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PubMed2006

Haplotype analysis of UDP-glucuronocyltransferase 2B7 gene (UGT2B7) polymorphisms in healthy Japanese subjects.

OBJECTIVES: UDP-glucuronocyltransferase 2B7 (UGT2B7) catalyzes glucuronidation of various types of endogenous compounds and drugs, but the genetic basis of interindividual variation in the metabolism of these substances has not yet been sufficiently elucidated. In addition, information about single nucleotide polymorphisms (SNPs) and haplotypes of the UGT2B7 gene that encode the enzyme in the Japanese population is still far from sufficient. DESIGN AND METHODS: We paid special attention to and performed an investigation on -327A > G, -161T > C, -138G > A, and -125T > C in the proximal promoter region, which is regarded as being important for the transcription of the UGT2B7 gene, and also on 211G > A and 802C > T, i.e., non-synonymous SNPs of exon 1 and exon 2 that encode the substrate binding domain. Their genotypes were determined by PCR-direct sequencing. RESULTS: As a result of genotyping, the minor allele frequencies in 160 Japanese individuals were found to be as follows: -327SNP A allele, 0.244; -161SNP T allele, 0.244; -138SNP A allele, 0; -125SNP C allele, 0.078; 211SNP T allele, 0.148 and 802SNP T allele, 0.244. By computational haplotype analysis, it was found that these regions formed a linkage disequilibrium block, and the presence of five haplotypes was demonstrated. CONCLUSIONS: These results suggest that the haplotype structure in the Japanese population is different from that of other ethnic groups.

Asian People↗

PubMed2005

Systematic linkage disequilibrium analysis of SLC12A8 at PSORS5 confirms a role in susceptibility to psoriasis vulgaris.

The gene for solute carrier family 12 member A8 has recently been proposed as a candidate gene for psoriasis susceptibility (PSORS5) on chromosome 3q based on association of five single nucleotide polymorphisms (SNP) in Swedish patients. To investigate whether this locus is relevant for German psoriasis vulgaris (PsV) patients, we analyzed a group of 210 trios and a case-control group including 375 patients. Based on our investigation of the linkage disequilibrium (LD) structure of SLC12A8, we assayed 35 haplotype tag SNP and grouped them into nine LD-blocks. In the case-control study, we detected an association for six SNP and three LD-based haplotypes. Association was strongest for ss35527511 (chi2 = 11.224, p = 0.0008) and haplotype E-2 (chi2 = 11.788, p = 0.00059) and independent of the presence of an HLA-associated PSORS1 risk allele. Through extended haplotype analysis, we could show that two independent association signals exist in SLC12A8, suggesting allelic heterogeneity. None of the SNP showed association in trios, apart from a weak association of rs2228674 (transmission disequilibrium test statistics p = 0.048), probably due to insufficient power. We conclude that SLC12A8 is a susceptibility locus for PsV. In order to establish the exact nature of this association, efforts to identify the disease-causing variants are ongoing.

Alleles↗

PubMed2002

Polymorphisms in the human xeroderma pigmentosum group A gene and their impact on cell survival and nucleotide excision repair.

