TaqMan OpenArray Research Articles

Associations between maternal asthma and atopy and breast milk-derived extracellular vesicle microRNA profiles in the PRISM pregnancy cohort

BACKGROUND AND AIM: Maternal asthma is associated with adverse infant and child health including preterm birth and respiratory outcomes. Breast milk contains extracellular vesicle (EV)-encapsulated microRNAs, a form of mother-infant biochemical communication and may represent one pathway by which maternal health impacts child development and health. In this study we investigated associations between maternal asthma and atopy and EV-microRNA profiles in the PRogramming of Intergenerational Stress Mechanisms (PRISM) pregnancy cohort. METHODS: EVs were isolated from breast milk collected from N=80 mothers 6.1±5.9 weeks postnatally. RNA was extracted and profiled using the TaqMan OpenArray Human miRNA panel. We analyzed associations between maternal asthma and atopy (active during pregnancy and ever) and microRNA detection (yes/no; N=172 microRNAs identified in 20-80% of samples) using logistic regression and expression levels (N=205 microRNA identified in 50% of samples) using robust linear regression adjusted for infant sex, maternal race, education, and postpartum week of breast milk collection. RESULTS:Asthma and atopy during pregnancy were associated with detection of 3 and 6 microRNAs (p 0.05), and expression levels of 27 and 12 microRNAs (a priori criteria: p0.05 and |Bregression|0.2), respectively. Enriched KEGG pathways for microRNAs differentially expressed with asthma or atopy during pregnancy included glycosaminoglycan biosynthesis, extracellular matrix-receptor remodeling, and PI3K-Akt signaling pathway. There was a trend toward fewer microRNAs associated with maternal asthma and atopy ever (detection: N=3 associated with asthma ever, N=9 associated with atopy ever; expression levels: N=14 associated with asthma ever; N=9 associated with atopy ever). CONCLUSIONS:Maternal asthma and atopy were associated with detection and expression of breast-milk derived EV-microRNA. Atopy was associated with detection of a greater number of microRNAs, whereas asthma was associated with expression of a greater number of microRNAs. Further research is needed to understand how breast milk microRNAs may affect biological pathways and child health outcomes. KEYWORDS: children's environmental health, asthma, birth outcomes, epigenomics

Influence of FPGS, ABCC4, SLC29A1, and MTHFR genes on the pharmacogenomics of fluoropyrimidines in patients with gastrointestinal cancer from the Brazilian Amazon.

Fluoropyrimidines are one of the most used drug class to treat cancer patients, although they show high levels of associated toxicity. This study analyzed 33 polymorphisms in 17 pharmacogenes involved with the pharmacogenomics of fluoropyrimidines, in gastrointestinal cancer patients undergoing fluoropyrimidine-based treatment in the Brazilian Amazon. The study population was composed of 216 patients, 92 of whom have an anatomopathological diagnosis of gastric cancer and 124 of colorectal cancer. The single nucleotide polymorphisms (SNP) were genotyped by allelic discrimination using the TaqMan OpenArray Genotyping technology, with a panel of 32 customized assays, run in a QuantStudio ™ 12K Flex Real-Time PCR System (Applied Biosystems, Life Technologies, Carlsbad USA). Ancestry analysis was performed using 61 autosomal ancestry informative markers (AIMs). The study population show mean values of 48.1% European, 31.1% Amerindian, and 20.8% African ancestries. A significant risk association for general and severe toxicity was found in the rs4451422 of FPGS (p = 0.001; OR 3.40; CI 95% 1.65-7.00 and p = 0.006; OR 4.63; CI 95% 1.56-13.72, respectively) and the rs9524885 of ABCC4 (p = 0.023; OR 2.74; CI 95% 1.14-6.65 and p = 0.024; OR 5.36; IC 95% 1.24-23.11, respectively) genes. The rs760370 in the SLC29A1 gene (p = 0.009; OR 6.71; CI 95% 1.16-8.21) and the rs1801133 in the MTHFR toxicity (p = 0.023; OR 3.09; CI 95% 1.16-8.21) gene also demonstrated to be significant, although only for severe toxicity. The results found in this study did not have statistics analysis correction. Four polymorphisms of the ABCC4, FPGS, SLC29A1, and MTHFR genes are likely to be potential predictive biomarkers for precision medicine in fluoropyrimidine-based treatments in the population of the Brazilian Amazon, which is constituted by a unique genetic background.

Serum circulating miRNA-342-3p as a potential diagnostic biomarker in parathyroid carcinomas: A pilot study.

