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PubMed2003

Detection of single nucleotide polymorphism (SNP) controlling the waxy character in wheat by using a derived cleaved amplified polymorphic sequence (dCAPS) marker.

We investigated a single nucleotide polymorphism (SNP) in the Wx-D1 gene, which was found in a mutant waxy wheat, and which expressed the Wx-D1 protein (granule-bound starch synthase I) as shown by immunoblot analysis. We also assayed starch synthase activity of granule-bound proteins. Using 22 doubled-haploid (DH) lines and 172 F(5) lines derived from the wild type x the mutant, we detected SNP via a PCR-based (dCAPS) marker. Amplified PCR products from Wx-D1 gene-specific primers, followed by mismatched primers designed for dCAPS analysis, were digested with the appropriate restriction enzyme. The two alleles, and the heterozygote genotype were easily and rapidly discriminated by gel-electrophoresis resolution to reveal SNP. All progeny lines that have the SNP of the mutant allele were waxy. Integrating the results of dCAPS analysis, immunoblot analysis and assays of starch synthase activity of granule-bound proteins indicates that the SNP in the Wx-D1 gene was responsible for its waxy character. This dCAPS marker is therefore useful as a marker to introduce the mutant allele into elite breeding lines.

Alleles↗

PubMedDate not supplied

C to A single nucleotide polymorphism in intron 18 of the human MST1R (RON) gene that maps at 3p21.3.

The MST1R (RON) gene, that maps at 3p21.3, encodes a protein tyrosine kinase receptor comprised of an extra-cellular domain that contains the ligand binding pocket and an intracellular region where the kinase domain is located. It controls cell survival and motility programs related to invasive growth. With the single strand conformation polymorphism (SSCP) method, a C to A nucleotide polymorphism (SNP) was found in intron 18 of the gene. The SNP has a frequency of 0.28 among African-American, 0.25 among Caucasian CEPH and 0.09 among Asian healthy individuals. During these studies, an alternatively spliced cDNA of MST1R, lacking exon 19, was also found that may result from this change.

Alternative Splicing↗

PubMed2002

TNF-alpha promoter single nucleotide polymorphisms are markers of human ancestry.

We present a map of single nucleotide polymorphisms (SNPs) in the human tumor necrosis factor (TNF)-alpha promoter based upon exploratory sequencing of 333 human TNF-alpha gene promoters from individuals of distinct ancestral backgrounds. We detect 10 TNF-alpha promoter SNPs that occur with distinct frequencies in populations of different ancestry. Consistent with these findings, we show that two TNF-alpha SNPs, the -243 SNP and the -856 SNP, are the first SNP markers of a sub-Saharan African-derived extended haplotype and an Amerindian HLA haplotype, respectively. Comparisons of TNF-alpha promoter SNP allele frequencies can thus help elucidate variation of HLA haplotypes and their distribution among existing ethnic groups and shed light into the history of human populations.

Evolution, Molecular↗

PubMed2000

Linkage disequilibrium mapping using single nucleotide polymorphisms--which population?

There is considerable interest in the potential of single nucleotide polymorphisms (SNPs) for mapping complex traits which are determined by genes of small individual effect (oligogenes). It is thought likely that many oligogenes are themselves common polymorphisms, perhaps biallelic, for which there is effectively neutral selection reflected in late age of onset. The extent of detectable linkage disequilibrium between SNP x SNP pairs and SNP x oligogene pairs is of considerable interest, particularly in the context of identifying 'favourable' populations. Unfortunately data are sparse and few populations have been extensively sampled. Polymorphisms with the appropriate characteristics that have been studied are blood groups in the Rhesus and MNS systems for which there are extensive data on four pairs of biallelics. These might be regarded as surrogates for SNP-SNP or SNP-oligogene pairs. By developing and applying an approach, previously used for major genes, to evaluate association (rho) in SNP haplotypes, it is evident that, with some exceptions, there is little difference between isolates and large populations. Furthermore it is apparent that there is useful linkage disequilibrium even for the MN-Ss locus pair (0.195 cM apart), in both large populations and isolates. This is somewhat more favourable to linkage disequilibrium mapping than a recent simulation suggests.

