A review of methods for detecting single-nucleotide polymorphisms in the Toll-like receptor gene family

The Toll-like receptors play an essential role in immunity through targeting the pathogen-associated molecular patterns. Nucleotide variations in TLR genes, especially single-nucleotide polymorphisms, have been shown to alter host immune susceptibility to several infections and diseases. Since TLR genes’ polymorphisms can be a promising biomarker, ongoing investigations aim to develop, optimize and validate SNP detection methods. This review discusses various TLR SNP detection methods, either used extensively or occasionally, but having a vast potential in high-throughput settings. Methods such as PCR-restriction fragment length polymorphism, TaqMan® assay, direct sequencing and matrix-assisted laser desorption ionization – time of flight mass spectroscopy MS are frequently used methods whereas Illumina GoldenGate® assay, reverse hybridization technology, PCR–confronting two-pair primers, KBiosciences KASPar® SNP assay, SNP stream®, PCR-fluorescence hybridization and SNaPshot® are powerful but sporadically used methods. We suggest that, for individual laboratories, the detection method of choice depends on a combination of factors such as throughput volume, reproducibility, feasibility and cost–effectiveness.

SNP discovery by amplicon sequencing and multiplex SNP genotyping in the allopolyploid species Brassica napusThis article is one of a selection of papers from the conference “Exploiting Genome-wide Association in Oilseed Brassicas: a model for genetic improvement of major OECD crops for sustainable farming”.

Oilseed rape ( Brassica napus ) is an allotetraploid species consisting of two genomes, derived from B. rapa (A genome) and B. oleracea (C genome). The presence of these two genomes makes single nucleotide polymorphism (SNP) marker identification and SNP analysis more challenging than in diploid species, as for a given locus usually two versions of a DNA sequence (based on the two ancestral genomes) have to be analyzed simultaneously during SNP identification and analysis. One hundred amplicons derived from expressed sequence tag (ESTs) were analyzed to identify SNPs in a panel of oilseed rape varieties and within two sister species representing the ancestral genomes. A total of 604 SNPs were identified, averaging one SNP in every 42 bp. It was possible to clearly discriminate SNPs that are polymorphic between different plant varieties from SNPs differentiating the two ancestral genomes. To validate the identified SNPs for their use in genetic analysis, we have developed Illumina GoldenGate assays for some of the identified SNPs. Through the analysis of a number of oilseed rape varieties and mapping populations with GoldenGate assays, we were able to identify a number of different segregation patterns in allotetraploid oilseed rape. The majority of the identified SNP markers can be readily used for genetic mapping, showing that amplicon sequencing and Illumina GoldenGate assays can be used to reliably identify SNP markers in tetraploid oilseed rape and to convert them into successful SNP assays that can be used for genetic analysis.

Variation at 8q24 and 9p24 and Risk of Epithelial Ovarian Cancer

The chromosome 8q24 region (specifically, 8q24.21.a) is known to harbor variants associated with risk of breast, colorectal, prostate, and bladder cancers. In 2008, variants rs10505477 and rs6983267 in this region were associated with increased risk of invasive ovarian cancer (p < 0.01); however, three subsequent ovarian cancer reports of 8q24 variants were null. Here, we used a multi-site case-control study of 940 ovarian cancer cases and 1,041 controls to evaluate associations between these and other single-nucleotide polymorphisms (SNPs) in this 8q24 region, as well as in the 9p24 colorectal cancer associated-region (specifically, 9p24.1.b). A total of 35 SNPs from previous reports and additional tagging SNPs were assessed using an Illumina GoldenGate array and analyzed using logistic regression models, adjusting for population structure and other potential confounders. We observed no association between genotypes and risk of ovarian cancer considering all cases, invasive cases, or invasive serous cases. For example, at 8q24 SNPs rs10505477 and rs6983267, analyses yielded per-allele invasive cancer odds ratios of 0.95 (95% confidence interval (CI) 0.82–1.09, p trend 0.46) and 0.97 (95% CI 0.84–1.12, p trend 0.69), respectively. Analyses using an approach identical to that of the first positive 8q24 report also yielded no association with risk of ovarian cancer. In the 9p24 region, no SNPs were associated with risk of ovarian cancer overall or with invasive or invasive serous disease (all p values > 0.10). These results indicate that the SNPs studied here are not related to risk of this gynecologic malignancy and that the site-specific nature of 8q24.21.a associations may not include ovarian cancer.

