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OR19 MIRNA mediated post transcriptional regulation of HLA transcripts
Abstracts / Human Immunology 78 (2017) 1–50
Wednesday, September 13, 2017 2:00 PM–3:30 PM Abstract Session 3: Genetic and Epigenetics of HLA
OR19
MIRNA MEDIATED POST TRANSCRIPTIONAL REGULATION OF HLA TRANSCRIPTS. Peter M. Clark a, Dimitri S. Monos a,b. aChildren’s Hospital of Philadelphia, Philadelphia, PA, United States; b University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, United States. Aim: MicroRNAs (miRNAs) regulate the post-transcriptional expression of targeted mRNA transcripts, however little is known about the influence of HLA polymorphisms on altered miRNA target specificity & allele specific post-transcriptional regulation. We utilize a custom developed bioinformatics platform, utilizing both miRNA transcript expression data and computational miRNA target site prediction to study the influence of HLA polymorphisms on altered miRNA target specificity and differential post-transcriptional regulation of HLA alleles within B and T lymphocytes. Methods: Computational predicted miRNA target sites for all known miRNA transcripts (miRBase release 0 21) within the 3 UTR of every known full-length HLA allele for HLA-A, HLA-B, HLA-C, HLA-DQA1, HLADQB1, HLA-DPB1 and HLA-DRB1 (IMGT version 3.27) were determined using miRanda with a minimum heteroduplex energy of 20 Kcal/mol. Tissue specific miRNA expression profiles were subsequently used to filter miRNA-target interactions in order to determine the tissue specific differential miRNA target specificity HLA alleles. Results: Our data demonstrate that endogenously expressed miRNA within both B and T lymphocytes dis0 play differential target specificity across HLA alleles, driven by polymorphisms present within the 3 UTR of HLA alleles. Interestingly, we find differential targeting of endogenously expressed miRNA within polymorphic regions of HLA-DRB1 and HLA-G that have been associated with altered HLA expression and implicated in GVHD, preeclampsia, oncogenesis and allograft rejection. We also observe that several endogenously 0 expressed miRNAs target the 3 UTR of numerous HLA genes, acting as a potential master, universal regulator of HLA expression with B and T lymphocytes. Conclusions: The developed bioinformatics platform identifies target sites of all known miRNA on the 0 3 UTR of every annotated full-length HLA allele. This atlas is further refined using tissue specific miRNA expression patterns to generate tissue specific target maps, revealing altered miRNA target specificity both across tissue types and across HLA alleles, driven by polymorphisms across HLA alleles. This powerful platform may be used to test novel hypothesis related to altered HLA expression as it relates to a variety of disease states.
OR20
DE NOVO GENERATION OF MHC HAPLOTYPES USING SINGLE MOLECULE SEQUENCING. Peter M. Clark a, Timothy Mosbruger a, Dimitri S. Monos a,b. aChildren’s Hospital of Philadelphia, Philadelphia, PA, United States; bUniversity of Pennsylvania Perelman School of Medicine, Philadelphia, PA, United States.. Aim: Utilizing a previously developed long-fragment DNA capture methodology, PacBio single molecule real-time (SMRT) sequencing and a custom de novo assembly analysis pipeline, we aim to resolve the longrange, fully phased MHC haplotypes for a single heterozygous individual. Methods: Long fragment DNA enrichment for the MHC was performed on fresh blood obtained from a heterozygous individual using our previously described method, RSE. Captured DNA was sequenced on the PacBio RSII sequencing platform. Raw reads were selected to include only those reads that map to annotated MHC haplotype sequences (n = 8). De novo assembly was performed using the CANU pipeline with tuned algorithm parameters to optimize continuity and accuracy of haplotigs (phased contigs) as compared to phased SNP genotyping data. Results: Following de novo assembly, PacBio reads were assembled into 27 contiguous fragments (contigs) covering 97% of the 4Mbp MHC (as aligned to the COX MHC haplotype). Phased contigs (haplotigs) were found to agree with phased SNP genotyping data at 78% of interrogated positions (n = 4,779 probes) indicating that the majority of haplotigs are properly phased. Phased SNP genotyping data was also used to phase across breaks in the assembly, resulting in the generation of two distinct MHC haplotypes for the sequenced sample. Conclusions: Our results demonstrate the capacity to generate de novo assembled, fully phased MHC haplotypes from PacBio single molecule sequencing reads. Although phasing across contig breaks caused by coverage gaps remains a challenge, we hope to resolve this issue using BioNano genomics optical maps, so as not to rely on phased SNP genotyping data. In essence, this method has the capacity to resolve full-length MHC haplotype sequences without the need for any reference genome or familial trio analysis.
