Commercially available SARS-CoV-2 RT-qPCR diagnostic tests need obligatory internal validation.

Although infection with severe acute respiratory syndrome coronavirus 2 (SARS CoV-2) does not appear to be as serious a threat to public health as it was in 2020-2021, the increased transmissibility of multiple Omicron descendants may constitute a continuous challenge for health care systems, and reliable detection of new variants is still imperative. This study evaluates the performance of three SARS-CoV-2 diagnostic tests: Novel Coronavirus (2019-nCoV) Real Time Multiplex RT-PCR Kit (Liferiver); Vitassay qPCR SARS-CoV-2 (Vitaassay) and TaqPath COVID­19 CE-IVD RT-PCR Kit (Thermo Fisher Scientific). The analytical sensitivity of the assays as well as their specificity were determined with the use of synthetic nucleic acid standards and clinical samples. All assays appeared to be 100% specific for SARS-CoV-2 RNA in general and the Omicron variant in particular. The LOD determined during this validation was 10 viral RNA copies/reaction for Liferiver and TaqPath and 100 viral RNA copies for Vitassay. We cannot exclude that the LOD for the Vitassay might be lower and close to the manufacturer's declared value of ≥ 20 genome copies/reaction, as we obtained 90% positive results for 10 viral RNA copies/reaction. Mean Ct values at the concentration of 10 viral RNA copies/reaction for the Liferiver, Vitassay and TaqPath kits (35, 37 and 33, respectively) were significantly lower than the cutoff values declared by the manufacturers (≤ 41, ≤ 40 and ≤ 37, respectively). We suggest reporting outcomes based on LOD and cutoff Ct values determined during internal validation rather than those declared by the assays' producers.

An association between copy number variation of enhancer involved in craniofacial development and biogeographic ancestry.

Human facial morphology is a combination of many complex traits and is determined by a large number of genes and enhancers. Here, we report a Copy Number Variation (CNV) study of enhancer hs1431 in populations of Central European and South Siberian ancestry. Central European samples included 97 Poles, while South Siberian samples included 78 Buryats and 27 Tuvinians. CNVs were detected by real-time PCR, using ViiA™ 7 Real-Time PCR System (Applied Biosystems). We revealed significant differences in CNV of hs1431 enhancer between Polish and Buryat population (p=0.0378), but not between Central European and South Siberian population (p=0.1225). Our results suggest that an increase in copy number variation of hs1431 enhancer is associated with biogeographic ancestry. However, this result needs extending and replicating in larger cohorts. This is the first study revealing the presence of copy number variation of enhancer hs1431 in humans.

CLU Polymorphisms in Patients with Pseudoexfoliation Syndrome in Polish Population.

PURPOSE: To evaluate CLU polymorphisms in patients with pseudoexfoliation syndrome. MATERIALS AND METHODS: We studied 81 patients (23 males and 58 females, the median age 76 years) and 91 control subjects (27 males and 64 females, the median age 75 years). Genotypes of the CLU polymorphisms (SNPs), rs3087554 and rs2279590, were determined using a commercially available validated genotyping assays. The χ 2 test was performed to compare patient and control groups for possible associations between SNP genotype/allele frequency and disease state. RESULTS: There were no significant differences for both allele and genotype frequencies between PEX patients and controls for rs3087554 and rs2279590 polymorphisms. The haplotypes distribution shows statistically significant difference between groups (p=0.03). The haplotype (CT) more often was found in controls than in PEX patients, conferring an 18-fold decreased risk to the disease. CONCLUSION: Our results indicate that CLU variants may contribute to the risk of PEX in the Polish population.

An Analysis of the Association between Epilepsy-Related Genes and Vertigo in the Polish Population.

Considering the possibility of a common genetic background of vertigo and epilepsy, we genotyped an affected group of individuals with vertigo and an unaffected group, by studying 26 single-nucleotide polymorphisms (SNPs) in 14 genes which were previously reported to be of particular importance for epilepsy. Significant differences were found between the patients and the control group (χ2 = 38.3, df = 3, p = 1.6 × 10-7) for the frequencies of haplotypes consist ing of 2 SNPs located in chromosome 11 (rs1939012 and rs1783901 within genes MMP8 and SCN3B, respectively). The haplotype rs1939012:C-rs1783901:A, consisting of the minor-frequency alleles was found to be associated with a higher risk of vertigo (OR = 5.0143, 95% CI = 1.6991-14.7980, p = 0.0035). In contrast, the haplotype rs1939012:T-rs1783901:A showed a significant association with a decreased risk of the disease (OR = 0.0597, 95% CI = 0.0136-0.2620, p = 0.0002). Our results suggest that the SNPs rs1939012 and rs1783901 may play a potential role of gene regulation and/or epistasis in a complex etiology of vertigo.

