SNAP: predict effect of non-synonymous polymorphisms on function

Many genetic variations are single nucleotide polymorphisms (SNPs). Non-synonymous SNPs are 'neutral' if the resulting point-mutated protein is not functionally discernible from the wild type and 'non-neutral' otherwise. The ability to identify non-neutral substitutions could significantly aid targeting disease causing detrimental mutations, as well as SNPs that increase the fitness of particular phenotypes. Here, we introduced comprehensive data sets to assess the performance of methods that predict SNP effects. Along we introduced SNAP (screening for non-acceptable polymorphisms), a neural network-based method for the prediction of the functional effects of non-synonymous SNPs. SNAP needs only sequence information as input, but benefits from functional and structural annotations, if available. In a cross-validation test on over 80,000 mutants, SNAP identified 80% of the non-neutral substitutions at 77% accuracy and 76% of the neutral substitutions at 80% accuracy. This constituted an important improvement over other methods; the improvement rose to over ten percentage points for mutants for which existing methods disagreed. Possibly even more importantly SNAP introduced a well-calibrated measure for the reliability of each prediction. This measure will allow users to focus on the most accurate predictions and/or the most severe effects. Available at http://www.rostlab.org/services/SNAP.     

A simple procedure for the analysis of single nucleotide polymorphisms facilitates map-based cloning in Arabidopsis

We developed a modified allele-specific PCR procedure for assaying single nucleotide polymorphisms (SNPs) and used the procedure (called SNAP for single-nucleotide amplified polymorphisms) to generate 62 Arabidopsis mapping markers. SNAP primers contain a single base pair mismatch within three nucleotides from the 3' end of one allele (the specific allele) and in addition have a 3' mismatch with the nonspecific allele. A computer program called SNAPER was used to facilitate the design of primers that generate at least a 1,000-fold difference in the quantity of the amplification products from the specific and nonspecific SNP alleles. Because SNAP markers can be readily assayed by electrophoresis on standard agarose gels and because a public database of over 25,000 SNPs is available between the Arabidopsis Columbia and Landsberg erecta ecotypes, the SNAP method greatly facilitates the map-based cloning of Arabidopsis genes defined by a mutant phenotype.     

Cloning of two classes of PR genes and the development of SNAP markers for powdery mildew resistance loci in chestnut rose (<Emphasis Type="Italic">Rosa roxburghii</Emphasis> Tratt)

Pathogenesis-related (PR) genes were isolated from chestnut rose (Rosa roxburghii Tratt) using a PCR approach with degenerate primers designed for the conserved regions of two PR gene families: class 2 (β-1,3-glucanase) and class 5 (osmotin). Thirteen PR2 and ten PR5 genes were obtained, with a nucleotide identity that ranged from 40.1 to 99.7% and from 99.2 to 99.8%, respectively. Sequence comparison revealed the presence of single nucleotide polymorphisms (SNPs) in these sequences with, on an average, one SNP in every 64-bp fragment for the PR2 genes and one in every 68-bp fragment for the PR5 genes. A total of 23 primers were used to genotype these SNPs for use in developing single nucleotide-amplified polymorphisms (SNAP) markers. One marker (Glu7) was found to be linked to powdery mildew resistance loci. Based on genetic mapping of a segregating F₁ population, we determined that 16 of the 23 SNAP markers formed one group and subsequently detected a quantitative trait locus that accounted for 12% of the variation in the powdery mildew resistance phenotype. The results of this study provide a first insight into the genomic structure of PR genes and show that the candidate gene approach in combination with SNAP markers is an attractive strategy to search for powdery mildew resistance loci in chestnut rose.     

PolyPhred: automating the detection and genotyping of single nucleotide substitutions using fluorescence-based resequencing.

Fluorescence-based sequencing is playing an increasingly important role in efforts to identify DNA polymorphisms and mutations of biological and medical interest. The application of this technology in generating the reference sequence of simple and complex genomes is also driving the development of new computer programs to automate base calling (Phred), sequence assembly (Phrap) and sequence assembly editing (Consed) in high throughput settings. In this report we describe a new computer program known as PolyPhred that automatically detects the presence of heterozygous single nucleotide substitutions by fluorescencebased sequencing of PCR products. Its operations are integrated with the use of the Phred, Phrap and Consed programs and together these tools generate a high throughput system for detecting DNA polymorphisms and mutations by large scale fluorescence-based resequencing. Analysis of sequences containing known DNA variants demonstrates that the accuracy of PolyPhred with single pass data is >99% when the sequences are generated with fluorescent dye-labeled primers and approximately 90% for those prepared with dye-labeled terminators.     

