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Nurse Practitioners↗
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Melting curve analysis of SNPs (McSNP): a gel-free and inexpensive approach for SNP genotyping.
High-throughput methods for assaying DNA variation require two important steps: (i) discriminating the variation and (ii) detecting the signal. In this report, we describe a novel SNP genotyping method that we refer to as melting curve analysis of SNPs (McSNP). McSNP combines a classic approach for discriminating alleles, restriction enzyme digestion, with a more recent method for detecting DNA fragments, melting curve analysis. Melting curve analysis is performed by slowly heating DNA fragments in the presence of the dsDNA-specific fluorescent dye SYBR Green I. As the sample is heated, fluorescence rapidly decreases when the melting temperature of a particular fragment is reached. We show that it is possible to determine the composition of simple mixtures of DNA fragments, such as those that result from restriction enzyme digestions of short PCR products. McSNP is well suited for high-throughput genotyping because 96 samples can be analyzed and automatically scored in 20 min. Our results clearly demonstrate that McSNP is a simple, inexpensive, and accurate means of genotyping SNP variation.
Genotype↗
Selection at linked sites in the partial selfer Caenorhabditis elegans.
Natural selection can produce a correlation between local recombination rates and levels of neutral DNA polymorphism as a consequence of genetic hitchhiking and background selection. Theory suggests that selection at linked sites should affect patterns of neutral variation in partially selfing populations more dramatically than in outcrossing populations. However, empirical investigations of selection at linked sites have focused primarily on outcrossing species. To assess the potential role of selection as a determinant of neutral polymorphism in the context of partial self-fertilization, we conducted a multivariate analysis of single-nucleotide polymorphism (SNP) density throughout the genome of the nematode Caenorhabditis elegans. We based the analysis on a published SNP data set and partitioned the genome into windows to calculate SNP densities, recombination rates, and gene densities across all six chromosomes. Our analyses identify a strong, positive correlation between recombination rate and neutral polymorphism (as estimated by noncoding SNP density) across the genome of C. elegans. Furthermore, we find that levels of neutral polymorphism are lower in gene-dense regions than in gene-poor regions in some analyses. Analyses incorporating local estimates of divergence between C. elegans and C. briggsae indicate that a mutational explanation alone is unlikely to explain the observed patterns. Consequently, we interpret these findings as evidence that natural selection shapes genome-wide patterns of neutral polymorphism in C. elegans. Our study provides the first demonstration of such an effect in a partially selfing animal. Explicit models of genetic hitchhiking and background selection can each adequately describe the relationship between recombination rate and SNP density, but only when they incorporate selfing rate. Clarification of the relative roles of genetic hitchhiking and background selection in C. elegans awaits the development of specific theoretical predictions that account for partial self-fertilization and biased sex ratios.
Animals↗
Assessment of positional candidate genes myf5 and igf1 for growth on bovine chromosome 5 in commercial lines of Bos taurus.
Quantitative trait loci for growth traits in beef cattle have been previously reported and fine-mapped in three chromosomal regions of 0 to 30 cM, 55 to 70 cM, and 70 to 80 cM of bovine chromosome 5. In this study, we further examined the association between gene-specific single nucleotide polymorphisms (SNP) of two positional candidate genes, bovine myogenic factor 5 (myf5) and insulin-like growth factor-1 (igf1), in the QTL regions and the birth weight (BWT), preweaning average daily gain (PWADG), and average daily gain on feed (ADGF) in commercial lines of Bos taurus. The QTL regions for the growth traits identified using a haplotype association analysis, which included the gene-specific SNP markers for both genes in this study, were in agreement with previous studies. The gene-specific SNP marker association analysis indicated that the SNP in myf5 had a significant additive effect on PWADG in the M1 line of Beefbooster Inc. (P < 0.10), and a significant additive effect (P < 0.05) and a significant dominance effect (P < 0.10) on ADGF in the M3 line of Beefbooster Inc. When the data from the two commercial lines were pooled, the SNP in myf5 showed a significant association with PWADG (P < 0.10) and with ADGF (P < 0.05). The association between the SNP and BWT, however, did not reach a significance level in the M1 line, the M3 line, or across the lines. For igf1, no significant association between the SNP and the growth traits was detected in either the M1 line or the M3 line, whereas there was only a significant dominance effect (P < 0.10) on BWT detected for the SNP in igfl when the data from the two commercial lines were pooled. These results suggest that myf5 is a strong candidate gene that influences PWADG and ADGF in beef cattle. The SNP of igf1 may not be a causative or close to the causative mutation that affects the three growth traits in the populations of beef cattle examined in this study. Other SNP of igf1 and myf5 or other genes in their respective chromosomal regions, however, should also be studied.
