SNP Scoring Research Articles

ASSIsT: an automatic SNP scoring tool for in- and outbreeding species.

ASSIsT (Automatic SNP ScorIng Tool) is a user-friendly customized pipeline for efficient calling and filtering of SNPs from Illumina Infinium arrays, specifically devised for custom genotyping arrays. Illumina has developed an integrated software for SNP data visualization and inspection called GenomeStudio® (GS). ASSIsT builds on GS-derived data and identifies those markers that follow a bi-allelic genetic model and show reliable genotype calls. Moreover, ASSIsT re-edits SNP calls with null alleles or additional SNPs in the probe annealing site. ASSIsT can be employed in the analysis of different population types such as full-sib families and mating schemes used in the plant kingdom (backcross, F1, F2), and unrelated individuals. The final result can be directly exported in the format required by the most common software for genetic mapping and marker–trait association analysis. ASSIsT is developed in Python and runs in Windows and Linux.Availability and implementation: The software, example data sets and tutorials are freely available at http://compbiotoolbox.fmach.it/assist/.Contact: eric.vandeweg@wur.nl

Genetic determinants of telomere length and risk of common cancers: a Mendelian randomization study.

Epidemiological studies have reported inconsistent associations between telomere length (TL) and risk for various cancers. These inconsistencies are likely attributable, in part, to biases that arise due to post-diagnostic and post-treatment TL measurement. To avoid such biases, we used a Mendelian randomization approach and estimated associations between nine TL-associated SNPs and risk for five common cancer types (breast, lung, colorectal, ovarian and prostate cancer, including subtypes) using data on 51 725 cases and 62 035 controls. We then used an inverse-variance weighted average of the SNP-specific associations to estimate the association between a genetic score representing long TL and cancer risk. The long TL genetic score was significantly associated with increased risk of lung adenocarcinoma (P = 6.3 × 10−15), even after exclusion of a SNP residing in a known lung cancer susceptibility region (TERT-CLPTM1L) P = 6.6 × 10−6). Under Mendelian randomization assumptions, the association estimate [odds ratio (OR) = 2.78] is interpreted as the OR for lung adenocarcinoma corresponding to a 1000 bp increase in TL. The weighted TL SNP score was not associated with other cancer types or subtypes. Our finding that genetic determinants of long TL increase lung adenocarcinoma risk avoids issues with reverse causality and residual confounding that arise in observational studies of TL and disease risk. Under Mendelian randomization assumptions, our finding suggests that longer TL increases lung adenocarcinoma risk. However, caution regarding this causal interpretation is warranted in light of the potential issue of pleiotropy, and a more general interpretation is that SNPs influencing telomere biology are also implicated in lung adenocarcinoma risk.

Limitations of Human Genetic Studies on Osteoarthritis

1 (control; mAF=15.2%) [10]. The mAF of the omitted KL=2 subgroup was 12.3%, confirming an inverse relationship between mAF of the SNP and KL scores. This clearly indicates that inclusion of the KL=2 subjects in the case group had caused a decrease in the detection power. In fact, this association was not reproduced by conventional Japanese and Chinese studies that include KL=2 in the case group [11]. Considering that prevalence of the KL=2 subgroup is shown to be fairly high in representative epidemiologic studies (17.3-41.3%; difference between KL ≥ 2 and KL ≥ 3 in Table 2), removal of this subgroup may inevitably cause a decrease in the total sample size. Generally, a lack of objective and quantitative measure for the disease definition remains a fatal limitation of clinical OA studies. The ROAD study has recently established the fully automatic program KOACAD (knee OA computer-aided diagnosis) to quantify the major OA parameters (joint space, osteophyte, etc.) on plain radiographs [8]. We believe that the KOACAD system as well as magnetic resonance image systems [12] will serve as optimal measures for the definition of OA in the near future, just as bone mineral density does in osteoporosis.

Refinement of variant selection for the LDL cholesterol genetic risk score in the diagnosis of the polygenic form of clinical familial hypercholesterolemia and replication in samples from 6 countries.