Should We Have Blind Faith in Bioinformatics Software? Illustrations from the SNAP Web-Based Tool

Bioinformatics tools have gained popularity in biology but little is known about their validity. We aimed to assess the early contribution of 415 single nucleotide polymorphisms (SNPs) associated with eight cardio-metabolic traits at the genome-wide significance level in adults in the Family Atherosclerosis Monitoring In earLY Life (FAMILY) birth cohort. We used the popular web-based tool SNAP to assess the availability of the 415 SNPs in the Illumina Cardio-Metabochip genotyped in the FAMILY study participants. We then compared the SNAP output with the Cardio-Metabochip file provided by Illumina using chromosome and chromosomal positions of SNPs from NCBI Human Genome Browser (Genome Reference Consortium Human Build 37). With the HapMap 3 release 2 reference, 201 out of 415 SNPs were reported as missing in the Cardio-Metabochip by the SNAP output. However, the Cardio-Metabochip file revealed that 152 of these 201 SNPs were in fact present in the Cardio-Metabochip array (false negative rate of 36.6%). With the more recent 1000 Genomes Project release, we found a false-negative rate of 17.6% by comparing the outputs of SNAP and the Illumina product file. We did not find any ‘false positive’ SNPs (SNPs specified as available in the Cardio-Metabochip by SNAP, but not by the Cardio-Metabochip Illumina file). The Cohen’s Kappa coefficient, which calculates the percentage of agreement between both methods, indicated that the validity of SNAP was fair to moderate depending on the reference used (the HapMap 3 or 1000 Genomes). In conclusion, we demonstrate that the SNAP outputs for the Cardio-Metabochip are invalid. This study illustrates the importance of systematically assessing the validity of bioinformatics tools in an independent manner. We propose a series of guidelines to improve practices in the fast-moving field of bioinformatics software implementation.     

Positive and negative selection on the human genome.

The distinction between deleterious, neutral, and adaptive mutations is a fundamental problem in the study of molecular evolution. Two significant quantities are the fraction of DNA variation in natural populations that is deleterious and destined to be eliminated and the fraction of fixed differences between species driven by positive Darwinian selection. We estimate these quantities using the large number of human genes for which there are polymorphism and divergence data. The fraction of amino acid mutations that is neutral is estimated to be 0.20 from the ratio of common amino acid (A) to synonymous (S) single nucleotide polymorphisms (SNPs) at frequencies of > or =15%. Among the 80% of amino acid mutations that are deleterious at least 20% of them are only slightly deleterious and often attain frequencies of 1-10%. We estimate that these slightly deleterious mutations comprise at least 3% of amino acid SNPs in the average individual or at least 300 per diploid genome. This estimate is not sensitive to human population history. The A/S ratio of fixed differences is greater than that of common SNPs and suggests that a large fraction of protein divergence is adaptive and driven by positive Darwinian selection.     

Molecular characterisation of camptothecin-induced mutations at the hprt locus in Chinese hamster cells

The capacity of the topoisomerase I inhibitor camptothecin (CPT) to induce single locus mutations at the hypoxanthine-guanine phosphoribosyltransferase (hprt) gene and the DNA changes underlying induced mutations were analysed in Chinese hamster ovary cells. Camptothecin treatments increased hprt mutations up to 50-fold over the spontaneous levels at highly cytotoxic doses. Genomic DNA was isolated from 6-thioguanine resistant clones and subjected to multiplex PCR to screen for gross alterations in the gene structure. The molecular analysis revealed that deletion mutants represented 80% of the analysed clones, including total hprt deletion, multiple and single exon deletions. Furthermore, a fraction of the analysed clones showed deletions of more than one exon that were characterised by the absence of non-contiguous exons. These data show that single locus mutations induced by camptothecin are characterised by large deletions or complex rearrangements rather than single base substitutions and suggest that the recombinational repair of camptothecin-induced strand breaks at replication fork may be involved in the generations of these alterations at the chromatin structure level.     

Mutation analysis in patients with congenital adrenal hyperplasia in the Spanish population: identification of putative novel steroid 21-hydroxylase deficiency alleles associated with the classic form of the disease.

