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PubMed2001

A high-throughput SNP typing system for genome-wide association studies.

One of the most difficult issues to be solved in genome-wide association studies is to reduce the amount of genomic DNA required for genotyping. Currently available technologies require too large a quantity of genomic DNA to genotype with hundreds or thousands of single-nucleotide polymorphisms (SNPs). To overcome this problem, we combined the Invader assay with multiplex polymerase chain reaction (PCR), carried out in the presence of antibody to Taq polymerase, as well as using a novel 384-well card system that can reduce the required reaction volume. We amplified 100 genomic DNA fragments, each containing one SNP, in a single tube, and analyzed each SNP with the Invader assay. This procedure correctly genotyped 98 of the 100 SNP loci examined in PCR-amplified samples from ten individuals: the genotypes were confirmed by direct sequencing. The reproducibility and universality of the method were confirmed with two additional sets of 100 SNPs. Because we used 40 ng of genomic DNA as a template for multiplex PCR, the amount needed to assay one SNP was only 0.4 ng; therefore, theoretically, more than 200,000 SNPs could be genotyped at once when 100 microg of genomic DNA is available. Our results indicate the feasibility of undertaking genome-wide association studies using blood samples of only 5-10 ml.

Alleles↗

PubMed2026

SeqQC-former: A sequence-quality fusion framework for QC-aware review prioritization of candidate somatic SNVs in cancer genomics.

The accurate prioritization of candidate somatic single-nucleotide variants (SNVs) remains a challenge due to the substantial variability in sequencing quality across genomic loci. SeqQC-Former is a sequence-quality fusion framework that integrates the local nucleotide context with read-level quality-control (QC) covariates derived from matched tumor-normal sequencing data. This integration generates QC-aware prioritization scores for the downstream review of candidate variants. Unlike conventional variant callers, SeqQC-Former is designed not to infer biological truth but to support post-calling review and prioritization under heterogeneous sequencing conditions. The framework was trained and evaluated on a SEQC2-derived dataset comprising 89,447 candidate loci, including 1378 positive and 88,069 negative loci. In chromosome-held-out validation, which aims to reduce potential genomic-position leakage, SeqQC-Former demonstrated strong discrimination (AUROC = 0.9479; AUPRC = 0.9448), indicating good generalization to previously unseen chromosomes. Given that the SEQC2-derived labels contain QC-associated information; these results should be interpreted as an evaluation of QC-aware prioritization capability rather than an independent validation of biological variant correctness. Ablation analyses revealed that structured QC covariates provided the dominant predictive signal under the current SEQC2-derived labeling regime. SeqQC-Former achieved a significantly higher AUROC than classical machine-learning baselines, as determined by DeLong's test (p < 0.01). Application to 53,164 glioblastoma variants demonstrated that external predictions were sensitive to QC scaling and threshold selection, underscoring that model outputs should be interpreted as QC-dependent prioritization scores rather than calibrated probabilities or definitive biological classifications. Overall, SeqQC-Former offers a reproducible post-calling QC-aware prioritization framework for large-scale somatic SNV review and underscores the importance of explicitly modeling sequencing-quality information when interpreting structured cancer genomics datasets.

Humans↗

PubMed2003

DNA polymerase X of African swine fever virus: insertion fidelity on gapped DNA substrates and AP lyase activity support a role in base excision repair of viral DNA.

DNA polymerase X (pol X) from African swine fever virus (ASFV) is the smallest naturally ocurring DNA-directed DNA polymerase (174 amino acid residues) described so far. Previous biochemical analysis has shown that ASFV pol X is a highly distributive, monomeric enzyme, lacking a proofreading 3'-5' exonuclease. Also, ASFV pol X binds intermediates of the single-nucleotide base excision repair (BER) process, and is able to efficiently repair single-nucleotide gapped DNA. In this work, we perform an extensive kinetic analysis of single correct and incorrect nucleotide insertions by ASFV pol X using different DNA substrates: (i) a primer/template DNA; (ii) a 1nt gapped DNA; (iii) a 5'-phosphorylated 1nt gapped DNA. The results obtained indicate that ASFV pol X exhibits a general preference for insertion of purine deoxynucleotides, especially dGTP opposite template C. Moreover, ASFV pol X shows higher catalytic efficiencies when filling in gapped substrates, which are increased when a phosphate group is present at the 5'-margin of the gap. Interestingly, ASFV pol X misinserts nucleotides with frequencies from 10(-4) to 10(-5), and the insertion fidelity varies depending on the substrate, being more faithful on a phosphorylated 1nt gapped substrate. We have analyzed the capacity of ASFV pol X to act on intermediates of BER repair. Although no lyase activity could be detected on preincised 5'-deoxyribose phosphate termini, ASFV pol X has lyase activity on unincised abasic sites. Altogether, the results support a role for ASFV pol X in reparative BER of damaged viral DNA during ASFV infection.

