Early Insights into Cancer Epigenetics: Gene Promoter Hypermethylation Emerges as a Potential Biomarker for Cancer Detection.

DNA methylation is one of the most intensely studied epigenetic modifications in mammals. In normal cells, it plays an essential role in core biologic processes by assuring the proper regulation of gene expression and stable gene silencing. In cancer cells, genome-wide DNA methylation patterns are altered and often represent an early and fundamental step in neoplastic transformation. The landmark study from Esteller and colleagues, published in Cancer Research in 2001, was the first to reveal high frequency promoter methylation across multiple cancer types. They highlighted that widespread alterations in DNA methylation may be a key characteristic of oncogenesis and proposed aberrant DNA methylation of gene promoters could provide markers for sensitive detection of nearly all cancer types. The authors used a candidate gene approach to show promoter hypermethylation occurred across 12 cancer-associated genes in DNA from over 600 primary tumor samples, representing 15 major tumor types. The profile of promoter hypermethylation differed in every tumor type, suggesting that alterations in DNA methylation are pervasive, but the genes affected may be tumor-specific and impact multiple signaling pathways. Over the past 20 years since this publication, the cancer epigenetics field has exploded to generate thousands of normal and cancer methylome maps and developed sophisticated informatic tools for genome-wide methylome analyses. These methylomes are providing roadmaps for the study of cancer biology and discovery of DNA methylation biomarkers for early detection and monitoring of cancer. See related article by Esteller and colleagues, Cancer Res 2001;61:3225-29.

Batch-effect detection, correction and characterisation in Illumina HumanMethylation450 and MethylationEPIC BeadChip array data.

BACKGROUND: Genomic technologies can be subject to significant batch-effects which are known to reduce experimental power and to potentially create false positive results. The Illumina Infinium Methylation BeadChip is a popular technology choice for epigenome-wide association studies (EWAS), but presently, little is known about the nature of batch-effects on these designs. Given the subtlety of biological phenotypes in many EWAS, control for batch-effects should be a consideration. RESULTS: Using the batch-effect removal approaches in the ComBat and Harman software, we examined two in-house datasets and compared results with three large publicly available datasets, (1214 HumanMethylation450 and 1094 MethylationEPIC BeadChips in total), and find that despite various forms of preprocessing, some batch-effects persist. This residual batch-effect is associated with the day of processing, the individual glass slide and the position of the array on the slide. Consistently across all datasets, 4649 probes required high amounts of correction. To understand the impact of this set to EWAS studies, we explored the literature and found three instances where persistently batch-effect prone probes have been reported in abstracts as key sites of differential methylation. As well as batch-effect susceptible probes, we also discover a set of probes which are erroneously corrected. We provide batch-effect workflows for Infinium Methylation data and provide reference matrices of batch-effect prone and erroneously corrected features across the five datasets spanning regionally diverse populations and three commonly collected biosamples (blood, buccal and saliva). CONCLUSIONS: Batch-effects are ever present, even in high-quality data, and a strategy to deal with them should be part of experimental design, particularly for EWAS. Batch-effect removal tools are useful to reduce technical variance in Infinium Methylation data, but they need to be applied with care and make use of post hoc diagnostic measures.

DNA methylation and body mass index from birth to adolescence: meta-analyses of epigenome-wide association studies.

