Noninvasive prenatal testing (NIPT) is able to gain fetal DNA which is circulating in maternal blood stream.
Over the last two decades numerous attempts have been made to find noninvasive techniques for diagnosing fetal aneuploidy (Pic. 1). Initial research focused on the isolation of fetal cells from the maternal blood. However the isolation of such cells proved to be technically difficult and inconsistent. In recent years the focus has shifted to cell free fetal DNA (cfDNA) in maternal circulation.
As a result of cell turnover, short (~200 bp) fragments of cfDNA are released into the bloodstream. During pregnancy, about 10% of cfDNA is of fetal origin (cffDNA). Cell free DNA was first used for non-invasive prenatal diagnosis of fetal sex using PCR amplification of Y-chromosome specific fragments. Subsequently, cfDNA was used to determine fetal Rhesus (Rh) status in Rh-negative women and to exclude paternally-derived mutations.
Recently cfDNA has been used to detect fetal chromosomal anomalies, by a technique generally referred to as Non-Invasive Prenatal Testing (NIPT). Most of these tests are based on sequencing and quantification of cfDNA in the maternal plasma. Non-invasive prenatal testing is based on the assumption that when the fetus has a normal constitution of 46 chromosomes, there is a constant ratio between the number of fragments derived from each chromosome. In contrast, when the fetus is affected by a chromosomal numeric aberration, there is a deviation from the expected ratio. For example, if the fetus has trisomy 21, more fetal cfDNA fragments from chromosome 21 are released into the maternal circulation. While the absolute increase in chromosome 21-derived fragments is quite low, sequencing and counting of numerous fragments provides statistical significant results. This approach only became feasible with the introduction of shot-gun or Massive Parallel Sequencing (MPS), which allows the simultaneous sequencing and counting of millions of cfDNA fragments.
cffDNA can be detected in maternal plasma as early as 5–7 weeks; however, test results are more accurate after 10 weeks because the amount of cffDNA increases over time. The cffDNA originates from the trophoblasts (Pic. 2) making up the placenta. An average of 11%-13.4% of cell-free DNA in maternal blood is of fetal origin, although this varies widely amongst patients. cffDNA diminishes quickly after the birth of the baby, so that it is no longer detectable in the maternal blood approximately 2 hours after birth. cffDNA is significantly smaller than the maternal DNA in the bloodstream, with fragments approximately 200bp in size. Many protocols to extract the fetal DNA from the maternal plasma use its size to distinguish it from the maternal DNA.
NIPT was first offered for trisomy (there are three instances of a particular chromosome, instead of the normal two) 21, but soon expanded to include trisomy 18 and 13 and sex chromosome anomalies such as Turner syndrome (45,X; a condition in which a female is partly or completely missing an X chromosome), Klinefelter syndrome (47,XXY; the set of symptoms that result from two or more X chromosomes in males), and others. Recently, NIPT has been reported as efficient in the detection of sub-chromosomal anomalies that are usually detected by chromosomal microarrays such as Velo-cardio-facial syndrome (VCF; a genetic condition characterized by abnormal pharyngeal arch development that results in defective development of the parathyroid glands, thymus, and region of the heart) caused by a microdeletion in chromosome 22q (Pic. 3). An alternative to MPS employs targeted sequencing of only a few chromosomes of clinical interest. An example of this approach based on quantification of pre-selected non-polymorphic loci (the place where chromosomal abnormality occurres) by digital analysis of selected regions (DANSRTM). Another targeted approach is based on sequencing of polymorphic loci on chromosomes of interest, which is then compared with the expected allele distribution based on maternal, and occasionally parental, genotypes. Since such targeted approaches sequence only specific fragments of interest, the cost is expected to decrease.
In summary, NIPT has now become a reality. High sensitivity and specificity already mean that fewer patients will require unnecessary invasive procedures. If the sensitivity and specificity of NIPT improve NIPT may ultimately replace invasive procedures. However at this juncture, these tests are to be regarded as very good screening tests, as their performance characteristics are superior to other screening modalities. Commercialization of NIPT has to be taken into consideration and thus utilization of NIPT should adhere to professional guidelines.
The performance of NIPT for other aneuploidies is lower than that of trisomy 21. The combined detection rate for trisomy 18 is 97.4% and for trisomy 13 is only 83.3%. These lower detection rates may be due to larger chromosome size and higher GC content of chromosome 13. Another explanation for the decreased sensitivity of NIPT for trisomies 13 and 18 may be due to the fact that in these cases there is a smaller placenta resulting in a lower concentration of cffDNA. As more conditions are tested (such as microdeletions), the cumulative false positive rates are expected to increase.
In addition to the limitations discussed above, there are a number of drawbacks that must be addressed before NIPT becomes common clinical practice:
False negative results: There have been several reports of false negative results, rates in the range of 0 to 1.4%. This fact must be conveyed to patients during pretest counseling.
False positive results: These are more common in trisomy 13, 18 and sex chromosome aneuploidy. As more conditions are added to NIPT, cumulative false positive rates are expected to increase.
Cost: The current cost of NIPT is too high to offer to the entire pregnant population. However, it is possible that NIPT may be utilized as a secondary screen for women determined to be at-risk by standard screening test, using a contingent screening approach.
Technical difficulties in using NIPT: In rare instances, the test fails to provide a result. This is mostly due to a low fraction of fetal DNA in the maternal plasma and technology used. Test failure occurs in approximately 0.7-3.8% of tests. However, redrawing blood from the patient will allow a result in the majority of these cases.
Because all NIPTs have potential false positive and false negative results, they are currently not considered diagnostic, but should be regarded as highly reliable screening tests. Therefore, abnormal NIPT results should be followed by invasive diagnostic testing (CVS or amniocentesis).
The only physical risks associated with the procedure itself are those normally associated with a blood draw and there is no risk of miscarriage.
People tend to overestimate the usefulness of genetic tests, and misinterpret their meaning. It is possible that pregnant women will interpret a positive NIPT test as positive diagnosis, and wish to abort a pregnancy on this basis. A clear summary of test accuracy for NIPT is necessary for use by doctors and patients for use in shared and informed decision-making.
The use of NIPT in clinical practice should be an informed patient choice. Pre-test counseling should focus not only on the benefits but also on the limitations of NIPT. It should be made clear that NIPT does not replace invasive diagnostic testing. Post-test counseling is also of great importance. Patients receiving positive results are recommended to have definite diagnostic testing because of potential false positive results. Patients receiving negative results should be counseled regarding the residual risk for a chromosomal anomaly. In addition, it must be made clear that NIPT does not reduce the risk for chromosome anomalies that are not included in the test. Patients manifesting major structural anomalies should not be reassured given a negative NIPT result but should be referred for genetic counseling and invasive testing, including chromosomal microarray analysis.