African Swine Fever↗

PubMed2000

Automated identification of amino acid sequence variations in proteins by HPLC/microspray tandem mass spectrometry.

Amino acid sequence variations resulting from single-nucleotide polymorphisms (SNPs) were identified using a novel mass spectrometric method. This method obtains 99+% protein sequence coverage for human hemoglobin in a single LC-microspray tandem mass spectrometry (microLC-MS/MS) experiment. Tandem mass spectrometry data was analyzed using a modified version of the computer program SEQUEST to identify the sequence variations. Conditions of sample preparation, chromatographic separation, and data collection were optimized to correctly identify amino acid changes in six variants of human hemoglobin (Hb C, Hb E, Hb D-Los Angeles, Hb G-Philadelphia, Hb Hope, and Hb S). Hemoglobin proteins were isolated and purified, dehemed, (S)-carboxyami-domethylated, and then subjected to a combination proteolytic digestion to obtain a complex peptide mixture with multiple overlaps in sequence. Reversed-phase chromatographic separation of peptides was achieved on-line with MS utilizing a robust fritless microelectrospray interface. Tandem mass spectrometry was performed on an ion trap mass spectrometer using automated data-dependent MS/MS procedures. Tandem mass spectra were collected from the five most abundant ions in each scan using dynamic and isotopic exclusion to minimize redundancy. The spectra were analyzed by a version of the SEQUEST algorithm modified to identify amino acid substations resulting from SNPs.

Amino Acid Sequence↗

PubMed2003

Multipoint linkage-disequilibrium mapping narrows location interval and identifies mutation heterogeneity.

Single-nucleotide polymorphism (SNP) genotypes were recently examined in an 890-kb region flanking the human gene CYP2D6. Single-marker and haplotype-based analyses identified, with genomewide significance (P < 10-7), a 403-kb interval displaying strong linkage disequilibrium (LD) with predicted poor-metabolizer phenotype. However, the width of this interval makes the location of causal variants difficult: for example, the interval contains seven known or predicted genes in addition to CYP2D6. We have developed the Bayesian fine-mapping software coldmap, which, applied to these genotype data, yields a 95% location interval covering only 185 kb and establishes genomewide significance for a causal locus within the region. Strikingly, our interval correctly excludes four SNPs, which individually display association with genomewide significance, including the SNP showing strongest LD (P < 10-34). In addition, coldmap distinguishes homozygous cases for the major CYP2D6 mutation from those bearing minor mutations. We further investigate a selection of SNP subsets and find that previously reported methods lead to a 38% savings in SNPs at the cost of an increase of <20% in the width of the location interval.

Bayes Theorem↗

PubMed2001

Loss of DNA polymerase beta stacking interactions with templating purines, but not pyrimidines, alters catalytic efficiency and fidelity.

Structures of DNA polymerases bound with DNA reveal that the 5'-trajectory of the template strand is dramatically altered as it exits the polymerase active site. This distortion provides the polymerase access to the nascent base pair to interrogate proper Watson-Crick geometry. Upon binding a correct deoxynucleoside triphosphate, alpha-helix N of DNA polymerase beta is observed to form one face of the binding pocket for the new base pair. Asp-276 and Lys-280 stack with the bases of the incoming nucleotide and template, respectively. To determine the role of Lys-280, site-directed mutants were constructed at this position, and the proteins were expressed and purified, and their catalytic efficiency and fidelity were assessed. The catalytic efficiency for single-nucleotide gap filling with the glycine mutant (K280G) was strongly diminished relative to wild type for templating purines (>15-fold) due to a decreased binding affinity for the incoming nucleotide. In contrast, catalytic efficiency was hardly affected by glycine substitution for templating pyrimidines (<4-fold). The fidelity of the glycine mutant was identical to the wild type enzyme for misinsertion opposite a template thymidine, whereas the fidelity of misinsertion opposite a template guanine was modestly altered. The nature of the Lys-280 side-chain substitution for thymidine triphosphate insertion (templating adenine) indicates that Lys-280 "stabilizes" templating purines through van der Waals interactions.

