Search Results - EGA European Genome-Phenome Archive

GWAS data on human pancreatic islets from 191 donors - Lund

Most signals detected by genome-wide association studies map to non-coding sequence and their tissue-specific effects influence transcriptional regulation. However, many key tissues and cell-types required for appropriate functional inference are absent from large-scale resources such as ENCODE and GTEx. We explored the relationship between genetic variants influencing predisposition to type 2 diabetes (T2D) and related glycemic traits, and human pancreatic islet transcription using RNA-Seq and genotyping data from 420 islet donors. We find: (a) eQTLs have a variable replication rate across the 44 GTEx tissues (<73%), indicating that our study captured islet-specific cis-eQTL signals; (b) islet eQTL signals show marked overlap with islet epigenome annotation, though eQTL effect size is reduced in the stretch enhancers most strongly implicated in GWAS signal location; (c) selective enrichment of islet eQTL overlap with the subset of T2D variants implicated in islet dysfunction; and (d) colocalization between islet eQTLs and variants influencing T2D or related glycemic traits, delivering candidate effector transcripts at 23 loci, including DGKB and TCF7L2. Our findings illustrate the advantages of performing functional and regulatory studies in tissues of greatest disease-relevance while expanding our mechanistic insights into complex traits association loci activity with an expanded list of putative transcripts implicated in T2D development.

Study EGAS00001004044

Prebiotic inulin-type fructans induce specific changes in the human gut microbiota

ObjectiveContrary to the long-standing prerequisite of inducing selective (i.e. bifidogenic) effects, recent findings suggest that prebiotic interventions lead to ecosystem-wide microbiota shifts. Yet, a comprehensive characterization of this process is still lacking. Here, we apply 16S rDNA microbiota profiling and matching (GC-MS) metabolomics to assess the consequences of inulin fermentation both on the composition of the colon bacterial ecosystem and fecal metabolites profiles.DesignFecal samples collected during a double blind, randomized, cross-over intervention study (NCT02548247) set up to assess the effect of inulin consumption on stool frequency in healthy adults with mild constipation were analyzed. Fecal microbiota composition and metabolite profiles were linked to the study’s clinical outcome as well as to quality-of-life measurements recorded.ResultsWhile fecal metabolite profiles were not significantly altered by inulin consumption, our analyses did detect a modest effect on global microbiota composition. At the same time, specific inulin-induced changes in relative abundances of Anaerostipes, Bilophila, and Bifidobacterium were identified. The observed decrease in Bilophila abundances following inulin consumption was associated with both softer stools and a favorable change in constipation-specific quality of life measures.ConclusionsEcosystem-wide analysis of the effect of a dietary intervention with prebiotic inulin-type fructans on the colon microbiota revealed that this effect is specifically associated to three genera, one of which (Bilophila) representing a promising novel target for mechanistic research.

Study EGAS00001002173

MutWP1__CRUK_Grand_Challenge_Mutographs_of_Cancer__Oesophageal_squamous_cell_carcinoma

The Mutographs project aims to advance our understanding of the causes of cancer through studies of mutational signatures. Led by Mike Stratton, together with Paul Brennan, Ludmil Alexandrov, Allan Balmain, David Phillips and Peter Campbell, this large-scale international research endeavour was awarded a Cancer Research UK Grand Challenge. Different patterns of somatic mutation are generated by the different environmental, lifestyle and genetic factors that cause cancer, many of them are still unknown. Within Mutographs, the International Agency for Research on Cancer is coordinating the recruitment of 5000 individuals with cancer (colorectal, renal, pancreatic, oesophageal adenocarcinoma or oesophageal squamous cancers) across 5 continents to explore whether different mutational signatures explain marked variation in incidence. In brief, through an international network of collaborators around the world, biological materials are collected, along with demographic, histological, clinical and questionnaire data. Whole genome sequences of tumour-germline DNA pairs are generated at the Wellcome Trust Sanger Institute (Illumina HiSeqX, 40X and 20X depth respectively). Somatic mutational signatures are subsequently extracted by non-negative matrix factorisation methods and correlated with risk factors data. Through an enhanced understanding of cancer aetiology, Mutographs unprecedented effort is anticipated to outline modifiable risk factors, lead to new approaches to prevent cancer, and provide opportunities to empower early detection, refine high-risk groups and contribute to further therapeutic development.

