Peter M. Clark

Analysis of 13 cell types reveals evidence for the expression of numerous novel primate- And tissue-specific microRNAs

Significance MicroRNAs (miRNAs) are small ∼22-nt RNAs that are important regulators of posttranscriptional gene expression. Since their initial discovery, they have been shown to be involved in many cellular processes, and their misexpression is associated with disease etiology. Currently, nearly 2,800 human miRNAs are annotated in public repositories. A key question in miRNA research is how many miRNAs are harbored by the human genome. To answer this question, we examined 1,323 short RNA sequence samples and identified 3,707 novel miRNAs, many of which are human-specific and tissue-specific. Our findings suggest that the human genome expresses a greater number of miRNAs than has previously been appreciated and that many more miRNA molecules may play key roles in disease etiology. Two decades after the discovery of the first animal microRNA (miRNA), the number of miRNAs in animal genomes remains a vexing question. Here, we report findings from analyzing 1,323 short RNA sequencing samples (RNA-seq) from 13 different human tissue types. Using stringent thresholding criteria, we identified 3,707 statistically significant novel mature miRNAs at a false discovery rate of ≤0.05 arising from 3,494 novel precursors; 91.5% of these novel miRNAs were identified independently in 10 or more of the processed samples. Analysis of these novel miRNAs revealed tissue-specific dependencies and a commensurate low Jaccard similarity index in intertissue comparisons. Of these novel miRNAs, 1,657 (45%) were identified in 43 datasets that were generated by cross-linking followed by Argonaute immunoprecipitation and sequencing (Ago CLIP-seq) and represented 3 of the 13 tissues, indicating that these miRNAs are active in the RNA interference pathway. Moreover, experimental investigation through stem-loop PCR of a random collection of newly discovered miRNAs in 12 cell lines representing 5 tissues confirmed their presence and tissue dependence. Among the newly identified miRNAs are many novel miRNA clusters, new members of known miRNA clusters, previously unreported products from uncharacterized arms of miRNA precursors, and previously unrecognized paralogues of functionally important miRNA families (e.g., miR-15/107). Examination of the sequence conservation across vertebrate and invertebrate organisms showed 56.7% of the newly discovered miRNAs to be human-specific whereas the majority (94.4%) are primate lineage-specific. Our findings suggest that the repertoire of human miRNAs is far more extensive than currently represented by public repositories and that there is a significant number of lineage- and/or tissue-specific miRNAs that are uncharacterized.

Argonaute CLIP-Seq reveals miRNA targetome diversity across tissue types

To date, analyses of individual targets have provided evidence of a miRNA targetome that extends beyond the boundaries of messenger RNAs (mRNAs) and can involve non-Watson-Crick base pairing in the miRNA seed region. Here we report our findings from analyzing 34 Argonaute HITS-CLIP datasets from several human and mouse cell types. Investigation of the architectural (i.e. bulge vs. contiguous pairs) and sequence (Watson-Crick vs. G:U pairs) preferences for human and mouse miRNAs revealed that many heteroduplexes are “non-canonical” i.e. their seed region comprises G:U and bulge combinations. The genomic distribution of miRNA targets differed distinctly across cell types but remained congruent across biological replicates of the same cell type. For some cell types intergenic and intronic targets were more frequent whereas in other cell types mRNA targets prevailed. The findings suggest an expanded model of miRNA targeting that is more frequent than the standard model currently in use. Lastly, our analyses of data from different cell types and laboratories revealed consistent Ago-loaded miRNA profiles across replicates whereas, unexpectedly, the Ago-loaded targets exhibited a much more dynamic behavior across biological replicates.

Molecular dynamics simulations of Ago silencing complexes reveal a large repertoire of admissible ‘seed-less’ targets

To better understand the recognition mechanism of RISC and the repertoire of guide-target interactions we introduced G:U wobbles and mismatches at various positions of the microRNA (miRNA) ‘seed’ region and performed all-atom molecular dynamics simulations of the resulting Ago-miRNA:mRNA ternary complexes. Our simulations reveal that many modifications, including combinations of multiple G:U wobbles and mismatches in the seed region, are admissible and result in only minor structural fluctuations that do not affect overall complex stability. These results are further supported by analyses of HITS-CLIP data. Lastly, introduction of disruptive mutations revealed a bending motion of the PAZ domain along the L1/L2 ‘hinge’ and a subsequent opening of the nucleic-acid-binding channel. Our findings suggest that the spectrum of a miRNAs admissible targets is different from what is currently anticipated by the canonical seed-model. Moreover, they provide a likely explanation for the previously reported sequence-dependent regulation of unintended targeting by siRNAs.

