Multiple Correction Test Research Articles

A combination of strongly associated prothrombotic single nucleotide polymorphisms could efficiently predict venous thrombosis risk.

Venous thrombosis (VT) is multifactorial trait that contributes to the global burden of cardiovascular diseases. Although abundant single nucleotide polymorphisms (SNPs) provoke the susceptibility of an individual to VT, research has found that the five most strongly associated SNPs, namely, rs6025 (F5 Leiden), rs2066865 (FGG), rs2036914 (F11), rs8176719 (ABO), and rs1799963 (F2), play the greatest role. Association and risk prediction models are rarely established by using merely the five strongly associated SNPs. This study aims to explore the combined VT risk predictability of the five SNPs and well-known non-genetic VT risk factors such as aging and obesity in the Hungarian population. SNPs were genotyped in the VT group (n = 298) and control group (n = 400). Associations were established using standard genetic models. Genetic risk scores (GRS) [unweighted GRS (unGRS), weighted GRS (wGRS)] were also computed. Correspondingly, the areas under the receiver operating characteristic curves (AUCs) for genetic and non-genetic risk factors were estimated to explore their VT risk predictability in the study population. rs6025 was the most prevalent VT risk allele in the Hungarian population. Its risk allele frequency was 3.52-fold higher in the VT group than that in the control group [adjusted odds ratio (AOR) = 3.52, 95% CI: 2.50-4.95]. Using all genetic models, we found that rs6025 and rs2036914 remained significantly associated with VT risk after multiple correction testing was performed. However, rs8176719 remained statistically significant only in the multiplicative (AOR = 1.33, 95% CI: 1.07-1.64) and genotypic models (AOR = 1.77, 95% CI: 1.14-2.73). In addition, rs2066865 lost its significant association with VT risk after multiple correction testing was performed. Conversely, the prothrombin mutation (rs1799963) did not show any significant association. The AUC of Leiden mutation (rs6025) showed better discriminative accuracy than that of other SNPs (AUC = 0.62, 95% CI: 0.57-0.66). The wGRS was a better predictor for VT than the unGRS (AUC = 0.67 vs. 0.65). Furthermore, combining genetic and non-genetic VT risk factors significantly increased the AUC to 0.89 with statistically significant differences (Z = 3.924, p < 0.0001). Our study revealed that the five strongly associated SNPs combined with non-genetic factors could efficiently predict individual VT risk susceptibility. The combined model was the best predictor of VT risk, so stratifying high-risk individuals based on their genetic profiling and well-known non-modifiable VT risk factors was important for the effective and efficient utilization of VT risk preventive and control measures. Furthermore, we urged further study that compares the VT risk predictability in the Hungarian population using the formerly discovered VT SNPs with the novel strongly associated VT SNPs.

Evaluation of genome-wide association signals for nonsyndromic cleft lip with or without cleft palate in a multiethnic Brazilian population

ObjectiveTo evaluate previous nonsyndromic cleft lip with or without cleft palate (NSCL±P) associated signals in 4p16.2, 8p11.23, 12q13.13, 12q13.2 and 17q21.32 in a multiethnic Brazilian cohort. DesignThe single nucleotide polymorphisms (SNPs) rs34246903 in 4p16.2, rs13317 in 8p11.23 (FGFR1, fibroblast growth factor receptor 1), rs3741442 in 12q13.13, rs705704 in 12q13.2 and rs4968247 in 17q21.32 were genotyped with TaqMan allelic discrimination assays in a case-control sample including 801 NSCL±P patients [233 nonsyndromic cleft lip (NSCLO) and 568 nonsyndromic cleft lip and palate (NSCLP)] and 881 healthy controls. Multiple logistic regression analyses, considering sex and genomic ancestry as covariates, were conducted, and the p value was adjusted with Bonferroni multiple correction testing (p ≤ 0.01). ResultsAlthough several associations were identified, those that resisted the multiple correction testing involved the alleles and genotypes of rs34246903 and rs13317. The NSCLO group had a lower frequency of the minor C allele of rs34246903 compared to controls, giving an odds ratio (OR) of 0.74 [95% confidence interval (CI): 0.59–0.93, p = 0.01]. The rs34246903 CC genotype (homozygous) and the recessive model revealed significant protective associations with NSCLO, yielding ORs of 0.50 (95% CI: 0.29–0.85, p = 0.005) and 0.55 (95% CI: 0.33–0.93, p = 0.01) respectively. The presence of C variant allele of rs13317 (OR: 0.81, 95% CI: 0.69–0.96, p = 0.01) as well the TC genotype (OR: 0.77, 95% CI: 0.62–0.94, p = 0.01) and the dominant model (OR: 0.77, 95% CI: 0.63–0.94, p = 0.009) showed significant associations with reduced risk of NSCL±P. ConclusionOur study is the first to support the association of rs34246903 (4p16.2) with NSCLO and rs13317 within FGFR1 with NSCL±P in the highly admixed Brazilian population. Further studies are needed to determine the functionality of those SNPs or to identify the causal markers in linkage disequilibrium with those susceptibility markers.