Study EGAS00001002725

MutWP1__CRUK_Grand_Challenge_Mutographs_of_Cancer__pancreas

The Mutographs project aims to advance our understanding of the causes of cancer through studies of mutational signatures. Led by Mike Stratton, together with Paul Brennan, Ludmil Alexandrov, Allan Balmain, David Phillips and Peter Campbell, this large-scale international research endeavour was awarded a Cancer Research UK Grand Challenge. Different patterns of somatic mutation are generated by the different environmental, lifestyle and genetic factors that cause cancer, many of them are still unknown. Within Mutographs, the International Agency for Research on Cancer is coordinating the recruitment of 5000 individuals with cancer (colorectal, renal, pancreatic, oesophageal adenocarcinoma or oesophageal squamous cancers) across 5 continents to explore whether different mutational signatures explain marked variation in incidence. In brief, through an international network of collaborators around the world, biological materials are collected, along with demographic, histological, clinical and questionnaire data. Whole genome sequences of tumour-germline DNA pairs are generated at the Wellcome Trust Sanger Institute (Illumina HiSeqX, 40X and 20X depth respectively). Somatic mutational signatures are subsequently extracted by non-negative matrix factorisation methods and correlated with risk factors data. Through an enhanced understanding of cancer aetiology, Mutographs unprecedented effort is anticipated to outline modifiable risk factors, lead to new approaches to prevent cancer, and provide opportunities to empower early detection, refine high-risk groups and contribute to further therapeutic development.

Study EGAS00001002726

Insular Celtic population structure and genomic footprints of migration

Previous studies of the genetic landscape of Ireland have suggested homogeneity, with population substructure undetectable using single-marker methods. Here we have harnessed the haplotype-based method fineSTRUCTURE in an Irish genome-wide SNP dataset, identifying 23 discrete genetic clusters which segregate with geographical provenance. Cluster diversity is pronounced in the west of Ireland but reduced in the east where older structure has been eroded by historical migrations. Accordingly, when populations from the neighbouring island of Britain are included, a west-east cline of Celtic-British ancestry is revealed along with a particularly striking correlation between haplotypes and geography across both islands. A strong relationship is revealed between subsets of Northern Irish and Scottish populations, where discordant genetic and geographic affinities reflect major migrations in recent centuries. Additionally, Irish genetic proximity of all Scottish samples likely reflects older strata of communication across the narrowest inter-island crossing. Using GLOBETROTTER we detected Irish admixture signals from Britain and Europe and estimated dates for events consistent with the historical migrations of the Norse-Vikings, the Anglo-Normans and the British Plantations. The influence of the former is greater than previously estimated from Y chromosome haplotypes. In all, we paint a new picture of the genetic landscape of Ireland, revealing structure which should be considered in the design of studies examining rare genetic variation and its association with traits.

Study EGAS00001002769

Single cell sequencing reveals the origin and the order of mutation acquisition in T-cell acute lymphoblastic leukemia.

Next-generation sequencing has provided a detailed overview of the various genomic lesions implicated in the pathogenesis of T-cell acute lymphoblastic leukemia (T-ALL). Typically, 10 to 20 genomic lesions are found in T-ALL cells at diagnosis. However, it is currently unclear in which order these mutations are acquired and in which progenitor cells this is initiated. To address these questions, we used targeted single-cell sequencing of total bone marrow cells and CD34+CD38- multipotent progenitor cells for 4 T-ALL cases. Hierarchical clustering detected a dominant leukemia clone at diagnosis, accompanied by a few subclones harboring only a fraction of the mutations. We developed a graph-based algorithm to determine the order of mutation acquisition. Two of the four patients had an early event in a known oncogene (MED12, STAT5B) amongst various pre-leukemic events. Intermediate events included loss of 9p21 (CDKN2A/B) and acquisition of fusion genes, while NOTCH1 mutations were typically late events. Analysis of CD34+CD38- cells and myeloid progenitors revealed that in half of the cases somatic mutations were detectable in multipotent progenitor cells. We demonstrate that targeted single cell sequencing can elucidate the order of mutation acquisition in T-ALL and that T-ALL development can start in a multipotent progenitor cell.

Study EGAS00001002830

Immune heterogeneity in small cell lung cancer and vulnerability to immune checkpoint blockade

