PDF(Pubmed)
Abstract：
Preimplantation genetic testing (PGT) for monogenic disorders (PGT-M) for germline mosaicism was previously highly dependent on polymerase chain reaction (PCR)-based directed mutation detection combined with linkage analysis of short tandem repeats (STRs). However, the number of STRs is usually limited. In addition, designing suitable probes and optimizing the reaction conditions for multiplex PCR are time-consuming and laborious. Here, we evaluated the effectiveness of next generation sequencing (NGS)-based haplotype linkage analysis in PGT of germline mosaicism.
PGT-M with NGS-based haplotype linkage analysis was performed for two families with maternal germline mosaicism for an X-linked Duchenne muscular dystrophy (DMD) mutation (del exon 45-50) or an autosomal TSC1 mutation (c.2074C > T). Trophectoderm biopsy and multiple displacement amplification (MDA) were performed for a total of nine blastocysts. NGS and Sanger sequencing were performed in genomic DNA of family members and embryonic MDA products to detect DMD deletion and TSC1 mutation, respectively. Single nucleotide polymorphism (SNP) sites closely linked to pathogenic mutations were detected with NGS and served in haplotype linkage analysis. NGS-based aneuploidy screening was performed for all embryos to reduce the risk of pregnancy loss.
All nine blastocytes showed conclusive PGT results. Each family underwent one or two frozen-thawed embryo transfer cycles to obtain a clinical pregnancy, and the prenatal diagnosis showed that the fetus was genotypically normal and euploid for both families.
NGS-SNP could effectively realize PGT for germline mosaicism. Compared with PCR-based methods, the NGS-SNP method with increased polymorphic informative markers can achieve a greater diagnostic accuracy. Further studies are warranted to verify the effectiveness of NGS-based PGT of germline mosaicism cases in the absence of surviving offsprings.
PGT-M with NGS-based haplotype linkage analysis was performed for two families with maternal germline mosaicism for an X-linked Duchenne muscular dystrophy (DMD) mutation (del exon 45-50) or an autosomal TSC1 mutation (c.2074C > T). Trophectoderm biopsy and multiple displacement amplification (MDA) were performed for a total of nine blastocysts. NGS and Sanger sequencing were performed in genomic DNA of family members and embryonic MDA products to detect DMD deletion and TSC1 mutation, respectively. Single nucleotide polymorphism (SNP) sites closely linked to pathogenic mutations were detected with NGS and served in haplotype linkage analysis. NGS-based aneuploidy screening was performed for all embryos to reduce the risk of pregnancy loss.
All nine blastocytes showed conclusive PGT results. Each family underwent one or two frozen-thawed embryo transfer cycles to obtain a clinical pregnancy, and the prenatal diagnosis showed that the fetus was genotypically normal and euploid for both families.
NGS-SNP could effectively realize PGT for germline mosaicism. Compared with PCR-based methods, the NGS-SNP method with increased polymorphic informative markers can achieve a greater diagnostic accuracy. Further studies are warranted to verify the effectiveness of NGS-based PGT of germline mosaicism cases in the absence of surviving offsprings.
摘要：
背景：种系镶嵌性单基因疾病（PGT-M）的植入前基因检测（PGT）以前高度依赖于基于聚合酶链反应（PCR）的定向突变检测和短串联重复序列（STR）的连锁分析。然而,STR的数量通常是有限的。此外,设计合适的探针和优化多重PCR的反应条件是费时费力的。这里,我们评估了基于下一代测序（NGS）的单倍型连锁分析在种系镶嵌PGT中的有效性。
方法：PGT-M与基于NGS的单倍型连锁分析对两个具有X连锁杜氏肌营养不良（DMD）突变（del外显子45-50）或常染色体TSC1突变（c.2074C>T）的母系种系镶嵌性的家庭进行。对总共9个囊胚进行了滋养外胚层活检和多重置换扩增（MDA）。对家族成员的基因组DNA和胚胎MDA产物进行NGS和Sanger测序，检测DMD缺失和TSC1突变，分别。用NGS检测到与致病性突变紧密相关的单核苷酸多态性（SNP）位点，并用于单倍型连锁分析。对所有胚胎进行基于NGS的非整倍性筛查以降低妊娠丢失的风险。
结果：所有9个胚母细胞均显示出决定性的PGT结果。每个家庭都经历了一个或两个冻融胚胎移植周期，以获得临床妊娠，产前诊断显示，胎儿基因型正常，两个家庭均为整倍体。
结论：NGS-SNP可有效实现种系镶嵌的PGT。与基于PCR的方法相比，增加多态性信息标记的NGS-SNP方法可以获得更高的诊断准确性。有必要进行进一步的研究，以验证在没有存活后代的情况下，基于NGS的PGT对种系镶嵌性病例的有效性。
方法：PGT-M与基于NGS的单倍型连锁分析对两个具有X连锁杜氏肌营养不良（DMD）突变（del外显子45-50）或常染色体TSC1突变（c.2074C>T）的母系种系镶嵌性的家庭进行。对总共9个囊胚进行了滋养外胚层活检和多重置换扩增（MDA）。对家族成员的基因组DNA和胚胎MDA产物进行NGS和Sanger测序，检测DMD缺失和TSC1突变，分别。用NGS检测到与致病性突变紧密相关的单核苷酸多态性（SNP）位点，并用于单倍型连锁分析。对所有胚胎进行基于NGS的非整倍性筛查以降低妊娠丢失的风险。
结果：所有9个胚母细胞均显示出决定性的PGT结果。每个家庭都经历了一个或两个冻融胚胎移植周期，以获得临床妊娠，产前诊断显示，胎儿基因型正常，两个家庭均为整倍体。
结论：NGS-SNP可有效实现种系镶嵌的PGT。与基于PCR的方法相比，增加多态性信息标记的NGS-SNP方法可以获得更高的诊断准确性。有必要进行进一步的研究，以验证在没有存活后代的情况下，基于NGS的PGT对种系镶嵌性病例的有效性。
