Objective: We report a case of application of third-generation sequencing (TGS) combined with preimplantation genetic testing (PGT) for successful prevention of hereditary spastic paraplegia (HSP) caused by SPAST gene mutations and assess the value of PGT-M and TGS in managing hereditary spastic paraplegia.
Methods: A family affected by HSP underwent whole exon sequencing (WES), and a c.1699G>T mutation in the SPAST gene was identified. The mutation site in the proband was confirmed through Sanger sequencing. A single nucleotide polymorphism (SNP) site flanking the SPAST gene mutation was selected as the genetic linkage marker, and a SNP haplotype carrying the mutated gene was constructed. Ovarian stimulation using an antagonist regimen was performed for oocyte retrieval, followed by intracytoplasmic sperm injection (ICSI) and embryo culture. Blastocyst trophectoderm cells were biopsied for preimplantation genetic testing for monogenic disorders (PGT-M) to allow the selection of disease-free embryos for transfer.
Results: In this cycle, a total of 20 oocytes were retrieved, among which 18 were successfully fertilized to result in 12 blastocysts eligible for biopsy. Genetic testing revealed that all the 12 blastocysts were successfully amplified and confirmed as euploidy. Among them, 8 blastocysts did not carry paternal mutations, and a high-quality euploid embryo was selected for frozen embryo transfer (FET). Subsequent amniotic fluid testing during pregnancy confirmed the absence of paternal mutations in the fetus, resulting in the birth of a healthy baby girl.
Conclusion: For cases of genetic diseases with missing pedigree data, the application of third-generation sequencing and PGT-M technique can effectively block vertical transmission of SPAST gene mutation to the offspring, avoid pregnancy with an aneuploid embryo, and help families with autosomal dominant HSP obtain healthy offsprings.
目的: 结合三代测序技术（TGS）与胚胎植入前遗传学检测（PGT）成功阻断一个由SPAST基因突变引起的遗传性痉挛性截瘫（HSP），探讨PGT-M与TGS在遗传性痉挛性截瘫中的应用价值。
方法: 选取一个遗传性痉挛性截瘫家系进行全外显子检测（WES），发现SPAST基因c.1699G>T突变，对该家系中先证者的SPAST基因突变位点c.1699G>T进行Sanger测序验证，同时应用三代测序技术在该家系SPAST基因突变位点两侧选择单核苷酸多态性（SNP）位点作为遗传连锁标记，进而构建携带基因突变的家系SNP单体型。行拮抗剂方案刺激卵巢以获取卵母细胞，并进行卵细胞质内单精子注射 （ICSI）及胚胎培养，对囊胚滋养层细胞进行活检。应用PGT-M技术后，选择其中不携带致病基因的胚胎进行移植。
结果: 本周期共获取卵母细胞20枚，其中正常受精18枚，最后形成可供活检的囊胚12枚。基因检测结果显示，12枚囊胚均成功扩增，且均为整倍体，12枚囊胚中8枚不携带父源突变，4枚胚胎携带父源突变。挑选其中1枚不携带父源突变的优质整倍体胚胎进行冻融胚胎移植（FET），成功妊娠后羊水检测显示胎儿不携带父源突变后产下一健康女婴。
结论: 对于家系不全的遗传病案例，应用三代测序及PGT-M技术可有效阻断SPAST基因突变向子代垂直传递，也可避免非整倍体胚胎妊娠导致的流产，帮助常染色体显性遗传性痉挛性截瘫家庭获得健康子代。
Keywords: SPAST gene; hereditary spastic paraplegia; preimplantation genetic diagnosis; single nucleotide polymorphisms; third-generation sequencing.