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Lo's Lab Publishes Non-invasive Prenatal Methylation Sequencing Method


Researchers from Dennis Lo's lab at The Chinese University of Hong Kong have adapted their non-invasive prenatal sequencing method for bisulfite sequencing to analyze the methylation status of fetal DNA in maternal plasma samples.

The group published a study describing the approach — and demonstrating how it could potentially be used as an alternative method for detecting trisomies — in Clinical Chemistry earlier this month.

Lo told Clinical Sequencing News that the method could potentially be used to monitor methylation status in developing pregnancies, or as an add-on to increase the accuracy of current trisomy tests.

"A lot of developmental processes are regulated by epigenetics, but up to now, since fetal tissue can only be obtained invasively, you could not do [this analysis] on healthy cases or in a serial manner," Lo said. "We thought if we could develop a method to profile the fetus by looking at mother's blood it would allow us to do that."

Lo's group pioneered a shotgun sequencing approach several years ago to noninvasively detect fetal aneuploidies from maternal plasma, licensing the discovery to Sequenom, which launched the first commercial test — called MaterniT21 — based on the technique in 2011.

Since then, several others have developed similar tests based on either shotgun or targeted sequencing.

In the new bisulfite sequencing paper, Lo and his colleagues demonstrated that with a related technique, using the Illumina HiSeq 2000 and methylated primers for whole-genome bisulfite sequencing, they could analyze the methylation of fetal DNA circulating in maternal blood across a fetal genome.

In the study, the team sequenced samples from a total of 13 pregnancies to develop and test two approaches to analyze fetal DNA methylation based on bisulfite sequencing products.

First, to demonstrate that the technique worked and to explore its robustness, Lo and his colleagues used known polymorphic differences between mother and fetus as anchor points to assess fetal DNA methylation.

"We wanted to make sure we were actually looking at the methylation status of a DNA molecule we know is coming from the fetus," Lo said. "The most powerful way of doing that is by looking at polymorphisms in which the mother is homozygous and the fetus is heterozygous and scanning around that molecule to find CpG sites and ask what their methylation status is."

But, Lo said, this method does not allow for analysis of all circulating fetal DNA. "The disadvantage is you can only interrogate molecules which contain that informative SNP, and there are many that do not," he said.

To address this, the group developed a second approach using the methylation profile of maternal blood cells and the fractional fetal DNA concentration in maternal plasma to create an algorithm to deduce which molecules are of fetal origin.

Based on the fact that placental DNA is hypomethylated relative to DNA from maternal blood cells, the group was able to calculate how much maternal samples were "diluted" by fetal DNA, to pick out the molecules of fetal origin.

"The advantage of this is that you can then scan the whole genome without limiting yourself to regions with these informative SNPs," Lo said.

Overall, the group demonstrated in the study that it was able to serially assess methylation from maternal blood collected in the first and third trimesters, as well as after delivery, and to indirectly predict fetal methylation profiles without having to use genotype differences between fetus and mother.

The team also demonstrated one potential clinical application of maternal plasma bisulfite sequencing with the successful detection of fetal trisomy 21 in five confirmed trisomy 21 pregnancies from a cohort of 12 pregnant women.

Though the results are preliminary, Lo said it's possible that bisulfite-based trisomy detection could be used in parallel with the trisomy detection methods his group has previously developed to increase the accuracy of such testing.

"We are very excited about that potential," he said.

Lo said his lab has applied for a patent covering the technique and is in conversations about potential licensing agreements, though he did not give any details about potential licensees.

Previous work by the group in non-invasive prenatal sequencing has been licensed to Sequenom.

Additionally, the method shows promise as a way to measure or monitor fetal DNA methylation for a range of other applications, Lo said. "From the literature it looks like there are a number of diseases that might be related to epigenetics, for example, there are hints that preeclampsia might have an epigenetic parameter."

"Also, people have looked at other maternal disease and behaviors that might affect the epigenetics — like smoking or maternal diabetes," Lo said. "All of this can be addressed by this technology."