Mitochondrial DNA variability and Covid-19 in the Slovak population.

Recent studies have shown that mitochondria are involved in the pathogenesis of Covid-19. Mitochondria play a role in production of reactive oxygen species and induction of an innate immune response, both important during infections. Common variability of mitochondrial DNA (mtDNA) can affect oxidative phosphorylation and the risk or lethality of cardiovascular, neurodegenerative diseases and sepsis. However, it is unclear whether susceptibility of severe Covid-19 might be affected by mtDNA variation. Thus, we have analyzed mtDNA in a sample of 446 Slovak patients hospitalized due to Covid-19 and a control population group consisting of 1874 individuals. MtDNA variants in the HVRI region have been analyzed and classified into haplogroups at various phylogenetic levels. Binary logistic regression was used to assess the risk of Covid-19. Haplogroups T1, H11, K and variants 16256C > T, 16265A > C, 16293A > G, 16311 T > C and 16399A > G were associated with an increased Covid-19 risk. On contrary, Haplogroup J1, haplogroup clusters H + U5b and T2b + U5b, and the mtDNA variant 16189 T > C were associated with decreased risk of Covid-19. Following the application of the Bonferroni correction, statistical significance was observed exclusively for the cluster of haplogroups H + U5b. Unsurprisingly, the most significant factor contributing to the mortality of patients with Covid-19 is the age of patients. Our findings suggest that mtDNA haplogroups can play a role in Covid-19 pathogenesis, thus potentially useful in identifying susceptibility to its severe form. To confirm these associations, further studies taking into account the nuclear genome or other non-biological influences are needed.

Population genetic analysis of 12 X-STR markers in Slovakia.

Background: During the last 20 years, X-chromosomal STR markers have become widely used in forensic genetics and paternity testing. Nevertheless, to exploit their full potential in any given population, a reliable reference dataset needs to be established. Since no relevant studies concerning these markers have been performed on the Slovak population so far, we decided to analyse several commonly used markers in this population.Aim: To create an informative set of Slovak population data concerning X-STR markers.Subjects and methods: We genotyped 378 individuals and analysed 12 loci (DXS10148, DX10135, DXS8378, DXS7132, DXS10079, DXS10074, DXS10103, HPRTB, DXS10101, DXS10146, DXS10134 and DXS742) localised in four distinct linkage groups.Results: Our analysis showed that the most informative marker is DXS10135 (PIC = 0,927) and the most informative linkage group (LG) is LG1 with 149 different haplotypes. This analysis also confirmed linkage disequilibrium for two pairs of markers (DX10101-DX10103 and DX10101-HPRTB) within LG3 in female samples. No statistically significant departure from HWE was observed for any locus. Moreover, the interpopulation comparison of 8 European populations based on haplotype frequencies showed no statistically significant FST values in any LG, except for LG2 in comparison with the German population.Conclusion: We created a haplotype database for forensic analyses and kinship testing in Slovakia, as well as the CE dataset which can be used to further increase the decision power in similar analyses in the future.

Pilot study of correlation of selected genetic factors with cribra orbitalia in individuals from a medieval population from Slovakia.

OBJECTIVE: The aim of this study is to investigate the potential genetic etiology of cribra orbitalia noted on human skeletal remains. MATERIALS: We obtained and analyzed ancient DNA of 43 individuals with cribra orbitalia. The analyzed set represented medieval individuals from two cemeteries in western Slovakia, Castle Devín (11th-12th century AD) and Cífer-Pác (8th-9th century AD). METHODS: We performed a sequence analysis of 5 variants in 3 genes associated with anemia (HBB, G6PD, PKLR), which are the most common pathogenic variants in present day of European populations, and one variant MCM6:c.1917 + 326 C>T (rs4988235) associated with lactose intolerance. RESULTS: DNA variants associated with anemia were not found in the samples. The allele frequency of MCM6:c.1917 + 326 C was 0.875. This frequency is higher but not statistically significant in individuals displaying cribra orbitalia compared to individuals without the lesion. SIGNIFICANCE: This study seeks to expand our knowledge of the etiology of cribra orbitalia by exploring the potential association between the lesion and the presence of alleles linked to hereditary anemias and lactose intolerance. LIMITATIONS: A relatively small set of individuals were analyzed, so an unequivocal conclusion cannot be drawn. Hence, although it is unlikely, a genetic form of anemia caused by rare variants cannot be ruled out. SUGGESTIONS FOR FURTHER RESEARCH: Genetic research based on larger sample sizes and in more diverse geographical regions.

