GeNeViSTA

1 Introduction

Chromosomal abnormalities, both numerical and structural, are known to occur in approximately 1 in 200 live births. The diagnosis for chromosomal abnormalities in the antenatal period is usually done by conducting cytogenetic analysis of amniotic, chorionic or fetal blood cells obtained by invasive procedures. Karyotyping is a well-established cytogenetic technique which has been extensively used as a diagnostic tool for pregnant women undergoing these procedures. The technique is considered 100% sensitive and specific and the gold standard for the detection of autosomal trisomies and sex chromosome aneuploidies, against which all other techniques are compared. In addition to chromosomal aneuploidies, structural rearrangements and triploidy can also be detected with a resolution of 10 million DNA base pairs. The various indications for fetal cytogenetic testing include: 1) abnormal ultrasound scan, 2) abnormal maternal serum biochemical results, 3) advanced maternal age (≥35 years at the expected time of confinement) 4) family history of chromosomal aberrations or other genetic disorders.

The rapid rise in the number of pregnancies undergoing maternal biochemical screening and targeted anomaly scans has led to increase in the number of invasive prenatal diagnostic procedures as well. Rapid diagnosis of aneuploidies in these cases is warranted for appropriate management of pregnancy as well as to relieve anxiety for the family. The legal limit of 20 weeks for pregnancy termination in India is a unique legal circumstance which calls for early detection of these abnormalities.

The autosomal trisomies constitute 80% of the chromosomal aberrations. Hence, Rapid Aneuploidy Detection (RAD) methods, which are targeted methods for the diagnosis of common autosomal trisomies (13, 18, 21) and sex chromosome aneuploidies are offered in cases of invasive prenatal testing. Three methods i.e. Fluorescent In Situ Hybridization (FISH), Quantitative Fluorescent Polymerase Chain Reaction (QF-PCR), and Multiplex Ligation Dependent Probe Amplification (MLPA) have been validated for use in prenatal diagnosis (Mann et al., 2004).
  ∙  Fluorescent In Situ Hybridization (FISH) technique: It is usually performed on uncultured interphase cells with probes designed specifically for chromosome 13, 18, 21, X and Y. The number of fluorescent signals per cell gives the number of copies of the targeted chromosome (Mann et al., 2004). The technique is known to be almost 100% sensitive and specific for detection of aneuploidies (Grimshaw et al., 2003). Another advantage is the capacity to detect triploidy where an extra set of chromosomes is present in the cell. However, the FISH technique is non-automated, time consuming and necessitates a skilled technician Mann et al., 2004; Grimshaw et al., 2003).
  ∙  Quantitative Fluorescent Polymerase Chain Reaction (QF-PCR): This assay has been widely used for the past 20 years for rapid aneuploidy detection. It is a PCR based molecular method which uses fluorescent labeled primers to amplify specific DNA markers which are polymorphic (STRs) and unique for chromosomes 13, 18, 21, X and Y. The amplified products are separated through capillary electrophoresis. The copy number of a specific sequence of each chromosome is determined based on the intensity of the fluorescent signal. The sensitivity and specificity of the assay is in the range of 95% -100% (Grimshaw et al., 2003; Cirigliano et al., 2009). There are several commercially available QF-PCR kits (Aneufast TM, Chromoquant aneuploidy detection kit) (Allingham-Hawkins et al., 2011). Detection of maternal cell contamination, triploidy and mosaicism as low as 15% are important advantages of these techniques (Mann et al., 2004; Cirigliano et al., 2009). However, extensive blood staining of specimens interferes with the results and interpretation of this test.
  ∙  Multiplex Ligation Dependent Probe Amplification (MLPA): It is also a PCR based method. It is relatively cheaper and less labor intensive than the FISH technique. The technique involves the use of two fluorescent labeled probes which are hybridized to specific DNA sequences and are then joined by the enzyme DNA ligase. The free ends of the ligated probes are complementary to the primer which enables the amplification of target sites. The amplified products are separated based on size using capillary electrophoresis. Each peak is considered to be the amplified product of a specific probe. The technique has a capacity to quantify up to 40-50 different target sequences in one reaction. The commercially available kit MLPA P095 kit is useful for the detection of chromosome 13, 18, 21, X and Y chromosome aneuploidies. For detection of aneuploidies, a sensitivity and specificity of 100% is attained by employing this technique (Van Opstal et al., 2009). One of the major drawbacks of this technique is the failure to detect triploidies especially in a female fetus. It is a completely automated method, and is being increasingly used as a method for RAD especially where large scale testing of samples is required. Additional newer techniques are available such as the chromosomal microarray technique (CMA) which has the capacity to detect targeted submicroscopic deletions and duplications other than aneuploidies in the prenatal samples.

2 Non-invasive prenatal testing (NIPT)

In addition to invasive procedures there are non-invasive screening methods which are gaining importance in recent times. Non-invasive prenatal testing (NIPT) has been widely accepted as a part of routine care for pregnant women in many countries. The testing involves analysis of cell-free DNA fragments which circulate in the blood of the pregnant women. It can be offered after 10 weeks of gestation. This technique has a sensitivity of 99% and a specificity of 99.92% for trisomy 21. For trisomy 18, the test has 96.8% sensitivity and 99.85% specificity and for trisomy 13, 92.1% and 99.80% respectively (Gil et al., 2014). The positive predictive value of this technique is approximately 45% (10 times better than the other maternal biochemical screening tests) for low risk women. The obvious advantage of this technique is that it provides an alternative for invasive testing and the related complications. However, currently NIPT is still considered a screening test and the aneuploidy should be confirmed by an invasive definitive test. It can be used as a first tier test in the first trimester or after abnormal results of the biochemical screening tests for women unwilling to opt for invasive procedures. However, there are numerous factors including twin/triplet pregnancies, inadequate fetal fraction of DNA, higher maternal body mass index, gestational age of less than 10 weeks etc. which can result in either failure or inaccurate results. Also, the test is unable to detect triploidy in the fetus. To date this testing is available in the US, Europe and some Asian countries including India. High cost and availability are also limitations for the use of this technique.

Table 1 summarizes the principles, techniques, advantages and limitations of the important rapid aneuploidy detection tests. Prior to ordering any of these tests, appropriate pretest counseling is essential.