Peripheral Blood RNA Research Articles

Detection of Low Frequency T-Cell Clones Via a Next Generation Sequencing (NGS) Based Immune-Oncology Research Assay

Introduction: The ability to detect and quantitate low frequency T-cell clones enables numerous hematology/oncology research applications, including identification and assessment of biomarkers associated minimal residual disease (MRD). Rare clone detection via NGS requires highly efficient library preparation and accurate sequencing methodologies, because single nucleotide substitution sequencing errors mimic the natural variation in the T-cell repertoire, resulting in detection of artifactual low frequency clones. Here we present an experimental framework and corresponding performance of rare clone detection utilizing the OncomineTM TCR Beta short read (TCRb-SR) assay, using Ampliseq-based library preparation targeting the highly variable CDR3 region of TCRb using either DNA or RNA as input, with sample-to-result in 2 days. Methods: To evaluate detection sensitivity of the TCRB-SR assay, we utilized Jurkat cell line DNA and RNA because the presence of a single T cell clone enables precise control of dilution studies. Commercially procured Jurkat gDNA or RNA was spiked into peripheral blood leukocyte gDNA or RNA from 10-1 to 10-6 absolute clone frequency to create specimens with a known T-cell clone at frequencies commonly observed in MRD research applications. Peripheral blood leukocyte gDNA or RNA was used as the background for spike in studies due to its high T-Cell diversity. Six to twenty technical replicates were analyzed per dilution point, with DNA inputs ranging from 100ng to 1ug and RNA inputs ranging from 25ng to 100ng to evaluate the minimum detectable clone frequency as a function of nucleic acid input. Libraries were prepared following the TCRB-SR manufacturer's instructions for both DNA and RNA, followed by templating and sequencing using Ion Chef and S5 systems. Data processing was performed in Torrent Suite software (v5.10) followed by read alignment to the IMGT database of variable, diversity, and joining genes using Ion Reporter software (v5.10). For the 10-6 target frequency with gDNA as the input, four 1ug libraries were combined for analysis in Ion Reporter. Analytical sensitivity was calculated at each target clone frequency by detection of the Jurkat clone as defined by V-gene, Joining gene, and CDR3 nucleotide sequence. Results: Detection sensitivity was dependent on the amount gDNA or RNA input. For gDNA inputs, we observed 100% sensitivity at 10-3 with 100ng input, 100% sensitivity at 10-4 with 250ng input, 95% sensitivity at 10-5 with 1ug input, and 100% sensitivity at 10-6 with 4ug input. For RNA inputs, we observed 100% sensitivity at 10-5 with 25ng input and 100% sensitivity at 10-6 with 100ng input. In addition, we observe a strong linearity of observed clone frequencies at each dilution level, with an r-squared of 0.97. Conclusions: Here we demonstrate the ability to detect T-cell clones down to 10-6 from gDNA or RNA inputs with high sensitivity and linearity utilizing the OncomineTM TCR Beta short read assay. We present data demonstrating detection of clones with absolute frequencies of 10-6 utilizing 4ug gDNA input or 100ng RNA input, highlighting strong performance at nucleic acid input levels typically seen in clinical research samples. Taken together, we show feasibility for rare clone detection in either gDNA or RNA enabling research and development for T-cell minimal residual disease applications. For research use only, not for use in diagnostic procedures. Disclosures Williamson: Thermo Fisher Scientific: Employment. Looney:Thermo Fisher Scientific: Employment. Lowman:Thermo Fisher Scientific: Employment. Sidhu:Thermo Fisher Scientific: Employment. Solano:Thermo Fisher Scientific: Employment. Salazar:Thermo Fisher Scientific: Employment. Tian:Thermo Fisher Scientific: Employment. Ramsamooj:Thermo Fisher Scientific: Consultancy.

Identification of Serum Exosomal hsa-circ-0004771 as a Novel Diagnostic Biomarker of Colorectal Cancer.