BACKGROUND: DNA methylation has been shown to be associated with adiposity in adulthood. However, whether similar DNA methylation patterns are associated with childhood and adolescent body mass index (BMI) is largely unknown. More insight into this relationship at younger ages may have implications for future prevention of obesity and its related traits. METHODS: We examined whether DNA methylation in cord blood and whole blood in childhood and adolescence was associated with BMI in the age range from 2 to 18 years using both cross-sectional and longitudinal models. We performed meta-analyses of epigenome-wide association studies including up to 4133 children from 23 studies. We examined the overlap of findings reported in previous studies in children and adults with those in our analyses and calculated enrichment. RESULTS: DNA methylation at three CpGs (cg05937453, cg25212453, and cg10040131), each in a different age range, was associated with BMI at Bonferroni significance, P < 1.06 × 10-7, with a 0.96 standard deviation score (SDS) (standard error (SE) 0.17), 0.32 SDS (SE 0.06), and 0.32 BMI SDS (SE 0.06) higher BMI per 10% increase in methylation, respectively. DNA methylation at nine additional CpGs in the cross-sectional childhood model was associated with BMI at false discovery rate significance. The strength of the associations of DNA methylation at the 187 CpGs previously identified to be associated with adult BMI, increased with advancing age across childhood and adolescence in our analyses. In addition, correlation coefficients between effect estimates for those CpGs in adults and in children and adolescents also increased. Among the top findings for each age range, we observed increasing enrichment for the CpGs that were previously identified in adults (birth Penrichment = 1; childhood Penrichment = 2.00 × 10-4; adolescence Penrichment = 2.10 × 10-7). CONCLUSIONS: There were only minimal associations of DNA methylation with childhood and adolescent BMI. With the advancing age of the participants across childhood and adolescence, we observed increasing overlap with altered DNA methylation loci reported in association with adult BMI. These findings may be compatible with the hypothesis that DNA methylation differences are mostly a consequence rather than a cause of obesity.

DNA methylation enables transposable element-driven genome expansion.

Multicellular eukaryotic genomes show enormous differences in size. A substantial part of this variation is due to the presence of transposable elements (TEs). They contribute significantly to a cell's mass of DNA and have the potential to become involved in host gene control. We argue that the suppression of their activities by methylation of the C-phosphate-G (CpG) dinucleotide in DNA is essential for their long-term accommodation in the host genome and, therefore, to its expansion. An inevitable consequence of cytosine methylation is an increase in C-to-T transition mutations via deamination, which causes CpG loss. Cytosine deamination is often needed for TEs to take on regulatory functions in the host genome. Our study of the whole-genome sequences of 53 organisms showed a positive correlation between the size of a genome and the percentage of TEs it contains, as well as a negative correlation between size and the CpG observed/expected (O/E) ratio in both TEs and the host DNA. TEs are seldom found at promoters and transcription start sites, but they are found more at enhancers, particularly after they have accumulated C-to-T and other mutations. Therefore, the methylation of TE DNA allows for genome expansion and also leads to new opportunities for gene control by TE-based regulatory sites.

Specificity of bispecific T cell receptors and antibodies targeting peptide-HLA.

Tumor-associated peptide-human leukocyte antigen complexes (pHLAs) represent the largest pool of cell surface-expressed cancer-specific epitopes, making them attractive targets for cancer therapies. Soluble bispecific molecules that incorporate an anti-CD3 effector function are being developed to redirect T cells against these targets using 2 different approaches. The first achieves pHLA recognition via affinity-enhanced versions of natural TCRs (e.g., immune-mobilizing monoclonal T cell receptors against cancer [ImmTAC] molecules), whereas the second harnesses an antibody-based format (TCR-mimic antibodies). For both classes of reagent, target specificity is vital, considering the vast universe of potential pHLA molecules that can be presented on healthy cells. Here, we made use of structural, biochemical, and computational approaches to investigate the molecular rules underpinning the reactivity patterns of pHLA-targeting bispecifics. We demonstrate that affinity-enhanced TCRs engage pHLA using a comparatively broad and balanced energetic footprint, with interactions distributed over several HLA and peptide side chains. As ImmTAC molecules, these TCRs also retained a greater degree of pHLA selectivity, with less off-target activity in cellular assays. Conversely, TCR-mimic antibodies tended to exhibit binding modes focused more toward hot spots on the HLA surface and exhibited a greater degree of crossreactivity. Our findings extend our understanding of the basic principles that underpin pHLA selectivity and exemplify a number of molecular approaches that can be used to probe the specificity of pHLA-targeting molecules, aiding the development of future reagents.

Epigenetic aging in newborns: role of maternal diet.

