Depletion of tRNA CCA-adding enzyme in Mycobacterium tuberculosis leads to polyadenylation of transcripts and precursor tRNAs.

In reference to gene annotation, more than half of the tRNA species synthesized by Mycobacterium tuberculosis require the enzymatic addition of the cytosine-cytosine-adenine (CCA) tail, which is indispensable for amino acid charging and tRNA functionality. It makes the mycobacterial CCA-adding enzyme essential for survival of the bacterium and a potential target for novel pipelines in drug discovery avenues. Here, we described the rv3907c gene product, originally annotated as poly(A)polymerase (rv3907c, PcnA) as a functional CCA-adding enzyme (CCAMtb) essential for viability of M. tuberculosis. The depletion of the enzyme affected tRNAs maturation, inhibited bacilli growth, and resulted in abundant accumulation of polyadenylated RNAs. We determined the enzymatic activities displayed by the mycobacterial CCAMtb in vitro and studied the effects of inhibiting of its transcription in bacterial cells. We are the first to properly confirm the existence of RNA polyadenylation in mycobacteria, a previously controversial phenomenon, which we found promoted upon CCA-adding enzyme downexpression.

The functional response of human monocyte-derived macrophages to serum amyloid A and Mycobacterium tuberculosis infection.

Introduction: In the course of tuberculosis (TB), the level of major acute phase protein, namely serum amyloid A (hSAA-1), increases up to a hundredfold in the pleural fluids of infected individuals. Tubercle bacilli infecting the human host can be opsonized by hSAA-1, which affects bacterial entry into human macrophages and their intracellular multiplication. Methods: We applied global RNA sequencing to evaluate the functional response of human monocyte-derived macrophages (MDMs), isolated from healthy blood donors, under elevated hSAA-1 conditions and during infection with nonopsonized and hSAA-1-opsonized Mycobacterium tuberculosis (Mtb). In the same infection model, we also examined the functional response of mycobacteria to the intracellular environment of macrophages in the presence and absence of hSAA-1. The RNASeq analysis was validated using qPCR. The functional response of MDMs to hSAA-1 and/or tubercle bacilli was also evaluated for selected cytokines at the protein level by applying the Milliplex system. Findings: Transcriptomes of MDMs cultured in the presence of hSAA-1 or infected with Mtb showed a high degree of similarity for both upregulated and downregulated genes involved mainly in processes related to cell division and immune response, respectively. Among the most induced genes, across both hSAA-1 and Mtb infection conditions, CXCL8, CCL15, CCL5, IL-1ß, and receptors for IL-7 and IL-2 were identified. We also observed the same pattern of upregulated pro-inflammatory cytokines (TNFα, IL-6, IL-12, IL-18, IL-23, and IL-1) and downregulated anti-inflammatory cytokines (IL-10, TGFß, and antimicrobial peptide cathelicidin) in the hSAA-1 treated-MDMs or the phagocytes infected with tubercle bacilli. At this early stage of infection, Mtb genes affected by the inside microenvironment of MDMs are strictly involved in iron scavenging, adaptation to hypoxia, low pH, and increasing levels of CO2. The genes for the synthesis and transport of virulence lipids, but not cholesterol/fatty acid degradation, were also upregulated. Conclusion: Elevated serum hSAA-1 levels in tuberculosis enhance the response of host phagocytes to infection, including macrophages that have not yet been in contact with mycobacteria. SAA induces antigen processing and presentation processes by professional phagocytes reversing the inhibition caused by Mtb infection.

The Orphan Response Regulator Rv3143 Modulates the Activity of the NADH Dehydrogenase Complex (Nuo) in Mycobacterium tuberculosis via Protein-Protein Interactions.

