Sequence Single Nucleotide Polymorphism Research Articles

MultiPSQ: A Software Solution for the Analysis of Diagnostic n-Plexed Pyrosequencing Reactions

BackgroundPyrosequencing can be applied for Single-Nucleotide-Polymorphism (SNP)-based pathogen typing or for providing sequence information of short DNA stretches. However, for some pathogens molecular typing cannot be performed relying on a single SNP or short sequence stretch, necessitating the consideration of several genomic regions. A promising rapid approach is the simultaneous application of multiple sequencing primers, called multiplex pyrosequencing. These primers generate a fingerprint-pyrogram which is constituted by the sum of all individual pyrograms originating from each primer used.MethodsTo improve pyrosequencing-based pathogen typing, we have developed the software tool MultiPSQ that expedites the analysis and evaluation of multiplex-pyrograms. As a proof of concept, a multiplex pyrosequencing assay for the typing of orthopoxviruses was developed to analyse clinical samples diagnosed in the German Consultant Laboratory for Poxviruses.ResultsThe software tool MultiPSQ enabled the analysis of multiplex-pyrograms originating from various pyrosequencing primers. Thus several target regions can be used for pathogen typing based on pyrosequencing. As shown with a proof of concept assay, SNPs present in different orthopoxvirus strains could be identified correctly with two primers by MultiPSQ.ConclusionsSoftware currently available is restricted to a fixed number of SNPs and sequencing primers, severely limiting the usefulness of this technique. In contrast, our novel software MultiPSQ allows analysis of data from multiplex pyrosequencing assays that contain any number of sequencing primers covering any number of polymorphisms.

A Decade of Plague in Mahajanga, Madagascar: Insights into the Global Maritime Spread of Pandemic Plague

ABSTRACTA cluster of human plague cases occurred in the seaport city of Mahajanga, Madagascar, from 1991 to 1999 following 62 years with no evidence of plague, which offered insights into plague pathogen dynamics in an urban environment. We analyzed a set of 44 Mahajanga isolates from this 9-year outbreak, as well as an additional 218 Malagasy isolates from the highland foci. We sequenced the genomes of four Mahajanga strains, performed whole-genome sequence single-nucleotide polymorphism (SNP) discovery on those strains, screened the discovered SNPs, and performed a high-resolution 43-locus multilocus variable-number tandem-repeat analysis of the isolate panel. Twenty-two new SNPs were identified and defined a new phylogenetic lineage among the Malagasy isolates. Phylogeographic analysis suggests that the Mahajanga lineage likely originated in the Ambositra district in the highlands, spread throughout the northern central highlands, and was then introduced into and became transiently established in Mahajanga. Although multiple transfers between the central highlands and Mahajanga occurred, there was a locally differentiating and dominant subpopulation that was primarily responsible for the 1991-to-1999 Mahajanga outbreaks. Phylotemporal analysis of this Mahajanga subpopulation revealed a cycling pattern of diversity generation and loss that occurred during and after each outbreak. This pattern is consistent with severe interseasonal genetic bottlenecks along with large seasonal population expansions. The ultimate extinction of plague pathogens in Mahajanga suggests that, in this environment, the plague pathogen niche is tenuous at best. However, the temporary large pathogen population expansion provides the means for plague pathogens to disperse and become ecologically established in more suitable nonurban environments.

Effect of Single Nucleotide Polymorphism in the Gene RBM8A Together with the Microdeletion 1q21 in Thrombocytopenia-Absent Radii Syndrome.

AbstractAbstract 2372Thrombocytopenia-absent radii syndrome (TAR) is a rare congenital disorder characterized by bilateral radius aplasia and thrombocytopenia due to virtual absence of bone marrow megakaryocytes (MKs). As other blood lineages are not affected, TAR is considered to be a bone marrow failure syndrome. At birth, platelet counts are often below 50/nL, but ameliorate in many patients during the first two years of life, however, without reaching the lower norm. Platelet reactivity in reponse to thrombopoietin is abrogated, mostly when patients are young while it is restored to normal in older patients. Interestingly, this patterning is not correlated with platelet counts. In 2007 we described a microdeletion on chromosome 1q21 in all patients analyzed as necessary, but not sufficient to cause TAR, as it was also present in unaffected family members. Using next generation sequencing, we recently identified the presence of one of two single nucleotide polymorphisms (SNPs) in non-coding, regulatory regions of the gene RBM8A whose compound inheritance together with the microdeletion causes TAR syndrome (Nature Genetics, 44, 435). All patients inherited one of the two SNPs from one of one parents and the microdeletion from the other. In 25% of cases the microdeletion was acquired de novo. This gene, which is located within the microdeleted region, encodes for Y14, a major subunit of the exon junction complex. In the German/Swiss/Polish cohort of 28 patients, 20 patients had the more frequent SNP in the 5’UTR, whereas 8 patients had a previously undescribed SNP in intron 1. The 5’UTR G->A introduces a novel binding site for transcriptional repressor Evi1. This factor is expressed in megakaryocytes and still present in platelets. Using electrophoretic mobility shift assays (EMSA) with lysates from platelets we found a complex exclusively present when a labeled oligonucleotide with the 5’UTR-SNP G->C was used and not with the wildtype sequence. This complex could be partially supershifted with an anti-Evi1 antibody, suggesting that an additional factor might bind to this complex. In contrast, the intronic SNP is predicted to abrogate a binding site for Mzf1. We used EMSA with oligonucleotides of both intronic SNP and wildtype sequence and indeed found a complex binding to the wildtype sequence as well as to a consensus sequence for Mzf1, but no or reduced binding to the intronic SNP. This complex could not be supershifted by adding an anti-Mzf1 antibody. However, when nuclear extracts from Meg01 cells were used, we found that one of three subcomplexes showed reduced binding in the presence of the antibody, even when the intronic SNP was used. As both SNPs showed different binding properties we carefully evaluated clinical data and found that platelet counts were more severely reduced in patients with the 5’UTR compared to patients harboring the intronic SNP. Although case numbers for the intronic SNP are low, in a direct comparison of age-matched patients, platelet counts were always higher in TAR patients with the intronic SNP. However, we did not find any obvious differences in skeletal features between the two groups. Further work is ongoing to better understand the causative role of either SNP for impaired megakaryopoiesis and thrombocytopenia in TAR syndrome. Disclosures:No relevant conflicts of interest to declare.