Steroid 21-hydroxylase deficiency, due to the genetic impairment of the CYP21 gene, is a major cause of congenital adrenal hyperplasia (CAH). In about 80% of the cases, the defect is related with the transfer of deleterious point mutations from the CYP21P pseudogene to the active CYP21 gene. Sixteen different point mutations have been searched for in 60 Spanish patients with the classic form of CAH and 171 unaffected family members, using selective amplification of the CYP21 gene followed by allele-specific oligonucleotide hybridization (PCR-ASOH) and sequencing analysis. While 31.9% of the disease alleles carry CYP21 deletions or large gene conversions, around 58% of the alleles carry single point mutations. Corresponding segregation of mutations was found in every case indicating that none of them has apparently appeared de novo. The most frequent mutations found in our sample are i2G, V281L, R356W, Q318X, P453S and F306+t, with rates of 30, 14.2, 10, 9.2, 9.2 and 7. 5%, respectively. We found similar frequencies for the A and C polymorphism at position 656 (40 and 31.5%, respectively) in wild-type alleles for the i2G mutation. Around 10% of the alleles, for which no mutations were identified by searching for the sixteen previously known mutations, are currently being sequenced and new possible mutations and polymorphisms have been identified.     

Dual actions of nitric oxide on angiogenesis: possible roles of PKC, ERK, and AP-1

Regulation of angiogenesis by nitric oxide (NO) is controversial since NO has been shown to have both pro- and anti-angiogenic effects. In this study, we examined the effect of the NO donor, S-nitro-N-acetyl-penicillamine (SNAP), on in vitro angiogenesis, and the mechanisms involved: PKC activity, ERK and c-Jun phosphorylation, and AP-1 DNA binding activity, in microvascular endothelial cells. SNAP, at 0.5-4 mM, significantly and dose-dependently inhibited angiogenesis, PKC activity, and ERK and c-Jun phosphorylation up to 80%, 83%, and 63% and 73%, respectively. SNAP at concentrations > 2mM also abolished AP-1 binding activity. Lower concentrations of SNAP (0.1-0.3 mM) significantly increased angiogenesis, PKC activity, and ERK and c-Jun phosphorylation up to 46%, 60%, and 61% and 180%, respectively. These findings indicate that the dual pro- and anti-angiogenic actions of NO are dose-dependent and suggest that they are mediated by PKC and ERK acting on AP-1.     

Automated correction of genome sequence errors

By using information from an assembly of a genome, a new program called AutoEditor significantly improves base calling accuracy over that achieved by previous algorithms. This in turn improves the overall accuracy of genome sequences and facilitates the use of these sequences for polymorphism discovery. We describe the algorithm and its application in a large set of recent genome sequencing projects. The number of erroneous base calls in these projects was reduced by 80%. In an analysis of over one million corrections, we found that AutoEditor made just one error per 8828 corrections. By substantially increasing the accuracy of base calling, AutoEditor can dramatically accelerate the process of finishing genomes, which involves closing all gaps and ensuring minimum quality standards for the final sequence. It also greatly improves our ability to discover single nucleotide polymorphisms (SNPs) between closely related strains and isolates of the same species.     

Mutational analysis of the serotonin receptor 5HT2c in severe early-onset human obesity

Deletion of the serotonin receptor 5HT2c in mice results in increased food intake and obesity. We screened 95 individuals with severe early-onset obesity for mutations in the coding sequence of this gene. We found a novel missense variant c.1255A > G (Thr419Ala) in a single Caucasian subject that was not found in 192 Caucasian control subjects. In transiently-transfected COS cells, the Thr419Ala variant was indistinguishable from the wild-type receptor in its ability to generate inositol phosphate, although differences in coupling to other pathways were not excluded. Three previously unreported silent variants: IVS3 + 30G > A, IVS3 + 80C > G and IVS4 - 31A > G were found with prevalences of 11.5%, 0.5% and 17.9%, respectively. In conclusion, mutations in 5HT2c are unlikely to be a common cause of severe early-onset human obesity. The identification of several novel polymorphisms at this locus may aid future genetic epidemiological studies.     

上肢长骨的性别判别分析研究

本文对100例(男女各50)国人上肢骨进行了36项测量。统计结果显示所有测量项目均值男性都大于女性并具有显著性的差异。本研究表明可以采用单一指标对破损严重的肢骨进行性别鉴定。36个测量项目中有23项单一指标性别判别率达75％以上,其中9项在80％以上。本文采用Fisher判别分析法建立了56项单一肢骨性别判别函数,判别率为80％—87％。为进一步提高判别效果,采用逐步判别分析法建立了四项逐步性别判别函数,判别率达90％以上。     

Denaturing high performance liquid chromatography (DHPLC) used in the detection of germline and somatic mutations.

