Department of Laboratory Medicine, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, People's Republic of China.
Collaborative Innovation Center of Hematology, Shanghai Jiaotong University School of Medicine, Shanghai, People's Republic of China.
Hum Genomics. 2022 Jul 27;16(1):28. doi: 10.1186/s40246-022-00400-4.
High-cost, time-consuming and complex processes of several current approaches limit the use of noninvasive prenatal diagnosis (NIPD) for monogenic disorders in clinical application. Thus, a more cost-effective and easily implementable approach is required.
We established a low-cost and convenient test to noninvasively deduce fetal genotypes of the mutation and single nucleotide polymorphisms (SNPs) loci by means of targeted amplification combined with deep sequencing of maternal genomic and plasma DNA. The sequential probability ratio test was performed to detect the allelic imbalance in maternal plasma. This method can be employed to directly examine familial pathogenic mutations in the fetal genome, as well as infer the inheritance of parental haplotypes through a group of selected SNPs linked to the pathogenic mutation.
The fetal mutations in 17 families with different types of monogenic disorders including hemophilia A, von Willebrand disease type 3, Duchenne muscular dystrophy, hyper-IgM type 1, glutaric acidemia type I, Nagashima-type palmoplantar keratosis, and familial exudative vitreoretinopathy were identified in the study. The mutations included various forms: point mutations, gene inversion, deletions/insertions and duplication. The results of 12 families were verified by sequencing of amniotic fluid samples, the accuracy of the approach in fetal genotyping at the mutation and SNPs loci was 98.85% (172/174 loci), and the no-call rate was 28.98% (71/245 loci). The overall accuracy was 12/12 (100%). Moreover, the approach was successfully applied in plasma samples with a fetal fraction as low as 2.3%.
We have shown in this study that the approach is a cost-effective, less time consuming and accurate method for NIPD of monogenic disorders.
目前几种方法存在成本高、耗时耗力且过程复杂的问题，限制了其在临床应用中对单基因疾病的非侵入性产前诊断（NIPD）。因此，需要一种更具成本效益且易于实施的方法。
我们建立了一种低成本、便捷的测试方法，通过靶向扩增结合母体外周血基因组和血浆 DNA 的深度测序，无创推断胎儿突变和单核苷酸多态性（SNP）位点的基因型。采用序列似然比检验（sequential probability ratio test）检测母体外周血中的等位基因失衡。该方法可直接检测胎儿基因组中的家族致病性突变，并通过一组与致病性突变相关的选定 SNP 推断出亲本单倍型的遗传情况。
在 17 个不同类型的单基因疾病（包括血友病 A、血管性血友病 3 型、杜氏肌营养不良症、高免疫球蛋白 M 血症 1 型、戊二酸血症 1 型、长谷川型手掌足底角化病和家族性渗出性玻璃体视网膜病变）的家系中，鉴定出胎儿突变。突变包括各种形式：点突变、基因倒位、缺失/插入和重复。12 个家系的结果通过羊水样本测序得到验证，该方法在突变和 SNP 位点的胎儿基因分型中的准确性为 98.85%（172/174 个位点），无结果率为 28.98%（71/245 个位点）。总体准确率为 12/12（100%）。此外，该方法在胎儿比例低至 2.3%的血浆样本中也取得了成功。
本研究表明，该方法是一种具有成本效益、耗时较短且准确的单基因疾病 NIPD 方法。
