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Noninvasive prenatal testing for fetal aneuploidy and single gene disorders

https://doi.org/10.1016/j.bpobgyn.2017.02.007Get rights and content

Highlights

  • Cell free DNA – the biological basis and the sequencing techniques behind NIPT.

  • Clinical performance of NIPT for the common automosomal trisomies and beyond.

  • Essential pre-test counselling for NIPT.

  • When NIPT is not be the preferred test.

  • “No-call” and discordant results.

Our concept of nucleic acid biology has advanced dramatically over the past two decades, with a growing appreciation that cell-free DNA (cfDNA) fragments are present in all body fluids including plasma. In no other field has plasma DNA been as rapidly translated into clinical practice as in noninvasive prenatal testing (NIPT) for fetal chromosome abnormalities. NIPT is a screening test that requires confirmation with diagnostic testing, but other applications of cfDNA provide diagnostic information and do not require invasive testing. These applications are referred to as noninvasive prenatal diagnosis (NIPD) and include determination of fetal sex, blood group and some single gene disorders. As technology advances, noninvasive tests based on cell-free nucleic acids will continue to expand. This review will outline the technical and clinical aspects of NIPT and NIPD relevant to the daily practice of maternity carers.

Introduction

Cell-free DNA (cfDNA) and RNA fragments are present in all body fluids in a dynamic state of constant turnover. CfDNA is comprised of small fragments of extracellular DNA that circulate freely in the bloodstream [1]. Technological progress in molecular genetics has enabled scientists to utilize this uniquely accessible genetic material for a whole new generation of blood tests that detect and monitor disease. In the field of obstetrics, this circulating DNA can be used for the noninvasive detection of fetal genetic abnormalities such as trisomy 21. In what has been a very rapid translation into clinical practice, noninvasive prenatal testing (NIPT) has revolutionised prenatal screening. Providers of prenatal care need to be familiar with the technical and clinical aspects of NIPT and these will be reviewed here.

Voluntary prenatal screening for fetal anomalies is a key component of antenatal care in developed countries and offers couples the option of obtaining more genetic information about their pregnancy. Although for some this information is not desired, for others, screening for common chromosome conditions such as Down syndrome (trisomy 21) provides an important opportunity to inform management. As diagnostic testing with amniocentesis or chorionic villus sampling (CVS) carries a cost and a procedural risk of miscarriage, screening tests are offered to better target diagnostic testing. Traditional screening approaches for trisomy 21 have considerable room for improvement, with detection rates of approximately 80–90%, for an average false positive rate of 5%. These performance characteristics translate to a substantial proportion of missed diagnoses and false positive “high risk” results leading to unnecessary invasive diagnostic testing [2].

Research efforts over the past two decades have therefore focused on developing a noninvasive yet highly accurate screening test that would increase detection and reduce false positive results. The combination of the discovery of cfDNA of fetal/placental origin in 1997 [3] and the rapid advances in DNA sequencing technology have combined to make this noninvasive approach a clinical reality in less than 15 years.

Section snippets

Biology of cfDNA

The majority of cfDNA in plasma is derived from haematopoietic cells that release fragments of DNA during cell turnover, but a large variety of solid organs also contribute to the circulating plasma pool [4]. In the non-pregnant state, the genomic profile (i.e. the proportional representation of each chromosome) in plasma cfDNA simply reflects the individual's karyotype and the size of each chromosome. During pregnancy however, cfDNA from the placenta is also released into the maternal plasma

Nomenclature

Prenatal testing based on analysis of cffDNA in the maternal plasma has been called noninvasive prenatal testing (NIPT) or noninvasive prenatal screening (NIPS) to distinguish it from traditional diagnostic methods of directly assessing the fetal karyotype using cells obtained via amniocentesis or CVS. Testing for fetal aneuploidy with cffDNA in maternal plasma is a screening test – it does not achieve diagnostic accuracy for aneuploidy and requires confirmation with pre or postnatal