Arginine↗

PubMed2004

Association of protein kinase C alpha (PRKCA) gene with multiple sclerosis in a UK population.

Twin, family and adoption studies suggest that susceptibility to multiple sclerosis is substantially mediated by genetic factors. Linkage to human chromosome 17q, homologous to a locus linked to experimental animal models of multiple sclerosis, has been widely replicated and the region likely to harbour a multiple sclerosis susceptibility gene has recently been refined to a 2.5 Mb region of 17q22-24. The candidate multiple sclerosis susceptibility gene, protein kinase C alpha (PRKCA), maps within this interval and association with 35 single-nucleotide polymorphism (SNP) markers, spanning the gene with a median spacing of 7.8 kb, was tested using a case-control approach. Single-marker genotype and estimated haplotype frequencies were compared in UK unrelated cases with multiple sclerosis (n = 184) and healthy controls (n = 340) in order to investigate association with susceptibility to disease. A haplotype of two SNPs mapping to the proximal region of the gene showed evidence for association with susceptibility (Bonferroni-corrected P value = 1.1 x 10(-5)). These findings suggest that further investigation of the PRKCA gene is warranted, particularly in cohorts with evidence of linkage to 17q22. Most of the SNPs investigated in this study were intronic and screening to identify disease-associated functional mutations is now required. Our results suggest that the promoter and proximal gene region should be not only included but prioritized in any screening strategy.

Adult↗

PubMed2005

Non-cross-linking gold nanoparticle aggregation as a detection method for single-base substitutions.

Aggregation of DNA-modified gold nanoparticles in a non-cross-linking configuration has extraordinary selectivity against terminal mismatch of the surface-bound duplex. In this paper, we demonstrate the utility of this selectivity for detection of single-base substitutions. The samples were prepared through standard protocols: DNA extraction, PCR amplification and single-base primer extension. Oligonucleotide-modified nanoparticles correctly responded to the unpurified products from the primer extension: aggregation for the full match and dispersion for all the mismatches. Applicability of this method to genomic DNA was tested with five human tumor cell lines, and verified by conventional technologies: mass spectrometry and direct sequencing. Unlike the existing methods for single-base substitution analysis, this method does not need specialized equipments, and opens up a new possibility of point-of-care diagnosis for single-nucleotide polymorphisms.

Biosensing Techniques↗

PubMed2000

A microsphere-based assay for multiplexed single nucleotide polymorphism analysis using single base chain extension.

A rapid, high throughput readout for single-nucleotide polymorphism (SNP) analysis was developed employing single base chain extension and cytometric analysis of an array of fluorescent microspheres. An array of fluorescent microspheres was coupled with uniquely identifying sequences, termed complementary ZipCodes (cZipCodes), which allowed for multiplexing possibilities. For a given assay, querying a polymorphic base involved extending an oligonucleotide containing both a ZipCode and a SNP-specific sequence with a DNA polymerase and a pair of fluoresceinated dideoxynucleotides. To capture the reaction products for analysis, the ZipCode portion of the oligonucleotide was hybridized with its cZipCodes on the microsphere. Flow cytometry was used for microsphere decoding and SNP typing by detecting the fluorescein label captured on the microspheres. In addition to multiplexing capability, the ZipCode system allows multiple sets of SNPs to be analyzed by a limited set of cZipCode-attached microspheres. A standard set of non-cross reactive ZipCodes was established experimentally and the accuracy of the system was validated by comparison with genotypes determined by other technologies. From a total of 58 SNPs, 55 SNPs were successfully analyzed in the first pass using this assay format and all 181 genotypes across the 55 SNPs were correct. These data demonstrate that the microsphere-based single base chain extension (SBCE) method is a sensitive and reliable assay. It can be readily adapted to an automated, high-throughput genotyping system. [Primer sequences used in this study are available as online supplementary materials at www.genome.org.]

