Detection of DNA base variation and cytosine methylation at a single nucleotide site using a highly sensitive fluorescent probe

A fluorescent DNA probe containing an anthracene group attached via an anucleosidic linker can identify all four DNA bases at a single site as well as the epigenetic modification C/5-MeC via a hybridisation sensing assay.

Association of polymorphisms in the hepatocyte growth factor gene promoter with keratoconus

Purpose. Keratoconus is a progressive disorder of the cornea that can lead to severe visual impairment or blindness. Although several genomic regions have been linked to rare familial forms of keratoconus, no genes have yet been definitively identified for common forms of the disease. Methods. Two genome-wide association scans were undertaken in parallel. The first used pooled DNA from an Australian cohort, followed by typing of top-ranked single-nucleotide polymorphisms (SNPs) in individual DNA samples. The second was conducted in individually genotyped patients, and controls from the USA. Tag SNPs around the hepatocyte growth factor (HGF) gene were typed in three additional replication cohorts. Serum levels of HGF protein in normal individuals were assessed with ELISA and correlated with genotype. Results. The only SNP observed to be associated in both the pooled discovery and primary replication cohort was rs1014091, located upstream of the HGF gene. The nearby SNP rs3735520 was found to be associated in the individually typed discovery cohort (P = 6.1 × 10 ). Genotyping of tag SNPs around HGF revealed association at rs3735520 and rs17501108/rs1014091 in four of the five cohorts. Meta-analysis of all five datasets together yielded suggestive P values for rs3735520 (P = 9.9 × 10 ) and rs17501108 (P = 9.9 × 10 ). In addition, SNP rs3735520 was found to be associated with serum HGF level in normal individuals (P = 0.036). Conclusions. Taken together, these results implicate genetic variation at the HGF locus with keratoconus susceptibility. © 2011 The Association for Research in Vision and Ophthalmology, Inc.

Family-based association studies of lipid gene polymorphisms in coronary artery disease

Dysfunction of lipid-metabolizing proteins is implicated in the pathogenesis of coronary artery disease. Single nucleotide polymorphisms in genes that encode sterol regulatory binding protein-la, adenosine triphosphate binding cassette-A1, hepatic lipase, lipoprotein lipase, and cholesteryl ester transfer protein were assessed as potential markers of disease susceptibility in a family-based study of 1,012 patients from 386 families. Association between single nucleotide polymorphisms and coronary artery disease was tested by the combined transmission disequilibrium test/sib transmission disequilibrium test and pedigree disequilibrium test. After Bonferroni's correction, the pedigree disequilibrium test demonstrated significant excess transmission (p < 0.0083) to affected patients of the hepatic lipase -514 T allele, which suggests that this may constitute a novel disease-susceptibility locus. (c) 2005 Elsevier Inc. All rights reserved.

Caveolin-1 single nucleotide polymorphism in antineutrophil cytoplasmic antibody associated vasculitis

Immunosuppression is cornerstone treatment of antineutrophil cytoplasmic antibody associated vasculitis (AAV) but is later complicated by infection, cancer, cardiovascular and chronic kidney disease. Caveolin-1 is an essential structural protein for small cell membrane invaginations known as caveolae. Its functional role has been associated with these complications. For the first time, caveolin-1 (CAV1) gene variation is studied in AAV.

GWAS identifies an NAT2 acetylator status tag single nucleotide polymorphism to be a major locus for skin fluorescence

Skin fluorescence (SF) is a non-invasive marker of AGEs and is associated with the long-term complications of diabetes. SF increases with age and is also greater among individuals with diabetes. A familial correlation of SF suggests that genetics may play a role. We therefore performed parallel genome-wide association studies of SF in two cohorts.

High-throughput single-nucleotide polymorphism-based typing of shared Pseudomonas aeruginosa strains in cystic fibrosis patients using the Sequenom iPLEX platform