Mutational analysis of 16 STR markers in the Slovak population.

BACKGROUND: Short tandem repeats (STRs) are genetic markers frequently used for human identification and paternity testing. They are highly mutable, which may occasionally lead to inconsistencies between the genotypes of parents and their children. As the mutation rates of individual STR markers can vary among populations, population-specific data are of high importance. AIM: To investigate the mutation rates of 16 STR markers in the Slovak population. SUBJECTS AND METHODS: In this study, we analysed the germline mutation rates of 16 STR markers (TH01, D3S1358, vWA, D21S11, D16S539, D1S1656, D19S433, SE33, D10S1248, D22S1045, D12S391, D8S1179, D2S1338, D2S441, D18S51 and FGA) in the Slovak population. At these loci, we analysed 42 096 allelic transfers and identified 61 mutation events. RESULTS: The loci with the highest overall mutation rates were SE33 and FGA, while no mutations were identified in TH01, D19S433 and D22S1045. The average paternal mutation rate was higher than the maternal mutation rate. All but one mutation consisted of gains or losses of a single repeat unit and the overall mutation rate was estimated to be 1.45 x10-3 per meiosis. CONCLUSION: This study provides data which can be used to further strengthen the correct paternity index calculations and reliability of paternity testing in Slovakia.

The results of multigene panel sequencing in Slovak HBOC families.

Hereditary breast and ovarian cancer (HBOC) is primarily associated with mutations in the BRCA1/2 genes. However, causal variants in other high, moderate, and low penetrance genes proportionally increase the risk of breast/ovarian cancer. This study aims to provide data about the mutation spectrum of HBOC-associated genes in Slovak HBOC families and estimate the ratio of BRCA versus non-BRCA causal variants. We used panel sequencing containing 22 high/moderate-risk susceptibility genes and parallel MLPA analysis of BRCA1/2, CHEK2 genes, to analyze 94 individuals with a strong family/personal history of breast and/or ovarian cancer. The analyzed group consisted of 80 patients diagnosed with cancer (85.1%) and 14 healthy individuals (14.9%) with a positive family history of HBOC syndrome. In total, we have identified 22 causal DNA variants (23.4%) showing 15 primary findings in BRCA1/2 genes (68.2%) and 7 positive secondary findings in CHEK2, PALB2, CDH1, and MUTYH genes (31.8%). The most frequent pathogenic alterations were BRCA1 mutations c.181T>G and CNV variant (c.5573-?_c.5701+?)del, known as deletion of exons 21-22. Besides known mutations, the BRCA1 variant c.2794del (p.Val932Leufs*68) and variant c.2480dup (p.Tyr827*) in the CDH1 gene represent the novel, previously unpublished variants that might be population-specific. In conclusion, we provide the first report of multigene panel testing in Slovak HBOC families demonstrating that almost one-third of pathogenic mutations are situated in susceptibility genes other than BRCA1/2. Although multigene panel testing requires precise data filtration and interpretation, it might bring the relevant data for clinical management of the patients.

Serious chronic disease of the cervical spine and trauma in a young female from the middle ages (Czech Republic).

