Biochemistry, Riphah International University Islamabad, Rawalpindi, Pakistan.
Hematology, Armed Forces Institute of Transfusion, Rawalpindi, Pakistan.
Hematology. 2022 Dec;27(1):353-359. doi: 10.1080/16078454.2022.2045052.
The discovery of circulating cell-free fetal DNA (cff-DNA) in maternal plasma has inspired the noninvasive prenatal testing (NIPT) approaches for various genetic fetal screening including rhesus D typing, sex determination, aneuploidies, and single-gene disorders.
Noninvasive determination of paternally inherited beta-thalassemia mutations in maternal total cell-free DNA (cf-DNA) by using allele-specific amplification refractory mutation system (ARMS) real-time PCR (RT-PCR) in concordance with the conventional invasive method.
An observational study was conducted at the Armed Forces Institute of Blood Transfusion in collaboration with the genetics resource center from March 2021 to August 2021. A total number of 26 couples were selected having a history of previously affected children with beta-thalassemia. A routine chorionic villus sampling (CVS) invasive procedure was carried out, and the mutation analysis was done using conventional PCR. To assess NIPT, a total cf-DNA was also extracted from maternal plasma and analyzed using allele-specific ARMS RT-PCR.
Based on conventional PCR testing, 13 of 26 couples were found having beta-thalassemia carriers with homozygous mutation, and 13 couples were carriers with heterozygous mutations. Further to assess NIPT, the cf-DNA of 13 pregnant females among the couples with different mutational patterns was analyzed by allele-specific ARMS RT-PCR to detect paternally inherited mutations. In comparison with conventional PCR, 11 cases (84.6%) were matched successfully, while two cases (15.4%) had no concordance with conventional invasive prenatal testing (IPT).
NIPT using maternal cf-DNA by allele-specific ARMS RT-PCR can be feasible to screen paternal inherited mutant alleles to rule out pregnant women from invasive procedures where the test would be negative for paternal inheritance. However, a low amount of fetal DNA in maternal plasma is a limiting factor and required further improvement to enrich fetal cf-DNA for complete concordance with conventional IPT.
母体血浆中循环无细胞胎儿 DNA (cff-DNA) 的发现激发了各种遗传胎儿筛查的非侵入性产前检测 (NIPT) 方法，包括 RhD 型鉴定、性别鉴定、非整倍体和单基因疾病。
利用等位基因特异性扩增阻滞突变系统 (ARMS) 实时 PCR (RT-PCR) ，在与传统侵入性方法一致的情况下，从母体总无细胞游离 DNA (cf-DNA) 中无创性确定父系遗传性β-地中海贫血突变。
2021 年 3 月至 2021 年 8 月，武装部队血液输血研究所与遗传资源中心合作进行了一项观察性研究。共选择了 26 对有先前受影响的β-地中海贫血儿童病史的夫妇。进行了常规绒毛膜绒毛取样 (CVS) 侵入性程序，并使用常规 PCR 进行突变分析。为了评估 NIPT，还从母体血浆中提取总 cf-DNA，并使用等位基因特异性 ARMS RT-PCR 进行分析。
根据传统 PCR 检测，26 对夫妇中有 13 对被发现携带纯合突变的β-地中海贫血携带者，13 对夫妇携带杂合突变。为了进一步评估 NIPT，对夫妇中不同突变模式的 13 名孕妇的 cf-DNA 进行了等位基因特异性 ARMS RT-PCR 分析，以检测父系遗传突变。与传统 PCR 相比，11 例（84.6%）成功匹配，而 2 例（15.4%）与传统侵入性产前检测 (IPT) 不相符。
使用母体 cf-DNA 通过等位基因特异性 ARMS RT-PCR 的 NIPT 可以对父系遗传突变等位基因进行筛查，从而使孕妇免于侵入性程序，在这些程序中，该测试对父系遗传呈阴性。然而，母体血浆中胎儿 DNA 的含量较少是一个限制因素，需要进一步改进以富集胎儿 cf-DNA，从而与传统 IPT 完全一致。