BACKGROUND: Epigenetic aging is associated with higher risk of cardiovascular disease, cancer, and all-cause mortality and may be a mechanistic link between early-life exposures, such as maternal dietary characteristics during pregnancy, and risk of adult disease. OBJECTIVES: We sought to determine the early-life risk factors for newborn epigenetic aging, specifically maternal dietary macronutrient intake, and whether epigenetic aging is associated with cardiovascular health markers in the newborn. METHODS: Epigenetic age acceleration of 169 newborns was measured from saliva using the Horvath age calculator. Maternal diet during pregnancy was assessed using food-frequency questionnaires. RESULTS: Newborns with positive age acceleration were more likely to be female and have greater body fatness. Maternal intakes of saturated fat [6.2 wk epigenetic age acceleration (95% CI: 1.0, 11.3) per 5% of energy; P = 0.02] and monounsaturated fat [12.4 wk (95% CI: 4.2, 20.5) per 5% of energy; P = 0.003] were associated with higher epigenetic age acceleration in the newborn. The strongest association of individual fatty acids were for palmitoleic acid (25.3 wk; 95% CI: 11.4, 39.2; P = 0.0004), oleic acid (2.2 wk; 95% CI: 0.8, 3.6; P = 0.002), and palmitic acid (2.9 wk; 95% CI: 1.0, 4.9; P = 0.004) per 1% of energy intake. Vitamin D supplementation was associated with lower epigenetic age acceleration (-8.1 wk; 95% CI: -14.5, -1.7; P = 0.01). Epigenetic age acceleration was associated with aortic intima-media thickness in preterm infants [1.0 µm (95% CI: 0.2, 1.8) per week of epigenetic age acceleration; P = 0.01], but not among those born at term (P = 0.78). Epigenetic age acceleration was not associated with heart rate variability in either preterm or term born infants (both P > 0.2). CONCLUSIONS: This study provides evidence of maternal dietary characteristics that are associated with epigenetic aging in the offspring. Prospective intervention studies are required to determine whether such associations are causal.

Methylome and transcriptome maps of human visceral and subcutaneous adipocytes reveal key epigenetic differences at developmental genes.

Adipocytes support key metabolic and endocrine functions of adipose tissue. Lipid is stored in two major classes of depots, namely visceral adipose (VA) and subcutaneous adipose (SA) depots. Increased visceral adiposity is associated with adverse health outcomes, whereas the impact of SA tissue is relatively metabolically benign. The precise molecular features associated with the functional differences between the adipose depots are still not well understood. Here, we characterised transcriptomes and methylomes of isolated adipocytes from matched SA and VA tissues of individuals with normal BMI to identify epigenetic differences and their contribution to cell type and depot-specific function. We found that DNA methylomes were notably distinct between different adipocyte depots and were associated with differential gene expression within pathways fundamental to adipocyte function. Most striking differential methylation was found at transcription factor and developmental genes. Our findings highlight the importance of developmental origins in the function of different fat depots.

Mother-child transmission of epigenetic information by tunable polymorphic imprinting.

Genomic imprinting mediated by DNA methylation restricts gene expression to a single allele determined by parental origin and is not generally considered to be under genetic or environmental influence. Here, we focused on a differentially methylated region (DMR) of approximately 1.9 kb that includes a 101-bp noncoding RNA gene (nc886/VTRNA2-1), which is maternally imprinted in ∼75% of humans. This is unlike other imprinted genes, which demonstrate monoallelic methylation in 100% of individuals. The DMR includes a CTCF binding site on the centromeric side defining the DMR boundary and is flanked by a CTCF binding site on the telomeric side. The centromeric CTCF binding site contains an A/C polymorphism (rs2346018); the C allele is associated with less imprinting. The frequency of imprinting of the nc886 DMR in infants was linked to at least two nongenetic factors, maternal age at delivery and season of conception. In a separate cohort, nc886 imprinting was associated with lower body mass index in children at 5 y of age. Thus, we propose that the imprinting status of the nc886 DMR is "tunable" in that it is associated with maternal haplotype and prenatal environment. This provides a potential mechanism for transmitting information, with phenotypic consequences, from mother to child.

Helper-Dependent Chain Reaction (HDCR) for Selective Amplification of Methylated DNA Sequences.