Denaturing high performance liquid chromatography (DHPLC) has been described recently as a method for screening DNA samples for single nucleotide polymorphisms and inherited mutations. Thirty-eight DNAs, 22 of which were heterozygous for previously characterized rearranged transforming gene (RET) or cystic fibrosis transmembrane conductance regulator gene (CFTR) mutations or polymorphisms, were examined using DHPLC analysis to assess the accuracy of this scanning method. Ninety-one per cent (20/22) of the PCR amplicons from specimens with heterozygous RET or CFTR sequence showed elution profiles distinct from corresponding homozygous normal patterns; whether the profiles for two amplicons containing heterozygous RET sequence were distinct from homozygous cases was equivocal. To investigate the usefulness of this method for detecting mutations in tumor DNAs, each of the phosphatase and tensin homologue deleted on chromosome ten gene (PTEN) exons were examined for mutations in 63 malignant gliomas. Seventeen PTEN PCR products from this series of brain tumors showed elution profiles indicating sample heterozygosity and in each instance conventional sequencing confirmed the presence of a mutation. PTEN amplicons containing exons 1, 3 and 5 were sequenced for each of the 63 tumor DNAs to determine whether any mutations may have escaped DHPLC detection, and this analysis identified one such alteration in addition to the eight mutations that DHPLC had revealed. In total, DHPLC identified 37 of 40 (92.5%) PCR products containing defined sequence variation and no alterations were indicated among 196 amplicons containing homozygous normal sequence.     

In Vivo Validation of Simultaneous Non-Contrast Angiography and intraPlaque Hemorrhage (SNAP) Magnetic Resonance Angiography: An Intracranial Artery Study

Objectives

Simultaneous Non-contrast Angiography and intraPlaque hemorrhage (SNAP) technique was recently proposed for joint MRA and intraplaque hemorrhage (IPH) imaging. The purpose of this study is to validate SNAP’s MRA performance in patients with suspected intracranial artery disease.

Methods

SNAP and time-of-flight (TOF) techniques with matched field of view and resolution were applied on 15 patients with suspected intracranial artery disease. Both techniques were evaluated based on their detection of luminal stenosis of bilateral middle cerebral arteries (MCA) and the delineation of smallest visible branches (SVB) of the MCA. Statistical analysis was conducted on the artery level.

Results

The SNAP MRA was found to provide similar stenosis detection performance when compared with TOF (Cohen’s κ 0.79; 95% Confidence Interval: 0.56–0.99). For the SVB comparison, SNAP was found to provide significantly better small artery delineation than TOF (p = 0.017). Inter-reader reproducibility for both measurements on SNAP was over 0.7. SNAP also detected IPH lesions on 13% of the patients.

Conclusions

The SNAP technique’s MRA performance was optimized and compared against TOF for intracranial artery atherosclerosis imaging and was found to provide comparable stenosis detection accuracy. Along with its IPH detection capability, SNAP holds the potential to become a first-line screening tool for high risk intracranial atherosclerosis disease evaluation.     

Evolutionary dynamics of viral attenuation

The genetic trajectory leading to viral attenuation was studied in a canine parvovirus (CPV) strain grown on dog kidney cells for 115 transfers. Consensus sequences of viral populations at passages 0, 3, 30, 50, 80, and 115 were obtained from PCR products covering 86% of the genome; clones from each of the 80th and 115th passages were also sequenced, covering 69% of the genome. Sixteen changes were fixed in the 115th-passage virus sample. Levels of polymorphism were strikingly different over time, in part because of a plaque-cloning step at passage 112 that reduced variation: passage 80 had 19 variants common among the clones, but passage 115 had only a single common variant. Several mutations increased in the culture at the same time, with most reaching fixation only after the 80th passage. The pattern of evolution was consistent with recombination and not with separate selective sweeps of individual mutations. Thirteen of the changes observed were identical to or at the same positions as changes observed in other isolates of CPV or feline panleukopenia virus.     

Pitfalls in the molecular genetic diagnosis of Leber hereditary optic neuropathy (LHON).

Pathogenetic mutations in mtDNA are found in the majority of patients with Leber hereditary optic neuropathy (LHON), and molecular genetic techniques to detect them are important for the diagnosis. A false-positive molecular genetic error has adverse consequences for the diagnosis of this maternally inherited disease. We found a number of mtDNA polymorphisms that occur adjacent to known LHON-associated mutations and that confound their molecular genetic detection. These transition mutations occur at mtDNA nt 11779 (SfaNI site loss, 11778 mutation), nt 3459 (BsaHI site loss, 3460 mutation), nt 15258 (AccI site loss, 15257 mutation), nt 14485 (mismatch primer Sau3AI site loss, 14484 mutation), and nt 13707 (BstNI site loss, 13708 mutation). Molecular genetic detection of the most common pathogenetic mtDNA mutations in LHON, using a single restriction enzyme, may be confounded by adjacent polymorphisms that occur with a false-positive rate of 2%-7%.     