Evaluation of TGF-Beta 2 and VEGFα Gene Expression Levels in Epiretinal Membranes and Internal Limiting Membranes in the Course of Retinal Detachments, Proliferative Diabetic Retinopathy, Macular Holes, and Idiopathic Epiretinal Membranes.

PURPOSE: To evaluate the expression profiles of the VEGFα and TGFß in the ERMs and ILMs in retinal disorders. METHODS: In this nonrandomized prospective study, 75 patients (34 females and 41 males) referred to pars plana vitrectomy (PPV) due to different retinal diseases were enrolled to the study. The samples of ERMs and ILMs collected during PPV were immediately put in TRIzol® Reagent (Life Technologies, USA) and stored at -70°C until RNA extraction. Gene expression analysis was done with TaqMan® Gene Expression Assays (Applied Biosystems, USA) following the manufacturer's instructions. RESULTS: The gene expression levels of VEGFα as well as of TGFß2 were significantly higher in ERMs than in ILMs in all studied groups. The level of TGFß2 expression exhibits a significantly lower values in iERMs as compared with the RRD group (p = 0.043). There were differences in TGFß2 expression in ILM in groups studied: DR versus RRD, p = 0.003; DR versus iERM, p = 0,047; and iERM versus RRD, p = 0.004. CONCLUSIONS: Our results revealed that factors associated with angiogenesis and wound healing processes in eyes with RRD, PDR, iERM, and MH were more upregulated in ERMs than in ILMs. This may indicate that ILM is not responsible for reproliferation and its peeling should be avoided in routine PPV.

The Association of Apolipoprotein E Gene Polymorphism With Cognitive Performance in Nondemented Polish Adults Aged 55 to 75.

The ε4 allele of the apolipoprotein E (APOE) gene is known as a risk factor for dementia. How APOE ε polymorphism affects cognitive performance in nondemented aging subjects remains less clear. In this study, the relationship between APOE status and cognitive performance across various cognitive domains in adults aged 55 to 75 years ( n = 74) without dementia was investigated. E4 carriers ( n = 11) performed worse versus noncarriers on forward Digit Span and delayed recall of the Rey-Osterrieth complex figure. General linear model analysis revealed a small but significant main effect of ε4 on Rey-Osterrieth complex figure delayed recall. Comparing ε2 carriers, ε3 homozygotes, and ε4 carriers, ε3/ε3 performed significantly better on Trail Making Test part B and derived score Trail Making Test B-A. The findings support the relation between the APOE ε polymorphism and visual memory, short-term auditory memory, visuospatial attention, and executive functions in an aging sample without dementia.

Searching for association of the CAG repeat polymorphism in the mitochondrial DNA polymerase gamma gene (POLG) with colorectal cancer.

Mitochondrial DNA polymerase gamma (POLG) is the only DNA polymerase involved in maintaining the mitochondrial genome. Recent studies demonstrated an association of CAG repeat polymorphism in the second exon of POLG gene with the risk of cancer. We investigated the CAG repeat variability in the POLG gene in tumor and non-tumor tissues from colorectal cancer patients and in DNA samples isolated from blood obtained from age-matched healthy persons. Somatically occuring CAG-repeat alterations in cancer tissues have been observed in 10% of patients, but no association has been found between the CAG repeat variants in the POLG gene and colorectal cancer risk.

Mitochondrial DNA Polymerase γ Mutations and Their Implications in mtDNA Alterations in Colorectal Cancer.

Mitochondrial DNA was found to be highly mutated in colorectal cancer cells. One of the key molecules involved in the maintenance of the mitochondrial genome is the nuclear-encoded polymerase gamma. The aim of our study was to determine if there is a link between polymorphisms within the polymerase gamma gene (POLG) and somatic mutations within the mitochondrial genome in cancer cells. We investigated POLG sequence variability in 50 colorectal cancer patients whose complete mitochondrial genome sequences were determined. Relative mtDNA copy number was also determined. We identified 251 sequence variants in the POLG gene. Most of them were germline-specific (∼92%). Twenty-one somatic changes in POLG were found in 10 colorectal cancer patients. We have found no association between the occurrence of mtDNA somatic mutations and the somatically occurring variants in POLG. MtDNA content was reduced in patients carrying somatic variants in POLG or germline nucleotide variants located in the region encoding the POLG polymerase domain, but the difference did not reach statistical significance. Our findings suggest that somatic mtDNA mutations occurring in colorectal cancer are not a consequence of somatic mutations in POLG. Nevertheless, POLG nucleotide variants may lead to a decrease in mtDNA content, and consequently result in mitochondrial dysfunction.