ObjectiveTo compare the serum miRNA expression profiles between patients with benign and malignant parathyroid tumours.BackgroundDespite recent advances in molecular biology, a histological tissue biopsy is still the method of choice used to diagnose most cancers. The preoperative cytology is not an applicable method for diagnosis of parathyroid cancer (PC); therefore, huge interest exists in terms of finding alternative methodologies to seek specific cancer biomarkers.DesignA retrospective cross‐sectional study.Patients and MethodsSerum samples of patients with PC (n = 13) and parathyroid adenoma (PA) (n = 11), age (p = .999) and sex (p = .999) were matched and examined via the simultaneous comparative expression analysis of 754 microRNAs (miRNAs). The «TaqMan OpenArray Human MicroRNA Panel» (Applied Biosystems) was used to conduct real‐time PCRs using the «QuantStudio 12К Flex» station (Life Technologies).ResultsAccording to the results of a pilot study, significant changes in expression levels between the PC group and the PA group (control) (p < .05) were observed for 17 miRNAs. Among them, the downregulation of miRNA‐342‐3p met the Benjamini‐Hochberg adjustment criteria for multiple comparisons (p = .02).ConclusionsSerum miRNA‐342‐3p could be a promising biomarker for PC to improve diagnosis and prognosis.

Amniotic fluid microRNA profiles in twin-twin transfusion syndrome with and without severe recipient cardiomyopathy

Twin-twin transfusion syndrome presents many challenges for clinicians, and the optimal means of identifying pregnancies that will benefit most from intervention is controversial. There is currently no clinically available biomarker to detect twin-twin transfusion syndrome or to stratify cases based on the risk factors. microRNAs are small RNAs that regulate gene expression and are biomarkers for various disease processes, including adult and pediatric heart failure. To date, no studies have investigated amniotic fluid microRNAs as biomarkers for disease severity, specifically for severe recipient cardiomyopathy in twin-twin transfusion syndrome cases. This study aimed to assess whether amniotic fluid microRNAs could be useful as biomarkers to identify pregnancies at greatest risk for severe recipient cardiomyopathy associated with twin-twin transfusion syndrome. Amniotic fluid was collected at the time of amnioreduction or selective fetoscopic laser photocoagulation from monochorionic diamniotic twin pregnancies with twin-twin transfusion syndrome at any stage. Fetal echocardiography was performed on all twins before the procedure, and severe cardiomyopathy was defined as a right ventricular myocardial performance index of the recipient fetus of >4 Z-scores. microRNA was extracted from the amniotic fluid samples and analyzed using an array panel assessing 379 microRNAs (TaqMan Open Array, ThermoFisher). Student t tests were performed to determine significant differences in microRNA expression between pregnancies with severe recipient cardiomyopathy and those with preserved cardiac function. A stringent q value of <.0025 was used to determine differential microRNA expression. Random forest plots identified the top 3 microRNAs that separated the 2 groups, and hierarchical cluster analysis was used to determine if these microRNAs properly segregated the samples according to their clinical groups. A total of 14 amniotic fluid samples from pregnancies with twin-twin transfusion syndrome with severe cardiomyopathy were compared with samples from 12 twin-twin transfusion syndrome control cases with preserved cardiac function. A total of 110 microRNAs were identified in the amniotic fluid samples. Twenty microRNAs were differentially expressed, and the top 3 differentiating microRNAs were hsa-miR-200c-3p, hsa-miR-17-5p, and hsa-miR-539-5p. Hierarchical cluster analysis based on these top 3 microRNAs showed a strong ability to differentiate severe cardiomyopathy cases from controls. The top 3 microRNAs were used to investigate the sensitivity and specificity of these microRNAs to differentiate between the 2 groups with a receiver operating characteristic curve demonstrating sensitivity and specificity of 80.8%. All 20 differentially expressed microRNAs were down-regulated in the group with severe cardiomyopathy. Amniotic fluid microRNAs demonstrated differential expression between twin-twin transfusion syndrome recipient fetuses with severe cardiomyopathy and those without and have the potential to be important biomarkers of disease severity in this population.

Mutant p53 Mediates Sensitivity to Cancer Treatment Agents in Oesophageal Adenocarcinoma Associated with MicroRNA and SLC7A11 Expression.

TP53 gene mutations occur in 70% of oesophageal adenocarcinomas (OACs). Given the central role of p53 in controlling cellular response to therapy we investigated the role of mutant (mut-) p53 and SLC7A11 in a CRISPR-mediated JH-EsoAd1 TP53 knockout model. Response to 2 Gy irradiation, cisplatin, 5-FU, 4-hydroxytamoxifen, and endoxifen was assessed, followed by a TaqMan OpenArray qPCR screening for differences in miRNA expression. Knockout of mut-p53 resulted in increased chemo- and radioresistance (2 Gy survival fraction: 38% vs. 56%, p < 0.0001) and in altered miRNA expression levels. Target mRNA pathways analyses indicated several potential mechanisms of treatment resistance. SLC7A11 knockdown restored radiosensitivity (2 Gy SF: 46% vs. 73%; p = 0.0239), possibly via enhanced sensitivity to oxidative stress. Pathway analysis of the mRNA targets of differentially expressed miRNAs indicated potential involvement in several pathways associated with apoptosis, ribosomes, and p53 signaling pathways. The data suggest that mut-p53 in JH-EsoAd1, despite being classified as non-functional, has some function related to radio- and chemoresistance. The results also highlight the important role of SLC7A11 in cancer metabolism and redox balance and the influence of p53 on these processes. Inhibition of the SLC7A11-glutathione axis may represent a promising approach to overcome resistance associated with mut-p53.