Alleles↗

PubMed2000

Full flexibility genotyping of single nucleotide polymorphisms by the GOOD assay.

Recently a facile method for genotyping single nucleotide polymorphisms (SNPs) using MALDI mass spectrometry, termed the GOOD assay, was developed. It does not require any purification and is performed with simple liquid handling, thermal incubation and cycling steps. Although this method is well suited to automation and high-throughput analysis of SNPs, it did not allow full flexibility due to lack of certain reagents. A complete set of ss-cyanoethyl phosphoramidites is presented herein that give this SNP genotyping method full sequence and multiplex capabilities. Applications to SNP genotyping in the prion protein gene, the ss-2-adrenergic receptor gene and the angiotensin converting enzyme gene using the GOOD assay are demonstrated. Because SNP genotyping technologies are generally very sensitive to varying DNA quality, the GOOD assay has been stabilised and optimised for low quality DNA. A template extraction method is introduced that allows genotyping from tissue that was taken while placing an ear tag on an animal. This dramatically facilitates the application of genotyping to animal agricultural applications, as it demonstrates that expensive and cumbersome DNA extraction procedures prior to genotyping can be avoided.

Animals↗

PubMed2004

Microarray-based method for genotyping of functional single nucleotide polymorphisms using dual-color fluorescence hybridization.

Screening disease-related single nucleotide polymorphism (SNP) markers in the whole genome has great potential in complex disease genetics and pharmacogenetics researches. It has led to a requirement for high-throughput genotyping platforms that can maximize the efficient screening functional SNPs with respect to accuracy, speed and cost. In this study, we attempted to develop a microarray-based method for scoring a number of genomic DNA in parallel for one or more molecular markers on a glass slide. Two SNP markers localized to the methylenetetrahydrofolate reductase gene (MTHFR) were selected as the investigated targets. Amplified PCR products from nine genomic DNA specimens were spotted and immobilized onto a poly-l-lysine coated glass slide to fabricate a microarray, then interrogated by hybridization with dual-color probes to determine the SNP genotype of each sample. The results indicated that the microarray-based method could determine the genotype of 677 and 1298 MTHFR polymorphisms. Sequencing was performed to validate these results. Our experiments successfully demonstrate that PCR products subjected to dual-color hybridization on a microarray could be applied as a useful and a high-throughput tool to analyze molecular markers.

Fluorescent Dyes↗

PubMed2004

Coronary artery disease and the thrombospondin single nucleotide polymorphisms.

GeneQuest was a high throughput, large-scale analysis of single nucleotide polymorphisms (SNPs) to identify gene associated with familial, premature coronary artery disease and myocardial infarction. The three SNPs showing the highest and most significant associations with disease were all members of the thrombospondin gene family, thrombospondin-1, thrombospondin-2 and thrombospondin-4. These unanticipated associations have kindled efforts to understand how the three SNPs influence the structures and functions of the thrombospondins. The SNP in thrombospondin-1 and thrombospondin-4 reside in their coding regions and result in single amino acid changes: in thrombospondin-1, the predominant asparagine at position 700 is changed to a serine while, in thrombospondin-4, it is a change of an alanine to a proline at position 387. The SNP in thrombospondin-2 is a base change in the 3'-untranslated region of the mRNA. At this early stage of investigation, predictive analyses suggest that the substitutions in thrombospondin-2 and thrombospondin-4 should alter structure, and there is direct evidence to indicate that the thrombospondin-1 SNP alters conformational stability. In addition, profound differences in the function of the thrombospondin-4 SNP variants have been identified with respect to their capacity to support endothelial cell adhesion and proliferation. While substantial additional information is needed to understand if and how the polymorphic forms of the thrombospondins affect coronary artery disease, the data assembled to date suggest marked effects of these SNPs on the structures and functions of the thrombospondins, which are consistent with induction of a proatherogenic and prothrombotic phenotype.

