By Ben Butkus
A group of scientists from Cyprus has combined methylated DNA immunoprecipitation and quantitative real-time PCR into a non-invasive, prenatal test that can diagnose Down syndrome with 100 percent sensitivity and specificity, according to a paper published online this week in Nature Medicine.
The researchers, from the Cyprus Institute of Neurology and Genetics, claim that their method is simpler, faster, and cheaper than other non-invasive methods for diagnosing Down syndrome, also known as trisomy 21, and suitable for routine employment in diagnostic labs.
As such, CING has formed a spin-off company called NIPD Genetics with the hope of commercializing the assay within two years, according to a research team member. In the meantime, the group is planning a larger-scale study using samples obtained from laboratories worldwide to further refine the test and investigate variability among different ethnic groups, the researcher said.
Several research groups and companies are developing non-invasive methods for diagnosing Down syndrome and other common aneuploidies from maternal blood in an effort to replace commonly used invasive procedures such as amniocentesis or chorionic villus sampling.
Perhaps most notably, separate teams led by Stephen Quake at Stanford University and Dennis Lo of the Chinese University of Hong Kong have been cultivating high-throughput sequencing-based methods for prenatal testing of Down syndrome. Sequenom has licensed exclusive rights to the Lo group's technology. Meantime, Quake's group has filed patents on screening methods that use massively parallel digital PCR technology (PCR Insider, 2/17/2011).
In addition, Spanish molecular diagnostics firm Molgentix hopes to seek US regulatory approval for its CE Marked test called Aneufast, which uses quantitative fluorescent PCR but has not yet been made completely non-invasive (PCR Insider, 2/17/2011); and Dutch researchers are working on a test that uses multiplex ligation-dependent probe amplification technology but may not be clinically viable until 2015 (PCR Insider, 7/1/2010).
The Cypriot team has instead opted to develop a trisomy 21 diagnostic strategy combining a previously developed technique called methylated DNA immunoprecipitation, or MeDiP, with qRT-PCR, citing the high expense and technical difficulty of many of the aforementioned methods.
Next-generation sequencing and other methodologies "have been shown to be of high cost to perform and require expensive equipment and infrastructure, which is not available in the majority of the diagnostic laboratories worldwide," Philippos Patsalis, a researcher at CING and corresponding author on the Nature Medicine paper, wrote in an e-mail to PCR Insider.
"On the other hand, our method is relatively simple, fast, and low-cost," Patsalis said. "Furthermore, it uses basic molecular biology equipment and methods; therefore, it can be introduced easily in laboratories around the world."
The group's method involves collecting 10 mL of peripheral blood from a pregnant woman, then isolating the DNA. This is followed by MeDiP, which uses an antibody specific for 5-methylcytidine to capture methylated sites and enrich for DNA regions that are specifically hypermethylated in the fetal DNA and hypomethylated in the maternal DNA.
"It is important at this point to clarify that we know exactly which regions of the DNA demonstrate these characteristics, as we have previously identified these regions," Patsalis said. "Therefore our study is focused on only those regions specifically located on chromosome 21, which is the chromosome associated with the development of Down syndrome."
After methylation enrichment of the fetal DNA in maternal circulation, the researchers apply qRT-PCR to quantify the amount of fetal DNA in the test sample. By comparing the amount of fetal DNA present in an unknown sample to that of a control sample from a pregnant woman known to carry a normal fetus, the technique can quantify the extra copy of chromosome 21and distinguish Down syndrome pregnancies from normal pregnancies.
As detailed in the Nature Medicine paper, the researchers used their method to correctly diagnose 14 cases of Down syndrome and 26 normal cases from maternal peripheral blood samples.
"The success of the test relies on the fact that the analysis is not based solely by quantifying the copy number of a single region, but the results are retrieved by combining multiple regions, therefore allowing us to reach 100 percent sensitivity and 100 percent specificity," Patsalis said.
For the qRT-PCR portion of their test, the group used a SYBR Green assay, "where our controls are a set of normal pregnancies and not a genetic locus on a different chromosome than chromosome 21," Patsalis said. In addition, the group has been using instruments and reagents from different companies with the same success, he said.
The work described in the Nature Medicine paper was primarily performed at CING, although collaborators from Mitera Hospital and the National and Kapodistraian University of Athens in Greece provided the group with the majority of their samples for testing.
The commercial rights to the method belong to CING, according to Patsalis, which through a spin-off company called NIPD Genetics plans to develop, produce, and distribute a commercially available kit. "We believe the kit will be introduced to the market in less than two years and will be viable," Patsalis said.
Before that happens, the researchers plan to conduct a much larger study of some 1,000 samples obtained from different ethnic groups in order to confirm their recent findings and investigate the possibility of variability between different ethnic groups.
This researchers will conduct this study in collaboration with at least 10 diagnostic laboratories or universities from the US, Australia, and Europe, Patsalis said.
"Once the clinical study of the additional 1,000 samples is completed, the test will be ready to be introduced in clinical practice," he added.
"In parallel, we are planning to initiate a study for the development of other common fetal abnormalities such as trisomy 13 [and] 18; and aneuploidies of chromosomes X and Y," Patsalis said. "We already have the required background and the plans in hand to initiate this study. The final aim is to develop a test that will include the testing of all the above abnormalities."
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