基于胎儿游离DNA和断裂点捕获的DMD基因产前诊断机制研究

Duchenne and Becker muscular atrophy (DMD/BMD) is a frequently seen Mendelian disease which is inherited in an X-linked recessive manner. There is no effective therapy available for DMD/BMD patients at presence, thus prevention is rather important for disease recurrence. Though noninvasive tests for fetal DMD gene mutation have been reported, this study will consider a thoroughly new procedure which is completely different from haplotype analyses, a method used in recent researches alike. Haplotype dosage analysis could not directly find the mutation; Linkage information was used and calculated for the final results. In addition, samples of the entire family were critical for haplotype analyses, not to mention the limitation from recombination. This study will focus on exon deletions and duplications. First, a sequencing chip covering the whole DMD gene, including exons and introns, will be designed for capturing breakpoints of deleted and duplicated exons, assisted by special data analyses following the sequencing procedures. These data could help finding fundamental rules of the occurrence and inheritance of fragments disruption in the DMD gene. Then the distribution of fetal cell free DNA will be measured and analyzed. Last but not least, DNA extracted from maternal plasma will be sequenced by next generation sequencing, targeting the whole DMD gene and the small regions flanking the breakpoints relatively. Fetal DMD gene mutation will be calculated using the two kinds of sequencing data mentioned above, along with the cell free DNA distribution features. This research treats the copy number variation of DMD exons as information about breakpoints, which is a brand new idea. This research will concentrate on explaining how the breakpoints inherited within DMD gene, and how could fetal genotype be extracted by cell free DNA and breakpoints capture.

进行性肌营养不良（DMD/BMD）是严重危害健康的常见X连锁遗传病。目前已有采用单倍型分析方法通过胎儿游离DNA对DMD基因进行无创产前诊断的报道。单倍型分析并不能直接判断突变是否存在，而是一种间接的基因型分析方法，并且受到家系完整性和基因重组的局限。本项目考虑采用一种区别于单倍型分析的全新策略，利用断裂点信息开展针对DMD基因外显子拷贝数异常的无创产前诊断机制研究。本研究将设计覆盖DMD全长基因的测序芯片，捕获外显子缺失区域的精确断裂点，通过跨断裂点研究分析DMD基因断裂的遗传特点和内在遗传机制；利用高精度定量技术研究胎儿游离DNA的片段分布规律；探索利用断裂点信息和胎儿游离DNA分布数据进行产前诊断的可行性。本项目将拷贝数变异转化为断裂点信息，是一项现阶段具有高度原创性和创新性的工作。本项目将致力于阐明DMD基因断裂遗传机制以及基于断裂点的胎儿游离DNA检测机制。

进行性肌营养不良（DMD/BMD）是严重危害健康的常见X连锁遗传病。目前已有利用胎儿游离DNA对DMD基因进行无创产前诊断的报道。利用单倍型分型技术的DMD基因无创产前检测只有在明确孕妇DMD携带状态、并且在明确致病等位基因的的情况下才有效，因此在临床应用方面存在局限。本项目采用高通量测序方法捕获位于整个DMD基因区域的断裂点位置,以对DMD基因外显子拷贝数变异机制进行研究，建立了针对DMD全基因捕获测序的方法，通过生信算法建立了检测DMD基因断裂位点的方法，对外显子拷贝数变异的随机断裂进行了研究。分析DMD基因断裂点在遗传过程中的稳定性,以探讨DMD基因重组过程中的遗传机制，分析了复杂DMD基因重组案例，阐明了DMD基因复杂重组的遗传致病机制。利用多重PCR扩增结合NGS的方法建立了胎儿游离DNA DMD基因外显子片段富集、捕获和检测的方法，并研究了该检测的效能。在25%胎儿浓度情况下，可以实现DMD基因单个外显子异常的可靠检出；在检测限下降到12.5%时则无法获得检出。本研究还进一步探索了使用胎儿细胞分离和10×Genomics技术用于实现游离胎儿DNA DMD基因片段检测的策略。本研究获得的结果对于未来进一步探索单基因疾病的无创产前检测研究具有一定价值。本研究共发表SCI收录研究论文4篇。