Association between genetic variants on chromosome 15q25 locus and several nicotine dependence traits in Polish population: a case-control study.

Tobacco smoking continues to be a leading cause of disease and mortality. Recent research has confirmed the important role of nicotinic acetylcholine receptor (nAChR) gene cluster on chromosome 15q 24-25 in nicotine dependence and smoking. In this study we tested the association of smoking initiation, age at onset of daily smoking, and heaviness of smoking with five single nucleotide polymorphisms (SNPs) within the CHRNA5-CHRNA3-CHRNB4 cluster. The group of 389 adult subjects of European ancestry from the north of Poland, including 212 ever (140 current and 72 former) and 177 never smokers with mean age 49.26, was genotyped for rs16969868, rs1051730, rs588765, rs6495308, and rs578776 polymorphisms. Distributions of genotypes for rs16969868 and rs1051730 were identical so they were analyzed together. Further analysis revealed the association between rs16969868-1051730 (OR = 2.66; 95% CI: 1.30-5.42) and number of cigarettes smoked per day (CPD) with heaviness of nicotine addiction measured by the Fagerström Test for Nicotine Dependence (FTND) (OR = 2.60; 95% CI: 1.24-5.43). No association between these polymorphisms and other phenotypes was found. Similarly, the association between rs588765, rs6495308, rs578776, and analyzed phenotypes was not confirmed. This study provides strong evidence for the role of the CHRNA5-CHRNA3-CHRNB4 cluster in heaviness of nicotine addiction.

Interplay between genetic and clinical variables affecting platelet reactivity and cardiac adverse events in patients undergoing percutaneous coronary intervention.

Several clinical and genetic variables are associated with influencing high on treatment platelet reactivity (HTPR). The aim of the study was to propose a path model explaining a concurrent impact among variables influencing HTPR and ischemic events. In this prospective cohort study polymorphisms of CYP2C19*2, CYP2C19*17, ABCB1, PON1 alleles and platelet function assessed by Multiple Electrode Aggregometry were assessed in 416 patients undergoing percutaneous coronary intervention treated with clopidogrel and aspirin. The rates of major adverse cardiac events (MACE) were recorded during a 12-month follow up. The path model was calculated by a structural equation modelling. Paths from two clinical characteristics (diabetes mellitus and acute coronary syndrome (ACS)) and two genetic variants (CYP2C19*2 and CYP2C19*17) independently predicted HTPR (path coefficients: 0.11 0.10, 0.17, and -0.10, respectively; p<0.05 for all). By use of those four variables a novel score for prediction of HTPR was built: in a factor-weighted model the risk for HTPR was calculated with an OR of 3.8 (95%CI: 3.1-6.8, p<0.001) for a score level of ≥1 compared with a score of <1. While MACE was independently predicted by HTPR and age in the multivariate model (path coefficient: 0.14 and 0.13, respectively; p<0.05), the coexistence of HTPR and age ≥75 years emerged as the strongest predictor of MACE. Our study suggests a pathway, which might explain indirect and direct impact of variables on clinical outcome: ACS, diabetes mellitus, CYP2C19*2 and CYP2C19*17 genetic variants independently predicted HTPR. In turn, age ≥75 years and HTPR were the strongest predictors of MACE.

[The application of mitochondrial genomics to forensic investigations based on human mitochondrial DNA testing]. / Wykorzystanie osiagniec genomiki mitochondrialnej w badaniach genetyczno-sadowych opartych na analizie sekwencji ludzkiego mitochondrialnego DNA.

In this study we present two forensic cases where mitochondrial DNA HVS I and HVS II haplotypes of evidentiary hairs match reference samples. Based on the information retrieved from mtDNA coding region of reference material, we selected single nucleotide polymorphisms (SNPs) located outside the HVS I and HVS II regions that could increase the informativeness of mtDNA analysis. The SNPs were typed via SNaPshot or dideoxy sequencing technology. In both cases the SNP results allowed for unambiguous exlusion of the evidence and for determining that reference samples originated from the same person.