Clinical Exome Performance for Reporting Secondary Genetic Findings

BACKGROUND Reporting clinically actionable incidental genetic findings in the course of clinical exome testing is recommended by the American College of Medical Genetics and Genomics (ACMG). However, the performance of clinical exome methods for reporting small subsets of genes has not been previously reported. METHODS In this study, 57 exome data sets performed as clinical (n = 12) or research (n = 45) tests were retrospectively analyzed. Exome sequencing data was examined for adequacy in the detection of potentially pathogenic variant locations in the 56 genes described in the ACMG incidental findings recommendation. All exons of the 56 genes were examined for adequacy of sequencing coverage. In addition, nucleotide positions annotated in HGMD (Human Gene Mutation Database) were examined. RESULTS The 56 ACMG genes have 18 336 nucleotide variants annotated in HGMD. None of the 57 exome data sets possessed a HGMD variant. The clinical exome test had inadequate coverage for >50% of HGMD variant locations in 7 genes. Six exons from 6 different genes had consistent failure across all 3 test methods; these exons had high GC content (76%-84%). CONCLUSIONS The use of clinical exome sequencing for the interpretation and reporting of subsets of genes requires recognition of the substantial possibility of inadequate depth and breadth of sequencing coverage at clinically relevant locations. Inadequate depth of coverage may contribute to false-negative clinical exome results.

The dichotomy between disease phenotype databases and the implications for understanding complex diseases involving the major histocompatibility complex

Many genes related to innate and adaptive immunity reside within the major histocompatibility complex (MHC) and have been associated with a multitude of complex, immune‐related disorders. Despite years of genetic study, this region has seen few causative determinants discovered for immune‐mediated diseases. Reported associations have been curated in various databases including the Genetic Association Database, NCBI database of clinically relevant variants (ClinVar) and the Human Gene Mutation Database and together capture genetic associations and annotated pathogenic loci within the MHC and across the genome for a variety of complex, immune‐mediated diseases. A review of these three distinct databases reveals disparate annotations between associated genes and pathogenic loci, alluding to the polygenic, multifactorial nature of immune‐mediated diseases and the pleiotropic character of genes within the MHC. The technical limitations and inherent biases imposed by current approaches and technologies in studying the MHC create a strong case for the need to perform targeted deep sequencing of the MHC and other immunologically relevant loci in order to fully elucidate and study the causative elements of complex immune‐mediated diseases.

N-BLR, a primate-specific non-coding transcript leads to colorectal cancer invasion and migration.

BackgroundNon-coding RNAs have been drawing increasing attention in recent years as functional data suggest that they play important roles in key cellular processes. N-BLR is a primate-specific long non-coding RNA that modulates the epithelial-to-mesenchymal transition, facilitates cell migration, and increases colorectal cancer invasion.ResultsWe performed multivariate analyses of data from two independent cohorts of colorectal cancer patients and show that the abundance of N-BLR is associated with tumor stage, invasion potential, and overall patient survival. Through in vitro and in vivo experiments we found that N-BLR facilitates migration primarily via crosstalk with E-cadherin and ZEB1. We showed that this crosstalk is mediated by a pyknon, a short ~20 nucleotide-long DNA motif contained in the N-BLR transcript and is targeted by members of the miR-200 family. In light of these findings, we used a microarray to investigate the expression patterns of other pyknon-containing genomic loci. We found multiple such loci that are differentially transcribed between healthy and diseased tissues in colorectal cancer and chronic lymphocytic leukemia. Moreover, we identified several new loci whose expression correlates with the colorectal cancer patients’ overall survival.ConclusionsThe primate-specific N-BLR is a novel molecular contributor to the complex mechanisms that underlie metastasis in colorectal cancer and a potential novel biomarker for this disease. The presence of a functional pyknon within N-BLR and the related finding that many more pyknon-containing genomic loci in the human genome exhibit tissue-specific and disease-specific expression suggests the possibility of an alternative class of biomarkers and therapeutic targets that are primate-specific.

An Expanded Role for HLA Genes: HLA-B Encodes a microRNA that Regulates IgA and Other Immune Response Transcripts

We describe a novel functional role for the HLA-B locus mediated by its intron-encoded microRNA (miRNA), miR-6891-5p. We show that in vitro inhibition of miR-6891-5p impacts the expression of nearly 200 transcripts within the B-lymphoblastoid cell line (B-LCL) COX, affecting a large number of metabolic pathways, including various immune response networks. The top affected transcripts following miR-6891-5p inhibition are those encoding the heavy chain of IgA. We identified a conserved miR-6891-5p target site on the 3′UTR of both immunoglobulin heavy chain alpha 1 and 2 (IGHA1 and IGHA2) transcripts and demonstrated that this miRNA modulates the expression of IGHA1 and IGHA2. B-LCLs from IgA-deficient patients expressed significantly elevated levels of miR-6891-5p when compared with unaffected family members. Upon inhibition of miR-6891-5p, IgA mRNA expression levels were increased, and IgA secretion was restored in the B-LCL of an IgA-deficient patient. These findings indicate that miR-6891-5p regulates IGHA1 and IGHA2 gene expression at the posttranscriptional level and suggest that increase in miR-6891-5p levels may contribute to the etiology of selective IgA deficiency.