DNA, Complementary↗

PubMed2005

Haplotyping of TNFalpha gene promoter using melting temperature analysis: detection of a novel -856(G/A) mutation.

Tumor necrosis factor alpha (TNFalpha) is a potent cytokine with a wide range of pro-inflammatory activities and plays a critical role in the pathogenesis of a number of infectious, inflammatory, autoimmune, and metabolic diseases. We determined four single-nucleotide polymorphisms (SNPs), -1031(C/T), -863(C/A), -857(C/T), and -308(G/A) in the TNFalpha promoter region using melting temperature analysis, among 1451 geriatric autopsy samples. Two adjacent SNPs, -863(C/A) and -857(C/T), were directly assayed by a single probe reaction, which correctly determined three of four expected haplotypes. Sequence confirmation related that the most rare haplotype (8/2902 chromosomes, frequency: 0.26%) contains a novel mutation of -856(G/A), instead of the predicted haplotype. These results indicate that melting temperature analysis provides a robust method to determine the polymorphisms in the TNFalpha promoter.

Haplotypes↗

PubMed2006

Polymorphisms of the low-density lipoprotein receptor-related protein 5 (LRP5) gene are associated with obesity phenotypes in a large family-based association study.

BACKGROUND: The low-density lipoprotein receptor-related protein 5 (LRP5) gene, essential for glucose and cholesterol metabolism, may have a role in the aetiology of obesity, an important risk factor for diabetes. PARTICIPANTS AND METHODS: To investigate the association between LRP5 polymorphisms and obesity, 27 single-nucleotide polymorphisms (SNPs), spacing about 5 kb apart on average and covering the full transcript length of the LRP5 gene, were genotyped in 1873 Caucasian people from 405 nuclear families. Obesity (defined as body mass index (BMI) >30 kg/m(2)) and three obesity-related phenotypes (BMI, fat mass and percentage of fat mass (PFM)) were investigated. RESULTS: Single markers (12 tagging SNPs and 4 untaggable SNPs) and haplotypes (5 blocks) were tested for associations, using family-based designs. SNP4 (rs4988300) and SNP6 (rs634008) located in block 2 (intron 1) showed significant associations with obesity and BMI after Bonferroni correction (SNP4: p<0.001 and p = 0.001, respectively; SNP6: p = 0.002 and 0.003, respectively). The common allele A for SNP4 and minor allele G for SNP6 were associated with an increased risk of obesity. Significant associations were also observed between common haplotype A-G-G-G of block 2 with obesity, BMI, fat mass and PFM with global empirical values p<0.001, p<0.001, p = 0.003 and p = 0.074, respectively. Subsequent sex-stratified analyses showed that the association in the total sample between block 2 and obesity may be mainly driven by female subjects. CONCLUSION: Intronic variants of the LRP5 gene are markedly associated with obesity. We hypothesise that such an association may be due to the role of LRP5 in the WNT signalling pathway or lipid metabolism. Further functional studies are needed to elucidate the exact molecular mechanism underlying our finding.

Adult↗

PubMed2006

Association study with 33 single-nucleotide polymorphisms in 11 candidate genes for hypertension in Chinese.

Essential hypertension is considered to be a typical complex disease with multifactorial etiology, which leads to inconsistent findings in genetic studies. One possibility of failure to replicate some single-locus results is that the underlying genetics of hypertension are not only based on multiple genes with minor effects but also on gene-gene interactions. To test this hypothesis, a case-control study was constructed in Chinese subjects, detecting both single locus and multilocus effects. Eleven candidate genes were selected from biochemical pathways that have been implicated in the development and progression of hypertension, and 33 polymorphisms were evaluated in 503 hypertension patients and 490 age- and gender-matched controls. Single-locus associations, using traditional logistic regression analyses, and multilocus associations, using classification and regression trees and multivariate adaptive regression splines, were both explored in this study. Final models were selected using either Bonferroni correction or cross-validation. Three polymorphisms, TH*rs2070762, ADRB2*Q27E, and GRK4*A486V, were found to be independently associated with essential hypertension in Chinese subjects. In addition to these individual predictors, a potential interaction of CYP11B2-AGTR1 is also involved in the etiology of hypertension. These findings support the multigenic nature of the etiology of essential hypertension and propose a potential gene-gene interactive model for future studies.

