Noninvasive prenatal diagnosis of fetal trisomy 21 by allelic ratio analysis using targeted massively parallel sequencing of maternal plasma DNA

Abstract

Background: Plasma DNA obtained from a pregnant woman contains a mixture of maternal and fetal DNA. The fetal DNA proportion in maternal plasma is relatively consistent as determined using polymorphic genetic markers across different chromosomes in euploid pregnancies. For aneuploid pregnancies, the observed fetal DNA proportion measured using polymorphic genetic markers for the aneuploid chromosome would be perturbed. In this study, we investigated the feasibility of analyzing single nucleotide polymorphisms using targeted massively parallel sequencing to detect such perturbations in mothers carrying trisomy 21 fetuses.

Methodology/principal findings: DNA was extracted from plasma samples collected from fourteen pregnant women carrying singleton fetuses. Hybridization-based targeted sequencing was used to enrich 2 906 single nucleotide polymorphism loci on chr7, chr13, chr18 and chr21. Plasma DNA libraries with and without target enrichment were analyzed by massively parallel sequencing. Genomic DNA samples of both the mother and fetus for each case were genotyped by single nucleotide polymorphism microarray analysis. For the targeted regions, the mean sequencing depth of the enriched samples was 225-fold higher than that of the non-enriched samples. From the targeted sequencing data, the ratio between fetus-specific and shared alleles increased by approximately 2-fold on chr21 in the paternally-derived trisomy 21 case. In comparison, the ratio is decreased by approximately 11% on chr21 in the maternally-derived trisomy 21 cases but with much overlap with the ratio of the euploid cases. Computer simulation revealed the relationship between the fetal DNA proportion, the number of informative alleles and the depth of sequencing.

Conclusions/significance: Targeted massively parallel sequencing of single nucleotide polymorphism loci in maternal plasma DNA is a potential approach for trisomy 21 detection. However, the method appears to be less robust than approaches using non-polymorphism-based counting of sequence tags in plasma.

Figure 1. Schematic of T21 detection by F-S ratio calculation.

Assuming the fractional fetal DNA concentration in chrRef is f, the F-S ratio would be f/(2-f) on chrRef irrespective of the aneuploidy status of the fetus. On the other hand, the F-S ratio on chr21 would be f/(2-f) if the mother is carrying a euploid fetus, 2f/(2-f) if the mother is carrying a paternally-derived T21 fetus, and f/2 if the mother is carrying a maternally-derived T21 fetus. Therefore, the would be 1 if the mother is carrying a euploid fetus, would become 2 if the mother is carrying a paternally-derived T21 fetus, and would become (1-f/2) if the mother is carrying a maternally-derived T21 fetus.

Figure 2. T21 detection by F-S ratio in non-targeted and targeted sequencing data.

values were calculated to differentiate the paternally- and maternally-derived T21 from the euploid fetuses in non-targeted (A) and targeted (B) sequencing data. (Ma-T21: maternally-derived T21. Pa-T21: paternally-derived T21.).

Figure 3. Computer simulation for T21 detection for fractional fetal DNA concentrations of 5% and 15%.

In order to obtain a specificity of greater than 99%, the cutoffs for T21 differentiation were chosen at 3 standard deviations above and below the mean F-S ratio of the euploid group. The sensitivity for paternally- and maternally-derived T21 detection was investigated on different numbers of informative allelic counts on chr21 and chrRef, respectively, for a fractional fetal DNA concentration of 15% (A). Similar analysis was performed for a fractional fetal DNA concentration of 5% (B). (Ma-T21: maternally-derived T21. Pa-T21: paternally-derived T21. Sen=sensitivity. Fe%=fractional fetal DNA concentration. Info AC=informative allelic counts on each of chr21 and chrRef. Info SNP=informative SNPs on each of chr21 and chrRef. Seq depth=sequencing depth. Info AC=Info SNP×Seq depth).

Figure 4. Computer simulation to investigate the minimal number of informative allelic counts for T21 detection.

The solid curve represents the minimal number of informative allelic counts required on each of chr21 and chrRef (Y axis), in order to achieve a reliable detection in maternally-derived T21 (sensitivity >99%, specificity >99%) according to a given fractional fetal DNA concentration (X axis). The dash curve represents the paternally-derived T21 model.