Familial hypercholesterolemia (FH) is an autosomal-dominant disorder caused by mutations in 1 of 3 genes. In the 60% of patients who are mutation negative, we have recently shown that the clinical phenotype can be associated with an accumulation of common small-effect LDL cholesterol (LDL-C)-raising alleles by use of a 12-single nucleotide polymorphism (12-SNP) score. The aims of the study were to improve the selection of SNPs and replicate the results in additional samples. We used ROC curves to determine the optimum number of LDL-C SNPs. For replication analysis, we genotyped patients with a clinical diagnosis of FH from 6 countries for 6 LDL-C-associated alleles. We compared the weighted SNP score among patients with no confirmed mutation (FH/M-), those with a mutation (FH/M+), and controls from a UK population sample (WHII). Increasing the number of SNPs to 33 did not improve the ability of the score to discriminate between FH/M- and controls, whereas sequential removal of SNPs with smaller effects/lower frequency showed that a weighted score of 6 SNPs performed as well as the 12-SNP score. Metaanalysis of the weighted 6-SNP score, on the basis of polymorphisms in CELSR2 (cadherin, EGF LAG 7-pass G-type receptor 2), APOB (apolipoprotein B), ABCG5/8 [ATP-binding cassette, sub-family G (WHITE), member 5/8], LDLR (low density lipoprotein receptor), and APOE (apolipoprotein E) loci, in the independent FH/M- cohorts showed a consistently higher score in comparison to the WHII population (P < 2.2 × 10(-16)). Modeling in individuals with a 6-SNP score in the top three-fourths of the score distribution indicated a >95% likelihood of a polygenic explanation of their increased LDL-C. A 6-SNP LDL-C score consistently distinguishes FH/M- patients from healthy individuals. The hypercholesterolemia in 88% of mutation-negative patients is likely to have a polygenic basis.

Whole exome sequencing of familial hypercholesterolaemia patients negative for LDLR/APOB/PCSK9 mutations

BackgroundFamilial hypercholesterolaemia (FH) is an autosomal dominant disease of lipid metabolism, which leads to early coronary heart disease. Mutations in LDLR, APOB and PCSK9 can be detected in 80% of...

Molecular genetic evidence for overlap between general cognitive ability and risk for schizophrenia: a report from the Cognitive Genomics consorTium (COGENT).

It has long been recognized that generalized deficits in cognitive ability represent a core component of schizophrenia, evident prior to full illness onset and independent of medication. The possibility of genetic overlap between risk for schizophrenia and cognitive phenotypes has been suggested by the presence of cognitive deficits in first-degree relatives of patients with schizophrenia; however, until recently, molecular genetic approaches to test this overlap have been lacking. Within the last few years, large-scale genome-wide association studies (GWAS) of schizophrenia have demonstrated that a substantial proportion of the heritability of the disorder is explained by a polygenic component consisting of many common SNPs of extremely small effect. Similar results have been reported in GWAS of general cognitive ability. The primary aim of the present study is to provide the first molecular genetic test of the classic endophenotype hypothesis, which states that alleles associated with reduced cognitive ability should also serve to increase risk for schizophrenia. We tested the endophenotype hypothesis by applying polygenic SNP scores derived from a large-scale cognitive GWAS meta-analysis (~5000 individuals from 9 non-clinical cohorts comprising the COGENT consortium) to four schizophrenia case-control cohorts. As predicted, cases had significantly lower cognitive polygenic scores compared to controls. In parallel, polygenic risk scores for schizophrenia were associated with lower general cognitive ability. Additionally, using our large cognitive meta-analytic dataset, we identified nominally significant cognitive associations for several SNPs that have previously been robustly associated with schizophrenia susceptibility. Results provide molecular confirmation of the genetic overlap between schizophrenia and general cognitive ability, and may provide additional insight into pathophysiology of the disorder.

Abstract ES02-2: Who should receive preventive therapy?

Abstract Several options are now becoming available for preventive therapy in women at increased risk for breast cancer. A major challenge is to determine which women are at sufficiently high risk to warrant treatment, and who will benefit from such treatment. Standard risk factors have been combined in several models to develop a risk score, and the spread in 10-year risk associated with these models will be examined. Two new features which offer improved prognostic discrimination are breast density and SNP profiles. The former can be routinely read visually, but there is a substantial inter-reader variability, and a goal is to develop automated testing which is reproducible and highly prognostic for risk. SNP profiles consist of panels (now over 70) of low risk but common genes that individually are not useful, but as a panel may add useful information. A discussion of the relative amount of information in classic models, mammographic density, and SNP scores will be presented along with initial estimates of their combined utility. Lastly we look at markers which may be able to predict response to endocrine prophylactic treatment. Currently change in breast density offers the most promise in this arena. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr ES02-2.