Alanine↗

PubMed2005

Searching for epistatic interactions in nuclear families using conditional linkage analysis.

BACKGROUND: Genomic screens generally employ a single-locus strategy for linkage analysis, but this may have low power in the presence of epistasis. Ordered subsets analysis (OSA) is a method for conditional linkage analysis using continuous covariates. METHODS: We used OSA to evaluate two-locus interactions in the simulated Genetic Analysis Workshop 14 dataset. We used all nuclear families ascertained by Aipotu, Karangar, and Danacaa. Using the single-nucleotide polymorphism map, multipoint affected-sibling-pair (ASP) linkage analysis was performed on all 100 replicates for each chromosome using SIBLINK. OSA was used to examine linkage on each chromosome using LOD scores at each 3-cM location on every other chromosome as covariates. Two methods were used to identify positive results: one searching across the entire covariate chromosome, the other conditioning on location of known disease loci. RESULTS: Single-locus linkage analysis revealed very high LOD scores for disease loci D1 through D4, with mean LOD scores over 100 replicates ranging from 4.0 to 7.8. Although OSA did not obscure this linkage evidence, it did not detect the simulated interactions between any of the locus pairs. We found inflated type I error rates using the first OSA method, highlighting the need to correct for multiple comparisons. Therefore, using "null chromosome pairs" without simulated disease loci, we calculated a corrected alpha-level. CONCLUSION: We were unable to detect two-locus interactions using OSA. This may have been due to lack of incorporation of phenotypic subgroups, or because linkage evidence as summarized by LOD scores performs poorly as an OSA covariate. We found inflated type I error rates, but were able to calculate a corrected alpha-level for future analyses employing this strategy to search for two-locus interactions.

Chromosome Mapping↗

PubMed2003

Multiplex minisequencing screen for common Southeast Asian and Indian beta-thalassemia mutations.

BACKGROUND: Beta-thalassemia is endemic to many regions in Southeast Asia and India, and <20 beta-globin gene mutations account for > or =90% of beta-thalassemia alleles in these places. We describe a multiplex minisequencing assay to detect these common mutations. METHODS: Gap-PCR was used to simultaneously amplify the beta-globin gene from genomic DNA and to detect the Delta619bp deletion mutation. Multiplex minisequencing was then performed on the amplified beta-globin fragment to detect an additional 15 common Southeast Asian and Indian beta-thalassemia mutations. Site-specific primers of different lengths were subjected to multiple rounds of annealing and single-nucleotide extension in the presence of thermostable DNA polymerase and the four dideoxynucleotides, each labeled with a different fluorophore. Minisequencing products were separated and detected by capillary electrophoresis, followed by automated genotyping. The optimized assay was subjected to a double-blind validation analysis of 89 beta-thalassemia and wild-type DNA samples of known genotype. RESULTS: Homozygous wild-type or mutant DNA samples produced electropherograms containing only a single colored peak for each mutation site, whereas samples heterozygous for a specific mutation displayed two different-colored peaks for that mutation site. Samples were automatically genotyped based on color and position of primer peaks in the electropherogram. In the double-blind validation analysis, all 89 DNA samples were genotyped correctly (100% assay specificity). CONCLUSIONS: The described semiautomated multiplex minisequencing assay can detect the most common Southeast Asian and Indian beta-thalassemia mutations, is amenable to high-throughput scale up, and may bring population-based screening of beta-thalassemia in endemic regions a step closer to implementation.

Asia, Southeastern↗

PubMed2003

Typing single-nucleotide polymorphisms using a gel-based sequencer: a new data analysis tool and suggestions for improved efficiency.

