Periconceptional diet may persistently influence DNA methylation levels with phenotypic consequences. However, a comprehensive assessment of the characteristics of prenatal malnutrition-associated ...differentially methylated regions (P-DMRs) is lacking in humans. Here we report on a genome-scale analysis of differential DNA methylation in whole blood after periconceptional exposure to famine during the Dutch Hunger Winter. We show that P-DMRs preferentially occur at regulatory regions, are characterized by intermediate levels of DNA methylation and map to genes enriched for differential expression during early development. Validation and further exploratory analysis of six P-DMRs highlight the critical role of gestational timing. Interestingly, differential methylation of the P-DMRs extends along pathways related to growth and metabolism. P-DMRs located in INSR and CPT1A have enhancer activity in vitro and differential methylation is associated with birth weight and serum LDL cholesterol. Epigenetic modulation of pathways by prenatal malnutrition may promote an adverse metabolic phenotype in later life.
Identification of imprinted genes, demonstrating a consistent preference towards the paternal or maternal allelic expression, is important for the understanding of gene expression regulation during ...embryonic development and of the molecular basis of developmental disorders with a parent-of-origin effect. Combining allelic analysis of RNA-Seq data with phased genotypes in family trios provides a powerful method to detect parent-of-origin biases in gene expression.
We report findings in 296 family trios from two large studies: 165 lymphoblastoid cell lines from the 1000 Genomes Project and 131 blood samples from the Genome of the Netherlands (GoNL) participants. Based on parental haplotypes, we identified > 2.8 million transcribed heterozygous SNVs phased for parental origin and developed a robust statistical framework for measuring allelic expression. We identified a total of 45 imprinted genes and one imprinted unannotated transcript, including multiple imprinted transcripts showing incomplete parental expression bias that was located adjacent to strongly imprinted genes. For example, PXDC1, a gene which lies adjacent to the paternally expressed gene FAM50B, shows a 2:1 paternal expression bias. Other imprinted genes had promoter regions that coincide with sites of parentally biased DNA methylation identified in the blood from uniparental disomy (UPD) samples, thus providing independent validation of our results. Using the stranded nature of the RNA-Seq data in lymphoblastoid cell lines, we identified multiple loci with overlapping sense/antisense transcripts, of which one is expressed paternally and the other maternally. Using a sliding window approach, we searched for imprinted expression across the entire genome, identifying a novel imprinted putative lncRNA in 13q21.2. Overall, we identified 7 transcripts showing parental bias in gene expression which were not reported in 4 other recent RNA-Seq studies of imprinting.
Our methods and data provide a robust and high-resolution map of imprinted gene expression in the human genome.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The chromosomal gene expression profiles established by the Human Transcriptome Map (HTM) revealed a clustering of highly expressed genes in about 30 domains, called ridges. To physically ...characterize ridges, we constructed a new HTM based on the draft human genome sequence (HTMseq). Expression of 25,003 genes can be analyzed online in a multitude of tissues (http://bioinfo.amc.uva.nl/HTMseq). Ridges are found to be very gene-dense domains with a high GC content, a high SINE repeat density, and a low LINE repeat density. Genes in ridges have significantly shorter introns than genes outside of ridges. The HTMseq also identifies a significant clustering of weakly expressed genes in domains with fully opposite characteristics (antiridges). Both types of domains are open to tissue-specific expression regulation, but the maximal expression levels in ridges are considerably higher than in antiridges. Ridges are therefore an integral part of a higher order structure in the genome related to transcriptional regulation.
Drawing genotype-to-phenotype maps in tumors is of paramount importance for understanding tumor heterogeneity. Assignment of single cells to their tumor clones of origin can be approached by matching ...the genotypes of the clones to the mutations found in RNA sequencing of the cells. The confidence of the cell-to-clone mapping can be increased by accounting for additional measurements. Follicular lymphoma, a malignancy of mature B cells that continuously acquire mutations in parallel in the exome and in B cell receptor loci, presents a unique opportunity to join exome-derived mutations with B cell receptor sequences as independent sources of evidence for clonal evolution.
Here, we propose CACTUS, a probabilistic model that leverages the information from an independent genomic clustering of cells and exploits the scarce single cell RNA sequencing data to map single cells to given imperfect genotypes of tumor clones.