Urinary microRNAs as non-invasive biomarkers for toxic acute kidney injury in humans

MicroRNAs in biofluids are potential biomarkers for detecting kidney and other organ injuries. We profiled microRNAs in urine samples from patients with Russell’s viper envenoming or acute self-poisoning following paraquat, glyphosate, or oxalic acid [with and without acute kidney injury (AKI)] and on healthy controls. Discovery analysis profiled for 754 microRNAs using TaqMan OpenArray qPCR with three patients per group (12 samples in each toxic agent). From these, 53 microRNAs were selected and validated in a larger cohort of patients (Russell’s viper envenoming = 53, paraquat = 51, glyphosate = 51, oxalic acid = 40) and 27 healthy controls. Urinary microRNAs had significantly higher expression in patients poisoned/envenomed by different nephrotoxic agents in both discovery and validation cohorts. Seven microRNAs discriminated severe AKI patients from no AKI for all four nephrotoxic agents. Four microRNAs (miR-30a-3p, miR-30a-5p, miR-92a, and miR-204) had > 17 fold change (p < 0.0001) and receiver operator characteristics area-under-curve (ROC-AUC) > 0.72. Pathway analysis of target mRNAs of these differentially expressed microRNAs showed association with the regulation of different nephrotoxic signaling pathways. In conclusion, human urinary microRNAs could identify toxic AKI early after acute injury. These urinary microRNAs have potential clinical application as early non-invasive diagnostic AKI biomarkers.

Antioxidants-Related Superoxide Dismutase (SOD), Catalase (CAT), Glutathione Peroxidase (GPX), Glutathione-S-Transferase (GST), and Nitric Oxide Synthase (NOS) Gene Variants Analysis in an Obese Population: A Preliminary Case-Control Study.

Oxidative stress and antioxidants play an important role in obesity etiopathology. Genetic variants, including single nucleotide polymorphisms (SNPs) of the antioxidant-related genes, may impact disease risk in several populations. This preliminary study aimed to explore the association of 12 SNPs related to superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), glutathione-S-transferase (GST), and nitric oxide synthase (NOS) genes with obesity susceptibility in a Saudi population. A total of 384 unrelated participants, including 154 (40.1%) obese individuals, were enrolled. TaqMan OpenArray Genotyping assays were used. Six SNPs were significantly more prevalent in obese cohorts: (1) GSTM1 rs1056806*C/T; (2) SOD1 rs2234694*A; (3) SOD2 rs4880*G; (4) SOD3 rs2536512*A; (5) GPX1 rs1800668*A; (6) NOS3 rs1799983*G. Four SNPs were associated with higher obesity risk under heterozygote and dominant models for GSTM1 rs1056806 (C/T), homozygote model for SOD2 rs4880 (A/G), and homozygote and recessive models for GPX1 rs1800668 (A/G). In contrast, SOD3 rs2536512 (A/G) were less likely to be obese under heterozygote and dominant models. The CGAG, CAAA, TGGG, and CGAG combined genotypes showed a higher risk of obesity. In conclusion, the present results suggest that oxidative-stress-related genetic determinants could significantly associate with obesity risk in the study population.

Glutathione S-Transferase (GSTT1 rs17856199) and Nitric Oxide Synthase (NOS2 rs2297518) Genotype Combination as Potential Oxidative Stress-Related Molecular Markers for Type 2 Diabetes Mellitus.

