Background: Mature B cell differentiation provides an important mechanism for the acquisition of adaptive immunity. Malignancies derived from mature B cells are common and constitute the majority of ...leukemias and lymphomas. MicroRNAs are known to play a role in oncogenesis, lineage-selection, and immune cell function, including early B cell differentiation. However, the full extent and function of microRNA expression during mature B cell differentiation and in B cell malignancies are not known.
Methods: From normal young patients undergoing tonsillectomies, we sorted the mature B cell subsets (naive, germinal center, memory and plasma) using FACS, based on their expression of CD19, CD38, IgD and CD27. These sorted B cells were profiled for microRNA expression using a highly sensitive multiplexed real-time PCR assay, as well as for gene expression at the whole genome level using Affymetrix U133plus microarrays. miRNA targets can be predicted based on seed sequence matching of their 2–8 nt to the 3′UTR of gene transcripts. For each B cell stage, we experimentally validated microRNA regulation of predicted target genes of interest, LMO2, MYBL1 and PRDM1, by microRNA over-expression experiments and luciferase assays.
Results: We found that microRNAs have a characteristic expression pattern that defines each mature B cell stage. Examination of both microRNA and mRNA expression showed that in each B cell population, the target genes predicted based on seed matching were expressed at lower levels, results that were highly significant (P<1E-10). We found that differential microRNA expression is important at every B cell stage transition, and differentially expressed microRNAs frequently target differentially expressed transcription factors. In the naive to germinal center B cell and germinal center B cell to memory cell transitions, we found that miR-223 had an inverse relationship with its predicted target genes LMO2 and MYBL1. To test this relationship predicted based on seed pairing, in Germinal Center-derived BJAB cells, we over-expressed miR-223 by introducing its precursor, and saw a subsequent knockdown of LMO2 and MYBL1 at both the mRNA and protein level. We confirmed seed sequence specificity by comparing miR-223 knockdown of luciferase reporter activity on the LMO2 3′UTR compared to its seed sequence mutant. We further found that miR-9 and miR-30 family members directly regulate PRDM1 (blimp1), a master regulator of the GC to PC transition. In U266 cells (PC-derived), introduction of miR-9 and miR-30 family precursor resulted in decreased PRDM1 protein expression, although transcript levels were not changed, consistent with previous evidence that miRNA can regulate at the post-transcriptional steps. We further profiled over 50 tumors derived from various B cell malignancies (small lymphocytic lymphoma, Burkitt lymphoma, and the molecular subsets of diffuse large B cell lymphoma) and found that these malignancies maintain the expression patterns of their respective lineage; microRNA expression profiles of normal B cells could correctly classify the lineage of these tumors in over 80% of the cases. In contrast to other malignancies, common lymphomas do not down-regulate microRNAs, but rather maintain the microRNA-expression patterns of their normal B-cell counterparts.
Conclusion: Through concomitant microRNA and mRNA-profiling, we demonstrate a regulatory role for microRNAs at every stage in mature B-cell differentiation. Further, we have experimentally identified a direct role for the microRNA-regulation of key transcription factors in B-cell differentiation: LMO2, MYBL1 and PRDM1 (Blimp1). Thus, our data demonstrate that microRNAs may be important in maintaining the mature B-cell phenotype in normal and malignant B-cells.
Abstract 2403
Poster Board II-380
MicroRNAs are 18-22 nucleotide-long RNA molecules that regulate expression of genes. We and others have previously demonstrated a role for microRNAs in the ...pathogenesis of B cell malignancies. Computational predictions suggest that the human genome encodes several thousand microRNAs. Thus far, about 700 microRNAs have been discovered in humans, including over 200 new microRNAs in the past year alone. The ongoing discovery of microRNAs makes it difficult to comprehensively study their role in a disease group. The advent of high throughput sequencing allows the simultaneous identification of millions of transcripts, thereby providing a sensitivity that is several orders of magnitude higher than conventional methods. We hypothesized that high throughput sequencing would be an effective tool to comprehensively identify microRNAs in normal and malignant B cells.
While there is an overlap between diffuse large B-cell lymphoma (DLBCL) and Burkitt lymphoma (BL) in morphology, immunophenotype and cytogenetics, distinguishing between BL and DLBCL is critical because there are important differences in their clinical management. We investigated whether microRNA expression could be used to reliably distinguish BL from DLBCL.
We carefully chose 31 human samples to represent the spectrum of normal and malignant B cells including FACS-sorted naive, germinal center, memory, plasma cells, EBV transformed and activated B cells. Samples derived from B cell malignancies included B-lymphoblastic lymphoma, chronic lymphocytic leukemia (immunoglobulin gene mutated and unmutated), mantle cell lymphoma, marginal zone lymphomas, HIV-related lymphoma, BL, DLBCL (activated and germinal center type), primary mediastinal B cell lymphoma, Hodgkin lymphoma, and multiple myeloma.
We applied massively parallel, high-throughput sequencing of the 18-22 nt RNAs from these cases and generated a total of 255,624,785 sequences (∼5 billion bases). Using a computational approach that we have previously validated with normal B cells, we identified the expression of 429 known microRNAs in normal and malignant B cells, a number that is over three times higher than previously recognized in any tissue type. We also identified the expression of 302 novel microRNAs in normal and malignant B cells. The vast majority of these microRNAs were highly conserved in multiple species.