DELISHUS: an efficient and exact algorithm for genome-wide detection of deletion polymorphism in autism

Motivation: The understanding of the genetic determinants of complex disease is undergoing a paradigm shift. Genetic heterogeneity of rare mutations with deleterious effects is more commonly being viewed as a major component of disease. Autism is an excellent example where research is active in identifying matches between the phenotypic and genomic heterogeneities. A considerable portion of autism appears to be correlated with copy number variation, which is not directly probed by single nucleotide polymorphism (SNP) array or sequencing technologies. Identifying the genetic heterogeneity of small deletions remains a major unresolved computational problem partly due to the inability of algorithms to detect them.Results: In this article, we present an algorithmic framework, which we term DELISHUS, that implements three exact algorithms for inferring regions of hemizygosity containing genomic deletions of all sizes and frequencies in SNP genotype data. We implement an efficient backtracking algorithm—that processes a 1 billion entry genome-wide association study SNP matrix in a few minutes—to compute all inherited deletions in a dataset. We further extend our model to give an efficient algorithm for detecting de novo deletions. Finally, given a set of called deletions, we also give a polynomial time algorithm for computing the critical regions of recurrent deletions. DELISHUS achieves significantly lower false-positive rates and higher power than previously published algorithms partly because it considers all individuals in the sample simultaneously. DELISHUS may be applied to SNP array or sequencing data to identify the deletion spectrum for family-based association studies.Availability: DELISHUS is available at http://www.brown.edu/Research/Istrail_Lab/.Contact: Eric_Morrow@brown.edu and Sorin_Istrail@brown.eduSupplementary information: Supplementary data are available at Bioinformatics online.

Identification of polymorphisms in ultraconserved elements associated with clinical outcomes in locally advanced colorectal adenocarcinoma

Ultraconserved elements (UCEs) are noncoding genomic sequences that completely identical among human, mouse, and rat species and harbor critical biologic functions. The authors hypothesized that single nucleotide polymorphisms (SNPs) within UCEs are associated with clinical outcomes in patients with colorectal cancer (CRC). Forty-eight SNPs within UCEs were genotyped in 662 patients with stage I through III CRC. The associations between genotypes and recurrence and survival were analyzed in patients with stage II or III CRC who received fluoropyrimidine-based adjuvant chemotherapy using a training and validation design. The training set included 115 patients with stage II disease and 170 patients with stage III disease, and the validation set included 88 patients with stage II disease and 112 patients with stage III disease. Eight SNPs were associated with clinical outcomes stratified by disease stage. In particular, for patients with stage II CRC who had at least 1 variant allele of reference SNP sequence 7849 (rs7849), a consistent association with increased recurrence risk was observed in the training set (hazard ratio [HR], 2.39; 95% confidence interval [CI], 1.04-5.52), in the replication set (HR, 3.70; 95% CI, 1.42-9.64), and in a meta-analysis (HR, 2.89; 95% CI, 1.54-5.41). Several other SNPs were significant in the training set but not in the validation set. These included rs2421099, rs16983007, and rs10211390 for recurrence and rs6590611 for survival in patients with stage II disease; and SNPs rs6124509 and rs11195893 for recurrence in patients with stage III disease. In addition, a significant cumulative effect was observed of multiple risk genotypes and potential gene-gene interactions on recurrence risk. To the authors' knowledge, this is the first study to evaluate the association between SNPs within UCEs and clinical outcome in patients with CRC. The results suggested that SNPs within UCEs may be valuable prognostic biomarkers for patients with locally advanced CRC who receive 5-fluorouracil-based chemotherapy.