A Phase I Biological Study of Azacitidine (Vidaza) to Determine the Optimal Biological Dose and Route of Administration.

Abstract 1753 Poster Board I-779 Background Azacitidine (5-azacytidine, Vidaza) is a DNA methylation inhibitor with used to treat myelodysplastic syndrome (MDS). The studies which led to FDA approval based dosing and administration guidelines on clinical response. In vitro studies have demonstrated that azacitidine exerts its effect by inhibiting DNA methyltransferase in hypermethylated tumor suppressor genes in malignant cells. Research to date has not linked azacitidine dosing with biochemical and clinical response in vivo. The degree of DNA repetitive element sequence methylation (such as LINE-1) has been demonstrated to correlate with global DNA methylation and may be used to determine DNA methylation changes after treatment with azacitidine. We have conducted a phase I study to link clinical and biologic response to azacitidine. This study aims to determine the optimal dose and route of administration for azacitidine to inhibit global DNA methylation levels in the peripheral blood of patients with hematologic malignancies. Methods Patients with hematologic malignancy who provided informed consent were eligible for study inclusion, with enrollment criteria based on the specific malignancy. Patients were enrolled into one of five dose level treatment groups (25mg, 50mg, 75mg, 100mg or 150mg IV per m2 per day for 5 days) for the first course of therapy. On day 28, all patients received a course of 75mg/m2/day IV for 5 days. Subcutaneous dosing of 75mg/m2/day for 5 days was used for course three. Patients received 75mg/m2/day either SQ or IV x 5 days every 4 weeks for course four and beyond. Peripheral blood was collected on days 1, 3, and 5 during each course, and global DNA methylation was measured using bisulfite-PCR Pyrosequencing of the 6 DNA repetitive elements (LINE1, AluYb8, AluSq, Sat-alpha, D4Z4, NBL-2). Additionally, gene promoter specific DNA methylation was assessed in a subset of patients using the Illumina GoldenGate Bead Array DNA Methylation Assay which measures DNA methylation of 1505 CpG sites (807 genes). Results Seventeen patients were treated (3 at 25mg, 4 at 50mg, 4 at 75mg, 3 at 100mg, and 3 at 150mg/m2). Diagnosis included 5 patients with MDS, 10 patients with AML (2 untreated older patients, 7 relapsed or refractory patients), 1 patient with CML (Imatinib refractory), and 1 patient with non-Hodgkin's lymphoma (relapsed disease). At the time of submission, 14 patients were evaluable for response with 4 CR (1 mCr, 1 CRp), 1 PR, 6 SD and 3 PD reported. The median number of cycles given was 3 (range 1-14+). LINE1 DNA methylation decreased by 1.4, 2.3, 4.8, 1.9 and 4.0% on day 5 for the 25mg, 50mg, 75mg, 100mg, and 150mg/m2 course one dose levels respectively. Mean decrease in LINE1 DNA methylation with 75mg/m2 IV was 3.7% and only 2.6% by 75mg/m2 of azacitidine SQ. There was a large amount of inter-patient variability but less intra-patient variability in DNA methylation response to azacitidine. Conclusion Azacitidine is effective at inhibiting DNA methylation at multiple dose levels for both IV and SQ routes of administration. There is a high degree of patient-to-patient variability in DNA methylation changes, although 75mg/m2 lead to the greatest mean decrease in DNA methylation by a 5 day IV regimen. Measurement of DNA methylation of LINE1 and AluYb8 repetitive elements were the best surrogate markers for measuring overall changes in gene specific promoter DNA methylation when compared with 807 genes assessed by the Illumina GoldenGate platform. High-throughput gene promoter DNA methylation analysis revealed subtle changes in DNA methylation, though gene specific changes could not be linked to therapeutic activity. Disclosures Off Label Use: Azacitidine in hematologic malignancies other than MDS. Mohrbacher:Celgene: Honoraria, Speakers Bureau. Gorospe:Novartis: Honoraria, Speakers Bureau. Yang:Celgene: Honoraria, Research Funding, Speakers Bureau.