Genotyping Performance Assessment of Whole Genome Amplified DNA with Respect to Multiplexing Level of Assay and Its Period of Storage

Whole genome amplification can faithfully amplify genomic DNA (gDNA) with minimal bias and substantial genome coverage. Whole genome amplified DNA (wgaDNA) has been tested to be workable for high-throughput genotyping arrays. However, issues about whether wgaDNA would decrease genotyping performance at increasing multiplexing levels and whether the storage period of wgaDNA would reduce genotyping performance have not been examined. Using the Sequenom MassARRAY iPLEX Gold assays, we investigated 174 single nucleotide polymorphisms for 3 groups of matched samples: group 1 of 20 gDNA samples, group 2 of 20 freshly prepared wgaDNA samples, and group 3 of 20 stored wgaDNA samples that had been kept frozen at −70°C for 18 months. MassARRAY is a medium-throughput genotyping platform with reaction chemistry different from those of high-throughput genotyping arrays. The results showed that genotyping performance (efficiency and accuracy) of freshly prepared wgaDNA was similar to that of gDNA at various multiplexing levels (17-plex, 21-plex, 28-plex and 36-plex) of the MassARRAY assays. However, compared with gDNA or freshly prepared wgaDNA, stored wgaDNA was found to give diminished genotyping performance (efficiency and accuracy) due to potentially inferior quality. Consequently, no matter whether gDNA or wgaDNA was used, better genotyping efficiency would tend to have better genotyping accuracy

A combined genome-wide linkage and association approach to find susceptibility loci for platelet function phenotypes in European American and African American families with coronary artery disease

<p>Abstract</p> <p>Background</p> <p>The inability of aspirin (ASA) to adequately suppress platelet aggregation is associated with future risk of coronary artery disease (CAD). Heritability studies of agonist-induced platelet function phenotypes suggest that genetic variation may be responsible for ASA responsiveness. In this study, we leverage independent information from genome-wide linkage and association data to determine loci controlling platelet phenotypes before and after treatment with ASA.</p> <p>Methods</p> <p>Clinical data on 37 agonist-induced platelet function phenotypes were evaluated before and after a 2-week trial of ASA (81 mg/day) in 1231 European American and 846 African American healthy subjects with a family history of premature CAD. Principal component analysis was performed to minimize the number of independent factors underlying the covariance of these various phenotypes. Multi-point sib-pair based linkage analysis was performed using a microsatellite marker set, and single-SNP association tests were performed using markers from the Illumina 1 M genotyping chip from deCODE Genetics, Inc. All analyses were performed separately within each ethnic group.</p> <p>Results</p> <p>Several genomic regions appear to be linked to ASA response factors: a 10 cM region in African Americans on chromosome 5q11.2 had several STRs with suggestive (p-value < 7 × 10^-4) and significant (p-value < 2 × 10^-5) linkage to post aspirin platelet response to ADP, and ten additional factors had suggestive evidence for linkage (p-value < 7 × 10^-4) to thirteen genomic regions. All but one of these factors were aspirin <it>response </it>variables. While the strength of genome-wide SNP association signals for factors showing evidence for linkage is limited, especially at the strict thresholds of genome-wide criteria (N = 9 SNPs for 11 factors), more signals were considered significant when the association signal was weighted by evidence for linkage (N = 30 SNPs).</p> <p>Conclusions</p> <p>Our study supports the hypothesis that platelet phenotypes in response to ASA likely have genetic control and the combined approach of linkage and association offers an alternative approach to prioritizing regions of interest for subsequent follow-up.</p

Identification of a Novel ZIC3 Isoform and Mutation Screening in Patients with Heterotaxy and Congenital Heart Disease