BackgroundDeregulation of the antioxidant enzymes was implicated in pathogenesis and complications of type 2 diabetes mellitus (T2DM). The data relate the genetic variants of these enzymes to T2DM are inconsistent among various populations.PurposeWe aimed to explore the association of 13 genetic variants of “superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione-S-transferase (GST) and nitric oxide synthase (NOS)” with T2DM susceptibility and the available clinical laboratory data.Subjects and MethodsA total of 384 individuals were enrolled in this work. Different genotypes of the genes mentioned above were characterized using TaqMan OpenArray Genotyping assays on a Real-Time polymerase chain reaction system.ResultsAfter age- and sex-adjustment, among the studied 13 variants, GSTT1 rs17856199 was associated with T2DM under homozygote (OR=3.42; 95% CI:1.04–11.2, p=0.031), and recessive (OR=3.57; 95% CI: 1.11–11.4, p=0.029) comparison models. The NOS2 rs2297518*A allele was more frequent among the T2DM cohort (58.1% vs 35.4%, p<0.001) and showed a dose-response effect; being heterozygote was associated with higher odds for developing DM (OR=4.06, 95% CI=2.13–7.73, p<0.001), whereas being AA homozygote had double the risk (OR=9.06, 95% CI=3.41–24.1, p<0.001). Combined NOS2 rs2297518*A and either GSTT1 rs17856199*A or *C genotype carriers were more likely to develop T2DM. Different associations with sex, BMI, hyperglycemia, and/or hyperlipidemia were evident. The principal component analysis revealed NOS2 rs2297518*G, old age, dyslipidemia, high systolic blood pressure, and elevated HbA1c were the main classifiers of T2DM patients.ConclusionThe oxidative stress-related molecular markers, GSTT1 rs17856199 and NOS2 rs2297518 variants were significantly associated with T2DM risk and phenotype in the study population.

86 Amniotic fluid microRNA profiles in twin-twin transfusion syndrome with and without severe cardiomyopathy

Twin-twin transfusion syndrome (TTTS) presents many challenges for clinicians, and the optimal means of identifying which pregnancies will benefit most from intervention is controversial. There is currently no clinically significant biomarker to detect or risk-stratify TTTS. microRNAs (miRs) are small RNAs that regulate gene expression and are biomarkers in various disease processes, including adult and pediatric heart failure. To date no studies have investigated amniotic fluid (AF) miR as a biomarker for severe TTTS. This study aims to assess whether AF miRs could be useful as biomarkers to identify pregnancies at greatest risk of severe cardiomyopathy due to TTTS. AF was collected at the time of amniocentesis or selective fetoscopic laser photocoagulation from monochorionic diamniotic twin pregnancies with TTTS at any stage and stored at -80° C. Fetal echocardiography was available for all subjects, and right ventricular Tei index of the recipient fetus ≥ 0.64 was used to define severe cardiomyopathy. RNA was extracted from the AF samples using the miRNeasy Mini Kit (Qiagen) and miR arrays were performed using the TaqMan Open Array miR panel (ThermoFisher). Student’s t-test was performed between groups and a stringent q-value of <0.005 was used to determine differentially expressed miRs. Hierarchical cluster analysis (HCA) was used to determine if these miRs properly separated the two groups. A total of 11 AF samples from pregnancies with severe cardiomyopathy were compared to 12 control TTTS samples without severe cardiomyopathy. 40 miRs were differentially expressed. HCA (Fig 1) based on these 40 miRs show a strong ability to differentiate the two groups. All differentially expressed miRs were downregulated in the severe cardiomyopathy group with the exception of upregulation of miR-208 (Fig 2), which has previously been shown to be associated with adverse outcomes in dilated cardiomyopathy. AF miRs demonstrate differential expression in TTTS with and without severe cardiomyopathy, and can be an important biomarker of disease severity in this population.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

MicroRNA Profiling in Oesophageal Adenocarcinoma Cell Lines and Patient Serum Samples Reveals a Role for miR-451a in Radiation Resistance.

Many patients with Oesophageal Adenocarcinoma (OAC) do not benefit from chemoradiotherapy treatment due to therapy resistance. To better understand the mechanisms involved in resistance and to find potential biomarkers, we investigated the association of microRNAs, which regulate gene expression, with the response to individual treatments, focusing on radiation. Intrinsic radiation resistance and chemotherapy drug resistance were assessed in eight OAC cell lines, and miRNA expression profiling was performed via TaqMan OpenArray qPCR. miRNAs discovered were either uniquely associated with resistance to radiation, cisplatin, or 5-FU, or were common to two or all three of the treatments. Target mRNA pathway analyses indicated several potential mechanisms of treatment resistance. miRNAs associated with the in vitro treatment responses were then investigated for association with pathologic response to neoadjuvant chemoradiotherapy (nCRT) in pre-treatment serums of patients with OAC. miR-451a was associated uniquely with resistance to radiation treatment in the cell lines, and with the response to nCRT in patient serums. Inhibition of miR-451a in the radiation resistant OAC cell line OE19 increased radiosensitivity (Survival Fraction 73% vs. 87%, p = 0.0003), and altered RNA expression. Pathway analysis of effected small non-coding RNAs and corresponding mRNA targets suggest potential mechanisms of radiation resistance in OAC.

