ALBERT

Description: Abstract 3298 The anucleate platelets play a critical role in the formation of thrombi and prevention of bleeding. While the repertoire of platelet transcripts is a reflection of the megakaryocyte at the time of platelet differentiation, post-transcriptional events are known to occur. Furthermore, a strong correlation between the expressed mRNAs and proteome has been identified. Having a complete understanding of the platelet transcriptome is important for generating insights into the genetic basis of platelet disease traits. To capture the complexity of the platelet transcriptome, we performed RNA sequencing (RNA-seq) in leukocyte-depleted platelets from 10 males, with median age of 24.5 yrs and unremarkable medical history. Their short and long RNA platelet transcriptomes were analyzed on the SOLiD 5500xl sequencing platform. We generated ∼3.5 billion sequence reads ∼40% of which could be mapped uniquely to the human genome. Our analysis revealed that ∼9,000 distinct protein-coding mRNAs and ∼800 microRNAs (miRNAs) were present in the transcriptome of each of the 10 sequenced individuals. Comparison of the levels of mRNA expression across the 10 individuals showed an exceptional level of consistency with pair-wise Pearson correlation values ≥0.98. The miRNA expression profiles across the 10 individuals showed a similar consistency with pair-wise Pearson correlation values ≥0.98. Surprisingly, we found that these mRNAs and miRNAs accounted for a little over 1/2 of all of the uniquely mapped sequence reads suggesting the abundant presence of additional non-protein coding RNA (ncRNA) transcripts. Using the annotated entries of the latest release of the ENSEMBL database, we investigated the genetic make-up of these other transcripts. We found that ∼25% of each individual's uniquely mapped reads corresponded to non-protein coding transcripts from mRNA-coding loci. These reads accounted for more than 10,000 distinct such transcripts. In addition, each of the individuals in our cohort expressed an average of ∼1,500 pseudogenes and ∼200 long intergenic non-coding RNAs (lincRNAs). The short RNA profiles of the ten individuals revealed an abundance of diverse categories of ncRNAs including the signal recognition particle RNA (srpRNA), small nuclear RNA (snRNA) and small cytoplasmic RNAs (scRNA). These ncRNAs are involved in the processing of pre-mRNAs and their presence and prevalence in the anucleate platetet suggests the existence of a complex network of mRNA processing that persists after the megakaryocyte fragmentation. We also investigated the RNA-omes of the ten individuals for evidence of transcription of the pyknon category of ncRNAs. Pyknons are of particular interest because each has numerous intergenic and intronic copies whereas nearly all known human protein-coding genes contain one or more pyknons in their mRNA. Recent experimental work has shown that intergenic instances of the pyknons are transcribed in a tissue- and cell-state specific manner. An average of ∼100,000 pyknons are transcribed in each of the 10 sequenced individuals suggesting the possibility of a far-reaching network of interactions that link exonic space to distant non-exonic regions and are active in platelets. Lastly, we found that a large variety of distinct repeat element categories are expressed in the RNA-omes (both short and long) of these individuals. Among the most abundantly represented categories of repeat elements were DNA transposons, long terminal repeat (LTR) retrotransposons, and non-LTR retrotransposons such as long interspersed elements (LINEs) and short interspersed elements (SINEs). In summary, our RNA-seq analyses have revealed a spectrum of platelet transcripts that transcends protein-coding genes and miRNAs. Indeed, the transcripts that have their source in genomic features not previously discussed or analyzed in the platelet context represent a very significant portion of all platelet transcripts. This in turn suggests an unanticipated richness, and presumably commensurate complexity, for the platelet transcriptome. While the role of these novel non-protein coding RNAs is currently unknown it is expected that at least some of them may be of functional significance which will in turn permit a better understanding of the molecular mechanisms that regulate platelet physiology and may contribute to processes beyond thrombosis and hemostasis. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 390 A conspicuous lesson that has emerged from the 1000 Genomes Project is the greater genetic variation in the population than previously appreciated. Transcriptomics is rapidly assuming a prominent role in the understanding of basic molecular mechanisms accounting for variation within the normal population and disease states. Besides protein-coding RNAs, the importance of non-coding RNAs (ncRNAs) – primarily as regulators of gene expression – is well recognized but largely unexplored. The platelet transcriptome reflects megakaryocyte RNA content at the time of proplatelet release, subsequent splicing events, selective packaging and platelet RNA stability. An accurate understanding of the platelet transcriptome has both biological (improved understanding of platelet protein translation and the mechanisms of megakaryocyte/platelet gene expression) and clinical (novel biomarkers of disease) relevance. We carried out transcriptome sequencing of total RNA isolated from leukocyte-depleted platelet preparations from four