Patients with heterotaxy have characteristic cardiovascular malformations, abnormal arrangement of their visceral organs, and midline patterning defects that result from abnormal left-right patterning during embryogenesis. Loss of function of the transcription factor ZIC3 causes X-linked heterotaxy and isolated congenital heart malformations and represents one of the few known monogenic causes of congenital heart disease. The birth incidence of heterotaxy-spectrum malformations is significantly higher in males, but our previous work indicated that mutations within ZIC3 did not account for the male over-representation. Therefore, cross species comparative sequence alignment was used to identify a putative novel fourth exon, and the existence of a novel alternatively spliced transcript was confirmed by amplification from murine embryonic RNA and subsequent sequencing. This transcript, termed Zic3-B, encompasses exons 1, 2, and 4 whereas Zic3-A encompasses exons 1, 2, and 3. The resulting protein isoforms are 466 and 456 amino acid residues respectively, sharing the first 407 residues. Importantly, the last two amino acids in the fifth zinc finger DNA binding domain are altered in the Zic3-B isoform, indicating a potential functional difference that was further evaluated by expression, subcellular localization, and transactivation analyses. The temporo-spatial expression pattern of Zic3-B overlaps with Zic3-A in vivo, and both isoforms are localized to the nucleus in vitro. Both isoforms can transcriptionally activate a Gli binding site reporter, but only ZIC3-A synergistically activates upon co-transfection with Gli3, suggesting that the isoforms are functionally distinct. Screening 109 familial and sporadic male heterotaxy cases did not identify pathogenic mutations in the newly identified fourth exon and larger studies are necessary to establish the importance of the novel isoform in human disease

Association of Common Polymorphisms in GLUT9 Gene with Gout but Not with Coronary Artery Disease in a Large Case-Control Study

BACKGROUND: Serum uric acid (UA) levels have recently been shown to be genetically influenced by common polymorphisms in the GLUT9 gene in two genome-wide association analyses of Italian and British populations. Elevated serum UA levels are often found in conjunction with the metabolic syndrome. Hyperuricemia is the major risk factor for gout and has been associated with increased cardiovascular morbidity and mortality. The aim of the present study was to further elucidate the association of polymorphisms in GLUT9 with gout and coronary artery disease (CAD) or myocardial infarction (MI). To test our hypotheses, we performed two large case-control association analyses of individuals from the German MI Family Study. METHODS AND FINDINGS: First, 665 patients with gout and 665 healthy controls, which were carefully matched for age and gender, were genotyped for four single nucleotide polymorphisms (SNPs) within or near the GLUT9 gene. All four SNPs demonstrated highly significant association with gout. SNP rs6855911, located within intron 7 of GLUT9, showed the strongest signal with a protective effect of the minor allele with an allelic odds ratio of 0.62 (95% confidence interval 0.52-0.75; p = 3.2*10(-7)). Importantly, this finding was not influenced by adjustment for components of the metabolic syndrome or intake of diuretics. Secondly, 1,473 cases with severe CAD or MI and 1,241 healthy controls were tested for the same four GLUT9 SNPs. The analyses revealed, however, no significant association with CAD or with MI. Additional screening of genome-wide association data sets showed no signal for CAD or MI within the GLUT9 gene region. CONCLUSION: Thus, our results provide compelling evidence that common genetic variations within the GLUT9 gene strongly influence the risk for gout but are unlikely to have a major effect on CAD or MI in a German population

Single-feature polymorphism discovery by computing probe affinity shape powers

<p>Abstract</p> <p>Background</p> <p>Single-feature polymorphism (SFP) discovery is a rapid and cost-effective approach to identify DNA polymorphisms. However, high false positive rates and/or low sensitivity are prevalent in previously described SFP detection methods. This work presents a new computing method for SFP discovery.</p> <p>Results</p> <p>The probe affinity differences and affinity shape powers formed by the neighboring probes in each probe set were computed into SFP weight scores. This method was validated by known sequence information and was comprehensively compared with previously-reported methods using the same datasets. A web application using this algorithm has been implemented for SFP detection. Using this method, we identified 364 SFPs in a barley near-isogenic line pair carrying either the wild type or the mutant <it>uniculm2 </it>(<it>cul2</it>) allele. Most of the SFP polymorphisms were identified on chromosome 6H in the vicinity of the <it>Cul2 </it>locus.</p> <p>Conclusion</p> <p>This SFP discovery method exhibits better performance in specificity and sensitivity over previously-reported methods. It can be used for other organisms for which GeneChip technology is available. The web-based tool will facilitate SFP discovery. The 364 SFPs discovered in a barley near-isogenic line pair provide a set of genetic markers for fine mapping and future map-based cloning of the <it>Cul2 </it>locus.</p