Abstract 15986: Plasma Microrna Profiling Reveals Potential Biomarkers of Thiazide Response

Introduction: Thiazide diuretics (TZDs) are the second most frequently prescribed class of antihypertensives. But &lt;50% patients achieve goal blood pressure (BP) on TZD monotherapy, mostly because therapy is chosen overlooking the heterogeneity in patient responses. Circulating microRNAs (miRNAs) remain attractive as non-invasive biomarkers to predict drug response and tailor therapy for optimal outcomes. Hypothesis: Plasma miRNAs may play a role in TZD response; we aim to identify those associated with hydrochlorothiazide (HCTZ) response. Methods: We profiled 754 miRNAs in baseline (untreated) plasma samples of 36 uncomplicated hypertensive adults (18 Responders (R) and 18 Non-responders (NR) based on their diastolic BP (DBP) response to HCTZ therapy) from the Pharmacogenomic Evaluation of Antihypertensive Responses clinical trial, using the real-time qPCR based TaqMan OpenArray Human microRNA panel. We filtered out miRNAs with low amplification scores and Cq&gt;38 and samples with low miRNA expression. Combination of miR-223 and miR-19b (identified as most stably expressed miRNAs by NormFinder Software) was used to normalize expression across samples. We used MannWhitney U test to identify microRNAs differentially expressed between R and NR; linear and logistic regressions for associations of miRNA expression with BP response. FDA corrected p&lt;0.05 was used as significance threshold, unadjusted p&lt;0.05 as suggestive threshold. Results: Mean systolic BP (SBP)/DBP change in R was 14/10 mmHg and in NR 0/1 mmHg. We found 77 miRNAs to be consistently expressed across samples, of which 11 were upregulated and 14 downregulated with &gt;2-fold difference between R and NR. Though no miRNAs reached FDR p&lt;0.05, miR-376a (fold change(fc)= 10.1, p= 0.035) and miR-17 (fc= -0.45, p= 0.017) were differentially expressed between R and NR and their expression associated with DBP and SBP change, along with miR-10b, miR-24, miR-134 associated with SBP change at p&lt;0.05. Conclusions: In this first ever genome-wide miRNA study of antihypertensive drug response, we discovered circulating miRNAs associated with BP response to HCTZ. Future studies are planned to validate these findings in a larger cohort, across different race group and across different TZD.

The Physiological MicroRNA Landscape in Nipple Aspirate Fluid: Differences and Similarities with Breast Tissue, Breast Milk, Plasma and Serum.

Background: MicroRNAs (miRNAs) target 60% of human messenger RNAs and can be detected in tissues and biofluids without loss of stability during sample processing, making them highly appraised upcoming biomarkers for evaluation of disease. However, reporting of the abundantly expressed miRNAs in healthy samples is often surpassed. Here, we characterized for the first time the physiological miRNA landscape in a biofluid of the healthy breast: nipple aspirate fluid (NAF), and compared NAF miRNA expression patterns with publically available miRNA expression profiles of healthy breast tissue, breast milk, plasma and serum. Methods: MiRNA RT-qPCR profiling of NAF (n = 41) and serum (n = 23) samples from two healthy female cohorts was performed using the TaqMan OpenArray Human Advanced MicroRNA 754-Panel. MiRNA quantification data based on non-targeted or multi-targeted profiling techniques for breast tissue, breast milk, plasma and serum were retrieved from the literature by means of a systematic search. MiRNAs from each individual study were orderly ranked between 1 and 50, combined into an overall ranking per sample type and compared. Results: NAF expressed 11 unique miRNAs and shared 21/50 miRNAs with breast tissue. Seven miRNAs were shared between the five sample types. Overlap between sample types varied between 42% and 62%. Highly ranked NAF miRNAs have established roles in breast carcinogenesis. Conclusion: This is the first study to characterize and compare the unique physiological NAF-derived miRNA landscape with the physiological expression pattern in breast tissue, breast milk, plasma and serum. Breast-specific sources did not mutually overlap more than with systemic sources. Given their established role in carcinogenesis, NAF miRNA assessment could be a valuable tool in breast tumor diagnostics.

Analysis of the interaction effect of 48 SNPs and obesity on type 2 diabetes in Chinese Hans

IntroductionBoth environmental and genetic factors contribute to type 2 diabetes (T2D) risk. Dozens of T2D susceptibility loci have been identified by genome-wide association study. However, these loci account for only...

Cumulative lifetime maternal stress and stress in pregnancy are differentially associated with extracellular vesicle encapsulated microRNA profiles in breast milk: Findings from the PRogramming of Intergenerational Stress Mechanisms (PRISM) pregnancy coho