As a proof of principle, we generated a custom microarray that included all the known human, and viral microRNAs, as well as 302 novel microRNAs identified by sequencing, and applied it to the clinically important distinction of BL from DLBCL. Biopsy samples were collected from 104 patients (BL, N=25, DLBCL, N=79) treated at 9 institutions that comprise an international consortium. All cases were reviewed for pathology diagnosis and profiled for microRNA expression. We constructed a Bayesian predictor to distinguish BL from DLBCL based on the microRNA expression. The predictor performance was tested using leave-one-out cross-validation. We also applied gene expression profiling to the cases of DLBCL to identify the molecular subsets of DLBCL: activated B cell like and germinal center B cell like DLBCL. The microRNA profiles of these cases were equally efficacious in distinguishing the DLBCL subsets.
The predictor constructed based on microRNA expression was over 90% accurate in distinguishing BL from DLBCL, using pathology diagnosis as the gold standard. Further, microRNA-based predictor was also over 90% accurate in the distinction of the molecular subsets of DLBCL, compared to the gold standard of gene expression-profiling.
As additional validation, we performed in situ hybridization of selected microRNAs to directly visualize their expression using methods that are easily accessible in conventional pathology laboratories. We found excellent concordance between the expression results derived from microarrays and in situ hybridization suggesting a ready path to clinical translation.
Our study represents the first comprehensive delineation of microRNA expression in B cell malignancies using high throughput sequencing. Our data suggest that microRNAs are a promising marker for the distinction of aggressive lymphomas.
No relevant conflicts of interest to declare.
Expression quantitative trait loci (eQTL) analysis identifies genetic markers associated with the expression of a gene. Most existing eQTL analyses and methods investigate association in a single, ...readily available tissue, such as blood. Joint analysis of eQTL in multiple tissues has the potential to improve, and expand the scope of, single-tissue analyses. Large-scale collaborative efforts such as the Genotype-Tissue Expression (GTEx) program are currently generating high quality data in a large number of tissues. However, computational constraints limit genome-wide multi-tissue eQTL analysis. We develop an integrative method under a hierarchical Bayesian framework for eQTL analysis in a large number of tissues. The model fitting procedure is highly scalable, and the computing time is a polynomial function of the number of tissues. Multi-tissue eQTLs are identified through a local false discovery rate approach, which rigorously controls the false discovery rate. Using simulation and GTEx real data studies, we show that the proposed method has superior performance to existing methods in terms of computing time and the power of eQTL discovery. We provide a scalable method for eQTL analysis in a large number of tissues. The method enables the identification of eQTL with different configurations and facilitates the characterization of tissue specificity.
Ethiopia is a country which suffers with one of the highest levels of unintended pregnancies and unsafe abortion practices. The aim of this study was to assess the utilization of post-abortion family ...planning and associated factors among women seeking abortion services in Asella town health facilities. A facility based cross-sectional study design was conducted among women who came for abortion services from July 15 to October 15, 2019. Two hundred seventy-six participants were included using a systematic random sampling technique. Both descriptive and logistic regression analysis were conducted. In multivariate analysis, variables which had a p-value < 0.05 was considered as significantly associated with the outcome variable. Postabortion family planning utilization among study participants was 146 (53.7%) (95% CI=47.4, 59.2). Formal education (AOR=4.45: 95% CI: (1.18, 16.74), previous history of abortion (AOR=0.35; 95% CI: (0.14, 0.85), positive attitude to towards family planning (AOR=2.62; 95% CI: (1.09, 6.27), counseled on postabortion family planning utilization (AOR=3.12; 95% CI: (1.30, 7.51) were significantly associated with post abortion family planning utilization. In this study, nearly fifty percent of the respondents did not utilize Postabortion Family Planning (PAFP). Educational status, history of pervious abortion, decision when to have a child, attitude toward PAFP utilization and counseling about PAFP were significantly associated with post-abortion family planning use. The health care providers who give abortion service should give counseling for all women who get abortion service. More accents should be given to misperception of PAFP to change the negative attitude of utilization of PAFP.
Background
Malaria is a leading cause of morbidity in Ethiopia. However, its transmission varies in both space and time, and large areas of the country are hypoendemic and epidemic-prone. The ...Ethiopia National Malaria Indicator Survey 2007 is a cross-sectional, nationally-representative household survey. The objective of the analyses presented here were to use the survey's data to identify factors associated with anemia presence in children under 6 years of age (U6); specifically, investigate the association between malaria and anemia; and discuss using anemia as a malaria proxy biomarker in the Ethiopian hypo-endemic transmission setting.
Methods
The survey sampled 4185 households in 347 enumeration areas ≤2500 m above sea level. Primary outcome was increasing anemia severity in sampled children: no anemia (Hb: ≥11g/dl); mild anemia (Hb: ≥8g/dl and <11g/dl); and moderate-severe anemia (Hb: <8g/dl). Secondary outcomes were positive malaria rapid diagnostic test (RDT) or blood slide microscopy.
Results
The analysis included 6054 (92.0%) children U6 in 3962 households. The proportion of children with no anemia, mild anemia, and moderate-severe anemia was 63.6%, 31.3%, and 5.1%, respectively. The overall prevalence of anemia (Hb <11g/dl) was 36.4% (95% CI 34.4-38.4). Factors independently associated with reduced relative odds of anemia categories were age (OR=0.7, 95% CI 0.7-0.7) and female sex (OR=0.9, 95% CI 0.8-1.0); malaria RDT positivity was associated with increased relative odds of a more severe anemia category (OR=5.8, 95% CI 3.7-9.2).
Conclusions
We conclude that at altitudes ≤2500 m malaria appears to be a significant risk factor for anemia; potentially anemia could be used as a useful proxy biomarker for malaria and its control in Ethiopia.
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