37 research outputs found
Assessing the utilization of high-resolution 2-field HLA typing in solid organ transplantation.
HLA typing in solid organ transplantation (SOT) is necessary for determining HLA-matching status between donor-recipient pairs and assessing patients\u27 anti-HLA antibody profiles. Histocompatibility has traditionally been evaluated based on serologically defined HLA antigens. The evolution of HLA typing and antibody identification technologies, however, has revealed many limitations with using serologic equivalents for assessing compatibility in SOT. The significant improvements to HLA typing introduced by next-generation sequencing (NGS) require an assessment of the impact of this technology on SOT. We have assessed the role of high-resolution 2-field HLA typing (HR-2F) in SOT by retrospectively evaluating NGS-typed pre- and post-SOT cases. HR-2F typing was highly instructive or necessary in 41% (156/385) of the cases. Several pre- and posttransplant scenarios were identified as being better served by HR-2F typing. Five different categories are presented with specific case examples. The experience of another center (Temple University Hospital) is also included, whereby 21% of the cases required HR-2F typing by Sanger sequencing, as supported by other legacy methods, to properly address posttransplant anti-HLA antibody issues
The impact of patterns in linkage disequilibrium and sequencing quality on the imprint of balancing selection
Regions under balancing selection are characterized by dense polymorphisms and multiple persistent haplotypes, along with other sequence complexities. Successful identification of these patterns depends on both the statistical approach and the quality of sequencing. To address this challenge, at first, a new statistical method called LD-ABF was developed, employing efficient Bayesian techniques to effectively test for balancing selection. LD-ABF demonstrated the most robust detection of selection in a variety of simulation scenarios, compared against a range of existing tests/tools (Tajima\u27s D, HKA, Dng, BetaScan, and BalLerMix). Furthermore, the impact of the quality of sequencing on detection of balancing selection was explored, as well, using: (i) SNP genotyping and exome data, (ii) targeted high-resolution HLA genotyping (IHIW), and (iii) whole-genome long-read sequencing data (Pangenome). In the analysis of SNP genotyping and exome data, we identified known targets and 38 new selection signatures in genes not previously linked to balancing selection. To further investigate the impact of sequencing quality on detection of balancing selection, a detailed investigation of the MHC was performed with high-resolution HLA typing data. Higher quality sequencing revealed the HLA-DQ genes consistently demonstrated strong selection signatures otherwise not observed from the sparser SNP array and exome data. The HLA-DQ selection signature was also replicated in the Pangenome samples using considerably less samples but, with high-quality long-read sequence data. The improved statistical method, coupled with higher quality sequencing, leads to more consistent identification of selection and enhanced localization of variants under selection, particularly in complex regions
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Active BRAF-V600E is the key player in generation of a sessile serrated polyp-specific DNA methylation profile
Background: Sessile serrated polyps (SSPs) have emerged as important precursors for a large number of sporadic colorectal cancers. They are difficult to detect during colonoscopy due to their flat shape and the excessive amounts of secreted mucin that cover the polyps. The underlying genetic and epigenetic basis for the emergence of SSPs is largely unknown with existing genetic studies confined to a limited number of oncogenes and tumor suppressors. A full characterization of the genetic and epigenetic landscape of SSPs would provide insight into their origin and potentially offer new biomarkers useful for detection of SSPs in stool samples. Methods: We used a combination of genome-wide mutation detection, exome sequencing and DNA methylation profiling (via methyl-array and whole-genome bisulfite sequencing) to analyze multiple samples of sessile serrated polyps and compared these to familial adenomatous polyps. Results: Our analysis revealed BRAF-V600E as the sole recurring somatic mutation in SSPs with no additional major genetic mutations detected. The occurrence of BRAF-V600E was coincident with a unique DNA methylation pattern revealing a set of DNA methylation markers showing significant (~3 to 30 fold) increase in their methylation levels, exclusively in SSP samples. These methylation patterns effectively distinguished sessile serrated polys from adenomatous polyps and did so more effectively than parallel gene expression profiles. Conclusions: This study provides an important example of a single oncogenic mutation leading to reproducible global DNA methylation changes. These methylated markers are specific to SSPs and could be of important clinical relevance for the early diagnosis of SSPs using non-invasive approaches such as fecal DNA testing