Association of GZMB polymorphisms and susceptibility to non-segmental vitiligo in a Korean population

Non-segmental vitiligo (NSV) is the most common type of vitiligo, which is characterized by chronic and progressive loss of melanocytes. Genetic factors have been shown to play a key role in NSV in association and family studies. Granzyme B is a serine protease found in the cytoplasmic granules of cytotoxic T lymphocytes and natural killer cells that play an important role in inducing apoptotic changes of target cells. Several recent studies have provided evidence that polymorphism in the GZMB gene might be associated with autoimmune disease. A total of 249 NSV patients and 455 healthy controls were recruited to determine whether single nucleotide polymorphisms (SNPs) [rs2236337 (3′ untranslated region, UTR), rs2236338 (Tyr247His), rs11539752 (Pro94Ala), rs10909625 (Lys80Lys), rs8192917 (Arg55Gln), and rs7144366 (5′ near gene)] in GZMB gene contribute to the risk of developing NSV. Genotyping was performed using a single 192.24 Dynamic Array IFC. Data were analyzed using EP1 SNP Genotyping Analysis software to obtain genotype calls. Among the six SNPs tested, five SNPs (rs2236337, rs2236338, rs11539752, rs10909625, and rs8192917) showed significant association with NSV susceptibility. Among them, rs2236338, rs11539752, rs10909625, and rs8192917 remained a statistically significant association following multiple correction test. The five SNPs were located within a block of linkage disequilibrium. Haplotypes T–A–G–T–T and C–G–C–C–C consisting of rs2236337, rs2236338, rs11539752, rs10909625, and rs8192917 demonstrated significant association with NSV. Our results suggest that GZMB polymorphisms are associated with the development of NSV.

Characterization of Intrinsic Radiosensitivity and Immune Cell Infiltrates in Bladder Cancer

Bladder cancer is managed by surgery, systemic therapy and radiation (RT) depending on clinicopathologic features and physician/patient choice. Various RT doses are used in bladder preservation, which are guided by normal tissue tolerance, not intrinsic biology. Emerging data supports the use of immunotherapy (IO) in bladder cancer, but optimal patient selection and therapy sequencing are not defined. This study characterizes the immune cell infiltrate (ICI) and intrinsic RT-sensitivity of bladder cancer in an institutional cohort and 4 public datasets with gene expression data to understand the relationship between RT-sensitivity and the ICI. With an IRB-approved prospective tissue collection protocol, 223 bladder cancer patients profiled with Affymetrix Hu-RSTA microarray chips (Affymetrix; Santa Clara, CA) were identified. Also, 4 public bladder cancer datasets from GEO with various clinicopathologic features were analyzed: GSE48075 (n = 142) and GSE32984 (n = 308) without neoadjuvant chemotherapy (NAC); GSE87304 (n = 305) with NAC; GSE120736 (primary/recurrent tumors; n = 145). In each dataset, tumors were grouped as non-invasive (NMIBC) or invasive (MIBC) by T category. Intrinsic tumor RT-sensitivity was inferred by the radiosensitivity index (RSI), a cancer-type agnostic gene expression algorithm. RSI values were split at the median and grouped as RSIlo (i.e. RT-sensitive) or RSIhi. Tumor ICI composition was inferred by 2 algorithms: CIBERSORT (22 distinct ICI types) and XCell (64 distinct immune and stromal cell types). Groups were compared with the Mann-Whitney U test. Multiple correction testing was performed. The institutional cohort median RSI is 0.42 (range 0.02-0.86). The median RSI values for the public datasets are: [GSE48075] 0.50 (range 0.22-0.69), [GSE32894] 0.23 (range -0.40-0.45), [GSE87304] 0.33 (range -0.07-0.48) and [GSE120736] 0.40 (range 0.06-0.72). No difference was seen in the median RSI between primary and recurrent bladder tumors (p = 0.38). Only one dataset had a difference in the median RSI between NMIBC and MIBC (GSE32894, p <0.001), with a higher RSI in MIBC. Next, using CIBERSORT and XCell, we found that resting or naïve ICIs (e.g. NK, CD8+ T, CD4+ T, B, CD4+ memory T cells) were higher in NMIBC in 3 out of 4 datasets (q <0.05). Several ICIs (e.g. activated NK, M1/M2 macrophages, CD4+ memory T cells) were higher in MIBC in all datasets (q <0.05). In 3 out of 4 datasets and the institutional cohort, RSIlo vs RSIhi tumors had higher CD4+ T, M1 macrophages, activated (NK, dendritic, CD4+ memory T cells) and CD8+ T cell types (q <0.05). Bladder tumors have a range of RT-sensitivities in this analysis, implying uniform RT dosing may lead to over and under treatment. Also, NMIBC has higher resting or naïve ICIs compared to MIBC and RT-sensitive tumors are enriched with specific effector ICIs. Integration of ICI and RT-sensitivity data may assist with optimizing patient selection for certain IO and RT combinations.