Following the pivotal Phase III IMpower133 study, atezolizumab (anti–PD-L1), combined with carboplatin and etoposide (CE), was the first immune checkpoint inhibitor approved for first-line treatment of extensive-stage small cell lung cancer (ES-SCLC) and is now a standard of care. A clearer understanding of therapeutically relevant SCLC subsets is needed to improve outcomes and to identify rational combination strategies. Transcriptomic analyses and nonnegative matrix factorization were conducted on 271 pre-treatment patient tumor samples from IMpower133 and four subsets with general concordance to previously reported SCLC subtypes were identified. Deeper investigation into the immune heterogeneity relevant to clinical outcomes uncover two subsets with differing neuroendocrine (NE) versus non-neuroendocrine (non-NE) phenotypes that demonstrated hallmarks of immune cell infiltration. The balance of tumor-associated macrophage (TAM) to T-effector signals distinguished these inflamed subsets. Tumors with low TAM but high T-effector signals had a NE phenotype and demonstrated longer overall survival with PD-L1 blockade and CE versus CE alone than did tumors with high TAM and high T-effector signal, which had a non-NE phenotype. The delineation of SCLC immune heterogeneity offers a clinically relevant approach to discriminate SCLC patients likely to benefit most from immunotherapeutic approaches and highlights the complex mechanisms underlying response and resistance to immune checkpoint blockade.

Study EGAS50000000138

Targeted_replication_of_LVOTO_genes

Non-syndromic cases of congenital heart defects (CHD) exhibit variable modes of inheritance (Mendelian and non-Mendelian). Several studies have identified strong candidates in humans by taking a candidate gene approach as well as by using whole exome next generation sequencing (NGS). So far these studies could only explain a minor fraction of the observed phenotype in humans, most of them in syndromic cases and no single study has focused on the subset of cases with left ventricular outflow tract obstruction (LVOTO). To discover novel disease-causing genes a large cohort of patients with LVOTO, approximately 100 cases, 25 families and 100 trios have been exome sequenced. This study based on NGS sequencing data yielded several known and novel compelling candidate genes, such as MYH6, NR2F2 and MYH11, but also novel ones, such as ITGB4. To evaluate the significance of our findings in a replication cohort we assembled another 1614 cases with an LVOTO phenotype from our collaborators in Toronto, Berlin and Amsterdam. Targeted resequencing in this additional cohort will help to find additional cases with mutations in the identified candidate genes to strengthen genotype-phenotype association. We will use control data from the INTERVAL project for case/control analyses The pulldowns will be performed as 24-plex ISC with 192 or greater indexes, and the sequencing will be performed with 192 samples per lane, requiring 9 lanes of sequencing.

Study EGAS00001001238

Hyperhaploid Multiple Myeloma

Hyperhaploid multiple myeloma is a rare numerical aberration group defined by a range of 24-34 chromosomes which is associated with a poor prognosis. Hyperhaploid samples were sequenced by either exome or targeted panel sequencing. Samples had a median of 13 monosomies (range 12-14), which in general were those not associated with trisomies in hyperdiploid samples. The chromosomes traditionally trisomic in hyperdiploid myeloma were disomic in hyperhaploid myeloma with retention of heterodisomy. We examined the hyperhaploid samples for frequently mutated genes and found that 9/9 (100%) hyperhaploid samples had a mutation in TP53, exceeding the overall rate of mutation in newly diagnosed patients (5.5%), indicating an oncogenic dependency in this group. All samples with TP53 mutation also had monosomy of chromosome 17, indicating bi-allelic inactivation of TP53. As such, this high risk group is part of Double-Hit myeloma. DNA was obtained from either CD138+ cells from the bone marrow of multiple myeloma patients (tumor) or from stem cell harvests from the same patient (control). 100 ng of DNA was fragmented, end-repaired, and adapters ligated using the HyperPlus kit (KAPA Biosystems). After PCR amplification the libraries were hybridized with probes against either the entire exome (MedExome, Nimblegen) or a targeted panel of 140 genes using SeqCap reagents (Nimblegen). Hybridized libraries underwent further amplification before being sequenced on a NextSeq500 (Illumina) using 75 bp paired end reads.

Study EGAS00001003203

PAX5 biallelic genomic alterations define a novel subgroup of B cell precursor acute lymphoblastic leukemia

Chromosomal rearrangements and specific aneuploidy patterns are initiating events and define subgroups in B cell precursor acute lymphoblastic leukemia (BCP-ALL). Here we analyzed 250 BCP-ALL cases and identified a novel subgroup (‘PAX5-plus’, n=19) by distinct DNA methylation and gene expression profiles. All patients in this subgroup harbored mutations in the B-lineage transcription factor PAX5, with p.P80R as hotspot. Mutations either affected two independent codons, consistent with compound heterozygosity, or suffered LOH predominantly through chromosome 9p aberrations. These biallelic events resulted in disruption of PAX5 transcriptional programs regulating B cell differentiation and tumor suppressor functions. Homozygous CDKN2A/B deletions and RAS activating hotspot mutations were highly enriched as cooperating events in the genomic profile of PAX5-plus ALL. Together this defined a specific pattern of triple alterations, exclusive to the novel subgroup. PAX5-plus ALL was observed in pediatric and adult patients. Although restricted by the limited sample size, a tendency for more favorable clinical outcome was observed with 10 of 12 adult PAX5-plus patients achieving long-term survival. PAX5-plus represents the first BCP-ALL subgroup defined by sequence alterations in contrast to gross chromosomal events and exemplifies how deregulated differentiation (PAX5), impaired cell cycle control (CDKN2A/B) together with sustained proliferative signaling (RAS) cooperatively drives leukemogenesis.

