PROLIFERATIVE VITREORETINOPATHY.

PURPOSE: To investigate differences in genotype distributions of single nucleotide polymorphisms within genes, encoding inflammatory mediators, among patients with rhegmatogenous retinal detachment (RRD) and patients with proliferative vitreoretinopathy (PVR). METHODS: A genetic association study was performed on 191 Slovenian patients, divided into 2 groups: 113 RRD patients with PVR and 78 RRD patients without PVR. Genotype distributions were investigated within the following 13 single nucleotide polymorphisms: rs3760396 (CCL2), rs9990554 (FGF2), rs17561 (IL1A), rs2069763 (IL2), rs1800795 (IL6), rs1800871 (IL10), rs3008 (JAK3), rs2229094 (LTA), rs1042522 (TP53), rs7656613 (PDGFRA), rs7226855 (SMAD7), rs1800471 (TGFB1), and rs1800629 (TNF). RESULTS: Differences in genotype distributions between patients with RRD with or without PVR were detected in rs1800795 (IL6) (P = 0.04), rs1800871 (in the vicinity of the IL10) (P = 0.034), and rs1800471 (TGFB1) (P = 0.032). After adjustment none of the 13 analyzed single nucleotide polymorphisms showed statistically significant associations in single nucleotide polymorphism genotype distributions between patients with RRD with and without PVR. CONCLUSION: Further research is needed, particularly expanded multicentric population-based studies, to clarify the issue of genetic contribution to PVR from different genetic, clinical, and population-based aspects.

Clinical and Haplotypic Variability of Slovenian USH2A Patients Homozygous for the c. 11864G>A Nonsense Mutation.

PURPOSE: to determine a detailed clinical and haplotypic variability of the Slovenian USH2A patients with homozygous c.11864G>A (p.Trp3955Ter) nonsense mutation and to develop sensitive, accurate and rapid screening test. METHODS: Ten unrelated homozygous patients with detailed ophthalmological exam were included in our study. The High-Resolution Melting (HRM) method was developed for fast and reliable detection of the c.11864G>A mutation. RESULTS: The c.11864G>A mutation represents the vast majority of pathogenic alleles in Slovenian USH2A-Usher syndrome population (84%). The median age of onset of nyctalopia was 16 years and all patients younger than 40 years had hyperautofluorescent rings on fundus autofluorescence imaging. The Kaplan Meier survival analysis showed a decline of central vision after the age of 40, with 50% patients reaching visual acuity (VA) ≤ 0.05 at the average age of 66 years visual field diameter less than 20° at the average age of 59 years. There was a relatively large phenotypic variability in the retinal and audiological phenotype. Analysis of the p.Trp3955Ter-homozygous patients revealed four different haplotypes, with the frequency of the most common haplotype ~65%. Disease severity did not correlate with the haplotype. CONCLUSIONS: According to the natural history of homozygous p.Trp3955Ter patients any therapy aimed to slow disease progression in these patients would be best started before the age of 40. Phenotypic variability suggests the presence of cis and/or trans factors outside the USH2A gene that are able to affect disease severity. High frequency of p.Trp3955Ter mutation in Slovenian USH2A gene pool appears to be initiated from different unrelated founders because of migrations from neighboring populations. The mutation on haplotype 2 seems to be the major founder allele.

Double Hyperautofluorescent Rings in Patients with USH2A-Retinopathy.

USH2A mutation is the most common cause of retinitis pigmentosa, with or without hearing impairment. Patients most commonly exhibit hyperautofluorescent ring on fundus autofluorescence imaging (FAF) and rod-cone dystrophy on electrophysiology. A detailed study of three USH2A patients with a rare pattern of double hyperautofluorescent rings was performed. Twenty-four patients with typical single hyperautofluorescent rings were used for comparison of the ages of onset, visual fields, optical coherence tomography, electrophysiology, and audiograms. Double rings delineated the area of pericentral retinal degeneration in all cases. Two patients exhibited rod-cone dystrophy, whereas the third had a cone-rod dystrophy type of dysfunction on electrophysiology. There was minimal progression on follow-up in all three. Patients with double rings had significantly better visual acuity, cone function, and auditory performance than the single ring group. Double rings were associated with combinations of null and missense mutations, none of the latter found in the single ring patients. According to these findings, the double hyperautofluorescent rings indicate a mild subtype of USH2A disease, characterized by pericentral retinal degeneration, mild to moderate hearing loss, and either a rod-cone or cone-rod pattern on electrophysiology, the latter expanding the known clinical spectrum of USH2A-retinopathy.

