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PubMed2006

High-throughput single-nucleotide polymorphisms genotyping: TaqMan assay and pyrosequencing assay.

Single-nucleotide polymorphisms (SNPs) are DNA sequence variations that occur at a single base in the genome sequence. SNPs are valuable markers for identifying genes responsible for susceptibility to common diseases, and in some cases, they are the causes of human diseases. A genetic study of a complex disease usually involves a case-control association study that requires genotyping of a large number of SNPs in hundreds of patients (cases) and matched controls. A significant difference of the allele frequency or genotypic frequency of a SNP between the two populations is considered to be the evidence for the association between the SNP and disease. A key to a fast and effective case-control association study requires high-throughput genotyping of SNPs. Two assays-the TaqMan SNP genotyping assay and the pyrosequencing assay-have been developed for this purpose and proven to be particularly useful. Here, we present the operative protocol, clarify the key technical issues, and highlight certain cautionary notes for high throughput SNP genotyping using TaqMan and pyrosequencing assays.

Gene Frequency↗

PubMed2001

Human calcitonin receptor-like receptor for adrenomedullin: genomic structure, eight single-nucleotide polymorphisms, and haplotype analysis.

Adrenomedullin (ADM), a peptide characterized by persistent hypotensive activity, is thought to be involved when the control mechanism of blood pressure is deranged, because its plasma concentration is upregulated in hypertensive patients. The receptor for ADM, a molecular complex consisting of calcitonin-receptor-like receptor (CRLR) and receptor-activity-modifying protein 2 (RAMP2), is activated through a unique intracellular transport mechanism. By analyzing the nucleotide sequences of bacterial artificial chromosome (BAC) clones, we have established that the gene encoding CRLR is spread over a genomic distance of 103,145 bases; it contains 15 exons interrupted by 14 introns, including 1 that spans more than 60 kilobases. Exons 1-3 constitute the 5' noncoding region; exons 4 through 15 are coding elements, of which exons 8 to 14 encode seven transmembrane domains. Eight novel single-nucleotide polymorphisms (SNPs) and their allelic frequencies in the Japanese population were found by direct sequencing of 32 alleles; two SNPs were in the 5' flanking region, one in exon 2, and the other five around intron-exon junctions. Eight haplotypes were constructed using these alleles in our Japanese population sample. The data establish a basis for investigations to detect molecular variants in the ADM receptor that might alter control of blood pressure and confer on individuals a predisposition to essential hypertension.

Alleles↗

PubMed2006

The GT to GC single nucleotide polymorphism at the beginning of an alternative exon 2C of human MTH1 gene confers an amino terminal extension that functions as a mitochondrial targeting signal.

Human MTH1 protein hydrolyzes oxidized purine nucleotides 8-oxo-2'-deoxyguanosine triphosphate (8-oxo-dGTP), 2-OH-dATP or their ribo-forms to their monophosphates, thus minimizing replicational and transcriptional errors both in the nuclei and mitochondria. MTH1 suppresses mitochondrial dysfunction and cell death caused by H(2)O(2). Furthermore, MTH1 suppresses the transient increase in 8-oxoguanine in mitochondrial DNA in the dopaminergic nerve terminals in mouse striatum after 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine administration, and it protects the nerve terminals. We previously reported that a novel MTH1 allele with a single nucleotide polymorphism (SNP) in its exon 2c segment encodes the fourth MTH1 isoform, namely, MTH1a (p26), in addition to the three known isoforms, MTH1b (p22), c (p21), and d (p18). Another SNP located in exon 4 of the MTH1 gene, which is closely linked to the SNP in exon 2c, substitutes the Val83 residue in MTH1d with Met83. We herein show that all MTH1 isoforms efficiently hydrolyzed 2-OH-dATP and 8-oxo-dGTP. The amino terminal region of MTH1a functioned as a mitochondrial targeting signal when it was expressed in the HeLa cells as a fusion protein with enhanced green fluorescent protein. The cellular fractionation revealed that MTH1a(Met83) was localized in the mitochondria to the same extent as was MTH1d(Val83). However, the mitochondrial translocation of MTH1d(Met83) was less efficient than that of MTH1d(Val83).