Two-component signal transduction systems enable mycobacterial cells to quickly adapt and adequately respond to adverse environmental conditions encountered at various stages of host infection. We attempted to determine the role of the Rv3143 "orphan" response regulator in the physiology of Mycobacterium tuberculosis and its orthologue Msmeg_2064 in Mycobacterium smegmatis. We identified the Rv3143 protein as an interaction partner for NuoD, a member of the type I NADH dehydrogenase complex involved in oxidative phosphorylation. The mutants Δrv3143 and Δmsmeg_2064 were engineered in M. tuberculosis and M. smegmatis cells, respectively. The Δmsmeg_2064 strain exhibited a significant reduction in growth and viability in the presence of reactive nitrogen species. The Rv3143-deficient strain was sensitive to valinomycin, which is known to reduce the electrochemical potential of the cell and overexpressed genes required for nitrate respiration. An increased level of reduction of the 2,3,5-triphenyltetrazolium chloride (TTC) electron acceptor in Δrv3143 and Δmsmeg_2064 cells was also evident. The silencing of ndh expression using CRISPRi/dCas9 affected cell survival under limited oxygen conditions. Oxygen consumption during entry to hypoxia was most severely affected in the double-mutant Δmsmeg_2064 ndhCRISPRi/dCas9 . We propose that the regulatory protein Rv3143 is a component of the Nuo complex and modulates its activity.

Cortisol concentration affects fat and muscle mass among Polish children aged 6-13 years.

BACKGROUND: Cortisol is a steroid hormone acting as a stress hormone, which is crucial in regulating homeostasis. Previous studies have linked cortisol concentration to body mass and body composition. METHODS: The investigations were carried out in 2016-2017. A total of 176 children aged 6-13 years in primary schools in central Poland were investigated. Three types of measurements were performed: anthropometric (body weight and height, waist and hip circumferences), body composition (fat mass FM (%), muscle mass - MM (%), body cellular mass - BCM (%), total body water - TBW (%)), and cortisol concentration using saliva of the investigated individuals. Information about standard of living, type of feeding after birth, parental education and maternal trauma during pregnancy was obtained with questionnaires. RESULTS: The results of regression models after removing the environmental factors (parental education, standard of living, type of feeding after birth, and maternal trauma during pregnancy) indicate a statistically significant association between the cortisol concentration and fat mass and muscle mass. The cortisol concentration was negatively associated with FM (%) (Beta=-0.171; p = 0.026), explaining 2.32 % of the fat mass variability and positively associated with MM (%) (Beta = 0.192; p = 0.012) explaining 3.09 % of the muscle mass variability. CONCLUSIONS: Cortisol concentration affects fat and muscle mass among Polish children. TRIAL REGISTRATION: The Ethical Commission at the University of Lodz (nr 19/KBBN-UL/II/2016).

The association between polymorphisms near TMEM18 and the risk of obesity: a meta-analysis.

BACKGROUND: Many studies have proposed that the pathogenesis of obesity has a genetic basis, with an important risk factor being the presence of polymorphisms in the region of the TMEM18 gene, which plays a significant role in feeding behaviour; however, subsequent studies among different ethnic populations and age groups have shown inconsistent results. Therefore, this present meta-analysis examines the relationship between TMEM18 polymorphisms with the risk of obesity with regard to age group and ethnic population. METHODS: A literature database search was conducted for available relevant studies investigating the association between obesity risk and the presence of rs6548238, rs4854344, rs11127485, rs2867125 and rs7561317 polymorphisms in TMEM18. Pooled odds ratio (OR) and 95% confidence intervals (95% CI) were calculated by either a fixed-effects model or random effect model based on a heterogeneity test. The meta-analysis of rs6548238 and its surrogates examined the relationships between 53 395 obesity cases and 123 972 healthy controls from 27 studies and published data from the POPULOUS collection (Poland). RESULTS: A significant association is observed between rs6548238 (and surrogate) and obesity risk, with OR = 1.25 (95% CI: 1.08-1.45). Regarding population type, a significant association was revealed among groups of Europeans with OR = 1.32 (1.10-1.59) and Mexicans with OR = 1.39 (1.13-1.73). However, a lack of statistical significance was noticed in groups in Asia with OR = 1.11 (95% CI: 0.86-1.42). Regarding age, a significant association was observed among children with OR = 1.28 (95% CI: 1.18-1.39) but not in adults OR = 1.21 (95% CI: 0.92-1.58). CONCLUSIONS: The polymorphisms near TMEM18 appear to play a role in the development of obesity. Our findings indicate that differences exist between ethnic populations and age groups, supporting those of a previous study showing the various effects of genetic factors on age and ethnic groups.