Abstract LB-174: Genome-wide DNA methylation signatures predict the early asymptomatic doxorubicin-induced cardiotoxicity in breast cancer

Abstract Background: Doxorubicin (DOX)-induced cardiotoxicity is cumulative-dose-dependent that begins with the first dose, but epigenetic changes after the first dose have not been examined. This study had two aims: Aim 1: to examine whether DNA methylation profile of peripheral blood cells (PBCs) induced by the first cycle of DOX-based chemotherapy can predict the risk of cardiotoxicity; and Aim 2 to determine if there are pretreatment methylation signatures at baseline to predict the risk of cardiotoxicity. Methods: Whole-genome DNA methylation of PBCs from 19 breast cancer patients was examined prior to the start of DOX-based chemotherapy and after the first cycle using Infinium HumanMethylation 450 BeadChip. ChAMP R package was used for the data normalization by the BMIQ algorithm and the COMBAT algorithm to adjust for batch effects. Chemotherapy-induced cardiotoxicity was determined by the assessment of left ventricle ejection fraction (LVEF) with multigated acquisition (MUGA) scan before chemotherapy and at its completion, and any LVEF decline by &amp;gt;10% was considered abnormal. Results: Pre- and post- DOX treatment were compared between patients with abnormal LVEF decline and those who maintained normal LVEF to assess the differences in the cytosine methylation states. The multiple correction testing resulted in a total of 515 differentially methylated CpGs between the patients with abnormal versus normal LVEF post-DOX treatment. There were 379 differentially methylated CpGs at baseline and 136 CpGs after the first cycle which significantly correlated with LVEF status after applying the Holm-Bonferroni method for multiple testing correction. The intersection of the probes resulted in 38 differentially methylated genes (DMG) that when used in hierarchical clustering, correctly clustered baseline samples into those that would result in normal and abnormal LVEF after treatment. DMG genes included SEPT5, IRF6, KCNQ1, NELL1, SIX6, HOXC9 and BCAN. IRF6, which encodes interferon regulatory factor 6, was identified as being significant in the subsequent analysis at the probe and region level. Comparisons of the samples before and after DOX treatment did not result in any probes associated with the treatment that were significant after multiple testing correction. Conclusions: The results from this study provide evidence that DNA methylation profile of peripheral blood cells has the potential to predict the risk of DOX-induced cardiotoxicity. The important finding was that the extent of methylation at baseline correlated with the post-DOX LVEF reduction, indicating that it may have the potential to predict the subsequent development of cardiotoxicity. Further studies with larger cohort of patients are needed to confirm these findings as well as narrowing down candidate methylation markers that can be implemented in a blood test. These finding may help guide treatment or identify patients that need to be followed closely to mitigate heart damage after DOX treatment. Citation Format: Michael A. Bauer, Issam Makhoul, Ping-Ching Hsu, Jeanne Wei, L. Joseph Su, Annjanette Stone, Weleetka Carter, Valentina K. Todorova. Genome-wide DNA methylation signatures predict the early asymptomatic doxorubicin-induced cardiotoxicity in breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-174.