Background: Exosomal circular RNAs (circRNAs) in peripheral blood are considered as emerging diagnostic biomarkers of cancers. Owing to the lack of sensitive and specific biomarkers, a large number of colorectal cancer (CRC) patients were diagnosed in advanced stages leading to high mortality. This study aimed to identify circulating exosomal circRNAs as novel diagnostic biomarkers of CRC.Materials and Methods: Candidate circRNA was selected by integrating analysis of Gene Expression Omnibus (GEO) database with online program GEO2R. A total of 170 patients and 45 healthy controls were enrolled to assess the diagnostic value of circRNAs for CRC. Exosomes isolated from the serum of participants and cell cultured media were confirmed by transmission electron microscope (TEM), Nanoparticle Tracking Analysis and western blot. The expression and the diagnostic utility of circRNA were tested by qRT-PCR and receiver operating characteristic (ROC) analysis, respectively.Results: The circulating exosomal hsa-circ-0004771 with most abundant among the top ten differentially expressed circRNAs (fold change ≥1.5) was selected for further study based on the results of GEO dataset analysis. The up-regulated exosomal hsa-circ-0004771 was verified in serum of CRC patients compared to healthy controls (HCs) and patients with benign intestinal diseases (BIDs) by qRT-PCR. The area under the ROC curves (AUCs) of circulating exosomal hsa-circ-0004771 were 0.59 (95%CI, 0.457–0.725), 0.86 (95%CI, 0.785–0.933) and 0.88 (95%CI, 0.815–0.940) to differentiate BIDs, stage I/II CRC patients and CRC patients from HCs, respectively. The AUC was 0.816 (95%CI, 0.728–0.9) to differentiate stage I/II CRC patients from patients with BIDs. In addition, the elevated expression of exosomal hsa-circ-0004771 in the serum of CRC patients was tumor-derived. It was found that the expression of exosomal hsa-circ-0004771 was down-regulated expression of in the serum of postoperative CRC patients as well as cultured media of CRC cells treated with GW4869.Conclusions: Circulating exosomal hsa-circ-0004771 was significantly up-regulated in CRC patients and served as a novel potential diagnostic biomarker of CRC.

Differential expression of long non-coding RNAs in peripheral blood of type 2 diabetic patients

Objective To screen and verify the differential expression profiles of long non-coding RNAs(LncRNAs) in peripheral blood of patients with type 2 diabetes mellitus(T2DM), to identify the potential molecular specific markers of early T2DM. Methods The blood samples of 4 type 2 diabetic patients and 4 normal control subjects were collected for microarray analysis. Then six candidate markers of LncRNAs screened from the differential expression profile were tested by qRT-PCR among the subjects (80 cases in the T2DM group and 50 cases in the control group). The possibility of these LncRNAs as molecular diagnostic markers was analyzed, and finally two of them were carried out by receiver operating characteristic (ROC) analysis. Results Compared with control subjects, there were differentially expressed 133 LncRNAs in type 2 diabetic patients, among which 5 were up-regulated with the maximum up-regulated fold 3.29 and 128 were down-regulated with the maximum down-regulated fold 8.99. Six down-regulated LncRNAs were selected for validation and revealed a similar result to that of microarray.The expressions of two LncRNAs(NONHSAT160746 and NONHSAT140069) in peripheral blood of diabetic patients were significantly lower than those of control subjects (P<0.01). The areas under the ROC curve of the two LncRNAs were 0.734 and 0.703, respectively(P<0.01). Conclusion LncRNAs NONHSAT160746 and LncRNAs NONHSAT140069 are the potential molecular specific markers for the diagnosis of diabetes mellitus. Key words: Diabetes mellitus, type 2; Long non-coding RNAs; Microarray analysis; Biological markers

Integrated genome-wide methylation and expression analyses reveal functional predictors of response to antidepressants

Major depressive disorder (MDD) is primarily treated with antidepressants, yet many patients fail to respond adequately, and identifying antidepressant response biomarkers is thus of clinical significance. Some hypothesis-driven investigations of epigenetic markers for treatment response have been previously made, but genome-wide approaches remain unexplored. Healthy participants (n = 112) and MDD patients (n = 211) between 18–60 years old were recruited for an 8-week trial of escitalopram treatment. Responders and non-responders were identified using differential Montgomery-Åsberg Depression Rating Scale scores before and after treatment. Genome-wide DNA methylation and gene expression analyses were assessed using the Infinium MethylationEPIC Beadchip and HumanHT-12 v4 Expression Beadchip, respectively, on pre-treatment peripheral blood DNA and RNA samples. Differentially methylated positions (DMPs) located in regions of differentially expressed genes between responders (n = 82) and non-responders (n = 95) were identified, and technically validated using a targeted sequencing approach. Three DMPs located in the genes CHN2 (cg23687322, p = 0.00043 and cg06926818, p = 0.0014) and JAK2 (cg08339825, p = 0.00021) were the most significantly associated with mRNA expression changes and subsequently validated. Replication was then conducted with non-responders (n = 76) and responders (n = 71) in an external cohort that underwent a similar antidepressant trial. One CHN2 site (cg06926818; p = 0.03) was successfully replicated. Our findings indicate that differential methylation at CpG sites upstream of the CHN2 and JAK2 TSS regions are possible peripheral predictors of antidepressant treatment response. Future studies can provide further insight on robustness of our candidate biomarkers, and greater characterization of functional components.

Circular RNA expression profile in peripheral whole blood of lung adenocarcinoma by high: Throughput sequencing.

