preparation-frozen-non-human-primate-fetal-islets-for-combined-single

Preparation of Frozen Non-Human Primate Fetal Islets for Combined Single Nuclei RNA-Sequencing and ATAC-Sequencing, and Bulk Metabolomics

One challenge in studies using tissue collected from multiple cohorts is avoiding batch effects when preparing for large-scale multi-omic experiments, ...

Vanderbilt University Medical Center

Candidate variant association studies have been largely unsuccessful in identifying common breast cancer susceptibility variants, although most studies have been underpowered to detect associations of a realistic magnitude. We assessed 41 common non-synonymous single-nucleotide polymorphisms (nsSNPs) for which evidence of association with breast cancer risk had been previously reported. Case-control data were combined from 38 studies of white European women (46 450 cases and 42 600 controls) and analyzed using unconditional logistic regression. Strong evidence of association was observed for three nsSNPs: ATXN7-K264R at 3p21 [rs1053338, per allele OR = 1.07, 95% confidence interval (CI) = 1.04-1.10, P = 2.9 × 10(-6)], AKAP9-M463I at 7q21 (rs6964587, OR = 1.05, 95% CI = 1.03-1.07, P = 1.7 × 10(-6)) and NEK10-L513S at 3p24 (rs10510592, OR = 1.10, 95% CI = 1.07-1.12, P = 5.1 × 10(-17)). The first two associations reached genome-wide statistical significance in a combined analysis of available data, including independent data from nine genome-wide association studies (GWASs): for ATXN7-K264R, OR = 1.07 (95% CI = 1.05-1.10, P = 1.0 × 10(-8)); for AKAP9-M463I, OR = 1.05 (95% CI = 1.04-1.07, P = 2.0 × 10(-10)). Further analysis of other common variants in these two regions suggested that intronic SNPs nearby are more strongly associated with disease risk. We have thus identified a novel susceptibility locus at 3p21, and confirmed previous suggestive evidence that rs6964587 at 7q21 is associated with risk. The third locus, rs10510592, is located in an established breast cancer susceptibility region; the association was substantially attenuated after adjustment for the known GWAS hit. Thus, each of the associated nsSNPs is likely to be a marker for another, non-coding, variant causally related to breast cancer risk. Further fine-mapping and functional studies are required to identify the underlying risk-modifying variants and the genes through which they act.

Common non-synonymous SNPs associated with breast cancer susceptibility: findings from the Breast Cancer Association Consortium.

Common variants in the hepatocyte nuclear factor 1 homeobox B (HNF1B) gene are associated with the risk of Type II diabetes and multiple cancers. Evidence to date indicates that cancer risk may be mediated via genetic or epigenetic effects on HNF1B gene expression. We previously found single-nucleotide polymorphisms (SNPs) at the HNF1B locus to be associated with endometrial cancer, and now report extensive fine-mapping and in silico and laboratory analyses of this locus. Analysis of 1184 genotyped and imputed SNPs in 6608 Caucasian cases and 37 925 controls, and 895 Asian cases and 1968 controls, revealed the best signal of association for SNP rs11263763 (P = 8.4 × 10(-14), odds ratio = 0.86, 95% confidence interval = 0.82-0.89), located within HNF1B intron 1. Haplotype analysis and conditional analyses provide no evidence of further independent endometrial cancer risk variants at this locus. SNP rs11263763 genotype was associated with HNF1B mRNA expression but not with HNF1B methylation in endometrial tumor samples from The Cancer Genome Atlas. Genetic analyses prioritized rs11263763 and four other SNPs in high-to-moderate linkage disequilibrium as the most likely causal SNPs. Three of these SNPs map to the extended HNF1B promoter based on chromatin marks extending from the minimal promoter region. Reporter assays demonstrated that this extended region reduces activity in combination with the minimal HNF1B promoter, and that the minor alleles of rs11263763 or rs8064454 are associated with decreased HNF1B promoter activity. Our findings provide evidence for a single signal associated with endometrial cancer risk at the HNF1B locus, and that risk is likely mediated via altered HNF1B gene expression.

Fine-mapping of the HNF1B multicancer locus identifies candidate variants that mediate endometrial cancer risk.

