Breveglieri Giulia, Travan Anna, D'Aversa Elisabetta, Cosenza Lucia Carmela, Pellegatti Patrizia, Guerra Giovanni, Gambari Roberto, Borgatti Monica
Department of Life Sciences and Biotechnology, Biochemistry and Molecular Biology Section, University of Ferrara, Ferrara, Italy.
Biotechnology Center, University of Ferrara, Ferrara, Italy.
PLoS One. 2017 Feb 24;12(2):e0172756. doi: 10.1371/journal.pone.0172756. eCollection 2017.
The β-thalassemias are genetic disorder caused by more than 200 mutations in the β-globin gene, resulting in a total (β0) or partial (β+) deficit of the globin chain synthesis. The most frequent Mediterranean mutations for β-thalassemia are: β039, β+IVSI-110, β+IVSI-6 and β0IVSI-1. Several molecular techniques for the detection of point mutations have been developed based on the amplification of the DNA target by polymerase chain reaction (PCR), but they could be labor-intensive and technically demanding. On the contrary, TaqMan® genotyping assays are a simple, sensitive and versatile method suitable for the single nucleotide polymorphism (SNP) genotyping affecting the human β-globin gene. Four TaqMan® genotyping assays for the most common β-thalassemia mutations present in the Mediterranean area were designed and validated for the genotype characterization of genomic DNA extracted from 94 subjects comprising 25 healthy donors, 33 healthy carriers and 36 β-thalassemia patients. In addition, 15 specimens at late gestation (21-39 gestational weeks) and 11 at early gestation (5-18 gestational weeks) were collected from pregnant women, and circulating cell-free fetal DNAs were extracted and analyzed with these four genotyping assays. We developed four simple, inexpensive and versatile genotyping assays for the postnatal and prenatal identification of the thalassemia mutations β039, β+IVSI-110, β+IVSI-6, β0IVSI-1. These genotyping assays are able to detect paternally inherited point mutations in the fetus and could be efficiently employed for non-invasive prenatal diagnosis of β-globin gene mutations, starting from the 9th gestational week.
β地中海贫血是由β珠蛋白基因中的200多种突变引起的遗传性疾病，导致珠蛋白链合成完全（β0）或部分（β+）缺陷。β地中海贫血最常见的地中海突变是：β039、β+IVSI-110、β+IVSI-6和β0IVSI-1。基于聚合酶链反应（PCR）对DNA靶标的扩增，已经开发了几种检测点突变的分子技术，但这些技术可能劳动强度大且技术要求高。相反，TaqMan®基因分型检测是一种简单、灵敏且通用的方法，适用于影响人类β珠蛋白基因的单核苷酸多态性（SNP）基因分型。针对地中海地区存在的最常见β地中海贫血突变，设计了四种TaqMan®基因分型检测，并对从94名受试者中提取的基因组DNA进行了基因分型鉴定，这些受试者包括25名健康供体、33名健康携带者和36名β地中海贫血患者。此外，从孕妇中收集了15份妊娠晚期（妊娠21 - 39周）和11份妊娠早期（妊娠5 - 18周）的样本，提取循环游离胎儿DNA并用这四种基因分型检测进行分析。我们开发了四种简单、廉价且通用的基因分型检测方法，用于产后和产前鉴定地中海贫血突变β039、β+IVSI-110、β+IVSI-6、β0IVSI-1。这些基因分型检测能够检测胎儿中父系遗传的点突变，并且从妊娠第9周开始就可以有效地用于β珠蛋白基因突变的非侵入性产前诊断。