Single-nucleotide polymorphisms (SNPs) are increasingly used as genetic markers. Although a high number of SNP-genotyping techniques have been described, most techniques still have low throughput or require major investments. For laboratories that have access to an automated sequencer, a single-base extension (SBE) assay can be implemented using the ABI SNaPshot trade mark kit. Here we present a modified protocol comprising multiplex template generation, multiplex SBE reaction, and multiplex sample analysis on a gel-based sequencer such as the ABI 377. These sequencers run on a Macintosh platform, but on this platform the software available for analysis of data from the ABI 377 has limitations. First, analysis of the size standard included with the kit is not facilitated. Therefore a new size standard was designed. Second, using Genotyper (ABI), the analysis of the data is very tedious and time consuming. To enable automated batch analysis of 96 samples, with 10 SNPs each, we developed SNPtyper. This is a spreadsheet-based tool that uses the data from Genotyper and offers the user a convenient interface to set parameters required for correct allele calling. In conclusion, the method described will enable any lab having access to an ABI sequencer to genotype up to 1000 SNPs per day for a single experimenter, without investing in new equipment.

Computational Biology↗

PubMed2005

Apolipoprotein E haplotyping by denaturing high-performance liquid chromatography.

The apolipoprotein E ( APOE ) gene in humans contains two single-base polymorphisms in exon 4, which result in three common alleles, conventionally named epsilon2 , epsilon3 and epsilon4 . Numerous studies have shown an important association between the epsilon4 variant and an increased risk of Alzheimer's disease; other data suggest a possible linkage of APOE genetic heterogeneity with lipid profile and an increased risk of atherothrombotic stroke and coronary heart disease. APOE genotyping is therefore an increasingly common assay in laboratory medicine. The most widely used technique for APOE genotyping is based upon restriction isotyping, i.e., amplification of the fragment of exon 4 containing the most common sequence variations, followed by enzymatic digestion of the amplicon and fragments analysis by gel electrophoresis. We developed a novel, reliable and fast method that exploits the sensitivity and specificity of denaturing high-performance liquid chromatography in detecting single-nucleotide polymorphisms. We show that, in most cases, with a single chromatographic separation it is possible to correctly identify the six different APOE allelic patterns, without any manipulation after the polymerase chain reaction amplification. When compared to restriction isotyping, our method is much faster, less labor intensive and similarly inexpensive.

Alleles↗

PubMed2005

New adjustment factors and sample size calculation in a DNA-pooling experiment with preferential amplification.

In the post-genome era, disease gene mapping using dense genetic markers has become an important tool for dissecting complex inheritable diseases. Locating disease susceptibility genes using DNA-pooling experiments is a potentially economical alternative to those involving individual genotyping. The foundation of a successful DNA-pooling association test is a precise and accurate estimation of allele frequency. In this article, we propose two new adjustment methods that correct for preferential amplification of nucleotides when estimating the allele frequency of single-nucleotide polymorphisms. We also discuss the effect of sample size when calibrating unequal allelic amplification. We conducted simulation studies to assess the performance of different adjustment procedures and found that our proposed adjustments are more reliable with respect to the estimation bias and root mean square error compared with the current approach. The improved performance not only improves the accuracy and precision of allele frequency estimations but also leads to more powerful disease gene mapping.

Algorithms↗

PubMed2005

The effect of polymorphisms in the enhancer of split gene complex on bristle number variation in a large wild-caught cohort of Drosophila melanogaster.

The Enhancer of split complex [E(spl)-C] in Drosophila encompasses a variety of functional elements controlling bristle patterning and on the basis of prior work is a strong candidate for harboring alleles having subtle effects on bristle number variation. Here we extend earlier studies identifying associations between complex phenotypes and polymorphisms segregating among inbred laboratory lines of Drosophila and test the influence of E(spl)-C on bristle number variation in a natural cohort. We describe results from an association mapping study using 203 polymorphisms spread throughout the E(spl)-C genotyped in 2000 wild-caught Drosophila melanogaster. Despite power to detect associations accounting for as little as 2% of segregating variation for bristle number, and saturating the region with single-nucleotide polymorphisms (SNPs), we identified no single SNP marker showing a significant (additive over loci) effect after correcting for multiple tests. Using a newly developed test we conservatively identify six regions of the E(spl)-C in which the insertion of transposable elements as a class contributes to variation in bristle number, apparently in a sex- or trait-limited fashion. Finally, we carry out all possible 20,503 two-way tests for epistasis and identify a slight excess of marginally significant interactions, although none survive multiple-testing correction. It may not be straightforward to extend the results of laboratory-based association studies to natural populations.

Animal Structures↗