Background:Lung adenocarcinoma (LA) is a most common form of non-small cell lung cancer (NSCLC). To date, there are still no effective early diagnosis methods for patients to be cured in time. Noncoding RNA plays an important role in oncogenesis and tumor development. The expression profile of circular RNA (circRNA) in peripheral whole blood (PWB) of LA has not been systematically investigated. In this study, we identified the differentially expressed (DE) circRNAs in PWB of LA by high-throughput sequencing.Methods:Five paired LA and normal participants PWB samples were chosen to investigate the expression profile of circRNAs by high-throughput sequencing. Twenty LA and 10 normal controls PWB samples were subjected to reverse-transcription polymerase chain reaction (RT-PCR) for validation of circRNAs expression profile. Gene Ontology (GO) functional analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, and circRNA-miRNA network analysis was also performed to predict the function of circRNAs in PWB.Results:A total of 10566 circRNAs were identified and annotated, most of the circRNAs were exonic (78.14%). Statistical analysis revealed 4390 DE circRNAs, in which were 3009 upregulated circRNAs and1381downregulated circRNAs in LA. RT-PCR results showed that circRNA expression in LA was higher than that in controls. GO functional analysis, KEGG pathway analysis, and circRNA-miRNA network analysis all showed that circRNAs correlated with tumor development and progression to a certain degree. The current study is the first to systematically characterize and annotate circRNA expression in PWB of LA. Some host genes of the DE circRNAs were involved in tumor signaling pathway and had complicated correlations with tumor related miRNAs, indicating that circRNAs might involve in development and progression of LA.Conclusions:Our study revealed that circRNAs were abnormally expressed in PWB of LA, which might offer potential targets for the early diagnosis of the disease and new genetic insights into LA.

Decreased expression of microRNAs targeting type-2 diabetes susceptibility genes in peripheral blood of patients and predisposed individuals.

Certain microRNA molecules (miRNAs) that target genes involved in beta-cell growth and insulin resistance are found deregulated in patients with type-2 diabetes mellitus (T2D) and correlate with its complications. However, the expression profile of miRNAs that regulate genes bearing T2D-related single-nucleotide polymorphisms has been hardly studied. We recently reported that the mRNA patterns of specific T2D-susceptibility genes are impaired in patients, and associate with disease parameters and risk factors. The aim of this study was to explore the levels of miRNAs that target those genes, in peripheral blood of patients versus controls. A panel of 14 miRNAs validated to target the CDKN2A, CDK5, IGF2BP2, KCNQ1, and TSPAN8 genes, was developed upon combined search throughout the DIANNA TarBase v7.0, miRTarBase, miRSearch v3.0-Exiqon, miRGator v3.0, and miRTarget Link Human algorithms. Specifically developed poly(A)polyadenylation(PAP)-reverse transcription(RT)-qPCR protocols were applied in peripheral blood RNA samples from patients and controls. Possible correlations with the disease, clinicopathological parameters and/or risk factors were evaluated. T2D patients expressed decreased levels of let-7b-5p, miR-1-3p, miR-24-3p, miR-34a-5p, miR-98-5p, and miR-133a-3p, compared with controls. Moreover, these levels correlated with certain disease features including insulin and % HbA1c levels in patients, as well as BMI, triglycerides' levels and family history in controls. A T2D-specific expression profile of miRNAs that target disease-susceptibility genes is for the first time described. Future studies are needed to elucidate the associated transcription-regulatory mechanisms, perchance involved in T2D pathogenesis, and to evaluate the potential of these molecules as possible biomarkers for this disorder.

A pilot study of a novel molecular host response assay to diagnose infection in patients after high-risk gastro-intestinal surgery

PurposeSeptiCyte LAB measures the expression of four host-response RNAs in peripheral blood to distinguish sepsis from sterile inflammation. This study evaluates whether sequential monitoring of this assay has diagnostic utility in patients after esophageal surgery. Materials and methodsPatients who developed a complication within 30 days following esophageal surgery and a random sample of 100 patients having an uncomplicated course. SeptiCyte LAB scores (ranging 0–10 reflecting increasing likelihood of infection) were compared to post-hoc physician adjudication of infection likelihood. ResultsAmong 370 esophagectomy patients, 120 (32%) subjects developed a complication requiring ICU (re)admission, 63 (53%) of whom could be analyzed. Immediate postoperative SeptiCyte LAB scores were highly variable, yet similar for patients having a complicated and uncomplicated postoperative course (median score of 2.4 (IQR 1.6–3.3) versus 2.2 (IQR 1.3–3), respectively). In a direct comparison of patients developing a confirmed infectious (n = 34) and non-infectious complication (n = 12), addition of SeptiCyte LAB to CRP improved diagnostic discrimination of infectious complications (AUC 0.88 (95%CI 0.77–0.99)) compared to CRP alone (AUC 0.76 (95%CI 0.61–0.91); p = .04). ConclusionsSequential measurement of SeptiCyte LAB may have diagnostic value in the monitoring of surgical patients at high risk of postoperative infection, but its clinical performance in this setting needs to be validated.