healthy adults using an AB/LT SOLiD™ system. For each individual, we constructed 3 libraries: a) long (≥ 40 nucleotides) total RNA, b) long RNA depleted of rRNA, and c) short (〈 40 nucleotides) RNA. ∼1 billion reads from the 12 datasets were mapped on each chromosome and strand of the human genome. About one-third mapped uniquely, similar to other unbiased methods like SAGE. Normalizing for transcript length and scale using ß-actin expression level provided the ability to appropriately scale expression within a read-set and to compare expression levels across read-sets. Of the known protein-coding loci, ∼9,500 were present in human platelets. Plotting the number of protein-coding genes as a function of the level of normalized expression underscored different gene estimates between total and rRNA-depleted RNA preparations, and substantial inter-individual variation in the less abundant genes. RT-PCR validated the RNA-seq estimates of transcript levels exhibiting a range of 〉3 orders of magnitude of normalized read counts (r=0.7757; p=0.0001). A strong correlation was measured between mRNAs identified by RNA-seq and 3 published microarray datasets for well-expressed mRNAs, although RNA-seq identified many more transcripts of lower abundance. Unexpectedly, ribosomal RNA depletion significantly and adversely affected estimates of the relative abundance of transcripts including members of the RNA interference pathway DGCR8, DROSHA, XPO5, DICER1, EIF2C1-4, which exhibited large differences (up to 32-fold) between the total and rRNA-depleted preparations. A rigorous and highly stringent approach identified bona fide intronic regions that gave rise to 6,992 and 1,236 currently uncharacterized long and short RNA transcripts, respectively. We discovered numerous previously unreported antisense transcripts: 1) to known protein-coding regions of the genome, 2) 10 miRNA precursors where each locus generated 1–2 distinct antisense transcripts, presumably mature and “star” miRNAs, and 3) long and short RNAs antisense to several known repeat families. We did not observe enrichment of long-intergenic ncRNAs. We considered various possible explanations for the ∼60% sequence reads that could not be mapped on the genome. Much more lenient parameter settings only accounted for only ∼6.5% sequenced reads. An even smaller fraction of reads was observed when considering all possible combinations of exon-exon junctions in the genome (12,382,819 junctions) and the highly polymorphic HLA region of chr 6, indicating these did not contribute in any substantive manner to the platelet transcriptome. Lastly, RNA-seq was highly reproducible (〉97 for 1 subject studied on 4 occasions). In summary, our work reveals a richness and diversity of platelet RNA molecules, suggesting a context where platelet biology transcends protein- and mRNA-centric descriptions. We will provide a publicly available web tool of these data embedded in a local mirror of the UCSC genome browser, facilitating the elucidation of previously unappreciated molecular species and molecular interactions. This will eventually permit an improved understanding of the molecular mechanisms that regulate platelet physiology and that contribute to disorders of thrombosis, hemostasis and inflammation. Disclosures: No relevant conflicts of interest to declare.

Description: The anucleate platelets play a critical role in the formation of thrombi and prevention of bleeding. In recent years, next-generation RNA sequencing (RNA-seq) has proven very useful in shedding light on the specifics of the platelet transcriptome. For example, RNA-seq of the long RNAs in platelets has revealed many non-coding RNAs (ncRNAs) as well as a diverse set of protein-coding genes whose mRNAs are highly correlated amongst individuals but only weakly linked to the currently available platelet proteome. By comparison, the short RNA transcriptome has not been as thoroughly characterized. As a matter of fact, these studies have so far focused on the 100’s of microRNAs (miRNAs) that are present in platelets leaving large swaths of the short RNA-ome uncharacterized. To gauge the complexity of the platelet short RNA-ome we performed short RNA-seq of leukocyte-depleted platelets from 10 healthy males (5 white and 5 black). The sequencing was done on the SOLiD 5500 XL platform and generated over 1.5 billion sequenced reads. To comprehensively characterize the complete short RNA-ome we only considered sequence reads that mapped on the genome without any mismatches but allowed a read to map to as many as 10,000 locations within the genome. This approach gave us the ability to simultaneously examine both the uniquely-present and the repeat-derived expressed elements of the genome. Using this approach, we were able to map ~50% of the sequenced reads. We found that for ~55% of the mapped reads their sequences are present at multiple genomic locations whereas the remaining ~45% originated from unique locations. Of the RNAs with unique genomic origins: ~50% correspond to miRNAs (with miR-223-3p being the most abundant miRNA across all 10 individuals), ~20% originate from various classes of repeat elements, and, the remaining 30% correspond to non-annotated regions of the genome that were non-annotated a of Release 75 of the ENSEMBL database. By comparison, of the RNAs with ambiguous genomic origins: ~20% belong to miRNAs (with miR-103a-3p, a miRNA present in two locations in the genome, being the most abundant miRNA across all 10 