Development of the MLPA technique as a non-invasive prenatal diagnostic test to detect fetal chromosomal abnormalities and infections in the maternal circulation in early pregnancy

Approximately, 10-20% of pregnancies end in miscarriage. The major causes of death are chromosomal abnormalities or first/second trimester infections. Despite nature’s selection, 3-5% of the newborns have congenital malformations. These include 0.5-1% chromosomal abnormalities (0.3% being trisomy 21), and 1-4% fetal/intrauterine infections. Chromosomal abnormalities and/or infections can be diagnosed through chorionic villus sampling (CVS) or mainly through amniocentesis (AC). However, the procedure-related risk of abortion is between 0.5-1% for AC and up to 2% for CVS. Currently, no safe, reliable, cost-effective, non-invasive prenatal diagnostic test exists to detect e.g. trisomy 21. It is therefore challenging to develop such a non-invasive prenatal diagnostic test. The main indications for invasive prenatal investigation are ultrasound abnormalities, high maternal age or increased risk for trisomy 21 on the basis of first trimester screening (using the combination test). The number of false-positive test results after prenatal diagnosis based on prenatal screening risk calculations is still high, although lower than for maternal age as a “screening instrument” only. So, still too much invasive procedures are done unnecessary and may lead to unwanted abortion due to the invasive procedure. Our general aim is to develop a reliable non-invasive prenatal diagnostic test on cell-free fetal DNA/RNA present in maternal plasma, using the “Multiplex Ligation-dependent Probe Amplification” (MLPA) technique. To achieve this goal we will use cell-free fetal DNA/RNA which circulates in maternal blood from 5-6 weeks pregnancy. During pregnancy the amount of cell-free fetal DNA/RNA is gradually increasing which makes it detectable through PCR-based techniques from more or less 8-9 weeks of pregnancy. Because the MLPA technique allows precise quantification, it is a good candidate method to detect highly fragmented small amounts of cell-free fetal DNA/RNA within maternal plasma. The MLPA-test provides rapid detection and the combination of probes makes it possible to detect multiple genomic regions at once. Different MLPA-kits (www.mrc-holland.com) are already on the market to detect the most frequent chromosomal abnormalities and (lung) infections (www.pathofinder.nl). The non-invasive prenatal MLPA test we develop further contains MLPA probes to detect the most common trisomies 13, 18 and 21. In parallel with this non-invasive MLPA analysis we try to develop a combined non-invasive prenatal MLPA test that also detects the most important intrauterine/fetal and perinatal TORCH infections (Toxoplasmosis, Other infections such as syphilis, hepatitis B, varicella-zoster virus, human immunodeficiency virus, parvovirus B19, Rubella, Cytomegalovirus and Herpes simplex virus) at the same time in pregnancy. This “TORCH infections” part needs further proof of principle and will be developed in close collaboration with the Pathofinder Company research-team. The specificity, sensitivity and reproducibility of the non-invasive MLPA test will be determined and compared with existing methods of prenatal diagnosis, first/ second trimester screening and with biochemical and/or molecular investigations such as quantitative real-time fluorescent PCR, targeted sequencing and TORCH-serology. The current study and MLPA method is (already) approved by the local medical ethical committee and is divided in three phases. Faze 1, consists of continuation of the MLPA proof of principle for chromosomes 13, 18 and for development of MLPA-probes to detect prenatal TORCH infections. Surplus maternal and cell-free fetal DNA/RNA will be used for this development. In Faze 2, a pilot study will be performed in 40 pregnant women with a low risk for fetal trisomy 21. They will be recruited at the time of first trimester screening (group 2a). Maternal blood EDTA samples from group 2a are compared with samples from group 2b consisting of 10 proven fetal trisomy 21, five trisomy 18 and two trisomy 13 pregnancies. In Faze 3, 715 women will be included from a high risk group (age ≥ 36 years, risk for trisomy 21 ≥ 1:250 after prenatal screening or ultrasound abnormalities pointing to a chromosomal abnormality). After informed consent from the pregnant woman and her partner, 2x20 ml EDTA blood will be (repeatedly) taken. Maternal and cell-free fetal DNA/RNA will be isolated in duplicate following standard procedure (QIAamp MinElute Virus kit from Qiagen). Samples are immediately used for MLPA detection after specific amplification in one tube-experiment. Results obtained in faze III with the non-invasive MLPA test will be compared at the end of the study with the results obtained from invasive prenatal testing or pregnancy outcome. Efficiency, validity (sensitivity and specificity), reliability (reproducibility) and actual costs to further implement this non-invasive MLPA test into a multi-centre prospective cohort study will be determined.