Here, we report the electrochemical detection of single-point mutations using solid-phase isothermal primer elongation with redox-labeled oligonucleotides. A single-base mutation associated with resistance to rifampicin, an antibiotic commonly used for the treatment of Mycobacterium tuberculosis, was used as a model system to demonstrate a proof-of-concept of the approach. Four 5'-thiolated primers, designed to be complementary with the same fragment of the target sequence and differing only in the last base, addressing the polymorphic site, were self-assembled via chemisorption on individual gold electrodes of an array. Following hybridization with single-stranded DNA, Klenow (exo-) DNA polymerase-mediated primer extension with ferrocene-labeled 2'-deoxyribonucleoside triphosphates (dNFcTPs) was only observed to proceed at the electrode where there was full complementarity between the surface-tethered probe and the target DNA being interrogated. We tested all four ferrocenylethynyl-linked dNTPs and optimized the ratio of labeled/natural nucleotides to achieve maximum sensitivity. Following a 20 min hybridization step, Klenow (exo-) DNA polymerase-mediated primer elongation at 37 °C for 5 min was optimal for the enzymatic incorporation of a ferrocene-labeled nucleotide, achieving unequivocal electrochemical detection of a single-point mutation in 14 samples of genomic DNA extracted from Mycobacterium tuberculosis strains. The approach is rapid, cost-effective, facile, and can be extended to multiplexed electrochemical single-point mutation genotyping.

• Keywords
• Klenow (exo-) DNA polymerase, ferrocene-labeled nucleotides, nucleoside triphosphates, single-nucleotide polymorphism (SNP), single-point mutation, solid-phase primer elongation,
• MeSH
• Polymorphism, Single Nucleotide MeSH
• Metallocenes MeSH
• Mycobacterium tuberculosis * genetics   MeSH
• Oxidation-Reduction MeSH
• Rifampin pharmacology   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Metallocenes MeSH
• Rifampin MeSH

Establishing links between phenotypes and molecular variants is of central importance to accelerate genetic improvement of economically important plant species. Our work represents the first genome-wide association study to the inherently complex and currently poorly understood genetic architecture of industrially relevant wood traits. Here, we employed an Illumina Infinium 34K single nucleotide polymorphism (SNP) genotyping array that generated 29,233 high-quality SNPs in c. 3500 broad-based candidate genes within a population of 334 unrelated Populus trichocarpa individuals to establish genome-wide associations. The analysis revealed 141 significant SNPs (α ≤ 0.05) associated with 16 wood chemistry/ultrastructure traits, individually explaining 3-7% of the phenotypic variance. A large set of associations (41% of all hits) occurred in candidate genes preselected for their suggested a priori involvement with secondary growth. For example, an allelic variant in the FRA8 ortholog explained 21% of the total genetic variance in fiber length, when the trait's heritability estimate was considered. The remaining associations identified SNPs in genes not previously implicated in wood or secondary wall formation. Our findings provide unique insights into wood trait architecture and support efforts for population improvement based on desirable allelic variants.

• Keywords
• Populus, association genetics, cellulose, lignin, single nucleotide polymorphism (SNP), wood traits, wood ultrastructure,
• MeSH
• Alleles MeSH
• Cell Wall MeSH
• Wood * growth & development   metabolism   ultrastructure   MeSH
• Phenotype * MeSH
• Genetic Association Studies MeSH
• Genome, Plant * MeSH
• Genotype * MeSH
• Polymorphism, Single Nucleotide * MeSH
• Populus genetics   growth & development   metabolism   ultrastructure   MeSH
• Genes, Plant * MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH

