SNP-based Approach Research Articles

Validation of mass spectrometry analysis of mitochondrial DNA

The analysis of mitochondrial DNA (mtDNA) is used routinely to assist in determining the source of old bones, teeth, hair shafts, and other challenged biological samples. Sequencing is the method of choice for typing mtDNA. However, the method is laborious, time consuming, costly and currently not a practical approach for typing single nucleotide polymorphisms (SNPs) contained within the coding region of the mtDNA genome. Electrospray ionization mass spectrometry (ESI-MS) has several advantages for mtDNA analysis including speed, sensitivity and the ability to interrogate mixtures. As with sequencing and unlike many other SNP-based approaches, individual polymorphic positions do not need to be specifically targeted, as all variation within amplified regions is assayed simultaneously. Since each amplicon is assayed as an individual component, the technique easily resolves length heteroplasmy and is capable of quantitatively analyzing heteroplasmic and mixed samples. The ESI-MS approach originally described for mtDNA analysis has been improved using the Ibis T5000™ and a 24 amplicon tiling format and validation studies are underway. Results from these studies support that the use of mass spectrometry is a robust and reliable method for mtDNA analysis that offers resolution approaching that of sequencing, the ability to deconvolve mixtures and analyze heteroplasmy effectively, and a sensitivity of detection equivalent to current methods, with at least an order of magnitude increased throughput and a reduction in cost.

Translation of an STR-based biomarker into a clinically compatible SNP-based platform for loss of heterozygosity

Loss of heterozygosity (LOH) has been shown to be a promising biomarker of cancer risk in patients with premalignant conditions. In this study we describe analytical validation in clinical biopsy samples of a SNP-based pyrosequencing panel targeting regions of LOH on chromosomes 17p and 9p including TP53 and CDKN2A tumor suppressor genes. Assays were tested for analytic specificity, sensitivity, efficiency, and reproducibility. Accuracy was evaluated by comparing SNP-based LOH results to those obtained by previously well-studied short tandem repeat polymorphisms (STRs) in DNA derived from different tissue sources including fresh-frozen endoscopic biopsies, samples from surgical resections, and formalin-fixed paraffin-embedded sections. A 17p/9p LOH panel comprised of 43 SNPs was designed to amplify with universal assay conditions in a two-step PCR and sequence-by-synthesis reaction that can be completed in two hours and 10 minutes. The methods presented can be a model for developing a SNP-based LOH approach targeted to any chromosomal region of interest for other premalignant conditions and this panel could be incorporated as part of a biomarker for cancer risk prediction, early detection, or as entry criteria for randomized trials.

Single Nucleotide Polymorphism-Based System Improves the Applicability of Quantitative PCR for Chimerism Monitoring

Recently, several studies demonstrated the feasibility of a real-time quantitative PCR (qPCR) approach for chimerism monitoring. qPCR offers a fast, sensitive, and elegant quantification of genotypes. However, before it becomes an established method for routine chimerism monitoring, a qPCR marker set for every transplant pair should be available. This requirement poses a major challenge since the genetic markers for qPCR--short insertions/deletions (Indels) and single nucleotide polymorphisms (SNPs)--published to-date do not guarantee applicability for every transplant pair. The aim of our study was to design and validate a new SNP allele-specific system to supplement an already existing Indel primer panel and improve applicability of the qPCR approach for chimerism status monitoring. Here, we present an approach for an economical in-house design of SNP allele-specific qPCR primers/probe sets with a locus-individualized reference system that allows for the accurate quantification of the respective informative locus using a simple DeltaDeltaCt method. We designed primers/probe sets specific for seven biallelic SNP loci and validated them in a population of 30 transplant pairs. Repeatability varied depending on the amount of quantifiable genotype. The combination of our SNP-qPCR system and Indel primers increased recipient genotype identification from 86.6% to 96.6% when tested in a population of our transplant pairs. These results demonstrate the feasibility of our SNP-based qPCR approach to improve the applicability of a qPCR for chimerism monitoring.