Discovery and cross-validation of peripheral blood and renal biopsy gene expression signatures from ethnically diverse kidney transplant populations.

We determined peripheral blood (PB) and biopsy (Bx) RNA expression signatures in a Brazilian and US cohort of kidney transplant patients. Phenotypes assigned by precise histology were: acute rejection (AR), interstitial fibrosis/tubular atrophy/chronic rejection (CR), excellent functioning transplants (TX), and glomerulonephritis recurrence (GN). Samples were analyzed on microarrays and profiles from each cohort were cross-validated on the other cohort with similar phenotypes. We discovered signatures for each tissue: (1) AR vs TX, (2) CR vs TX, and (3) GN vs TX using the Random Forests algorithm. We validated biopsies signatures of AR vs TX (area under the curve [AUC] 0.97) and CR vs TX (AUC 0.87). We also validated both PB and Bx signatures of AR vs TX and CR vs TX with varying degrees of accuracy. Several biological pathways were shared between AR and CR, suggesting similar rejection mechanisms in these 2 clinical phenotypes. Thus, we identified gene expression signatures for AR and CR in transplant patients and validated them in independent cohorts of significantly different racial/ethnic backgrounds. These results reveal that there are strong unifying immune mechanisms driving transplant diseases and identified in the signatures discovered in each cohort, suggesting that molecular diagnostics across populations are feasible despite ethnic and environmental differences.

PB1724 FLT3 MUTATED AML: THE ROLE OF CLADRIBINE AND MIDOSTAURIN ADDED TO THE STANDARD INTENSIVE CHEMOTHERAPY ‐ SINGLE CENTRE EXPERIENCE FROM UMC LJUBLJANA, SLOVENIA

Background:The FLT3 ITD mutation in a patient with acute myeloid leukemia (AML) is associated with a poor outcome. Midostaurin is the first multikinase inhibitor approved for the treatment of adults with newly diagnosed FLT3 positive AML. The drug has been available in Slovenia from July 2017 and used in all eligible FLT3 patients. Furthermore, following encouraging results shown by the Polish Adult Leukemia Group, we have been adding cladribine to standard combination DA3+7 induction (namely DAC) for all AML patients aged 60 years or less since 2014.Aims:To compare the outcome of FLT3 positive AML patients treated with DA3+7 or DAC with further addition of midostaurin.Methods:We retrospectivelly analyzed all consecutive FLT3 positive AML patients, aged 60 years or less, treated with intensive chemotherapy between years 2012‐2018. Bone marrow (or in some cases peripheral blood) RNA was used for FLT3 mutation detection. FLT3 ITD mutation was detected by reverse transcription PCR (RT‐PCR) and gel electrophoresis analysis. FLT3 D836 mutation detection was carried out by RT‐PCR and Restriction Fragment Length Polymorphism technique using restriction enzyme EcoRV. From 01/2012 – 04/2014 DA3 + 7 was used, while from 05/2014 – 12/2018 DAC (addition of 5 mg/m2 of cladribine in a 3‐hour infusion on days 1‐5 to DA3 + 7) was used for all AML patients aged 60 years or less eligible for intensive chemotherapy. Remission was assessed by bone marrow aspiration in the aplastic phase and before the first consolidation therapy. Consolidation treatment was intermediate or high dose cytosar. Midostaurin was added according to indication no later than in the first consolidation phase (2x50 mg from day 8‐21). All patients with FLT3 ITD mutation and available donor were transplanted. Patients who had not achieved remission after induction chemotherapy proceeded with rescue chemotherapy and allogeneic transplantation. Patients who were not transplanted continued maintenance treatment with midostaurin 2x50 mg continuously for one year.Results:There were 39 FLT3 positive AML patients aged 60 years or less who were treated with intensive chemotherapy in the period 2012‐2018: 16 with DA3 + 7 and 23 with DAC. There was no difference between the baseline characteristics between the groups (Table 1). Remission after induction was achieved in 9/16 patients in the DA group (1 patient was treated with midostaurin during induction) and 19/23 in the DAC group (5 patients received midostaurin during the induction), p = 0,072. Survival was significantly better in the DAC group in which overall 12/23 patients were treated with midostaurin compared to DA3 + 7 where only 1 patient was treated with midostaurin; p = 0,028. The proportion of patients with allogeneic transplant was similar (7/16 in the DA and 11/23 in the DAC group; p = 0,802). When comparing patients with or without midostaurin treatment the survival was significantly better in patients treated with midostaurin (p = 0,005), however, the median observation time was only 11 months. The addition of midostaurin to DAC did not increase toxicity.Summary/Conclusion:Addition of cladribine and midostaurin to standard DA3 + 7 improved survival in FLT3 positive AML patients. No new safety signals were noted in patients receiving DAC + midostaurin. Further follow‐up is required to evaluate influence of midostaurin in combination with DAC induction chemotherapy on progression free survival and overall survival.image

Comprehensive analysis of differentially expressed profiles of non‑coding RNAs in peripheral blood and ceRNA regulatory networks in non‑syndromic orofacial clefts.