UNLABELLED: The BsmI polymorphism in the VDR gene has been extensively investigated by PCR and restriction digestion in bone genetics. A SNP within the corresponding region for the previously published reverse primer was observed and confirmed by DNA sequencing. BsmI mis-genotyping caused by this SNP could confound genetic findings. INTRODUCTION: By analyzing the FokI, BsmI, ApaI, and TaqI polymorphisms in the vitamin D receptor (VDR) gene, we observed a significantly different genotype distribution in the BsmI polymorphic locus with a deviation from Hardy-Weinberg equilibrium. One of the reasons for polymerase chain reaction (PCR) non-amplification may be a mismatched base at the primer binding region. Therefore, the aim of this study was to analyse whether a single nucleotide polymorphism (SNP), which has been recently described as TruI, is responsible for the discrepancy between expected and observed genotype frequencies. MATERIALS AND METHODS: The VDR genotypes were identified in a cohort of 165 peri- and postmenopausal women of white origin. PCR amplification was carried out using the originally published primers and followed by restriction cleavage. The BsmI genotypes were further verified with a reverse primer external to the original binding site. The presence of the TruI polymorphism under the previously published reverse primer was confirmed by a restriction digestion and DNA sequencing. In Bb subjects, the colocalization of b allele with the TruI restriction site on the same chromosome was confirmed by a simultaneous digestion of the PCR product with both BsmI and TruI restriction enzymes. RESULTS: The BsmI reanalysis with an external primer provided a higher number of heterozygous subjects with a proportionally smaller number of BB subjects, and the changed genotype distribution was under Hardy-Weinberg equilibrium (BB, 31; Bb, 80; bb, 54; r = 0.0203; p = 0.90). In our primary analysis, the presence of the TruI polymorphism led to a drop out of b allele during PCR amplification and thus to the false prevalence of BB genotypes (BB, 50; Bb, 61; bb, 54; r = 11.17; p = 0.01). CONCLUSION: The SNP in the region corresponding to the reverse primer may lead to BsmI mis-genotyping, which may have confounded some previous genetic studies.

• MeSH
• Alleles MeSH
• Genotype MeSH
• Heterozygote MeSH
• Homozygote MeSH
• Polymorphism, Single Nucleotide * MeSH
• Humans MeSH
• Models, Genetic MeSH
• Polymorphism, Genetic MeSH
• Receptors, Calcitriol genetics   MeSH
• Deoxyribonucleases, Type II Site-Specific metabolism   MeSH
• Sequence Analysis, DNA MeSH
• Protein Binding MeSH
• Binding Sites MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• endodeoxyribonuclease BsmI MeSH Browser
• Receptors, Calcitriol MeSH
• Deoxyribonucleases, Type II Site-Specific MeSH

Reduced DNA repair capacity and DNA damage accumulation may lead to cancer development. Regulation of and coordination between genes involved in DNA repair pathways is fundamental for maintaining genome stability, and post-transcriptional gene regulation by microRNAs (miRNAs) may therefore be of particular relevance. In this context, the presence of single nucleotide polymorphisms (SNPs) within the 3'untranslated regions of target DNA repair genes could alter the binding with specific miRNAs, modulating gene expression and ultimately affecting cancer susceptibility. In this study, we investigated the role of genetic variations in miRNA-binding sites of nucleotide excision repair (NER) genes in association with colorectal cancer (CRC) risk. From 28 NER genes, we screened among SNPs residing in their 3'untranslated regions and simultaneously located in miRNA-binding sites, with an in silico approach. Through the calculation of different binding free energy according to both alleles of identified SNPs, and with global binding free energies median providing a threshold, we selected nine NER gene variants. We tested those SNPs in 1098 colorectal cancer cases and 1469 healthy controls from the Czech Republic. Rs7356 in RPA2 and rs4596 in GTF2H1 were associated with colorectal cancer risk. After stratification for tumor location, the association of both SNPs was significant only for rectal cancer (rs7356: OR 1.52, 95% CI 1.02-2.26, P = 0.04 and rs4596: OR 0.69, 95% CI 0.50-0.94, P = 0.02; results not adjusted for multiple testing). Variation in miRNA target binding sites in the 3'untranslated region of NER genes may be important for modulating colorectal cancer risk, with a different relevance according to tumor location.

• MeSH
• 3' Untranslated Regions MeSH
• Genetic Predisposition to Disease * MeSH
• Polymorphism, Single Nucleotide * MeSH
• Colorectal Neoplasms genetics   MeSH
• Humans MeSH
• DNA Repair genetics   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• 3' Untranslated Regions MeSH

During a survey on grapevine yellows disease complex in vineyards of Lombardy region (northern Italy), phytoplasmas associated with Flavescence dorée disease were identified in symptomatic grapevines. Polymerase chain reaction and restriction fragment length polymorphism (RFLP) analyses of 16S rDNA revealed the prevalence of phytoplasmal subgroup 16SrV-D. Bioinformatic analyses of nucleotide sequences of rplV and rpsC genes, amplified from 16SrV-D phytoplasma infected grapevines and cloned, underscored the presence of five confirmed rpsC single nucleotide polymorphism (SNP) lineages, determined by different combination of SNPs at nucleotide positions 29, 365, 680, and 720 of rpsC gene. Virtual and actual RFLP analyses with the enzyme TaqI validated the presence of these SNPs. Co-infections by up to four distinct rpsC SNP lineages of 16SrV-D phytoplasma were found in grapevines. These results could open new perspectives for the study of the ecology and the epidemiology of Flavescence dorée.