Adenosine Triphosphate↗

PubMed2004

Haplotype tag single nucleotide polymorphism analysis of the human orthologues of the rat type 1 diabetes genes Ian4 (Lyp/Iddm1) and Cblb.

The diabetes-prone BioBreeding (BB) and Komeda diabetes-prone (KDP) rats are both spontaneous animal models of human autoimmune, T-cell-associated type 1 diabetes. Both resemble the human disease, and consequently, susceptibility genes for diabetes found in these two strains can be considered as potential candidate genes in humans. Recently, a frameshift deletion in Ian4, a member of the immune-associated nucleotide (Ian)-related gene family, has been shown to map to BB rat Iddm1. In the KDP rat, a nonsense mutation in the T-cell regulatory gene, Cblb, has been described as a major susceptibility locus. Following a strategy of examining the human orthologues of susceptibility genes identified in animal models for association with type 1 diabetes, we identified single nucleotide polymorphisms (SNPs) from each gene by resequencing PCR product from at least 32 type 1 diabetic patients. Haplotype tag SNPs (htSNPs) were selected and genotyped in 754 affected sib-pair families from the U.K. and U.S. Evaluation of disease association by a multilocus transmission/disequilibrium test (TDT) gave a P value of 0.484 for IAN4L1 and 0.692 for CBLB, suggesting that neither gene influences susceptibility to common alleles of human type 1 diabetes in these populations.

Adaptor Proteins, Signal Transducing↗

PubMed2001

A novel single nucleotide polymorphism altering stability and activity of CYP2a6.

CYP2A6 is known as a major cytochrome P450 (CYP) responsible for the oxidation of nicotine and coumarin in humans. In this study, we explored genetic polymorphisms, which reduce CYP2A6 activity in Japanese. Two novel mutations in exon 9 of the CYP2A6 gene were found. A single nucleotide polymorphism of T1412C and G1454T resulted in Ile471Thr and Arg485Leu substitution, respectively. The frequency of the former variant allele was considerably high (15.7%), while the latter variant appeared to be a rare polymorphism. Heterologous expression of CYP2A6 using a cDNA possessing C instead of T-base at codon 471 in Escherichia coli caused remarkable reduction of the stability of holoenzyme at 37 degrees C. Furthermore, this variant enzyme almost lacked nicotine C-oxidase activity, although coumarin 7-hydroxylase activity was still observed. These data suggest that individuals homozygous for the T1412C variant allele or heterozygous for this and a defect allele such as the CYP2A6*4 may be poor metabolizer of nicotine, but not coumarin.

Aryl Hydrocarbon Hydroxylases↗

PubMed2001

Characterization of a single nucleotide polymorphism in the coding sequence of the bovine transferrin gene.

A single nucleotide polymorphism was identified in the coding sequence of the bovine transferrin gene. Two alleles (SSCP1 and SSCP2) were detected by SSCP analysis and the mutation point was identified and confirmed by direct sequencing of the PCR products. The relationship between protein and DNA polymorphism was established. Protein variants A, D1 and E correspond to SSCP allele 1 and variant D2 corresponds to SSCP allele 2. DNA sequences from genotypes AA, AE, AD2, D1E, D2E and D2D2 reveal an A/G substitution at position 1455 of the cDNA which causes a Gly/Glu substitution which could be responsible for the mobility difference between D1 and D2 variants. Because of the number of variants, this suggests that other SNPs exist in the bovine transferrin gene. A linkage analysis between the SSCPs and two microsatellites (UWCA46 and CSSM019) mapped the transferrin gene to BTA1. Two-point analysis revealed a tight linkage within the transferrin protein variants and the SSCPs.

Animals↗

PubMed2001

Using molecular beacons to detect single-nucleotide polymorphisms with real-time PCR.