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Wednesday, September 13, 2017 2:00 PM–3:30 PM Abstract Session 3: Genetic and Epigenetics of HLA
OR19
MIRNA MEDIATED POST TRANSCRIPTIONAL REGULATION OF HLA TRANSCRIPTS. Peter M. Clark a, Dimitri S. Monos a,b. aChildren’s Hospital of Philadelphia, Philadelphia, PA, United States; b University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, United States. Aim: MicroRNAs (miRNAs) regulate the post-transcriptional expression of targeted mRNA transcripts, however little is known about the influence of HLA polymorphisms on altered miRNA target specificity & allele specific post-transcriptional regulation. We utilize a custom developed bioinformatics platform, utilizing both miRNA transcript expression data and computational miRNA target site prediction to study the influence of HLA polymorphisms on altered miRNA target specificity and differential post-transcriptional regulation of HLA alleles within B and T lymphocytes. Methods: Computational predicted miRNA target sites for all known miRNA transcripts (miRBase release 0 21) within the 3 UTR of every known full-length HLA allele for HLA-A, HLA-B, HLA-C, HLA-DQA1, HLADQB1, HLA-DPB1 and HLA-DRB1 (IMGT version 3.27) were determined using miRanda with a minimum heteroduplex energy of 20 Kcal/mol. Tissue specific miRNA expression profiles were subsequently used to filter miRNA-target interactions in order to determine the tissue specific differential miRNA target specificity HLA alleles. Results: Our data demonstrate that endogenously expressed miRNA within both B and T lymphocytes dis0 play differential target specificity across HLA alleles, driven by polymorphisms present within the 3 UTR of HLA alleles. Interestingly, we find differential targeting of endogenously expressed miRNA within polymorphic regions of HLA-DRB1 and HLA-G that have been associated with altered HLA expression and implicated in GVHD, preeclampsia, oncogenesis and allograft rejection. We also observe that several endogenously 0 expressed miRNAs target the 3 UTR of numerous HLA genes, acting as a potential master, universal regulator of HLA expression with B and T lymphocytes. Conclusions: The developed bioinformatics platform identifies target sites of all known miRNA on the 0 3 UTR of every annotated full-length HLA allele. This atlas is further refined using tissue specific miRNA expression patterns to generate tissue specific target maps, revealing altered miRNA target specificity both across tissue types and across HLA alleles, driven by polymorphisms across HLA alleles. This powerful platform may be used to test novel hypothesis related to altered HLA expression as it relates to a variety of disease states.
OR20
DE NOVO GENERATION OF MHC HAPLOTYPES USING SINGLE MOLECULE SEQUENCING. Peter M. Clark a, Timothy Mosbruger a, Dimitri S. Monos a,b. aChildren’s Hospital of Philadelphia, Philadelphia, PA, United States; bUniversity of Pennsylvania Perelman School of Medicine, Philadelphia, PA, United States.. Aim: Utilizing a previously developed long-fragment DNA capture methodology, PacBio single molecule real-time (SMRT) sequencing and a custom de novo assembly analysis pipeline, we aim to resolve the longrange, fully phased MHC haplotypes for a single heterozygous individual. Methods: Long fragment DNA enrichment for the MHC was performed on fresh blood obtained from a heterozygous individual using our previously described method, RSE. Captured DNA was sequenced on the PacBio RSII sequencing platform. Raw reads were selected to include only those reads that map to annotated MHC haplotype sequences (n = 8). De novo assembly was performed using the CANU pipeline with tuned algorithm parameters to optimize continuity and accuracy of haplotigs (phased contigs) as compared to phased SNP genotyping data. Results: Following de novo assembly, PacBio reads were assembled into 27 contiguous fragments (contigs) covering 97% of the 4Mbp MHC (as aligned to the COX MHC haplotype). Phased contigs (haplotigs) were found to agree with phased SNP genotyping data at 78% of interrogated positions (n = 4,779 probes) indicating that the majority of haplotigs are properly phased. Phased SNP genotyping data was also used to phase across breaks in the assembly, resulting in the generation of two distinct MHC haplotypes for the sequenced sample. Conclusions: Our results demonstrate the capacity to generate de novo assembled, fully phased MHC haplotypes from PacBio single molecule sequencing reads. Although phasing across contig breaks caused by coverage gaps remains a challenge, we hope to resolve this issue using BioNano genomics optical maps, so as not to rely on phased SNP genotyping data. In essence, this method has the capacity to resolve full-length MHC haplotype sequences without the need for any reference genome or familial trio analysis.
21