Genetic association study of adiposity and melanocortin-4 receptor (MC4R) common variants: Replication and functional characterization of non-coding regions

Common genetic variants 3′ of MC4R within two large linkage disequilibrium (LD) blocks spanning 288 kb have been associated with common and rare forms of obesity. This large association region has not been refined and the relevant DNA segments within the association region have not been identified. In this study, we investigated whether common variants in the MC4R gene region were associated with adiposity-related traits in a biracial population-based study. Single nucleotide polymorphisms (SNPs) in the MC4R region were genotyped with a custom array and a genome-wide array and associations between SNPs and five adiposity-related traits were determined using race-stratified linear regression. Previously reported associations between lower BMI and the minor alleles of rs2229616/Val103Ile and rs52820871/Ile251Leu were replicated in white female participants. Among white participants, rs11152221 in a proximal 3′ LD block (closer to MC4R) was significantly associated with multiple adiposity traits, but SNPs in a distal 309 LD block (farther from MC4R ) were not. In a case-control study of severe obesity, rs11152221 was significantly associated. The association results directed our follow-up studies to the proximal LD block downstream of MC4R. By considering nucleotide conservation, the significance of association, and proximity to the MC4R gene, we identified a candidate MC4R regulatory region. This candidate region was sequenced in 20 individuals from a study of severe obesity in an attempt to identify additional variants, and the candidate region was tested for enhancer activity using in vivo enhancer assays in zebrafish and mice. Novel variants were not identified by sequencing and the candidate region did not drive reporter gene expression in zebrafish or mice. The identification of a putative insulator in this region could help to explain the challenges faced in this study and others to link SNPs associated with adiposity to altered MC4R expression. © 2014 Evans et al

A Whole-Genome SNP Association Study of NCI60 Cell Line Panel Indicates a Role of Ca2+ Signaling in Selenium Resistance

Epidemiological studies have suggested an association between selenium intake and protection from a variety of cancer. Considering this clinical importance of selenium, we aimed to identify the genes associated with resistance to selenium treatment. We have applied a previous methodology developed by our group, which is based on the genetic and pharmacological data publicly available for the NCI60 cancer cell line panel. In short, we have categorized the NCI60 cell lines as selenium resistant and sensitive based on their growth inhibition (GI50) data. Then, we have utilized the Affymetrix 125K SNP chip data available and carried out a genome-wide case-control association study for the selenium sensitive and resistant NCI60 cell lines. Our results showed statistically significant association of four SNPs in 5q33–34, 10q11.2, 10q22.3 and 14q13.1 with selenium resistance. These SNPs were located in introns of the genes encoding for a kinase-scaffolding protein (AKAP6), a membrane protein (SGCD), a channel protein (KCNMA1), and a protein kinase (PRKG1). The knock-down of KCNMA1 by siRNA showed increased sensitivity to selenium in both LNCaP and PC3 cell lines. Furthermore, SNP-SNP interaction (epistasis) analysis indicated the interactions of the SNPs in AKAP6 with SGCD as well as SNPs in AKAP6 with KCNMA1 with each other, assuming additive genetic model. These genes were also all involved in the Ca2+ signaling, which has a direct role in induction of apoptosis and induction of apoptosis in tumor cells is consistent with the chemopreventive action of selenium. Once our findings are further validated, this knowledge can be translated into clinics where individuals who can benefit from the chemopreventive characteristics of the selenium supplementation will be easily identified using a simple DNA analysis

