Wednesday, 2 January 2013

Cell-free fetus DNA sequencing in the era of NGS: New opportunities for pre-natal diagnosis

The idea that free fetal DNA is present in the maternal blood is quite old and dates back to the end of 90s when, starting from the observation that cancer cell DNA could be found freely circulating in the human blood serum, Lo et al. demonstrated that fetal cell-free DNA could be detected as well starting from a specimen of mother blood (see the original paper). A nice overview of prenatal technology advances can be found on this article published in Wired.

After this discovery several studies emerged and confirmed the possibility to use this easy accessible fetus DNA to detect well-known chromosome abnormalities previously diagnosed by invasive and risky procedures such as amniocentesis or chorionic villus sampling (CVS). In the first years of the new millenium techniques based on free circulating fetal DNA have attracted increasing attention for Rhesus D (RhD) genotyping and detection of chr 21 and cur 18 trysomies (see this review on Nature). Since they have proved to be as accurate as the classical tests requiring either amniocentesis or CVS but as low-risk as a blood draw, a commercial test kit based on this principle first hit the market in October 2011. Known as cell-free fetal DNA testing, it’s now offered by Sequenom, Verinata, and Ariosa Diagnostics

The main advantages in detecting free fetal DNA from maternal blood are the quick and early response and the total absence of risk for the fetus itself. In fact fetal DNA could be detected as early as the 6th week of of pregnancy and the test simply requires a sample of mother's blood. This is a great achievement in the management of difficult pregnancies, thus easing off stress from parents' mind due to the doubtful choice between the risk of having a severely affected child and the risk of impairing a healthy fetus with an invasive procedure. Moreover an early response leaves more time for counseling and decision making process.
The cell-free fetal DNA tests are by now limited to the detection of some well-known trisomy related conditions (Down, Edwards and Patau syndromes) and few other chromosomal abnomalities such as Klineferter or Turner syndromes. Moreover, despite its advantages and reliability, cell-free fetal DNA testing is still not diffused as a clinical practice and sometimes it's even not mentioned by physicians, so that the requests are often driven by the mothers themselves, looking online for a safer test. At the end of November 2012, the American Congress of Obstetricians and Gynecologists released a long-awaited opinion on the cell-free fetal DNA test. The group recommended it for patients at an increased risk for chromosomal defects, including those over age 35 and those with a history of trisomy pregnancies. These guidelines hopefully will make doctors more aware of the new test (many ob-gyns apparently have never even heard of it).

New and rapid improvements in cell-free fetal DNA analysis will now come from applying NGS sequencing techniques, potentially leading to a new era of prenatal genomic diagnosis: deeper, faster, and risk free. In the 2012 a paper published on Science Translational Medicine demonstrated the feasibility of Whole-Genome Sequencing of fetal DNA extracted from mother's blood. The investigators integrated the haplotype-resolved genome sequence of the mother, the shotgun sequence of the father, and the deep sequencing of cell-free DNA in maternal plasma (maternal and fetal) to non-invasively predict the whole-genome sequence of a fetus. This approach requires a paternal sample to determine the fetal genome, which is a practical limitation to its clinical application. Another paper on Nature goes even further, eliminating the need for a paternal sample. Shotgun sequencing of the plasma cell-free DNA was performed, and the relative amounts of parental haplotypes were measured by counting the number of alleles specific to each parental haplotype (‘markers’). The paternally inherited haplotypes were reconstructed by detection of paternal-specific alleles, followed by imputation at linked positions using reference haplotypes from the 1000 Genomes project. This method allowed deduction of the inheritance of each parental haplotype and construction of the full inherited fetal genome. Moreover, the authors were able to detect in the DNA of the fetus a deletion on chr 22 responsible for Di George syndrome. Although technical and analytical challenges remain, in particular to correctly detect de-novo fetal genetic variations, these studies open new possibilities to apply cell-free fetal DNA test also for the identification of point mutations and other small rearrangements.
A complete review on recent advancements in genomic prenatal diagnosis is given in this review published on Trends in Genetics.

There are no doubts that prenatal DNA-based diagnosis will develop rapidly and that WGS of fetal DNA will became feasible and accurate in short times. This rises one more ethical and practical questions on how the genetic data will be managed and stored and on which information should be reported. The leading opinion by now is that only proved disease causing variations will be reported and many suggest to limit the list even further to only those mutations with severe consequences. The main concern in fact is to avoid that reproductive selection is applied also in the case of mild pathologies, or even worst based on unwanted characters based on cultural background. On the other hand, one advantage of having whole genome information is the possibility to adopt therapies or special life habits for conditions for which an early intervention could limit or avoid symptoms. Prenatal genome sequencing also poses the question on who is the owner of the data, or at least who is responsible for it. The test actually acquire information on a subject that can't give his consent and that potentially may be unwilling to know its DNA code at all when its grown up. But these information could also have a great benefic impact for the subject, since they one day may reveal details essential for his health as research constantly upgrade our knowledge about functional impact of genetic variants. So who is eventually in charge for the updates and who will decide on genetic information access? A possible solution could be some kind of encryption strategy (with a decryption code available to the owner only) that guarantees information security. For example I found an interesting solution (developed by Emiliano de Cristofano when he was at the University of California) that also allows for selective decryption of DNA sequence, giving access to a specific region while leaving the rest of the genome unrevealed.

Promises and questions!

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