Shared strains of Pseudomonas aeruginosa are now well recognized in people with cystic fibrosis (CF), and suitable P. aeruginosa laboratory typing tools are pivotal to understanding their clinical significance and guiding infection control policies in CF clinics. We therefore compared a single-nucleotide polymorphism (SNP)-based typing method using Sequenom iPLEX matrix-assisted laser desorption ionization with time-of-flight mass spectrometry (MALDI-TOF MS) with typing methods used routinely by our laboratory. We analysed 617 P. aeruginosa isolates that included 561 isolates from CF patients collected between 2001 and 2009 in two Brisbane CF clinics and typed previously by enterobacterial repetitive intergenic consensus (ERIC)-PCR, as well as 56 isolates from non-CF patients analysed previously by multilocus sequence typing (MLST). The isolates were tested using a P. aeruginosa Sequenom iPLEX MALDI-TOF (PA iPLEX) method comprising two multiplex reactions, a 13-plex and an 8-plex, to characterize 20 SNPs from the P. aeruginosa housekeeping genes acsA, aroE, guaA, mutL, nuoD, ppsA and trpE. These 20 SNPs were employed previously in a real-time format involving 20 separate assays in our laboratory. The SNP analysis revealed 121 different SNP profiles for the 561 CF isolates. Overall, there was at least 96% agreement between the ERIC-PCR and SNP analyses for all predominant shared strains among patients attending our CF clinics: AUST-01, AUST-02 and AUST-06. For the less frequently encountered shared strain AUST-07, 6/25 (24%) ERIC-PCR profiles were misidentified initially as AUST-02 or as unique, illustrating the difficulty of gel-based analyses. SNP results for the 56 non-CF isolates were consistent with previous MLST data. Thus, the PA iPLEX format provides an attractive high-throughput alternative to ERIC-PCR for large-scale investigations of shared P. aeruginosa strains.

Rapid single-nucleotide polymorphism-based identification of clonal Pseudomonas aeruginosa isolates from patients with cystic fibrosis by the use of real-time PCR and high-resolution melting curve analysis

Pseudomonas aeruginosa genotyping relies mainly upon DNA fingerprinting methods, which can be subjective, expensive and time-consuming. The detection of at least three different clonal P. aeruginosa strains in patients attending two cystic fibrosis (CF) centres in a single Australian city prompted the design of a non-gel-based PCR method to enable clinical microbiology laboratories to readily identify these clonal strains. We designed a detection method utilizing heat-denatured P. aeruginosa isolates and a ten-single-nucleotide polymorphism (SNP) profile. Strain differences were detected by SYBR Green-based real-time PCR and high-resolution melting curve analysis (HRM10SNP assay). Overall, 106 P. aeruginosa sputum isolates collected from 74 patients with CF, as well as five reference strains, were analysed with the HRM10SNP assay, and the results were compared with those obtained by pulsed-field gel electrophoresis (PFGE). The HRM10SNP assay accurately identified all 45 isolates as members of one of the three major clonal strains characterized by PFGE in two Brisbane CF centres (Australian epidemic strain-1, Australian epidemic strain-2 and P42) from 61 other P. aeruginosa strains from Australian CF patients and two representative overseas epidemic strain isolates. The HRM10SNP method is simple, is relatively inexpensive and can be completed in <3 h. In our setting, it could be made easily available for clinical microbiology laboratories to screen for local P. aeruginosa strains and to guide infection control policies. Further studies are needed to determine whether the HRM10SNP assay can also be modified to detect additional clonal strains that are prevalent in other CF centres.

Polymorphisms near TBX5 and GDF7 are associated with increased risk for Barrett's esophagus

BACKGROUND & AIMS: Barrett's esophagus (BE) increases the risk of esophageal adenocarcinoma (EAC). We found the risk to be BE has been associated with single nucleotide polymorphisms (SNPs) on chromosome 6p21 (within the HLA region) and on 16q23, where the closest protein-coding gene is FOXF1. Subsequently, the Barrett's and Esophageal Adenocarcinoma Consortium (BEACON) identified risk loci for BE and esophageal adenocarcinoma near CRTC1 and BARX1, and within 100 kb of FOXP1. We aimed to identify further SNPs that increased BE risk and to validate previously reported associations.

METHODS: We performed a genome-wide association study (GWAS) to identify variants associated with BE and further analyzed promising variants identified by BEACON by genotyping 10,158 patients with BE and 21,062 controls.

RESULTS: We identified 2 SNPs not previously associated with BE: rs3072 (2p24.1; odds ratio [OR] = 1.14; 95% CI: 1.09-1.18; P = 1.8 × 10(-11)) and rs2701108 (12q24.21; OR = 0.90; 95% CI: 0.86-0.93; P = 7.5 × 10(-9)). The closest protein-coding genes were respectively GDF7 (rs3072), which encodes a ligand in the bone morphogenetic protein pathway, and TBX5 (rs2701108), which encodes a transcription factor that regulates esophageal and cardiac development. Our data also supported in BE cases 3 risk SNPs identified by BEACON (rs2687201, rs11789015, and rs10423674). Meta-analysis of all data identified another SNP associated with BE and esophageal adenocarcinoma: rs3784262, within ALDH1A2 (OR = 0.90; 95% CI: 0.87-0.93; P = 3.72 × 10(-9)).