Abstract 2525: Comprehensive analysis of thymic epithelial tumors using an integrated bioinformatics platform reveals novel biological characteristics

Abstract Introduction:Thymic epithelial tumors (TETs) are rare malignancies. Among TETs, thymoma is the most predominant, characterized by a unique association with myasthenia gravis, followed by thymic carcinoma which is less common but more clinically aggressive. The TCGA recently published a comprehensive analysis of 117 TETs using molecular profiling by exome-seq, RNA-seq, RPPA, and methylation. Using an integrated informatics platform we re-analyzed the TCGA TET data to identify novel biological characteristics of these rare tumors. Methods: Molecular and clinical data from the TCGA TET project was downloaded from the cbioportal database and imported into the tag.bio informatics platform. Tag.bio is a commercial cloud-based analysis platform that combines data, algorithms and question-based analysis protocols. It is used to for the rapid and reproducible statistical comparison of cohorts.Statistical analysis for differential expression, methylation, RPPA and mutational enrichment was performed using the Mann-Whitney test with multiple correction testing. Pathway and upstream regulator analysis were performed using Ingenuity Pathway Analysis. Results: Differential expression analysis of myasthenia gravis (MG) patients (N=32) compared to those with no history of MG (N=85), revealed the most significant gene as PPARGC1A (overexpressed, p&amp;lt;1e-8). This gene has not been previously associated with MG. PPARGC1A is highly expressed in skeletal muscle and mediates mitochondrial biogenesis. Supporting this observation, upstream regulator analysis was performed which identifies regulators that can explain the observed gene expression changes in a dataset. The results demonstrated that PPARGC1A as the most activated transcriptional regulator in MG+ tumors and may represent a novel autoantigen for MG. TETs are relatively indolent tumors, and only a small subset metastasizes. Differential expression in a subset of Stage IV tumors (N=6) identified overexpressed FGFR3. Of note, a previous study identified two patients with activating mutations in FGFR3. A genomic hallmark of Thymic Carcinoma (TC) is Chr16q loss. Several tumor suppressors reside on 16q, including:CYLD, CBFB, CDH1, CDH11, CTCF, ZFHX3, but the exact causative candidate is unknown. Analysis of the mutational data identified 2 of 10 patients with concurrent 16q loss and deleterious mutations in CYLD, congruent with previous reports of recurrent mutations in CYLD in TC. CYLD has been previously demonstrated to regulate STAT3. Differential expression followed by upstream regulator analysis of TC vs Thymoma demonstrated activated STAT3 in TC. Conclusion:Re-analysis of the TCGA TET dataset using a novel integrated bioinformatics platform identified novel biology of TETs including: PPARGC1A association with MG; FGFR3 expression in TET metastasis; and CYLD/STAT3 axis in TC. Citation Format: Milan Radovich, Jeffrey P. Solzak, Wade Webster, Jesse Paquette. Comprehensive analysis of thymic epithelial tumors using an integrated bioinformatics platform reveals novel biological characteristics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2525.

Association of recurrent venous thromboembolism and circulating microRNAs

BackgroundPatients with unprovoked first venous thromboembolism (VTE) are at a high risk of recurrence. Although circulating microRNAs (miRNAs) have been found to be associated with VTE and are markers of hypercoagulability, this study is the first to examine whether circulating miRNAs are associated with the risk of VTE recurrence.ResultsA nested case-control study design was used where plasma samples were obtained from 78 patients with unprovoked VTE from the Malmö Thrombophilia Study (MATS). A total of 39 VTE patients with recurrent VTE (cases) were matched with 39 VTE patients without recurrent VTE (controls) defined by age and sex (MATS population). Plasma levels of 179 different miRNAs were evaluated in the 78 samples (after anticoagulant treatment was stopped) using qPCR. A total of 110 miRNAs were detected in all samples. Among those, 12 miRNAs (miR-15b-5p, miR-106a-5p, miR-197-3p, miR-652-3p, miR-361-5p, miR-222-3p, miR-26b-5p, miR-532-5p, miR-27b-3p, miR-21-5p, miR-103a-3p, and miR-30c-5p) were found to be associated with recurrent VTE after multiple correction test and conditional logistic regression analysis. A further analysis showed that miR-15b-5p, miR-197-3p, miR-27b-3p, and miR-30c-5p exhibited a trend over time, with a larger difference in miRNA levels between cases and controls for earlier recurrence. Of these 12 miRNAs, 8 miRNAs significantly correlated with circulating transforming growth factor β1/2 (TGFβ1/2). Three of them correlated with platelet count.ConclusionWe have identified 12 plasma miRNAs that may have the potential to serve as novel, non-invasive predictive biomarkers for VTE recurrence.