Over the last few years a number of restriction enzymes that cut DNA only if cytosines within their recognition sequences are methylated have been characterized and become commercially available. Cleavage with these enzymes to release DNA fragments in a methylation-dependent manner can be combined with a novel method of amplification, Helper Dependent Chain Reaction (HDCR), to selectively amplify these fragments. HDCR uses "Helper" oligonucleotides as templates for extension of the free 3' end of target fragments to incorporate tag sequences at the ends of fragments. These tag sequences are then used for priming of amplification of target fragments. Modifications to the amplification primers (Drivers) and the Helpers ensure that there is selection for the sequences within target fragments with each cycle of amplification. The combination of methylation-dependent enzymes and HDCR allows the sensitive and selective amplification of methylated DNA sequences without the need for bisulfite treatment.

Smoke-Induced Changes to the Epigenome Provide Fertile Ground for Oncogenic Mutation.

How genetic and epigenetic events synergize to generate the oncogenic state is not well understood. In this issue of Cancer Cell, Vaz et al. provide compelling evidence that exposure to chronic cigarette smoke causes progressive epigenetic alterations that prime for key genetic events to drive the development of lung cancer.

Modulation of Hepatitis C Virus-Specific CD8 Effector T-Cell Function with Antiviral Effect in Infectious Hepatitis C Virus Coculture Model.

The antiviral effects of hepatitis C virus (HCV)-specific CD8 T cells have been shown in an HCV replicon system but not in an authentic infectious HCV cell culture (HCVcc) system. Here, we developed tools to examine the antigenicity of HCV-infected HLA-A2-positive Huh7.5 hepatoma cells (Huh7.5A2 cells) in activating HCV-specific CD8 T cells and the downstream antiviral effects. Infectious HCV epitope mutants encoding the well-defined genotype 1a-derived HLA-A2-restricted HCV NS3-1073 or NS5-2594 epitope were generated from a genotype 2a-derived HCV clone (Jc1Gluc2A) by site-directed mutagenesis. CD8 T-cell lines specific for NS3-1073 and NS5-2594 were expanded from HCV-seropositive persons by peptide stimulation in vitro or engineered from HCV-seronegative donor T cells by transduction of a lentiviral vector expressing HCV-specific T-cell receptors. HCV-specific CD8 T cells were cocultured with Huh7.5 cells that were pulsed with titrating doses of HCV epitope peptides or infected with HCV epitope mutants. HCV-specific CD8 T-cell activation (CD107a, gamma interferon, macrophage inflammatory protein 1ß, tumor necrosis factor alpha) was dependent on the peptide concentrations and the relative percentages of HCV-infected Huh7.5A2 cells. HCV-infected Huh7.5A2 cells activated HCV-specific CD8 T cells at levels comparable to those achieved with 0.1 to 2 µM pulsed peptides, providing a novel estimate of the level at which endogenously processed HCV epitopes are presented on HCV-infected cells. While HCV-specific CD8 T-cell activation with cytolytic and antiviral effects was blunted by PD-L1 expression on HCV-infected Huh7.5A2 cells, resulting in the improved viability of Huh7.5A2 cells, PD-1 blockade reversed this effect, producing enhanced cytolytic elimination of HCV-infected Huh7.5A2 cells. Our findings, obtained using an infectious HCVcc system, show that the HCV-specific CD8 T-cell function is modulated by antigen expression levels, the percentage of HCV-infected cells, and the PD-1/PD-L1 pathways and has antiviral and cytotoxic effects.IMPORTANCE We developed several novel molecular and immunological tools to study the interactions among HCV, HCV-infected hepatocytes, and HCV-specific CD8 T cells. Using these tools, we show the level at which HCV-infected hepatoma cells present endogenously processed HCV epitopes to HCV-specific CD8 T cells with antiviral and cytotoxic effects. We also show the marked protective effect of PD-L1 expression on HCV-infected hepatoma cells against HCV-specific CD8 T cells.

BRAF V600E Mutant Colorectal Cancer Subtypes Based on Gene Expression.