Novel and Haplotype Specific MicroRNAs Encoded by the Major Histocompatibility Complex

The MHC is recognized for its importance in human health and disease. However, many disease-associated variants throughout the region remain of unknown significance, residing predominantly within non-coding regions of the MHC. The characterization of non-coding RNA transcripts throughout the MHC is thus central to understanding the genetic contribution of these variants. Therefore, we characterize novel miRNA transcripts throughout the MHC by performing deep RNA sequencing of two B lymphoblastoid cell lines with completely characterized MHC haplotypes. Our analysis identifies 89 novel miRNA transcripts, 48 of which undergo Dicer-dependent biogenesis and are loaded onto the Argonaute silencing complex. Several of the identified mature miRNA and pre-miRNA transcripts are unique to specific MHC haplotypes and overlap common SNPs. Furthermore, 43 of the 89 identified novel miRNA transcripts lie within linkage disequilibrium blocks that contain a disease-associated SNP. These disease associated SNPs are associated with 65 unique disease phenotypes, suggesting that these transcripts may play a role in the etiology of numerous diseases associated with the MHC. Additional in silico analysis reveals the potential for thousands of putative pre-miRNA encoding loci within the MHC that may be expressed by different cell types and at different developmental stages.

Post-transcriptional Regulation of BRCA2 through Interactions with miR-19a and miR-19b

Breast cancer type 2, early onset susceptibility gene (BRCA2) is a major component of the homologous recombination DNA repair pathway. It acts as a tumor suppressor whose function is often lost in cancers. Patients with specific mutations in the BRCA2 gene often display discrete clinical, histopathological, and molecular features. However, a subset of sporadic cancers has wild type BRCA2 and display defects in the homology-directed repair pathway, which is the hallmark of ‘BRCAness.’ The mechanisms by which BRCAness arises are not well understood but post-transcriptional regulation of BRCA2 gene expression by microRNAs (miRNAs) may contribute to this phenotype. Here, we examine the post-transcriptional effects that some members of the six-miRNA cluster known as the miR-17/92 cluster have on the abundance of BRCA2’s messenger RNA (mRNA) and protein. We discuss two interactions involving the miR-19a and miR-19b members of the cluster and the 3′UTR of BRCA2’s mRNA. We investigated these miRNA:mRNA interactions in 15 cell lines derived from pancreatic, breast, colon, and kidney tissue. We show that over-expression of these two miRNAs results in a concomitant decrease of BRCA2’s mRNA and protein expression in a subset of the tested cell lines. Additionally, using luciferase reporter assays we identified direct interactions between miR-19a/miR-19b and a miRNA response element (MRE) in BRCA2’s 3′UTR. Our results suggest that BRCA2 is subject to a complex post-transcriptional regulatory program that has specific dependencies on the genetic and phenotypic background of cell types.

Clinical Genomics: When Whole Genome Sequencing Is like a Whole-body CT Scan

Although we have witnessed major improvements in genomic testing in the past 5 years, multiple challenges need to be solved before clinical genomic testing becomes routine (1). The clinical implementation of genomic testing parallels the challenges we have faced with other transformative technologies. For the past decade, the medical community has been discussing the impact on healthcare and society of direct-to-consumer marketed whole-body computed tomography (CT)8 scanning (2). The CT scan is a technology that uses X-rays to produce cross-sectional images of the human body. There are clinical scenarios in which the CT scan is invaluable: disease diagnosis, evaluation after trauma, and monitoring response to oncologic therapy. However, CT scan is also being marketed as a preventive diagnostic for healthy individuals (3). Genomics can parallel CT scanning in scenarios of ( a ) first line diagnostic testing, ( b ) last ditch diagnosis, and ( c ) inappropriate clinical use. Genomic testing implies the examination of the nucleic acid sequence of multiple, if not all, known genes. Broadly, this includes microarray tests that survey changes in single nucleotide polymorphisms (SNPs) and copy-number variation across the entire genome, sequencing the exome or whole genome by next-generation sequencing, and sequencing large panels of clinically significant genes. Genomic testing is currently moving from academic research practice to commercialization and direct-to-consumer marketing. Although genomic testing is not yet routine, DNA testing targeted to single genes, viruses, or bacteria is commonplace and performed in many clinical laboratories. The use of genetic information has evolved over the past decade from targeted single-gene tests to broader genomic tests that encompass thousands of genes. Numerous ethical, economic, and …