Alleles↗
Association of Phosphodiesterase 4D with ischemic stroke: a population-based case-control study.
BACKGROUND AND PURPOSE: The Phosphodiesterase 4D (PDE4D) gene was reported recently to be associated with ischemic stroke in an Icelandic population. The association was found predominately with large vessel and cardioembolic stroke. However, 2 recent reports were unable to confirm this association, although a trend toward association with cardioembolic stroke was reported. None of the reports included significant proportions of blacks. We tested for genotype and haplotype association of polymorphisms of the PDE4D gene with ischemic stroke in a population-based, biracial, case-control study. METHODS: A total of 357 cases of ischemic stroke and 482 stroke-free controls from the same community were examined. Single nucleotide polymorphisms (SNPs) were chosen based on significant associations reported previously. Linkage disequilibrium (LD), SNP, and haplotype association analysis was performed using PHASE 2.0 and Haploview 3.2. RESULTS: Although several univariate associations were identified, only 1 SNP (rs2910829) was found to be significantly associated with cardioembolic stroke among both whites and blacks. The rs152312 SNP was associated with cardioembolic stroke among whites after multiple comparison corrections. The same SNP was not associated with cardioembolic stroke among blacks. However, significant haplotype association was identified for both whites and blacks for all ischemic stroke, cardioembolic stroke, and stroke of unknown origin. Haplotype association was identified for small vessel stroke among whites. CONCLUSIONS: PDE4D is a risk factor for ischemic stroke and, in particular, for cardioembolic stroke, among whites and blacks. Further study of this gene is warranted.
3',5'-Cyclic-AMP Phosphodiesterases↗
A novel procedure for efficient genotyping of single nucleotide polymorphisms.
Due to the surge in interest in using single nucleotide polymorphisms (SNPs) for genotyping a facile and affordable method for this is an absolute necessity. Here we introduce a procedure that combines an easily automatable single tube sample preparation with an efficient high throughput mass spectrometric analysis technique. Known point mutations or single nucleotide polymorphisms are easily analysed by this procedure. It starts with PCR amplification of a short stretch of genomic DNA, for example an exon of a gene containing a SNP. By shrimp alkaline phosphatase digest residual dNTPs are destroyed. Allele-specific products are generated using a special primer, a conditioned set of alpha-S-dNTPs and alpha-S-ddNTPs and a fresh DNA polymerase in a primer extension reaction. Unmodified DNA is removed by 5'-phospho-diesterase digestion and the modified products are alkylated to increase the detection sensitivity in the mass spectrometric analysis. All steps of the preparation are simple additions of solutions and incubations. The procedure operates at the lowest practical sample volumes and in contrast to other genotyping protocols with mass spectrometric detection requires no purification. This reduces the cost and makes it easy to implement. Here it is demonstrated in a version using positive ion detection on described mutations in exon 17 of the amyloid precursor protein gene and in a version using negative ion detection on three SNPs of the granulocyte-macrophage colony stimulating factor gene. Preparation and analysis of SNPs is shown separately and simultaneously, thus demonstrating the multiplexibility of this genotyping procedure. The preparation protocol for genotyping is adapted to the conditions used for the SNP discovery method by denaturing HPLC, thus demonstrating a facile link between protocols for SNP discovery and SNP genotyping. Results corresponded unanimously with the control sequencing. The procedure is useful for high throughput genotyping as it is required for gene identification and pharmacogenomics where large numbers of DNA samples have to be analysed. We have named this procedure the 'GOOD Assay' for SNP analysis.