Prediction of solvent accessibility and sites of deleterious mutations from protein sequence

Residues that form the hydrophobic core of a protein are critical for its stability. A number of approaches have been developed to classify residues as buried or exposed. In order to optimize the classification, we have refined a suite of five methods over a large dataset and proposed a metamethod based on an ensemble average of the individual methods, leading to a two-state classification accuracy of 80%. Many studies have suggested that hydrophobic core residues are likely sites of deleterious mutations, so we wanted to see to what extent these sites can be predicted from the putative buried residues. Residues that were most confidently classified as buried were proposed as sites of deleterious mutations. This proposition was tested on six proteins for which sites of deleterious mutations have previously been identified by stability measurement or functional assay. Of the total of 130 residues predicted as sites of deleterious mutations, 104 (or 80%) were correct.     

Antiangiogenesis efficacy of nitric oxide donors

Angiogenesis is a complex process involving endothelial cell migration, proliferation, invasion, and tube formation. Inhibition of these processes might have implications in various angiogenesis‐mediated disorders. Because nitric oxide (NO) is known to play a key role in various vascular diseases, the present study was undertaken to determine the role of NO in angiogenesis‐mediated processes using the NO donor, S‐nitroso N‐acetyl penicillamine (SNAP) and S‐nitroso N‐acetyl glutathione (SNAG). The antiangiogenic efficacy of these NO donors was examined using in vivo and in vitro model systems. The in vitro studies demonstrated the ability of SNAP to inhibit cytokine fibroblast growth factor (FGF2)‐stimulated tube formation and serum‐induced cell proliferation. The inhibitory effect on cell proliferation by SNAP concentrations above the millimolar range was associated with significant shifts in the concentration of NO metabolites. Furthermore, using the mouse Matrigel implant model and the chick chorioallantoic membrane (CAM) models, SNAP demonstrated maximal inhibitory efficacy (85–95% inhibition) of cytokine (FGF2)‐induced neovascularization in both in vivo models. SNAP and SNAG resulted in 85% inhibition of FGF2‐induced neovascularization in the mouse Matrigel model when given at 5 mg/kg/day infusion in minipumps during 14 days and 87% inhibition of angiogenesis induced by FGF2 in the CAM when administered a single dose of 50 μg. Thus, NO donors might be a useful tool for the inhibition of angiogenesis associated with human tumor growth, or neovascular, ocular, and inflammatory diseases. J. Cell. Biochem. 80:104–114, 2000. © 2000 Wiley‐Liss, Inc.     

Mapping SNPs to protein sequence and structure data

MOTIVATION: Data on both single nucleotide polymorphisms and disease-related mutations are being collected at ever-increasing rates. To understand the structural effects of missense mutations, we consider both classes under the term single amino acid polymorphisms (SAAPs) and we wish to map these to protein structure where their effects can be analyzed. Our initial aim therefore is to create a completely automatically maintained database of SAAPs mapped to individual residues in the Protein Data Bank (PDB) updated as new mutations or structures become available. RESULTS: We present an integrated pipeline for the automated mapping of SAAP data from HGVbase to individual PDB residues. Achieving this in a completely automated and reliable manner is a complex task. Data extracted from HGVbase are mapped to EMBL entries to confirm whether the mutation occurs in an exon and, if so, where in the sequence it occurs. From there we map to Swiss-Prot entries and thence to the PDB. AVAILABILITY: The resulting database may be accessed over the web at http://www.bioinf.org.uk/saap/ or http://acrmwww.biochem.ucl.ac.uk/saap/ CONTACT: a.martin@biochem.ucl.ac.uk.     

Prediction of protein stability changes for single-site mutations using support vector machines

Accurate prediction of protein stability changes resulting from single amino acid mutations is important for understanding protein structures and designing new proteins. We use support vector machines to predict protein stability changes for single amino acid mutations leveraging both sequence and structural information. We evaluate our approach using cross-validation methods on a large dataset of single amino acid mutations. When only the sign of the stability changes is considered, the predictive method achieves 84% accuracy-a significant improvement over previously published results. Moreover, the experimental results show that the prediction accuracy obtained using sequence alone is close to the accuracy obtained using tertiary structure information. Because our method can accurately predict protein stability changes using primary sequence information only, it is applicable to many situations where the tertiary structure is unknown, overcoming a major limitation of previous methods which require tertiary information. The web server for predictions of protein stability changes upon mutations (MUpro), software, and datasets are available at http://www.igb.uci.edu/servers/servers.html.