Dissecting the RecA-(In)dependent Response to Mitomycin C in Mycobacterium tuberculosis Using Transcriptional Profiling and Proteomics Analyses.

Mycobacteria exploit at least two independent global systems in response to DNA damage: the LexA/RecA-dependent SOS response and the PafBC-regulated pathway. Intracellular pathogens, such as Mycobacterium tuberculosis, are exposed to oxidative and nitrosative stress during the course of infection while residing inside host macrophages. The current understanding of RecA-independent responses to DNA damage is based on the saprophytic model of Mycobacterium smegmatis, a free-living and nonpathogenic mycobacterium. The aim of the present study was to identify elements of RecA-independent responses to DNA damage in pathogenic intracellular mycobacteria. With the help of global transcriptional profiling, we were able to dissect RecA-dependent and RecA-independent pathways. We profiled the DNA damage responses of an M. tuberculosis strain lacking the recA gene, a strain with an undetectable level of the PafBC regulatory system, and a strain with both systems tuned down simultaneously. RNA-Seq profiling was correlated with the evaluation of cell survival in response to DNA damage to estimate the relevance of each system to the overall sensitivity to genotoxic agents. We also carried out whole-cell proteomics analysis of the M. tuberculosis strains in response to mitomycin C. This approach highlighted that LexA, a well-defined key element of the SOS system, is proteolytically inactivated during RecA-dependent DNA repair, which we found to be transcriptionally repressed in response to DNA-damaging agents in the absence of RecA. Proteomics profiling revealed that AlkB was significantly overproduced in the ΔrecA pafBCCRISPRi/dCas9 strain and that Holliday junction resolvase RuvX was a DNA damage response factor that was significantly upregulated regardless of the presence of functional RecA and PafBC systems, thus falling into a third category of DNA damage factors: RecA- and PafBC-independent. While invisible to the mass spectrometer, the genes encoding alkA, dnaB, and dnaE2 were significantly overexpressed in the ΔrecA pafBCCRISPRi/dCas9 strain at the transcript level.

Associations between second to fourth digit ratio, cortisol, vitamin D, and body composition among Polish children.

Associations between body characteristics (body composition: fat mass, muscle mass, cell, and water mass as well as body proportion-BMI), the 2D:4D digit ratio, and the concentrations of cortisol and vitamin (25-OH)D among Polish children have not been studied before. A total of 133 (73 girls and 60 boys) healthy Polish children aged 7-11 years were examined. The investigation was divided into three parts: measuring (the length of the second and fourth fingers in both hands, body composition, and body height and mass), questionnaires (socioeconomic status), and laboratory investigations (25-OH vitamin D and cortisol concentrations in saliva measured with ELISA methods). Boys with digit ratios below 1 had lower vitamin D concentration than those with digit ratios equal to or higher than 1 (Z = - 2.33; p = 0.019). Only boys with the male-typical pattern of 2D:4D digit ratio tended to have a lower 25-OH vitamin D concentration in saliva. Thus, it might indicate an effect of prenatal programming on the concentrations of steroid hormones in later life. Neither vitamin D, 2D:4D digit ratio nor the cortisol level was associated with the body components or proportions. More studies are needed to evaluate the molecular and genetic background of this phenomenon.

Association Between rs12037447, rs146732504, rs151078858, rs55723436, and rs6094136 Polymorphisms and Kawasaki Disease in the Population of Polish Children.