• MeSH
• Bacterial Proteins genetics   MeSH
• DNA, Bacterial chemistry   genetics   MeSH
• DNA Fingerprinting MeSH
• Phylogeny MeSH
• Polymorphism, Single Nucleotide * MeSH
• Molecular Sequence Data MeSH
• Plant Diseases microbiology   MeSH
• Phytoplasma classification   genetics   isolation & purification   MeSH
• Polymerase Chain Reaction MeSH
• Polymorphism, Restriction Fragment Length MeSH
• DNA, Ribosomal genetics   MeSH
• Ribosomal Proteins genetics   MeSH
• RNA, Ribosomal, 16S genetics   MeSH
• Sequence Analysis, DNA MeSH
• Sequence Homology MeSH
• Cluster Analysis MeSH
• Vitis microbiology   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Geographicals
• Italy MeSH
• Names of Substances
• Bacterial Proteins MeSH
• DNA, Bacterial MeSH
• DNA, Ribosomal MeSH
• Ribosomal Proteins MeSH
• RNA, Ribosomal, 16S MeSH

Estimated breeding values (EBV) for first-lactation milk production of Holstein cattle in the Czech Republic were calculated using a conventional animal model and by single-step prediction of the genomic enhanced breeding value. Two overlapping data sets of milk production data were evaluated: (1) calving years 1991 to 2006, with 861,429 lactations and 1,918,901 animals in the pedigree and (2) calving years 1991 to 2010, with 1,097,319 lactations and 1,906,576 animals in the pedigree. Global Interbull (Uppsala, Sweden) deregressed proofs of 114,189 bulls were used in the analyses. Reliabilities of Interbull values were equivalent to an average of 8.53 effective records, which were used in a weighted analysis. A total of 1,341 bulls were genotyped using the Illumina BovineSNP50 BeadChip V2 (Illumina Inc., San Diego, CA). Among the genotyped bulls were 332 young bulls with no daughters in the first data set but more than 50 daughters (88.41, on average) with performance records in the second data set. For young bulls, correlations of EBV and genomic enhanced breeding value before and after progeny testing, corresponding average expected reliabilities, and effective daughter contributions (EDC) were calculated. The reliability of prediction pedigree EBV of young bulls was 0.41, corresponding to EDC=10.6. Including Interbull deregressed proofs improved the reliability of prediction by EDC=13.4 and including genotyping improved prediction reliability by EDC=6.2. Total average expected reliability of prediction reached 0.67, corresponding to EDC=30.2. The combination of domestic and Interbull sources for both genotyped and nongenotyped animals is valuable for improving the accuracy of genetic prediction in small populations of dairy cattle.

• MeSH
• Breeding methods   statistics & numerical data   MeSH
• Genetic Markers genetics   MeSH
• Genomics methods   MeSH
• Polymorphism, Single Nucleotide genetics   MeSH
• Quantitative Trait, Heritable MeSH
• Lactation genetics   MeSH
• Dairying methods   statistics & numerical data   MeSH
• Pedigree MeSH
• Cattle genetics   physiology   MeSH
• Records veterinary   MeSH
• Animals MeSH
• Check Tag
• Male MeSH
• Cattle genetics   physiology   MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Genetic Markers MeSH

Graft-versus-host disease (GVHD) represents a significant cause of mortality after allogeneic hematopoietic stem cell transplantation (HSCT). NF-kB system is a master regulator of innate immunity responses. It controls the expression of various cytokines and chemokines many of which are involved in GVHD pathogenesis. Chemo(radio) therapy administered during conditioning induces DNA damage and activates DNA damage response (DDR) signaling resulting in irreversible cell cycle arrest - cellular senescence which has been described to be associated with robust pro-inflammatory secretion mostly controlled by NF-kB. The NFKB1 gene encodes the DNA-binding subunit of the NF-kB complex. Using the candidate gene approach, we analyzed possible association of two single-nucleotide polymorphisms (SNPs) rs3774937 C/T and rs3774959 A/G of the NFKB1 gene with GVHD and transplant-related mortality (TRM) occurrence in 109 recipients allografted from HLA-identical donor. Both SNPs in recipients were found to be strongly associated with acute GVHD. Nevertheless, no significant association with chronic GVHD and TRM was found. Presented pilot results contribute to pre-clinical observations and suggest that NF-kB may be an important regulator of HSCT-related inflammatory reactions such as acute GVHD. Novel pathogenic mechanisms of GVHD may arise from perspectives of DDR and cellular senescence where NF-kB plays an essential role.

