COMPETENCIAS TIC

1.8.1 Recent developments in cell-free DNA markers for non-invasive prenatal diagnosis

Putative epigenetic markers for cff DNA have been discovered on chromosomes 18, 3 and 21, using the model system first proposed by Chim and colleagues in 2005 (Chim et al, 2005; Chan et al, 2006 and Chim et al, 2008). On the basis of the early discovery of the MASPIN marker, and by use of the model system I discovered several novel candidate epigenetic biomarkers on Chromosome 21 (Old et al, 2007). However it is unlikely that any of the cff DNA markers discovered so far either by the Lo group or by my own research could form part of a non-invasive diagnostic test.

So far I have discovered that after testing CEBs on plasma DNA from pregnant women (Old et al, 2007), the methylation signature of placenta DNA is not maintained throughout pregnancy (see chapter 8). In addition to this the markers published by the Lo group are unlikely to be used in diagnostic test as they are not true biomarkers for first trimester plasma DNA. The methylation signature of placenta DNA for the MASPIN marker is significantly different to that of maternal blood cell DNA at the third trimester. However, the two tissue types are less easily distinguished during the first trimester, with completely unmethylated placenta DNA making up a smaller proportion of circulating cff DNA in the first trimester (Chim et al, 2005).

The discovery of an additional potential marker of cell-free fetal DNA was published in 2006. Hypermethylation of the RASSF1A gene is associated with an increased risk of many types of cancer and is located on chromosome 3. RASSF1A was published as a universal fetal DNA marker epigenetic biomarker by Chan and colleagues (Chan et al, 2006); but has been shown to be variable in it’s specificity for cff DNA (data presented by Aitcha Ait Soussan at SAFE PhD Workshop, Bologna 2008).

The latest proposed markers on Chromosome 21 are PDE9A and U-CGI137 markers have been also only been shown to match the epigenetic profile of placenta DNA in the third trimester. So far they have not been shown to be concordant during either the first or second trimesters, also the paucity of methylated CpG sites in the U- CGI137 putative marker is noted by the authors (Chim et al, 2008).

Having conducted my own research using the model system and by testing candidate epigenetic biomarkers with plasma DNA from pregnant women (see chapter 8), one conclusion which can be drawn is that the model system is not appropriate for the discovery of epigenetic biomarkers as the peripheral blood leukocyte DNA and term placenta DNA do not accurately represent the DNA populations present in the plasma of pregnant women (see chapter 9).

1.8.2 Recent developments in cell-free fetal RNA markers for non- invasive prenatal diagnosis

On 04/06/2008 Sequenom a US company, which has purchased the intellectual property rights with regard to cell-free fetal nucleic acids, issued a press release. The release stated that from a small study of 200 plasma samples from pregnant women that 100% of trisomy 21 cases were detected with 0% false positive results. The company also reported that the test offered coverage of 93% (for the US Caucasian population). In a blind study out of 180 samples 3 trisomy 21 samples were detected with 0% false positive results, and of a smaller blind study 7/21 trisomy 21 cases were detected with 0% false positive results (www.sequenom.com).

These results look promising, however it is still desirable to develop DNA based NIPD as it relies on much simpler and cost effective technology, to give an even more rapid response from sample collection to result; as the measurement of cff DNA does not rely on reverse transcription to be assessed. Also using cff DNA would eliminate the small risk of misdiagnosis caused by cases of undetected confined placental mosaicism.

1.9 Aims

The main aim of the project is to develop fetal specific DNA markers for the detection of cff DNA sequences originating from fetal Chromosome 21 in the maternal circulation. This will require the use molecular techniques in order to distinguish epigenetic modifications (CpG methylation) between cff DNA and cfm DNA. This will be achieved by the identification of a number of sites of differential methylation, between term placenta genomic DNA and adult female leukocyte genomic DNA as candidates for epigenetic biomarkers.

Once candidate epigenetic biomarkers have been identified, further testing and validation using plasma DNA samples from pregnant and non-pregnant women will be required.

To determine the utility of analysing DNA methylation in the circulation, the ratio of methylated to unmethylated CpG DNA will be assessed in plasma DNA. This is in order to end any speculation as to the preferential clearance of unmethylated CpG DNA plasma. This work will require the measurement of the allelic ratio, of an imprinted DNA sequence, in order to determine if either methylated or unmethylated CpG DNA are cleared preferentially.

Further to the suitability of using DNA in the circulation as a biomarker, the relative abundances of specific DNA sequences will be assessed by real-time PCR, in order to determine if the general assumption that all DNA sequences are represented equally in the circulating genome (Chan et al, 2005 b). The PCR efficiency of each sequence will be analysed to give quantitative data on the relative abundances of each sequence in plasma DNA.