Polymorphisms in DNA repair genes may contribute to defects in DNA repair and increased susceptibility to cancer. The xeroderma pigmentosum group A (XPA) gene is required for nucleotide excision repair (NER) and mutations in XPA highly predispose humans to skin cancer. We examined DNA samples from 189 individuals for polymorphisms in the XPA gene. First, SSCP analysis was used to examine each of the six exons and their intron boundaries. One frequent single nucleotide polymorphism (SNP) in the untranslated region of exon 1 and two rare SNPs which produce the changes Arg228Gln and Val234Leu in the coding region of exon 6 were identified. Quite surprisingly, no sequence variants were found within the coding regions or the adjacent intron boundaries of exons 1-5. Ecdysone-inducible expression vectors containing wild type XPA cDNA or cDNAs representing the two polymorphisms that we identified in exon 6 were created and independently introduced into the XPA deficient cell line XP12RO-SV. Transcription-coupled repair (TCR), global genome repair (GGR) and cell survival following UV irradiation were studied in each cell line in the absence or presence of the ecdysone hormone analog, ponasterone A. No substantial difference in repair or cell survival was found in cells complemented with wild type or polymorphic alleles of XPA. A 10-fold increase in the expression of XPA by addition of ponasterone A resulted in faster removal of 6-4 photoproducts from the total genomes of cells complemented with wild type or polymorphic alleles of XPA but had no significant impact on TCR or global genome repair of cyclobutane pyrimidine dimers (CPDs). Since our SSCP analysis failed to detect significant numbers of polymorphisms we directly sequenced exons 4-6 in a subset of our samples. One additional rare SNP, which produces the change Leu252Val was found in exon 6 and four rare SNPs and one rare single nucleotide deletion were found in intron 4. Hence, the XPA gene appears to be a cold spot for genetic variation and rare polymorphisms in the coding region of the gene do not reduce NER or cell survival after UV irradiation.

Cell Survival↗

PubMed2004

Searching for cancer-associated gene polymorphisms: promises and obstacles.

Low-penetrance genetic variations appear to form the most essential component of the heritability of cancer risk. Search for relevant polymorphic candidates faces significant obstacles, due to both the high number of potentially promising single nucleotide polymorphisms (SNPs) and the intrinsic difficulties in identification of weak gene-disease interactions. At present, extensive case-control studies can be applied only to a limited number of gene polymorphisms. Therefore, the choice of SNPs that deserve an exhaustive populational analysis is of primary importance. Preferences are usually given to those genetic pathways, whose variability and role in cancer causation have been already shown by prior studies. The available electronic databases and software tools may allow further SNP sorting, based on functional predictions. The design for the pilot study may need to be different from the one for large-scale case-control analysis. Some investigations justify non-random patient selection for preliminary assessment of low-penetrance effects, with the emphasis on particularly susceptible individuals (familial, early onset, multiple cancer cases). Other presumably accelerating approaches suggest a decisive exclusion of SNP candidates showing only marginal effects, relaxed formats for rapid dissemination of preliminary data, use of more demonstrative controls such as elderly tumor-free subjects, etc. These short-cuts cannot be properly validated for the time being, due to the paucity of identified low-penetrance risk modifiers. It is expected that the increasing capacities of available DNA collections, coupled with the rapid development of high-throughput genotyping technologies, will vastly accelerate the research on polygenic cancer susceptibility.

Alleles↗

PubMed2026

Phylogenetic inconsistency of pairwise SNP clustering for inferring tuberculosis transmission in a high-burden, endemic setting: a case study from Thailand.