CONCLUSIONS: We identified 2 loci associated with risk of BE and provided data to support a further locus. The genes we found to be associated with risk for BE encode transcription factors involved in thoracic, diaphragmatic, and esophageal development or proteins involved in the inflammatory response.

Plastin Polymorphisms Predict Gender- and Stage-Specific Colon Cancer Recurrence after Adjuvant Chemotherapy

Tumor recurrence after curative resection remains a major problem in patients with locally advanced colorectal cancer treated with adjuvant chemotherapy. Genetic single-nucleotide polymorphisms (SNP) may serve as useful molecular markers to predict clinical outcomes in these patients and identify targets for future drug development. Recent in vitro and in vivo studies have demonstrated that the plastin genes PLS3 and LCP1 are overexpressed in colon cancer cells and play an important role in tumor cell invasion, adhesion, and migration. Hence, we hypothesized that functional genetic variations of plastin may have direct effects on the progression and prognosis of locally advanced colorectal cancer. We tested whether functional tagging polymorphisms of PLS3 and LCP1 predict time to tumor recurrence (TTR) in 732 patients (training set, 234; validation set, 498) with stage II/III colorectal cancer. The PLS3 rs11342 and LCP1 rs4941543 polymorphisms were associated with a significantly increased risk for recurrence in the training set. PLS3 rs6643869 showed a consistent association with TTR in the training and validation set, when stratified by gender and tumor location. Female patients with the PLS3 rs6643869 AA genotype had the shortest median TTR compared with those with any G allele in the training set [1.7 vs. 9.4 years; HR, 2.84; 95% confidence interval (CI), 1.32-6.1; P = 0.005] and validation set (3.3 vs. 13.7 years; HR, 2.07; 95% CI, 1.09-3.91; P = 0.021). Our findings suggest that several SNPs of the PLS3 and LCP1 genes could serve as gender- and/or stage-specific molecular predictors of tumor recurrence in stage II/III patients with colorectal cancer as well as potential therapeutic targets.

IL1B and DEFB1 Polymorphisms Increase Susceptibility to Invasive Mold Infection After Solid-Organ Transplantation.

BACKGROUND: Single-nucleotide polymorphisms (SNPs) in immune genes have been associated with susceptibility to invasive mold infection (IMI) among hematopoietic stem cell but not solid-organ transplant (SOT) recipients. METHODS: Twenty-four SNPs from systematically selected genes were genotyped among 1101 SOT recipients (715 kidney transplant recipients, 190 liver transplant recipients, 102 lung transplant recipients, 79 heart transplant recipients, and 15 recipients of other transplants) from the Swiss Transplant Cohort Study. Association between SNPs and the end point were assessed by log-rank test and Cox regression models. Cytokine production upon Aspergillus stimulation was measured by enzyme-linked immunosorbent assay in peripheral blood mononuclear cells (PBMCs) from healthy volunteers and correlated with relevant genotypes. RESULTS: Mold colonization (n = 45) and proven/probable IMI (n = 26) were associated with polymorphisms in the genes encoding interleukin 1β (IL1B; rs16944; recessive mode, P = .001 for colonization and P = .00005 for IMI, by the log-rank test), interleukin 1 receptor antagonist (IL1RN; rs419598; P = .01 and P = .02, respectively), and β-defensin 1 (DEFB1; rs1800972; P = .001 and P = .0002, respectively). The associations with IL1B and DEFB1 remained significant in a multivariate regression model (P = .002 for IL1B rs16944; P = .01 for DEFB1 rs1800972). The presence of 2 copies of the rare allele of rs16944 or rs419598 was associated with reduced Aspergillus-induced interleukin 1β and tumor necrosis factor α secretion by PBMCs. CONCLUSIONS: Functional polymorphisms in IL1B and DEFB1 influence susceptibility to mold infection in SOT recipients. This observation may contribute to individual risk stratification.