Genetic selection of embryos that later develop the metabolic syndrome

The Barker hypothesisIs an excellent explanation of the process where human and animal foetuses exposed to malnutrition, either by maternal malnutrition or placental insufficiency, are metabolically programmed, with selective stunting of cell differentiation and organ growth. With the postnatal excess of nutrition observed in developed countries, this irreversible programming causes metabolic syndrome, including obesity, type 2 diabetes, and hypertension. Metabolic programming involves epigenetic changes including imprinting which might be transmitted through more than one generation rather than being completely re-set or erased during reproduction. The Barker hypothesis was supported by epidemiological data that recognised no excess fetal or postnatal mortality when pregnant women were starved during the Dutch famine in World War II. This argued against the “thrifty genotype” theory introduced in 1962, which proposed that starvation selected against members of the population with less “thrifty” genes, but the survivors who had “thrifty” genes developed metabolic syndrome if they were subsequently over-nourished. Embryonic/fetal selectionEmbryos or early foetuses could be selected very early in pregnancy on the basis of their genotype, by maternal malnutrition, hypertension, obesity or other causes of placental insufficiency. The genotype that allows embryos, or cells within them, to survive a less hospitable environment in the decidua after implantation might contribute to the later development of metabolic syndrome.This article hypothesises that an adverse intrauterine environment, caused by maternal malnutrition or placental insufficiency, kills a proportion of embryos and selects a surviving population of early embryos whose growth in utero is retarded by their genotype, their environment or a combination of both. The metabolic syndrome follows if the offspring is over-nourished later in life.The embryonic selection hypothesis presented here could be tested by using single nucleotide polymorphism (SNP) microarrays to study adults who had a history of intrauterine growth retardation (IUGR) and subsequent metabolic syndrome. Their SNP array could be compared with their parents and unaffected unrelated or related controls. If there were no selection based on a “thrifty genotype”, all parental sequences would be expected to appear in their surviving children, whether or not they had IUGR or developed metabolic syndrome. SNP sequences present in parents or controls but missing from adult offspring with metabolic syndrome who had IUGR, could be associated with or linked to genes that influence susceptibility to metabolic syndrome. This hypothesis proposes that missing genotypes would be lost if the embryos that inherited them died very early in pregnancy.

Development of a TaqMan Allelic Discrimination Assay for detection of Single Nucleotides Polymorphisms associated with anti-malarial drug resistance

BackgroundAnti-malarial drug resistance poses a threat to current global efforts towards control and elimination of malaria. Several methods are used in monitoring anti-malarial drug resistance. Molecular markers such as single nucleotide polymorphism (SNP) for example are increasingly being used to identify genetic mutations related to anti-malarial drug resistance. Several methods are currently being used in analysis of SNP associated with anti-malarial drug resistance and although each one of these methods has unique strengths and shortcoming, there is still need to improve and/or develop new methods that will close the gap found in the current methods.MethodsTaqMan Allelic Discrimination assays for detection of SNPs associated with anti-malarial drug resistance were designed for analysis on Applied Biosystems PCR platform. These assays were designed by submitting SNP sequences associated with anti-malarial drug resistance to Applied Biosystems website. Eleven SNPs associated with resistance to anti-malarial drugs were selected and tested. The performance of each SNP assay was tested by creating plasmid DNAs carrying codons of interests and analysing them for analysis. To test the sensitivity and specificity of each SNP assay, 12 clinical samples were sequenced at codons of interest and used in the analysis. Plasmid DNAs were used to establish the Limit of Detection (LoD) for each assay.ResultsData from genetic profiles of the Plasmodium falciparum laboratory strains and sequence data from 12 clinical samples was used as the reference method with which the performance of the SNP assays were compared to. The sensitivity and specificity of each SNP assay was establish at 100%. LoD for each assay was established at 2 GE, equivalent to less than 1 parasite/μL. SNP assays performed well in detecting mixed infection and analysis of clinical samples.ConclusionTaqMan Allelic Discrimination assay provides a good alternative tool in detection of SNPs associated with anti-malarial drug.

Abstract C114: Androstenedione levels in postmenopausal women with resected early-stage breast cancer are associated with SNPs in CYP11B1 and CYP11B2 identified by a genome-wide association study (GWAS).

