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 pairedend 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.size profiling | fetal aneuploidy | next-generation sequencing | Down syndrome | Turner syndrome I n the plasma of pregnant women, cell-free fetal DNA is present in a large background of maternally derived DNA (1). Cell-free DNA molecules are mainly short fragments of less than 200 bp (2, 3). Early work based on real-time quantitative PCR has shown that fetal DNA is generally shorter than maternally derived DNA (2). Subsequently, researchers have taken advantage of such a size difference to enrich for fetal DNA in maternal plasma samples for noninvasive prenatal testing (4-6).More recently, the development of paired-end massively parallel sequencing has allowed the size distributions of fetally and maternally derived DNA to be studied at a single-base resolution (7,8). Both the size distributions of fetally and maternally derived DNA exhibit a series of peaks, including a major peak at 166 bp, a smaller peak at 143 bp, and a 10-bp periodicity below 143 bp (8). The most distinctive difference between fetal and maternal DNA in maternal plasma is that fetal DNA shows a reduced proportion of molecules of 166 bp and an increased proportion of molecules of less than 150 bp (8). In this study, we outlined the theoretical basis and explored the implementation of using molecular size analysis of plasma DNA as a diagnostic approach. We demonstrated the feasibility o...

Noninvasive detection of fetal subchromosomal abnormalities by semiconductor sequencing of maternal plasma DNA

“…The same sequencing depth of 3.5 million reads was used like in our previous study on trisomy 21,18, and 13 and sex chromosome aneuploidies (12). To confirm that our technique provided results consistent with our previous study, we first assessed the ability of SSP to detect aneuploidy in our cohort.…”

“…To identify the full sequences of cfDNA, we used in-house Perl scripts to exclude incomplete reads and determine the size distributions of plasma cfDNA molecules. Fetal DNA is generally shorter than maternal DNA (28), resulting in the feasibility of predicting the fetal DNA concentration in maternal plasma (18). Plasma samples with a higher fetal DNA fraction would have a higher proportion of short plasma DNA fragments (∼130-140 bp; region A) and a lower proportion of long plasma DNA fragments (∼155-175 bp; region B).…”

“…Sequencing SNPs was considered as the primary method of determining fetal DNA fraction (17). Recently, Lo et al (18) developed a novel method that relies on the fact that circulating fetal DNA molecules are generally shorter than the corresponding maternal DNA molecules to estimate the fraction of fetal DNA in maternal plasma. This approach can be adapted for an SSP, which is capable of sequencing entire molecules of fetal DNA to obtain the size of the DNA fragments.…”

Non-invasive prenatal testing (NIPT): limitations on the way to become diagnosis

“…Studies have shown that ccffDNA can first be observed as early as 4 gestation weeks and the amount of ccffDNA increases as the pregnancy progresses [22] but diminishes quickly after the birth of the baby, so that it is no longer detectable in the maternal blood approximately 2 h after birth. The fetal DNA is significantly smaller than the maternal DNA in the bloodstream, with fragments with a median size of 146 bp and makes its way into the maternal bloodstream via shedding of the placental microparticles into the maternal bloodstream [23]. Fetal ccffDNA is heavily diluted with maternal plasma and this "fetal fraction" is positively correlated with gestational age while preliminary evidence suggests that it is negatively correlated with maternal weight [7,24,25].…”

“…In general, the fetal fraction percentage has to be provided and to be estimated with real-time quantitative PCR [22,26]: over 8% is considered satisfactory for most NIPT methods, while a 4-8% is considered marginal [11]. There are now research efforts ongoing for the extraction of the fetal DNA from the maternal plasma based on its size in order to distinguish it from the maternal DNA and/or accurately estimate it [23].…”