PURPOSE: Mutation of BRAF at the valine 600 residue occurs in approximately 10% of colorectal cancers, a group with particularly poor prognosis. The response of BRAF mutant colorectal cancer to recent targeted strategies such as anti-BRAF or combinations with MEK and EGFR inhibitors remains limited and highly heterogeneous within BRAF V600E cohorts. There is clearly an unmet need in understanding the biology of BRAF V600E colorectal cancers and potential subgroups within this population. EXPERIMENTAL DESIGN: In the biggest yet reported cohort of 218 BRAF V600E with gene expression data, we performed unsupervised clustering using non-negative matrix factorization to identify gene expression-based subgroups and characterized pathway activation. RESULTS: We found strong support for a split into two distinct groups, called BM1 and BM2. These subtypes are independent of MSI status, PI3K mutation, gender, and sidedness. Pathway analyses revealed that BM1 is characterized by KRAS/AKT pathway activation, mTOR/4EBP deregulation, and EMT whereas BM2 displays important deregulation of the cell cycle. Proteomics data validated these observations as BM1 is characterized by high phosphorylation levels of AKT and 4EBP1, and BM2 patients display high CDK1 and low cyclin D1 levels. We provide a global assessment of gene expression motifs that differentiate BRAF V600E subtypes from other colorectal cancers. CONCLUSIONS: We suggest that BRAF mutant patients should not be considered as having a unique biology and provide an in depth characterization of heterogeneous motifs that may be exploited for drug targeting. Clin Cancer Res; 23(1); 104-15. ©2016 AACR.

Evaluation of Methylation Biomarkers for Detection of Circulating Tumor DNA and Application to Colorectal Cancer.

Solid tumors shed DNA into circulation, and there is growing evidence that the detection of circulating tumor DNA (ctDNA) has broad clinical utility, including monitoring of disease, prognosis, response to chemotherapy and tracking tumor heterogeneity. The appearance of ctDNA in the circulating cell-free DNA (ccfDNA) isolated from plasma or serum is commonly detected by identifying tumor-specific features such as insertions, deletions, mutations and/or aberrant methylation. Methylation is a normal cell regulatory event, and since the majority of ccfDNA is derived from white blood cells (WBC), it is important that tumour-specific DNA methylation markers show rare to no methylation events in WBC DNA. We have used a novel approach for assessment of low levels of DNA methylation in WBC DNA. DNA methylation in 29 previously identified regions (residing in 17 genes) was analyzed in WBC DNA and eight differentially-methylated regions (DMRs) were taken through to testing in clinical samples using methylation specific PCR assays. DMRs residing in four genes, BCAT1, GRASP, IKZF1 and IRF4, exhibited low positivity, 3.5% to 7%, in the plasma of colonoscopy-confirmed healthy subjects, with the sensitivity for detection of ctDNA in colonoscopy-confirmed patients with colorectal cancer being 65%, 54.5%, 67.6% and 59% respectively.

Effect of prenatal DHA supplementation on the infant epigenome: results from a randomized controlled trial.

BACKGROUND: Evidence is accumulating that nutritional exposures in utero can influence health outcomes in later life. Animal studies and human epidemiological studies have implicated epigenetic modifications as playing a key role in this process, but there are limited data from large well-controlled human intervention trials. This study utilized a large double-blind randomized placebo-controlled trial to test whether a defined nutritional exposure in utero, in this case docosahexaenoic acid (DHA), could alter the infant epigenome. Pregnant mothers consumed DHA-rich fish oil (800 mg DHA/day) or placebo supplements from 20 weeks' gestation to delivery. Blood spots were collected from the children at birth (n = 991) and blood leukocytes at 5 years (n = 667). Global DNA methylation was measured in all samples, and Illumina HumanMethylation450K BeadChip arrays were used for genome-wide methylation profiling in a subset of 369 children at birth and 65 children at 5 years. RESULTS: There were no differences in global DNA methylation levels between the DHA and control group either at birth or at 5 years, but we identified 21 differentially methylated regions (DMRs) at birth, showing small DNA methylation differences (<5%) between the treatment groups, some of which seemed to persist until 5 years. The number of DMRs at birth was greater in males (127 DMRs) and in females (72 DMRs) separately, indicating a gender-specific effect. CONCLUSION: Maternal DHA supplementation during the second half of pregnancy had small effects on DNA methylation of infants. While the potential functional significance of these changes remains to be determined, these findings further support the role of epigenetic modifications in developmental programming in humans and point the way for future studies. TRIAL REGISTRATION: Australian New Zealand Clinical Trials Registry (ANZCTR), ACTRN12605000569606 and ACTRN12611001127998.