Non-syndromic orofacial clefts (NSOC), which include cleft lip with or without cleft palate (CL/P) and cleft palate only (CPO), are common congenital birth defects in humans. Accumulating evidence indicates that long non-coding RNAs (lncRNAs) and microRNAs (miRNAs or miRs) play important roles in NSOC; however, the potential regulatory associations between them remain largely unknown. In this study, we performed next-generation RNA sequencing (RNA-seq) to identify transcriptome profiles, including mRNAs, lncRNAs and miRNAs, in patients with CL/P and CPO. A total of 36 lncRNAs, 1,341 mRNAs and 60 miRNAs were found to be differentially expressed in the CL/P group compared to the control group, and 57 lncRNAs, 1,255 mRNAs and 162 miRNAs were found to be differentially expressed in the CPO group compared to the control group. Subsequently, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was performed to validate the expression of selected lncRNAs, miRNAs and mRNAs. In addition, bioinformatics methods were employed to explore the potential functions of ncRNAs and to construct lncRNA-miRNA-mRNA regulatory networks. To the best of our knowledge, this is the first study to comprehensively analyze regulated non-coding RNAs (ncRNAs) in CL/P and CPO, providing a novel perspective on the etiology of NSOC and laying the foundation for future research into the potential regulatory mechanisms of ncRNAs and mRNAs in NSOC.

Effects of Rupture of Intracranial Aneurysms on Small RNAs in Peripheral Blood (S22.008)

Effects of Rupture of Intracranial Aneurysms on Small RNAs in Peripheral Blood (S22.008)

Diagnostic and Correlative Analysis of Circrnas in Bone Marrow and Peripheral Blood of Patients with Refractory Cytopenia with Multilineage Dysplasia

This study was to detect expression levels of circular RNA (circRNA) in bone marrow (BM) and peripheral blood (PB) of patients with refractory cytopenia with multilineage dysplasia (RCMD). The objective was to identify circRNAs with potential diagnostic value and investigate the correlation between these circRNAs in BM and PB of RCMD patients.We first analyzed circRNAs in the BM of 5 RCMD patients and 4 control subjects using circRNA arrays. We found 369 circRNAs were differentially expressed between these two groups; 145 were upregulated and 224 were down regulated. Using a set of stringent criteria, we selected 10 circRNAs as potential biomarkers and verified their expression levels in 115 RCMD patients and 20 control subjects. 6 circRNAs were increased in RCMD patients while 3 were decreased and 1 showed no difference. ROC analysis using the same cohort confirmed 8 of these 9 circRNAs could be potential diagnostic biomarkers. We then tested the expression levels of these 10 circRNAs in PB samples from same 115 RCMD patients. There was no correlation between expression levels of these 10 circRNAs in BM and PB samples. This suggested that 10 circRNAs identified in BM samples could not be insteaded by circRNAs in PB samples. Funding: This work was supported in part by grants from the National Natural Science Foundation of China (#81500099) and Shanghai Municipal Science and Technology Commission (#16ZR1404400). Declaration of Interest: The authors declare no conflict of interest. Ethical Approval: The study was approved by the institutional review board at Huashan Hospital of Fudan University and informed consents were obtained from each study participant.

Sex-based dimorphisms in expression of BDNF and BACE1 in bipolar patients

Bipolar disorder (BD) is a chronic, serious mental disorder distinguished by repeated episodes of mania and depression. Previous studies have demonstrated dysregulation of a number of transcripts in brain tissue or peripheral blood of BD patients. In the present study, we compared expression of two protein coding genes (brain-derived neurotrophic factor (BDNF) and beta-secretase 1 (BACE1)) and their natural occurring anti-sense (AS) RNAs (BDNF-AS and BACE1-AS) in peripheral blood of 50 BD patients (mean age ± standard deviation (SD) = 36.5 ± 9.32) and 50 healthy subjects (mean age ± SD = 33.62 ± 8.59). BDNF and BACE1 were significantly up-regulated in peripheral blood of total BD patients compared with total healthy subjects (Expression ratio = 2.2, P value = 0.003; Expression ratio = 2.2, P value = 0.002 respectively). However, comparison of their levels in sex-based subgroups showed their up-regulations only in male patients compared with male health subjects (Expression ratio = 2.48, P value = 0.006; Expression ratio = 2.1, P value = 0.01). No significant differences were found in expressions of BDNF-AS and BACE1-AS between BD and health subjects. We detected a significant correlation between BDNF expression and age at disease onset in BD group after adjustment of the effects of sex (R = 0.26, P value = 0.03). Moreover, there were trends toward correlations between BDNF expression and disease duration in BD group and between BDNF expression and age in health subjects (P values = 0.05). Combination of BDNF, BDNF-AS and BACE1 expression levels could differentiate BD patients from healthy subjects with 68% sensitivity and 82% specificity (area under curve = 0.72, P value = 0.0001). The current study suggests a sex-based dimorphic pattern in expression of BDNF and BACE1. Moreover, our results imply that expression pattern of these genes could be diagnostic markers in BD. Future studies are needed to assess this speculation in larger patient samples.