Binding Sites↗

PubMed2005

Single nucleotide polymorphism in transcriptional regulatory regions and expression of environmentally responsive genes.

Single nucleotide polymorphisms (SNPs) in the human genome are DNA sequence variations that can alter an individual's response to environmental exposure. SNPs in gene coding regions can lead to changes in the biological properties of the encoded protein. In contrast, SNPs in non-coding gene regulatory regions may affect gene expression levels in an allele-specific manner, and these functional polymorphisms represent an important but relatively unexplored class of genetic variation. The main challenge in analyzing these SNPs is a lack of robust computational and experimental methods. Here, we first outline mechanisms by which genetic variation can impact gene regulation, and review recent findings in this area; then, we describe a methodology for bioinformatic discovery and functional analysis of regulatory SNPs in cis-regulatory regions using the assembled human genome sequence and databases on sequence polymorphism and gene expression. Our method integrates SNP and gene databases and uses a set of computer programs that allow us to: (1) select SNPs, from among the >9 million human SNPs in the NCBI dbSNP database, that are similar to cis-regulatory element (RE) consensus sequences; (2) map the selected dbSNP entries to the human genome assembly in order to identify polymorphic REs near gene start sites; (3) prioritize the candidate polymorphic RE containing genes by searching the existing genotype and gene expression data sets. The applicability of this system has been demonstrated through studies on p53 responsive elements and is being extended to additional pathways and environmentally responsive genes.

Databases, Genetic↗

PubMed2006

Algorithm for automatic genotype calling of single nucleotide polymorphisms using the full course of TaqMan real-time data.

Single nucleotide polymorphisms (SNPs) are often determined using TaqMan real-time PCR assays (Applied Biosystems) and commercial software that assigns genotypes based on reporter probe signals at the end of amplification. Limitations to the large-scale application of this approach include the need for positive controls or operator intervention to set signal thresholds when one allele is rare. In the interest of optimizing real-time PCR genotyping, we developed an algorithm for automatic genotype calling based on the full course of real-time PCR data. Best cycle genotyping algorithm (BCGA), written in the open source language R, is based on the assumptions that classification depends on the time (cycle) of amplification and that it is possible to identify a best discriminating cycle for each SNP assay. The algorithm is unique in that it classifies samples according to the behavior of blanks (no DNA samples), which cluster with heterozygous samples. This method of classification eliminates the need for positive controls and permits accurate genotyping even in the absence of a genotype class, for example when one allele is rare. Here, we describe the algorithm and test its validity, compared to the standard end-point method and to DNA sequencing.

Algorithms↗

PubMed2005

Linkage disequilibrium grouping of single nucleotide polymorphisms (SNPs) reflecting haplotype phylogeny for efficient selection of tag SNPs.

Single nucleotide polymorphisms (SNPs) have been proposed to be grouped into haplotype blocks harboring a limited number of haplotypes. Within each block, the portion of haplotypes is expected to be tagged by a selected subset of SNPs; however, none of the proposed selection algorithms have been definitive. To address this issue, we developed a tag SNP selection algorithm based on grouping of SNPs by the linkage disequilibrium (LD) coefficient r(2) and examined five genes in three ethnic populations--the Japanese, African Americans, and Caucasians. Additionally, we investigated ethnic diversity by characterizing 979 SNPs distributed throughout the genome. Our algorithm could spare 60% of SNPs required for genotyping and limit the imprecision in allele-frequency estimation of nontag SNPs to 2% on average. We discovered the presence of a mosaic pattern of LD plots within a conventionally inferred haplotype block. This emerged because multiple groups of SNPs with strong intragroup LD were mingled in their physical positions. The pattern of LD plots showed some similarity, but the details of tag SNPs were not entirely concordant among three populations. Consequently, our algorithm utilizing LD grouping allows selection of a more faithful set of tag SNPs than do previous algorithms utilizing haplotype blocks.