The expected performance of single nucleotide polymorphism loci in paternity testing

We discuss the utility of single nucleotide polymorphism loci for full trio and mother-unavailable paternity testing cases, in the presence of population substructure and relatedness of putative and actual fathers. We focus primarily on the expected number of loci required to gain specified probabilities of mismatches, and report the expected proportion of paternity indices greater than three threshold values for these loci. (c) 2004 Elsevier Ireland Ltd. All rights reserved.

Evidence for associations between common polymorphisms of estrogen receptor beta gene with homocysteine and nitric oxide

Background Homocysteine and asymmetric dimethylarginine (ADMA) affect nitric oxide (NO) concentration, thereby contributing to cardiovascular disease (CVD). Both amino acids can be reduced in vivo by estrogen. Variation in the estrogen receptor (ER) may influence homocysteine and ADMA, yet no information is available on associations with single nucleotide polymorphisms in the estrogen receptor genes ER alpha (PvuII and XbaI) and ER beta (1730G -> A and cx+56 G -> A). Objective To find relationships between common polymorphisms associated with cardiovascular disease and cardiovascular risk factors homocysteine and ADMA. Methods In a cross-sectional study with healthy postmenopausal women (n = 89), homocysteine, ADMA, nitric oxide metabolites (NOx), plasma folate and ER alpha and beta polymorphisms ER alpha PvuII, ER alpha XbaI; ER beta 1730G -> A (AluI), ER beta cx+56 G -> A (Tsp5091) were analyzed. Results Women who are homozygotic for ER beta cx+56 G -> A A/A exhibited higher homocysteine (p = 0.012) and NOx (p = 0.056) levels than wildtype or heterozygotes. NOx concentration was also significantly affected by ER beta 1730 G -> A polymorphism (p = 0.025). The ER beta (p < 0.001) and ER alpha (p < 0.001) polymorphisms were in linkage disequilibrium. Conclusions Women who are homozygotic for ER beta cx+S6 G -> A A/A may be at increased risk for cardiovascular disease due to higher homocysteine levels.

Anticoagulant resistance in Norway rats (Rattus norvegicus Berk.) in Kent - a VKORC1 single nucleotide polymorphism, tyrosine139phenylalanine, new to the UK

A sample of 10 Norway rats (Rattus norvegicus) was taken for DNA resistance testing from an agricultural site in Kent where applications of the anticoagulant rodenticide bromadiolone had been unsuccessful. All animals tested were homozygous for the single nucleotide VKORC1 polymorphism tyrosine139phenylalanine, or Y139F. This is a common resistance mutation found extensively in France and Belgium but not previously in the UK. Y139F confers a significant level of resistance to first-generation anticoagulants, such as chlorophacinone, and to the second-generation compound bromadiolone. Another compound widely used in the UK, difenacoum, is also thought to be partially resisted by rats which carry Y139F. A silent VKORC1 mutation was also found in all rats tested. The presence of a third important VKORC1 mutation which confers resistance to anticoagulant rodenticides in widespread use in the UK, the others being Y139C and L120Q, further threatens the ability of pest control practitioners to deliver effective rodent control.

Neurotrophic gene polymorphisms and response to psychological therapy

Therapygenetics, the study of genetic determinants of response to psychological therapies, is in its infancy. Here, we investigate whether single-nucleotide polymorphisms in nerve growth factor (NGF) (rs6330) and brain-derived neutrotrophic factor (BDNF) (rs6265) genes predict the response to cognitive behaviour therapy (CBT). Neurotrophic genes represent plausible candidate genes: they are implicated in synaptic plasticity, response to stress, and are widely expressed in brain areas involved in mood and cognition. Allelic variation at both loci has shown associations with anxiety-related phenotypes. A sample of 374 anxiety-disordered children with white European ancestry was recruited from clinics in Reading, UK, and in Sydney, Australia. Participants received manualised CBT treatment and DNA was collected from buccal cells using cheek swabs. Treatment response was assessed at post-treatment and follow-up time points. We report first evidence that children with one or more copies of the T allele of NGF rs6330 were significantly more likely to be free of their primary anxiety diagnosis at follow-up (OR=0.60 (0.42–0.85), P=0.005). These effects remained even when other clinically relevant covariates were accounted for (OR=0.62 (0.41–0.92), P=0.019). No significant associations were observed between BDNF rs6265 and response to psychological therapy. These findings demonstrate that knowledge of genetic markers has the potential to inform clinical treatment decisions for psychotherapeutic interventions.