Analysis of the NAHR-associated de novo mutation rate using type-1 NF1 deletions as a model

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Non-allelic homologous recombination (NAHR) is an important mutational mechanism causing disease-associated deletions and duplications in the human genome. Despite its key role as one of the major mutational mechanism underlying recurrent genomic rearrangements, the molecular basis and characteristic features of NAHR are poorly understood. This project aims to study the NAHR-associated de novo mutation rate in the breakpoint-flanking regions of 30 type-1 NF1 deletions, the most frequently recurring mutations in neurofibromatosis type-1. The breakpoints of these deletions are located in the NAHR hotspots PRS1 and PRS2 within the low copy repeats NF1-REPa and NF1-REPc. To distinguish de novo mutations from inherited variants, we plan to compare the deletion breakpoint-flanking sequences with the non-recombinant haplotypes in PRS1 and PRS2 of the transmitting parents who are not themselves affected by NF1 and who do not harbour the NF1 deletion in their blood. In such cases, the NF1 deletion had occurred de novo during meiosis in the germlines of the transmitting parents before being passed on to the affected children. NF1 deletions are rare, occurring with an estimated frequency of 1:60000. Thus, our cohort of 30 type-1 NF1 deletion patients and their clinically unaffected transmitting parents is uniquely valuable for the analysis of the NAHR-associated de novo mutation rate in breakpoint-flanking regions. The NAHR-associated de novo mutation rate measured in breakpoint-flanking regions will be directly compared with the mutation rate detected in regions of the NF1-REPs that are not involved in NAHR-associated crossovers. The assessment of the NAHR-associated de novo mutation rate will be complemented by a high-resolution gene conversion analysis within breakpoint-flanking regions of type-1 NF1 deletions including not only paralog-specific variants (PSVs), as performed in our previous project, but also single nucleotide variants (SNVs). To this end, it will be necessary to investigate the parental haplotypes within the non-recombinant PRS1 and PRS2 regions of the transmitting parents and to determine the phase of these haplotypes. These analyses should indicate the relative extent to which NAHR-associated de novo mutations and gene conversion influence the sequence composition of NAHR hotspots and hence their activity which is sequence-dependent. NF1 deletions therefore serve as an excellent model for the investigation of the molecular basis of NAHR; findings from this analysis should also be applicable to other NAHR-mediated rearrangements. Indeed, we aim to identify an NAHR-associated molecular signature that would help to predict NAHR hotspots in other regions of the genome. The finding of an increased de novo mutation rate in crossover-flanking regions would indicate a hitherto unknown similarity between allelic homologous recombination (AHR) and NAHR during meiosis which would help us to understand the molecular basis of both key mechanisms.