Whole-genome sequence analysis is now widely used to delineate tuberculosis transmission clusters. A standard practice is to cluster bacterial isolates based on a fixed maximum genome-wide pairwise single nucleotide polymorphism (pwSNP) distance threshold. In this study, we evaluated the phylogenetic consistency of pwSNP-distance clustering with thresholds ranging between 1 and 25 single nucleotide polymorphisms (SNPs) using two contrasting data sets: (i) a data set from the UK (N = 390) published by T. M. Walker, C. L. C. Ip, R. H. Harrell, J. T. Evans, et al. (Lancet Infect Dis 13:137-146, 2013, https://doi.org/10.1016/S1473-3099(12)70277-3), which was foundational to the establishment of this method, and (ii) a data set from Thailand (N = 3,341), characterized by persistent transmission and sparse, non-systematic sampling. For the UK data set, the standard pwSNP-distance clustering using thresholds of ≥12 SNPs yielded entirely monophyletic clusters and showed high concordance with a comparative monophyly constrained, tree-based method. In contrast, for the Thai data set, pwSNP-distance clustering often generated non-monophyletic clusters, even by the 25-SNP threshold. The pwSNP-distance and comparative tree-based clustering methods only showed large consistency at thresholds of ≥22 SNPs. This suggests that SNP clusters defined by low distance thresholds (i.e., <12 SNPs for the UK data set, and <22 SNPs for the Thai data set) may lack robustness, and the problem is particularly severe for data sets characterized by persistent transmission, likely due to poorer cluster separation. Moreover, our findings indicate that large cluster sizes, high maximum intra-cluster genetic distances, and broad sample collection time spans may serve as useful indicators of potentially non-monophyletic clusters. We also demonstrate that mixed infections can produce spurious, phylogenetically long-range SNP linkages, underscoring the necessity of strict sequence quality control.IMPORTANCEFixed-threshold pairwise single nucleotide polymorphism (pwSNP)-distance clustering is commonly used to delineate tuberculosis transmission clusters. From an epidemiological perspective, a genuine transmission cluster must be monophyletic, originating from a single source. However, pwSNP-distance clustering is inherently simplistic and can therefore violate this principle, making the assessment of its phylogenetic consistency critical. Our results demonstrate that while this method effectively delineated complete transmission clusters for the data set from the UK, a low-burden and non-persistent transmission setting, it frequently generated non-monophyletic clusters when applied to the Thai data set, characterized by persistent transmission alongside sparse and non-systematic sampling. Furthermore, we found that clusters derived using low distance thresholds could notably vary between the pwSNP-distance and comparative tree-based clustering methods, suggesting limited reliability and robustness. To accurately delineate tuberculosis transmission clusters, especially for complex data from high-burden, endemic settings, we recommend transitioning from pwSNP-distance clustering toward more robust, phylogenetic clustering that respects evolutionary descent.

Mycobacterium tuberculosis↗

PubMed2005

Identification of functional candidate genes for body composition by expression analyses and evidencing impact by association analysis and mapping.

This study aims to identify hepatic genes affecting traits related to muscularity and obesity by combining expression analyses, association studies, and gene mapping. Functional candidate genes with trait-associated expression were obtained by hybridising custom made application-specific cDNA microarrays with targets of discordant sib pairs of a porcine experimental population. Out of 238 genes addressed, nine genes were regulated by the factor>or=2 between the sib pairs. Differential gene expression was independently confirmed for selected genes by real time RT-PCR. Transcript levels of four genes (APOH, PEDF, SLCO1B3, TBG) were significantly different between the phenotype groups. Screening for trait associated markers within TBG and APOH by comparative sequencing of discordant sib pairs revealed a SNP at position nt 778 (A>C) (N229H) of TBG. No polymorphism in APOH was detected. Association analysis confirmed effects of TBG on carcass traits statistically. Allocating TBG to a QTL region on chromosome X revealed genetic evidence for the effect. Moreover, our results indicate that there are probably two polymorphisms segregating-one (N229H) altering binding capability of TBG and another still to be detected altering the transcription rate of TBG.

Analysis of Variance↗

PubMed2007

A preliminary analysis of the DNA and diet of the extinct Beothuk: a systematic approach to ancient human DNA.

We have used a systematic protocol for extracting, quantitating, sexing and validating ancient human mitochondrial and nuclear DNA of one male and one female Beothuk, a Native American population from Newfoundland, which became extinct approximately 180 years ago. They carried mtDNA haplotypes, which fall within haplogroups X and C, consistent with Northeastern Native populations today. In addition we have sexed the male using a novel-sexing assay and confirmed the authenticity of his Y chromosome with the presence of the Native American specific Y-QM3 single nucleotide polymorphism (SNP). This is the first ancient nuclear SNP typed from a Native population in the Americas. In addition, using the same teeth we conducted a stable isotopes analysis of collagen and dentine to show that both individuals relied on marine sources (fresh and salt water fish, seals) with no hierarchy seen between them, and that their water sources were pooled or stored water. Both mtDNA sequence data and Y SNP data hint at possible gene flow or a common ancestral population for both the Beothuk and the current day Mikmaq, but more importantly the data do not lend credence to the proposed idea that the Beothuk (specifically, Nonosabasut) were of admixed (European-Native American) descent. We also analyzed patterns of DNA damage in the clones of authentic mtDNA sequences; there is no tendency for DNA damage to occur preferentially at previously defined mutational hotspots, suggesting that such mutational hotspots are not hypervariable because they are more prone to damage.