Abstract P108: Wnt Pathway Gene Expression is Associated With Greater Arterial Stiffness

The wingless (Wnt) pathway is known to regulate many systems, including embryonic development, bone mineralization, and cancer. Now there is emerging evidence from Basic and our own Population research that Wnt pathway activation is associated with cardiovascular disease (CVD) pathologies, including atherosclerosis and arterial stiffening. However, research on this association in humans is still under-studied and limited in scope. As such, we aimed to survey the entire Wnt signaling pathway for association with subclinical CVD, using a custom gene expression panel (Nanostring, Seattle, WA) of 43 Wnt genes with previous Basic research evidence for a role in vascular disease. We ran this panel in stabilized peripheral blood RNA from 369 African Ancestry men in the Tobago Health Study (mean age 64 years, range 51-89 years). Subclinical CVD assessments were completed at the same study visit as the blood draw, including: arterial stiffness measured by brachial-ankle pulse-wave velocity; atherosclerosis measures of carotid plaque and intima-media thickness via B-mode ultrasonography; and vascular calcification measured by computed tomography in the chest (coronary) and abdomen (aorta). Gene expression was measured in counts per sample, was normalized to housekeeping genes, and the background signal was subtracted. All gene expression and outcome measures were transformed to normality after excluding any extreme outliers. Fourteen Wnt genes showed variable expression in our sample and were tested individually as predictors of subclinical CVD using multiple linear or logistic regression, as appropriate. A conservative Bonferroni-corrected alpha (0.05/14 genes = 0.0036) was used to account for multiple comparisons in unadjusted models. Associations passing this threshold were then adjusted for common CVD risk factors including age, height, weight, hypertension, pulse, diabetes, smoking, alcohol intake, and sedentary lifestyle. After full adjustment, expression of APC, WNT signaling pathway regulator ( APC ) and transcription factor 4 ( TCF4 ) showed significant association with arterial stiffness (P=0.012 for both). When entered into a single model (Spearman r=0.14, P=0.007), APC and TCF4 expression were independently associated with arterial stiffness (P=0.004 for both), such that higher expression of APC , but lower expression of TCF4 , was associated with greater arterial stiffness. No other tested genes or subclinical CVD measures passed both the conservative unadjusted alpha level and the full model adjustment. This study represents the first test of the association between broad Wnt signaling pathway gene expression and subclinical CVD. While APC and TCF4 have shown some association with vascular pathologies in Basic research, this is the first study to establish the association of their expression with human arterial stiffness.

Susceptibility to ERAP1 gene single nucleotide polymorphism modulates the inflammatory cytokine setting in ankylosing spondylitis.

To evaluate the association of ERAP1 gene single nucleotide polymorphisms (SNPs) with the risk of ankylosing spondylitis (AS) and their role in modulation of the inflammatory interleukin (IL)-17/IL-23 axis in the disease. For genotyping, 190 AS cases and 190 healthy controls were enrolled. After DNA extraction, all the subjects were genotyped for rs17482078, rs469876, and rs27038 polymorphisms using single specific primer polymerase chain reaction (PCR) assay. After isolation of peripheral blood mononuclear cells, RNA extraction and complementary DNA synthesis, real-time PCR using SYBR Green master mix was employed to determine messenger RNA (mRNA) expression of IL-17A and IL-23 in PBMCs. Using enzyme-linked immunosorbent assay, the concentration of these cytokines was determined in serum samples. It was observed that the A allele of rs27038 polymorphism significantly increased AS risk (odds ratio [OR] = 1.53, 95% CI =1.11-2.12; P = 0.0096). Moreover, AA and AG genotypes of this SNP were associated with increased (OR = 2.89, 95% CI = 1.42-5.85; P=0.0031) and decreased (OR = 0.57, 95% CI = 0.36-0.92; P=0.021), respectively, risk of the disease. The rs27038 SNP was associated with C-reactive protein level. There were significantly increased mRNA and serum concentrations of both IL-17A and IL-23 in AS patients compared with controls. Furthermore, AS patients with the AA in comparison to other genotypes for rs27038 SNP indicated significantly increased mRNA and serum concentration levels for both cytokines. This study demonstrated the association of ERAP1 gene rs27038 polymorphism with the risk of AS in an Iranian population. Additionally, it seems that rs27038 is involved in the modulation of the inflammatory IL-17/IL-23 axis in AS.