Abstract Introduction: Androstenedione is a precursor for estrogen biosynthesis catalyzed by aromatase. Plasma androstenedione concentrations in postmenopausal women with early stage breast cancer display large interindividual variation (Ingle et al., Cancer Res. 70:3278–86, 2010). We performed a GWAS to identify single nucleotide polymorphisms (SNPs) associated with plasma levels of androstenedione in 776 postmenopausal women with resected early stage breast cancer. Methods: Plasma androstenedione concentrations were measured by GC-MS/MS. GWAS was performed using Illumina Human610-Quad BeadChips. The GWAS results were controlled for population stratification (PS) by Eigenstrat analyses. Plasma androstenedione concentrations were associated with genotype by using a quasi-likelihood model with adjustment for PS, BMI, age and other clinical variables associated with androstenedione concentrations (p<0.01). Imputation using HapMap2 data was performed within 200 kb on either side of regions containing SNPs with p<E-05. Results: 563,945 SNPs were analyzed, followed by imputation. The genotyped SNP with the lowest p value, rs4736317 (p=4.61E-06), was on chromosome 8 near the CYP11B1 and CYP11B2 genes. This SNP had a multiplicative effect of 1.128 (95% Cl: 1.07, 1.19) and a MAF of 0.40. After imputation, 20 SNPs with apparent p<E-06 were observed in this same region, with the lowest p value for rs9773022 (p=8.1E-08). We then performed functional genomic studies, and observed that two of the SNPs with low p values (rs4736312, p=1.49E-05 and rs4736350, p=6.34E-06) created apparent Oct-1 transcription factor binding sites (TFBS). Differential nuclear protein binding of Oct-1 to probes for these SNPs was verified with EMS and supershift assays using nuclear extract from A549 and NCI-H295R cells. Treatment of these two cell lines with 10 nM androstenedione led to the disruption of Oct-1 binding with the A (wild type) allele on rs4736312. rs4736312 is located in the 3'-UTR of CYP11B1 15 bp distant from response elements for the progesterone receptor and the glucocorticoid receptor. We also demonstrated that CYP11B1 and CYP11B2 were induced by estradiol in U2OS cell lines that express ER receptor and by androstenedione in H295R cells that express the androgen receptor. Transfection of a reporter gene construct with the rs4736312 SNP sequences into COS-1 and H295R cells suppressed reporter gene activity for the variant SNP sequence as compared to wild type, an observation that may correlate with the higher plasma levels of androstenedione in women (549× 328 vs 410×208 mean×SD, M/ml) with the variant allele. Conclusion: We have identified a series of SNPs in a region of chromosome 8 in and around CYP11B1 and CYP11B2 that are associated with elevated plasma androstenedione concentrations in postmenopausal women with resected early stage breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C114.

Association of toll‐like receptor 2 polymorphisms with somatic cell score in Xinjiang Brown cattle

This study aims to identify single nucleotide polymorphisms (SNPs) and haplotypes in the TLR2 gene, and analyze the association of SNPs or haplotypes and somatic cell scores in 151 Xinjiang Brown cattle and 138 Holsteins to evaluate the role of TLR2 during intramammary infections. TLR2 coding region was amplified by PCR and screened for SNP sequencing. Genotypes and frequencies of SNPs were identified. Finally, the associations of genotypes or haplotypes and somatic cell scores (SCS) were analyzed. The results showed that: (i) 15 SNPs (E+653, E+945, E+978, E+1010, E+1250, E+1688, E+1707, E+1779, E+1782, E+1891, E+1995, E+2025, E+2055, E+2214 and E+2295) were observed and detected from 289 cows; (ii) distribution of the 14 SNPs were significantly different from Xinjiang Brown cattle and Holstein (P<0.001) except for the E+945 (P>0.05); (iii) in 11 SNPs (E+945, E+978, E+1010, E+1688, E+1707, E+1779, E+1782, E+1995, E+2025, E+2055 and E+2214), the SCS of AB genotype was lower than AA (P<0.05) in Xinjiang Brown cattle; and (iv) haplotypes composed of the above-mentioned 11 SNPs were constructed. The SCS of cattle with Hap5 was lower than that of Hap3 (P<0.05). This suggests that Hap5 might play an important role in sub-mastitis resistance in Xinjiang Brown cattle.

Detection of Single Nucleotide Polymorphism Using Tension-Dependent Stochastic Behavior of a Single-Molecule Template

Single nucleotide polymorphism (SNP) is the most common genetic variation among individuals. The association of SNP with individual's response to pathogens, phenotypic variations, and gene functions emphasizes the importance of sensitive and reliable SNP detection for biomedical diagnosis and therapy. To increase sensitivity, most approaches employ amplification steps, such as PCR, to generate detectable signals that are usually ensemble-averaged. Introduction of amplification steps increases the complexity of a system, whereas ensemble averaging of signals often suffers from background interference. Here, we have exploited the stochastic behavior of a single-molecule probe to recognize SNP sequence in a microfluidic platform using a laser-tweezers instrument. The detection relies on on-off mechanical signals that provide little background interference and high specificity between wild type and SNP sequences. The microfluidic setting allows multiplex sensing and in situ recycling of the SNP probe. As a proof-of-concept, we have detected as low as 100 pM of an SNP target associated with coronary heart diseases within half an hour without any amplification steps. The mechanical signal permits the detection of single mutations involving either G/C or A/T pairs. We anticipate this system has the capacity to function as a highly sensitive generic biosensor after incorporation of a specific recognition element, such as an aptamer for example.