A functional alternative splicing mutation in human tryptophan hydroxylase-2

The brain serotonergic system has an essential role in the physiological functions of the central nervous system and dysregulation of serotonin (5-HT) homeostasis has been implicated in many neuropsychiatric disorders. The tryptophan hydroxylase-2 (TPH2) gene is the rate-limiting enzyme in brain 5-HT synthesis, and thus is an ideal candidate gene for understanding the role of dysregulation of brain serotonergic homeostasis. Here, we characterized a common, but functional single-nucleotide polymorphism (SNP rs1386493) in the TPH2 gene, which decreases efficiency of normal RNA splicing, resulting in a truncated TPH2 protein (TPH2-TR) by alternative splicing. TPH2-TR, which lacks TPH2 enzyme activity, dominant-negatively affects full-length TPH2 function, causing reduced 5-HT production. The predicted mRNA for TPH2-TR is present in postmortem brain of rs1386493 carriers. The rs13864923 variant does not appear to be overrepresented in either global or multiplex depression cohorts. However, in combination with other gene variants linked to 5-HT homeostasis, this variant may exhibit important epistatic influences

Redundant Mechanisms Prevent Mitotic Entry Following Replication Arrest in the Absence of Cdc25 Hyper-Phosphorylation in Fission Yeast

Following replication arrest the Cdc25 phosphatase is phosphorylated and inhibited by Cds1. It has previously been reported that expressing Cdc25 where 9 putative amino-terminal Cds1 phosphorylation sites have been substituted to alanine results in bypass of the DNA replication checkpoint. However, these results were acquired by expression of the phosphorylation mutant using a multicopy expression vector in a genetic background where the DNA replication checkpoint is intact. In order to clarify these results we constructed a Cdc25(9A)-GFP native promoter integrant and examined its effect on the replication checkpoint at endogenous expression levels. In this strain the replication checkpoint operates normally, conditional on the presence of the Mik1 kinase. In response to replication arrest the Cdc25(9A)-GFP protein is degraded, suggesting the presence of a backup mechanism to eliminate the phosphatase when it cannot be inhibited through phosphorylation

Expression of Linear and Novel Circular Forms of an INK4/ARF-Associated Non-Coding RNA Correlates with Atherosclerosis Risk

Human genome-wide association studies have linked single nucleotide polymorphisms (SNPs) on chromosome 9p21.3 near the INK4/ARF (CDKN2a/b) locus with susceptibility to atherosclerotic vascular disease (ASVD). Although this locus encodes three well-characterized tumor suppressors, p16INK4a, p15INK4b, and ARF, the SNPs most strongly associated with ASVD are ∼120 kb from the nearest coding gene within a long non-coding RNA (ncRNA) known as ANRIL (CDKN2BAS). While individuals homozygous for the atherosclerotic risk allele show decreased expression of ANRIL and the coding INK4/ARF transcripts, the mechanism by which such distant genetic variants influence INK4/ARF expression is unknown. Here, using rapid amplification of cDNA ends (RACE) and analysis of next-generation RNA sequencing datasets, we determined the structure and abundance of multiple ANRIL species. Each of these species was present at very low copy numbers in primary and cultured cells; however, only the expression of ANRIL isoforms containing exons proximal to the INK4/ARF locus correlated with the ASVD risk alleles. Surprisingly, RACE also identified transcripts containing non-colinear ANRIL exonic sequences, whose expression also correlated with genotype and INK4/ARF expression. These non-polyadenylated RNAs resisted RNAse R digestion and could be PCR amplified using outward-facing primers, suggesting they represent circular RNA structures that could arise from by-products of mRNA splicing. Next-generation DNA sequencing and splice prediction algorithms identified polymorphisms within the ASVD risk interval that may regulate ANRIL splicing and circular ANRIL (cANRIL) production. These results identify novel circular RNA products emanating from the ANRIL locus and suggest causal variants at 9p21.3 regulate INK4/ARF expression and ASVD risk by modulating ANRIL expression and/or structure