Determination of fetal chromosome aberrations from fetal DNA in maternal blood: has the challenge finally been met?

Abstract

The analysis of cell-free fetal nucleic acids in maternal blood for prenatal diagnosis has been transformed by several recent profound technology developments. The most noteworthy of these are 'digital PCR' and 'next-generation sequencing' (NGS), which might finally deliver the long-sought goal of noninvasive detection of fetal aneuploidy. Recent data, however, indicate that NGS might even be able to offer a much more detailed appraisal of the fetal genome, including paternal and maternal inheritance of point mutations for mendelian disorders such as β-thalassaemia. Although these developments are very exciting, in their current form they are still too complex and costly, and will need to be simplified considerably for their optimal translation to the clinic. In this regard, targeted NGS does appear to be a step in the right direction, although this should be seen in the context of ongoing progress with the isolation of fetal cells and with proteomic screening markers.

Figure 1

Detection of fetal aneuploidy using digital PCR. In this procedure, fluorescent PCR specific for sequences on chromosomes 12 and 21 is carried out in individual microreaction chambers. The amount of input template is titrated in such a fashion that each microreaction vessel contains >1 copy. After the plateau phase has been reached (approximately 40 cycles), the PCR reaction is terminated, and the number of positive reactions for each locus is counted. In euploid cases the ratio of blue (chromosome 12) to red (chromosome 21) signals should be 1, whereas in cases with Down syndrome the ratio of blue (chromosome 12) to red (chromosome 21) signals should be 1.5 (illustrated in more detail in Ref. 46). This method has to date not been successfully used for the detection of fetal aneuploidy using cell-free DNA in maternal plasma.

Figure 2

Detection of fetal aneuploidy using next-generation sequencing. In this procedure the cell-free DNA fragments in maternal plasma are isolated, and a library with special sequence tags is then made. These tags permit subsequent multiplex analysis. The library is examined by next-generation sequencing, which determines the sequence of each and every fragment. By bioinformatic analysis these sequences are ascribed to chromosomal locations. Following this, the number of sequence reads for each chromosome is counted. For chromosome 21 this is typically of the order of several thousand reads, which can then be compared with several million reads spread across the genome. If the fetus is affected by Down syndrome, then slightly more reads will be recorded for chromosome 21 compared with those from a euploid fetus. By comparing these data with a bank of reference samples, and by the use of predetermined cut-off values (Z score), the ploidy of the sample being examined can be determined (described in more detail in Refs 53, 54).

Figure 3

Schematic representation of a targeted sequencing approach using the SureSelect^TM Target Enrichment System. In this procedure the cell-free DNA fragments are isolated and a library is generated as per the standard next-generation sequencing protocol. Prior to sequencing, however, this library is hybridised to the SureSelect^TM Oligo Capture Library, which is manufactured in such a manner that it will recognise a specific chromosome, such as chromosome 21. These oligo sequences contain magnetic particles to permit their retrieval in a magnetic field. Hence, following hybridisation (65°C, 24 h), captured sequences are selected by magnetic selection, and unselected sequences are washed away. The bound fragments are then purified and prepared for sequencing and examined as described for Figure 2. This procedure was recently shown to permit a 213-fold enrichment of the targeted X chromosome (Ref. 57).