Background/Aims: While maternal stress has been linked to adverse child health outcomes, mechanistic links have not been fully elucidated. Maternal microRNAs encapsulated in Extracellular Vesicles (EVs) reach the infant through breastmilk and are a novel biochemical communication pathway for early-life programming. We leverage the PRogramming of Intergenerational Stress Mechanisms (PRISM) pregnancy cohort to investigate associations between maternal stress and breast milk EV-microRNAs. Methods: We assessed maternal lifetime stress using the Life Stressor Checklist-Revised (LSCR) survey and negative life events (NLEs) experienced in the past 6 months during pregnancy using the Crisis in Family Systems-Revised (CRISYS-R) survey. Extracellular vesicles were isolated from N=80 breastmilk samples collected at 6.1±5.9 weeks postnatally. Total RNA was extracted and microRNAs were profiled using the TaqMan OpenArray Human miRNA panel. Logistic regression assessed associations between continuous LSCR and NLE scores and EV-microRNA detection (outcome yes/no);associations with EV-microRNA expression levels were assessed using robust linear regression (N=74 with EV-microRNA and maternal stress data). Models were adjusted for infant sex, maternal race/ethnicity, education, and week of breast milk collection. Results: Among 345 EV-microRNAs detected in &gt;10% of samples, detection of 127 (47%) was associated with LSCR score and detection of 97 (28%) was associated with NLE score (p &lt; 0.05). Among 205 EV-microRNAs detected in &gt;50% of samples, expression of 8 was associated with LSCR scores and expression of 17 was associated with NLE score at our a priori criteria of p &lt; 0.05 and |Bregression| &gt; 0.2. MicroRNAs associated with LSCR and NLE scores were involved in KEGG pathways related to fatty acid metabolism and steroid biosynthesis. Conclusions: Maternal lifetime cumulative stress and stress during pregnancy were associated with EV-microRNAs in breast milk although microRNA profiles differed. Further research is needed to identify biological pathways impacted by differentially expressed microRNAs and investigate relationships with child health outcomes.

A Data-Driven Approach to Carrier Screening for Common Recessive Diseases.

Genetic screening is an advanced tool for reducing recessive disease burden. Nowadays, it is still unclear as to the number of genes or their variants that are necessary for effective screening. This paper describes the development of a carrier screening custom panel for cystic fibrosis, phenylketonuria, alpha-1 antitrypsin deficiency, and sensorineural hearing loss consisting of 116 variants in the CFTR, PAH, SERPINA1, and GJB2 genes. The approach is based on the cheapest and fastest method, on using a small number of genes, and on the estimation of the effectiveness of carriers’ detection. The custom panel was tested on a population-based cohort that included 1244 participants. Genotypes were determined by the TaqMan OpenArray Genotyping platform on the QuantStudio 12K Flex Real-Time PCR System. The frequency of heterozygotes in the Russian population was 16.87% or 1:6 (CI95%: 14.76–19.00% by Clopper-Pearson exact method): in CFTR—2.81% (1:36), PAH—2.33% (1:43), SERPINA1—4.90% (1:20), and GJB2—6.83% (1:15). The data on allele frequencies were obtained for the first time on a Russian population. The panel allows us to identify the vast majority of carriers of recessive diseases in the population. It is an effective approach to carrier screening for common recessive diseases.

Correlation between Expression Profiles of Key Signaling Genes in Colorectal Cancer Samples from Type 2 Diabetic and Non-Diabetic Patients.

Several lines of epidemiological and biochemical evidence support the association of type 2 diabetes mellitus (T2DM) and colorectal cancer (CRC). T2DM has been shown to impinge on the transcriptome of colon tumor cells, promoting their proliferation and invasion. In order to gain insight into diabetes-specific modulation of colon cancer signaling, we analyzed gene expression patterns of more than five hundred genes encoding signaling proteins on TaqMan OpenArray panels from colonoscopic colorectal tumor samples of type 2 diabetic and non-diabetic patients. In total, 48 transcripts were found to be differentially expressed in tumors of T2DM patients as compared to healthy colon samples. Enrichment analysis with the g:GOSt (Gene Ontology Statistics) functional profiling tool revealed that the underlying genes can be classified into five signaling pathways (in decreasing order of significance: Wnt (wingless-type)/β-catenin; Hippo; TNF (tumor necrosis factor); PI3K/Akt (phosphoinositide-3 kinase/protein kinase B), and platelet activation), implying that targeted downregulation of these signaling cascades might help combat CRC in diabetic patients. Transcript levels of some of the differentially expressed genes were also measured from surgically removed diabetic and non-diabetic CRC specimens by individual qPCR (quantitative real-time PCR) assays using the adjacent normal tissue mRNA levels as an internal control. The most significantly altered genes in diabetic tumor samples were largely different from those in non-diabetic ones, implying that T2DM profoundly alters the expression of signaling genes and presumably the biological characteristics of CRC.

Brief Report: Demographic and Genetic Associations With Markers of Small and Large Fiber Sensory Neuropathy in HIV Patients Treated Without Stavudine.