The skeletal remains of the young female (20-24 years) from Grave JP/106, discovered in the Southern Suburb of the Breclav - Pohansko Stronghold (Early Middle Ages, 9th century-beginning of the 10th century, present day Czech Republic) display several noteworthy pathologies. The first is deformation of the mandible, which was most probably caused by a fracture of the ramus in combination with a subcondylar fracture. The spine of this young woman also exhibits a probable traumatic injury of the cervical spine in combination with a slowly growing structure situated inside the spinal canal, which caused deformation centered upon C7. The cervical and thoracic spine together with internal surfaces of several ribs exhibit infectious changes of advanced stage, in all likelihood of tuberculous origin, but osteomyelitis cannot be excluded. Histological analysis of the new bone formation in the ribs confirmed infectious origin, as does Micro CT of C5 and C6. Analyses conducted by two different departments with different methods (PCR amplification of 123 bp long section from IS6110 and Next Generation shotgun sequencing) failed to identify DNA of Mycobacterium tuberculosis from the first rib.

Detection of mitochondrial haplogroups in a small avar-slavic population from the eigth-ninth century AD.

OBJECTIVES: In the sixth century AD, Avars came to Central Europe from middle Eurasian steppes and founded a strong Empire called the Avar Khagante (568-799/803 AD) in the Pannonian basin. During the existence of this empire, they undertook many military and pugnacious campaigns. In the seventh century, they conquered the northern territory inhabited by Slavs, who were further recruited in Avar military and were commissioned with obtaining food supplies. During almost 200 years of Avar domination, a significant influence by the Avar culture (especially on the burial rite) and assimilation with indigenous population (occurrence of "East Asian"cranial features) could be noticed in this mixed area, which is supported by achaeological and anthropologcal research. Therefore we expected higher incidence of east Eurasian haplogroups (introduced by Avars) than the frequencies detected in present-day central European populations. MATERIALS AND METHODS: Mitochondrial DNA from 62 human skeletal remains excavated from the Avar-Slavic burial site Cífer-Pác (Slovakia) dated to the eighth and ninth century was analyzed by the sequencing of hypervariable region I and selected parts of coding region. Obtained haplotypes were compared with other present-day and historical populations and genetic distances were calculated using standard statistical method. RESULTS AND DISCUSSION: In total, the detection of mitochondrial haplogroups was possible in 46 individuals. Our results prooved a higher frequency of east Eurasian haplogroups in our analyzed population (6.52%) than in present-day central European populations. However, it is almost three times lower than the frequency of east Eurasian haplogroups detected in other medieval Avar populations. The statistical analysis showed a greater similarity and the lowest genetic distances between the Avar-Slavic burial site Cifer-Pac and medieval European populations than the South Siberian, East and Central Asian populations. CONCLUSION: Our results indicate that the transfer of Avar genetic variation through their mtDNA was rather weak in the analyzed mixed population.

Comprehensive genetic study of cystic fibrosis in Slovak patients in 25 years of genetic diagnostics.

INTRODUCTION: Cystic fibrosis (CF) has one of the longest histories in hereditary disease molecular diagnostics. However, identification of causative mutations in the CFTR gene is complicated by over 2000 currently identified mutations; with more still being discovered. Knowledge of mutation spectrum may improve effective routine diagnostics and is obligatory in mutation-specific treatment. OBJECTIVES: This study presents comprehensive mutation screening of the CFTR gene; with 275 unrelated, clinically confirmed and treated cystic fibrosis (CF) patients diagnosed in 25 years genetic testing in Slovakia. METHODS: Detection of the most common CFTR mutations was performed by ELUCIGENE 29 and ELUCIGENE CF EU2 kits. HRM and dHPLC mutation screening methods with subsequent Sanger sequencing were applied for minor mutation screening, and MLPA analysis for deletion/duplication detection. RESULTS: A total of 70 different mutations were identified, from which the most common mutation F508del accounted for 60.36% of all disease alleles and 8 mutations have currently been observed only in Slovak patients. Two large deletions identified on chromosomes 2 and 22 were further characterized to identify breakpoints. Based on mutation screening results and neonatal screening we estimated incidence in Slovakian newborns at approximately 1:6000-7000. CONCLUSION: In our study, we identified mutations in 98.54% of all disease chromosomes, while 86.54% were identified using ELUCIGENE kits, 0.54% by MLPA analysis and 11.46% by sequencing analysis. Knowledge of the mutation spectrum in genetically diagnosed patients improves possibilities of genetic counseling and cascade screening in the affected families and Slovak population.