Marked impact of IL28B genotype in the natural clearance of hepatitis C virus in patients with haemoglobinopathies

Patients with transfusion-dependent haemoglobinopathies, such as thalassaemia major (TM) and sickle/β-thalassaemia (S/βThal), have a high risk of hepatitis C virus (HCV) infection. Chronic hepatitis C (CHC) is the major cause of liver disease (Di Marco et al, 2010). Genome-wide association studies (GWAS) detected a single nucleotide polymorphism (SNP) on chromosome 19q13, 3 kilobases (kb) upstream of the IL28B gene (−3 kbC>T, IL28B rs12979860) (Suppiah et al, 2009; Tanaka et al, 2009; Thompson et al, 2010). This SNP is associated with HCV-RNA spontaneous clearance and is in linkage disequilibrium with a non-synonymous variant in IL28B exon 2 (K70RA>G) (Ge et al, 2009; O’Brien, 2009; Thomas et al, 2009; Rauch et al 2010). Treatment of chronic HCV infection in TM and S/βThal patients with pegylated-interferon-α2 and ribavirin (PEG-IFN-α/RBV) could be compromised by severe adverse side effects (SAEs), i.e. ribavirin-associated haemolysis (Sherker et al, 2003; Inati et al, 2005; Di Marco et al, 2010). Therefore, in this cohort of patients, the prediction of spontaneous C virus clearance could be crucial. This study analysed the variants IL28B rs12979860 and K70R to evaluate their allele frequencies and to correlate them to HCV clearance in a cohort of 42 not consecutive patients with TM and S/βThal who were evaluated for possible antiviral Interferon-Ribavirin treatment. HCV natural clearance was defined as the lack of HCV-RNA detection in the serum of the patient in absence of antiviral therapy. All of the patients studied had been previously infected, and all patients who cleared HCV were HCV-RNA positive before becoming negative. It was not possible to calculate the median range of time elapsed from infection to the clearance of HCV in the serum because all of the patients were infected before 1990, when HCV had not been detected and the serum test was not available worldwide. The sensitivity of the polymerase chain reaction (PCR) used to determine serum HCV- RNA concentration was 15 iu/ml. Forty-two patients who had not been treated with PEG-IFN-α/RBV were included in this study. The demographic and clinical characteristics of the 42 studied patients are summarized in Table I. Genomic DNA was extracted from peripheral blood. The DNA variants, IL28B rs12979860 and K70R, were investigated by direct genomic sequencing, endonuclease digestion and reverse dot-blot hybridization. Specific PCR primers were developed in-house on the basis of the IL28B gene map (GENATLAS) and FASTA SNP sequences. According to the literature, the genotype IL28B rs12979860CC/K70RAA was considered to be most frequently associated with spontaneous HCV-RNA clearance, whereas the genotypes IL28B rs12979860TT/K70RGG and IL-28B rs12979860CT/K70RAG were most frequently associated with a persistent infection (Ge et al, 2009; O’Brien, 2009; Thomas et al, 2009; Rauch et al 2010). Allele frequencies of IL28B rs12979860 C>T polymorphism were 63·1% for the C allele and 36·9% for the T allele. Allele frequencies for the K70R variant were 63·1% for the A allele and 36·9% for the G allele. Twenty out of 42 patients (47·6%) showed a spontaneous HCV-RNA clearance: 15 out of 20 (75%) had the genotype IL28B rs12979860 CC/K70RAA, five out of 20 (25%) had IL28B rs12979860CT/K70RAG (Table II). Six out of 20 patients who showed HCV-RNA clearance were also anti-HCV negative: five out of six had the genotype IL28B rs12979860 CC/K70RAA, and the remaining patient had IL28B rs12979860 CT/K70RAG. Twenty-two of 42 patients (52·4%) showed HCV-RNA persistence: 13 out of 22 (59·1%) showed a viral genotype 1b and IL28B rs12979860 CT/K70RAG; four out of 22 (18·2%) showed a viral genotype 1b and IL28B rs12979860 CC/K70RAA; four out of 22 (18·2%) showed IL28B rs12979860 TT/K70RGG (three with viral genotype 2a2c and one with viral genotype 1b); one patient had a viral genotype 2a2c and IL28B rs12979860 CC/K70RAG (Table II). About 30% of individuals who acquire HCV infection resolve their viraemia, leaving only the antibody response as a marker of prior exposure (O’Brien, 2009). In our study, 47·6% of patients had a spontaneous HCV-RNA clearance showing a discrepancy with the natural clearance rate in the general population, which includes mainly non-thalassaemic subjects Therefore, the difference in the rate of HCV-RNA clearance could be due to the disease in which infection occurred. Moreover, it may be possible that chelation treatment, in determining binding of iron, which is essential for the function of polymerase enzymes, could facilitate HCV-RNA clearance. In conclusion, IL28B genotype seems to play a role in natural clearance of HCV-RNA: 15 out of 20 (75%) untreated patients who showed a spontaneous HCV-RNA clearance had a genotype favouring viral clearance (IL28B rs12979860CC/K70RAA) (Table II). Our data suggest that, having confirmed the mean time from infection to HCV clearance, a wait-and-watch approach should be used in patients with haemoglobinopathies and a favourable IL28B genotype until liver damage occurs, defined as an increase of serum ALT levels >2 times normal limits for 6 months that is not related to iron overloading.

Exploiting genome structure in association analysis.