Black or African American↗

PubMed2026

IFNL1 gene promoter single nucleotide polymorphism rs7247086 enhances transcription through a STAT-binding site.

A single nucleotide polymorphism (SNP) within the human interferon lambda 1 (IFN-L1, IFN-λ1) gene promoter, rs7247086 (C/T) has been reported to be associated with severe dengue and possibly with psoriasis and COVID-19. However, its functional nature is unknown. The present study was undertaken to examine the effect of rs7247086 on transcription, by utilizing promoter and enhancer-reporter assays. We see that the T allele completes a consensus signal transducer and activator of transcription (STAT)-binding site. While we did not find strong evidence to show that the STAT-binding site drove transcription from the IFNL1 gene promoter, we saw that it acts like an enhancer in reporter assays. The T allele of rs7247086 within the STAT-binding site significantly increased transcription of the reporter gene compared to the C allele when incorporated into enhancer-reporter constructs in both HEK293 and A549 cell lines. Mechanistically, we obtained evidence from electrophoretic mobility shift assays to show that the T allele binds to STAT proteins more strongly than the C allele. In a cohort of healthy individuals, we saw that the T allele carriers, specifically males but not females, had significantly increased secretion of IFN-λ1 from their peripheral blood mononuclear cells after stimulation. Lastly, rs7247086 significantly associated with psoriasis, only in males but not in females.

Humans↗

PubMed2005

An analysis of identical single-nucleotide polymorphisms genotyped by two different platforms.

The overlap of 94 single-nucleotide polymorphisms (SNP) among the 4,720 and 11,120 SNPs contained in the linkage panels of Illumina and Affymetrix, respectively, allows an assessment of the discrepancy rate produced by these two platforms. Although the no-call rate for the Affymetrix platform is approximately 8.6 times greater than for the Illumina platform, when both platforms make a genotypic call, the agreement is an impressive 99.85%. To determine if disputed genotypes can be resolved without sequencing, we studied recombination in the region of the discrepancy for the most discrepant SNP rs958883 (typed by Illumina) and tsc02060848 (typed by Affymetrix). We find that the number of inferred recombinants is substantially higher for the Affymetrix genotypes compared to the Illumina genotypes. We illustrate this with pedigree 10043, in which 3 of 7 versus 0 of 7 offspring must be double recombinants using the genotypes from the Affymetrix and the Illumina platforms, respectively. Of the 36 SNPs with one or more discrepancies, we identified a subset that appears to cluster in families. Some of this clustering may be due to the presence of a second segregating SNP that obliterates a XbaI site (the restriction enzyme used in the Affymetrix platform), resulting in a fragment too long (>1,000 bp) to be amplified.

Female↗

PubMed2006

Electrophysiological characterization of three non-synonymous single nucleotide polymorphisms (R87Q, A251T, and P307S) found in hKv1.5.

Non-synonymous single nucleotide polymorphisms (SNPs) in the KCNA5/hKv1.5 gene, which encodes for a voltage-gated K+ channel responsible for the I (Kur) current in the human atria, have been recently reported. To gain further knowledge on potential influence of hKv1.5 SNPs, we searched for their presence in a specific population of 96 French-Canadians and characterized electrophysiological properties of the variants in two cell lines. The presumed promoter (-83 bp) and coding regions were sequenced. We found three heterozygous SNPs: R87Q, A251T, and P307S. Functional analysis of SNPs transfected in Chinese hamster ovary (CHO) cells showed that both R87Q and P307S diminished the inactivation amplitude (e.g., at +60 mV, amplitudes were 89+/-26, 23+/-4, and 22+/-7 pA/pF for the wild type, R87Q and P307S, respectively; n=8, 6, and 8, respectively). Inactivation was slowed with these variants (e.g., tau (fast) at +50 mV were 270+/-48, 490+/-66, and 340+/-45 ms for the wild type, R87Q, and P307S, respectively) while R87Q additionally accelerated the rate of hKv1.5 channel opening. A dominant-negative effect was observed for R87Q but not for P307S. SNPs properties were not reproduced when expressed in the HEK293 cell line, suggesting that the regulatory beta-subunit present in CHO cells (and the human heart) is essential for the SNPs effects that we have observed.