• Keywords
• Allogeneic hematopoietic stem cell transplantation, Cellular senescence, Graft-versus-host disease, NFKB1 gene, Senescence-associated secretory phenotype, Single-nucleotide polymorphism,
• MeSH
• Allografts MeSH
• Adult MeSH
• Polymorphism, Single Nucleotide * MeSH
• Middle Aged MeSH
• Humans MeSH
• Survival Rate MeSH
• Graft vs Host Disease genetics   mortality   therapy   MeSH
• NF-kappa B p50 Subunit genetics   MeSH
• Pilot Projects MeSH
• Disease-Free Survival MeSH
• Hematopoietic Stem Cell Transplantation * MeSH
• Check Tag
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Male MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Observational Study MeSH
• Names of Substances
• NF-kappa B p50 Subunit MeSH
• NFKB1 protein, human MeSH Browser

CYP2D6 is a member of cytochrome P450 enzymes that metabolise over 25% of commonly used drugs. Genetic polymorphisms can cause insufficient drug efficacy at usually administered doses or can be the cause of adverse drug reaction. CYP2D6 genotyping can be used to predict CYP2D6 phenotype and thereby explain some abnormalities in drug response and thus optimize pharmacotherapy. The aim of this study was to investigate the frequency of functionally important variant alleles of the CYP2D6 gene throughout the Czech population to predict the prevalence of ultra-rapid and poor metabolizer phenotypes. The DNA of 223 unrelated, healthy volunteers was analysed to detect the presence of CYP2D6*6, *5, *4, *3 and gene duplication. The variant allele frequencies in our population were 0.22%, 3.14%, 22.87%, 1.12% and 3.14% for CYP2D6*6, CYP2D6*5, CYP2D6*4, CYP2D6*3 and CYP2D6*MxN, respectively. Fifteen subjects carried two variant alleles leading to predicted poor type of metabolism, 84 subjects were heterozygous extensive metabolizers (het-EM). The full-text contains detailed comparison with European white populations. The distribution of variant alleles complies with the Hardy-Weinberg equilibrium. The frequencies of functional variant alleles of CYP2D6 in Czech population are in concordance with other Caucasian populations.

• MeSH
• Cytochrome P-450 CYP2D6 genetics   physiology   MeSH
• Adult MeSH
• Pharmacogenetics MeSH
• Gene Frequency * MeSH
• Genotype MeSH
• Polymorphism, Single Nucleotide * MeSH
• Pharmaceutical Preparations metabolism   MeSH
• Middle Aged MeSH
• Humans MeSH
• Adolescent MeSH
• Genetics, Population MeSH
• Aged MeSH
• Check Tag
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Adolescent MeSH
• Male MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Geographicals
• Czech Republic MeSH
• Names of Substances
• Cytochrome P-450 CYP2D6 MeSH
• Pharmaceutical Preparations MeSH

