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

非等位基因同源重组(NAHR)是导致人类基因组疾病相关缺失和复制的重要突变机制。尽管Nahr是反复发生的基因组重排的主要突变机制之一，但其分子基础和特征却鲜为人知。该项目旨在研究30个1型NF1缺失的断裂点侧翼区域中Nahr相关的从头突变率，这些缺失是1型神经纤维瘤病中最常见的复发突变。这些缺失的断裂点位于低拷贝重复序列NF1-REPA和NF1-REPc内的Nahr热点Prs1和Prs2。为了区分从头突变和遗传变异，我们计划将缺失断裂点两侧的序列与传播父母的PRS1和PRS2中的非重组单倍型进行比较，这些单倍型本身不受NF1的影响，也不在他们的血液中含有NF1缺失。在这种情况下，在遗传给受影响的孩子之前，NF1基因的缺失在传播父母的生殖系中发生了从头开始。Nf1基因缺失很少见，估计发生的频率为1：60000。因此，我们的30名1型NF1缺失患者及其临床未受影响的传递父母的队列对于分析断点侧翼区域Nahr相关的从头突变比率具有独特的价值。在断点两侧区域测量的Nahr相关从头突变率将直接与在未参与Nahr相关交叉的NF1-REP区域中检测到的突变率进行比较。对Nahr相关从头突变率的评估将得到1型NF1缺失断裂点侧翼区域内的高分辨率基因转换分析的补充，不仅包括我们先前项目中执行的Paralog特定变种(PSV)，而且还包括单核苷酸变种(SNV)。为此，有必要调查传输亲本的非重组PRS1和PRS2区域内的亲本单倍型，并确定这些单倍型的阶段。这些分析应该表明Nahr相关从头突变和基因转换影响Nahr热点的序列组成从而影响其活性的相对程度，这是序列依赖的。因此，NF1缺失是研究Nahr分子基础的一个很好的模型；这一分析的结果也应该适用于Nahr介导的其他重排。事实上，我们的目标是识别与Nahr相关的分子签名，这将有助于预测基因组其他区域的Nahr热点。在交叉侧翼区域发现更高的从头突变率将表明在减数分裂过程中等位基因同源重组(AHR)和NaHR之间存在迄今未知的相似性，这将有助于我们理解这两个关键机制的分子基础。

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.