Deficiency in diacylglycerol acyltransferase (DGAT1) is a rare cause of neonatal diarrhea, without a known mechanism or in vitro model. A patient presenting at our institution at 7 weeks of life with failure to thrive and diarrhea was found by whole-exome sequencing to have a homozygous DGAT1 truncation mutation. Duodenal biopsies showed loss of DGAT1 and deficits in apical membrane transporters and junctional proteins in enterocytes. When placed on a very low-fat diet, the patient's diarrhea resolved with normalization of brush border transporter localization in endoscopic biopsies. DGAT1 knockdown in Caco2-BBe cells modeled the deficits in apical trafficking, with loss of apical DPPIV and junctional occludin. Elevation in cellular lipid levels, including diacylglycerol (DAG) and phospholipid metabolites of DAG, was documented by lipid analysis in DGAT1 knockdown cells. Culture of the DGAT1 knockdown cells in lipid-depleted media led to re-establishment of occludin and return of apical DPPIV. DGAT1 loss appears to elicit global changes in enterocyte polarized trafficking that could account for deficits in absorption seen in the patient. The in vitro modeling of this disease should allow for investigation of possible therapeutic targets.

Reversible deficits in apical transporter trafficking associated with deficiency in diacylglycerol acyltransferase.

The increasing demand of single-cell RNA-sequencing (scRNA-seq) experiments, such as the number of experiments and cells queried per experiment, necessitates higher sequencing depth coupled to high data quality. New high-throughput sequencers, such as the Illumina NovaSeq 6000, enables this demand to be filled in a cost-effective manner. However, current scRNA-seq library designs present compatibility challenges with newer sequencing technologies, such as index-hopping, and their ability to generate high quality data has yet to be systematically evaluated.

Dual indexed library design enables compatibility of in-Drop single-cell RNA-sequencing with exAMP chemistry sequencing platforms.

Colorectal cancers (CRCs) arise from precursor polyps whose cellular origins, molecular heterogeneity, and immunogenic potential may reveal diagnostic and therapeutic insights when analyzed at high resolution. We present a single-cell transcriptomic and imaging atlas of the two most common human colorectal polyps, conventional adenomas and serrated polyps, and their resulting CRC counterparts. Integrative analysis of 128 datasets from 62 participants reveals adenomas arise from WNT-driven expansion of stem cells, while serrated polyps derive from differentiated cells through gastric metaplasia. Metaplasia-associated damage is coupled to a cytotoxic immune microenvironment preceding hypermutation, driven partly by antigen-presentation differences associated with tumor cell-differentiation status. Microsatellite unstable CRCs contain distinct non-metaplastic regions where tumor cells acquire stem cell properties and cytotoxic immune cells are depleted. Our multi-omic atlas provides insights into malignant progression of colorectal polyps and their microenvironment, serving as a framework for precision surveillance and prevention of CRC.

Differential pre-malignant programs and microenvironment chart distinct paths to malignancy in human colorectal polyps.

Integrins - the principal extracellular matrix (ECM) receptors of the cell - promote cell adhesion, migration, and proliferation, which are key events for cancer growth and metastasis. To date, most integrin-targeted cancer therapeutics have disrupted integrin-ECM interactions, which are viewed as critical for integrin functions. However, such agents have failed to improve cancer patient outcomes. We show that the highly expressed integrin β1 subunit is required for lung adenocarcinoma development in a carcinogen-induced mouse model. Likewise, human lung adenocarcinoma cell lines with integrin β1 deletion failed to form colonies in soft agar and tumors in mice. Mechanistically, we demonstrate that these effects do not require integrin β1-mediated adhesion to ECM but are dependent on integrin β1 cytoplasmic tail-mediated activation of focal adhesion kinase (FAK). These studies support a critical role for integrin β1 in lung tumorigenesis that is mediated through constitutive, ECM binding-independent signaling involving the cytoplasmic tail.

Ligand-independent integrin β1 signaling supports lung adenocarcinoma development.

SET domain-containing protein 2 () is commonly mutated in renal cell carcinoma. SETD2 methylates histone H3 as well as a growing list of non-histone proteins.

SETD2 regulates the methylation of translation elongation factor eEF1A1 in clear cell renal cell carcinoma.

Colorectal cancer exhibits dynamic cellular and genetic heterogeneity during progression from precursor lesions toward malignancy. Analysis of spatial multi-omic data from 31 human colorectal specimens enabled phylogeographic mapping of tumor evolution that revealed individualized progression trajectories and accompanying microenvironmental and clonal alterations. Phylogeographic mapping ordered genetic events, classified tumors by their evolutionary dynamics, and placed clonal regions along global pseudotemporal progression trajectories encompassing the chromosomal instability (CIN+) and hypermutated (HM) pathways. Integrated single-cell and spatial transcriptomic data revealed recurring epithelial programs and infiltrating immune states along progression pseudotime. We discovered an immune exclusion signature (IEX), consisting of extracellular matrix regulators DDR1, TGFBI, PAK4, and DPEP1, that charts with CIN+ tumor progression, is associated with reduced cytotoxic cell infiltration, and shows prognostic value in independent cohorts. This spatial multi-omic atlas provides insights into colorectal tumor-microenvironment co-evolution, serving as a resource for stratification and targeted treatments.

