Size-based molecular diagnostics using plasma DNA for noninvasive prenatal testing

Abstract

Noninvasive prenatal testing using fetal DNA in maternal plasma is an actively researched area. The current generation of tests using massively parallel sequencing is based on counting plasma DNA sequences originating from different genomic regions. In this study, we explored a different approach that is based on the use of DNA fragment size as a diagnostic parameter. This approach is dependent on the fact that circulating fetal DNA molecules are generally shorter than the corresponding maternal DNA molecules. First, we performed plasma DNA size analysis using paired-end massively parallel sequencing and microchip-based capillary electrophoresis. We demonstrated that the fetal DNA fraction in maternal plasma could be deduced from the overall size distribution of maternal plasma DNA. The fetal DNA fraction is a critical parameter affecting the accuracy of noninvasive prenatal testing using maternal plasma DNA. Second, we showed that fetal chromosomal aneuploidy could be detected by observing an aberrant proportion of short fragments from an aneuploid chromosome in the paired-end sequencing data. Using this approach, we detected fetal trisomy 21 and trisomy 18 with 100% sensitivity (T21: 36/36; T18: 27/27) and 100% specificity (non-T21: 88/88; non-T18: 97/97). For trisomy 13, the sensitivity and specificity were 95.2% (20/21) and 99% (102/103), respectively. For monosomy X, the sensitivity and specificity were both 100% (10/10 and 8/8). Thus, this study establishes the principle of size-based molecular diagnostics using plasma DNA. This approach has potential applications beyond noninvasive prenatal testing to areas such as oncology and transplantation monitoring.

Keywords: Down syndrome; Turner syndrome; fetal aneuploidy; next-generation sequencing; size profiling.

Fig. 1.

Size distributions of DNA molecules in two maternal plasma samples with different fetal DNA fractions. Solid line represents the sample (case 99633) with a lower fetal DNA fraction, whereas dashed line represents the sample (case 94884) with a higher fetal DNA fraction.

Fig. 2.

(A) Correlation between size ratios and fetal DNA fractions for the 36 samples in the training group. The fetal DNA fraction is determined from the proportion of chromosome Y sequences in the maternal plasma samples. The solid line is the linear regression line through the data, and the corresponding regression equation is given on the graph. (B) Plot of the fetal DNA fraction deduced from the size ratio using the above regression equation against that determined from the proportion of chromosome Y sequences for the 37 samples in the validation group.

Fig. 3.

(A) A representative Bioanalyzer electropherogram of a maternal plasma DNA sequencing library. Areas A and B correspond to library fragment sizes of 200–265 bp and 285–290 bp, respectively. The library fragment size is the size of the plasma DNA molecule plus the size of the sequencing adaptor (122 bp). (B) Correlation between size ratios (area A/area B) derived from electropherograms and fetal DNA fractions for the 36 samples in the training group. The fetal DNA fraction is determined from the proportion of chromosome Y sequences in the maternal plasma samples. The solid line is the linear regression line through the data, and the corresponding regression equation is given on the graph. (C) Plot of the fetal DNA fraction deduced from the electropherogram-derived size ratio using the above regression equation against the fetal DNA fraction determined from the proportion of chromosome Y sequences for the 37 samples in the validation group.

Fig. 4.

Size-based z-scores for (A) chromosome 21, (B) chromosome 18, and (C) chromosome 13 for the 124 test samples in the first sample set (which include 40 euploid, 36 trisomy 21, 27 trisomy 18, and 21 trisomy 13 cases). Dashed line in each panel indicates the z-score cutoff value of 3.

Fig. 5.

(A) Size-based and (B) count-based z-scores for chromosome X of the 18 cases in the second sample set, including 8 cases with euploid female fetuses and 10 cases with monosomy X fetuses. Another 8 cases with euploid female fetuses from the second sample set were used as reference controls. Dashed lines indicate the z-score cutoff value of −3.