Detection of single-nucleotide polymorphisms (SNPs) in high-throughput studies promises to be an expanding field of molecular medicine in the near future. Highly specific, simple, and accessible methods are needed to meet the rigorous requirements of single-nucleotide detection needed in pharmacogenomic studies, linkage analysis, and the detection of pathogens. Molecular beacons present such a solution for the high-throughput screening of SNPs in homogeneous assays using the polymerase chain reaction (PCR). Molecular beacons are probes that fluoresce on hybridization to their perfectly complementary targets. In recent years they have emerged as a leading genetic analysis tool in a wide range of contexts from quantification of RNA transcripts, to probes on microarrays, to single-nucleotide polymorphism detection. The majority of these methods use PCR to obtain sufficient amounts of sample to analyze. The use of molecular beacons with other amplification schemes has been reliably demonstrated, though PCR remains the method of choice. Here we discuss and present how to design and use molecular beacons to achieve reliable SNP genotyping and allele discrimination in real-time PCR. In addition, we provide a new means of analyzing data outputs from such real-time PCR assays that compensates for differences between sample condition, assay conditions, variations in fluorescent signal, and amplification efficiency. The mechanisms by which molecular beacons are able to have extraordinary specificity are also presented.

Alleles↗

PubMed1994

Genetic Bit Analysis: a solid phase method for typing single nucleotide polymorphisms.

A new method for typing single nucleotide polymorphisms in DNA is described. In this method, specific fragments of genomic DNA containing the polymorphic site(s) are first amplified by the polymerase chain reaction (PCR) using one regular and one phosphorothioate-modified primer. The double-stranded PCR product is rendered single-stranded by treatment with the enzyme T7 gene 6 exonuclease, and captured onto individual wells of a 96 well polystyrene plate by hybridization to an immobilized oligonucleotide primer. This primer is designed to hybridize to the single-stranded target DNA immediately adjacent from the polymorphic site of interest. Using the Klenow fragment of E. coli DNA polymerase I or the modified T7 DNA polymerase (Sequenase), the 3' end of the capture oligonucleotide is extended by one base using a mixture of one biotin-labeled, one fluorescein-labeled, and two unlabeled dideoxynucleoside triphosphates. Antibody conjugates of alkaline phosphatase and horseradish peroxidase are then used to determine the nature of the extended base in an ELISA format. This paper describes biochemical features of this method in detail. A semi-automated version of the method, which we call Genetic Bit Analysis (GBA), is being used on a large scale for the parentage verification of thoroughbred horses using a predetermined set of 26 diallelic polymorphisms in the equine genome.

Autoanalysis↗

PubMed2006

Left ventricular mass and +276 G/G single nucleotide polymorphism of the adiponectin gene in uncomplicated obesity.

The single nucleotide polymorphism at position 276 in the adiponectin gene (APM1/ACDC +276 G>T) and left ventricular mass (LVM) have been associated with increased cardiovascular risk. We sought to evaluate whether +276 G>T variants in the adiponectin gene are correlated with LVM in uncomplicated obese subjects. APM1/ACDC +276 G>T single nucleotide polymorphism, echocardiographic indexed LVM (LVM/body surface area and LVM/height(2.7)), insulin sensitivity by euglycemic clamp, and plasma adiponectin levels were analyzed in 62 obese subjects without complications (51 women and 11 men; mean age, 34.2 +/- 10.2 years; BMI, 38.6 +/- 9.1 kg/m2). Forty lean subjects formed the control group for LVM evaluation. We found 23 (37%) uncomplicated obese subjects with the APM1/ACDC +276 G/G genotype, 25 (40%) with the G/T genotype, and 14 (23%) with the T/T genotype. G/G uncomplicated obese subjects showed significant higher LVM/body surface area and LVM/height(2.7) (within the normal range in the majority of them) than uncomplicated obese subjects carrying the G/T and T/T genotypes (p < 0.01 and p < 0.05, respectively). This study showed that LVM is significantly higher in uncomplicated obese subjects carrying the G/G genotype at position 276 of the human adiponectin gene.

Adiponectin↗

PubMed2004

Single nucleotide polymorphisms in the ovine casein genes detected by polymerase chain reaction-single strand conformation polymorphism.