The microRNA network is altered in anterior cingulate cortex of patients with unipolar and bipolar depression

MicroRNAs (miRNAs) are small, non-coding RNAs acting as post-transcriptional regulators of gene expression. Though implicated in multiple CNS disorders, miRNAs have not been examined in any psychiatric disease state in anterior cingulate cortex (AnCg), a brain region centrally involved in regulating mood. We performed qPCR analyses of 29 miRNAs previously implicated in psychiatric illness (major depressive disorder (MDD), bipolar disorder (BP) and/or schizophrenia (SZ)) in AnCg of patients with MDD and BP versus controls. miR-132, miR-133a and miR-212 were initially identified as differentially expressed in BP, miR-184 in MDD and miR-34a in both MDD and BP (although none survived multiple correction testing and must be considered preliminary). In silico target prediction algorithms identified putative targets of differentially expressed miRNAs. Nuclear Co-Activator 1 (NCOA1), Nuclear Co-Repressor 2 (NCOR2) and Phosphodiesterase 4B (PDE4B) were selected based upon predicted targeting by miR-34a (with NCOR2 and PDE4B both targeted by miR-184) and published relevance to psychiatric illness. Luciferase assays identified PDE4B as a target of miR-34a and miR-184, while NCOA1 and NCOR2 were targeted by miR-34a and 184, respectively. qPCR analyses were performed to determine whether changes in miRNA levels correlated with mRNA levels of validated targets. NCOA1 showed an inverse correlation with miR-34a in BP, while NCOR2 demonstrated a positive correlation. In sum, this is the first study to demonstrate miRNA changes in AnCg in psychiatric illness and validate miR-34a as differentially expressed in CNS in MDD. These findings support a mechanistic role for miRNAs in the regulation of stress-responsive genes disrupted in psychiatric illness.

Association of in vitro fertilization with global and IGF2/H19 methylation variation in newborn twins.

In vitro fertilization (IVF) and its subset intracytoplasmic sperm injection (ICSI), are widely used medical treatments for conception. There has been controversy over whether IVF is associated with adverse short- and long-term health outcomes of offspring. As with other prenatal factors, epigenetic change is thought to be a molecular mediator of any in utero programming effects. Most studies focused on DNA methylation at gene-specific and genomic level, with only a few on associations between DNA methylation and IVF. Using buccal epithelium from 208 twin pairs from the Peri/Postnatal Epigenetic Twin Study (PETS), we investigated associations between IVF and DNA methylation on a global level, using the proxies of Alu and LINE-1 interspersed repeats in addition to two locus-specific regulatory regions within IGF2/H19, controlling for 13 potentially confounding factors. Using multiple correction testing, we found strong evidence that IVF-conceived twins have lower DNA methylation in Alu, and weak evidence of lower methylation in one of the two IGF2/H19 regulatory regions and LINE-1, compared with naturally conceived twins. Weak evidence of a relationship between ICSI and DNA methylation within IGF2/H19 regulatory region was found, suggesting that one or more of the processes associated with IVF/ICSI may contribute to these methylation differences. Lower within- and between-pair DNA methylation variation was also found in IVF-conceived twins for LINE-1, Alu and one IGF2/H19 regulatory region. Although larger sample sizes are needed, our results provide additional insight to the possible influence of IVF and ICSI on DNA methylation. To our knowledge, this is the largest study to date investigating the association of IVF and DNA methylation.

Correlation analysis between period circadian clock 2 gene and the prognosis of pancreatic ductal adenocarcinoma