Molecular cartography uncovers evolutionary and microenvironmental dynamics in sporadic colorectal tumors.

Clonal hematopoiesis of indeterminate potential (CHIP) is a common age-related phenomenon in which hematopoietic stem cells acquire mutations in a select set of genes commonly mutated in myeloid neoplasia which then expand clonally. Current sequencing assays to detect CHIP mutations are not optimized for the detection of these variants and can be cost-prohibitive when applied to large cohorts or to serial sequencing. In this study, an affordable (approximately US $8 per sample), accurate, and scalable sequencing assay for CHIP is introduced and validated. The efficacy of the assay was demonstrated by identifying CHIP mutations in a cohort of 456 individuals with DNA collected at multiple time points in Vanderbilt University's biobank and quantifying clonal expansion rates over time. A total of 101 individuals with CHIP/clonal cytopenia of undetermined significance were identified, and individual-level clonal expansion rate was calculated using the variant allele fraction at both time points. Differences in clonal expansion rate by driver gene were observed, but there was also significant individual-level heterogeneity, emphasizing the multifactorial nature of clonal expansion. Additionally, mutation co-occurrence and clonal competition between multiple driver mutations were explored.

Cost-Effective and Scalable Clonal Hematopoiesis Assay Provides Insight into Clonal Dynamics.

Heat is a cardinal feature of inflammation, yet its impacts on immune cells remain uncertain. We show that moderate-grade fever temperatures (39°C) increased murine CD4 T cell metabolism, proliferation, and inflammatory effector activity while decreasing regulatory T cell suppressive capacity. However, heat-exposed T helper 1 (T1) cells selectively developed mitochondrial stress and DNA damage that activated Trp53 and stimulator of interferon genes pathways. Although many T1 cells subjected to such temperatures died, surviving T1 cells exhibited increased mitochondrial mass and enhanced activity. Electron transport chain complex 1 (ETC1) was rapidly impaired under fever-range temperatures, a phenomenon that was specifically detrimental to T1 cells. T1 cells with elevated DNA damage and ETC1 signatures were also detected in human chronic inflammation. Thus, fever-relevant temperatures disrupt ETC1 to selectively drive apoptosis or adaptation of T1 cells to maintain genomic integrity and enhance effector functions.

Article	Year
Common non-synonymous SNPs associated with breast cancer susceptibility: findings from the Breast Cancer Association Consortium. Human molecular genetics\| PubMed ID: 24943594	2014
Fine-mapping of the HNF1B multicancer locus identifies candidate variants that mediate endometrial cancer risk. Human molecular genetics\| PubMed ID: 25378557	2015
Reversible deficits in apical transporter trafficking associated with deficiency in diacylglycerol acyltransferase. Traffic (Copenhagen, Denmark)\| PubMed ID: 30095213	2018
Dual indexed library design enables compatibility of in-Drop single-cell RNA-sequencing with exAMP chemistry sequencing platforms. BMC genomics\| PubMed ID: 32616006	2020
Differential pre-malignant programs and microenvironment chart distinct paths to malignancy in human colorectal polyps. Cell\| PubMed ID: 34910928	2021
Ligand-independent integrin β1 signaling supports lung adenocarcinoma development. JCI insight\| PubMed ID: 35763345	2022
SETD2 regulates the methylation of translation elongation factor eEF1A1 in clear cell renal cell carcinoma. Kidney cancer journal : official journal of the Kidney Cancer Association\| PubMed ID: 36684483	2022
Molecular cartography uncovers evolutionary and microenvironmental dynamics in sporadic colorectal tumors. Cell\| PubMed ID: 38065082	2023
Cost-Effective and Scalable Clonal Hematopoiesis Assay Provides Insight into Clonal Dynamics. The Journal of molecular diagnostics : JMD\| PubMed ID: 38588769	2024
Subset-specific mitochondrial stress and DNA damage shape T cell responses to fever and inflammation. Science immunology\| PubMed ID: 39303018	2024

Angela Jones

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