Identification of a novel susceptibility locus for juvenile idiopathic arthritis by genome-wide association analysis.

ObjectiveJuvenile idiopathic arthritis (JIA) is a chronic rheumatic disease of childhood. Two well-established genetic factors known to contribute to JIA susceptibility, HLA and PTPN22, account for less than half of the genetic susceptibility to disease; therefore, additional genetic factors have yet to be identified. The purpose of this study was to perform a systematic search of the genome to identify novel susceptibility loci for JIA.MethodsA genome-wide association study using Affymetrix GeneChip 100K arrays was performed in a discovery cohort (279 cases and 184 controls). Single-nucleotide polymorphisms (SNPs) showing the most significant differences between cases and controls were then genotyped in a validation sample of cases (n = 321) and controls, combined with control data from the 1958 UK birth cohort (n = 2,024). In one region in which association was confirmed, fine-mapping was performed (654 cases and 1,847 controls).ResultsOf the 112 SNPs that were significantly associated with JIA in the discovery cohort, 6 SNPs were associated with JIA in the independent validation cohort. The most strongly associated SNP mapped to the HLA region, while the second strongest association was with a SNP within the VTCN1 gene. Fine-mapping of that gene was performed, and 10 SNPs were found to be associated with JIA.ConclusionThis study is the first to successfully apply a SNP-based genome-wide association approach to the investigation of JIA. The replicated association with markers in the VTCN1 gene defined an additional susceptibility locus for JIA and implicates a novel pathway in the pathogenesis of this chronic disease of childhood.

BAC-end sequence-based SNPs and Bin mapping for rapid integration of physical and genetic maps in apple

A genome-wide BAC physical map of the apple, Malus × domestica Borkh., has been recently developed. Here, we report on integrating the physical and genetic maps of the apple using a SNP-based approach in conjunction with bin mapping. Briefly, BAC clones located at ends of BAC contigs were selected, and sequenced at both ends. The BAC end sequences (BESs) were used to identify candidate SNPs. Subsequently, these candidate SNPs were genetically mapped using a bin mapping strategy for the purpose of mapping the physical onto the genetic map. Using this approach, 52 (23%) out of 228 BESs tested were successfully exploited to develop SNPs. These SNPs anchored 51 contigs, spanning ∼ 37 Mb in cumulative physical length, onto 14 linkage groups. The reliability of the integration of the physical and genetic maps using this SNP-based strategy is described, and the results confirm the feasibility of this approach to construct an integrated physical and genetic maps for apple.

Systematic Identification of Genes that Regulate Neuronal Wiring in the Drosophila Visual System

Forward genetic screens in model organisms are an attractive means to identify those genes involved in any complex biological process, including neural circuit assembly. Although mutagenesis screens are readily performed to saturation, gene identification rarely is, being limited by the considerable effort generally required for positional cloning. Here, we apply a systematic positional cloning strategy to identify many of the genes required for neuronal wiring in the Drosophila visual system. From a large-scale forward genetic screen selecting for visual system wiring defects with a normal retinal pattern, we recovered 122 mutations in 42 genetic loci. For 6 of these loci, the underlying genetic lesions were previously identified using traditional methods. Using SNP-based mapping approaches, we have now identified 30 additional genes. Neuronal phenotypes have not previously been reported for 20 of these genes, and no mutant phenotype has been previously described for 5 genes. The genes encode a variety of proteins implicated in cellular processes such as gene regulation, cytoskeletal dynamics, axonal transport, and cell signalling. We conducted a comprehensive phenotypic analysis of 35 genes, scoring wiring defects according to 33 criteria. This work demonstrates the feasibility of combining large-scale gene identification with large-scale mutagenesis in Drosophila, and provides a comprehensive overview of the molecular mechanisms that regulate visual system wiring.

