Middha, Sumit

Abstract Abstract 709 Background: A recent analysis of merged genome-wide and candidate gene genotypes in VTE cases and controls identified multiple tag SNPs that were strongly associated with VTE. Objective: To identify rare and/or novel functional variants by sequencing the implicated genes. Methods: Cases (n=1488) were Mayo Clinic European-American patients of non-Hispanic ancestry with objectively-diagnosed VTE in the absence of active cancer, venous catheter or antiphospholipid antibodies. Controls (n=1439) were Mayo Clinic outpatients without VTE who were frequency-matched on case age, gender, race, MI/stroke status and state of residence. For this analysis, we selected a subset of these cases and controls for sequencing to take advantage of the joint configuration of two ABO SNPs of primary interest, rs8176719 (ABO exon 6 deletion determining type O blood group) and rs2519093 (ABO intron 1 tag SNP), which were previously shown to be strongly associated with VTE (p=5.7E-12 and p=3.0E-16, respectively). We randomly sampled 82 cases and 14 controls within 3 of the 9 potential allele frequency cells (Figure). The rs8176719 alleles are -−/−- (double deletion is the common allele), –/G, and G/G (the rare allele). The rs2519093 alleles are GG (G is the common allele), AG, and AA (A is the rare allele). For each SNP, the genotypes are represented as 0, 1, or 2 copies of the minor allele. We represented the joint allelic configuration of the two SNPs with the number of copies of the rs8176719 given first as 0/0 (both with 0 copies of the minor allele), 0/1, 0/2 (0 copies of the rs8176719 SNP), 1/0 (1 copy of the rare rs8176719 SNP), 1/1/, and 1/2, and 2/0 (2 copies of the rare allele for the rs8176719 SNP and 0 copies of the rare allele of the rs2519093 SNP), 2/1, and 2/2. From the Figure one observes discrepancies between cases and controls at the 0/0, 1/1 and 2/2 combinations. We randomly sampled from these three combinations, taking one third of the case series. For each SNP, we had 28 cases with 0/0 copies of the rare allele, 27 cases with 1/1 copies of the rare allele; and 27 cases with the combination of 2/2 copies of the rare allele. We compared these 82 cases with 14 controls that do not have any of these combinations. Sixteen genes were selected for deep sequencing, including 5 genes harboring SNPs significantly associated with VTE (F5, SLC19A2, ABO, NME7, ATP1B1), 10 genes with SNPs marginally associated with VTE (C1orf114, KLKB1, SELP, F11, SCUBE1, PRKCB1, CD44, ITPR1, GFRA1, BLZF1), and CYP4V2 which reportedly confounds F11 and KLKB1. Agilent SureSelect probes were designed to capture and enrich the ∼2 Mb genomic regions of these 16 genes. Samples were multiplexed (12-plex) and sequenced using Illumina HiSeq 2000. The sequence reads were aligned to the human genome build 36 using Burrows-Wheeler Aligner, and the single nucleotide variants (SNVs) and small INDELs were called using SNVMix and GATK, respectively. For this analysis, novel ABO SNVs were tested for an association with VTE using age-, sex-adjusted logistic regression and Fisher's Exact Test. Results: 98% of the targeted regions were sequenced with > 20X coverage. On average, ∼2500 SNVs and ∼200 INDELs were detected in each sample. Fifteen novel SNVs in intron 6 and 3' of the ABO gene were associated with VTE (p<E-06) and belonged to 3 distinctive LD blocks; none were in LD with the coding or tag ABO SNPs (rs8176719; rs2519093). SNVs inside the middle LD block at the 3' of ABO are located within an enhancer and promoter histone marked with putative transcription factor binding sites. In addition, strong evidence from both ENCODE and dbEST support the middle LD block as lying within a novel transcript, probably an extension of the 3' of ABO. In addition, we discovered a novel, significant, protective, frame-shifting single base (G) deletion at ABO chr9:135120877. Conclusion: Novel ABO functional variants that are associated with VTE were identified by deep sequencing. Disclosures: Heit: Daiichi Sankyo: Consultancy, Honoraria.