Casein genetic polymorphisms are important and well known due to their effects on quantitative traits and technological properties of milk. At the DNA level, polymerase chain reaction (PCR)-single-strand conformation polymorphism (SSCP) allows for the simultaneous typing of several alleles at casein loci, as well as the detection of unknown polymorphisms. Here we describe the usefulness of the PCR-SSCP technique for casein typing in sheep. In particular, three single-nucleotide polymorphisms (SNP) are described at CSN1S1, CSN2, and CSN3, all resulting in amino acid exchanges. At CSN1S1, a transition T-->C was found, resulting in the deduced amino acid exchange Ile186-->Thr186. A transition A-->G resulting in the deduced amino acid exchange Met183-->Val183 was identified at CSN2. The 2 SNP showed a rather high frequency (ranging from 0.12 to 0.26) in 3 Italian breeds (Sarda, Comisana, Sopravissana). Another transition C-->T (Ser104-->Leu104) was found at CSN3 in one heterozygous animal.

Alleles↗

PubMed2005

5,10-Methylenetetrahydrofolate reductase polymorphisms and pharmacogenetics: a new role of single nucleotide polymorphisms in the folate metabolic pathway in human health and disease.

Knowledge about the role of folate, a water-soluble B vitamin, and single nucleotide polymorphisms (SNPs) in the folate metabolic pathway in human health and disease has been rapidly expanding. Recently, functionally significant SNPs in 5,10-methylenetetrahydrofolate reductase (MTHFR), a critical enzyme for intracellular folate homeostasis and metabolism, have been identified and characterized. An emerging body of in vitro and clinical evidence suggests that these MTHFR SNPs may be an important pharmacogenetic determinant of predicting response to and toxicity of methotrexate and 5-fluorouracil-based cancer and anti-inflammatory treatments because of their well-defined and highly relevant biochemical effects on intracellular folate composition and one-carbon transfer reactions.

5,10-Methylenetetrahydrofolate Reductase (FADH2)↗

PubMed2001

[The single nucleotide polymorphisms and its application to forensic medicine].

Single-nucleotide polymorphisms (SNPs) are the most abundant forms of human genetic variation. These variable sites are present at high density in the genome, making them powerful tool for the diagnosis of genetic and genetic-related diseases, population genetics research and drug development. They are also found widespread application to the forensic medicine. This report mainly describe the SNPs characteristics and its potential applications to the forensic medicine including the possibility, the problems and high-throughput automation detection methods.

Forensic Medicine↗

PubMed2004

CD177 polymorphisms: correlation between high-frequency single nucleotide polymorphisms and neutrophil surface protein expression.

BACKGROUND: Human neutrophil antigen-2a (NB1) is located on NB1 glycoprotein, which is expressed on subpopulations of neutrophils. PRV-1 is a gene that is over-expressed in neutrophils from patients with polycythemia rubra vera. The gene encoding NB1 differs from PRV-1 at four nucleotides. The purpose of this study was to determine if PRV-1 and NB1 are alleles of the same gene or two separate genes; and, moreover, if they are alleles, to explore potential correlations to NB1 glycoprotein expression. STUDY DESIGN AND METHODS: Primer pairs were used to amplify WBC DNA in the regions surrounding the four NB1 polymorphic sites. The four resulting amplicons were sequenced. The size of the neutrophil population in each donor that stained brightly with CD177 antibody was assessed by flow cytometry. RESULTS: If PRV-1 and NB1 are separate genes, then all people should be heterozygous for the PRV-1/NB1 polymorphisms. Because 11 of 23 donors were homozygous for PRV-1 polymorphisms at all four sites, PRV-1 and NB1 are alleles of the same gene, CD177. When the sequenced exons were compared with PRV-1, 13 single nucleotide polymorphisms (SNPs) that result in amino acid changes were found. The G42C NB1 polymorphism, found in 10 donors, was the most common SNP. The size of the CD177 bright-staining neutrophil population was greater in 42C/C donors than in 42G/G donors (66 +/- 20% vs. 41 +/- 21%, p = 0.004). CONCLUSIONS: PRV-1 and NB1 are alleles of the polymorphic gene CD177. The most common SNP in bp 42 predicted an amino acid change that may have an effect on protein expression.

Black or African American↗

PubMed2001

Four single-nucleotide polymorphisms in the human BUB1 gene.