Amyloid beta-Protein Precursor↗
Automatic scoring and quality assessment using accuracy bounds for FP-TDI SNP genotyping data.
BACKGROUND: Human diversity, namely single nucleotide polymorphisms (SNPs), is becoming a focus of biomedical research. Despite the binary nature of SNP determination, the majority of genotyping assay data need a critical evaluation for genotype calling. We applied statistical models to improve the automated analysis of 2-dimensional SNP data. METHODS: We derived several quantities in the framework of Gaussian mixture models that provide figures of merit to objectively measure the data quality. The accuracy of individual observations is scored as the probability of belonging to a certain genotype cluster, while the assay quality is measured by the overlap between the genotype clusters. RESULTS: The approach was extensively tested with a dataset of 438 nonredundant SNP assays comprising >150,000 datapoints. The performance of our automatic scoring method was compared with manual assignments. The agreement for the overall assay quality is remarkably good, and individual observations were scored differently by man and machine in 2.6% of cases, when applying stringent probability threshold values. CONCLUSION: Our definition of bounds for the accuracy for complete assays in terms of misclassification probabilities goes beyond other proposed analysis methods. We expect the scoring method to minimise human intervention and provide a more objective error estimate in genotype calling.
Algorithms↗
[Effects of exogenous nitric oxide donor on germination and activities of hydrolytic enzymes in wheat seed under osmotic stress].
Results in this paper showed that the effect of nitric oxide (NO) donor, sodium nitroprusside (SNP), which could promote the germination of wheat seeds under osmotic stress, was through enhancing imbibition using water content determination and imbibition analysis of wheat seed. SNP could also enhance amylase isozyme I activities, accelerate the liquefaction of endosperms, while have no effect on esterase. Moreover, SNP could strongly increase the sugars, such as the fructose, glucose and sucrose contents in wheat seeds during early germination (12 h) under normal conditions. When the seeds were treated with SNP with or without PTIO, an NO scavenger, and different exogenous sugars, i.e. fructose, glucose and sucrose, it was found that these sugars might activate amylase isozyme I activity mediated by the signal molecule NO to start the early stage of germination.
Amylases↗
Nitric oxide induces stomatal closure and enhances the adaptive plant responses against drought stress.
Nitric oxide (NO) is a very active molecule involved in many and diverse biological pathways where it has proved to be protective against damages provoked by oxidative stress conditions. In this work, we studied the effect of two NO donors, sodium nitroprusside (SNP) and S-nitroso-N-acetylpenicillamine SNP-treated on the response of wheat (Triticum aestivum) to water stress conditions. After 2 and 3 h of drought, detached wheat leaves pretreated with 150 microM SNP retained up to 15% more water than those pretreated with water or NO(2)(-)/NO(3)(-). The effect of SNP treatment on water retention was also found in wheat seedlings after 7 d of drought. These results were consistent with a 20% decrease in the transpiration rate of SNP-treated detached wheat leaves for the same analyzed time. In parallel experiments, NO was also able to induce a 35%, 30%, and 65% of stomatal closure in three different species, Tradescantia sp. (monocotyledonous) and two dicotyledonous, Salpichroa organifolia and fava bean (Vicia faba), respectively. In SNP-treated leaves of Tradescantia sp., the stomatal closure was correlated with a 10% increase on RWC. Ion leakage, a cell injury index, was 25% lower in SNP-treated wheat leaves compared with control ones after the recovery period. Carboxy-PTIO (2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide), a specific NO scavenger, reverted SNP action by restoring the transpiration rate, stomatal aperture, and the ion leakage to the level found in untreated leaves. Northern-blot analysis showed that SNP-treated wheat leaves display a 2-fold accumulation of a group three late embryogenesis abundant transcript with respect to control leaves both after 2 and 4 h of drought periods. All together, these results suggest that the exogenous application of NO donors might confer an increased tolerance to severe drought stress conditions in plants.
Adaptation, Physiological↗
A novel T-77C polymorphism in DNA repair gene XRCC1 contributes to diminished promoter activity and increased risk of non-small cell lung cancer.