Abstract Human immunity exhibits remarkable heterogeneity among individuals, which engenders variable responses to immune perturbations in human populations. Population studies reveal that, in addition to interindividual heterogeneity, systemic immune signatures display longitudinal stability within individuals, and these signatures may reliably dictate how given individuals respond to immune perturbations. We hypothesize that analyzing relationships among these signatures at the population level may uncover baseline immune phenotypes that correspond with response outcomes to immune stimuli. To test this, we quantified global gene expression in peripheral blood CD4+ cells from healthy individuals at baseline and following CD3/CD28 stimulation at two time points 1 mo apart. Systemic CD4+ cell baseline and poststimulation molecular immune response signatures (MIRS) were defined by identifying genes expressed at levels that were stable between time points within individuals and differential among individuals in each state. Iterative differential gene expression analyses between all possible phenotypic groupings of at least three individuals using the baseline and stimulated MIRS gene sets revealed shared baseline and response phenotypic groupings, indicating the baseline MIRS contained determinants of immune responsiveness. Furthermore, significant numbers of shared phenotype-defining sets of determinants were identified in baseline data across independent healthy cohorts. Combining the cohorts and repeating the analyses resulted in identification of over 6000 baseline immune phenotypic groups, implying that the MIRS concept may be useful in many immune perturbation contexts. These findings demonstrate that patterns in complex gene expression variability can be used to define immune phenotypes and discover determinants of immune responsiveness.

Abstract African American Hereditary Prostate Cancer Study (AAHPC) was developed as a national collaboration to explore the role of genetics in the causation of hereditary prostate cancer (HPC) in African American (AA) men. AAHPC is in partnership with the International Consortium for Prostate Cancer Genetics (ICPCG), which conducts collaborative studies of HPC genetics in multiplex families. As part of an ICPCG sequencing study of 539 affected individuals from 366 HPC pedigrees, we performed whole exome sequencing in 21 ICPCG AA families, of which there were 14 AAHPC affected men from 11 pedigrees. The combined ICPCG AA cohort consisted of N=26 affected members. Post-variant calling quality control (QC) was implemented using Golden Helix SVS 8 software with filters set for removal of variants with Read Depth &lt; 10, Quality Score &lt; 20, Quality Score: Read Depth Ratio &lt; 0.5, Call Rate &lt; 0.75. Variants were additionally filtered by MAF based on the NHLBI ESP650051-V2 exomes variant frequencies for the AA population using a MAF threshold of 5%. Following QC, 176/299 SNVs and 20/39 INDELs remained for further analysis. In these analyses, we focused on 13 known cancer genes (MSR1, AR, BRCA1, BRCA2, BTNL2, EPHB2, CDH1, RNASEL, ELAC2, HOXB13, CHEK2, TP53 and NBN). Three sequenced families had &gt; 1 affected members sequenced (2 or 3 per family) and the remaining 18 families had one member sequenced. Under the dominant model, our preliminary results show that no rare variants in the 13 candidate genes were found in 3/3 affecteds in two families. Rare SNVs in seven candidate genes (AR, CDH1, ELAC2, HOXB13, RNASEL, BRCA2 and EPHB2) were found in 2/3 affecteds for two families and 2/2 affecteds in one family. Several of the remaining 18 affected men (1 sequenced per pedigree) shared the same rare SNV in these candidate genes. For INDELs, rare variants in three candidate genes were found in pedigrees with ≥ 2 affecteds. Several of the remaining 18 affected men (one sequenced per pedigree) shared the same rare INDEL. Additional QC is underway to validate these variants and bioinformatic analyses are being used to predict effects of the variants in an effort to unravel the complex genetic heterogeneity of HPC in AA. Citation Format: Cheryl D. Cropp, Shannon K. McDonnell, Sumit Middha, Melissa DeRycke, Danielle M. Karyadi, Daniel Schaid, Stephen N. Thibodeau, William B. Isaacs, Elaine A. Ostrander, Janet Stanford, Kathleen A. Cooney, Joan E. Bailey-Wilson, John D. Carpten. Rare variant discovery in known cancer genes from whole-exome sequencingof African American hereditary prostate cancer families. [abstract]. In: Proceedings of the Eighth AACR Conference on The Science of Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; Nov 13-16, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2016;25(3 Suppl):Abstract nr B40.