Base Sequence↗

PubMed2002

Family-based analysis using a dense single-nucleotide polymorphism-based map defines genetic variation at PSORS1, the major psoriasis-susceptibility locus.

Psoriasis is a common skin disorder of multifactorial origin. Genomewide scans for disease susceptibility have repeatedly demonstrated the existence of a major locus, PSORS1 (psoriasis susceptibility 1), contained within the major histocompatibility complex (MHC), on chromosome 6p21. Subsequent refinement studies have highlighted linkage disequilibrium (LD) with psoriasis, along a 150-kb segment that includes at least three candidate genes (encoding human leukocyte antigen-C [HLA-C], alpha-helix-coiled-coil-rod homologue, and corneodesmosin), each of which has been shown to harbor disease-associated alleles. However, the boundaries of the minimal PSORS1 region remain poorly defined. Moreover, interpretations of allelic association with psoriasis are compounded by limited insight of LD conservation within MHC class I interval. To address these issues, we have pursued a high-resolution genetic characterization of the PSORS1 locus. We resequenced genomic segments along a 220-kb region at chromosome 6p21 and identified a total of 119 high-frequency SNPs. Using 59 SNPs (18 coding and 41 noncoding SNPs) whose position was representative of the overall marker distribution, we genotyped a data set of 171 independently ascertained parent-affected offspring trios. Family-based association analysis of this cohort highlighted two SNPs (n.7 and n.9) respectively lying 7 and 4 kb proximal to HLA-C. These markers generated highly significant evidence of disease association (P<10-9), several orders of magnitude greater than the observed significance displayed by any other SNP that has previously been associated with disease susceptibility. This observation was replicated in a Gujarati Indian case/control data set. Haplotype-based analysis detected overtransmission of a cluster of chromosomes, which probably originated by ancestral mutation of a common disease-bearing haplotype. The only markers exclusive to the overtransmitted chromosomes are SNPs n.7 and n.9, which define a 10-kb PSORS1 core risk haplotype. These data demonstrate the power of SNP haplotype-based association analyses and provide high-resolution dissection of genetic variation across the PSORS1 interval, the major susceptibility locus for psoriasis.

Case-Control Studies↗

PubMed2007

Single nucleotide polymorphism array analysis of cancer.

PURPOSE OF REVIEW: Classifying tumors and identifying therapeutic targets requires a description of the genetic changes underlying cancer. Single nucleotide polymorphism (SNP) arrays provide a high-resolution platform for describing several types of genetic changes simultaneously. With the resolution of these arrays increasing exponentially, they are becoming increasingly powerful tools for describing the genetic events underlying cancer. RECENT FINDINGS: The ability to map loss of heterozygosity (LOH) and overall copy number variations using SNP arrays is known. Techniques have recently been developed to map LOH at high resolution in the absence of paired normal data. Copy number variations described by SNP array studies are now reaching resolutions enabling the identification of novel oncogenes and tumor suppressor genes. The ability to determine allele-specific copy number changes has only recently been described. Moreover, SNP arrays offer a high-throughput platform for large-scale association studies that are likely to lead to the identification of multiple germline variants that predispose to cancer. SUMMARY: SNP arrays are an ideal platform for identifying both somatic and germline genetic variants that lead to cancer. They provide a basis for DNA-based cancer classification and help to define the genes being modulated, improving understanding of cancer genesis and potential therapeutic targets.

Chromosome Aberrations↗

PubMed2006

Intra- and interpopulation genotype reconstruction from tagging SNPs.