X-ray repair cross-complementing 1 (XRCC1) plays a key role in DNA base excision repair and cells lacking its activity are hypersensitive to DNA damage. Recently, we reported a SNP (rs3213245, -77T>C) in the XRCC1 gene 5' untranslated region (UTR) was significantly associated with the risk of developing esophageal squamous-cell carcinoma. Computer analysis predicted that this SNP was in the core of Sp1-binding motif, which suggested its functional significance. Gel shift and super shift assays confirmed that -77T>C polymorphic site in the XRCC1 promoter was within the Sp1-binding motif and the T>C substitution greatly enhanced the binding affinity of Sp1 to this region. Luciferase assays indicated that the Sp1-high-affinity C-allelic XRCC1 promoter was associated with a reduced transcriptional activity. The association between -77T>C and three other amino-acid substitution-causing polymorphisms in XRCC1 and risk of lung cancer was examined in 1024 patients and 1118 controls and the results showed that only the -77T>C polymorphism was significantly associated with an increased risk of developing lung cancer. Multivariate logistic regression analysis found that an increased risk of lung cancer was associated with the variant XRCC1 -77 genotypes (TC and CC) compared with the TT genotype (OR=1.46, 95% CI=1.18-1.82; P=0.001) and the increased risk was more pronounced in smokers (OR=1.63, 95% CI=1.20-2.21) than in non-smokers (OR=1.28, 95% CI=0.94-1.76). Taken together, these results showed that the functional SNP -77T>C in XRCC1 5'UTR was associated with cancer development owing to the decreased transcriptional activity of C-allele-containing promoter with higher affinity to Sp1 binding.
Adult↗
A synonymous SNP of the corneodesmosin gene leads to increased mRNA stability and demonstrates association with psoriasis across diverse ethnic groups.
Psoriasis is a chronic skin disorder with multifactorial aetiology. Genome-wide scans have provided unambiguous evidence for a major disease susceptibility locus on chromosome 6p21 (PSORS1). A minimal PSORS1 interval has been defined which encompasses three genes (HLA-C, HCR and CDSN) carrying psoriasis-associated SNPs. On the basis of this genetic evidence, we have undertaken an assessment of CDSN allele functional impact. A comparison of CDSN intragenic haplotypes showed that SNPs exclusive to disease-associated chromosomes are located in regions implicated in the stabilization of RNA transcripts. As CDSN is over-expressed in psoriatic lesions, we hypothesised that disease-associated intragenic SNPs may alter the rate of its mRNA decay. Here, we demonstrate that mRNAs transcribed from a CDSN risk haplotype present a 2-fold increase in stability, compared with those transcribed from a neutral haplotype (t-test P=0.004). Site-directed mutagenesis revealed that a single synonymous SNP (CDSN*971T) accounts for the observed increase in RNA stability. CDSN*971T maps to a RNA stability motif and UV cross-linking analysis demonstrated that the SNP affects the transcript affinity for a 39 kDa RNA binding protein. Association analyses show that haplotypes bearing CDSN*971T confer psoriasis susceptibility in a wide range of ethnic groups. These results demonstrate the effect of synonymous variation upon allele specific gene expression, a finding of relevance to future studies of the pathogenesis of common and complex traits.
Animals↗
Analysis of the factor V Leiden mutation using the READIT Assay.
BACKGROUND: A variety of methods exist for the detection of single-nucleotide polymorphisms (SNPs) present in amplified segments of genomic DNA. We show the application of a novel SNP scoring tool for analysis of the factor V Leiden mutation. METHODS AND RESULTS: We have developed a novel method for analyzing SNPs. The luciferase-based technique, known as the READIT Technology (Promega Corp, Madison, WI), was used to analyze 510 residual human samples sent for factor V Leiden testing from three independent testing laboratories. A blinded retrospective analysis of the factor V Leiden mutation was used to determine the accuracy and throughput capabilities of the technology. One hundred percent concordance was observed between the READIT Assay and genotype assignments made in the testing laboratories. In addition, greater than 6 SDs of separation were observed between the means of wild-type and heterozygote sample populations. Repetitive sample measurements with representative wild-type, heterozygote, and mutant samples showed that greater than 9 SDs separated the means of heterozygote and homozygote sample populations. Confidence intervals based on the means of wild-type, heterozygote, and mutant sample populations were determined. CONCLUSION: Perfect concordance using the READIT Assay showed its effectiveness as a SNP scoring tool. The design of the factor V READIT Assay was straightforward, requiring the design of two unmodified oligonucleotides that differ at the 3' penultimate position to form perfect hybrids with the wild-type or Leiden form of the factor V sequence. The use of previously published amplification primers and conditions minimized the time needed to optimize and validate the assay. The READIT Calculator supplied with the assay allowed automated genotype assignments and statistical analysis from the READIT Assay data. Confidence-interval analysis validated the ability to distinguish between wild-type, heterozygote, and mutant samples using the READIT Assay.
