Diagnosis of fetal aneuploidy

Introduction Fetal Aneuploidy is not as common as other birth defects, affecting 9 in 1000 births and increasing with age. Amniocentesis and chorionic villus sampling are two of the most popular methods used to detect Fetal Aneuploidy. Since these procedures are invasive, they may result in an adverse effect on the health of the unborn fetus and/or the mother. Recently, there has been much research on the development of noninvasive methods to detect Fetal Aneuploidy (1).

Laboratory Techniques for Prenatal Diagnosis Currently, laboratory techniques are being developed that are more efficient and accurate than before for detecting Fetal Aneuploidy and other chromosomal aberrations. Interphase-fluorescence in situ hybridization, quantitative fluorescence polymerase chain reaction and direct preparation of chromosomes from chorionic villi are all current methods being used that are the most effective for detecting Fetal Aneuploidy (2). Early Diagnosis: During the first trimester of pregnancy, chorionic villi sampling has become an effective and common procedure where a piece of placental tissue is taken for examination. Another common method is amniocentesis, where amniotic fluid is drawn from the uterus. These amniotic cells can be cultured as early as the second trimester of pregnancy. Both of these methods have risks because they are both invasive. Risks include infection of the amniotic fluid, leaking of the amniotic sac, and even miscarriage in the most serious cases (2). Rapid Diagnosis: Cell culturing usually takes up to one to two weeks in the case of chorionic villi sampling and amniocentesis. Fluorescence in situ hybridization (FISH) is used for rapid diagnosis. It detects the presence of specific DNA sequences by using fluorescent probes that bind to the DNA sequences. FISH involves three basic steps. The first step entails the fixation of a sample on a slide. The second step is the hybridization of a labeled probe to fragments of DNA. The last step is the detection of the tagged DNA sequence (2). The following diagram summarizes FISH:

(National Human Genome Research Institute (NHGRI) http://www.genome.gov/12514471 by artist Darryl Leja)

Noninvasive Diagnosis: Due to the detection of fetal cells and fetal DNA circulating in maternal blood, noninvasive diagnosis of Fetal Aneuploidy is becoming more promising (2).

Maternal Plasma Nucleic Acid Analysis The development of a variety of screening methods for Fetal Aneuploidy and other chromosomal aberrations is now a prominent research area because of the discovery of circulating fetal nucleic acid in maternal blood plasma. However, the key problem is that circulating fetal nucleated cells comprise only three to six percent of maternal blood plasma DNA. Therefore, two effective approaches have been developed that can be used for the detection of Fetal Aneuploidy. The first involves the measuring of the allelic ratio of SNP’s (single nucleotide polymorphisms) in the mRNA coding region in the placenta. The next approach is analyzing both maternal and fetal DNA and looking for differences in the DNA methylation patterns (3). The following diagram summarizes these approaches:

Developments in Fetal Aneuploidy Detection Using Plasma RNA

2000 It was found that in pregnant women carrying male fetuses, the mRNA transcribed from the Y chromosome was found in maternal blood plasma.

2002 Plasma RNA is found to be stable.

2003 Fetal RNA in maternal bloods plasma was found to have it’s origins in the placenta.

2004 mRNA markers found in the placenta can be detected in the maternal blood plasma using microarrays.

2007 Using microarrays, a specific mRNA from the placenta was found in maternal blood plasma. This mRNA disappeared from the maternal blood plasma following delivery of the baby (3).

Digital PCR Recently, it has been proposed that digital PCR can used for detection of Fetal Aneuploidy using fetal DNA and RNA found in maternal blood plasma. Research has shown that digital PCR can be used to differentiate between normal and aneuploid DNA using fetal DNA in the maternal blood plasma. “The arrival of digital PCR brings possibilities for the noninvasive detection of Fetal Aneuploidy and Mendelian disorders. It is hopeful that these developments in research will be furthered in the future (4).

Noninvasive Diagnosis of Fetal Aneuploidy Fetal cell DNA in maternal blood plasma is elevated in pregnancies complicated by fetal trisomy 21, 13 and 18. This is good news because more fetal DNA is available in the maternal blood plasma to test for Fetal Aneuploidy. Fetal cell DNA has been directly sequenced using shotgun sequencing technology. This DNA was obtained from the blood plasma of eighteen pregnant women. This was followed by mapping the chromosome using the quantification of fragments. This was done using advanced methods in DNA sequencing resulting in the parallel sequencing of the fetal DNA. The amount of sequence tags mapped to each chromosome was counted. If there was a surplus or deficiency in any of the chromosomes, this meant that there was a fetal aneuploid. Using this method of shotgun sequencing, the successful identification of trisomy 21 (Down syndrome), trisomy 18 (Edward syndrome), and trisomy 13 (Patau syndrome) was possible. This method of noninvasive diagnosis is now starting to be heavily used and researched further (1).

The Attitude of Women toward Noninvasive Diagnosis of Fetal Aneuploid Using Maternal Blood

Research was conducted to determine how women felt about noninvasive diagnosis of fetal aneuploid using maternal blood. This study was conducted using surveys. It was reported that eight-two percent of pregnant women and seventy-nine percent of female medical students view this type of diagnosis in a positive light, agreeing that it is important for prenatal care. Overall, women responded optimistically that this form of diagnosis will be the future (5).

References 1. Noninvasive diagnosis of fetal aneuploidy by shotgun sequencing DNA from maternal blood. Fan HC, Blumenfeld YJ, Chitkara U, Hudgins L, Quake SR. Proc Natl Acad Sci U S A. 2008 Oct 21;105(42):16266-71. Epub 2008 Oct 6.

2. Developments in laboratory techniques for prenatal diagnosis. Miny P, Tercanli S, Holzgreve W. Curr Opin Obstet Gynecol. 2002 Apr;14(2):161-8. Review.

3. Noninvasive prenatal detection of fetal chromosomal aneuploidies by maternal plasma nucleic acid analysis: a review of the current state of the art. Lo YM. BJOG. 2009 Jan;116(2):152-7. Review.

4. Digital PCR: a powerful new tool for noninvasive prenatal diagnosis? Zimmermann BG, Grill S, Holzgreve W, Zhong XY, Jackson LG, Hahn S. Prenat Diagn. 2008 Dec;28(12):1087-93. Review.

5. The attitude of women toward current and future possibilities of diagnostic testing in maternal blood using fetal DNA. Kooij L, Tymstra T, Berg P. Prenat Diagn. 2009 Feb;29(2):164-8.