The optimal method to be used for tSNP selection, the applicability of a reference LD map to unassayed populations, and the scalability of these methods to genome-wide analysis, all remain subjects of debate. We propose novel, scalable matrix algorithms that address these issues and we evaluate them on genotypic data from 38 populations and four genomic regions (248 SNPs typed for approximately 2000 individuals). We also evaluate these algorithms on a second data set consisting of genotypes available from the HapMap database (1336 SNPs for four populations) over the same genomic regions. Furthermore, we test these methods in the setting of a real association study using a publicly available family data set. The algorithms we use for tSNP selection and unassayed SNP reconstruction do not require haplotype inference and they are, in principle, scalable even to genome-wide analysis. Moreover, they are greedy variants of recently developed matrix algorithms with provable performance guarantees. Using a small set of carefully selected tSNPs, we achieve very good reconstruction accuracy of "untyped" genotypes for most of the populations studied. Additionally, we demonstrate in a quantitative manner that the chosen tSNPs exhibit substantial transferability, both within and across different geographic regions. Finally, we show that reconstruction can be applied to retrieve significant SNP associations with disease, with important genotyping savings.

Algorithms↗

PubMed2026

Genetically Predicted Gene Expression and Circulating Metabolites Associated with Cervical High-Grade Squamous Intraepithelial Lesion: A Mendelian Randomization Study.

BACKGROUND: High-grade squamous intraepithelial lesion (HSIL) is a precancerous condition of the cervix. Identifying risk factors associated with HSIL and understanding their potential mechanisms may inform prevention strategies. This study aimed to investigate the associations of genetically predicted gene expression and circulating metabolites with HSIL risk using Mendelian randomization (MR). METHODS: We performed two-sample MR analysis to evaluate the associations of genetically predicted gene expression (eQTLGen consortium, N=31,684) and circulating metabolites (genome-wide association study [GWAS], N=8,299) with HSIL risk (FinnGen R12, N=293,218; 8,291 cases). Mediation analysis was conducted to explore whether metabolites might mediate the associations between genes and HSIL. Sensitivity analyses, including Mendelian randomization pleiotropy residual sum and outlier (MR-PRESSO), leave-one-out, and colocalization, were performed to assess the robustness of the findings. All GWAS data used in this study were derived from European-ancestry populations. RESULTS: Eleven genes showed significant associations with HSIL after false discovery rate (FDR) correction (q<0.05), including VWA7, PAX8, GUSBP1, IKZF3, PAX8-AS1, NFKBIL1 (interpret with caution due to an influential single nucleotide polymorphism [SNP]), ERBB2, COL11A2, SKIV2L, TCF19, and PGAP3. Eleven circulating metabolites were also significantly associated with HSIL. Mediation analysis suggested that two phospholipid metabolites (GCST90200685 and GCST90200692) might mediate a small proportion of the total protective association of COL11A2 with HSIL (1.46% and 1.45%, respectively), indicating that the protective association of COL11A2 is largely independent of these circulating metabolites. Colocalization analysis showed strong evidence of shared causal variants for eight genes (PP.H4>0.98), while COL11A2 showed weak evidence of colocalization (PP.H4=1.58×10-15). Functional enrichment analysis indicated that COL11A2-related genes were enriched in extracellular matrix (ECM)-receptor interaction and PI3K-Akt signaling pathways. CONCLUSION: This MR study identified 11 genes and 11 circulating metabolites associated with HSIL risk. Among these, COL11A2 showed a protective association that appeared to be largely independent of circulating phospholipid metabolites, suggesting potential local mechanisms. These findings provide genetic and metabolic clues for future studies on HSIL etiology.

COL11A2↗

PubMed2006

Integrating genetic and gene expression data: application to cardiovascular and metabolic traits in mice.