Background: Kawasaki disease (KD) is an acute self-limited febrile vasculitis that mainly affects young children. Coronary artery involvement is the most serious complication in children with KD. It is currently the leading cause of acquired cardiac disease in children from developed countries. Literature data indicate a significant role of genetic susceptibility to KD. Objective: The aim of this study was to perform the first Genome-Wide Association Study (GWAS) in a population of Polish children with KD and identify susceptible genes involved in the pathogenesis of KD. Materials and Methods: The blood samples of Kawasaki disease patients (n = 119) were collected between 2016 and 2020, isolated and stored at the Department of Pediatrics, Nutrition and Metabolic Diseases, Children's Memorial Health Institute in Warsaw. The control group was based on Polish donors (n = 6,071) registered as the POPULOUS collection at the Biobank Lab of The Department of Molecular Biophysics in University of Lodz. DNA samples were genotyped for 558,231 Single Nucleotide Polymorphisms (SNPs) using the 24 × 1 Infinium HTS Human Core Exome microarrays according to the protocol provided by the manufacturer. In order to discover and verify genetic risk-factors for KD, association analysis was carried out using PLINK 1.9. Results: Of all 164,395 variants, 5 were shown to occur statistically (padjusted < 0.05) more frequent in Kawasaki disease patients than in controls. Those are: rs12037447 in non-coding sequence (padjusted = 8.329 × 10-4, OR = 8.697, 95% CI; 3.629-20.84) and rs146732504 in KIF25 (padjusted = 0.007354, OR = 11.42, 95% CI; 3.79-34.43), rs151078858 in PTPRJ (padjusted = 0.04513, OR = 8.116, 95% CI; 3.134-21.01), rs55723436 in SPECC1L (padjusted = 0.04596, OR = 5.596, 95% CI; 2.669-11.74), rs6094136 in RPN2 (padjusted = 0.04755, OR = 10.08, 95% CI; 3.385-30.01) genes. Conclusion: Polymorphisms of genes KIF25, PTRPJ, SPECC1L, RNP2 may be linked with the incidence of Kawasaki disease in Polish children.

Y-Chromosome Genetic Analysis of Modern Polish Population.

The study presents a full analysis of the Y-chromosome variability of the modern male Polish population. It is the first study of the Polish population to be conducted with such a large set of data (2,705 individuals), which includes genetic information from inhabitants of all voivodeships, i.e., the first administrative level, in the country and the vast majority of its counties, i.e., the second level. In addition, the available data were divided into clusters corresponding to more natural geographic regions. Genetic analysis included the estimation of F ST distances, the visualization with the use of multidimensional scaling plots and analysis of molecular variance. Y-chromosome binary haplogroups were classified and visualized with the use of interpolation maps. Results showed that the level of differentiation within Polish population is quite low, but some differences were indicated. It was confirmed that the Polish population is characterized by a high degree of homogeneity, with only slight genetic differences being observed at the regional level. The use of regional clustering as an alternative to counties and voivodeships provided a more detailed view of the genetic structure of the population. Those regional differences identified in the present study highlighted the need for additional division of the population by cultural and ethnic criteria in such studies rather than just by geographical or administrative regionalization.

Comprehensive Analysis of ABCG2 Genetic Variation in the Polish Population and Its Inter-Population Comparison.

ATP-binding cassette sub-family G member 2 (ABCG2), also known as breast cancer resistance protein (BCRP), is one of the key efflux ATP-binding cassette (ABC) transporters of xenobiotics, their metabolites and endogenous compounds such as urate. Some of its genetic variants have been found to influence protein functioning, resulting in serious clinical implications concerning chemotherapy response, as well as gout or blood group phenotype Jr(a-). Previous reports have suggested that the frequencies of certain crucial polymorphisms, such as c.34G>A (p.Val12Met) and c.421C>A (p.Gln141Lys) differ significantly between the Polish population and other Caucasian populations. Thus, to clarify this issue, the present study performs a complete analysis of the genetic variation of ABCG2 coding sequence in the Polish population. The genetic variation in 14 out of 15 coding exons of the ABCG2 gene, as well as their flanking intron sequences, were examined among 190 healthy representatives of the Polish population using scanning with High Resolution Melting (HRM). HRM scanning revealed 17 polymorphisms: eight in the exons (including five missense variants and one point-nonsense mutation) and nine in the intron sequences (eight single nucleotide polymorphisms (SNPs) and one deletion variant). These included variants correlating with the presence of gout and phenotype Jr(a-). Linkage disequilibrium, haplotype blocks and haplotype analyses were also performed. The frequencies of the most common polymorphisms in the Polish population did not differ significantly to those observed for other Caucasian populations, but demonstrated divergence from non-Caucasian populations. We hope that our findings may be helpful for other researchers and clinicians, evaluating the pharmacogenetic role of ABCG2.