Study EGAS00001003209

Mid-pass Whole-genome Sequencing in a Malagasy Cohort Uncovers Body Composition Associations

The majority of human genomic research studies have been conducted in European-ancestry cohorts, reducing the likelihood of detecting potentially novel and globally impactful findings. Here, we present mid-pass whole-genome sequencing data and a genome-wide association study in a cohort of 264 self-reported Malagasy individuals from three locations on the island of Madagascar. We describe genetic variation in this Malagasy cohort, providing insight into the shared and unique patterns of genetic variation across the island. We observe phenotypic variation by location, and find high rates of hypertension particularly in the Southern Highlands sampling site as well as elevated self-reported malaria prevalence in the West Coast site relative to other sites. After filtering to a subset of 214 minimally-related individuals, we find a number of genetic associations with body composition traits, including many variants that are only observed in African populations or populations with admixed African ancestry from the 1000 Genomes Project. This study highlights the importance of including diverse populations in genomic research for the potential to gain novel insights, even with small cohort sizes. This project was conducted in partnership and consultation with local stakeholders in Madagascar and serves as an example of genomic research that prioritizes community engagement and that has potential impacts on our understanding of human health and disease.

Study EGAS50000000496

Identification and characterisation of pathogenic and non-pathogenic FGF14 repeat expansions

Repeat expansions in FGF14 cause autosomal dominant late-onset cerebellar ataxia (SCA27B) with estimated pathogenic thresholds of 250 (incomplete penetrance) and 300 AAG repeats (full penetrance), but the sequence of pathogenic and non-pathogenic expansions remains unexplored. Here, we demonstrate that STRling and ExpansionHunter accurately detect FGF14 expansions from short-read genomic data using outlier approaches. By combining long-range PCR and nanopore sequencing in 169 patients with cerebellar ataxia and 802 controls, we compare FGF14 expansion alleles, including interruptions and flanking regions. Uninterrupted AAG expansions are significantly enriched in patients with ataxia from a lower threshold (180-200 repeats) than previously reported based on expansion size alone. Conversely, AAGGAG hexameric expansions are equally frequent in patients and controls. Distinct 5' flanking regions, interruptions and pre-repeat sequences correlate with repeat size. Furthermore, pure AAG (pathogenic) and AAGGAG (non-pathogenic) repeats form different secondary structures. Regardless of expansion size, SCA27B is a recognizable clinical entity characterized by frequent episodic ataxia and downbeat nystagmus, similar to the presentation observed in a family with a previously unreported nonsense variant (SCA27A). Overall, this study suggests that SCA27B is a major overlooked cause of adult-onset ataxia, accounting for 23-31% of unsolved patients. We strongly recommend re-evaluating pathogenic thresholds and integrating expansion sequencing into the molecular diagnostic process.

Study EGAS50000000481

Landscape of somatic mutations and clonal evolution in mantle cell lymphoma

Mantle cell lymphoma (MCL) is an aggressive tumor, but a subset of patients may follow an indolent clinical course. To understand the mechanisms underlying this biological heterogeneity, we performed whole-genome and/or whole-exome sequencing on 29 MCL cases and their respective matched normal DNA, as well as 6 MCL cell lines. Recurrently mutated genes were investigated by targeted sequencing in an independent cohort of 172 MCL patients. We identified 25 significantly mutated genes, including known drivers such as ataxia-telangectasia mutated (ATM), cyclin D1 (CCND1), and the tumor suppressor TP53; mutated genes encoding the anti-apoptotic protein BIRC3 and Toll-like receptor 2 (TLR2); and the chromatin modifiers WHSC1, MLL2, and MEF2B. We also found NOTCH2 mutations as an alternative phenomenon to NOTCH1 mutations in aggressive tumors with a dismal prognosis. Analysis of two simultaneous or subsequent MCL samples by whole-genome/whole-exome (n = 8) or targeted (n = 19) sequencing revealed subclonal heterogeneity at diagnosis in samples from different topographic sites and modulation of the initial mutational profile at the progression of the disease. Some mutations were predominantly clonal or subclonal, indicating an early or late event in tumor evolution, respectively. Our study identifies molecular mechanisms contributing to MCL pathogenesis and offers potential targets for therapeutic intervention.