We apply CACTUS to two follicular lymphoma patient samples, integrating three measurements: whole exome, single-cell RNA, and B cell receptor sequencing. CACTUS outperforms a predecessor model by confidently assigning cells and B cell receptor-based clusters to the tumor clones.
The integration of independent measurements increases model certainty and is the key to improving model performance in the challenging task of charting the genotype-to-phenotype maps in tumors. CACTUS opens the avenue to study the functional implications of tumor heterogeneity, and origins of resistance to targeted therapies. CACTUS is written in R and source code, along with all supporting files, are available on GitHub ( https://github.com/LUMC/CACTUS ).
In genome-wide association studies (GWAS) of complex traits, single SNP analysis is still the most applied approach. However, the identified SNPs have small effects and provide limited biological ...insight. A more appropriate approach to interpret GWAS data of complex traits is to analyze the combined effect of a SNP set grouped per pathway or gene region. We used this approach to study the joint effect on human longevity of genetic variation in two candidate pathways, the insulin/insulin-like growth factor (IGF-1) signaling (IIS) pathway and the telomere maintenance (TM) pathway. For the analyses, we used genotyped GWAS data of 403 unrelated nonagenarians from long-lived sibships collected in the Leiden Longevity Study and 1,670 younger population controls. We analyzed 1,021 SNPs in 68 IIS pathway genes and 88 SNPs in 13 TM pathway genes using four self-contained pathway tests (PLINK set-based test, Global test, GRASS and SNP ratio test). Although we observed small differences between the results of the different pathway tests, they showed consistent significant association of the IIS and TM pathway SNP sets with longevity. Analysis of gene SNP sets from these pathways indicates that the association of the IIS pathway is scattered over several genes (
AKT1
,
AKT3
,
FOXO4
,
IGF2
,
INS
,
PIK3CA
,
SGK
,
SGK2
, and
YWHAG
), while the association of the TM pathway seems to be mainly determined by one gene (
POT1
). In conclusion, this study shows that genetic variation in genes involved in the IIS and TM pathways is associated with human longevity.
Patients undergoing allogeneic stem cell transplantation as treatment for hematological diseases face the risk of Graft-versus-Host Disease as well as relapse. Graft-versus-Host Disease and the ...favorable Graft-versus-Leukemia effect are mediated by donor T cells recognizing polymorphic peptides, which are presented on the cell surface by HLA molecules and result from single nucleotide polymorphism alleles that are disparate between patient and donor. Identification of polymorphic HLA-binding peptides, designated minor histocompatibility antigens, has been a laborious procedure, and the number and scope for broad clinical use of these antigens therefore remain limited. Here, we present an optimized whole genome association approach for discovery of HLA class I minor histocompatibility antigens. T cell clones isolated from patients who responded to donor lymphocyte infusions after HLA-matched allogeneic stem cell transplantation were tested against a panel of 191 EBV-transformed B cells, which have been sequenced by the 1000 Genomes Project and selected for expression of seven common HLA class I alleles (HLA-A
01:01, A
02:01, A
03:01, B
07:02, B
08:01, C
07:01, and C
07:02). By including all polymorphisms with minor allele frequencies above 0.01, we demonstrated that the new approach allows direct discovery of minor histocompatibility antigens as exemplified by seven new antigens in eight different HLA class I alleles including one antigen in HLA-A
24:02 and HLA-A
23:01, for which the method has not been originally designed. Our new whole genome association strategy is expected to rapidly augment the repertoire of HLA class I-restricted minor histocompatibility antigens that will become available for donor selection and clinical use to predict, follow or manipulate Graft-versus-Leukemia effect and Graft-versus-Host Disease after allogeneic stem cell transplantation.
