Single cell RNA sequencing to uncover intestinal cell-type specific cis-eQTL driving inherited predisposition to IBD

IBD is characterized by a chronic idiopathic inflammation of the gastrointestinal (GI) tract and consist of two main forms: ulcerative colitis and Crohn’s disease. The importance of genetic susceptibility has been well established through Genome Wide Association Studies (GWAS), which have identified over 200 risk loci for IBD. However, the « true causative » genes in these loci have been identified for only few on the basis of independently associated coding variants. Fine-mapping studies suggested that most risk variants cause “cis”-eQTL in disease relevant cell types, but recent post-GWAS studies could not find matching cis-eQTLs for the majority of risk loci (137/200). This indicates that the relevant cell types were either not present amongst the analyzed cell populations or under-represented. In this study, we performed cis-eQTL analysis with single cell RNA-seq of human gut biopsies to uncover the truly relevant cell types with higher resolution and unbiased approach. Biopsies were collected from three GI locations (ileum, transverse colon, rectum) from the same individuals. Cell suspensions were prepared, tagged by location and cell fraction using TotalseqB hashtag antibodies for multiplexing and processed to the 10X Genomics Chromium. Data were analyzed using Cellranger and Seurat to identify the cell clusters and marker genes. In total, 50 individuals’ biopsies data were integrated. Simultaneously, genotype was analyzed with Infinium OmniExpress-24v1 chip from 1 ml blood and imputed. Both scRNA-seq data and imputed genotypes were input to qtltools v1.3.1 for cis-eQTL anlaysis. Analysis are actually ongoing and will certainly generate new set of cell-based eQTL and determine whether some of these drive inherited predisposition to IBD by comparing the corresponding expression association patterns with disease association patterns using methods developed in our laboratory

Transcriptome analysis reveals tumor microenvironment changes in glioblastoma

A better understanding of transcriptional evolution of IDH-wild-type glioblastoma may be crucial for treatment optimization. Here, we perform RNA sequencing (RNA-seq) (n = 322 test, n = 245 validation) on paired primary-recurrent glioblastoma resections of patients treated with the current standard of care. Transcriptional subtypes form an interconnected continuum in a two-dimensional space. Recurrent tumors show preferential mesenchymal progression. Over time, hallmark glioblastoma genes are not significantly altered. Instead, tumor purity decreases over time and is accompanied by co-increases in neuron and oligodendrocyte marker genes and, independently, tumor-associated macrophages. A decrease is observed in endothelial marker genes. These composition changes are confirmed by single-cell RNA-seq and immunohistochemistry. An extracellular matrix-associated gene set increases at recurrence and bulk, single-cell RNA, and immunohistochemistry indicate it is expressed mainly by pericytes. This signature is associated with significantly worse survival at recurrence. Our data demonstrate that glioblastomas evolve mainly by microenvironment (re-)organization rather than molecular evolution of tumor cells

Transcriptome analysis reveals tumor microenvironment changes in glioblastoma

A better understanding of transcriptional evolution of IDH-wild-type glioblastoma may be crucial for treatment optimization. Here, we perform RNA sequencing (RNA-seq) (n = 322 test, n = 245 validation) on paired primary-recurrent glioblastoma resections of patients treated with the current standard of care. Transcriptional subtypes form an interconnected continuum in a two-dimensional space. Recurrent tumors show preferential mesenchymal progression. Over time, hallmark glioblastoma genes are not significantly altered. Instead, tumor purity decreases over time and is accompanied by co-increases in neuron and oligodendrocyte marker genes and, independently, tumor-associated macrophages. A decrease is observed in endothelial marker genes. These composition changes are confirmed by single-cell RNA-seq and immunohistochemistry. An extracellular matrix-associated gene set increases at recurrence and bulk, single-cell RNA, and immunohistochemistry indicate it is expressed mainly by pericytes. This signature is associated with significantly worse survival at recurrence. Our data demonstrate that glioblastomas evolve mainly by microenvironment (re-)organization rather than molecular evolution of tumor cells

Alcohol and dietary folate intake and promoter CpG island methylation in clear-cell renal cell cancer

We investigated whether alcohol and dietary folate intakes were associated with promoter methylation in clear-cell renal cell carcinoma (ccRCC). The Netherlands Cohort Study with a case-cohort design included 120,852 subjects aged 55-69yr in 1986. Diet was measured with a food-frequency questionnaire. After 20.3yr of follow-up, paraffin-embedded tumor blocks were collected. Methylation-specific polymerase chain reaction (MSP) was used to analyze promoter methylation of 11 genes. ccRCC cases were classified into low (0-19% of the genes), intermediate (20-39%), and high (40%+) methylation. Multivariable Cox regression analyses were conducted, stratified according to methylation, including 3980 subcohort members and 297 ccRCC cases. Increasing alcohol intake was associated with decreased ccRCC risk, but was not statistically significant; multivariable adjusted hazard ratio (HR) for 30g alcohol/day versus 0 g/day was 0.78 [95% confidence interval (CI): 0.48-1.24], and P-value for trend was 0.46. In strata according to methylation index, no significant heterogeneity was observed. Dietary folate intake was not associated with ccRCC risk. There was no significant heterogeneity between strata according to methylation index. There was no effect modification of alcohol and dietary folate intake on ccRCC risk, nor in strata according to methylation index. Our findings do not support the hypothesis that alcohol and dietary folate intakes are involved in ccRCC

Frequency of mosaicism points towards mutation-prone early cleavage cell divisions in cattle.

