Carrera, P. | bioRxiv Science

bioRxiv2026

Single genome analysis of normal cells identifies the early steps of kidney cancer.

Whole genome analyses have shown that every cell of the body accumulates thousands of genetic changes during a lifetime, with implications for cancer initiation. We have analyzed normal kidney cells from both healthy individuals and patients with the Von Hippel Lindau (VHL) syndrome, which predisposes to kidney cancer. Using a clonal-expansion method that enables the collection of high confidence genetic and gene expression data from the same cell, we define a new mutational signature that marks a specific subset of damage-activated proximal tubule cells, as well as 96% of clear cell renal cell carcinomas. The underlying mutational process is specifically active in highly transcribed DNA and impacts the occurrence of cancer driver variants in the VHL gene. Our data from normal cells, supported by in vitro experiments, point to perturbations of oxygen availability as a causative factor. These findings link hypoxia, a frequent endogenous stress in the normal renal tissue, to excessive mutation and cancer initiation.

cancer biology↗

bioRxiv2023

A WFS1 variant disrupting acceptor splice site uncovers the impact of alternative splicing on ER-stress independent β cell apoptosis in a patient with Wolfram syndrome.

Aims/hypothesisWolfram Syndrome 1 (WS1) is an inherited condition mainly manifesting in childhood-onset diabetes mellitus and progressive optic nerve atrophy. The causative gene, WFS1, encodes for Wolframin, a master regulator of several cellular responses, whose mutations associate with clinical variability. Indeed, nonsense/frameshift variants correlate with more severe symptoms than missense/in-frame ones. As achieving a genotype-phenotype correlation is crucial to deal with disease outcome, works investigating the impact of transcriptional and translational landscapes stemming from such mutations are needed. Therefore, we sought to elucidate the molecular determinants behind the pathophysiological alterations in a WS1 patient carrying compound heterozygous mutations in WFS1 gene: c.316-1G>A, affecting the acceptor splice site (ASS) upstream exon 4, and c.757A>T, introducing a premature termination codon (PTC) in exon 7. MethodsBioinformatic analysis was carried out to infer the alternative splicing events occurring after disruption of ASS, followed by RNAseq and PCR to validate the transcriptional landscape. Patient-derived induced Pluripotent Stem Cells (iPSCs) were used as an in vitro model of WS1 and to investigate the WFS1 alternative splicing isoforms into pancreatic {beta} cells. CRISPR/Cas9 technology was employed to correct ASS mutation and generate a syngeneic control for the ER-stress induction and immunotoxicity assays. ResultsWe showed that patient-derived iPSCs retained the ability to differentiate into pancreatic {beta} cells. We demonstrated that the allele carrying the ASS mutation c.316-1G>A originates two PTC-containing alternative splicing transcripts (c.316del and c.316-460del), and two ORF-conserving mRNAs (c.271-513del and c.316-456del) leading to N-terminally truncated polypeptides. By retaining the C-terminal domain, these isoforms sustained the endoplasmic reticulum (ER)-stress response in {beta} cells. Otherwise, PTC-carrying transcripts were regulated by the nonsense mediated decay (NMD) in basal conditions. Exposure to cell stress inducers and pro-inflammatory cytokines affected the NMD-related gene SMG7 expression levels (>2 fold decrease; p<0.001) without eliciting a robust unfolded protein response in WFS1 {beta} cells, thus resulting in a dramatic accumulation of the PTC-containing isoforms c.316del (>100-fold increase over basal; p<0.001) and c.316-460del (>20-fold increase over basal; p<0.001) and predisposing affected {beta} cells to undergo apoptosis. Cas9-mediated recovery of ASS retrieved the canonical transcriptional landscape, rescuing the normal phenotype in patient-derived {beta} cells. Conclusions/interpretationThis study represents a new model to study Wolframin, highlighting how each single mutation of WFS1 gene can determine dramatically different functional outcomes. Our data point to increased vulnerability of WFS1 {beta} cells to stress and inflammation, and we postulate that this is triggered by escaping NMD and accumulation of mutated transcripts and truncated proteins. These findings pave the way for further studies on the molecular basis of genotype-phenotype relationship in WS1, to uncover the key determinants that might be targeted to ameliorate the clinical outcome of patients affected by this rare disease.

genetics↗

bioRxiv2023

The CD36 scavenger receptor Bez regulates lipid redistribution from fat body to oocytes in Drosophila

Class B scavenger receptors of the CD36 family are important for lipid mobilization from mammalian adipose tissue. This protein family has 3 members in mammals, but 14 in Drosophila melanogaster. Lipid distribution in Drosophila is mediated by homologs of the LDL receptors, while little is known about the function of scavenger receptors for this process in invertebrates. Here we unravel a role for the so far uncharacterized scavenger receptor Bez in lipid export from Drosophila adipocytes. Bez shares the lipid binding residue with CD36 and is expressed at the plasma membrane of the embryonic, larval and adult fat body. Bez loss-of-function lowers the organismal content of storage lipids while they accumulate in the fat body, concomitant with female sterility and degeneration of ovaries. Using an alkyne-labeled fatty acid tracer, we demonstrate that Bez interacts with the apoB homolog Lipophorin at the plasma membrane of adipocytes, thereby enabling lipid transfer. Our study shows how a scavenger receptor interacts with lipoproteins to distribute storage lipids from the fat body to the ovaries and thereby contributes to the metabolic control of development.

developmental biology↗