Antigen-independent, autonomous signalling of the B-cell receptor (BCR) is an oncogenic driver common to all cases and subtypes of chronic lymphocytic leukemia (CLL). We have recently shown that the ...dominant clonal population of low-count (<500 cells/µL) monoclonal B lymphocytosis (MBL) with CD5 +CD20 lowCD79B low CLL phenotype identified prospectively in healthy siblings of CLL patients also exhibit autonomous BCR signalling, albeit with lower signalling strength than CLL BCR (Quinten et al., Haematologica 2023). To gain further insight into the mechanisms of clonal expansion within the CD5 + B-cell compartment as the possible origin of CLL, we performed single-cell gene expression profiling and exemplary analyses of BCR function in normal CD5 + B cells (CD20 hiCD79B hi) and in CLL-phenotype B cells (CD5 +CD20 lowCD79B low) of healthy siblings of CLL patients in comparison to CLL and high-count MBL cells. Screening of blood samples of 16 sibling stem cell donors of CLL patients by flow cytometry revealed the presence of CD5 +CD20 lowCD79B low B cells at low counts in 5 donors. These cells and normal CD5 +CD20 hiCD79B hi B cells were sorted in parallel, pooled at equal ratios, and analyzed together with CLL B cells from their sibling stem cell recipients and 2 unrelated high-count MBL B cells on two 10X Genomics chips for single-cell 5‘ gene expression and BCR sequencing. Data deconvolution per individual was performed based on expressed SNPs and on the individuals’ HLA types. In a combined 2-dimensional t-SNE plot based on the 2000 most variably expressed genes, the normal CD5 +CD20 hiCD79B hi B cells from all 5 donors clustered distinctly from CLL-phenotype cells from all sources (dotted lines, Figure) and indeed showed significantly higher expression of CD20 and CD79B. Gene set enrichment analysis revealed significant upregulation of the hallmark oxidative phosphorylation gene set in all CLL samples compared to non-CLL B-cell populations. BCR analysis demonstrated clonal dominance of >99% for both Ig heavy and light chains in the 5 CLL samples. In accordance with the indication for allogeneic stem cell transplantation, these CLL BCR were unmutated. In marked contrast, normal CD5 +CD20 hiCD79B hi B cells from the healthy SC donors were also unmutated but completely polyclonal, i.e. no identical BCR was detected in any two cells of this compartment. In 4/5 donors, the CLL-phenotype CD5 +CD20 lowCD79B low compartment contained multiple clonal expansions of various degrees. In one high-count MBL, 17 other B-cell clones with various degrees of low-level expansion coexisted with the >90% dominant MBL clone. BCR from the most dominant and one or more of the less expanded CD5 +CD20 lowCD79B low clones of the healthy donors were tested for autonomous signaling as quantified by BCR-induced calcium flux without experimental cross-linking in murine TKO pre-B cells and compared to BCR from nonexpanded CD5 +CD20 hiCD79B hi cells and from their CLL siblings. Autonomous BCR signaling strength was calculated from the fraction of cells with spontaneous calcium flux above test background and calibrated against calcium flux after BCR crosslinking. CLL and high-count MBL BCR had robust median autonomous BCR signaling of 0.326. Expanded CD5 +CD20 lowCD79B low clones of the healthy donors showed significantly weaker BCR signalling strength of 0.163 (p<0.01; Scatter Plot). BCR signalling in nonexpanded CD5 + B cells appeared weaker than in expanded clones but was clearly above the background of BCR from mature B cells isolated from lymph node biopsies expressing a mutated IgG. In conclusion, the low-count CD5 +CD20 lowCD79B low CLL-phenotype B-cell compartment in healthy adults is composed of multiple, predominantly mutated B-cell clones. Therefore, the term “monoclonal B lymphocytosis” is highly misleading for these B-cell populations and should be avoided. Normal CD5 +CD20 hiCD79B hi B cells are exclusively unexpanded and express unmutated BCR, indicating a naïve status. Unexpectedly, these nonexpanded CD5 + B cells express BCR with relatively weak but quantifiable autonomous signalling. Globally, autonomous BCR signalling strength across the CD5 + B-cell compartment appears to be correlated to the degree of clonal expansion. Fully malignant CLL cells overexpress genes of the oxidative phosphorylation pathway, presumably to meet increased energy demand from alternative sources.