Real-Time PCR Assays of Single-Nucleotide Polymorphisms Defining the Major Brucella Clades

Members of the genus Brucella are known worldwide as pathogens of wildlife and livestock and are the most common organisms of zoonotic infection in humans. In general, brucellae exhibit a range of host specificity in animals that has led to the identification of at least seven Brucella species. The genomes of the various Brucella species are highly conserved, which makes the differentiation of species highly challenging. However, we found single-nucleotide polymorphisms (SNPs) in housekeeping and other genes that differentiated the seven main Brucella species or clades and thus enabled us to develop real-time PCR assays based around these SNPs. Screening of a diverse panel of 338 diverse isolates with these assays correctly identified each isolate with its previously determined Brucella clade. Six of the seven clade-specific assays detected DNA concentrations of less than 10 fg, indicating a high level of sensitivity. This SNP-based approach places samples into a phylogenetic framework, allowing reliable comparisons to be made among the lineages of clonal bacteria and providing a solid basis for genotyping. These PCR assays provide a rapid and highly sensitive method of differentiating the major Brucella groups that will be valuable for clinical and forensic applications.

Polymorphisms in Interleukin-15 Gene on Chromosome 4q31.2 Are Associated with Psoriasis Vulgaris in Chinese Population

Through a series of linkage analyses in a large Chinese family cohort of psoriasis, we previously identified and confirmed a non-HLA psoriasis linkage locus PSORS9 within a small region at 4q31.2-32.1. Within the critical region of the PSORS9 locus, IL-15 has been long recognized as a strong candidate gene for psoriasis. In this study, we investigated the association between IL-15 genetic polymorphisms and psoriasis in a large Chinese sample. Highly significant evidence for association was identified at a single-nucleotide polymorphism (SNP) (g.96516A --> T) within the 3'-untranslated region (UTR) of the IL-15 gene (P=0.00006, after correction for multiple testing). Haplotype analysis using the SNPs within the 3'UTR region also provided strong supporting evidence for association (P=0.00005), where we identified a haplotype of the 3'UTR region of IL-15 associated with increased risk to psoriasis (odds ratio=1.65). This association was also supported by the results of our expression activity analyses, where we demonstrated that the identified risk haplotype is associated with an increased activity of IL-15. Therefore, we provided early evidence for the important role of IL-15 genetic variants in the pathogenesis of psoriasis, probably by increasing interleukin production and inflammation in the lesions of psoriasis.

Brain-specific tryptophan hydroxylase 2 (TPH2): a functional Pro206Ser substitution and variation in the 5'-region are associated with bipolar affective disorder

The neurotransmitter serotonin [5-hydroxytryptamine (5-HT)] controls a broad range of biological functions that are disturbed in affective disorder. In the brain, 5-HT production is controlled by tryptophan hydroxylase 2 (TPH2). In order to assess the possible contribution of TPH2 genetic variability to the aetiology of bipolar affective disorder (BPAD), we systematically investigated common and rare genetic variation in the TPH2 gene through a sequential sequencing and SNP-based genotyping approach. Our study sample comprised two cohorts of BPAD from Germany and Russia, totalling 883 patients and 1300 controls. SNPs located in a haplotype block covering the 5' region of the gene as well as a rare, non-synonymous SNP, resulting in a Pro206Ser substitution, showed significant association with bipolar disorder. The odds ratio for the minor allele in the pooled sample was 1.5 (95% CI 1.2-1.9) for rs11178997 (in the 5'-associated haplotype block) and 4.8 (95% CI 1.6-14.8) for rs17110563 encoding the Pro206Ser substitution. Examination of the functional effects of TPH2 Pro206Ser provided evidence for a reduced thermal stability and solubility of the mutated enzyme, suggesting reduced 5-HT production in the brain as a pathophysiological mechanism in BPAD.