Four single-nucleotide polymorphisms have been found in the human BUB1 gene, which encodes a kinase involved in the mitotic spindle checkpoint. A cytosine-to-thymine change in exon 10, corresponding to codon 375 (c.1124C>T), causes an amino acid substitution of serine to phenylalanine. A guanine/cytosine polymorphism in exon 4 (c.279G>C) and a thymine/cytosine polymorphism in exon 12 (c.1293T>C) do not cause amino acid substitution. The other polymorphism, of thymine/cytosine (IVS9-8T>C), is found at 8bp upstream of exon 10. As mutations of the hBUB1 gene were reported in a subset of human cancers, these polymorphisms could provide useful tools for the genetic study of susceptibility to various human cancers.

Alleles↗

PubMed2001

Selection of single nucleotide polymorphisms for association studies in candidate genes.

Comparison of polymorphic sites such as single nucleotide polymorphisms (SNPs) within a gene between cases and controls may be useful for establishing a role of this gene in disease susceptibility. The approach includes two steps: the first step is the discovery of the different SNPs within the candidate gene and the second step is the association testing per se that can be done on the entire set of sites discovered or on a subset of these sites only. Selecting a subset of sites may increase the power to detect the association with the candidate gene since a smaller number of tests would then be performed. We proposed a strategy to select sites within a candidate gene and applied it on the Genetic Analysis Workshop 12 candidate gene data. Using these selected sites, we detected an association with candidate genes 1 and 6.

Alleles↗

PubMed2002

Integration of DNA ligation and rolling circle amplification for the homogeneous, end-point detection of single nucleotide polymorphisms.

Association studies using common sequence variants or single nucleotide polymorphisms (SNPs) may provide a powerful approach to dissect the genetic inheritance of common complex traits. Such studies necessitate the development of cost-effective, high throughput technologies for scoring SNPs. The method described in this paper for the co-detection of both alleles of a SNP in a single homogeneous reaction combines the specificity of a high fidelity DNA ligation step with the power of rolling circle amplification. The incorporation of Amplifluor energy transfer primers enables signal detection in a homogeneous format, making this approach highly amenable to automation. The adaptation of the genotyping method for high throughput screening using conventional liquid handling systems is described.

Alleles↗

PubMed2006

Analysis of population differentiation in North Eurasian cattle (Bos taurus) using single nucleotide polymorphisms in three genes associated with production traits.

Single nucleotide polymorphisms (SNPs) in growth hormone 1 (GH1), insulin-like growth factor 1 (IGF1) and leptin (LEP), all candidates for production traits in cattle, were characterized in North Eurasian cattle breeds. Allele frequencies of IGF1 exhibited significant (P < 0.05) deviation from neutral expectation and therefore, might be associated with divergence in North Eurasian cattle because of genetic selection. Allele frequencies and lower heterozygosity of LEP may indicate a recent introduction of an alternative allele in this geographic region. Locus F(ST) estimates were highest for IGF1 (0.151, sigma = 0.042) and lowest for GH (0.062, sigma = 0.020). Our results suggest a slightly higher population differentiation across the candidate genes (FST = 0.108) than across microsatellites (FST = 0.095), possibly because of selection and stochastic effects.

Animals↗

PubMed2001

L-RCA (ligation-rolling circle amplification): a general method for genotyping of single nucleotide polymorphisms (SNPs).

A flexible, non-gel-based single nucleotide polymorphism (SNP) detection method is described. The method adopts thermostable ligation for allele discrimination and rolling circle amplification (RCA) for signal enhancement. Clear allelic discrimination was achieved after staining of the final reaction mixtures with Cybr-Gold and visualisation by UV illumination. The use of a compatible buffer system for all enzymes allows the reaction to be initiated and detected in the same tube or microplate well, so that the experiment can be scaled up easily for high-throughput detection. Only a small amount of DNA (i.e. 50 ng) is required per assay, and use of carefully designed short padlock probes coupled with generic primers and probes make the SNP detection cost effective. Biallelic assay by hybridisation of the RCA products with fluorescence dye-labelled probes is demonstrated, indicating that ligation-RCA (L-RCA) has potential for multiplexed assays.

Alleles↗