Critical evaluation of the Illumina MethylationEPIC BeadChip microarray for whole-genome DNA methylation profiling.

BACKGROUND: In recent years the Illumina HumanMethylation450 (HM450) BeadChip has provided a user-friendly platform to profile DNA methylation in human samples. However, HM450 lacked coverage of distal regulatory elements. Illumina have now released the MethylationEPIC (EPIC) BeadChip, with new content specifically designed to target these regions. We have used HM450 and whole-genome bisulphite sequencing (WGBS) to perform a critical evaluation of the new EPIC array platform. RESULTS: EPIC covers over 850,000 CpG sites, including >90 % of the CpGs from the HM450 and an additional 413,743 CpGs. Even though the additional probes improve the coverage of regulatory elements, including 58 % of FANTOM5 enhancers, only 7 % distal and 27 % proximal ENCODE regulatory elements are represented. Detailed comparisons of regulatory elements from EPIC and WGBS show that a single EPIC probe is not always informative for those distal regulatory elements showing variable methylation across the region. However, overall data from the EPIC array at single loci are highly reproducible across technical and biological replicates and demonstrate high correlation with HM450 and WGBS data. We show that the HM450 and EPIC arrays distinguish differentially methylated probes, but the absolute agreement depends on the threshold set for each platform. Finally, we provide an annotated list of probes whose signal could be affected by cross-hybridisation or underlying genetic variation. CONCLUSION: The EPIC array is a significant improvement over the HM450 array, with increased genome coverage of regulatory regions and high reproducibility and reliability, providing a valuable tool for high-throughput human methylome analyses from diverse clinical samples.

Elimination of Latently HIV-infected Cells from Antiretroviral Therapy-suppressed Subjects by Engineered Immune-mobilizing T-cell Receptors.

Persistence of human immunodeficiency virus (HIV) in a latent state in long-lived CD4+ T-cells is a major barrier to eradication. Latency-reversing agents that induce direct or immune-mediated cell death upon reactivation of HIV are a possible solution. However, clearance of reactivated cells may require immunotherapeutic agents that are fine-tuned to detect viral antigens when expressed at low levels. We tested the antiviral efficacy of immune-mobilizing monoclonal T-cell receptors against viruses (ImmTAVs), bispecific molecules that redirect CD8+ T-cells to kill HIV-infected CD4+ T-cells. T-cell receptors specific for an immunodominant Gag epitope, SL9, and its escape variants were engineered to achieve supraphysiological affinity and fused to a humanised CD3-specific single chain antibody fragment. Ex vivo polyclonal CD8+ T-cells were efficiently redirected by immune-mobilising monoclonal T-cell receptors against viruses to eliminate CD4+ T-cells from human histocompatibility leukocyte antigen (HLA)-A*0201-positive antiretroviral therapy-treated patients after reactivation of inducible HIV in vitro. The efficiency of infected cell elimination correlated with HIV Gag expression. Immune-mobilising monoclonal T-cell receptors against viruses have potential as a therapy to facilitate clearance of reactivated HIV reservoir cells.

Relative telomere lengths in tumor and normal mucosa are related to disease progression and chromosome instability profiles in colorectal cancer.