OR41 Novel HLA alleles identified by next generation sequencing using blood-derived DNA of patients with hematologic disease may influence the search process for the appropriate donor in hematopoietic stem cell transplantation
Mannose Binding Lectin Genotypes Influence Recovery from Hepatitis B Virus Infection
Mannose binding lectin (MBL) is a central component of the innate immune response and thus may be important for determining hepatitis B virus (HBV) persistence. Since single-nucleotide polymorphisms (SNPs) in the gene encoding MBL (mbl2) alter the level of functional MBL, we hypothesized that mbl2 genotypes are a determinant of HBV persistence or recovery from viral infection. We tested this hypothesis by using a nested case control design with 189 persons with HBV persistence matched to 338 individuals who had naturally recovered from HBV infection. We determined genotypes of two promoter and three exon 1 SNPs in mbl2 and grouped these genotypes according to the amount of functional MBL production. We found that the promoter SNP −221C, which leads to deficient MBL production, was more common in those subjects with viral persistence (odds ratio [OR], 1.38; 95% confidence interval [CI], 1.01 to 1.89; P = 0.04). Those subjects homozygous for the combination of promoter and exon 1 genotypes associated with the highest amount of functional MBL had significantly increased odds of recovery from infection (OR, 0.55; 95% CI, 0.37 to 0.84; P = 0.005). Conversely, those homozygous for the combination of promoter and exon 1 genotypes which produce the lowest amount of functional MBL were more likely to have viral persistence (OR, 1.76; 95% CI, 1.02 to 3.01; P = 0.04). These data are consistent with the hypothesis that functional MBL plays a central role in the pathogenesis of acute hepatitis B
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Genome-Wide CRISPR-Cas9 Screen Identifies MicroRNAs That Regulate Myeloid Leukemia Cell Growth.
Mammalian microRNA expression is dysregulated in human cancer. However, the functional relevance of many microRNAs in the context of tumor biology remains unclear. Using CRISPR-Cas9 technology, we performed a global loss-of-function screen to simultaneously test the functions of individual microRNAs and protein-coding genes during the growth of a myeloid leukemia cell line. This approach identified evolutionarily conserved human microRNAs that suppress or promote cell growth, revealing that microRNAs are extensively integrated into the molecular networks that control tumor cell physiology. miR-155 was identified as a top microRNA candidate promoting cellular fitness, which we confirmed with two distinct miR-155-targeting CRISPR-Cas9 lentiviral constructs. Further, we performed anti-correlation functional profiling to predict relevant microRNA-tumor suppressor gene or microRNA-oncogene interactions in these cells. This analysis identified miR-150 targeting of p53, a connection that was experimentally validated. Taken together, our study describes a powerful genetic approach by which the function of individual microRNAs can be assessed on a global level, and its use will rapidly advance our understanding of how microRNAs contribute to human disease
An Analysis of Tumor Necrosis Factor α Gene Polymorphisms and Haplotypes with Natural Clearance of Hepatitis C Virus Infection
The cytokine tumor necrosis factor alpha (TNF-α) is important in generating an immune response against a hepatitis C virus (HCV) infection. The functions of TNF-α may be altered by single-nucleotide polymorphisms (SNPs) in its gene, TNF. We hypothesized that SNPs in TNF may be important in determining the outcome of an HCV infection. To test this hypothesis, we typed nine TNF SNPs in a cohort of individuals with well-defined HCV outcomes. Three of these SNPs were typed in a second cohort. Data were analyzed using logistic regression stratifying by ethnicity, since rates of HCV clearance differ in black subjects versus white subjects. The SNP -863A was associated with viral clearance in black subjects (odds ratios (OR) 0.52, 95% confidence interval (CI) 0.29–0.93). Furthermore, the common wild-type haplotype -863C/-308G was associated with viral persistence in black subjects (OR 1.91, 95% CI 1.24–2.95). These findings were independent of linkage with human leukocyte antigen (HLA) alleles. Further study of this polymorphism and haplotype is needed to understand these associations and the role of TNF-α in determining outcomes of HCV infection