Results from a Genome-Wide Association Study (GWAS) in Mastocytosis Reveal New Gene Polymorphisms Associated with WHO Subgroups.

Mastocytosis is rare disease in which genetic predisposition is not fully understood. The aim of this study was to analyze associations between mastocytosis and single nucleotide polymorphisms (SNPs) by a genome-wide association study (GWAS) approach. A total of 234 patients were enrolled in our study, including 141 with cutaneous mastocytosis (CM; 78 children and 63 adults) and 93 with systemic mastocytosis (SM, all adults). The control group consisted of 5606 healthy individuals. DNA samples from saliva or blood were genotyped for 551 945 variants using DNA microarrays. The prevalence of certain SNPs was found to vary substantially when comparing patients and healthy controls: rs10838094 of 5OR51Q1 was less frequently detected in CM and SM patients (OR = 0.2071, p = 2.21 × 10-29), rs80138802 in ABCA2 (OR = 5.739, p = 1.98 × 10-28), and rs11845537 in OTX2-AS1 (rs11845537, OR = 6.587, p = 6.16 × 10-17) were more frequently detected in CM in children and adults. Additionally, we found that rs2279343 in CYP2B6 and rs7601511 in RPTN are less prevalent in CM compared to controls. We identified a number of hitherto unknown associations between certain SNPs and CM and/or SM. Whether these associations are clinically relevant concerning diagnosis, prognosis, or prevention remains to be determined in future studies.

Association of saliva 25(OH)D concentration with body composition and proportion among pre-pubertal and pubertal Polish children.

OBJECTIVES: Due to increasing problems with obesity and vitamin D deficiency among children, studies that tackle both problems together are needed. METHODS: Data were collected from 182 randomly selected children aged 6-13 years in primary schools in central Poland. Measures included anthropometric dimensions, body composition, questionnaires completed by participants' parents, and saliva samples. The level of 25(OH)D was assessed from the saliva samples using an enzyme-linked immunosorbent assay kit. The children were divided into two groups: pre-pubertal (girls below 10 years and boys below 11 years) and pubertal individuals (girls above 10 years and boys above 11 years). RESULTS: The 25(OH)D concentrations were higher in late spring (June) among pre-pubertal children than in the autumn (November-December) among pubertal children. The level of 25(OH)D was positively correlated with body cell mass (BCM,%) among all children (pubertal: R = 0.20, P = .044; pre-pubertal: R = 0.23, P = .041) and inversely associated with waist-to-hip ratio (WHR) among pubertal children of both sexes (R = -0.25; P = .031). The stepwise regression analysis revealed that investigation in spring (June) and breastfeeding was associated with increased muscle mass (MM, %) (beta = 0.253, P = .003 and beta = 0.225, P = .005, respectively) and total body water (TBW, %) (beta = 0.276, P = .004 and beta = 0.246, P = .011, respectively) and was associated with decreased body mass index (BMI; beta = -0.222, P = .024 and beta = -0.269, P = .009, respectively) and fat mass (%) (beta = -0.288, P = .003 and beta = -0.266, P = .005, respectively). CONCLUSIONS: Season of salivary sampling and breastfeeding status were more strongly associated with body components, BMI and WHR, than 25(OH)D concentrations.