Circular RNAs as Potential Blood Biomarkers in Amyotrophic Lateral Sclerosis.

Circular RNAs (circRNAs) are emerging as a novel, yet powerful player in many human diseases. They are involved in several cellular processes and are becoming a noteworthy type of biomarkers. Among other functions, circRNAs can serve as RNA sponges or as scaffolds for RNA-binding proteins. Here, we investigated a microarray expression profile of circRNAs in leukocyte samples from ALS patients and age- and sex-matched healthy controls to identify differentially expressed circRNAs. We selected 10 of them for a qPCR validation of expression on a larger set of samples, identification of their associations with clinical parameters, and evaluation of their diagnostic potential. In total, expression of 7/10 circRNAs was significant in a larger cohort of ALS patients, compared with age- and sex-matched healthy controls. Three of them (hsa_circ_0023919, hsa_circ_0063411, and hsa_circ_0088036) showed the same regulation as in microarray results. These three circRNAs also had AUC > 0.95, and sensitivity and specificity for the optimal threshold point > 90%, showing their potential for using them as diagnostic biomarkers.

Association between histopathological changes and expression of selected microRNAs in skin of adult patients with IgA vasculitis.

AIMS: IgA vasculitis (IgAV) is a common small-vessel systemic vasculitisthat is histologically characterised by granulocyte infiltration and IgA deposition in vessel walls. Information on microRNA (miRNA) involvement inIgAVis limited. The aim of this study was to analyse the association between histopathological changes and expression profiles of 14 miRNAs in the affected skin of 70 adult patients with IgAV. METHODS AND RESULTS: miRNA expression analysis was performed by quantitative real-time polymerase chain reaction and evaluation of histopathological changes by light and immunofluorescence microscopy on formalin-fixed paraffin-embedded skin excision samples. In IgAV-affected skin, granulocyte infiltration was significantly associated with vessel fibrinoid necrosis. Of the analysed miRNAs, four showed two-fold increased expression (let-7d, let-7f, miR-21-5p, and miR-203-3p), five showed five-fold increased expression (let-7b, miR-17-5p, miR-155-5p, miR-423-5p, and miR-451a), and threeshowed 15-fold increased expression (let-7a, miR-21-3p, miR-223-3p), as compared with controls (all P < 0.001). miR-146a-5p and miR-148b-3p showed three-fold decreased expression (P = 0.981 and P < 0.001). The expression of miR-223-3p also showed a significant positive association with granulocyte infiltration and fibrinoid necrosis. CONCLUSIONS: Altered miRNA expression, especially of miRNA-223-3p, may be associated with the skin inflammatory state in IgAV. The majority of aberrantly expressed miRNAs in IgAV-affected skin are known to influence the nuclear factor-κB signalling pathway, which is crucial for activation of key proinflammatory genes, including those encoding tumour necrosis factor-α, interleukin (IL)-6, and IL-8. Furthermore, miR-146a-5p and miR-148b-3p, which are negative regulators of inflammatory gene expression, showed decreased expression and could contribute to the exaggerated inflammation. Further investigation of miRNA expression in the affected tissues could improve our knowledge of IgAV pathogenesis, and possibly help to identify novel biomarkers in body fluids.

