平衡性染色体结构变异(BCSV)包括倒位和易位,是一类多见的遗传变异,可导致出生缺陷等多种遗传病。其致病性取决于断裂点的位置及对基因结构和功能的影响,精确定位断裂点是研究BCSV致病机制的关键。申请人前期建立了二代测序检测BCSV断裂点的研究方法,比核型分析和FISH有很大改善并成为研究致病基因的新途径,但该方法尚存在费时费钱且需大量DNA的缺点,不适合对大样本做快速检测。本研究拟通过改进文库构建和半导体测序等措施缩短分析周期(11天降到2天);通过改进文库和油包水PCR减少DNA用量(30ug降到1ug);通过Proton测序和多样本条码平行测序减低成本(几千美金降到几百美金)。进一步利用新技术分析50例智力落后的临床BCSV样本,根据断裂点是位于基因间还是基因内设计相应的实验方法研究断裂点对基因结构与表达的影响,评估致病性，鉴定新的候选致病基因,为智力落后新的致病机制研究提供线索。

Balanced chromosome structural variants (BCSVs) which involve translocations and inversions are common genetic defects in human genome. Depending on whether or not such a rearrangement affects a critical gene, it may result in a serious disease on one scenario or a benign consequence on another. The pathogenicity of BCSV is hard to predict without knowing the genes that are disrupted by the rearrangement breakpoints. Precisely mapping the breakpoints is the key step towards a better assessment of BCSV's disease causality. We have previously developed a next generation sequencing based breakpoint mapping strategy which has better efficiency than the old karyotyping-FISH method. Yet the new approach is still quite time and DNA consuming, not suitable for routine research and clinical applications. In order to develop a faster, cheaper and more applicable method, we plan to utilize semiconductor sequencer in this project. We will develop new protocols for library construction, paired-end sequencing and sample multiplexing. So the whole process takes only 2 days instead of 13 days.require only 1-3ug DNA instead of 30ug,it costs sevaral hundren dollars instead of sevaral thousand.We will evaluate and improve the performance of the new technique and apply it to 50 ascertained patient cases with BCSV and intellectual disability. We will be able to map the breakpoints at single nucleotide level and identify genes at or near the breakpoints. We will further analyze the structure integrity and expression level of these genes. The clinical significance will be assessed based on the identity of the genes and their degree of structural or functional disruption by the rearrangements. The genes identified by breakpoint mapping provide new leads towards understanding disease mechanisms of intellectual disability.

高通量测序技术随着成本下降和应用的推广，在分子诊断领域已经被广泛应用，最常用的全外显子测序和临床外显子测序对于罕见遗传病的诊断效率也获得高度认同。在此基础上，越来越多的研究希望拓展高通量测序的应用前景，使其能够检测更多类型的基因组变异，如结构性变异等。目前，以测序深度评估为主要算法可以检测全基因组范围内的拷贝数变异，但其他类型的结构性变异，如平衡易位和染色体重复片段的插入性重复，则没有可应用的高通量测序技术和分析方法。早前开发的mate-pair文库从原理上可以检测各种类型的结构性变异，但仍存在费时、费力、分析复杂等缺点。本课题基于先前应用于肿瘤中融合基因检测而设计的多重锚定PCR技术，针对不同类型染色体结构性变异的断裂位点，利用类似的单侧引物设计算法，设计间隔确定且保证扩增片段能覆盖断裂位点的锚定引物，从而达到捕获断裂位点另一端位置的序列信息，该方法称为覆盖断裂位点的锚定PCR技术（Breakpoint Spreading Anchored PCR, BSA-PCR）。该技术可以在断裂位点一端序列信息未知的情况下，有方向性的扩增断裂位点区域，从来保证能够成功捕获到需要的区域来进行测序，得到断裂位点的具体信息，我们利用该技术对多例染色体拷贝数增加的片段进行了可能包含断裂位点的区域进行捕获和高通量测序，成功检测了平衡易位和染色体重复片段的插入性重复的断裂位点。对临床患者在遗传检测后临床意义位置的结构性变异，通过开发的高通量测序技术进行进一步检测分析，不仅能为患者的临床诊断提供更加可靠的遗传学证据，也为发现新致病基因和研究疾病致病机制提供了重要切入点和研究基础。