DNA↗
[Susceptibility genes for the development of autoimmune thyroid disease].
Autoimmune thyroid disease (AITD) is caused by an immune response to self thyroid antigens. Twin studies and familial aggregation indicated that AITD is a complex disease with genetic factors and major histocompatibility complex is the most clearly established genetic factor. We confirmed the association of the co-receptor CTLA4 with AITD. We have identified the association of the SNP in the Fc receptor-like 3 with several autoimmune diseases, including AITD, SLE and rheumatoid arthritis, which SNP affected the level of autoantibody production. Furthermore, we have identified ZFAT as a susceptibility gene in 8q24 for AITD by whole-genome linkage analysis and further SNP-based dense mapping. The associated SNP in ZFAT affected the antisense transcript of ZFAT, which in turn affected the ZFAT expression.
Antigens, CD↗
Unravelling genetic data by arrayed primer extension.
We have developed a method for arrayed primer extension (APEX) on an oligonucleotide microchip together with the 4-color fluoresence imaging equipment and supporting software, that allows analysis of the DNA sequence and changes in it. Mutation analysis of BRCA1 gene and single nucleotide polymorphism (SNP) chip for genotyping were used as a model system. Chip surface chemistry, template preparation and APEX reaction conditions were optimised and the assay is ready to be implemented in variety of DNA analysis from SNP testing to DNA resequencing.
Alleles↗
Fluorescent microsphere-based readout technology for multiplexed human single nucleotide polymorphism analysis and bacterial identification.
Large-scale human genotyping requires technologies with a minimal number of steps, high accuracy, and the ability to automate at a reasonable cost. In this regard, we have developed a rapid, cost-effective readout method for single nucleotide polymorphism (SNP) genotyping that combines an easily automatable single-tube allele-specific primer extension (ASPE) with an efficient high throughput flow cytometric analysis performed on a Luminex 100 flow cytometer. This robust technique employs an ASPE reaction using PCR-derived target DNA containing the SNP and a pair of synthetic complementary capture probes that differ at their 3' end-nucleotide defining the alleles. Each capture probe has been synthesized to contain a unique 25-nucleotide identifying sequence (ZipCode) at its 5' end. An array of fluorescent microspheres, covalently coupled with complementary ZipCode sequences (cZipCodes), was hybridized to biotin-labeled ASPE reaction products, sequestering them for flow cytometric analysis. ASPE offers both an advantage of streamlining the SNP analysis protocol and an ability to perform multiplex SNP analysis on any mixture of allelic variants. All steps of the assay are simple additions of the solutions, incubations, and washes. This technique was used to assay 15 multiplexed SNPs on human chromosome 12 from 96 patients. Comparison of the microsphere-based ASPE assay results to gel-based oligonucleotide ligation assay (OLA) results showed 99.2% agreement in genotype assignments. In addition, the microsphere-based multiplex SNPs assay system was adapted for the identification of bacterial samples by both ASPE and single base chain extension (SBCE) assays. A series of probes designed for different variable sites of bacterial 16S rDNA permitted multiplex analysis and generated species- or genus-specific patterns. Seventeen bacterial species representing a broad range of gram-negative and gram-positive bacteria were analyzed within 16 variable sites of 16S rDNA sequence. The results were consistent with the published sequences and confirmed by direct DNA sequencing.