Genes CEP55, FOXD3, FOXF2, GNAO1, GRIA4, and KCNA5 as potential diagnostic biomarkers in colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) is one of the leading causes of death by cancer worldwide and in need of novel potential diagnostic biomarkers for early discovery. METHODS: We conducted a two-step study. We first employed bioinformatics on data from The Cancer Genome Atlas to obtain potential biomarkers and then experimentally validated some of them on our clinical samples. Our aim was to find a methylation alteration common to all clusters, with the potential of becoming a diagnostic biomarker in CRC. RESULTS: Unsupervised clustering of methylation data resulted in four clusters, none of which had a known common genetic or epigenetic event, such as mutations or methylation. The intersect among clusters and regulatory regions resulted in 590 aberrantly methylated probes, belonging to 198 differentially expressed genes. After performing pathway and functional analysis on differentially expressed genes, we selected six genes: CEP55, FOXD3, FOXF2, GNAO1, GRIA4 and KCNA5, for further experimental validation on our own clinical samples. In silico analysis demonstrated that CEP55 was hypomethylated in 98.7% and up-regulated in 95.0% of samples. Genes FOXD3, FOXF2, GNAO1, GRIA4 and KCNA5 were hypermethylated in 97.9, 81.1, 80.3, 98.4 and 94.0%, and down-regulated in 98.3, 98.9, 98.1, 98.1 and 98.6% of samples, respectively. Our experimental data show CEP55 was hypomethylated in 97.3% of samples and down-regulated in all samples, while FOXD3, FOXF2, GNAO1, GRIA4 and KCNA5 were hypermethylated in 100.0, 90.2, 100.0, 99.1 and 100.0%, and down-regulated in 68.0, 76.0, 96.0, 95.2 and 84.0% of samples, respectively. Results of in silico and our experimental analyses showed that more than 97% of samples had at least four methylation markers altered. CONCLUSIONS: Using bioinformatics followed by experimental validation, we identified a set of six genes that were differentially expressed in CRC compared to normal mucosa and whose expression seems to be methylation dependent. Moreover, all of these six genes were common in all methylation clusters and mutation statuses of CRC and as such are believed to be an early event in human CRC carcinogenesis and to represent potential CRC biomarkers.

MicroRNA expression in the affected skin of adult patients with IgA vasculitis.

IgA vasculitis (IgAV) represents a common systemic vasculitis in pediatric and adult population. Our current knowledge of disease pathogenesis is still very limited, without information on miRNAs in IgAV. The aim of our study was to determine the expression of five pre-selected miRNAs (miRNA-146a-5p, miRNA-148-3p, miRNA-155-5p, miRNA-223-3p, and let-7b) in the affected skin of adult IgAV patients. The study included 65 skin samples from consecutive, untreated IgAV patients (61.5% male, median age 67.6 years, range 29-91), diagnosed between October 2014 and September 2016, and 20 samples of normal skin from healthy volunteers. Total RNA was isolated from tissue sections of formalin-fixed, paraffin-embedded samples. Expression of miRNAs was measured using qRT-PCR. To present relative miRNA expression, the ΔΔCT method was used. Skin miRNA expression was correlated to clinical characteristics of adult IgAV patients. We found significantly higher levels of miRNA-155-5p, miRNA-223-3p, and let-7b in the affected skin compared to controls (18.6-fold, 6.4-fold, and 7.9-fold higher respectively). Contrary, the miRNA 148-3p expression was significantly lower (2.2-fold). The expression of the miRNA-146-5p showed near normal levels. Patients with necrotic skin lesions had significantly higher miRNA-223 tissue expression than those with non-necrotic purpura (p = 0.029). Gastrointestinal tract involvement inversely correlated with the expression of miRNA-155-5p and/or miRNA-146a-5p in affected skin. Altered expression of miRNA-148b-3p, miRNA-155-5p, miRNA-223-3p, and let-7b was found in vasculitic skin lesions in IgAV. Additionally, we found a positive association between the severity of purpura and skin miRNA-223-3p expression. Aberrantly expressed miRNAs could represent a biomarker in adult IgAV.

Genetic Landscape of Slovenians: Past Admixture and Natural Selection Pattern.

The Slovenian territory played a crucial role in the past serving as gateway for several human migrations. Previous studies used Slovenians as a source population to interpret different demographic events happened in Europe but not much is known about the genetic background and the demographic history of this population. Here, we analyzed genome-wide data from 96 individuals to shed light on the genetic role and history of the Slovenian population. Y chromosome diversity splits into two major haplogroups R1b and R1a with the latter suggesting a genetic contribution from the steppe. Slovenian individuals are more closely related to Northern and Eastern European populations than Southern European populations even though they are geographically closer. This pattern is confirmed by an admixture and clustering analysis. We also identified a single stream of admixture events between the Slovenians with Sardinians and Russians around ∼2630 BCE (2149-3112). Using ancient samples, we found a significant admixture in Slovenians using Yamnaya and the early Neolithic Hungarians as sources, dated around ∼1762 BCE (1099-2426) suggesting a strong contribution from the steppe to the foundation of the observed modern genetic diversity. Finally, we looked for signals of selection in candidate variants and we found significant hits in HERC2 and FADS responsible for blue eye color and synthesis of long-chain unsaturated fatty acids, respectively, when Slovenians were compared to Southern Europeans. While the comparison was done with Eastern Europeans, we identified significant signals in PKD2L1 and IL6R which are genes associated with taste and coronary artery disease, respectively.