Abstract
Cluster inference based on spatial extent thresholding is a popular analysis method multiple testing in spatial data, and is frequently used for finding activated brain areas in ...neuroimaging. However, the method has several well-known issues. While powerful for finding regions with some activation, the method as currently defined does not allow any further quantification or localisation of signal. In this paper, we repair this gap. We show that cluster-extent inference can be used (1) to infer the presence of signal in any region of interest and (2) to quantify the percentage of activation in such regions. These additional inferences come for free, i.e. they do not require any further adjustment of the alpha-level of tests, while retaining full family-wise error control. We achieve this extension of the possibilities of cluster inference by embedding the method into a closed testing procedure, and solving the graph-theoretic k-separator problem that results from this embedding. We demonstrate the usefulness of the improved method in a large-scale application to neuroimaging data from the Neurovault database.
Introduction
Follicular Lymphoma (FL) is one of the most prevalent B-cell neoplasms and despite recent advances remains incurable in most cases. FL cells are malignant counterparts of normal germinal ...center B-cells. At molecular level FL are characterized by the t(14;18) which results in the overexpression of the BCL-2. These apoptosis resistant cells accumulate somatic mutations associated with tumorigenesis and progression. Whereas several mutagenic mechanisms shape the FL genomic landscape, up to 22% of the mutations may be attributed to activation-induced cytidine deaminase (AID) activity.
Under physiological conditions AID is responsible for somatic hypermutation (SHM) in immunoglobulin genes (IG) of germinal center B-cells. In fact, it is accepted that ongoing SHM still occurs at relatively high rates in FL cells as compared with other germinal center lymphoid neoplasms. Moreover, we recently demonstrated the direct effect of AID overexpression inducing somatic mutations driving murine and human B-cell neoplasm progression in vivo. Therefore, unveiling the molecular basis of AID activity in lymphoma cells remains essential to understand lymphomagenesis and to develop novel targeted therapies.
The advent of single-cell high-throughput sequencing has enabled the analysis of molecular events at an unprecedented resolution. Therefore, we designed a study to capture AID-induced somatic hypermutation at the single cell level in FL.
Methods
Tumor samples derived from 14 FL patients were analyzed, as controls, we chose a B-cell malignancy with lower SHM rates and included 5 samples derived from chronic lymphocytic leukemia (CLL) and 2 from monoclonal B lymphocytosis (MBL). Single cell whole cDNA libraries were obtained by 10X Genomics. Immunoglobulin gene single cell libraries were prepared by enrichment with seminested amplification using 3 ′ constant domain primers followed by 10X Genomics prep. Both single-cell libraries were sequenced in paired-end mode (2 × 150 bp) on an Illumina Hiseq platform. We developed an immunoglobulin alignment tool to enable the analysis of highly mutated IG sequences derived from FL. A consensus sequence was obtained by transcript associated with a unique molecular identifier (UMI) for every cell. Then, the presence of variants in the IG by position in a particular cell was annotated. These observations were filtered using quality parameters (read depth => 25 , frequency of the event => 20%, UMIs supporting every variant => 5).
Results
We analyzed an average of 926 cells per case. Our data confirms at the single cell level previous reports on high clonal heterogeneity and hypermutation rate (mean 18.7%) in FL. When analyzing intracellular heterogeneity we observed single FL cells expressing simultaneously transcripts derived from the same immunoglobulin VDJ rearrangement but displaying high confidence single nucleotide variants. After applying strict filtering strategies to account for potential technical issues we defined the occurrence of “SHM snapshot events” when a single cell displayed a set of transcripts from a particular VDJ rearrangement with and without the occurring single nucleotide variant. Such events were detected in 8 of the 14 FL analyzed samples. In those 8 samples, 114 events were observed in which two different transcripts of one specific IG were found within a single cell. AID-related motifs (WRCY, WA and RCG) were found in 45% of the SHM snapshot events. SHM snapshot events were undetectable in CLL (5 samples) and 3 SHM snapshot events were detected in MBL (2 samples). In addition, the occurrence of SHM snapshot events was significantly associated with AID expression (Fisher exact test, p-value < 0.001). On the other hand, AID expression was not detectable on CLL and MBL samples.
Conclusion
Here we report for the first time the occurrence of ongoing somatic hypermutation at a single cell level in FL. The simultaneous detection of both the pre and post mutation IG mRNA transcripts within a single cell may be indicative of SHM occurring more recently than the lifespan of mRNA transcript. The detection of this phenomenon in 57% of FL samples and its association with AID expression suggests that AID-induced mutagenesis may be acting at a much higher rate than expected. This work highlights the role of AID in shaping the tumor heterogeneity in FL and the need to further understand the role of this enzyme in lymphomagenesis and tumor progression.