Genetic and Environmental Biomarkers Associated with Triglyceride Levels in Two Groups of Slovak Women.

OBJECTIVE: This study analyzed the association between the MLXIPL gene polymorphism (rs3812316) and triglyceride (TG) levels and selected environmental biomarkers in Slovak women at risk for cardiovascular disease compared to a reference sample. MATERIALS AND METHODS: The studied sample consisted of 200 women at cardiovascular risk (mean age 52.96 ± 6.01 years) and 244 healthy women (mean age 47.52 ± 5.34 years). Participants gave details of their health and lifestyle during their medical examination, and peripheral blood samples were used for biochemical analyses and DNA genotyping. A nested polymerase chain reaction-restriction fragment length polymorphism assay was used to detect the rs 3812316 SNP. RESULTS: We determined that there were significantly different genotype distributions in two TG categories: (1) subjects with normal TG values had a significantly higher G allele frequency than those with elevated TG levels (χ2 = 6.1556, df = 2, p = 0.046); and (2) the rare G allele frequency was 0.11 in the cardiovascular risk group and 0.15 in the reference group. Binary regression analysis showed that women with at least one G allele had a significantly lower relative risk of hypertriglyceridemia than women with the CC genotype (OR = 0.399, p = 0.022, 95% CI = 0.182-0.876). CONCLUSION: This cross-sectional study suggests that MLXIPL rs3812316 genotypes may be associated with TG levels. However, further analysis is advisable because of study limitations.

Analysis of Leucine-rich repeat kinase 2 (LRRK2) and Parkinson protein 2 (parkin, PARK2) genes mutations in Slovak Parkinson disease patients.

Parkinson disease (PD) is a chronic neurodegenerative movement disorder characterized by selective loss of nigrostriatal dopaminergic neurons and formation of Lewy bodies. Clinical manifestations include motor impairments involving tremor, bradykinesia, postural instability and rigidity. Using dHPLC method we screened exons 31, 35, 41, 48 of the Leucine-rich repeat kinase 2 (LRRK2) gene and exons 2, 6 and 7 of Parkinson protein 2 (parkin, PARK2) genes in a cohort of 216 consecutive, unrelated Slovak patients with familial or sporadic PD, including early and late onset. By this means we aimed to detect the most common pathogenic mutations within LRRK2 (Arg1441Cys, Arg1441Gly, Arg1628Pro, Tyr1699Cys, Gly2019Ser, Ile2020Thr, Gly2385Arg) and parkin genes responsible for late and early onset forms of disease, respectively. However, none of these mutations was identified in our cohort. Heterozygous point mutation p.Arg275Trp in exon 7 of parkin gene was identified in one patient with age at onset 61 years. Furthermore, we observed the presence of one exonic (LRRK2 ex 48: 7155A>G) and eight intronic polymorphisms (in LRRK2: IVS35+23T>A, IVS47-91insGCCAT, IVS47-91insGCAT, IVS47-41A>G, IVS47-9delT, IVS47-20C>T, IVS47-90A>G, in parkin: IVS2+25T>C), three of which were novel.

Mutation analysis of PMP22 in Slovak patients with Charcot-Marie-Tooth disease and hereditary neuropathy with liability to pressure palsies.