Multiplex PCR-based real-time invader assay (mPCR-RETINA): a novel SNP-based method for detecting allelic asymmetries within copy number variation regions

We report the development of a real-time Invader assay combined with multiplex PCR (mPCR-RETINA), an SNP-based approach that can measure the allelic ratio in copy number variation (CNV) regions of a genome. RETINA monitors the real-time fluorescence intensity of each allele during the Invader assay and detects allelic asymmetries caused by genomic duplication/multiplication in heterozygous individuals. By combining mPCR-RETINA and real-time quantitative PCR that detects total copy number, we can estimate the copy number of each allele in CNV regions, which should be useful for investigating the functional significance of allele copy number with disease susceptibilities and drug responses. Also, mPCR-RETINA can efficiently refine the detailed structures of CNV regions. Due to the combination of RETINA with multiplex PCR, mPCR-RETINA requires a very small amount of genomic DNA for analysis (0.1-0.38 ng/locus). Additionally, mPCR-RETINA has clear advantages in its simple protocol and target-specific reaction, even in nonunique regions. We believe mPCR-RETINA will provide a significant contribution to identifying functional alleles in CNV regions.

Traceability of the PGI product “Vitellone Bianco dell’Appennino Centrale” by SNP markers

Traceability of meat has become a key aspect of food-quality assurance and a priority for EU countries, to meet consumer demand for comprehensive and integrated food safety policies. In this context, the traceability of animals and animal products at the breed level might play a key role as it would enable the certification of regional products linked to particular breeds. Technologies based on DNA analysis have the potential to achieve this goal bypassing the large scale and systematic biological sampling necessary for individual fingerprinting.The objective of this work was to test the power of a SNP panel to trace PGI “Vitellone Bianco dell’Appennino Centrale”, a product linked to three breeds: Chianina, Marchigiana and Romagnola.A total of 180 unrelated animals belonging to PGI-allowed breeds (Chianina n=22; Romagnola n=22; and Marchigiana n=22) and PGI-not allowed breeds (Piemontese n=22, Maremmana n=24, Italian Red Pied n=24, Italian Brown n=22, Italian Friesian n=22) were sampled and genotyped with a panel of 130 SNP in 90 independent genes.Individual genotypes were assigned to breed by Bayesian based clustering approach. In particular the SNP data were analysed by the Rannala and Mountain approach implemented in the software Geneclass 2 (file:///C:/Software/GeneClass2/Help/index.htm).The SNP-based molecular approach in combination with Bayesian assignment algorithms allowed an overall correct assignment of more than 95% of individuals to their breeds of origins. This percentage increase to 96% grouping the breeds allowed in a unique cluster.Sorting the SNPs by their Gst values and using the 90 showing a value higher than 0.01, we maintain the same rate of allocation success indicating that selecting SNPs for their informative content will reduce the number of markers required for a reliable traceability at breed level, reducing the laboratory effort and consequently the cost for the large scale application of the methodOur results suggest that SNP markers in combination with Bayesian algorithm can be exploited in allocation studies and are able to trace “Vitellone Bianco dell’Appennino Centrale”. The main advantages of SNP are their abundance and the potential of full automation.

Pharmacogenetics in solid organ transplantation: present knowledge and future perspectives.

The promise of pharmacogenetics is to elucidate the inherited basis of differences between individual responses to drugs, in order to identify the right drug and dose for each patient. Genetic polymorphisms are implicated in the interindividual variability of the pharmacokinetic or pharmacodynamic characteristics of immunosuppressive drugs. The first pharmacogenetic trait identified was monogenic, and concerned the prototypic example of thiopurine methyltransferase (TPMT) implicated in azathioprine metabolism. Individuals with low TPMT activity, inherited in an autosomal codominant fashion, are at risk of drug-induced myelosuppression. TPMT activity determination and DNA-based tests are now used in clinical practice. It has been also demonstrated that there is a link between the polymorphisms of the cytochrome P450 3A5, 3A4 and the multidrug resistance-1 (MDR1) genes, and the daily dose necessary to achieve adequate blood tacrolimus levels. Analysis of MDR1 haplotypes or using the association of the different genes might further improve predictions. Since genotyping methods improve rapidly, it will soon be easy to test for thousands of single nucleotide polymorphisms in one assay. Present challenges are to determine the genes of interest and to validate such determination prospectively in clinical practice.