DataSheet2_Noninvasive Prenatal Testing of Methylmalonic Acidemia cblC Type Using the cSMART Assay for MMACHC Gene Mutations.docx

Noninvasive prenatal testing (NIPT) for monogenic disorders has been developed in recent years; however, there are still significant technical and analytical challenges for clinical use. The clinical feasibility of NIPT for methylmalonic acidemia cblC type (cblC type MMA) was investigated using our circulating single-molecule amplification and re-sequencing technology (cSMART). Trios molecular diagnosis was performed in 29 cblC type MMA-affected children and their parents by traditional Sanger sequencing. In the second pregnancy, invasive prenatal diagnosis (IPD) of the pathogenic MMACHC gene was used to determine fetal genotypes, and NIPT was performed using a novel MMACHC gene–specific cSMART assay. Maternal–fetal genotypes were deduced based on the mutation ratio in maternal plasma DNA. Concordance of fetal genotypes between IPD and NIPT, and the sensitivity and specificity of NIPT were determined. After removing two cases with a low P value or reads, the concordance ratio for NIPT and IPD was 100.00% (27/27), and the sensitivity and specificity were 100.00% (54.07–100.00%) and 100.00% (83.89–100.00%), respectively. This study demonstrates that NIPT using the cSMART assay for cblC type MMA was accurate in detecting fetal genotypes. cSMART has a potential clinical application as a prenatal diagnosis and screening tool for carrier and low-risk genotypes of cblC type MMA and other monogenic diseases.

近年来，针对单基因遗传病（monogenic disorders）的无创产前检测（Noninvasive prenatal testing, NIPT）已取得进展，但临床应用仍面临诸多重大技术与分析层面的挑战。本研究采用循环单分子扩增重测序技术（circulating single-molecule amplification and re-sequencing technology, cSMART），探究了cblC型甲基丙二酸血症（methylmalonic acidemia cblC type, cblC型MMA）的无创产前检测临床可行性。研究人员通过传统桑格测序（Sanger sequencing），对29名确诊cblC型MMA的患儿及其父母完成三人组分子诊断（Trios molecular diagnosis）。在二次妊娠期间，研究人员借助有创产前诊断（invasive prenatal diagnosis, IPD）对致病性MMACHC基因进行检测以明确胎儿基因型，同时采用新型MMACHC基因特异性cSMART检测技术开展无创产前检测。研究基于母血浆DNA中的突变比例推导母婴基因型，评估了有创产前诊断与无创产前检测的胎儿基因型一致性，并确定了无创产前检测的灵敏度与特异度。剔除2例P值偏低或测序读段（reads）不足的样本后，无创产前检测与有创产前诊断的基因型符合率达100.00%（27/27）；无创产前检测的灵敏度与特异度分别为100.00%（54.07–100.00%）与100.00%（83.89–100.00%）。本研究证实，采用cSMART检测技术开展的cblC型MMA无创产前检测，在胎儿基因型检测中具有精准性。cSMART技术有望作为产前诊断与筛查工具，应用于cblC型MMA及其他单基因遗传病的携带者与低风险基因型检测。