Neurotoxic antiretroviral therapy (ART) such as stavudine has been now replaced with safer therapies, reducing the prevalence of neuropathy from 34% to 15% in HIV+ Indonesians. However, it is unclear whether the residual cases display damage to small or large nerve fibers and whether both are influenced by known risk factors, including alleles of CAMKK2 associated with neuropathy in HIV patients. The encoded protein influences the growth and repair of nerve fibers. HIV-positive adults on ART for >12 months without exposure to stavudine were screened for neuropathy using the AIDS Clinical Trials Group Brief Peripheral Neuropathy Screen (BPNS). Large fiber neuropathy was assessed by nerve conduction (NC) and small fiber neuropathy using stimulated skin wrinkling (SSW) applied to the fingers. CAMKK2 alleles were assessed by TaqMan OpenArray technology. Neuropathy diagnoses were more common with SSW than BPNS (49/173 vs 26/185, χ; P = 0.0009), with poor alignment between these outcomes (P = 0.60). NC and BPNS diagnosed neuropathy at similar frequencies (29/151 vs 26/185; P = 0.12) and were aligned (P < 0.0001). In bivariate analyses, all diagnoses were associated with patients' age and persistent HIV replication, with minor effects from CD4 T-cell counts and time on ART. CAMKK2 alleles associated with neuropathy diagnosed with BPNS and SSW but not NC. Multivariable analyses confirmed the importance of age and HIV replication, with distinct CAMKK2 polymorphisms affecting BPNS and SSW. Paradoxically, height was protective against skin wrinkling. Overall the data link CAMKK2 genotypes with small rather than large fiber damage. SSW may reflect pathology distinct from that identified using BPNS.

Associations between maternal lifetime stressors and negative events in pregnancy and breast milk-derived extracellular vesicle microRNAs in the programming of intergenerational stress mechanisms (PRISM) pregnancy cohort

ABSTRACT Maternal stress is associated with adverse child health. Breast milk microRNAs encapsulated in extracellular vesicles (EVs) are involved in mother-infant biochemical communication during early-life programming. We leverage the PRogramming of Intergenerational Stress Mechanisms (PRISM) pregnancy cohort to investigate associations between maternal stress and breast milk EV-microRNAs. Lifetime stress and negative life events (NLEs) during pregnancy were assessed using the Life Stressor Checklist-Revised (LSCR) and the Crisis in Family Systems-Revised surveys, respectively. RNA was extracted from breast milk EVs (N = 80; collected 6.1 ± 5.9 weeks postnatally), and microRNAs were profiled using the TaqMan OpenArray Human miRNA panel. Associations between stress scores and detection (yes/no) of 173 microRNAs identified in 20–80% of samples were assessed using logistic regression; associations with expression levels of 205 EV-microRNAs identified in >50% of samples were assessed using linear regression. In adjusted models, detection of 60 and 44 EV-microRNAs was associated with higher LSCR and NLE scores, respectively (p < 0.05). Expression level of 8 and 17 EV-microRNAs was associated with LSCR and NLE scores, respectively, at our a priori criteria of p < 0.05 and |B regression|>0.2. Enriched KEGG pathways for microRNAs associated with stress scores included fatty acid metabolism and the Hippo signaling pathway. Maternal lifetime stress and NLEs during pregnancy were both associated with detection and expression level of breast milk EV-microRNAs, although associations with microRNA profiles differed between stress measures. Further research is needed to identify biological pathways impacted by associated microRNAs and investigate relationships with child health outcomes. Abbreviations: EV: extracellular vesicle; PRISM: PRogramming of Intergenerational Stress Mechanisms pregnancy cohort; LSCR: Life Stressor Checklist-Revised survey; NLE: negative life event; CRISYS-R: Crisis in Family Systems-Revised survey; KEGG: Kyoto Encyclopaedia of Genes and Genomes; NYC: New York City; SD: standard deviation; IQR: interquartile range; Cq: relative cycle threshold values; PCA: principal component analysis

Abstract 5716: miR-21 inhibition regulates mutant KRAS effector pathways and intercepts development of pancreatic ductal adenocarcinoma