BACKGROUND: The pharmacokinetics of tacrolimus (TAC) and mycophenolic acid (MPA) are highly variable. An impact of single-nucleotide polymorphisms (SNPs) of the genes coding for enzymes and transporters involved in the pharmacokinetics of TAC and/or MPA is intuitively conceivable. Accordingly, we sought to analyze the influence of different SNPs on TAC and MPA exposure in pediatric renal transplant recipients. METHODS: A subpopulation of 37 patients (median age: 12.8 years, range 2.2-18.3 years) participating in the TWIST study was included in the analysis of SNPs of CYP3A5, ABCB1 (MDR1), ABCG2, SLCO1B3 (coding for OATP2), ABCC2 (coding for cMOAT), and UGT1/2. TAC trough concentrations and abbreviated area under the concentration-time curves (AUC) of MPA were measured on days 7, 28, 91, and 183 after transplant. Both of these were adjusted to the respective dose the patient received. RESULTS: The allele frequencies of analyzed SNP's were comparable to those reported previously for white populations. Dose-adjusted trough concentrations of TAC were approximately 60% lower in patients with the CYP3A5*1/*3 allele as compared with the CYP3A5*3/*3 allele (P = 0.004). Steroid-free patients in CYP3A5*3/*3 and CYP3A5*1/*3 carrier subgroups had comparable dose-adjusted TAC concentrations to the subgroup on steroids (P = 0.13). Patients younger than 10 years had a significantly lower median dose-adjusted TAC C0 concentration than patients older than 10 years; this age effect was comparable in heterozygous and homozygous CYP3A5 carriers as well as in patients on and off steroid medication. As for MPA, the genetic variability of transporters or enzymes had no impact on dose-adjusted MPA-AUC due to the low allele frequencies. Patients off steroids had a higher dose-adjusted MPA-AUC (0.18 mg·h/L per mg/m, 0.012-0.27) compared with patients on steroids (0.12 mg·h·L·mg, 0.09-0.19; P = 0.04). CONCLUSIONS: Genetic variability of CYP3A5 has an impact on TAC metabolism in pediatric renal transplant recipients, contributing partly to the variability of TAC exposure. Therefore, adjusting initial TAC dosing to the genotype of CYP3A5 might be of clinical benefit.

• MeSH
• Alleles MeSH
• Time Factors MeSH
• Cytochrome P-450 CYP3A genetics   MeSH
• Child MeSH
• Pharmacogenetics MeSH
• Gene Frequency MeSH
• Genotype MeSH
• Immunosuppressive Agents administration & dosage   pharmacokinetics   MeSH
• Polymorphism, Single Nucleotide MeSH
• Mycophenolic Acid administration & dosage   pharmacokinetics   MeSH
• Humans MeSH
• Adolescent MeSH
• Area Under Curve MeSH
• Child, Preschool MeSH
• Multidrug Resistance-Associated Protein 2 MeSH
• Tacrolimus administration & dosage   pharmacokinetics   MeSH
• Kidney Transplantation methods   MeSH
• Dose-Response Relationship, Drug MeSH
• Check Tag
• Child MeSH
• Humans MeSH
• Adolescent MeSH
• Male MeSH
• Child, Preschool MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Multicenter Study MeSH
• Randomized Controlled Trial MeSH
• Comparative Study MeSH
• Names of Substances
• ABCC2 protein, human MeSH Browser
• CYP3A5 protein, human MeSH Browser
• Cytochrome P-450 CYP3A MeSH
• Immunosuppressive Agents MeSH
• Mycophenolic Acid MeSH
• Multidrug Resistance-Associated Protein 2 MeSH
• Tacrolimus MeSH

Hypertrophic cardiomyopathies (HCM) are the principal cause of sudden cardiac death in young athletes and it is estimated that 1 in 500 people have HCM. The aim of this work was to develop an electrochemical platform for the detection of HCM-associated SNP in the Myosin Heavy Chain 7 (MYH7) gene, in fingerprick blood samples. The platform exploits isothermal solid-phase primer elongation using recombinase polymerase amplification with either individual or a combination of four ferrocene-labelled nucleoside triphosphates. Four thiolated reverse primers containing a variable base at their 3' end were immobilised on individual gold electrodes of an array. Following hybridisation with target DNA, solid phase recombinase polymerase amplification was carried out and primer elongation incorporating the ferrocene labelled oligonucleotides was only detected at one of the electrodes, thus facilitating identification of the SNP under interrogation. The assay was applied to the direct detection of the SNP in fingerprick blood samples from eight different individuals, with the results obtained corroborating with next generation sequencing. The ability to be able to robustly identify the SNP using a 10 μL fingerprick sample, demonstrates that SNP discrimination is achieved using low femtomolar (ca. 8 × 105 copies DNA) levels of DNA.

• Keywords
• Biosensors, Electrochemical detection, Ferrocene-dNTPs, Fingerprick, SNP, Solid phase recombinase polymerase amplification,
• MeSH
• Biosensing Techniques * MeSH
• DNA genetics   MeSH
• Polymorphism, Single Nucleotide MeSH
• Humans MeSH
• Metallocenes MeSH
• Recombinases * genetics   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• DNA MeSH
• Metallocenes MeSH
• Recombinases * MeSH