Plasma 25-Hydroxyvitamin D and Its Genetic Determinants in Relation to Incident Myocardial Infarction and Stroke in the European Prospective Investigation into Cancer and Nutrition (EPIC)-Germany Study

Circulating 25-hydroxyvitamin D (25(OH)D) has been associated with cardiovascular disease (CVD) risk in observational studies. Also, SNPs to explain variation in 25(OH)D have been identified by genome-wide association studies. Detection of direct associations between SNPs that significantly affect 25(OH)D and CVD risk would indicate a causal role of vitamin D, as reverse causation could be excluded and confounding could be better controlled. Thus, a combined analysis of candidate SNPs in relation to circulating 25(OH)D and CVD risk was carried out. A case-cohort study within the EPIC-Germany study was conducted comprising a randomly drawn subcohort of 2,132 subjects (57.9% women, mean age: 50.6 years) and incident cases of myocardial infarction (n=559) and stroke (n=471) that occurred during a mean follow-up duration of 7.6 years. 25(OH)D concentrations were measured by LC-MS/MS in baseline plasma samples. Additionally, eight candidate SNPs were assayed. Associations between 25(OH)D, SNPs and the risks of myocardial infarction and stroke were assessed by multivariable regression analyses. Mean 25(OH)D level was 47.2 nmol/L in the subcohort. Four SNPs were associated with 25(OH)D (p<0.05). In subjects with 25(OH)D levels <25 nmol/L, the risks of CVD as composite endpoint (Hazard Ratio: 1.53, 95% confidence interval: 1.12–2.09), myocardial infarction, and stroke were significantly increased compared to subjects with levels ≥50 nmol/L, while no significant linear associations were observed. A SNP score was not related to the risks of total CVD (Hazard Ratio: 1.0, 95% confidence interval: 0.71–1.42), myocardial infarction, or stroke. The same was true concerning single SNPs. Given the lack of association between SNPs and the risks of stroke and myocardial infarction, the present findings do not point to a major causal role of vitamin D in the development of these diseases. However, a detection of modest associations between genetic markers and CVD risk in larger consortia cannot be ruled out.

Association between serum uric acid and the metabolic syndrome among a middle- and old-age Chinese population

Our aim was to study whether there is causal association between serum uric acid and metabolic syndrome (MetS). A cross-sectional study was performed, including a total of 27,009 subjects (23,345 subjects having uric acid data) from the Dongfeng-Tongji Cohort study. The MetS was defined by the International Diabetes Foundation criteria of 2005. Association analysis was performed by logistic regression. A genetic risk score was calculated by adding the uric acid increasing alleles in two SNPs (rs11722228 in SLC2A9 and rs2231142 in ABCG2) which were identified from our genome-wide association study on uric acid levels. The causal association was examined by mendelian randomization analysis. Among a middle- and old-age Chinese population, serum uric acid concentrations were strongly associated with the risk of MetS and its several components (P<0.0001). The effects were stronger in women than in men. Despite the lack of statistical significance, both SNPs exhibited a trend with increased MetS risk (rs11722228, OR=1.06, 95% CI 0.99-1.14; rs2231142, OR=1.02, 95% CI 0.95-1.10), consistent with their increasing uric acid effects. Each additional uric acid increasing allele in the genetic risk score was associated with 3% increased MetS risk (OR=1.03, 95% CI 0.98-1.09; P=0.23). Further adjustment for serum uric acid attenuated the trend of individual SNP and genetic risk score with increased MetS risk (all OR<1.0). These findings suggested that serum uric acid was associated with MetS risk in a middle- and old-age Chinese population. Whether this association was causal remained to be investigated in the future studies.