No relevant conflicts of interest to declare.
Objectives: Follicular lymphoma (FL) typically originates from premalignant mature B cells that carry the founder t(14;18) BCL2 translocation. Mutations in epigenetic modifiers and acquisition of ...N-glycosylation sites in CDR regions of the B-cell receptor (BCR) are recurrent secondary events in FL pathogenesis. Despite these oncogenic drivers, FL can remain indolent and clinically stable for years. The molecular events driving subclonal evolution into symptomatic progression and eventual transformation to aggressive lymphoma are insufficiently understood. FL cells are frozen in their B-cell development at the germinal center stage and undergo continuous somatic hypermutation mediated by expression of activation-induced deaminase (AID). We aim to identify crucial drivers of subclonal FL evolution by high-throughput mapping at single-cell resolution.
Methods: Viable FL cells were isolated and cryopreserved from 23 histologically or immunocytologically confirmed FL samples from 13 patients with informed consent. Full-length VDJ/VJ transcripts were isolated by unbiased template-switching ARTISAN PCR and massive parallel NGS sequencing on the PacBio platform. The clonal primordial FL BCR (pBCR) was reconstructed from unmutated IGV/IGJ sequences with the CDR3 of the least mutated BCR. Since the IgTree program was unable to process the obtained numbers of BCR sequences, we developed the WILLOW algorithm for analysis of BCR evolution based on the principle of maximum parsimony and on distance from the pBCR. Intraclonal BCR variability was quantified by Shannon's diversity index. 5' single cell transcriptomics and VDJ/VJ sequencing was performed on 2 pools of highly purified FL cells from 5 lymph node biopsies on the 10x Genomics platform. Data were deconvoluted based on expressed variants by the Single Cell Sample Matcher (SCSM) algorithm. Clustering based on gene expression profiles was performed by shared nearest neighbour (SNN) modularity optimization within the R Seurat package. Genes whose expression differed significantly (adjusted p<0.05) between clusters were assigned to gene ontology terms.
Results: ARTISAN PCR/PacBio NGS yielded a median of 743 full-length VDJ and VJ sequences (range 62-12782) per BCR chain with expected high intraclonal diversity (median 200 subclones, range 15-3301). WILLOW revealed dominant FL subclones with a subclonal hierarchy wherein multiple routes converged to offspring nodes with identical additional mutations rather than tree-like branching (Figure). In serial samples of 4 patients, lymph node biopsies had only marginally higher subclonal diversity than blood or bone marrow samples (p=0,055; Wilcoxon's matched-pairs signed rank test). Overall BCR mutational burden increased over time in sequential biopsies. Two cases of histological FL transformation were dominated by a single subclone (65% and 80% of all VDJ/VJ sequences, respectively) that was rare in the preceding FL BCR network (0.2% and 1.8%). Pooled transcriptomics data from 6050-6500 cells were assigned to individual samples by SCSM and revealed up to seven transcriptional clusters per FL. In 9 of 10 FL, genes assigned to immune function strongly contributed to separation into one or more clusters. Single cell VDJ/VJ sequencing yielded combined heavy and light chain BCR sequences for a median of 502 FL cells per biopsy (range 22 - 1919) that permitted mapping of subclonal evolution by WILLOW based on complete BCR information. Transcriptome clusters were not distributed evenly throughout the WILLOW FL BCR networks but rather statistically associated with distinct major FL subclones. Vice versa, major FL subclones within the same biopsy were distinguished by particular gene expression profiles.
Conclusions: WILLOW facilitates mapping of subclonal FL evolution based on high-throughput BCR sequencing. FL evolution proceeds in networks rather than tree-like branching, whereby acquisition of certain combinations of several BCR mutations can occur in parallel in different trajectories. Transcriptomic profiling of single FL cells identifies distinct clusters within a single biopsy. Mapping of these clusters to the FL cell position in the subclonal FL evolutionary network identifies putative mechanisms that are associated with subclonal progression. These mechanisms involve physiological B-cell signalling pathways.
Display omitted
No relevant conflicts of interest to declare.
PDF