Telomeric dysfunction is linked to colorectal cancer (CRC) initiation. However, the relationship of normal tissue and tumor telomere lengths with CRC progression, molecular features and prognosis is unclear. Here, we measured relative telomere length (RTL) by real-time quantitative PCR in 90 adenomas (aRTL), 419 stage I-IV CRCs (cRTL) and adjacent normal mucosa (nRTL). Age-adjusted RTL was analyzed against germline variants in telomere biology genes, chromosome instability (CIN), microsatellite instability (MSI), CpG island methylator phenotype (CIMP), TP53, KRAS, BRAF mutations and clinical outcomes. In 509 adenoma or CRC patients, nRTL decreased with advancing age. Female gender, proximal location and the TERT rs2736100 G allele were independently associated with longer age-adjusted nRTL. Adenomas and carcinomas exhibited telomere shortening in 79% and 67% and lengthening in 7% and 15% of cases. Age-adjusted nRTL and cRTL were independently associated with tumor stage, decreasing from adenoma to stage III and leveling out or increasing from stage III to IV, respectively. Cancer MSI, CIMP, TP53, KRAS and BRAF status were not related to nRTL or cRTL. Near-tetraploid CRCs exhibited significantly longer cRTLs than CIN- and aneuploidy CRCs, while cRTL was significantly shorter in CRCs with larger numbers of chromosome breaks. Age-adjusted nRTL, cRTL or cRTL:nRTL ratios were not associated with disease-free or overall survival in stage II/III CRC. Taken together, our data show that both normal mucosa and tumor RTL are independently associated with CRC progression, and highlight divergent associations of CRC telomere length with tumor CIN profiles.

Cholesteryl esters stabilize human CD1c conformations for recognition by self-reactive T cells.

Cluster of differentiation 1c (CD1c)-dependent self-reactive T cells are abundant in human blood, but self-antigens presented by CD1c to the T-cell receptors of these cells are poorly understood. Here we present a crystal structure of CD1c determined at 2.4 Å revealing an extended ligand binding potential of the antigen groove and a substantially different conformation compared with known CD1c structures. Computational simulations exploring different occupancy states of the groove reenacted these different CD1c conformations and suggested cholesteryl esters (CE) and acylated steryl glycosides (ASG) as new ligand classes for CD1c. Confirming this, we show that binding of CE and ASG to CD1c enables the binding of human CD1c self-reactive T-cell receptors. Hence, human CD1c adopts different conformations dependent on ligand occupancy of its groove, with CE and ASG stabilizing CD1c conformations that provide a footprint for binding of CD1c self-reactive T-cell receptors.

Direct intrahepatic portocaval shunt for treatment of portal thrombosis and Budd-Chiari syndrome.

Budd-Chiari syndrome (BCS) refers to hepatic venous outflow obstruction that in severe cases can lead to acute liver failure prompting consideration of revascularization or transplantation. Here, a 22 year old female with angiographically proven BCS secondary to JAK2/V617F positive Polycythemia vera on therapeutic warfarin presented with acute liver failure (ALF). Imaging revealed a new, near complete thrombotic occlusion of the main portal vein with extension into the superior mesenteric vein. An emergent direct intrahepatic portocaval shunt (DIPS) was created and liver function promptly normalized. She has been maintained on rivaroxaban since that time. Serial assessment over 1 year demonstrated continued shunt patency and improved flow in the mesenteric vasculature on ultrasound as well as normal liver function. DIPS is a viable alternative in the treatment of ALF from BCS when standard recanalization is not feasible. Improved blood flow may also improve portal/mesenteric clot burden. While further investigation is needed, new targeted anticoagulants may be viable as a long term anticoagulation strategy.

COBRA-Seq: Sensitive and Quantitative Methylome Profiling.

Combined Bisulfite Restriction Analysis (COBRA) quantifies DNA methylation at a specific locus. It does so via digestion of PCR amplicons produced from bisulfite-treated DNA, using a restriction enzyme that contains a cytosine within its recognition sequence, such as TaqI. Here, we introduce COBRA-seq, a genome wide reduced methylome method that requires minimal DNA input (0.1-1.0 mg) and can either use PCR or linear amplification to amplify the sequencing library. Variants of COBRA-seq can be used to explore CpG-depleted as well as CpG-rich regions in vertebrate DNA. The choice of enzyme influences enrichment for specific genomic features, such as CpG-rich promoters and CpG islands, or enrichment for less CpG dense regions such as enhancers. COBRA-seq coupled with linear amplification has the additional advantage of reduced PCR bias by producing full length fragments at high abundance. Unlike other reduced representative methylome methods, COBRA-seq has great flexibility in the choice of enzyme and can be multiplexed and tuned, to reduce sequencing costs and to interrogate different numbers of sites. Moreover, COBRA-seq is applicable to non-model organisms without the reference genome and compatible with the investigation of non-CpG methylation by using restriction enzymes containing CpA, CpT, and CpC in their recognition site.