Objective To explore the prognosis related genes of pancreatic ductal adenocarcinoma （PDAC）and investigate the molecular regulation mechanism.Methods Gene expression data of 102 PDAC patients with complete clinical survival data were selected from gene expression database of National Center for Biotechnology Information.The 106 transcription regulation gene collection was collected from Transfac database.The 715 microRNA （miRNA）target regulation gene collection was selected according to PicTar and TargetScanS method.Biological pathway data obtained from the Kyoto Encyclopedia of Genes and Genomes （KEGG）.The known cancer genes were collected from the cancer gene census （CGC） database.Univariate Cox proportional hazards model was used to analyze the correlation between gene expression data and survival time,then obtained survival related candidate genes from the whole genome. Then the enriched genes were analyzed by hypergeometric distribution algorithm from three databases. Multiple correction testing was performed by BH-FDR method （FDR 〈 0.05 ）.Kaplan-Meier was performed for survival curve analysis of PDAC.Results The results of data of 102 PDAC patients analyzed by univariate Cox proportional hazards model indicated that 273 genes were significantly related to the survival time of patients （P 〈0.000 1 ）.After 273 survival genes were enrichment analyzed in 106 transcription factor regulation gene collection,12 survival genes enriched transcription factor target gene sets were found.After 273 survival genes were enrichment analyzed in 715 miRNA target regulation gene collection,11 survival genes enriched miRNAs target sets were discovered.After 273 survival genes were enrichment analyzed in pathway data of KEGG,15 survival genes enriched pathways were obtained. Period circadian clock 2 （PER2 ）was regulated by CCAAT/enhancer binding protein （CEBPA）at transcription level and regulated by miRNA-32 after transcription.The prognosis of PDAC was affected by circadian rhythm pathway.The 102 patients with PDAC were ranked according to the expression of PER2 from high to low,the first 51 cases were included in PER2 higher expression group and the left were included in PER2 lower expression group.Kaplan-Meier survival analysis indicated that PER2 was significantly correlated with prognosis of PDAC （P 〈0.01 ）.Conclusion CEBPA/miRNA-32/PER2 and its related circadian clock pathway may be the target pathway in interfering the development of PDAC,and is correlated with the prognosis of PDAC. Key words: Pancreatic ductal adenocarcinoma; PER2; Prognosis; Molecule mechanism

Interactions between circadian gene variants and fatty acid intake for metabolic syndrome risk: a meta‐analysis from the Cohorts for Heart and Aging Research in Genomic Epidemiology Consortium (373.2)

The circadian system is the body’s endogenous clock that governs a wide range of physiological systems and behaviors, and is regulated primarily by light and food intake. Variants in genes encoding components of the system result in circadian dysregulation, and have recently been linked to metabolic syndrome (MetS). In order to identify modifiable factors to attenuate the risk of MetS in individuals susceptible to circadian dysregulation, we investigated relationships between diet and these genetic risk factors in eight studies from the CHARGE consortium (up to 10,495 European descent individuals). In fixed‐effects meta‐analyses, we evaluated interactions between fatty acid (FA) intake and 5 variants on MetS. No interaction reached significance after multiple correction testing. However, the rs1387153 variant near MTNR1B, which encodes a circadian hormone receptor, interacted nominally (P = 0.01) with polyunsaturated FA intake on HOMA‐IR (homeostasis model assessment of insulin resistance). Additionally, we explored the role of sleep duration as a modulator of dietary intake. Consistent with previous studies, sleep duration was associated with total (P=0.03) and saturated (P=0.02) FA intake, only in women. The current findings inform understanding of complex relationships between circadian and metabolic physiology and may be of application for personalized recommendations to reduce MetS burden.

Abstract B66: Chronic myeloid leukemic cells trigger poly(ADP-ribose) polymerase-dependent cell death in natural killer cells.