Analysis of LOXL1 single nucleotide polymorphisms in Polish population with pseudoexfoliation syndrome.

PURPOSE: To assess the possible association of lysyl oxidase-like 1 (LOXL1) gene variants with pseudoexfoliation syndrome (PEX) in Polish population. METHODS: The group studied comprised of 36 patients with PEX (men and women) who presented to Department of Ophthalmology Collegium Medicum UMK in Bydgoszcz, Poland, and 30 control subjects. Blood samples were obtained from each patient via peripheral venipuncture, and genomic DNA was isolated according to the standard procedures. Three LOXL1 single nucleotide polymorphisms (SNPs) rs1048661 (R141L), rs3825942 (G153D) and rs216524 were genotyped in patient sample. RESULTS: The significant association with PEX was found for the G allele of rs3825942 (p = 0.0047) and for the T allele of rs216541 (p = 0.021). The haplotype (GGT) consisting of all three risk alleles was significantly overrepresented (87.5%) in patients with PEX. CONCLUSION: Single nucleotide polymorphisms in LOXL1 are associated with PEX in Polish population which confirms the association previously reported for Icelandic, Swedish, Indian and other populations.

Search for association between suicide and 5-HTT, MAOA and DAT polymorphism in Polish males.

A better understanding of genetic determinants of suicidal behavior might be very useful in clinical practice. The objectives of the present study were to answer the question whether there is an association between functional polymorphic forms of 5-HTT, MAOA or DAT and suicidality, and to examine whether the combination of functional alleles in 5-HTT, MAOA and DAT genes would predict a predisposition to suicidal behavior. Functional polymorphisms in 5-HTT, MAOA and DAT genes were investigated in 66 male suicide completers and 51 male control subjects from the Polish population. There were no significant differences in the allele and genotype frequencies between the case and control group. In the individual genotype tests, examination of the distribution differences of each genotype showed that genotype (3;12-12;S-S;9-10) differed between the suicide victims and control subjects. This genotype existed only in the control sample and appeared with the frequency of 8% (p = 0.03).

[Consent to genetic paternity testing]. / Zgoda na badania genetyczne w celu ustalenia ojcostwa.

The present article aims at reviewing the legislation in Poland and other countries concerning the consent to DNA sample collection, with the special reference to genetic relatedness analyses (including paternity tests) in anonymous samples of biological materials. The Polish legislator has not regulated this issue in a direct manner. Therefore, in view of progressing commercialization of genetic paternity tests, it is necessary to undertake legislative actions towards regulation of DNA tests admissibility, both in civil proceedings and by commission of private individuals.

[Identification of semen in bloodstains with the use of alternative light source and biochemical screening tests]. / Identyfikacja nasienia w zaplamieniach krwawych z uzyciem alternatywnego zródla swiatta i przesiewowych testów biochemicznych.

The objective of the investigation was the verification of the presence of semen in stains constituting mixtures of semen and blood employing alternative light source (ALS) and commercially available biochemical screening tests based on the activity of acid phosphatase (AP) and prostate-specific antigen (PSA). The tests demonstrated that discrimination between particular components of a blood-semen mixture was impossible either with the naked eye, as well as with the use of ALS. White fluorescence was observed only in stains consisting of pure semen and semen-blood mixtures at a ratio of 100:1. The assay for PSA was positive in the case of all the examined semen dilutions and semen-blood mixtures, whereas the sensitivity of the AP-based test assay was lower by one order of magnitude.

[The application of minisequencing reactions for haplogroup assignment of mitochondrial DNA]. / Zastosowanie reakcji minisekwencjonowania do oznaczania przynaleznosci haplogrupowej mitochondrialnego DNA.

In the last few years, one could observe an increased interest in mitochondrial DNA (mtDNA) single nucleotide polymorphisms (SNPs) as a result of their numerous applications in population genetics and forensic science. Continuous progress in molecular technologies together with an increasing body of phylogenetic knowledge, based mainly on complete mitochondrial genome sequencing, allows both for selection and accurate typing of many SNPs in mitochondrial DNA. Among the SNP typing techniques, due to its high sensitivity and promptness of determinations, minisequencing appears to be one of the fastest and most frequently applied methods in forensic laboratories. This review presents currently available minisequencing systems used for haplogroup assignment of mtDNA in European, East Asian and Native American populations.