Castells-Esteve, C. | Search bioRxiv

bioRxiv2026

Unscheduled DNA synthesis reveals a DNA repair hotspot and biomarker of somatic instability at the expanded-CAG repeat tract in the huntingtin gene

Somatic instability (SI) of expanded DNA repeats is a hallmark of repeat expansion disorder (REDs) and drives onset and progression in Huntingtons disease (HD) yet the absence of target engagement (TE) biomarkers for SI-modulating therapies represents a critical gap to clinical development. Here, we describe the development of the unscheduled repair synthesis assay (URSA)--combining 5-ethynyl-2-deoxyuridine (EdU) pulse-labeling with digital PCR or sequencing--and show that the CAG-expanded huntingtin (HTT) exon 1 allele is a highly active DNA repair hotspot in cells from people with HD (PwHD). Repair activity increases with repeat length, is allele-specific, and depends strongly on MSH3, a central driver of somatic expansion. URSA robustly quantifies MSH3 modulation in preclinical models within days compared to weeks or months required by conventional repeat-length measurements. Critically, substantial repair activity is detectable in peripheral blood mononuclear cells (PBMCs) from PwHD, where signal correlates with CAG length and improves predictive models of somatic expansion (SE) beyond age and CAG length alone. Unlike repeat-length changes, which require years to accumulate in blood, URSA signal is measurable within days. These findings establish DNA repair activity at the mutant HTT locus as a mechanistically grounded pharmacodynamic biomarker, enabling TE monitoring on a clinically actionable timescale, and with broad applicability to REDs and other diseases where modulation of the DNA damage response is therapeutically targeted.

neuroscience↗

bioRxiv2023

Renal Cl-/H+ antiporter ClC-5 regulates collagen production and release in Dent Disease models

Mutations in the Cl-/H+ antiporter ClC-5 cause Dents Disease 1 (DD1), a rare primary tubulopathy that eventually progresses to renal failure. In fact, even with normal kidney function, DD1 patients present renal tubulointerstitial fibrosis. However, the link between ClC-5 loss-of-function and renal fibrosis remains unclear. Here, we have shown that DD1 mice models lacking ClC-5 present higher renal collagen deposition and fibrosis. Accordingly, deletion of ClC-5 in human renal proximal tubule epithelial cells (CLCN5 KD) recapitulates this effect. We have demonstrated that CLCN5 KD causes an increase of collagen I (Col I) and IV (Col IV) intracellular levels by promoting their transcription through {beta}-catenin pathway and impairing their lysosomal-mediated degradation. In addition, CLCN5 KD cells release more Col I and IV at the extracellular space that form fibres with altered properties and resistance to removal compared to control cells. Altogether, we describe a new regulatory mechanism for collagens production and release by ClC-5, which is altered in DD1 and provides a better understanding of disease progression to renal fibrosis. SIGNIFICANCE STATEMENTRenal fibrosis is a common pathologic process occurring as consequence of chronic kidney injury and leading to renal dysfunction. Dents Disease is a rare renal pathology that progresses to chronic kidney disease and tubulointerstitial fibrosis. Interestingly, it is caused by mutations in a single gene called CLCN5, therefore it can help understanding the cellular mechanisms of renal fibrosis. Using cellular and mice models of the disease, we describe a mechanism linking CLCN5 function, cell differentiation and regulation of collagen levels, major component of extracellular matrix and important player for renal fibrosis development. In conclusion, our results provide a link between CLCN5 and altered collagen deposition, which could be relevant for other renal Fanconi syndrome related diseases also progressing to fibrosis.

cell biology↗