An Index of SNPs in Immune Genes May Enhance the Prognostic Value of IPI in Patients with Aggressive Non-Hodgkin's Lymphoma

Abstract 2653 INTRODUCTION:Non-Hodgkin's lymphoma (NHL) is a heterogeneous disease. The current International Prognostic Index (IPI) is useful, but not perfectly predictive and there is a need for a more precise prognostic index. The host immune response to cancer has been shown to predict outcome in multi-variate analyses in many types of cancer. We hypothesized that its genetics may contribute to prognostication. We focused on an evaluation of inherited functional polymorphisms in key immune genes that have previously been shown by others to influence outcome in various cancers. The objective was to determine whether an index of SNPs that alter function of critical immune system genes would enhance the predictive value of the IPI in patients with aggressive NHL.PATIENTS AND METHODS: Our study cohort consisted of 192 patients with NHL enrolled at diagnosis between 1990 and 1996. Patients provided informed consent and demographic, clinical and outcome data as well as tumor and blood samples were collected and stored on all patients. Patients were treated as per the standard of care at the time. 124 were of the aggressive type while 68 were low grade. We focused on the aggressive cohort in this analysis. DNA was extracted from blood, bone marrow and biopsy tissue. We evaluated the following genes; IL1A rs1800587, CXCR2 rs1126580, IL4R rs2107356, TNF rs1800629, IL4R175V rs454078, IL8 rs4073, FCGR3A rs396991, P2RX7 rs3751143, IL12B rs3212227. Genotyping was performed using allele-specific PCR primers tagged with GC tails to enhance melting point discrimination. PCR reactions were set up with the aid of a robot and run on a real-time PCR instrument. Kaplan-Meyer curves were generated for the overall survival of each genotype. Univariate analysis was performed to compare genotypes with IPI score, IPI factors and disease bulk. RESULTS:We evaluated relationships to existing standard prognostic factors. We found a significant relationship between IL12B CC carriers and IPI score 0–1 (p=0.0012). Patients with IL12B CC had less extranodal disease, lower stage and bulk. We modeled the data to identify combinations of SNPs with the most predictive power. The most predictive genotype combination included the genes for IL8, IL12B, IL4R, CXCR2 and TNF. A SNP score, determined by the number of favourable genotypes, showed significance in survival, p=0.004 [fig.1]. The group of 68 patients with an IPI score of 2–3 was analyzed to determine whether the SNP score could distinguish outcome within this IPI group [fig 2]. Patients with 3 of these favourable alleles had significantly improved survival [HR= 2.791]. Scoring with an index made up of three genes-IL8, CXCR2 and TNF alone -was also significant, p=0.02 [not shown]. CONCLUSIONS:We evaluated 10 SNPs with functional consequences on the immune response in a population of previously untreated aggressive lymphoma patients undergoing standard management. We found that a combination of SNPs statistically predicted outcome of these 124 patients with aggressive lymphoma. Although Habermann et al [Blood 2008 112(7); 2694–2702] had created a SNP score based on IL1A, IL8RB (CXCR2), IL4R and TNF, we have found that outcome is best predicted in our cohort with IL8, IL12, CXCR2, IL4R and TNF. A useful model could also be obtained using 3 SNPS (IL8, CXCR2 and TNF). Our results are important in that we have shown that this combination of SNPs can further enhance prognostification beyond IPI. Specifically statistically significant overall survival differences in IPI 2,3 patients was found based upon the SNP score using IL8, IL12, CXCR2, IL4R and TNF and the three-SNP combination. Incorporating SNP scores into IPI may be a useful strategy to further identify patients with increased likelihood of improved or adverse outcomes. [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Multiplexed p53 Mutation Detection by Free-Solution Conjugate Microchannel Electrophoresis with Polyamide Drag-Tags

We report a new, bioconjugate approach to performing highly multiplexed single-base extension (SBE) assays, which we demonstrate by genotyping a large panel of point mutants in exons 5-9 of the p53 gene. A series of monodisperse polyamide "drag-tags" was created using both chemical and biological synthesis and used to achieve the high-resolution separation of genotyping reaction products by microchannel electrophoresis without a polymeric sieving matrix. A highly multiplexed SBE reaction was performed in which 16 unique drag-tagged primers simultaneously probe 16 p53 gene loci, with an abbreviated thermal cycling protocol of only 9 min. The drag-tagged SBE products were rapidly separated by free-solution conjugate electrophoresis (FSCE) in both capillaries and microfluidic chips with genotyping accuracy in excess of 96%. The separation requires less than 70 s in a glass microfluidic chip, or about 20 min in a commercial capillary array sequencing instrument. Compared to gel electrophoresis, FSCE offers greater freedom in the design of SBE primers by essentially decoupling the length of the primer and the electrophoretic mobility of the genotyping products. FSCE also presents new possibilities for the facile implementation of SBE on integrated microfluidic electrophoresis devices for rapid, high-throughput genetic mutation detection or SNP scoring.