individuals) and ~60% correspond to various classes of repeat elements (with members of the HY4 scRNA ncRNAs accounting for nearly a third of all sequence reads). These findings make it evident that the platelet transcriptome has a considerable richness in short RNAs that arise from repetitive elements. To further characterize those RNAs that map to regions of the genome that are not currently annotated, we considered the possibility that they may be novel miRNAs. Using the miRDeep2 algorithm, we sought novel miRNAs among the uncharacterized transcripts and identified 47 of them; the sequences for 18 of these 47 appear at multiple genomic locations in analogy to miR-103/107, miR-19a/19b, etc. Lastly, as our ten samples represented two races, we hypothesized that a subset of the identified sequences would be differentially expressed between the two groups. Using DESeq2, we identified over 157 sequences to be differentially expressed. The most highly differentially expressed sequences corresponded to a miRNA and a repeat element. In summary, our RNA-seq analyses have revealed a very diverse spectrum of platelet short RNAs that transcends the miRNA category. Indeed, we find that short transcripts that have their source in genomic loci that have not been previously discussed or analyzed in the platelet context represent a very significant portion of all short RNAs in platelets. This in turn highlights an unanticipated richness, and presumably commensurate complexity, for the platelet transcriptome. While the role of these novel non-protein coding short RNAs is currently unknown it is expected that at least some of them may be of functional significance. Consequently, they could contribute to processes beyond thrombosis and hemostasis and may permit a better understanding of the molecular mechanisms that regulate platelet physiology. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 481 Most successful DNA-based genome wide association studies identify genomic regions, not genes themselves, and the findings are often devoid of context or mechanism. To identify the genetic basis of disease and disease traits, it is imperative to characterize the quantity and forms of the genes that are expressed in the tissue of interest. It is not feasible to use primary megakaryocytes to profile mRNA from large numbers of subjects, but platelet RNA is easy to obtain. Others and we have previously surveyed genome-wide platelet RNA expression using microarrays, an approach that has had a major impact on systems biology. However, microarrays have a number of limitations, including the use of probes only to known transcripts, a limited dynamic range for quantifying very low and high levels of transcripts, high background levels from cross-hybridization, and complicated normalization schemes to compare expression levels across experiments. Novel high-throughput sequencing approaches that overcome the limitations of microarrays have recently become available. RNA sequencing (RNAseq) has a remarkable ability to quantify mRNAs and provide information about transcript sequence variations, including single nucleotide changes and alternately spliced exons. The goal of these studies was to apply RNAseq to capture platelet transcriptome complexity. Total RNA was prepared using leukocyte-depleted platelets (LDP; less than 1 WBC per 5 million platelets) from 4 donors; 2 were studied twice each. Analysis of this material showed that compared to nucleated cells (HeLa, Meg-01), platelets had 50%-90% less ribosomal RNA, and high levels of messenger and small RNAs (Agilent 2100). The major reduction in platelet rRNA was confirmed by RNA gel analysis. The platelet whole transcriptomes were analyzed via the Applied Biosystems (AB) SOLiD 3Plus next generation sequencing protocols and platform. A typical sequence run generated ∼250 million reads of 50 bp each. We observed more than 30,000 independent platelet mRNA-coding transcripts from about 10,000 genes, demonstrating substantial numbers of variant isoforms. The increased sensitivity of RNAseq for low copy number is clear from these results, because prior platelet transcriptome studies using microarrays have identified only 1500–6000 expressed genes. As an example, the platelet-specific transcript, ITGA2B, showed very high copy number in platelets, but no expression in HeLa cells and modest expression in the megakaryocyte cell line, Meg-01. As is expected for RNA-Seq data, the density of mapped reads varies by exon and local sequence. We also provide examples of newly discovered SNPs that encode non-conservative amino acid changes (AKT2 1209A/T; PIK3CB 837C/G) and alter consensus exon/intron splice junction sites (P2YR12 nt 65 G/A). We have also identified a major difference in the ratio of two splice variants of the FcRg chain, 4:1 in one human platelet donor and 49:1 in another. In summary, we have demonstrated that RNAseq can accurately and sensitively determine the quantity and quality of variations in individual platelet transcriptomes. It appears that the the platelet transcriptome is approximately 10 times more complex than previously thought. The major relative reduction in platelet rRNA may be an advantage for characterizing functional platelet transcripts. RNAseq should permit better understanding of the molecular mechanisms regulating platelet physiology and identify novel genetic variants that contribute to disorders of thrombosis and hemostasis. Disclosures: No relevant conflicts of interest to declare.

