第三代测序技术在ATR-X综合征家系胚胎植入前遗传学检测中的应用

Application of third-generation sequencing technology in preimplantation genetic testing of embryos in a family with ATR-X syndrome

目的探讨第三代测序技术在染色体微重复相关ATR-X综合征家系胚胎植入前遗传学检测中的临床应用价值。方法2022年10月就诊于江西省妇幼保健院辅助生殖中心的1例生育过疑似ATR-X综合征患儿的家系为研究对象。染色体拷贝数变异测序(copy number variation sequencing,CNV-Seq)检测女方携带Xq21.1区域550 kb临床意义不明确的杂合微重复,该重复累及ATRX基因部分序列。采用第三代长读长测序技术对女方基因组序列进行检测,确定上述重复插入基因组的物理位置,明确该重复的致病性,并获得与上述微重复连锁遗传的单核苷酸多态性位点(single nucleotide polymorphism,SNP)单倍型。夫妻双方签署知情同意书后行胚胎植入前遗传学检测(preimplantation genetic testing,PGT)助孕。挑选1枚不携带致病性微重复的整倍体胚胎移植,于孕中期羊水穿刺后进行产前诊断,验证是否与PGT结果一致,跟踪随访胎儿出生后情况。结果第三代长读长测序及Sanger测序验证结果显示,女方携带的Xq21.1微重复插入至基因组chrX:76804463-76804464(GRCh37/hg19),为ATRX基因内串联重复,预测可能导致ATRX蛋白正常功能受损。该家系经PGT治疗,获得27枚卵子,卵胞质内单精子注射(intracytoplasmic sperm injection,ICSI)受精后成功养成13枚囊胚。囊胚活检细胞经遗传学检测显示,2枚胚胎为不携带上述致病微重复的整倍体胚胎。冻融胚胎移植1枚正常胚胎,成功妊娠,孕17周羊水穿刺检测结果未见异常,2023年11月孕39^(+5)周顺产娩一女活婴,体健。结论第三代测序技术凭借其长读长的特点,对临床意义不明确微重复进行PGT检测时有显著优势,不仅能够明确微重复插入基因组的位置,判断其致病性,还能够获得与目标变异连锁遗传的SNP单倍型,为后续胚胎检测做准备。

Objective To explore the clinical application value of third-generation sequencing technology in preimplantation genetic testing(PGT)of an ATR-X syndrome pedigree with chromosomal microduplication.Methods The study selected a pedigree with a suspected ATR-X syndrome child at Assisted Reproductive Center of Jiangxi Maternal and Child Health Hospital in October 2022.After chromosome copy number variation sequencing(CNV Seq)detection,it was found that the female carried a 550 kb heterozygous microrepeat with unclear clinical significance in the Xq21.1 region,which involved partial sequences of the ATRX gene.The third generation long read sequencing technology was used to detect the female genome sequence,determine the physical location of the insertion of the above repeat into the genome,clarify the pathogenicity of the repeat,and obtain a single nucleotide polymorphism(SNP)haplotype linked to the above micro repeat inheritance.After the couple's full informed consent,PGT was performed to assist pregnancy.One haploid embryo without pathogenic microduplication was selected for transfer.To verify the consistency with PGT test results,amniocentesis prenatal diagnosis was performed in the second trimester after successful pregnancy,and the fetus was followed up after birth.Results The results of the third generation long read sequencing and Sanger sequencing verification showed that the Xq21.1 microrepeat carried by the female was inserted into the genome chrX:76804463-76804464(GRCh37/hg19),which is an intra tandem repeat of the ATRX gene,and it is predicted that it may cause damage to the normal function of the ATRX protein.After one cycle of PGT treatment,27 oocytes were obtained and 13 blastocysts were successfully developed after intracytoplasmic sperm injection(ICSI).Through genetic testing,it was found that two blastocysts were haploid embryos without carrying the aforementioned pathogenic microduplication.After thawing and transferring one of the blastocysts,the pregnancy was achieved,and the prenatal diagnosis results of amniocentesis in the second trimester were consistent with the PGT results.In November 2023,at 39^(+5) weeks of pregnancy,a female live baby was delivered by natural delivery,and she is in good health.Conclusion The third-generation sequencing technology has significant advantages in PGT detection of clinically ambiguous microreplicates with functional deficiency due to its long read length characteristics.It can not only determine the location of microreplicates inserted into the genome and determine their pathogenicity,but also obtain SNP haplotypes that are linked to the target mutation,thus preparing for subsequent embryo detection.