Abstract Introduction: Pancreatic ductal adenocarcinoma (PDAC) tumorigenesis is largely driven by an activating KRAS (mKRAS) mutation that is nearly ubiquitous in the earliest stages of disease, pancreatic intraepithelial neoplasias (PanINs). Targeting KRAS directly has, thus far, been unsuccessful due to compensatory pathways or associated toxicity of proposed treatments. MicroRNAs (miRNAs) are short non-coding RNAs that represent a novel avenue to target KRAS effector function through post-transcriptional regulation. Experimental Procedures: Transgenic KrasG12D/+;Trp53R172H/+;Pdx-1-Cre (KPC) mice develop early PanINs that progress to invasive PDAC, allowing investigation into early mechanisms of tumorigenesis. The TaqMan OpenArray platform was used to globally profile miRNA expression. miRNAs of interest were validated with dedicated qPCR and inhibited with complementary antisense oligonucleotides. Effects of miR inhibition on KRAS downstream function and tumorigenicity were catalogued using cell lines, KPC, and xenograft models. The Cancer Genome Atlas (TCGA) was used to translate our findings into a human cohort. Finally, we are using patient-derived organoid models to elucidate effects on KRAS effector pathways. Results: Global miRNA expression analysis and dedicated qPCR identified miR-21 as differentially expressed in early stage PanINs in the KPC mouse model. RNA-seq following inhibition of miR-21 revealed modulation of KRAS signaling with downregulation of MAPK, mTOR, and actin cytoskeleton pathways. Phenotypic analysis in the setting of miR-21 inhibition demonstrated reduced proliferation, migration, and invasion. Further, miR-21 inhibition delays PanIN progression to PDAC in KPC mice. Mechanistic study of miR-21 demonstrates activation of cancer associated fibroblasts (CAFs) in the tumor microenvironment (TME) and promotes resistance to chemotherapy. Analysis of TCGA PDAC cohort revealed a significant correlation between miR-21 expression and tumor epithelial cell content (P-value: hsa-miR-21-3p= 8.7e-7; hsa-miR-21-5p = 2.1e-13), prompting further studies in a human model. Organoid models with matched-isogenic CAF lines have been generated from five patients undergoing surgical resection for PDAC. Immortalized organoid models can be co-cultured with either early passage CAFs or transformed CAF lines. Organoid co-culture with CAFs are able to recapitulate the biology seen in other model systems. Conclusions: miR-21 appears to be an early and reliable molecular marker of pancreatic neoplasia. Manipulation of miR-21 expression augments downstream KRAS effector function with phenotypic changes evident in vitro and in vivo. Organoid co-culture models are a unique and powerful tool for the study of downstream molecular mechanisms responsible for phenotypic changes resulting from inhibition of miR-21. The identification of miR-21 as regulator of KRAS effector function and tumor/CAF cross-talk reveals a potential new target for future targeted therapies. Citation Format: Jacquelyn W. Zimmerman, Richard A. Burkhart, Nina J. Chu, Elana J. Fertig, Elizabeth M. Jaffee. miR-21 inhibition regulates mutant KRAS effector pathways and intercepts development of pancreatic ductal adenocarcinoma [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 5716.

Abstract 3694: Differential expressions of miR-17 and complement factor D in carfilzomib-related cardiotoxicity in multiple myeloma patients: Results of microRNA array and proteomics integrated analysis

Abstract Background: Carfilzomib (CFZ), a proteasome inhibitor used to treat multiple myeloma (MM), has been associated with risk for cardiotoxicity including heart failure (HF). There is a lack of data on the mechanism underlying CFZ-induced cardiotoxicity. Our study aims to better understand molecular mechanisms of CFZ-related cardiotoxicity by determining the differential expression of circulating miRNAs and proteins in MM patients. Methods: Plasma samples were collected from 13 MM patients after treated with CFZ (including 7 with cardiotoxicity and 6 with no cardiotoxicity) at the University of Florida Health Cancer Center. TaqMan OpenArray Human MicroRNA Panel containing 754 human miRNA was used for miRNA analysis, and iTRAQ analysis on LC/MS/MS platforms was used for global proteomic profiling. Results: The overall mean age was 61 and 38.5% were men. A total of 374 proteins were identified and 57 were differentially expressed between cardiotoxicity and non-cardiotoxicity groups (false discovery rate &amp;lt;0.05). Differential expression analysis of miRNAs profiling yielded 5 miRNAs that were dysregulated (p-value &amp;lt;0.05 and fold change (FC) &amp;lt; 0.5): miR-17, miR-30c, miR-30b, miR-106a, and miR-92a. Among the top proteins that differentiate the two groups were complement factor D (CFD) (P= 5.5*10-5, and FC= 6), which is targeted by miR-17. CFD circulates in an active form and cleaves complement factor B (FB) to Ba and Bb, which promote the alternative pathway complement (AP) activation, the complement activation has shown to occur in chronic HF, and is associated with adverse clinical events. Also, CFD has a function in the activation of notch signaling, which is required for the proliferation of cardiac stem cells and the survival of cardiomyocytes. Conclusion: These findings suggest that CFZ may induce cardiac dysfunction via increasing CFD activity that is regulated by miR-17, leading to activation of the complement system. Dysregulation in components of the AP of complement activation could impact the diastolic dysfunction associated with HF progression. Citation Format: Marwa Tantawy, Lakshmi Manasa Chekka, Taimour Langaee, Timothy J Garrett, Michael Fradley, Robert Frank Cornell, Rachid C. Baz, Sonal Singh, Lihui Yuan, Chintan P. Shah, Nida Waheed, Carl J. Pepine, Jan S. Moreb, Yan Gong. Differential expressions of miR-17 and complement factor D in carfilzomib-related cardiotoxicity in multiple myeloma patients: Results of microRNA array and proteomics integrated analysis [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 3694.