It has recently become possible to directly estimate the germ-line de novo mutation (dnm) rate by sequencing the whole genome of father-mother-offspring trios, and this has been conducted in human1–5, chimpanzee6, mice7, birds8and fish9. In these studiesdnm’s are typically defined as variants that are heterozygous in the offspring while being absent in both parents. They are assumed to have occurred in the germ-line of one of the parents and to have been transmitted to the offspring via the sperm cell or oocyte. This definition assumes that detectable mosaïcism in the parent in which the mutation occurred is negligible. However, instances of detectable mosaïcism or premeiotic clusters are well documented in humans and other organisms, including ruminants10–12. We herein take advantage of cattle pedigrees to show that as much as ∼30% to ∼50% ofdnm’s present in a gamete may occur during the early cleavage cell divisions in males and females, respectively, resulting in frequent detectable mosaïcism and a high rate of sharing of multiplednm’s between siblings. This should be taken into account to accurately estimate the mutation rate in cattle and other species.</jats:p

Colour-sidedness in Gloucester cattle is associated with a complex structural variant impacting regulatory elements downstream of KIT

Colour-sidedness is a striking coat colour pattern found in a number of cattle breeds, typically characterised by a white stripe that extends along the back, head and belly of the animal. This dominant phenotype is caused by two related translocations (Cs6 and Cs29) that alter a region downstream of the KIT gene. Gloucester cattle are native to the UK and are known for an unusual colour-sided pattern that does not extend to the head. We carried out whole-genome sequencing of two Gloucester bulls as well as colour-sided Irish Moiled, British White and Pustertaler Sprinzen for comparison. We found that the Gloucester cattle also have a complex structural variant downstream of KIT, which overlaps the regions involved in Cs6 and Cs29 .All three alleles potentially disrupt a number of putative regulatory elements downstream of KIT. These results complement and expand on the recently published work focused on the Pinzgauer breed from Austria, a carrier of the same colour-sided pattern as seen in Gloucester cattle.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

High resolution mapping of cross-over events in cattle using NGS data

peer reviewedHigh resolution mapping of cross-over events in cattle using NGS data Keywords: recombination, cattle, NGS Homologous recombination plays an important role in proper segregation of homologues in the first meiotic division. Failure in proper segregation results in aneuploidy, which is a leading cause for pregnancy loss in humans. Recently, global recombination rate has been studied in large cattle populations genotyped with SNP arrays ( 50K). However, the fine-scale resolution of these studies remained limited as a result of the relatively low marker density. Here we report high-resolution mapping of cross-over (CO) events in a cattle pedigree using whole genome sequence data. We carry out an extensive cleaning of our sequence data to remove errors (errors in the genome build, sequencing errors and presence of CNVs) that dramatically inflate CO counts. Using 5 million high quality sequence variants we identify 3,880 CO events in 155 male gametes and 3,088 CO events in 124 female gametes. The median resolution of the identified COs was 34 kb with about 70% of the events mapped to an interval less than 100 kb. The male and female map lengths were estimated at 27.5 M and 23.8 M respectively. Consistent with previous studies in cattle, we find higher recombination rate in males and higher frequency of COs at chromosome ends. Interestingly, compared to the map lengths estimated from SNP chip we find an increase of 3.7 and 2.7 M in male and female maps respectively. Despite the cleaning efforts, we cannot determine at this time whether the increased in map lengths correspond to CO missed with genotyping arrays, to spurious CO identified with NGS data (due to unidentified sources of errors) or both

INTER-INDIVIDUAL VARIATION OF DE NOVO ERV TRANSPOSITION RATE IN THE CATTLE GERMLINE

By studying a lethal recessive disease (cholesterol deficiency in Holstein-Friesian cattle breed), we recently discovered a family of endogenous retroviruses (ERV) still active in present-day cattle populations. To study the inter-individual variation of ERV de novo transposition rate (dnTR), we first used a pedigree-based method. As part of a larger project aimed at studying germline de novo mutation rate (Damona project), we generated a dataset of whole-genome sequences for 131 extended trios ((grand-) parents, proband and at least 5 grand-offspring). Here we mined them specifically for germline de novo ERV insertions - defined as insertions (i) detected in the proband, (ii) absent from its parents and (iii) transmitted to the next generation. Five de novo insertions were identified: 1 of maternal origin, 4 paternal, including 3 from the same sire and 2 within the same sperm cell. Thus, the ERV dnTR can be roughly estimated at 1 new insertion every ~50 gametes, yet with major differences between individuals (as 3 from the 5 detected events occurred in the same animal). To allow us to increase the number of studied gametes, we modified a method (Pooled CRISPR Inverse PCR sequencing: PCIP-seq) initially developed to detect somatic retroviral insertions (Artesi et al, 2020). We optimized it to directly locate and quantify de novo ERV insertional events in the male germline (sperm DNA). We are currently applying it to a large cohort of sires (n > 200). The resulting normalized sire-specific dnTR will be treated as a quantitative molecular phenotype to perform a genome-wide association study in order to pinpoint genomic loci influencing ERV dnTR in the cattle germline. This new quantitative method can easily be adapted to other families of active transposable elements in livestock