A genome-wide association study involves examining a large number of single-nucleotide polymorphisms (SNPs) to identify SNPs that are significantly associated with the given phenotype, while trying to reduce the false positive rate. Although haplotype-based association methods have been proposed to accommodate correlation information across nearby SNPs that are in linkage disequilibrium, none of these methods directly incorporated the structural information such as recombination events along chromosome. In this paper, we propose a new approach called stochastic block lasso for association mapping that exploits prior knowledge on linkage disequilibrium structure in the genome such as recombination rates and distances between adjacent SNPs in order to increase the power of detecting true associations while reducing false positives. Following a typical linear regression framework with the genotypes as inputs and the phenotype as output, our proposed method employs a sparsity-enforcing Laplacian prior for the regression coefficients, augmented by a first-order Markov process along the sequence of SNPs that incorporates the prior information on the linkage disequilibrium structure. The Markov-chain prior models the structural dependencies between a pair of adjacent SNPs, and allows us to look for association SNPs in a coupled manner, combining strength from multiple nearby SNPs. Our results on HapMap-simulated datasets and mouse datasets show that there is a significant advantage in incorporating the prior knowledge on linkage disequilibrium structure for marker identification under whole-genome association.

High Predictivity of IL28B Genotype for Natural Clearance of HCV-RNA in Patients with Hemoglobinopathies

AbstractAbstract 2070 Background and Aim.Patients with transfusion-dependent hemoglobinopathies, as thalassemia major (TM) and sickle/β-thalassemia (S/βThal), have an high risk to be infected by hepatitis C virus (HCV), developing chronic hepatitis C (CHC).Current standard-of-care therapy for CHC consists of pegylated interferon-α 2 and ribavirin (PEG-IFN- α/RBV). A genome-wide association study (GWAs) identified a single nucleotide polymorphism (SNP), on chromosome 19, 3 kilobases (kb) upstream of the IL28B gene (IL28B -3kb C>T). This SNP is associated to the natural clearance of HCV-RNA. The IL28B -3kb C>T SNP is in strong linkage disequilibrium with a non-synonymous variant in the exon2 of IL28B gene (213A>G, K70R). Because of in these patients the PEG-IFN-α/RBV treatment of chronic HCV infection could be complicated by severe side adverse events, i.e. ribavirin-associated hemolysis, it is noteworthy to predict the spontaneous C virus clearance by investigate these two IL28B DNA variants. Therefore, in a cohort of 42 patients with hemoglobinopathies, it was analyzed the two mentioned DNA variants to evaluate their allele frequencies and to eventually correlate them to the C virus clearance. Patients and Methods.Forty two patients with hemoglobinopathies, not-treated with PEG- IFN-α/RBV, were included in this study. Molecular analysi:. Genomic DNAs were extracted from peripheral blood mononuclear cells. DNA variants, IL28B -3kb C>T e K70R, were investigated by direct genomic sequencing (CEQ880-Beckman), endonuclease digestion and reverse dot-blot hybridization. PCR primers, specific for the investigated DNA regions, were designed in our laboratory on the basis of IL28B gene map (GENATLAS) and FASTA SNP sequences. According to literature, the genotype IL28B -3kbCC/K70RAA was considered most frequently associated with the spontaneous HCV-RNA clearance, whereas the genotypes IL28B 3kbTT/K70R GG and -3kbCT/K70R AG were considered most frequently associated with a persistent infection. Result:Allele frequencies of -3kbC>T polymorphism were 63,1% for the favourable C allele and 36,9% for the T allele. Allele frequencies for the K70R variant were 63.1% for the A allele and 36,9% for the G allele. Twenty of 42 studied patients (47,6%) showed a spontaneous HCV-RNA clearance: 15/20 (75%) had IL28B genotype -3kbCC/K70R AA, 5/20 (25%) had IL28B genotype -3kbCT/K70R AG. Twenty two of 42 studied patients (52,4%) showed HCV-RNA persistence: 13/22 (59,1%) showed viral genotype 1b and IL28B genotype -3kbCT/K70R AG; 4/22 (18,2%) showed viral genotype 1b and IL28B genotype -3kbCC/K70R AA; 4/22 (18,2%) showed IL28B genotype -3kbTT/K70R GG (3 had viral genotype 2a2c and one viral genotype 1b; one case had viral genotype 2a2c and was homozygous for the favourable -3kb C allele but it was heterozygous for the K70R mutation (Table 1). Conclusion:It seems that IL28B genotype has a marked impact on natural clearance of HCV-RNA: 15/20 (75%) not treated patients who showed a spontaneous HCV-RNA clearance had an IL28B genotype promoting the viral defence (IL28B-3kbCC/K70R AA) (Table 1). These data are in agreement with Thomas et al (Thomas et al. Nature 461,798-801-2009) who showed a -3kb CC genotype in the 60% of patients who clear HCV.Therefore, they may suggest that waiting and watch approach should be advised in patients with hemoglobinopathy and favourable IL28B genotype.Tab.1Effects of IL28B genotype on HCV-RNA spontaneous clearanceHCV genotype-3kbC>T genotypeK70R genotypeHCVclearance 20/42 (47′6%)15/20 (75%)1bCCAA5/20 (25%)1bCTAGHCV persistence22/42 (52,4%)3/22 (13,7%)2a2cTTGG1/22 (4,5%)1bTTGG13/22 (59,1%)1bCTAG4/22 (18,2%)1bCCAA1/22 (4,5%)2a2cCCAG Disclosures:No relevant conflicts of interest to declare.

