NTRK1基因新发突变致遗传性感觉和自主神经病4型的遗传学分析及出生缺陷阻断研究

Preimplantation genetic testing and birth defect prevention of hereditary sensory and autonomic neuropathyⅣcaused by novel NTRK1 mutation

目的探讨基于二代测序的植入前单体型分析在遗传性感觉和自主神经病4型(hereditary sensory and autonomic neuropathyⅣ,HSANⅣ)出生缺陷阻断中的应用价值。方法纳入1个HSANⅣ家系,采用高通量测序和Sanger测序相结合的方法对NTRK1基因进行突变分析,通过构建TrkA表达载体对突变进行致病性预测。对活检的囊胚滋养层细胞进行全基因组扩增,选择致病基因上下游数十至数百个单核苷酸多态性位点作为连锁遗传标记,通过基于二代测序的植入前单体型分析完成植入前遗传学检测。结果先证者NTRK1基因存在复合杂合突变,包括新发现的移码突变(c.963delG;p.Val321-Valfs*149)和已报道的错义突变(c.850+1G>A),新突变可导致编码蛋白截短与功能失调,其母亲和父亲分别为上述突变携带者。经植入前遗传学检测,活检的2个囊胚中1个携带母方致病基因,1个受累。选择携带母方致病基因的胚胎进行移植,未获妊娠。结论本研究是我国NTRK1基因突变行植入前遗传学检测的首例报道,新发现的c.963delG突变丰富了NTRK1基因的突变谱,同时为阻断单基因遗传病出生缺陷的发生提供有效的预防手段。

Objective To explore the application of next generation sequencing(NGS)-based single nucleotide polymorphism(SNP)haplotyping of preimplantaion genetic testing for monogenic(PGT-M)in the prevention of hereditary sensory and autonomic neuropathyⅣ(HSANⅣ).Methods A Chinese family of HSANⅣwas enrolled in this study.NGS and Sanger sequencing were combined to conduct mutational analysis of NTRK1 gene.Functional evaluation of mutation was performed through the cloning of expression construct.After whole genome amplification of biopsied trophectoderm cells,some high-frequency SNP markers were selected for NGS-based haplotyping,including SNPs located upstream and downstream of NTRK1 target region.Results Two compound heterozygous mutations of the NTRK1 gene,the previously reported mutation c.850+1G>A and the novel frameshift mutation(c.963delG;p.Val321-Valfs*149),were identified in the proband,inherited from maternal and paternal sides,respectively.The novel frameshift mutation created a new reading frame,leading to dysfunctional and truncated protein.According to the PGT-M results,one of the blastocysts,inherited the maternal pathogenic allele and the paternal normal allele,was thawed and transferred.While the other blastocyst inherited both pathogenic allele.Unfortunately,there was no subsequent pregnancy.Conclusion This is the first report of preimplantation genetic testing of NTRK1 gene mutations in China.The novel frameshift mutation c.963delG expands the gene mutation spectrum of HSANⅣ.NGS-based SNP haplotyping applied in PGT-M provides an effective way to prevent birth defects of single-gene inherited diseases.