A New Non-invasive Test for Down Syndrome:Trisomy 21 Diagnosis using Fetal-Specific DNA Methylation

Using a non-invasive technique developed by a group of investigators in Greece and UK, correct diagnosis of Down syndrome was achieved. This technical report was published on March 6, 2011 in the online version of Nature Medicine, a peer-reviewed scholarly publication. This technique has the potential to replace the currently used invasive prenatal diagnostic procedure to detect Down Syndrome (trisomy 21).

Down syndrome is a genetic condition in which a person has 47 chromosomes instead of the usual 46. In most cases, Down syndrome occurs when there is an extra copy of chromosome 21. This form of Down syndrome is called trisomy 21. This genetic defect causes problems with the way the body and brain develop. Down syndrome is the most common single cause of human birth defects, affecting around one in 700 births. A child with Down syndrome has three, rather than the normal two, copies of chromosome 21. Currently, Down syndrome is diagnosed by one or more of three invasive techniques that carry a risk to the fetus. The new work seeks to reduce this risk and to improve detection.


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In this study (Ref 1), the investigators were able to discriminate normal from trisomy 21 by comparing normal and trisomy 21 cases using fetal-specific methylated regions located on chromosome 21. DNA methylation, scientists call as an epigenetic phenomena, involves the addition of a methyl group to the 5 position of the cytosine (pyrimidine) ring or the number 6 nitrogen of the adenine (purine) ring of DNA. DNA is made up of purine-pyrimidine bases. Methylation of DNA can alter the gene expression pattern, but does not alter the primary sequence of the DNA.

The difference in methylation of specific DNA sequences between mother and fetus can be used to identify fetal specific DNA in the blood circulation of the mother. In the current study, using a technique called immunoprecipitation, the investigators were able to specifically separate methylated fetal DNA from unmethylated maternal DNA. The regions of interest on chromosome 21 in the DNA samples immunoprecipitated were amplified by another technique called polymerase chain reaction (PCR). The fetal-specific methylation ratio approach provided correct diagnosis of 14 trisomy 21 and 26 normal cases.

The new test requires only a sample of blood from the mother. In the mother's blood circulation are free DNA molecules - and about five per cent of that free DNA is derived from fetal cells.

Previous studies have found fetal DNA concentrations in the maternal circulation to be higher in women carrying fetuses with Down syndrome than in unaffected pregnancies. The seminal study that form the foundation of these findings was published in Lancet in 1997 by Lo and colleagues from the University of Oxford (Ref 2). They found circulating fetal DNA in maternal plasma and suggested that the finding may have implications for non-invasive prenatal diagnosis, and for improving our understanding of the feto-maternal relationship. The study was inspired by the demonstration of tumor-derived DNA in the plasma of cancer patients. Subsequently non-invasive diagnosis using free fetal DNA (ff DNA) has been utilized in the detection of paternally inherited alleles, sex-linked conditions and some single-gene disorders and is a viable indicator of predisposition to certain obstetric complications (Ref 3). Less than 10 years after the discovery ffDNA in the maternal circulation, the world's first non-invasive genotyping service was offered in Bristol for the diagnosis of RHD genotype and fetal gender (Ref 4).

According to the authors of the new study, their "diagnostic approach is not affected by fetal gender or the presence of informative polymorphic sites" and "this advantage is of high importance as the technology will be available to any pregnant woman."

"There is a real need to improve prenatal diagnosis," explains Professor Philippos Patsalis, senior author and leader of the research from the Cyprus Institute of Neurology and Genetics. "We have used our understanding of biology to develop a blood test for the mother that reports chromosome number in the baby.

"Our results were really exciting: we correctly determined every Down syndrome and every unaffected baby in our test."

The method is simple, fast and easy to perform in every genetic diagnostic lab worldwide because it does not require expensive equipment, software or special infrastructure. It is also currently considerably cheaper than techniques such as DNA sequencing.

The new test can pick up fetal DNA signals - even though this is in the minority in the mother's blood - because it detects changes called methylation that can occur more in fetal than in maternal DNA. The team targeted carefully chosen regions of chromosome 21. This allows them to measure how many copies of chromosome 21 are present.

The team suggests that the technique might also be used for other chromosome imbalances similar to Down syndrome. They also make clear the important point that a large-scale study is required to develop the test further and before it can be provided as a service.

References:
1. Papageorgiou EA, Karagrigoriou K, Tsaliki E, Velissariou V, Carter NP and Patsalis PC Fetal-specific DNA methylation ratio permits noninvasive prenatal diagnosis of trisomy 21. Nature Medicine 2011

2. Lo YMD, Corbetta N, Chamberlain PF, Rai V, Sargent IL and Redman CWG (1997) Presence of fetal DNA in maternal plasma and serum. Lancet 350, 485-487

3. Maddocks DG, Alberry MS, Attilakos G, Madgett TE, Choi K, Soothill PW, Avent ND.(2009) The SAFE project: towards non-invasive prenatal diagnosis.Biochem Soc Trans. 37:460-5.

4. Finning K, Martin P and Daniels G (2004) A clinical service in the UK to predict fetal Rh (rhesus) D blood group using free fetal DNA in maternal plasma. Ann. N.Y. Acad. Sci. 1022, 119-123.