Non-Invasive Prenatal Detection of Trisomy 21 Using Tandem Single Nucleotide Polymorphisms

BackgroundScreening tests for Trisomy 21 (T21), also known as Down syndrome, are routinely performed for the majority of pregnant women. However, current tests rely on either evaluating non-specific markers, which lead to false negative and false positive results, or on invasive tests, which while highly accurate, are expensive and carry a risk of fetal loss. We outline a novel, rapid, highly sensitive, and targeted approach to non-invasively detect fetal T21 using maternal plasma DNA.Methods and FindingsHighly heterozygous tandem Single Nucleotide Polymorphism (SNP) sequences on chromosome 21 were analyzed using High-Fidelity PCR and Cycling Temperature Capillary Electrophoresis (CTCE). This approach was used to blindly analyze plasma DNA obtained from peripheral blood from 40 high risk pregnant women, in adherence to a Medical College of Wisconsin Institutional Review Board approved protocol. Tandem SNP sequences were informative when the mother was heterozygous and a third paternal haplotype was present, permitting a quantitative comparison between the maternally inherited haplotype and the paternally inherited haplotype to infer fetal chromosomal dosage by calculating a Haplotype Ratio (HR). 27 subjects were assessable; 13 subjects were not informative due to either low DNA yield or were not informative at the tandem SNP sequences examined. All results were confirmed by a procedure (amniocentesis/CVS) or at postnatal follow-up. Twenty subjects were identified as carrying a disomy 21 fetus (with two copies of chromosome 21) and seven subjects were identified as carrying a T21 fetus. The sensitivity and the specificity of the assay was 100% when HR values lying between 3/5 and 5/3 were used as a threshold for normal subjects.ConclusionsIn summary, a targeted approach, based on calculation of Haplotype Ratios from tandem SNP sequences combined with a sensitive and quantitative DNA measurement technology can be used to accurately detect fetal T21 in maternal plasma when sufficient fetal DNA is present in maternal plasma.

A functional SNP in the regulatory region of the decay-accelerating factor gene associates with extraocular muscle pareses in myasthenia gravis

Complement activation in myasthenia gravis (MG) may damage muscle endplate and complement regulatory proteins such as decay-accelerating factor (DAF) or CD55 may be protective. We hypothesize that the increased prevalence of severe extraocular muscle (EOM) dysfunction among African MG subjects reported earlier may result from altered DAF expression. To test this hypothesis, we screened the DAF gene sequences relevant to the classical complement pathway and found an association between myasthenics with EOM paresis and the DAF regulatory region c.-198C>G SNP (odds ratio=8.6; P=0.0003). This single nucleotide polymorphism (SNP) results in a twofold activation of a DAF 5′-flanking region luciferase reporter transfected into three different cell lines. Direct matching of the surrounding SNP sequence within the DAF regulatory region with the known transcription factor-binding sites suggests a loss of an Sp1-binding site. This was supported by the observation that the c.-198C>G SNP did not show the normal lipopolysaccharide-induced DAF transcriptional upregulation in lymphoblasts from four patients. Our findings suggest that at critical periods during autoimmune MG, this SNP may result in inadequate DAF upregulation with consequent complement-mediated EOM damage. Susceptible individuals may benefit from anti-complement therapy in addition to immunosuppression.

Prim-SNPing: A Primer Designer for Cost-Effective SNP Genotyping

Many kinds of primer design (PD) software tools have been developed, but most of them lack a single nucleotide polymorphism (SNP) genotyping service. Here, we introduce the web-based freeware "Prim-SNPing," which, in addition to general PD, provides three kinds of primer design functions for cost-effective SNP genotyping: natural PD, mutagenic PD, and confronting two-pair primers (CTPP) PD. The natural PD and mutagenic PD provide primers and restriction enzyme mining for polymerase chain reaction-restriction fragment of length polymorphism (PCR-RFLP), while CTPP PD provides primers for restriction enzyme-free SNP genotyping. The PCR specificity and efficiency of the designed primers are improved by BLAST searching and evaluating secondary structure (such as GC clamps, dimers, and hairpins), respectively. The length pattern of PCR-RFLP using natural PD is user-adjustable, and the restriction sites of the RFLP enzymes provided by Prim-SNPing are confirmed to be absent within the generated PCR product. In CTPP PD, the need for a separate digestion step in RFLP is eliminated, thus making it faster and cheaper. The output of Prim-SNPing includes the primer list, melting temperature (Tm) value, GC percentage, and amplicon size with enzyme digestion information. The reference SNP (refSNP, or rs) clusters from the Single Nucleotide Polymorphism database (dbSNP) at the National Center for Biotechnology Information (NCBI), and multiple other formats of human, mouse, and rat SNP sequences are acceptable input. In summary, Prim-SNPing provides interactive, user-friendly and cost-effective primer design for SNP genotyping. It is freely available at http://bio.kuas.edu.tw/prim-snping.