Abstract # 3189 Weighted gene co-expression network analysis suggests interferon activity is associated with depression severity

To further the identification and treatment of patients with mood disorders having an immune component, we must identify the specific nature of immune processes associated with mood. We analyzed peripheral blood RNA sequencing data from the Depression Genes and Networks (DGN) Study using Weighted Gene Co-expression Network Analysis (WGCNA) from 441 adult subjects with a past or current diagnosis of Major Depressive Disorder. After filtering, 5778 genes were clustered in 19 modules. Overall, modules had weak clinical correlation; one module containing 63 genes showed the best correlation with scores on the 9-item Public–Health Questionnaire (PHQ-9) (r = 0.1, p = 0.03). This module was significantly enriched for the Gene Ontology Biological Process Type I Interferon Signaling (adjusted p? 0.001) and other pathways of anti-viral immunity. This module significantly overlapped with a module of 74 genes derived from 325 subjects from the DGN healthy control sample; it did not correlate significantly with any clinical trait in those subjects. One module of 52 genes from the depressed sample, containing primarily immunoglobulin chain genes, had no corresponding module in the healthy sample. This module, however, did not correlate significantly with any clinical trait. These findings replicate the previously published association of Interferon pathways with depression in this sample. Replication in an independent sample is necessary to validate the role of antiviral immunity in depression.

Aberrant Expression of Long Non-coding RNAs in Peripheral Blood of Autistic Patients.

Long non-coding RNAs (lncRNAs) constitute a large proportion of human transcriptome and are involved in fundamental aspects of neurodevelopment. In the present study, we evaluated expression levels of three lncRNAs, namely, Nuclear Paraspeckle Assembly Transcript 1 (NEAT1), p21-associated ncRNA DNA damage-activated (PANDA), and taurine-upregulated gene 1 (TUG1), in peripheral blood of autism spectrum disorder (ASD) patients compared with those in healthy subjects. We found a significant upregulation of NEAT1 and TUG1 in ASD patients. In addition, relative expressions of the pairs of lncRNAs were significantly correlated in both patients and healthy subjects. However, expressions of these lncRNAs were not correlated with age of study participants. The present study provides further evidences for dysregulation of lncRNAs in ASD patients.

Alternative transcript imbalance underlying breast cancer susceptibility in a family carrying PALB2 c.3201+5G>T.

Disruption of splicing motifs by genetic variants can affect the correct generation of mature mRNA molecules leading to aberrant transcripts. In some cases, variants may alter the physiological transcription profile composed of several transcripts, and an accurate in vitro evaluation is crucial to establish their pathogenicity. In this study, we have characterized a novel PALB2 variant c.3201+5G>T identified in a breast cancer family. Peripheral blood RNA was analyzed in two carriers and tencontrols by RT-PCR and Sanger sequencing. The splicing profile was also characterized by semi-quantitative capillary electrophoresis and quantitative PCR. RAD51 foci formation and PALB2 LOH status were evaluated in primary breast tumor samples from the carriers. PALB2 c.3201+5G>T disrupts intron 11 donor splice site and modifies the abundance of several alternative transcripts (∆11, ∆12, and ∆11,12), also present in control samples. All transcripts are predicted to encode for non-functional proteins. Semi-quantitative and quantitative analysis of PALB2 full-length transcript indicated haploinsufficiency in carriers. One tumor exhibited PALB2 LOH and RAD51 assay indicated homologous recombination deficiency in both tumors. Our results support a pathogenic classification for PALB2 c.3201+5G>T, highlighting the impact of variants causing an imbalanced expression of natural RNA isoforms in cancer susceptibility.

Integrated facial analysis and targeted sequencing identifies a novel KDM6A pathogenic variant resulting in Kabuki syndrome

Abstract Kabuki syndrome (KS) is a rare congenital mental retardation condition characterized by facial dysmorphia, visceral and skeletal malformations, and developmental delay. The integrated phenotype and genotype-based prioritization is critical for diagnoses of genetic diseases. In this study, a Chinese woman, presenting with characteristic facial features of KS, came for pre-pregnancy consultation. We aimed to clarify the diagnosis and provide pre-pregnancy genetic counseling. Facial dysmorphology analysis and next-generation sequencing-based multigene panel approach were used to identify candidate syndromes and causative variants, respectively. The candidate variant was verified by Sanger sequencing. We identified a novel de novo KDM6A pathogenic variant (c.3521G&gt;A) in the woman, which was in line with the Face2Gene analysis result. Peripheral blood RNA assay showed that the variant transcript underwent the nonsense-mediated mRNA decay and led to subsequent haploinsufficiency of KDM6A. Our study provides the genetic diagnosis method for KS type 2 and identifies the first KDM6A point variant in Chinese patient.