Proteomic and transcriptomic experiments reveal an essential role of RNA degradosome complexes in shaping the transcriptome of Mycobacterium tuberculosis.

The phenotypic adjustments of Mycobacterium tuberculosis are commonly inferred from the analysis of transcript abundance. While mechanisms of transcriptional regulation have been extensively analysed in mycobacteria, little is known about mechanisms that shape the transcriptome by regulating RNA decay rates. The aim of the present study is to identify the core components of the RNA degradosome of M. tuberculosis and to analyse their function in RNA metabolism. Using an approach involving cross-linking to 4-thiouridine-labelled RNA, we mapped the mycobacterial RNA-bound proteome and identified degradosome-related enzymes polynucleotide phosphorylase (PNPase), ATP-dependent RNA helicase (RhlE), ribonuclease E (RNase E) and ribonuclease J (RNase J) as major components. We then carried out affinity purification of eGFP-tagged recombinant constructs to identify protein-protein interactions. This identified further interactions with cold-shock proteins and novel KH-domain proteins. Engineering and transcriptional profiling of strains with a reduced level of expression of core degradosome ribonucleases provided evidence of important pleiotropic roles of the enzymes in mycobacterial RNA metabolism highlighting their potential vulnerability as drug targets.

Mitochondrial DNA variability of the Polish population.

The aim of the present study was to define the mtDNA variability of Polish population and to visualize the genetic relations between Poles. For the first time, the study of Polish population was conducted on such a large number of individuals (5852) representing administrative units of both levels of local administration in Poland (voivodeships and counties). Additionally, clustering was used as a method of population subdivision. Performed genetic analysis, included FST, MDS plot, AMOVA and SAMOVA. Haplogroups were classified and their geographical distribution was visualized using surface interpolation maps. Results of the present study showed that Poles are characterized by the main West Eurasian mtDNA haplogroups. Furthermore, the level of differentiation within the Polish population was quite low but the existing genetic differences could be explained well with geographic distances. This may lead to a conclusion that Poles can be considered as genetically homogenous but with slight differences, highlighted at the regional level. Some patterns of variability were observed and could be explained by the history of demographic processes in Poland such as resettlements and migrations of women or relatively weaker urbanisation and higher rural population retention of some regions.

The association between socioeconomic status, duration of breastfeeding, parental age and birth parameters with BMI, body fat and muscle mass among prepubertal children in Poland.

Objectives: We aimed to indicate simple determinants of abnormal body composition in children, such as socio-economic status (SES), duration of breastfeeding, parental age and birth parameters. Methods: The final data set consisted of 469 healthy prepubertal individuals (247 girls and 222 boys). We studied body mass, body height, and parameters of body composition such as muscle mass and fat mass. The birth parameters and gestational age were obtained from the children's medical record books held by the parents which were completed by medical personnel immediately following birth. Information about socio-economic status (SES), duration of breastfeeding and parental age was obtained by questionnaire. The statistical methods included forward multiple regression and generalized linear models (GLZ) or general linear model (GLM). Results: Higher fat mass (FM) (%) was connected with shorter duration of breastfeeding (< 2 months and lower SES (p < 0.05). Lower muscle mass (MM) (%) was linked with lower SES (p < 0.05) and lower birth weight (p < 0.05). Higher body mass index (BMI) was connected with higher birth weight (p < 0.05), shorter duration of breastfeeding (< 2 months) and lower SES (p < 0.05). Moreover interaction effects were observed in the case of the FM (%) (breastfeeding x SES; breastfeeding x parental age) and the BMI (breastfeeding x paternal age). Conclusions: Body composition can be linked with the duration of breastfeeding, SES, parental age, birth weight and birth length.

Association of the 2D:4D digit ratio with body composition among the Polish children aged 6-13 years.

