通过Bionano单分子光学图谱对携带相互易位或倒位罕见病例进行断裂点精准定位及致病机制研究

Reciprocal translocations and inversions are the main types of genomic structural variation, with an incidence ranging from about 1 in 500 to 1 in 2000 human newborns, and around 7% of carriers show abnormal phenotypes. It is of great significance to precisely locate the breakpoint for define the pathogenesis of this rare genetic disease. However, commonly applied methods for breakpoint locating are currently based on sanger sequencing and shotgun highthrough put sequencing, which can only generate short reads with single-end sequencing. Although mate-pair sequencing might be a solution for producing long reads, it remains limited by difficulties in alignment of some genome regions with duplicated sequences. To address this problem, we plan to bring BioNano’s next generation genome mapping, which is a powerful tool for genome-wide structural variation detection with very long reads(150kb-1.5Mb) and spanning repeated sequence or some complex components, into the field of breakpoint researches. In the present study, we will map the breakpoints precisely by BioNano’s next generation genome in 20 patients carrying reciprocal translocations or inversions collected by our research group. Candidate genes will undergo functional analysis using patient-specific iPSCs and the underlying pathogenic mechanisms will be explored. Potential findings of this study would also guide the prevention and therapy of rare genetic diseases.

染色体相互易位或倒位是基因组结构变异的主要类型，在人类新生儿中的发生率约1/2000至1/500，其中约7%的相互易位或倒位携带者会伴有临床表型，断裂点精确定位对于明确此类罕见遗传病的发病机制有着重要的意义。本研究中引入了国际最先进的Bionano单分子光学图谱，该方法对于基因组结构变异分析有着极大的技术优势，该技术具有超长序列读长（150kb-1.5Mb），可跨越重复片段和一些包含复杂元件的区域，是相互易位或倒位等SV断裂点定位和复杂基因组组装的利器。本项目拟通过前期收集整理的20例相互易位或倒位伴异常表型的罕见特殊家系，采用Bionano单分子光学图谱进行重排序列精确测定，对这些序列进行深入分析寻找候选致病基因，结合患者特异性的诱导性多潜能干细胞（iPSCs）行功能研究，明确致病机制，本项目对指导此类罕见遗传病的预防与治疗也有着非常重要的意义。

本项目建立了基于Bionano光学图谱技术(OTM)、单分子测序技术（SMRT）等结合的基因组结构变异（SVs）检测、功能研究、基因型-表型研究的方法。研究纳入了多例具有多发异常表型的新发染色体平衡染色体（ABCRs）病例，并采用OTM和SMRT与短片段序列相结合，开展 SVs检测。所有样本均首先行OTM检测，通过短读长测序验证SVs的具体断裂点，再进一步对两个复杂的染色体易位病例进行了SMRT。根据目标序列、可疑基因和重复单元的断点特征分析，讨论了基因型与SV间关系。在四例ABCR病例中，共有24个断点连接在单碱基水平上被确定。在两个ABCR病例中分析了与表型相关的断点连接，而另外两个ABCR病例中检测到更多不同的非失衡和复杂的染色体重排（CCR）。这些发现证明并强调了ABCR的复杂性，提示对于CCR病例，应进行全面的检测和深入研究。OTM和SMRT可以在基因组范围内对ABCR进行全面的SVs分析，尤其是对于伴有隐匿性不平衡和复杂SVs的CCR。此外，我们的研究也表明这些方法在发现SVs可能的形成机制、探索其致病性方面具有潜在前景。.

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