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DNA Size Differences Useful for NIPT; Have Potential in Cancer Dx, Transplant Monitoring

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NEW YORK (GenomeWeb) − Researchers led by Dennis Lo at the Chinese University of Hong Kong have shown that size differences between fetal DNA and maternal DNA in the mother's plasma can be exploited for non-invasive prenatal testing, providing an alternative to methods used in commercial tests that might boost overall specificity.

The approach, which relies on paired-end sequencing or capillary electrophoresis to measure the DNA fragment size and was published online in Proceedings of the National Academy of Sciences last month, also has potential applications outside the prenatal testing field, for example in cancer diagnostics and in transplant monitoring.

While current non-invasive prenatal aneuploidy tests offered by Sequenom, Ariosa, Natera, Illumina's Verinata, and others are more than 99 percent accurate, they are still considered by many as very good screening tests rather than diagnostic tests, according to Lo, in part because they rely on statistical approaches that might produce false-positive results.

"You need a second tool to further increase the specificity, and this size-based approach is one possible second tool to do that," he said. Another possibility would be to use differences in methylation between fetal and maternal DNA, he added, a strategy his team published for applications in cancer last year.

The new size-based method is covered by US Patent No., 8,620,593, "Size-based genomic analysis," and by US Patent application 20130237431, "Size-based analysis of fetal DNA fraction in maternal plasma," both assigned to the Chinese University of Hong Kong.

Sequenom has licensed the IP, and a company representative told Clinical Sequencing News that the firm will evaluate the utility of the approach and may integrate it into future product offerings "where useful."

Sequenom currently offers the MaterniT21 Plus test, which uses sequencing of maternal plasma DNA to determine the relative amount of each chromosome in order to detect fetal chromosomal abnormalities. It is based on another method that was developed by Lo and his colleagues.

The fact that fetally and maternally derived DNA in the plasma have different size distributions has been known for at least a decade, and has been used by researchers to enrich fetal DNA, Lo told CSN.

Recently, next-gen sequencing has allowed researchers to measure those differences more precisely. Overall, fetal DNA tends to have more fragments that are smaller than 150 base pairs, and fewer fragments around 166 base pairs in size.

Lo's team has now used the size differences to detect fetal chromosomal aneuploidies and to measure the fraction of fetal DNA in maternal plasma – sufficient fetal DNA is an important factor for the success of commercial non-invasive prenatal tests.

For their study, the researchers analyzed two sets of maternal plasma samples by paired-end sequencing on the Illumina Genome Analyzer or the HiSeq. One set of previously studied samples included 60 euploid fetuses, 36 cases of trisomy 21, 27 cases of trisomy 18, and 21 cases of trisomy 13. The other set contained 16 euploid fetuses and 10 cases of monosomy X.

They then looked at the size distribution of sequenced fragments from each chromosome. Their assumption was that for a fetus with trisomy 21, for example, with three instead of two chromosomes 21, they would find more small DNA fragments derived from chromosome 21 than from other chromosomes. Likewise, for a fetus with monosomy X, with one instead of two X chromosomes, they would find fewer small fragments from the X chromosome than from the other chromosomes.

Indeed, they were able to correctly identify all trisomy 21, trisomy 18, and monosomy X cases, as well as 20 out of 21 trisomy 13 cases.

In addition, the researchers were able to derive the fetal DNA fraction in maternal plasma without the use of sequencing. For that, they ran a sample of the sequencing library on an Agilent Bioanalyzer, which uses chip-based capillary electrophoresis to separate the DNA by size. From the size peaks they observed, they calculated the proportion of fetal DNA in the sample, which corresponded to the fetal DNA fraction they had previously calculated on the basis of the presence of Y chromosomal DNA from a male fetus.

Sequenom currently uses a methylation-based assay, and Ariosa a SNP assay to determine the fetal DNA fraction, which affects the accuracy of NIPT assays. Using capillary electrophoresis instead, which is already used by most labs to check the quality of sequencing libraries prior to running them, would be cheaper and easier, Lo said.

Differences in fragment size could also be exploited in areas other than prenatal testing. For example, there is some evidence that the size of tumor DNA in plasma differs from that of non-cancer DNA. And in transplant patients, DNA from the donor and the recipient appear to have different size profiles. An increase in short DNA fragments in the blood could indicate a heart attack, assuming that DNA released from heart cells is smaller in size than DNA from other sources, Lo said.

The results of the study are "very intriguing," according to Luis Diaz, director of translational medicine at the Ludwig Center for Cancer Genetics and Therapeutics at Johns Hopkins, who was not involved in the study. While size differences between plasma DNA of different origins have been known for a long time, "we now have a better magnifying glass" due to next-gen sequencing technologies, he said.

However, he cautioned that size-based results alone may lack in specificity. "When you deal with a qualitative readout, like size, it's not digital," he said. "While I think that it's a promising line of research, one of my concerns is the specificity that size will provide."

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