The 2D:4D digit ratio is formed as a result of genetic factors but also prenatal exposure to sex hormones. The higher index value the higher concentration of the prenatal oestrogen. It is commonly known that testosterone is a hormone connected with muscle mass growth and that oestrogen affects adipogenesis. The aim of this study was to find if the digit ratio can be an informative indicator of the fat mass and muscle mass and body proportions in prepubertal children. Material and methods The analysed cohort included 420 children (221 girls and 199 boys) aged 6-13 years. Pearson's and Spearman's tests were conducted to assess whether 2D:4D was significantly correlated with the body composition measurements. Multiple regression models and stepwise forward regression were applied to select the most important independent variables affecting fat mass (%) and muscle mass (%) as well as the BMI and the WHR. Results The study shows that the digit ratio is negatively correlated with muscle mass (MM%) among girls (p < 0.05).There was no similar relationship in the group of boys. The regression models showed a significant role in determining the body composition and body proportions played by maternal factors such as: maternal level of education and weight gain during pregnancy. Conclusions The 2D:4D digit ratio seems to be an informative indicator of the muscle mass development since girls' early childhood. Moreover, maternal environment is also important in forming the offspring's body composition and proportions.

Investigating the impact of age-depended hair colour darkening during childhood on DNA-based hair colour prediction with the HIrisPlex system.

Predictive DNA analysis of externally visible characteristics exerts an increasing influence on contemporary forensic and anthropological investigations, with pigmentation traits currently being the most advanced for predictive modelling. Since pigmentation prediction error in some cases may be due to the result of age-related hair colour darkening, and sex influence in eye colour, this study aims to investigate these less explored phenomena on a group of juvenile individuals. Pigmentation phenotypes of children between the age of 6-13 years old were evaluated, in addition to data about their hair colour during early childhood from a select number of these individuals. The HIrisPlex models for DNA-based eye and hair colour prediction were used with input from SNP genotyping using massive parallel sequencing. Analysis of the total group of 476 children showed high accuracy in blue (AUC = 0.89) and brown (AUC = 0.91) eye colour prediction, while hair colour was predicted with AUC = 0.64 for blond, AUC = 0.64 for brown and AUC = 0.97 for red. 70.8% (n = 143) of the total number of children phenotypically blond for hair colour during early childhood progressed to brown during advanced childhood. In 70.6% (n = 101) of those cases, an incorrect blond hair prediction was made during the time of analysis. A noticeable decline in AUC values for blond (from 0.76 to 0.65) and brown (from 0.72 to 0.64) were observed when comparing hair colour prediction outcomes for the phenotypes recorded for the two different time points (at the age of 2-3 and 6-13). The number of incorrect blond hair colour predictions was significantly higher in children with brown hair at age 6-13 who were blond at early childhood (n = 47, 32.9%), relative to children who had brown hair at both time points (n = 6, 9.4%). However, in 28.0% (n = 40) of children who did experience hair colour darkening, HIrisPlex provided the correct prediction for the darkened hair colour phenotype, despite them being blond in early childhood. Our study implies that HIrisPlex can correctly predict adult hair colour in some individuals who experience age-related hair colour darkening during adolescence. However, in most instances prediction seems to default to the pre-adolescent hair colour for individuals with this phenomenon. In the future, the full adolescent age range in which hair colour darkening can occur should be considered in the study samples used for training hair colour prediction models to obtain a more complete picture of the phenomenon and its impact on DNA-based hair colour prediction in adults.

High Resolution Melting (HRM) for High-Throughput Genotyping-Limitations and Caveats in Practical Case Studies.

High resolution melting (HRM) is a convenient method for gene scanning as well as genotyping of individual and multiple single nucleotide polymorphisms (SNPs). This rapid, simple, closed-tube, homogenous, and cost-efficient approach has the capacity for high specificity and sensitivity, while allowing easy transition to high-throughput scale. In this paper, we provide examples from our laboratory practice of some problematic issues which can affect the performance and data analysis of HRM results, especially with regard to reference curve-based targeted genotyping. We present those examples in order of the typical experimental workflow, and discuss the crucial significance of the respective experimental errors and limitations for the quality and analysis of results. The experimental details which have a decisive impact on correct execution of a HRM genotyping experiment include type and quality of DNA source material, reproducibility of isolation method and template DNA preparation, primer and amplicon design, automation-derived preparation and pipetting inconsistencies, as well as physical limitations in melting curve distinction for alternative variants and careful selection of samples for validation by sequencing. We provide a case-by-case analysis and discussion of actual problems we encountered and solutions that should be taken into account by researchers newly attempting HRM genotyping, especially in a high-throughput setup.