Cytotoxic T-Lymphocyte Antigen 4 Gene and Recovery from Hepatitis B Virus Infection
Cytotoxic T-lymphocyte antigen 4 (CTLA-4) is an inhibitory T-cell receptor expressed by activated and regulatory T cells. We hypothesized that single-nucleotide polymorphisms (SNPs) in the gene encoding CTLA-4 may affect the vigor of the T-cell response to hepatitis B virus (HBV) infection, thus influencing viral persistence. To test this hypothesis, we genotyped six CTLA4 SNPs, from which all frequent haplotypes can be determined, using a large, matched panel of subjects with known HBV outcomes. Haplotypes with these SNPs were constructed for each subject using PHASE software. The haplotype distribution differed between those with viral persistence and those with clearance. Two haplotypes were associated with clearance of HBV infection, which was most likely due to associations with the SNPs - 1722C (odds ratio [OR] = 0.60, P = 0.06) and +49G (OR = 0.73, P = 0.02). The wild-type haplotype, which contains an SNP leading to a decreased T-cell response (+6230A), was associated with viral persistence (OR = 1.32, P = 0.04). These data suggest that CTLA4 influences recovery from HBV infection, which is consistent with the emerging role of T regulatory cells in the pathogenesis of disease
Genome-Wide CRISPR-Cas9 Screen Identifies MicroRNAs That Regulate Myeloid Leukemia Cell Growth
<div><p>Mammalian microRNA expression is dysregulated in human cancer. However, the functional relevance of many microRNAs in the context of tumor biology remains unclear. Using CRISPR-Cas9 technology, we performed a global loss-of-function screen to simultaneously test the functions of individual microRNAs and protein-coding genes during the growth of a myeloid leukemia cell line. This approach identified evolutionarily conserved human microRNAs that suppress or promote cell growth, revealing that microRNAs are extensively integrated into the molecular networks that control tumor cell physiology. miR-155 was identified as a top microRNA candidate promoting cellular fitness, which we confirmed with two distinct miR-155-targeting CRISPR-Cas9 lentiviral constructs. Further, we performed anti-correlation functional profiling to predict relevant microRNA-tumor suppressor gene or microRNA-oncogene interactions in these cells. This analysis identified miR-150 targeting of p53, a connection that was experimentally validated. Taken together, our study describes a powerful genetic approach by which the function of individual microRNAs can be assessed on a global level, and its use will rapidly advance our understanding of how microRNAs contribute to human disease.</p></div
Anti-correlation functional profiling identifies relevant miRNA-target interactions, including miR-150 repression of p53, that regulate MV4-11 cell line growth.
<p>(A) Heat map indicating representative oncogenes whose loss leads to decreased cell growth according to Log2 Fold Change values from lentiCRISPRv2 library screen (first column), and functionally anti-correlated miRNAs that are predicted to target each oncogene. Grey boxes indicate that the miRNA is not predicted to bind the 3’UTR of the oncogene (NT = Not targeted). (B) Heat map indicating representative TSGs whose loss lead to increased cell growth according to our Log2 Fold Change values from lentiCRISPRv2 library screen (first column), and miRNAs predicted to target each TSG whose growth anti-correlated in library. Grey boxes indicates that the miRNA is not predicted to bind the 3’UTR of TSG (NT = Not targeted). (C) Schematic showing miR-150 targeting of the p53 3’UTR. (D) Schematic of the miR-150 hairpin sequence as annotated in miRBase and sgRNA design of the miR-150-targeting lentiCRISPRv2 construct (150-CR1). (E) Expression level of miR-150 in MV4-11 cells infected with EV control or 150-CR1 lentiCRISPRv2 constructs determined by qPCR. Expression normalized to 5s. (F) Western blot of p53 in p53-CR1, 150-CR1, and EV control infected MV4-11 cell lines with actin serving as load control. (G) Competitive growth curve of EV (GFP+), p53-CR1 (GFP+), or 150-CR1 (GFP+) infected MV4-11 cells mixed ~1:1 with WT MV4-11 cells at time point 0. Y-axis = (%GFP+ cells at indicated time point)/(%GFP+ cells initial). (A, B) Only expressed protein-coding genes and microRNAs with p-values <0.05 were analyzed. Data represented as mean +/- SEM. P-values as indicated: *≤0.05, **≤0.01, ***≤0.001, and ns p>0.05. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153689#pone.0153689.s004" target="_blank">S3 Table</a>.</p