Charcot-Marie-Tooth disease (CMT) and related peripheral neuropathies are the most commonly inherited neurological disorders in humans, characterized by clinical and genetic heterogeneity. The most prevalent clinical entities belonging to this group of disorders are CMT type 1A (CMT1A) and hereditary neuropathy with liability to pressure palsies (HNPP). CMT1A and HNPP are predominantly caused by a 1.5 Mb duplication and deletion in the chromosomal region 17p11.2, respectively, and less frequently by other mutations in the peripheral myelin protein 22 (PMP22) gene. Despite being relatively common diseases, they haven't been previously studied in the Slovak population. Therefore, the aim of this study was to identify the spectrum and frequency of PMP22 mutations in the Slovak population by screening 119 families with CMT and 2 families with HNPP for causative mutations in this gene. The copy number determination of PMP22 resulted in the detection of CMT1A duplication in 40 families and the detection of HNPP deletion in 7 families, 6 of which were originally diagnosed as CMT. Consequent mutation screening of families without duplication or deletion using dHPLC and sequencing identified 6 single base changes (3 unpublished to date), from which only c.327C>A (Cys109X) present in one family was provably causative. These results confirm the leading role of PMP22 mutation analysis in the differential diagnosis of CMT and show that the spectrum and frequency of PMP22 mutations in the Slovak population is comparable to that seen in the global population.

A major Y-chromosome haplogroup R1b Holocene era founder effect in Central and Western Europe.

The phylogenetic relationships of numerous branches within the core Y-chromosome haplogroup R-M207 support a West Asian origin of haplogroup R1b, its initial differentiation there followed by a rapid spread of one of its sub-clades carrying the M269 mutation to Europe. Here, we present phylogeographically resolved data for 2043 M269-derived Y-chromosomes from 118 West Asian and European populations assessed for the M412 SNP that largely separates the majority of Central and West European R1b lineages from those observed in Eastern Europe, the Circum-Uralic region, the Near East, the Caucasus and Pakistan. Within the M412 dichotomy, the major S116 sub-clade shows a frequency peak in the upper Danube basin and Paris area with declining frequency toward Italy, Iberia, Southern France and British Isles. Although this frequency pattern closely approximates the spread of the Linearbandkeramik (LBK), Neolithic culture, an advent leading to a number of pre-historic cultural developments during the past ≤10 thousand years, more complex pre-Neolithic scenarios remain possible for the L23(xM412) components in Southeast Europe and elsewhere.

Elevated immunoglobulin D levels in children with PFAPA syndrome.

BACKGROUND: The periodic fever, aphthous stomatitis, pharyngitis and cervical adenitis (PFAPA) syndrome appears to be more common than generally appreciated and should be differentiated from hereditary periodic fever syndromes, particularly from mevalonate kinase deficiency (MKD). PATIENTS AND METHODS: 14 unrelated patients (7 males, 7 females) met clinical criteria for both the PFAPA syndrome and MKD. Immunoglobulin D (IgD) levels, mevalonic aciduria and mevalonate kinase (MVK) genotype was determined in all patients. RESULTS: Children experienced their first febrile episode at the age of 24.5±5.9 months (mean±SD), the clinical diagnosis of PFAPA syndrome was established with delay at 42.7±11.7 months. The duration of febrile episodes was 3.4±0.2 days, the asymptomatic interval between them lasted 5.4±0.9 weeks. Accompanying symptoms included pharyngitis (92.8%), cervical lymphadenitis (85.7%), aphthous stomatitis (21.4%), arthralgia (14.3%) and skin erythema (35.7%). Neither mevalonic aciduria nor MVK gene mutations were found in any of the subjects, however, unexpectedly, increased plasma IgD (322.2±29.2 U/l) levels were detected in all patients. CONCLUSION: Raised IgD levels may represent a non-specific epiphenomenon, which frequently accompanies PFAPA syndrome as well as MKD. Because of the overlapping clinical and laboratory features, genetic testing of the MVK gene is indicated to differentiate these two conditions, if clinical criteria for both are fulfilled.

Separating the post-Glacial coancestry of European and Asian Y chromosomes within haplogroup R1a.