Study EGAS00001000510

Mutational signatures of aflatoxin

Aflatoxin B1 (AFB1) is a mutagen and IARC (International Agency for Research on Cancer) Group 1 carcinogen that causes hepatocellular carcinoma (HCC). Here we present the first whole genome data on the mutational signatures of AFB1 exposure from a total of > 40,000 mutations in four experimental systems: two different human cell lines, and in liver tumors in wild-type mice and in mice that carried a hepatitis B surface antigen transgene – this to model the multiplicative effects of aflatoxin exposure and hepatitis B in causing HCC. AFB1 mutational signatures from all four experimental systems were remarkably similar. We integrated the experimental mutational signatures with data from newly-sequenced HCCs from Qidong County, China, a region of well-studied aflatoxin exposure. This indicated that COSMIC mutational signature 24, previously hypothesized to stem from aflatoxin exposure, indeed likely represents AFB1 exposure, possibly combined with other exposures. Among published somatic mutation data, we found evidence of AFB1 exposure in 0.7% of HCCs treated in North America, 1% of HCCs from Japan, but 16% of HCCs from Hong Kong. Thus, aflatoxin exposure apparently remains a substantial public health issue in some areas. This aspect of our study exemplifies the promise of future widespread resequencing of tumor genomes in providing new insights into the contribution of mutagenic exposures to cancer incidence.

Study EGAS00001002490

Exon4-mutations in KRAS Affect MEK-ERK and Pi3K-AKT Signaling in Multiple Myeloma

Approximately 40% of MM cases harbor a point mutation in either KRAS or NRAS. However, it is still unclear whether RAS-mutations are associated with disease outcome. Specifically, no information exists whether KRAS-mutations have an impact on survival in MM patients at diagnosis that were treated with novel agents (e.g. bortezomib, lenalidomide). Targeting KRAS directly for therapeutic purposes is still impossible, but recently the first covalent mutant-specific inhibitor targeting KRASG12C made it into clinical trials. However, other KRAS-hotspot-mutations exist in MM patients, including the less common exon4-mutations. Within the current study, the coding regions of KRAS in 80 MM patients at diagnosis, uniformly treated with bortezomib and high-dose chemotherapy, were deep-sequenced. Moreover, the functional impact of KRASG12A as well as the two exon4-mutations A146T (AMO1) and A146V (MM patient) on different survival pathways was investigated. Specifically, an siRNA-mediated knockdown in AMO1 was performed and KRASWT, KRASG12A, KRAS146T and KRAS146V were overexpressed in the KRASWT cell lines JJN3 and OPM2 using lentiviral transduction and the transposase based Sleeping Beauty system. KRAS-mutations were not associated with survival. Yet, the expression of all KRAS-mutants had an impact on MEK/ERK-signaling in two independent MM cell lines and on AKT/PI3K-signaling in one MM cell line.

Study EGAS00001004110

Comprehensive Genomic Characterization of Gene Therapy-Induced T-cell Acute Lymphoblastic Leukemia (H007)

Six out of ten patients in a hematopoietic stem cell gene therapy (GT) trial for Wiskott-Aldrich syndrome (WAS) developed T-cell acute lymphoblastic leukemia (T-ALL) associated with gamma-retroviral vector integration at the LMO2 locus. We hypothesized that detailed molecular analysis of T-ALL driven by temporally defined first deregulation of LMO2 might offer valuable insights into the pathogenesis of GT-associated and sporadic T-ALL (HIPO project 007). In all six patients whole-genome and transcriptome sequencing demonstrated that in addition to oncogenic chromosomal translocations involving the T-cell receptor loci, retrovirally induced leukemias faithfully recapitulated secondary genomic events observed in sporadic T-ALL. Multiple genetic events culminated in the development of a rapidly proliferating leukemic clone and onset of clinical manifestation at 1.3 to 5 years after infusion of the gene corrected transplant. These genetic alterations affected NOTCH1 and PI3K signaling, cell cycle regulation as wells as T-cell transcription factors LEF1 and TCF1. Two of the six cases displayed a phenotype reminiscent of early immature T-ALLs, and lacked additional T-ALL typical alterations. Our analysis shows that insertional mutagenesis involving the LMO2 locus triggers T-ALL development due to diverse genetic alterations in large parts, but not exclusively, in known T-ALL driver genes, demonstrating the molecular diversity of GT-associated T-ALL.