Abstract Background and Purpose: Natural killer (NK) cells are commonly dysfunctional in chronic myeloid leukemia (CML) and their status may determine the course of disease. We aimed to define the molecular mechanisms of leukemia-induced NK cell inhibition with focus on the role of CML cell-derived reactive oxygen species (ROS) and the poly(ADP-ribose) polymerase-1 (PARP-1) pathway of cell death (parthanatos). Methods: Malignant granulocytes from patients with BCR-ABL+ CML were analyzed using flow cytometry for cell surface expression of the oxygen radical-producing NADPH/NOX-2 oxidase (determined by expression of the NADPH oxidase subunit gp91phox). ROS production by CML cells was assessed by chemiluminescence. In co-culture experiments, purified NK cells from healthy donors were exposed to malignant CML cells followed by analysis of NK cell viability. In these experiments, inhibitors of PARP-1 (PJ34 and EB-47) and the caspase cascade (Z-VAD.fmk) were added to determine the mechanisms of cell death in NK cells exposed to CML cells. Translocation of apoptosis-inducing factor (AIF) from mitochondria to nucleus was determined in sorted NK cells by immunoblot technique. Results: All BCR-ABL+ CML cells showed high cell surface expression of gp91phox, indicating the presence of a NADPH/NOX-2 oxidase. CML cells produced high amounts of ROS upon stimulation with the protein kinase C activator PMA or by the bacterial tripeptide fMLF suggestive of a functional ROS-forming oxidase. Unstimulated CML cells triggered extensive apoptosis-like cell death in co-cultured NK cells, which was prevented by the ROS-degrading enzyme catalase and by the NADPH oxidase/NOX-2 inhibitor diphenylene iodonium. The PARP-1 inhibitor PJ34 (0.5 microM) prevented CML-induced NK cell death at CML/NK cell ratios of 0.125: p&amp;lt;0.05 (using cells from five CML patients); 0.25: p&amp;lt;0.0001 (n=8), 0.5: p&amp;lt;0.0001 (n=6), and 1: p&amp;lt;0.0001 (n=8; analyzed by two-way Anova followed by Bonferroni's multiple correction test). A structurally unrelated PARP-1 inhibitor, EB-47, shared the NK cell-protective properties of PJ34 and EB-47 whereas the pan-caspase inhibitor Z-VAD.fmk failed to rescue NK cells from CML cell-induced death. After CML cell exposure, NK cells expressed JC-1 monomers suggestive of a disrupted mitochondrial membrane potential. AIF, a mitochondrial protein involved in PARP-1 dependent cell death, accumulated in NK cell nuclei after interaction with CML cells. Conclusions: Our results imply (1) that paracrine ROS production by CML cells followed by NK cell death may constitute a mechanism by which CML cells evade destruction by cytotoxic lymphocytes, and (2) that CML cells trigger NK cell death by inducing PARP-1 dependent parthanatos. These findings may have implications for the design of more efficient immunotherapeutic protocols in CML. Citation Format: Johan Aurelius, Anna Martner, Rebecca E. Riise, Ana I. Romero, Lars Palmqvist, Mats Brune, Kristoffer Hellstrand, Fredrik B. Thorén. Chronic myeloid leukemic cells trigger poly(ADP-ribose) polymerase-dependent cell death in natural killer cells. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr B66.

Association of limbic system-associated membrane protein (LSAMP) to male completed suicide

BackgroundNeuroimaging studies have demonstrated volumetric abnormalities in limbic structures of suicide victims. The morphological changes might be caused by some inherited neurodevelopmental defect, such as failure to form proper axonal connections due to genetically determined dysfunction of neurite guidance molecules. Limbic system-associated membrane protein (LSAMP) is a neuronal adhesive molecule, preferentially expressed in developing limbic system neuronal dendrites and somata. Some evidence for the association between LSAMP gene and behavior has come from both animal as well as human studies but further investigation is required. In current study, polymorphic loci in human LSAMP gene were examined in order to reveal any associations between genetic variation in LSAMP and suicidal behaviour.MethodsDNA was obtained from 288 male suicide victims and 327 healthy male volunteers. Thirty SNPs from LSAMP gene and adjacent region were selected by Tagger algorithm implemented in Haploview 3.32. Genotyping was performed using the SNPlex™ (Applied Biosystems) platform. Data was analyzed by Genemapper 3.7, Haploview 3.32 and SPSS 13.0.ResultsChi square test revealed four allelic variants (rs2918215, rs2918213, rs9874470 and rs4821129) located in the intronic region of the gene to be associated with suicide, major alleles being overrepresented in suicide group. However, the associations did not survive multiple correction test. Defining the haplotype blocks using confidence interval algorithm implemented in Haploview 3.32, we failed to detect any associated haplotypes.ConclusionDespite a considerable amount of investigation on the nature of suicidal behaviour, its aetiology and pathogenesis remain unknown. This study examined the variability in LSAMP gene in relation to completed suicide. Our results indicate that LSAMP might play a role in pathoaetiology of suicidal behaviour but further studies are needed to understand its exact contribution.

