Centre for Research into Circulating Fetal Nucleic Acids, Li Ka Shing Institute of Health Sciences, Hong Kong SAR, China.
Clin Chem. 2012 Oct;58(10):1467-75. doi: 10.1373/clinchem.2012.189589. Epub 2012 Aug 15.
A genomewide genetic and mutational profile of a fetus was recently determined via deep sequencing of maternal plasma DNA. This technology could have important applications for noninvasive prenatal diagnosis (NIPD) of many monogenic diseases. Relative haplotype dosage (RHDO) analysis, a core step of this procedure, would allow one to elucidate the maternally inherited half of the fetal genome. For clinical applications, the cost and complexity of data analysis might be reduced via targeted application of this approach to selected genomic regions containing disease-causing genes. There is thus a need to explore the feasibility of performing RHDO analysis in a targeted manner.
We performed target enrichment by using solution-phase hybridization followed by massively parallel sequencing of the β-globin gene region in 2 families undergoing prenatal diagnosis for β-thalassemia. We used digital PCR strategies to physically deduce parental haplotypes. Finally, we performed RHDO analysis with target-enriched sequencing data and parental haplotypes to reveal the β-thalassemic status for the fetuses.
A mean sequencing depth of 206-fold was achieved in the β-globin gene region by targeted sequencing of maternal plasma DNA. RHDO analysis was successful for the sequencing data obtained from the target-enriched samples, including a region in one of the families in which the parents had similar haplotype structures. Data analysis revealed that both fetuses were heterozygous carriers of β-thalassemia.
Targeted sequencing of maternal plasma DNA for NIPD of monogenic diseases is feasible.
最近通过对母体血浆 DNA 进行深度测序，确定了胎儿的全基因组遗传和突变特征。这项技术在许多单基因疾病的非侵入性产前诊断（NIPD）中可能具有重要的应用。相对单体型剂量（RHDO）分析是该程序的核心步骤，它可以阐明母体遗传的胎儿基因组的一半。为了临床应用，通过将这种方法有针对性地应用于包含致病基因的选定基因组区域，可以降低成本和数据分析的复杂性。因此，需要探索以靶向方式进行 RHDO 分析的可行性。
我们通过使用溶液相杂交，然后对正在接受β-地中海贫血症产前诊断的 2 个家庭的β-球蛋白基因区域进行大规模平行测序，进行靶向富集。我们使用数字 PCR 策略来物理推断出亲本单倍型。最后，我们使用靶向富集测序数据和亲本单倍型进行 RHDO 分析，以揭示胎儿的β-地中海贫血症状态。
通过对母体血浆 DNA 进行靶向测序，β-球蛋白基因区域的平均测序深度达到了 206 倍。对靶向富集样本获得的测序数据进行 RHDO 分析是成功的，包括一个具有相似单倍型结构的父母的家庭的一个区域。数据分析显示，两个胎儿都是β-地中海贫血症的杂合携带者。
对母体血浆 DNA 进行靶向测序以进行单基因疾病的 NIPD 是可行的。