Study EGAS00001003870

Intratumoural heterogeneity and immune modulation in lung adenocarcinoma of female smokers and never smokers

Lung cancer is still the leading cause of cancer death worldwide despite declining smoking prevalence in industrialised countries. Although lung cancer is highly associated with smoking status, a significant proportion of lung cancer cases develop in patients who never smoked, with an observable bias towards female never smokers. A better understanding of lung cancer heterogeneity and immune system involvement during tumour evolution and progression in never smokers is therefore highly warranted. We employed single nucleus transcriptomics of surgical lung adenocarcinoma (LADC) and normal lung tissue samples from patients with or without smoking history. Immune cells as well as fibroblasts and endothelial cells respond to tobacco smoke exposure by inducing a highly inflammatory state in normal lung tissue. In the presence of LADC, we identified differentially expressed transcriptional programmes in macrophages and cancer-associated fibroblasts, providing insight into how the niche favours tumour progression. Within tumours, we distinguished eight subpopulations of neoplastic cells in female smokers and never smokers. Through pseudotemporal ordering, we inferred a trajectory towards two differentiated tumour cell states implicated in cancer progression and invasiveness. A proliferating cell population sustaining tumour growth exhibits differential immune modulating signatures in both patient groups. Our results resolve cellular heterogeneity and immune interactions in LADC, with a special emphasis on female never smokers and implications for the design of therapeutic approaches.

Study EGAS00001006331

5- FU treated organoids

5-Fluorouracil (5-FU) is a chemotherapeutic drug component that is commonly used for the treatment of solid cancers. The anticancer properties of 5-FU have been attributed to interference with nucleotide synthesis and through direct incorporation into the DNA as being a pyrimidine analog. As both mechanisms of action may have a mutational impact on surviving tumor cells, we performed three independent analyses to characterize the genomic consequences of 5-FU treatment: i) in vitro treatment of intestinal organoids with 5-FU followed by whole genome sequencing, ii) genome-wide mutation analyses in tumor samples from 5-FU treated patients with breast or colorectal cancer, and iii) analysis of paired biopsies from patients treated with 5-FU between biopsies. Our results demonstrate that both in vitro and in vivo 5-FU causes a mutational pattern that is dominated by T>G substitutions in a CTT context and with strong resemblance to COSMIC signature 17. Interestingly, this signature is also found in non 5-FU treated patients, most prominently in esophageal tumors, indicating that distinct endogenous and exogenous triggers can converge into highly similar mutational signatures. Furthermore, our results suggest that 5-FU may have adverse mutagenic effects on healthy cells and contributes to genetic variability in surviving cancer cells thereby contributing to tumor evolution.

Study EGAS00001003592

Genetic insights into the biological mechanisms governing human ovarian ageing

Reproductive longevity is critical for fertility and impacts healthy ageing in women, yet insights into the underlying biological mechanisms and treatments to preserve it are limited. Here, we identify 290 genetic determinants of ovarian ageing, assessed using normal variation in age at natural menopause (ANM) in ~200,000 women of European ancestry. These common alleles influence clinical extremes of ANM; women in the top 1% of genetic susceptibility have an equivalent risk of premature ovarian insufficiency to those carrying monogenic FMR1 premutations. Identified loci implicate a broad range of DNA damage response (DDR) processes and include loss-of-function variants in key DDR genes. Integration with experimental models demonstrates that these DDR processes act across the life-course to shape the ovarian reserve and its rate of depletion. Furthermore, we demonstrate that experimental manipulation of DDR pathways highlighted by human genetics increase fertility and extend reproductive life in mice. Causal inference analyses using the identified genetic variants indicates that extending reproductive life in women improves bone health and reduces risk of type 2 diabetes, but increases risks of hormone-sensitive cancers. These findings provide insight into the mechanisms governing ovarian ageing, when they act across the life-course, and how they might be targeted by therapeutic approaches to extend fertility and prevent disease.

Study EGAS00001004947

Loss of LGR4/GPR48 causes severe neonatal salt-wasting due to disrupted WNT signaling altering adrenal zonation

