Department of Clinical Toxicological Analyzes, School of Pharmaceutical Sciences, University of São Paulo, São Paulo, Brazil.
Genomic Engenharia Molecular Ltda, São Paulo, Brazil.
BMC Med Genomics. 2020 Oct 23;13(1):157. doi: 10.1186/s12920-020-00806-w.
The use of noninvasive techniques to determine paternity prenatally is increasing because it reduces the risks associated with invasive procedures. Current methods, based on SNPs, use the analysis of at least 148 markers, on average.
To reduce the number of regions, we used microhaplotypes, which are chromosomal segments smaller than 200 bp containing two or more SNPs. Our method employs massively parallel sequencing and analysis of microhaplotypes as genetic markers. We tested 20 microhaplotypes and ascertained that 19 obey Hardy-Weinberg equilibrium and are independent, and data from the 1000 Genomes Project were used for population frequency and simulations.
We performed simulations of true and false paternity, using the 1000 Genomes Project data, to confirm if the microhaplotypes could be used as genetic markers. We observed that at least 13 microhaplotypes should be used to decrease the chances of false positives. Then, we applied the method in 31 trios, and it was able to correctly assign the fatherhood in cases where the alleged father was the real father, excluding the inconclusive results. We also cross evaluated the mother-plasma duos with the alleged fathers for false inclusions within our data, and we observed that the use of at least 15 microhaplotypes in real data also decreases the false inclusions.
In this work, we demonstrated that microhaplotypes can be used to determine prenatal paternity by using only 15 regions and with admixtures of DNA.
由于非侵入性技术可降低与侵入性程序相关的风险，因此越来越多地使用这些技术来确定产前亲子关系。目前基于 SNP 的方法平均使用了 148 个标记物的分析。
为了减少区域数量，我们使用了微单倍型，这是一种包含两个或更多 SNP 的小于 200bp 的染色体片段。我们的方法采用大规模平行测序和微单倍型作为遗传标记物的分析。我们测试了 20 个微单倍型，并确定其中 19 个符合 Hardy-Weinberg 平衡且相互独立，并且使用 1000 基因组计划的数据进行了群体频率和模拟分析。
我们使用 1000 基因组计划的数据进行了真实和虚假亲子关系的模拟，以确认微单倍型是否可用作遗传标记物。我们观察到，至少应使用 13 个微单倍型以降低假阳性的机会。然后，我们在 31 个三亲家庭中应用了该方法，并且在假定父亲是亲生父亲的情况下，该方法能够正确分配父亲身份，排除了不确定的结果。我们还交叉评估了母亲-血浆对与假定父亲的关系，以确定在我们的数据中是否存在虚假包含，并且我们观察到在真实数据中至少使用 15 个微单倍型也可以减少虚假包含。
在这项工作中，我们证明了微单倍型可以通过仅使用 15 个区域和 DNA 混合物来确定产前亲子关系。
