Personalized recurrence risk assessment following the birth of a child with a pathogenic de novo mutation.

Following the diagnosis of a paediatric disorder caused by an apparently de novo mutation, a recurrence risk of 1-2% is frequently quoted due to the possibility of parental germline mosaicism; but for any specific couple, this figure is usually incorrect. We present a systematic approach to providing individualized recurrence risk. By combining locus-specific sequencing of multiple tissues to detect occult mosaicism with long-read sequencing to determine the parent-of-origin of the mutation, we show that we can stratify the majority of couples into one of seven discrete categories associated with substantially different risks to future offspring. Among 58 families with a single affected offspring (representing 59 de novo mutations in 49 genes), the recurrence risk for 35 (59%) was decreased below 0.1%, but increased owing to parental mixed mosaicism for 5 (9%)-that could be quantified in semen for paternal cases (recurrence risks of 5.6-12.1%). Implementation of this strategy offers the prospect of driving a major transformation in the practice of genetic counselling.

Replication Fork Reversal during DNA Interstrand Crosslink Repair Requires CMG Unloading.

DNA interstrand crosslinks (ICLs) are extremely cytotoxic, but the mechanism of their repair remains incompletely understood. Using Xenopus egg extracts, we previously showed that repair of a cisplatin ICL is triggered when two replication forks converge on the lesion. After CDC45/MCM2-7/GINS (CMG) ubiquitylation and unloading by the p97 segregase, FANCI-FANCD2 promotes DNA incisions by XPF-ERCC1, leading to ICL unhooking. Here, we report that, during this cell-free ICL repair reaction, one of the two converged forks undergoes reversal. Fork reversal fails when CMG unloading is inhibited, but it does not require FANCI-FANCD2. After one fork has undergone reversal, the opposing fork that still abuts the ICL undergoes incisions. Our data show that replication fork reversal at an ICL requires replisome disassembly. We present a revised model of ICL repair that involves a reversed fork intermediate.

RPA activates the XPF-ERCC1 endonuclease to initiate processing of DNA interstrand crosslinks.

During replication-coupled DNA interstrand crosslink (ICL) repair, the XPF-ERCC1 endonuclease is required for the incisions that release, or "unhook", ICLs, but the mechanism of ICL unhooking remains largely unknown. Incisions are triggered when the nascent leading strand of a replication fork strikes the ICL Here, we report that while purified XPF-ERCC1 incises simple ICL-containing model replication fork structures, the presence of a nascent leading strand, modelling the effects of replication arrest, inhibits this activity. Strikingly, the addition of the single-stranded DNA (ssDNA)-binding replication protein A (RPA) selectively restores XPF-ERCC1 endonuclease activity on this structure. The 5'-3' exonuclease SNM1A can load from the XPF-ERCC1-RPA-induced incisions and digest past the crosslink to quantitatively complete the unhooking reaction. We postulate that these collaborative activities of XPF-ERCC1, RPA and SNM1A might explain how ICL unhooking is achieved in vivo.