Disorders of isolated mineralocorticoid deficiency causing potentially life-threatening salt-wasting crisis early in life have been associated with gene variants of aldosterone biosynthesis or resistance, but in some patients no such variants are found. WNT/β-catenin signaling is crucial for differentiation and maintenance of the aldosterone producing adrenal zona glomerulosa (zG). We describe a highly consanguineous family with multiple perinatal deaths or infants presenting at birth with failure to thrive, severe salt-wasting crises associated with isolated hypoaldosteronism, nail anomalies, short stature, and deafness. Whole exome sequencing revealed a homozygous splice variant in the R-SPONDIN receptor LGR4 gene (c.618-1G>C) regulating WNT signaling. The resulting transcripts affected protein function and stability, and resulted in loss of Wnt/β-catenin signaling in vitro. The impact of LGR4 inactivation was analyzed by adrenal cortex specific ablation of Lgr4, using Lgr4Flox/Flox mated with Sf1:Cre mice. Inactivation of Lgr4 within the adrenal cortex in the mouse model caused decreased WNT signaling, aberrant zonation with deficient zG and reduced aldosterone production. Thus, human LGR4 mutations establish a direct link between LGR4 inactivation and decreased canonical WNT signaling with abnormal zG differentiation and endocrine function. Therefore, variants in WNT signaling and its regulators should systematically be considered in familial hyperreninemic hypoaldosteronism.

Study EGAS00001006808

CYP2C19 long-read sequencing

Inter-individual differences in drug response based on genetic variations can lead to drug toxicity and treatment inefficacy. A large part of this variability is caused by genetic variants in pharmacogenes. Unfortunately, the Single Nucleotide Variant (SNV) panels currently used in clinical pharmacogenomic (PGx) testing are unable to detect all genetic variability in these genes. Long-read sequencing, on the other hand, has been shown to be able to resolve complex (pharmaco)genes. In this study we have assessed the added value of long-read sequencing for PGx focusing on the clinically important and highly polymorphic CYP2C19 gene. With a capture-based long-read sequencing panel we were able to characterize the entire region and assign variants to their allele of origin (phasing), resulting in the identification of 813 unique variants in 37 samples. To assess the clinical utility of this data we have compared the performance of three different *-allele tools (Aldy, PharmCat and PharmaKU) which are specifically designed to assign clinical haplotypes to pharmacogenes based on all input variants. We conclude that long-read sequencing can improve our ability to characterize the CYP2C19 locus, help to identify novel haplotypes and that *-allele tools are an useful asset in phenotype prediction. Ultimately, this approach could help to better predict an individual’s drug response and improve therapy outcomes.

Study EGAS00001006929

BMP4 and temozolomide synergize in the majority of patient derived glioblastoma cultures

The poor prognosis of patients with glioblastoma is partly due to resistance to standard treatment, which combines chemoradiation using temozolomide (TMZ). The concept of cancer stem cells provided new insight into tumor resistance and management, and isolated Glioblastoma stem-like cells (GSCs) which contribute to this therapy resistance. Bone morphogenetic protein 4 (BMP4) is a differentiation factor that stimulates astroglial differentiation of such GSCs, thereby reducing their self-renewal. Exposing GSCs to BMP4 could therefore sensitize them to chemotherapy with TMZ, but previous studies with various treatment paradigms have produced contradictory results in this matter. Therefore, we re-defined the optimal treatment paradigm for the combination of TMZ and BMP4 and subsequently re-assessed the inter-tumoral variability in response to co-treatment with TMZ+BMP4 in vitro. First, the simultaneous treatment was found more effective than sequential therapy. Second, after application of our optimized treatment protocol, 70% of a total of 20 patient-derived glioblastoma cultures showed synergy when the treatments were combined. The synergistic effect was a result of increased cellular apoptosis and not of decreased proliferation. Comparative RNA-sequencing suggested that combination treatment results in decreased MAPK signaling. We conclude that addition of BMP4 to standard therapy with TMZ could enlarge the group of patients with good therapeutic response.

Study EGAS00001007095

What is metadata?

What is metadata? Data represents units of information and metadata can be seen, simply put, as data about other data. For example, in a simple experiment in which a patient genome was sequenced, there are multiple and diverse fields of metadata: from the biological sex of the patient to the sequencing platform used. Another way to understand metadata, following our genomic experiment example, is going through the different layers of (meta)data: One of the basic units of information (data) would be the sequence of nucleotides (e.g. GCTGG and GCCGG). This DNA sequence can be contextualised within a chromosome by providing its locus (e.g. β-globin gene). This data, the locus, is about other data, the sequence of nucleotides, which would render it metadata. We can go one step further, and provide more context, by giving the associated phenotype of each of these sequences: GCTGG corresponds to a healthy individual, while GCCGG corresponds to a person with β-thalassaemia. Once again, this data is contextualising the data we have provided up to this point, which makes it metadata. With this simple example, what we are doing is adding layers of data that describe the ones below. Had we given the sequences alone, little would be possible from the researchers' point of view. But, now that the data has its accompanying metadata, a researcher could compare both sequences and their phenotypes to infer a possible cause of the disorder. Each layer of information broadens the possible applications of the data. Now that we know what metadata is, it is time to emphasise the importance of submitting good quality metadata. The quality of the metadata will determine whether the data is usable, discoverable, and interpretable by researchers and clinicians. One example that highlights the importance of good quality metadata is the description of biological sex of a patient: while the terms "female," "girl," or "woman" all represent the same concept, they are not harmonised. These different representations of a concept can create confusion and errors in data analysis. By applying standard definitions and terminologies to biological sex metadata, we can avoid misunderstandings and facilitate the integration of different datasets. In order to comply with the FAIR principles and, more specifically, to allow for the (meta)data to be interoperable (i.e. used universally and unambiguously), we need to place some standards onto the (meta)data EGA archives. These can be as simple as asserting that the given patient's biological sex is one of the correct terms (e.g. female or male); or as complex as multiple layers and combinations of validation steps with existing records. You can find further details on how EGA manages these standards on our metadata model page.