Animals↗

PubMed2005

Functional characterization of three naturally occurring single nucleotide polymorphisms in the CES2 gene encoding carboxylesterase 2 (HCE-2).

Twelve single nucleotide polymorphisms (SNPs) in the human CES2 gene, which encodes a carboxylesterase, hCE-2 [human carboxylesterase 2 (EC 3.1.1.1)], have been reported in the Japanese. In this report, we have examined functional alterations of three SNPs, a nonsynonymous SNP (100C>T, R34W), an SNP at the splice acceptor site in intron 8 (IVS8-2A>G), and one newly discovered nonsynonymous SNP (424G>A, V142M). For the two nonsynonymous SNPs, the corresponding variant cDNAs were expressed in COS-1 cells. Both the R34W and V142M variants showed little esterase activities toward the anticancer agent irinotecan and two typical carboxylesterase substrates, p-nitrophenol acetate and 4-methylumbelliferyl acetate, although increased levels of cDNA-mediated protein expression were observed by Western blotting as compared with the wild type. To investigate a possible splicing aberration in IVS8-2A>G, an in vitro splicing assay was utilized and transcripts derived from CES2 gene fragments of the wild type and IVS8-2A>G were compared. Sequence analysis of the cloned transcripts revealed that IVS8-2A>G yielded mostly aberrantly spliced transcripts, including a deleted exon or a 32-bp deletion proximal to the 5' end of exon 9, which resulted in truncated hCE-2 proteins. These results suggested that 100C>T (R34W), 424G>A (V142M), and IVS8-2A>G are functionally deficient SNPs.

Animals↗

PubMed2006

Association of the progesterone receptor gene with breast cancer risk: a single-nucleotide polymorphism tagging approach.

Association studies on susceptibility to breast cancer using single nucleotide polymorphisms (SNP) in the progesterone receptor (PGR) gene have been previously published, but the results have been inconclusive. We used a comprehensive SNP-tagging approach to search for low-penetrance susceptibility alleles in a study of up to 4,647 cases and 4,564 controls, in a two-stage study design. We identified seven tagging SNPs using genotype data from the National Institute of Environmental Health Sciences (NIEHS) Environmental Genome Project and typed these, and an additional three SNPs, in 2,345 breast cancer cases and 2,284 controls (set 1). Three SNPs showed no evidence for association and were not studied further, whereas seven SNPs (rs11571171, rs7116336, rs660149, rs10895068, rs500760, rs566351, and rs1042838) exhibited significant associations at P < 0.1 using either a heterogeneity or trend test and progressed to be genotyped in set 2. After both stages, only one SNP was significantly associated with an increased risk of breast cancer - the PGR-12 (rs1042638) V660L valine to leucine polymorphism [VL heterozygotes (odds ratio, 1.13; 95% confidence interval, 1.03-1.24) and the LL homozygotes (odds ratio, 1.30; 95% confidence interval, 0.98-1.73), P(het) = 0.008, P(trend) = 0.002]. Similar estimates were obtained in a combined analysis of our data with those from three other published studies. We conclude that the 660L allele may be associated with a moderately increased risk of breast cancer, but that other common SNPs in the PGR gene are unlikely to be associated with a substantial risk of breast cancer.

Aged↗

PubMed2002

Simultaneous genotyping of single nucleotide polymorphisms in the IL-6, IL-10, TNFalpha and TNFbeta genes.

