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.
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.
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.
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.
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.
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.
High-grade B-cell lymphomas with MYC and BCL2 and/or BCL6 rearrangements (HGBL-DH/THs) include a group of diffuse large B-cell lymphomas (DLBCLs) with inferior outcomes after standard chemoimmunotherapy. We recently described a gene expression signature that identifies 27% of germinal center B-cell DLBCLs (GCB-DLBCLs) as having a double-hit-like expression pattern (DHITsig) and inferior outcomes; however, only half of these cases have both MYC and BCL2 translocations identifiable using standard breakapart fluorescence in situ hybridization (FISH). Here, 20 DHITsig+ GCB-DLBCLs apparently lacking MYC and/or BCL2 rearrangements underwent whole-genome sequencing. This revealed 6 tumors with MYC or BCL2 rearrangements that were cryptic to breakapart FISH. Copy-number analysis identified 3 tumors with MYC and 6 tumors with MIR17HG gains or amplifications, both of which may contribute to dysregulation of MYC and its downstream pathways. Focal deletions of the PVT1 promoter were observed exclusively among DHITsig+ tumors lacking MYC translocations; this may also contribute to MYC overexpression. These results highlight that FISH fails to identify all HGBL-DH/THs, while revealing a range of other genetic mechanisms potentially underlying MYC dysregulation in DHITsig+ DLBCL, suggesting that gene expression profiling is more sensitive for identifying the biology underlying poor outcomes in GCB-DLBCL.
ATAC-seq dataset for the paper: Title Multi-omics analysis of human population variation in immune function and in vivo response to BCG vaccination Abstract Immune responses are tightly regulated, yet highly variable between individuals. To investigate human population variation of trained immunity, we immunized healthy individuals with Bacillus Calmette-Guérin (BCG). This live attenuated vaccine induces not only an adaptive immune response against tu-berculosis, but also triggers innate immune activation and memory. We established personal immune profiles and chromatin accessibility maps over a time course of BCG vaccination in 323 individuals. This large resource uncovered genetic and epigenetic predictors of baseline immunity and BCG vaccine response. We found that BCG vaccination enhances the innate immune response only in individuals with dormant immune states at baseline, suggesting that exogeneous induction of trained immunity is not a universal booster of innate immunity, but specifically elevates weak innate immune responses. This study advances our understanding of BCG’s heterologous immune-stimulatory effects and trained immunity in humans. Moreover, our results highlight the value of epigenetic cell states as an “endo-phenotype” that connects immune function with genotype and the environment.
We have developed orthotopic patient-derived xenograft models of HER2 positive breast cancer metastasized into the brain of patients to test novel therapeutic strategies. In this study, we identified a novel combinatorial therapeutic strategy that has resulted in a durable remission and markedly increased overall survival in majority of patient-derived xenograft (PDX) models tested. We performed whole exome sequencing analysis of these PDX tumors and their matched blood and patient samples to investigate drug sensitive and resistance mechanisms. Our sequencing data revealed an interesting association of genotyping and phenotyping with tumors responses to drug treatment.