High prevalence of carriers of variant c.1528G>C of HADHA gene causing long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency (LCHADD) in the population of adult Kashubians from North Poland.

BACKGROUND/OBJECTIVES: The mitochondrial ß-oxidation of fatty acids is a complex catabolic pathway. One of the enzymes of this pathway is the heterooctameric mitochondrial trifunctional protein (MTP), composed of four α- and ß-subunits. Mutations in MTP genes (HADHA and HADHB), both located on chromosome 2p23, cause MTP deficiency, a rare autosomal recessive metabolic disorder characterized by decreased activity of MTP. The most common MTP mutation is long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) deficiency caused by the c.1528G>C (rs137852769, p.Glu510Gln) substitution in exon 15 of the HADHA gene. SUBJECTS/METHODS: We analyzed the frequency of genetic variants in the HADHA gene in the adults of Kashubian origin from North Poland and compared this data in other Polish provinces. RESULTS: We found a significantly higher frequency of HDHA c.1528G>C (rs137852769, p.Glu510Gln) carriers among Kashubians (1/57) compared to subjects from other regions of Poland (1/187). We found higher frequency of c.652G>C (rs71441018, pVal218Leu) polymorphism in the HADHA gene within population of Silesia, southern Poland (1/107) compared to other regions. CONCLUSION: Our study indicate described high frequency of c.1528G>C variant of HADHA gene in Kashubian population, suggesting the founder effect. For the first time we have found high frequency of rs71441018 in the South Poland Silesian population.

New variants near RHOJ and C2, HLA-DRA region and susceptibility to endometriosis in the Polish population-The genome-wide association study.

OBJECTIVE: Endometriosis is a common gynaecological disease, associated with severe pelvic pain and reduced fertility; however, molecular mechanisms remain largely unknown. Genome-wide association studies (GWAS) are able to identify genetic loci, which can play significant role during endometriosis development. AIM: The study aimed at localisation of new genes and chromosomal loci, the nucleotide variants of which determine the level of susceptibility to endometriosis. STUDY DESIGN: Blood samples from 171 patients with endometriosis were used as material for studies. The patients were recruited to the study at the Department of Operative Gynaecology of the Institute of the Polish Mother's Memorial Hospital in Lodz. A control group (n=2934) came from the POPULOUS collection registered at Biobank Lab, Department of Molecular Biophysics, University of Lodz. DNA of the patients with endometriosis was compared with DNA of women free from that disease, the comparison being supported by GWAS. RESULTS: Genome-wide significant correlation was identified between one new, not previously described, single nucleotide polymorphism (SNP), rs10129516, localised on chromosome 14 in intergenic region between PARP1P2 and RHOJ genes (p=1.44×10-10, OR=3.104, 95% CI=2.329-4.136) and endometriosis. We have also identified significant association with endometriosis of 18 SNPs localised on chromosome 6 in position range 31883957 - 32681631 (C2 and HLA-DRA genes region) with the lowest observed p value for rs644045 in C2 gene (p=2.04×10-8, OR=1.955, 95% CI=1.541-2.480). CONCLUSION: Reported GWAS identified the novel loci associated with endometriosis in Polish women, not previously reported. The most interesting observation shown in our study are regions associated with susceptibility to endometriosis of loci located near C2, HLA-DRA and RHOJ genes. RESULTS: of that study did not correspond to previously published data about polymorphism in that regions and further evaluations are necessary in groups with higher numbers of patients to explain whether the above-mentioned genetic variant may be the risk factor for pathogenesis of endometriosis.