Single nucleotide polymorphisms (SNP) in the human IL-6, IL-10, TNFalpha and TNFbeta genes have been associated with gene function and susceptibility to disease. In this study, primers containing mismatches at 1-3 nucleotide positions were designed to incorporate a new restriction site recognized by endonucleases AlwNI, BcgI, BglI, BsaBI, BslI, BstXI, EcoNI or XcmI for genotyping SNPs in the IL-6 gene (position - 174), IL-10 gene (positions -592 and -1082), TNFalpha gene (positions -238, - 308 and -863) and TNFbeta gene (position + 249) by mismatched polymerase chain reaction and restriction fragment length polymorphism (PCR/RFLP). Our results show that appropriately designed BslI-based mismatched PCR/RFLP assays can be successfully used to determine the genotypes for approximately 40% of SNPs. The mismatched PCR strategy can be coupled with multiplex-amplification to enable simple and rapid determination of several SNP genotypes in a single reaction.

Cytokines↗

PubMed2006

Whole-genome genotyping of haplotype tag single nucleotide polymorphisms.

The International HapMap Consortium recently completed genotyping over 3.8 million single nucleotide polymorphisms (SNPs) in three major populations, and the results of studying patterns of linkage disequilibrium indicate that characterization of 300,000-500,000 tag SNPs is sufficient to provide good genomic coverage for linkage-disequilibrium-based association studies in many populations. These whole-genome association studies will be used to dissect the genetics of complex diseases and pharmacogenomic drug responses. As such, the development of a cost-effective genotyping platform that can assay hundred of thousands of SNPs across thousands of samples is essential. In this review, we describe the development of a whole-genome genotyping (WGG) assay that enables unconstrained SNP selection and effectively unlimited multiplexing from a single sample preparation. The development of WGG in concert with high-density BeadChips has enabled the creation of three different high-density SNP genotyping BeadChips: the Sentrix Human-1 Genotyping BeadChip containing over 109,000 exon-centric SNPs; the HumanHap300 BeadChip containing over 317,000 tag SNPs, and the HumanHap550 Beadchip containing over 550,000 tag SNPs.

Automation↗

PubMed2005

Association of transforming growth factor-beta1 single nucleotide polymorphism C-509T with allergy and immunological activities.

BACKGROUND: A single nucleotide polymorphism (SNP) C-509T within the tumor growth factor beta1 (TGFbeta1) gene has been associated with atopic asthma and asthma severity. To further understand the mechanisms involved, the association of C-509T with allergy, T-lymphocyte proliferation and plasma TGFbeta1 concentration has been explored in a case-control study with allergic and non-allergic subjects. METHODS: The recruited subjects including allergic (n = 38) and nonallergic (n = 25) participants have been genotyped for C-509T using allele discrimination assay. Association of C-509T with allergy status was examined using logistic regression analysis in both dominant and recessive models. Association of C-509T with T-cell proliferation in control and antigen-stimulated peripheral blood mononuclear cells (PBMCs), plasma TGFbeta1 and total IgE level were tested by multiple regression analysis. RESULTS: Individuals with homozygous mutant TT genotype showed a higher risk of allergy (TT: odds ratio = 5.099, 95% confidence limit: 1.355-19.190, p = 0.016) after covariates were adjusted. A trend to increased plasma TGFbeta1 in subjects with T allele has been discovered. In the meantime, the T allele is associated with lower T cell proliferation in controls and maximum response to above antigens. A low T-cell proliferation is correlated with higher plasma TGFbeta1 concentration (p < 0.01). The in vitro studies confirmed the suppressing effect of TGFbeta1 on T-cell proliferation at physiological range. A significant inhibitory effect on IL-4 production was also observed. CONCLUSIONS: A C to T base change in TGFbeta1 SNP C-509T has been associated with a higher risk of allergy. The mechanisms are not clear. Elevated TGFbeta1 levels associated with the C-509T polymorphism might suppress immune activation as well as Th2 cytokine production.

Alleles↗