5 TRANSCRIPTIONAL PROFILING OF PIG IN IN VIVO, IN VITRO-FERTILIZED, AND NUCLEAR TRANSFER-DERIVED BLASTOCYSTS AND THE DONOR SOMATIC CELL LINE

The objective of this study was to perform transcriptional profiling between in vivo (IVV), in vitro-fertilized (IVF), and nuclear transfer (NT) blastocyst stage embryos, along with the donor cell line used for NT, in order to identify candidate genes that may contribute to the suboptimal phenotypes of cloned pigs. IVV samples were collected surgically 8 days post-estrus. IVF and NT embryos were transferred into recipient gilts on Day 0 or 1 of estrus and were subsequently collected 6 days later by uterine flush. NT oocytes were activated using one of three methods:NT-1 (electrical activation/fusion), NT-2 (electrical activation/fusion + treatment with proteasomal inhibitor MG 132), or NT-3 (electrical fusion + thimerosal/dithiothreitol (DTT) activation). NT was performed by using pCAG-EGFP positive fetal fibroblast cells to avoid collection of parthenogenetic blastocysts. Donor cells were collected post-NT in pools of 100. Three pools of 10–15 embryos were collected for each treatment. Each pool was analyzed twice, resulting in three biological and two technical replicates. A reference design was used and the reference RNA represented a pool of both reproductive and non-reproductive tissues. Total RNA was isolated by using Trizol (Invitrogen, Carlsbad, CA, USA) and amplified by using an Ovation Ribo-SPIA linear amplification kit (NuGEN Technologies, Inc., San Carlos, CA, USA). Amplified cDNA from blastocysts or cells was labeled with Cy5 and compared to cDNA from the reference sample labeled with Cy3. The cDNAs were hybridized to an in-house printed pig reproductive tissue-specific 19 968 spot cDNA microarray. Microarray images were acquired using a GenePix� 4000B scanner. Spot quality was assessed and results files were constructed using GenePix Pro 4.0. Lowess normalization and analysis was performed in Genespring 7.3.1 (Agilent Technologies, Inc., Palo Alto, CA, USA). Two comparisons were made: IVF versus IVV, and a comparison of all treatments IVV, IVF, NT-1, NT-2, NT-3, and donor cell line. ANOVA (P &lt; 0.05) was performed with the Benjamini and Hochberg False Discovery Rate multiple correction test. The IVF and IVV comparison resulted in 0 differentially detected cDNAs. The IVV, IVF, NT-1, NT-2, NT-3, and donor cell line comparison detected 1477 differentially detected cDNAs, including heat shock proteins (HSPD1 and HSPE1), which are lowly expressed in the donor cell line, and X inactive-specific transcript (XIST), which has higher expression in IVV and IVF compared to that in NT blastocysts. A standard correlation was performed on both comparisons. The R2 value for the IVV and IVF comparison was 0.892, while the R2 value for all samples was 0.716. These results illustrate that IVV and IVF blastocysts, developed within the uterus, are nearly identical. However, a comparison of blastocysts in all treatments including NT and the donor cell line revealed many differentially expressed genes that can be further evaluated for biological function and usefulness as potential markers of quality embryo development after NT.

Transcriptional Profiling of Pig Embryogenesis by Using a 15-K Member Unigene Set Specific for Pig Reproductive Tissues and Embryos1

Differential mRNA expression patterns were evaluated between germinal vesicle oocytes (pgvo), four-cell (p4civv), blastocyst (pblivv), and in vitro-produced four-cell (p4civp) and in vitro-produced blastocyst (pblivp) stage embryos to determine key transcripts responsible for early embryonic development in the pig. Five comparisons were made: pgvo to p4civv, p4civv to pblivv, pgvo to pblivv, p4civv to p4civp, and pblivv to pblivp. ANOVA (P < 0.05) was performed with the Benjamini and Hochberg false-discovery-rate multiple correction test on each comparison. A comparison of pgvo to p4civv, p4civv to pblivv, and pgvo to pblivv resulted in 3214, 1989, and 4528 differentially detected cDNAs, respectively. Real-time PCR analysis on seven transcripts showed an identical pattern of changes in expression as observed on the microarrays, while one transcript deviated at a single cell stage. There were 1409 and 1696 differentially detected cDNAs between the in vitro- and in vivo-produced embryos at the four-cell and blastocyst stages, respectively, without the Benjamini and Hochberg false-discovery-rate multiple correction test. Real-time polymerase chain reaction (PCR) analysis on four genes at the four-cell stage showed an identical pattern of gene expression as found on the microarrays. Real-time PCR analysis on four of five genes at the blastocyst stage showed an identical pattern of gene expression as found on the microarrays. Thus, only 1 of the 39 comparisons of the pattern of gene expression exhibited a major deviation between the microarray and the real-time PCR. These results illustrate the complex mechanisms involved in pig early embryonic development.