Documentation submission/metadata/what-is-metadata

Target gene sequencing for human normal endometrial glands

The raw fastq files target sequencing of 112 genes for 1,298 endometrial glands and matched blood samples. The paired-end sequencing data sets (R1 and R2) are deposited. ABCC1, ACRC, ANK3, ARHGAP35, ARID1A, ARID5B, ATCAY, ATM, ATR, BARD1, BCOR, BRCA1, BRCA2, BRD4, BRIP1, CAMTA1, CDC23, CDYL, CFAP54, CHD4, CHEK1, CHEK2, CTCF, CTNNB1, CUX1, DGKA, DISP2, DYNC2H1, EMSY, FAAP24, FAM135B, FAM175A, FAM65C, FANCA, FANCB, FANCC, FANCD2, FANCE, FANCF, FANCG, FANCI, FANCL, FANCM, FAT1, FAT3, FBN2, FBXW7, FGFR2, FRG1, GPR50, HEATR1, HIST1H4B, HNRNPCL1, HOOK3, KIAA1109, KIF26A, KMT2B, KMT2C, KRAS, LAMA2, LRP1B, MLH1, MON2, MRE11A, MSH2, MSH6, MTOR, NBN, PALB2, PHEX, PIK3CA, PIK3R1, PLXNB2, PLXND1, PMS2, POLE, POLR3B, PPP2R1A, PTEN, PTPN13, RAD50, RAD51, RAD51B, RAD51C, RAD51D, RAD52, RAD54B, RAD54L, RICTOR, SACS, SIGLEC9, SLC19A1, SLX4, SPEG, STT3A, TAF1, TAF2, TAS2R31, TFAP2C, TNC, TONSL, TP53, TTC6, UBA7, VNN1, WT1, XIRP2, ZBED6, ZC3H13, ZFHX3, ZFHX4, ZMYM4.

Dataset EGAD00001008465

Polyclonal selection of immune checkpoint mutations in thyroid autoimmunity - ExomeNanoSeq

Our immune system contains multiple tolerance checkpoints to prevent the activation of self-reactive lymphocytes. How some lymphocytes escape these constraints to cause autoimmune disease remains poorly understood. A long-standing hypothesis in autoimmunity posits that somatic mutations in immune regulatory genes may enable self-reactive lymphocytes to bypass tolerance checkpoints. However, testing this hypothesis has proved challenging due to technical limitations in detecting somatic mutations in polyclonal cell populations. Here, we use deep whole-exome and targeted NanoSeq, a highly accurate single-molecule DNA sequencing protocol, to comprehensively search for driver mutations in autoimmune thyroid disease. This revealed a remarkably high number of B cell clones convergently acquiring loss-of-function somatic mutations in key immune checkpoint genes TNFRSF14 (also known as HVEM) and CD274 (encoding PD-L1), as well as less frequent driver mutations in a diversity of other immune genes. In highly inflamed biopsies, we detected tens to hundreds of independent immune-checkpoint mutant clones. Laser capture microdissection, methylation sequencing, spatial transcriptomics, immunohistochemistry and single-nucleus DNA sequencing localised these mutations to non-naive B cells and revealed frequent co-occurrence of drivers in single cells. We found widespread TNFRSF14 biallelic loss in mutant B cells, and several clones with as many as 4-6 driver mutations. Whilst each clone accounts for a small proportion of cells (typically <1%), the myriad mutant clones in each donor collectively amounted to a substantial fraction of B cells harbouring one or more driver mutations. Our results support the hypothesis that somatic mutations in autoimmune lymphocytes may allow them to escape tolerance constraints through a polyclonal cascade of somatic evolution. These findings provide new insights into the molecular basis of autoimmune disease and suggest novel diagnostic and therapeutic avenues.

Dataset EGAD00001016058

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