SNP‐SCALE: SNP scoring by colour and length exclusion

AbstractHere we present SNP‐SCALE, a new single nucleotide polymorphism genotyping protocol based on allele‐specific polymerase chain reaction (AS‐PCR), but with two critical modifications designed to maximize accuracy, efficiency and throughput and minimize costs. The first is the addition of a locked nucleic acid (LNA) at the 3′‐SNP position of each of the two allele‐specific forward primers. The second is the addition of size‐differentiating M13 tails at the 5′ end of each primer, which facilitates the use of universal fluorescent primers. The use of LNAs makes SNP‐SCALE significantly more sensitive and specific than standard AS‐PCR. Moreover, SNP‐SCALE is ideally suited to a touchdown‐like PCR protocol, such that PCRs can be standardized, PCR optimization is straightforward, and medium‐high throughput can be readily achieved. We anticipate that the SNP‐SCALE protocol will be useful to molecular ecologists for a broad range of studies.

East Asian mtDNA haplogroup determination in Koreans: Haplogroup-level coding region SNP analysis and subhaplogroup-level control region sequence analysis

The present study analyzed 21 coding region SNP markers and one deletion motif for the determination of East Asian mitochondrial DNA (mtDNA) haplogroups by designing three multiplex systems which apply single base extension methods. Using two multiplex systems, all 593 Korean mtDNAs were allocated into 15 haplogroups: M, D, D4, D5, G, M7, M8, M9, M10, M11, R, R9, B, A, and N9. As the D4 haplotypes occurred most frequently in Koreans, the third multiplex system was used to further define D4 subhaplogroups: D4a, D4b, D4e, D4g, D4h, and D4j. This method allowed the complementation of coding region information with control region mutation motifs and the resultant findings also suggest reliable control region mutation motifs for the assignment of East Asian mtDNA haplogroups. These three multiplex systems produce good results in degraded samples as they contain small PCR products (101-154 bp) for single base extension reactions. SNP scoring was performed in 101 old skeletal remains using these three systems to prove their utility in degraded samples. The sequence analysis of mtDNA control region with high incidence of haplogroup-specific mutations and the selective scoring of highly informative coding region SNPs using the three multiplex systems are useful tools for most applications involving East Asian mtDNA haplogroup determination and haplogroup-directed stringent quality control.

Applications for protein sequence-function evolution data: mRNA/protein expression analysis and coding SNP scoring tools

The vast amount of protein sequence data now available, together with accumulating experimental knowledge of protein function, enables modeling of protein sequence and function evolution. The PANTHER database was designed to model evolutionary sequence–function relationships on a large scale. There are a number of applications for these data, and we have implemented web services that address three of them. The first is a protein classification service. Proteins can be classified, using only their amino acid sequences, to evolutionary groups at both the family and subfamily levels. Specific subfamilies, and often families, are further classified when possible according to their functions, including molecular function and the biological processes and pathways they participate in. The second application, then, is an expression data analysis service, where functional classification information can help find biological patterns in the data obtained from genome-wide experiments. The third application is a coding single-nucleotide polymorphism scoring service. In this case, information about evolutionarily related proteins is used to assess the likelihood of a deleterious effect on protein function arising from a single substitution at a specific amino acid position in the protein. All three web services are available at .

High throughput SNP genotyping with two mini-sequencing assays.