The millions of common DNA variations that occur in the human population, or among inbred strains of mice and rats, perturb the expression (transcript levels) of a large fraction of the genes expressed in a particular tissue. The hundreds or thousands of common cis-acting variations that occur in the population may in turn affect the expression of thousands of other genes by affecting transcription factors, signaling molecules, RNA processing, and other processes that act in trans. The levels of transcripts are conveniently quantitated using expression arrays, and the cis- and trans-acting loci can be mapped using quantitative trait locus (QTL) analysis, in the same manner as loci for physiologic or clinical traits. Thousands of such expression QTL (eQTL) have been mapped in various crosses in mice, as well as other experimental organisms, and less detailed maps have been produced in studies of cells from human pedigrees. Such an integrative genetics approach (sometimes referred to as "genetical genomics") is proving useful for identifying genes and pathways that contribute to complex clinical traits. The coincidence of clinical trait QTL and eQTL can help in the prioritization of positional candidate genes. More importantly, mathematical modeling of correlations between levels of transcripts and clinical traits in genetic crosses can allow prediction of causal interactions and the identification of "key driver" genes. An important objective of such studies will be to model biological networks in physiologic processes. When combined with high-density single nucleotide polymorphism (SNP) mapping, it should be feasible to identify genes that contribute to transcript levels using association analysis in outbred populations. In this review we discuss the basic concepts and applications of this integrative genomic approach to cardiovascular and metabolic diseases.

Animals↗

PubMed2007

Genotyping of single-nucleotide polymorphisms by primer extension reaction in a dry-reagent dipstick format.

The primer extension (PEXT) reaction is the most widely used approach to genotyping of single-nucleotide polymorphisms (SNPs). Current methods for analysis of PEXT reaction products are based on electrophoresis, fluorescence resonance energy transfer, fluorescence polarization, pyrosequencing, mass spectrometry, microarrays, and spectrally encoded microspheres. We report the first dry-reagent dipstick method that enables rapid visual detection of PEXT products without instrumentation. The method is applied to the analysis of six SNPs in the mannose-binding lectin gene (MBL2). The genomic region that spans each SNP of interest is amplified by PCR. Two primer extension reactions are performed with allele-specific primers (for one or the other variant nucleotide), which contain an oligo(dA) segment at the 5'-end. Biotin-dUTP is incorporated in the extended strand. The product is applied to the strip followed by immersion in the appropriate buffer. As the DNA moves along the strip by capillary action, it hybridizes with oligo(dT)-functionalized gold nanoparticles, such that only extended products are captured by immobilized streptavidin at the test zone, generating a red line. A second red line is formed at the control zone of the strip by hybridization of the nanoparticles with immobilized oligo(dA). The dipstick test is complete within 10 min. We analyzed six SNPs of the mannose-binding lectin gene (MBL2) using genomic DNA from 27 patients, representing a total of 74 variant nucleotide positions. Patient genotypes showed 100% concordance with direct DNA sequencing data. The described PEXT-dipstick assay is rapid and highly accurate; it does not require specialized instrumentation or highly trained technical personnel. It is appropriate for a diagnostic laboratory where a few selected SNP markers are examined per patient with a low cost per assay.

DNA Primers↗

PubMed2003

Pyrosequencing for SNP genotyping.

Explore the source record for details and available documents.

Data Interpretation, Statistical↗

PubMed2006

Fine mapping of the keratoconus with cataract locus on chromosome 15q and candidate gene analysis.

PURPOSE: To report the fine mapping of the keratoconus with cataract locus on chromosome 15q and the mutational analysis of positional candidate genes. METHODS: Genotyping of two novel microsatellite markers and a single nucleotide polymorphism (SNP) in the critical region of linkage for keratoconus with cataract on 15q was performed. Positional candidate genes (MORF4L1, KIAA1055, ETFA, AWP1, REC14, KIAA1199, RCN2, FAH, IDH3A, MTHFS, ADAMTS7, MAN2C1, PTPN9, KIAA1024, ARNT2, BCL2A1, ISL2, C15ORF22 (P24B), DNAJA4, FLJ14594, CIB2 (KIP2), C15ORF5, and PSMA4) prioritized on the basis of ocular expression and probable function were screened by PCR-based DNA sequencing methods. RESULTS: We report the refinement of the linkage region for keratoconus with cataract to an interval of approximately 5.5 Mb flanked by the MAN2C1 gene and the D15S211 marker on chromosome 15q. Mutational analysis of positional candidate genes detected many sequence variations and single nucleotide polymorphisms. None of the sequence variants were considered pathogenic as they were also found in unaffected family members and normal control DNA samples. CONCLUSIONS: Fine mapping of the keratoconus with cataract locus on 15q has reduced the linked region to 5.5 Mb, thereby excluding 28 candidate genes. A further 23 candidate genes were excluded by direct sequencing methods, although a pathogenic genomic rearrangement or exonic deletion would not have been detected.