Selinexor, a First-in-Class XPO1 Inhibitor, Is Efficacious and Tolerable in Patients with Myelodysplastic Syndromes Refractory to Hypomethylating Agents

There is no standard therapy for patients with myelodysplastic syndromes (MDS) refractory to hypomethylating agents (HMA) and the median overall survival (OS) of HMA-refractory MDS patients with IPSS Intermediate-2/High-risk MDS is ~6 months. Here, we present data from a phase 2 clinical trial of selinexor, an oral, first-in-class Selective Inhibitor of Nuclear Export (SINE) compound that inhibits XPO1, in patients with MDS refractory to HMAs. XPO1 is the major nuclear export protein responsible for shuttling many key cellular regulators out of the nucleus and is overexpressed in many cancers. Preclinical studies of selinexor have shown that inhibition of XPO1 causes nuclear retention of wildtype p53 and disruption of the NF-κB pathway, both relevant targets for MDS. Moreover, selinexor has shown promising responses in acute myeloid leukemia, multiple myeloma, and Non-Hodgkin lymphoma. We therefore set out to determine the safety and efficacy of selinexor in MDS, and to study potential molecular correlates of response to selinexor.Patients with MDS or MDS/MPN who were refractory to azacitidine or decitabine were included (defined as either progression after initial response or if no response after >6 cycles). The primary endpoint was overall response rate (ORR = CR + mCR + PR + HI) and secondary endpoints included safety, response duration, and OS. The starting dose was 35 mg/m2 PO twice-weekly in 28-day cycles. Bone marrow biopsies, bone marrow DNA and peripheral blood RNA were collected from serial samples and used for correlative analyses including next-generation targeted gene sequencing, RNA-seq, immunohistochemistry, and proteomics. A total of 25 patients were evaluable for safety and 19 were evaluable for response. Six patients were not evaluable for response based on unrelated death from concurrent illness (n=5) or withdrawal of consent (n=1) before completion of one cycle. The median age was 77 (range, 50-85) and the majority of pts were High- or Very High-risk by IPSS-R (72%) with >10% bone marrow blasts (70%).The ORR was 32% (all mCRs) with an average response duration of 6.8 months. An additional 42% of pts achieved stable disease (SD) for an average response duration of 6.4 months for all patients receiving clinical benefit (Figure A). The median overall survival was 9.7 months with a median follow-up of 2.1 years. Those with mCR or SD had greater survival than non-responders (Figure B). Grade 3 or 4 adverse events (AEs) in the first 3 pts (thrombocytopenia (n=3) and hyponatremia (n=1)) led to reduction of the dose to 60mg flat dose twice-weekly for 2 weeks followed by 1 week off, resulting in significantly improved tolerability. The most frequently occurring AE thereafter was grade 2 or 3 fatigue. All other AEs were manageable with routine supportive care.The presence of an SF3B1 hotspot mutation was significantly associated with response to selinexor (p=0.01; Figure C). No other single mutation had a significant association, although patients with mutated TP53 and those with >3 mutations were numerically higher in the non-responders but did not reach statistical significance. The mRNA levels of a number of transcripts from the NF-κB pathway and of Myc were decreased after treatment with selinexor in responders, albeit with some variability. The levels of XPO1 mRNA increased with treatment within 4 hours of the fist dose, indicative of XPO1 target engagement with selinexor (Figure D). Further analyses are underway to determine if splicing is differentially modulated in SF3B1 mutated patients. Immunohistochemistry and proteomics for protein alterations are also ongoing.These data indicate that selinexor is safe and tolerable in MDS when given at 60mg twice-weekly for 2 weeks with 1 week off with a 32% response rate. Responses appear to be enriched in patients with SF3B1 mutations and selinexor appears to affect pathways that may be activated downstream of mutant-SF3B1, such as NF-κB and Myc. Detailed correlative and pre-clinical analyses are ongoing and should provide further illumination of the biological relevance of these markers, which will be tested prospectively in future clinical trials. Further studies of selinexor combined with targeted agents that disrupt similar or complementary oncogenic pathways in MDS could further improve responses. [Display omitted] DisclosuresRampal:Incyte: Honoraria, Research Funding; Stemline: Research Funding; Jazz: Consultancy, Honoraria; Constellation: Research Funding; Celgene: Honoraria. Park:Adaptive Biotechnologies: Consultancy; Kite Pharma: Consultancy; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy; Shire: Consultancy; AstraZeneca: Consultancy; Novartis: Consultancy; Juno Therapeutics: Consultancy, Research Funding. Stein:Agios: Consultancy; Bayer: Consultancy; Novartis: Consultancy; Celgene: Consultancy; Daiichi Sankyo: Consultancy; Pfizer: Consultancy. Tallman:AbbVie: Research Funding; Cellerant: Research Funding; ADC Therapeutics: Research Funding; Daiichi-Sankyo: Other: Advisory board; Orsenix: Other: Advisory board; BioSight: Other: Advisory board; AROG: Research Funding.