Two mini-sequencing methods, FP-TDI (template-directed dye-terminator incorporation with fluorescence-polarization) and MassArray (matrix assisted laser desorption ionization time of flight detection mass spectrometry), were optimized. A numeric standard was introduced to evaluate the SNP scoring quality of FP-TDI assay, thus made the optimization work easier. At the same time, using multi-PCR technology, 8-plex genotyping of MassArray assay was successfully carried out, some softwares were developed and the data process of MassArray was highly automated. Then these two methods were applied to high throughput SNP genotyping, the accuracy, efficiency and robustness were compared. The result shows FP-TDI is more sensitive to the concentration of SNPprimer and PCR product, as well as extension cycles, the SNPprimer length of FP-TDI should be 24 to 30 bp long, whereas MassArray assay prefers to be as short as only 16 bp. Altogether 6440 SNP sites of human chromosome 3 were genotyped in a sample of 90 individuals, 4792 sites by FP-TDI assay and 1648 sites by MassArray assay, the success rates of FP-TDI and MassArray were 67.7% and 93.6% respectively. The throughput of MassArray was higher than FP-TDI, and the cost of MassArray was lower, MassArray was more suitable for high throughput SNP genotyping.

Allele-specific Holliday junction formation: a new mechanism of allelic discrimination for SNP scoring.

We report here a new mechanism for allelic discrimination--allele-specific Holliday Junction formation. The Holliday Junction (HJ) is a unique DNA structure that can be formed in a sequence-nonspecific manner by routine PCR. To cause the PCR-based HJ formation to occur in an allele-specific manner, the PCR primers are manipulated such that an extra mismatch next to a SNP of interest is introduced between a target and a reference amplicon and a GC-clamp is added. Based on this new mechanism, novel SNP genotyping methods were developed, including a homogeneous fluorescence polarization (FP) competition assay that requires neither labeled primers/probes nor expensive enzymes/substrates. Using this novel genotyping technology, we were able to convert >95% of SNP sequences into genotyping assays that work well under a universal set of assay conditions and achieved 100% accuracy in clinical samples.

DHPLC scoring of a SNP between promoter sequences of HMW glutenin x-type alleles at the Glu-D1 locus in wheat.

The promoter regions of HMW glutenin x-type genes at the Glu-D1 locus were surveyed for SNPs within a subpopulation of German bread wheat cultivars. On the basis of the promoter sequences of HMW glutenin subunit genes Glu-A1-x1, Glu-A1-x2, Glu-B1-x1, Glu-B1-x7, Glu-D1-x2, and Glu-D1-x5, an amplification refractory mutation system assay was designed to selectively amplify Dx-specific PCR fragments. Comparative sequence analysis among seven Glu-D1-x2 and seven Glu-D1-x5 wheat cultivars only confirmed a G-A transition in the promoter sequence to be a true polymorphism. SNP scoring by DHPLC of 95 German bread wheat cultivars, with the exception of cv. Anemos, showed that the transition completely agreed with the presence of HMW glutenin subunits 1Dx5 + 1Dy10 in SDS-PAGE. Therefore, the developed DHPLC assay is suitable for high-throughput genotyping to assist the selection of HMW glutenin genes in wheat quality breeding programs.

Structure-specific DNA cleavage on surfaces.

The structure-specific invasive cleavage reaction is a useful means for sensitive and specific detection of single nucleotide polymorphisms, or SNPs, directly from genomic DNA without a need for prior target amplification. A new approach integrating this invasive cleavage assay and surface DNA array technology has been developed for potentially large-scale SNP scoring in a parallel format. Two surface invasive cleavage reaction strategies were designed and implemented for a model SNP system in codon 158 of the human ApoE gene. The upstream oligonucleotide, which is required for the invasive cleavage reaction, is either co-immobilized on the surface along with the probe oligonucleotide or alternatively added in solution. The ability of this approach to unambiguously discriminate a single base difference was demonstrated using PCR-amplified human genomic DNA. A theoretical model relating the surface fluorescence intensity to the progress of the invasive cleavage reaction was developed and agreed well with experimental results.

A stochastic volatility model specification with diagnostics for thinly traded equity markets

The majority of world equity markets exhibit non-synchronous- and non-trading for some quoted asset series. This investigation sets out to determine the complexity of illiquid markets applying versions of stochastic differential equation (SDE) specifications. Efficient method of moments (EMM) is used to estimate and evaluate the diffusion models. EMM estimation on SNP scores reveals that a standard SDE with first and second order drift, non-synchronous trading and constant diffusion are rejected. However, a simple two-factor stochastic volatility specification with non-synchronous trading and conditional heteroscedasticity, reports success for illiquid equity markets. Tracking portfolios may therefore not track derivative products perfectly and importantly; the stochastic volatility model seems to be the preferred volatility specification.