Case-Control Studies↗

PubMed2006

The T393C polymorphism in the gene GNAS1 of G protein is associated with survival of patients with invasive breast carcinoma.

The GNAS1 locus encodes the G(alpha)s protein which stimulates the formation of cyclic AMP (cAMP). Subsequently the cAMP pathway mediates various pleiotropic effects including regulation of apoptosis and proliferation. We have recently shown that genotypes of the single nucleotide polymorphism (SNP) T393C in the gene GNAS1 are associated with survival of patients suffering from bladder, renal cell and colorectal carcinoma. In the present study, the genotypes of the T393C SNP were determined in 279 patients with invasive breast carcinoma. Comparing the genotypes with the overall survival as well as important clinico-pathological parameters showed that carriers of the T allele had a significantly less favourable course of the disease when compared to carriers of the homozygous CC genotype. In multivariate analysis the homozygous TT genotype was independently associated with a decreased overall survival. Our results suggest that the GNAS1 T393C SNP is a novel genetic host factor for disease progression in patients with invasive breast carcinoma.

Adult↗

PubMed2003

Different applications of polymerases with and without proofreading activity in single-nucleotide polymorphism analysis.

With the completion of the human genome project, single-nucleotide polymorphisms (SNPs) have become the focus of intense study in biomedical research. Polymerase-mediated primer extension has been employed in a variety of SNP assays. However, these SNP assays using polymerase without proofreading function are compromised by their low reliability. Using a newly developed short amplicon harboring restriction enzyme site, EcoR-I, we were able to compare the single-base discrimination abilities of polymerases with and without proofreading function in primer extension in a broad range of annealing temperatures. Thermodynamic analysis demonstrated a striking single-nucleotide discrimination ability of polymerases with proofreading function. Using unmodified 3'-end allele-specific primers, only template-dependent products were generated by polymerase with proofreading activity. This powerful single-base discrimination ability of exo(+) polymerases was further evaluated in primer extension using three types of 3' terminally modified allele-specific primers. As compared with the poor fidelity in primer extension of polymerases lacking 3' exonuclease activity, this study provides convincing evidence that the use of proofreading polymerases in combination with 3'-end modified allele-specific primers can be a powerful new strategy for the development of SNP assays.

Base Sequence↗

PubMed2005

Association study of detoxification genes in age related macular degeneration.

BACKGROUND/AIMS: Age related macular degeneration (AMD) is a complex disorder leading to loss of central vision, and identification of risk factors associated with susceptibility to AMD has been a key objective for ophthalmic genetics research for almost a decade. This association study has examined genetic polymorphisms in 12 candidate genes as possible risk factors for predisposition to the development of the exudative variant of AMD in a Northern Irish population. The choice of genes was based on their function in the breakdown of industrial pollutants, cigarette smoke, defence against oxidative stress, or involvement in the general ageing process. METHODS: Up to five single nucleotide polymorphisms (SNPs) were typed for CYP1A1, CYP1A2, CYP2E1, CYP2D6, EPHX1, MnSOD, AhR, NAT2, CAT, GPX1, PON1, and ADPRT1 by multiplex snapshot single base primer extension method. Genes showing high linkage disequilibrium (LD) between SNPs were analysed by haplotype analysis. Genes showing low LD were assessed using individual SNPs based on genotypes. RESULTS: After correction for number of genes/SNPs tested, no significant association with AMD was found although several genes merit further investigation. This study suggests that a coding SNP in EPHX1 (Y113H) may be important in AMD and supports a previous observation of an association with exudative AMD. In addition, haplotype analysis highlighted ADPRT1, CYP2D6, and AhR as worthy of further study. CONCLUSION: This study has identified a number of genes requiring further investigation including EPHX1, ADPRT1, CYP2D6, and AhR.

Aged↗