Dynamic Programming for Single Nucleotide Polymorphism ID Identification in Systematic Association Studies

Single nucleotide polymorphisms (SNPs) play an important role in personalized medicine. However, the SNP data reported in many association studies provide only the SNP nucleotide/amino acid position, without providing the SNP ID recorded in National Center for Biotechnology Information databases. A tool with the ability to provide SNP ID identification, with a user-friendly interface, is needed. In this paper, a dynamic programming algorithm was used to compare homologs when the processed input sequence is aligned with the SNP FASTA database. Our novel system provides a web-based tool that uses the National Center for Biotechnology Information dbSNP database, which provides SNP sequence identification and SNP FASTA formats. Freely selectable sequence formats for alignment can be used, including general sequence formats (ACGT, [dNTP1/dNTP2] or IUPAC formats) and orientation with bidirectional sequence matching. In contrast to the National Center for Biotechnology Information SNP-BLAST, the proposed system always provides the correct targeted SNP ID (SNP hit), as well as nearby SNPs (flanking hits), arranged in their chromosomal order and contig positions. The system also solves problems inherent in SNP-BLAST, which cannot always provide the correct SNP ID for a given input sequence. Therefore, this system constitutes a novel application which uses dynamic programming to identify SNP IDs from the literature and keyed-in sequences for systematic association studies. It is freely available at http://bio.kuas.edu.tw/SNPosition/.

The SAAPdb web resource: A large-scale structural analysis of mutant proteins

The Single Amino Acid Polymorphism database (SAAPdb) is a new resource for the analysis and visualization of the structural effects of mutations. Our analytical approach is to map single nucleotide polymorphisms (SNPs) and pathogenic deviations (PDs) to protein structural data held within the Protein Data Bank. By mapping mutations onto protein structures, we can hypothesize whether the mutant residues will have any local structural effect that may "explain" a deleterious phenotype. Our prior work used a similar approach to analyze mutations within a single protein. An analysis of the contents of SAAPdb indicates that there are clear differences in the sequence and structural characteristics of SNPs and PDs, and that PDs are more often explained by our structural analysis. This mapping and analysis is a useful resource for the mutation community and is publicly available at http://www.bioinf.org.uk/saap/db/.

Rapid High Resolution Single Nucleotide Polymorphism–Comparative Genome Hybridization Mapping in Caenorhabditis elegans

We have developed a significantly improved and simplified method for high-resolution mapping of phenotypic traits in Caenorhabditis elegans using a combination of single nucleotide polymorphisms (SNPs) and oligo array comparative genome hybridization (array CGH). We designed a custom oligonucleotide array using a subset of confirmed SNPs between the canonical wild-type Bristol strain N2 and the Hawaiian isolate CB4856, populated with densely overlapping 50-mer probes corresponding to both N2 and CB4856 SNP sequences. Using this method a mutation can be mapped to a resolution of approximately 200 kb in a single genetic cross. Six mutations representing each of the C. elegans chromosomes were detected unambiguously and at high resolution using genomic DNA from populations derived from as few as 100 homozygous mutant segregants of mutant N2/CB4856 heterozygotes. Our method completely dispenses with the PCR, restriction digest, and gel analysis of standard SNP mapping and should be easy to extend to any organism with interbreeding strains. This method will be particularly powerful when applied to difficult or hard-to-map low-penetrance phenotypes. It should also be possible to map polygenic traits using this method.

A genetic linkage map for the ectomycorrhizal fungus Laccaria bicolor and its alignment to the whole‐genome sequence assemblies

A genetic linkage map for the ectomycorrhizal basidiomycete Laccaria bicolor was constructed from 45 sib-homokaryotic haploid mycelial lines derived from the parental S238N strain progeny. For map construction, 294 simple sequence repeats (SSRs), single-nucleotide polymorphisms (SNPs), amplified fragment length polymorphisms (AFLPs) and random amplified polymorphic DNA (RAPD) markers were employed to identify and assay loci that segregated in backcross configuration. Using SNP, RAPD and SSR sequences, the L. bicolor whole-genome sequence (WGS) assemblies were aligned onto the linkage groups. A total of 37.36 Mbp of the assembled sequences was aligned to 13 linkage groups. Most mapped genetic markers used in alignment were colinear with the sequence assemblies, indicating that both the genetic map and sequence assemblies achieved high fidelity. The resulting matrix of recombination rates between all pairs of loci was used to construct an integrated linkage map using JoinMap. The final map consisted of 13 linkage groups spanning 812 centiMorgans (cM) at an average distance of 2.76 cM between markers (range 1.9-17 cM). The WGS and the present linkage map represent an initial step towards the identification and cloning of quantitative trait loci associated with development and functioning of the ectomycorrhizal symbiosis.