Dynamic expression of 11 miRNAs in 83 consecutive primary and corresponding recurrent glioblastoma: correlation to treatment, time to recurrence, overall survival and MGMT methylation status.

Background Glioblastoma (GBM) is the most common and the most malignant glioma subtype. Among numerous genetic alterations, miRNAs contribute to pathogenesis of GBM and it is suggested that also to GBM recurrence and resistance to therapy. Based on publications, we have selected 11 miRNAs and analyzed their expression in GBM. We hypothesized that selected miRNAs are differentially expressed and involved in primary as well as in recurrent GBM, that show significant expressional differences when different treatment options are in question, and that are related to certain patients and tumor characteristics. Patients and methods Paraffin embedded tissues, obtained from primary and corresponding recurrent tumor from 83 patients with primary GBM were used. Eleven miRNAs (miR-7, miR-9, miR-15b, miR-21, miR-26b, miR-124a, miR-199a, let-7a, let-7b, let-7d, and let-7f) were selected for qPCR expression analysis. For patients who received temozolamide (TMZ) as chemotherapeutic drug, O6-methylguanine-DNA methyltransferase (MGMT) methylation status was defined using the methyl-specific PCR. Results There was a significant change in expression of miR-7, miR-9, miR-21, miR-26b, mirR-124a, miR-199a and let-7f in recurrent tumor compared to the primary. In recurrent tumor, miR-15b, let-7d and let-7f significantly changed comparing both treatment options. We also observed difference in progression free survival between patients that received radiotherapy and patients that received radiotherapy and chemotherapy, and longer survival for patients who received chemotherapy after second surgery compared to not treated patients. miR-26b showed correlation to progression free survival and let-7f to overall survival. We did not find any expression difference between the tumors with and without methylated MGMT. Conclusions Our data suggest that analyzed miRNAs may not only contribute to pathogenesis of primary GBM, but also to tumor progression and its recurrence. Moreover, expression of certain miRNAs appears to be therapy-dependent and as such they might serve as additional biomarker for recurrence prediction and potentially predict a therapy-resistance.

Comparison of SNP Genotypes Related to Proliferative Vitreoretinopathy (PVR) across Slovenian and European Subpopulations.

The present study investigated the distribution of genotypes within single nucleotide polymorphisms (SNPs) in genes, related to PVR pathogenesis across European subpopulations. Genotype distributions of 42 SNPs among 96 Slovenian healthy controls were investigated and compared to genotype frequencies in 503 European individuals (Ensembl database) and their subpopulations. Furthermore, a case-control status was simulated to evaluate effects of allele frequency changes on statistically significant results in gene-association studies investigating functional polymorphisms. In addition, 96 healthy controls were investigated within 4 SNPs: rs17561 (IL1A), rs2069763 (IL2), rs2229094 (LTA), and rs1800629 (TNF) in comparison to PVR patients. Significant differences (P < 0.05) in distribution of genotypes among 96 Slovenian participants and a European population were found in 10 SNPs: rs3024498 (IL10), rs315952 (IL1RN), rs2256965 (LST1), rs2256974 (LST1), rs909253 (LTA), rs2857602 (LTA), rs3138045 (NFKB1A), rs3138056 (NFKB1A), rs7656613 (PDGFRA), and rs1891467 (TGFB2), which additionally showed significant differences in genotype distribution among European subpopulations. This analysis also showed statistically significant differences in genotype distributions between healthy controls and PVR patients in rs17561 of the IL1A gene (OR, 3.00; 95% CI, 0.77-11.75; P = 0.036) and in rs1800629 of the TNF gene (OR, 0.48; 95% CI, 0.27-0.87; P = 0.014). Furthermore, we have shown that a small change (0.02) in minor allele frequency (MAF) significantly affects the statistical p value in case-control studies. In conclusion, the study showed differences in genotype distributions in healthy populations across different European countries. Differences in distribution of genotypes may have had influenced failed replication results in previous PVR-related SNP-association studies.