Human Y-chromosome haplogroup structure is largely circumscribed by continental boundaries. One notable exception to this general pattern is the young haplogroup R1a that exhibits post-Glacial coalescent times and relates the paternal ancestry of more than 10% of men in a wide geographic area extending from South Asia to Central East Europe and South Siberia. Its origin and dispersal patterns are poorly understood as no marker has yet been described that would distinguish European R1a chromosomes from Asian. Here we present frequency and haplotype diversity estimates for more than 2000 R1a chromosomes assessed for several newly discovered SNP markers that introduce the onset of informative R1a subdivisions by geography. Marker M434 has a low frequency and a late origin in West Asia bearing witness to recent gene flow over the Arabian Sea. Conversely, marker M458 has a significant frequency in Europe, exceeding 30% in its core area in Eastern Europe and comprising up to 70% of all M17 chromosomes present there. The diversity and frequency profiles of M458 suggest its origin during the early Holocene and a subsequent expansion likely related to a number of prehistoric cultural developments in the region. Its primary frequency and diversity distribution correlates well with some of the major Central and East European river basins where settled farming was established before its spread further eastward. Importantly, the virtual absence of M458 chromosomes outside Europe speaks against substantial patrilineal gene flow from East Europe to Asia, including to India, at least since the mid-Holocene.

A database of mitochondrial DNA hypervariable regions I and II sequences of individuals from Slovakia.

In order to identify polymorphic positions and to determine their frequencies and the frequency of haplotypes in the human mitochondrial control region, two hypervariable regions (HV1 and HV2) of the mitochondrial DNA (mtDNA) of 374 unrelated individuals from Slovakia were amplified and sequenced. Sequence comparison led to the identification of 284 mitochondrial lineages as defined by 163 variable sites. Genetic diversity (GD) was estimated at 0.997 and the probability of two randomly selected individuals from population having identical mtDNA types (random match probability, RMP) for the both regions is 0.60%.

Mutation analysis of the CFTR gene in Slovak cystic fibrosis patients by DHPLC and subsequent sequencing: identification of four novel mutations.

Cystic fibrosis (CF) is the most common lethal autosomal recessive disorder in Caucasians. Its incidence is approximately 1:2500 newborns. CF is caused by mutations in the transmembrane conductance regulator (CFTR) gene, which encodes an important chloride ion channel. The disease affects the respiratory, digestive and reproductive systems. To date more than 1550 mutations and polymorphisms have been identified throughout the CFTR gene, making the DNA diagnosis more difficult. Rapid accurate identification of CFTR gene mutations is important for confirming the clinical diagnosis, for cascade screening in families at risk for CF, for understanding the correlation between genotype and phenotype, and moreover it is also the only means for prenatal diagnosis. Individuals suspect of CF are in Slovakia presently screened for the presence of 30 common mutations, giving mutation detection rate only approximately 48%. To increase the detection rate we applied a gene scanning approach using DHPLC system for analysing specifically all CFTR exons. The fragments showing abnormal elution profiles were subsequently sequenced to characterize the DNA change. We have identified a total of 28 different mutations up to present not found in Slovak CF patients, and 17 different polymorphisms. Four mutations (G437D, H954P, H1375N, and 3120+33G>T) are novel, not yet found in any other CF patient all over the word.

A counter-clockwise northern route of the Y-chromosome haplogroup N from Southeast Asia towards Europe.

A large part of Y chromosome lineages in East European and East Asian human populations belong to haplogroup (hg) NO, which is composed of two sister clades N-M231 and O-M175. The O-clade is relatively old (around 30 thousand years (ky)) and encompasses the vast majority of east and Southeast Asian male lineages, as well as significant proportion of those in Oceanian males. On the other hand, our detailed analysis of hg N suggests that its high frequency in east Europe is due to its more recent expansion westward on a counter-clock northern route from inner Asia/southern Siberia, approximately 12-14 ky ago. The widespread presence of hg N in Siberia, together with its absence in Native Americans, implies its spread happened after the founder event for the Americas. The most frequent subclade N3, arose probably in the region of present day China, and subsequently experienced serial bottlenecks in Siberia and secondary expansions in eastern Europe. Another branch, N2, forms two distinctive subclusters of STR haplotypes, Asian (N2-A) and European (N2-E), the latter now mostly distributed in Finno-Ugric and related populations. These phylogeographic patterns provide evidence consistent with male-mediated counter-clockwise late Pleistocene-Holocene migratory trajectories toward Northwestern Europe from an ancestral East Asian source of Paleolithic heritage.