Description: Expression of fetal globin is silenced normally in adult life; however, determinants linked and/or unlinked to the globin-gene clusters could modify Hb F expression so it persists into adults. Increased expression in adults offers hope as a cure for sickle cell disease (SCD) and beta thalassemia, since formation of FS hybrids in SCD inhibits deoxy Hb S polymerization while increased fetal chain expression compensates partially for decreased adult beta-globin chains in beta thalassemia. Characterization and controlled manipulation of high Hb F determinants is critical to decreasing clinical severity of these life-threatening genetic diseases, which result in high morbidity and mortality worldwide. We report on analysis of a unique beta-thalassemia cohort from Sardinia who present with either a mild, non-transfusion-dependent (NTD) form expressing high Hb F, or with a severe, transfusion-dependent (TD) form expressing low Hb F. Both groups are homozygous for the beta39 chain-termination mutation and lack adult beta globin. Genome-wide DNA arrays were run on 42 TD and 33 NTD patients using the Affymetrix 500K (500,568 SNPs in a total of 23 individuals) or 6.0 (909,622 SNPs in a total of 52 individuals) SNP chip platforms. The average sample call rates were 96.3% for the 6.0 chip and 94.3% for the 500K chip. Data from the two chips were merged and a total of 482,243 SNPs that were present in both chips were considered for genetic association analysis using a case/control design (NTD versus TD). Quality control filters removed 42,166 SNPs (8.7%) with genotype call rates 〈 90%. An additional 54,683 SNPs (12.4%) were removed because they either failed a test of Hardy-Weinberg equilibrium at a p〈 0.0001 or had a minor allele frequency (MAF) of 〈 0.01. The genotype call rate for the remaining 385,394 SNPs was 97.3%. Preliminary analysis of difference in allele frequency distributions in the two groups of patients did not reveal any signal of association that remained statistically significant when corrected for multiple tests. The smallest p-value observed was 2x10-6 for SNP rs8028098 on chromosome 15. However, several SNPs in two candidate regions on chromosomes 2p16 and 6q23 were nominally significant (p

Description: Next generation sequencing of RNA (RNA-seq) is an emerging technology that has so far been used successfully to profile the transcriptomes of several cell types and cell states. For the platelet transcriptome, RNA-seq descriptions exist for only a few subjects. Additionally, there have been no studies of the same individual’s transcriptome using two different technologies. As such, it has been unclear how well platelet transcriptomes correlate among different donors or across different RNA platforms, and what the transcriptomes’ relationship is with the platelet proteome. We generated RNA-seq profiles of the long RNA transcriptomes from the platelets of 10 healthy young males (5 white and 5 black). In addition to RNA-seq, we profiled the platelet messenger RNAs of the same 10 individuals using the Affymetrix GeneChip System. We observed that the abundance of platelet mRNA transcripts was highly correlated across the 10 individuals, a finding that was independent of race and of the employed technology. Additionally, our RNA-seq data showed that these high inter-individual correlations extend beyond mRNAs to several categories of non-coding RNAs. However, there was a notable exception: the category of pseudogenes exhibited a clear difference in expression by race. Comparison of our mRNA signatures with the only publicly available quantitative platelet proteome data showed that most (87.5%) identified platelet proteins had a detectable corresponding mRNA. Interestingly, there was also a high number of mRNAs that were present in the transcriptomes of all 10 individuals but had no representation in the proteome. Spearman correlation of the relative abundances for those platelet genes that were represented by both an mRNA and a protein, revealed an unexpectedly weak correlation between the transcriptome and the proteome. Further analysis of the overlapping and non-overlapping platelet mRNAs and proteins identified groups of genes with very distinct characteristics. Gene Ontology analysis of the respective gene identifiers revealed that the gene groups corresponded to distinct cellular processes, an interesting finding that provides novel insights for platelet biology. The very high inter-individual correlations of the transcriptome signatures across 10 different subjects representing two races together with the results of our analyses indicate that it is feasible to assemble a platelet mRNA-ome that can serve as a reference for future platelet transcriptomic studies of human health and disease. Disclosures: No relevant conflicts of interest to declare.