Alleles↗
SNP identification in unamplified human genomic DNA with gold nanoparticle probes.
Single nucleotide polymorphisms (SNPs) comprise the most abundant source of genetic variation in the human genome. SNPs may be linked to genetic predispositions, frank disorders or adverse drug responses, or they may serve as genetic markers in linkage disequilibrium analysis. Thus far, established SNP detection techniques have utilized enzymes to meet the sensitivity and specificity requirements needed to overcome the high complexity of the human genome. Herein, we present for the first time a microarray-based method that allows multiplex SNP genotyping in total human genomic DNA without the need for target amplification or complexity reduction. This direct SNP genotyping methodology requires no enzymes and relies on the high sensitivity of the gold nanoparticle probes. Specificity is derived from two sequential oligonucleotide hybridizations to the target by allele-specific surface-immobilized capture probes and gene-specific oligonucleotide-functionalized gold nanoparticle probes. Reproducible multiplex SNP detection is demonstrated with unamplified human genomic DNA samples representing all possible genotypes for three genes involved in thrombotic disorders. The assay format is simple, rapid and robust pointing to its suitability for multiplex SNP profiling at the 'point of care'.
Genome, Human↗
Use of an electrochemically labeled nucleotide terminator for known point mutation analysis.
A novel single nucleotide polymorphism (SNP) assay utilizing an electrochemically tagged chain terminator is described. The system employs the single-base extension (SBE) technique coupled to capillary gel electrophoresis with end-column electrochemical detection. A redox-labeled chain terminator, ferrocene-acycloATP, is used in the SBE reaction. When the mutation site corresponds to the labeled chain terminator, the extension product is rendered electroactive. The reaction mixture is subsequently separated by capillary gel electrophoresis and the extension product detected at the separation anode with sinusoidal voltammetry. This work demonstrates the first known SNP assay utilizing redox-active chain terminators coupled to electrochemical detection. The methodology presented could lead to a fast, simple, and cost-effective SNP scoring system.
Adenosine Triphosphate↗
Association of the calpain-10 gene with type 2 diabetes in Europeans: results of pooled and meta-analyses.
We conducted pooled and meta-analyses of the association of the calpain-10 gene (CAPN10) polymorphisms SNP-43, Indel-19 and SNP-63 individually and as haplotypes with type 2 diabetes (T2D) in 3237 patients and 2935 controls of European ancestry. In the pooled analyses, the common SNP-43*G allele was associated with modest but statistically significant increased risk of T2D (odds ratio (OR)=1.11 (95% confidence interval (CI), 1.02-1.20), P=0.01). Two haplotype combinations were associated with increased risk of T2D (1-2-1/1-2-1, OR=1.20 (1.03-1.41), P=0.02; and 1-1-2/1-2-1, OR=1.26 (1.01-1.59), P=0.04) and one with decreased risk (1-1-1/2-2-1, OR=0.86 (0.75-0.99), P=0.03). The meta-analysis also showed a significant effect of the 1-2-1/1-2-1 haplogenotype on risk (OR=1.25 (1.05-1.50), P=0.01). However, there was evidence for heterogeneity with respect to this effect (P=0.06). The heterogeneity appeared to be due to data sets in which the cases were selected from samples used in linkage studies of T2D. Using only the population-based case-control samples removed the heterogeneity (P=0.89) and strengthened the evidence for association with T2D in both the pooled (SNP-43*G, OR=1.19 (1.07-1.32), P=0.001; 1-2-1/1-2-1 haplogenotype, OR=1.46 (1.19-1.78), P=0.0003; 1-1-2/1-2-1 haplogenotype, OR=1.52 (1.12-2.06), P=0.007; and 1-1-1/2-2-1 haplogenotype, OR=0.83 (0.70-0.99), P=0.03) and the meta-analysis (SNP-43*G, OR=1.18 (1.05-1.32), P=0.005; 1-2-1/1-2-1 haplogenotype, OR=1.68 (1.33-2.11), P=0.00001). The pooled and meta-analyses as well as the linkage disequilibrium and haplotype diversity studies suggest a role for genetic variation in CAPN10 affecting risk of T2D in Europeans.
Calpain↗