Traditional aneuploidy screening methods

The autosomal aneuploidies trisomy 21 (Down syndrome), trisomy 18 (Edward syndrome) and trisomy 13 (Patau syndrome) are the three most common chromosome abnormalities to affect ongoing pregnancies. Trisomy 21 makes up approximately half of all prenatally diagnosed chromosome abnormalities and individuals with this condition have intellectual disability and a range of medical morbidities. Maternal age is the most important risk factor for trisomy 21. By the maternal age of 40, the risk of

NIPT for detection of trisomy 21, 18 and 13

The fundamental principle of NIPT for trisomy 21 is the detection of an excess number of DNA fragments from chromosome 21 in the maternal plasma (i.e. a counting approach). A fetus affected with trisomy 21 will release relatively more chromosome 21 DNA fragments into the maternal circulation compared to other chromosomes because of the presence of a third copy of chromosome 21 in the trophoblast. DNA sequencing of maternal plasma cfDNA, which contains a mixture of maternal and fetal DNA, allows

Fetal fraction

An important factor influencing the laboratory performance of NIPT is the fetal fraction, which is the proportion of total cfDNA in maternal plasma that is derived from the fetus/placenta [26]. If the fetal fraction is low, it is more difficult for NIPT to detect an aneuploid fetus. There are several biological factors that influence fetal fraction, including gestational age, maternal weight, and fetal aneuploidy [27]. Maternal weight has a well-recognized negative impact on fetal fraction:

Clinical performance of NIPT

  • a)

    Sensitivity and specificity

The performance of NIPT for aneuploidy screening is highly accurate and far better than standard prenatal screening approaches. A systematic review and meta-analysis published in 2016 found the pooled sensitivity of NIPT for trisomy 21 was 99.3%, for trisomy 18, 97.4% and for trisomy 13, 97.4% [29]. The American College of Medical Genetics and Genomics accordingly recommends informing all pregnant women that NIPT is the most sensitive screening option for trisomies

Clinical implementation models for NIPT

Overall, three models have been proposed for incorporation of NIPT into current screening programs. The first model uses NIPT as a second tier screening test where women with a high risk result from conventional screening are subsequently offered NIPT or diagnostic testing. The advantage here is that only ∼5% of women would be offered NIPT, thus reducing cost [45]. The second model is as a universal primary screening test in combination with a 12 week ultrasound [46]. This provides the highest

When NIPT should not be the recommended test

NIPT for chromosomal aneuploidies is an excellent screening test for common chromosome conditions, but does not provide the detail and range of genomic information gained by invasive testing. An early ultrasound must be performed prior to considering NIPT as up to 16% of high risk women will have an ultrasound finding at 10–14 weeks that alters the prenatal counselling, such as correction of gestational age, detection of multiple pregnancy, fetal demise or a structural abnormality [54].

Women

Screening beyond the common trisomies

Trisomies 21, 13 and 18 represent only 71% of all chromosomal abnormalities [60]. Unlike conventional forms of serum screening, NIPT is capable of assessing the dosage of any chromosome in the genome including the X and Y chromosomes.

  • a)

    NIPT for sex chromosome aneuploidy (SCA)

Monosomy X or Turner's syndrome occurs in 1–1.5% of pregnancies [61], has a high rate of spontaneous miscarriage, and presents a characteristic phenotype in liveborn infants as well as a number of medical complications

Discordant results – high risk NIPT result with normal amniocentesis

The karyotype obtained from amniocentesis is considered to most closely reflect the true fetal karyotype since it is obtained directly from fetal cells. However, since NIPT analyses both maternal and fetal/placental DNA, false positive results can occur due to biological abnormalities in the mother or placenta that are not present in the fetus. Reasons for “biological false positives”, where there is an abnormal NIPT result but a normal fetal karyotype on amniocentesis include, placental causes

Essential components of pre-test counselling

Counselling for prenatal screening has become more complex since the introduction of NIPT due to the increase in patient choice and the rapidly evolving technology. Education for medical professionals must be a high priority in health policy to ensure the safe and responsible provision of new screening tests. Essential counselling points for pre-test consultations on NIPT are summarized in Table 1 [84].

NIPD for single gene disorders

In separate developments to NIPT for chromosome disorders, the use of maternal plasma cffDNA to develop noninvasive tests for single gene disorders is the other major application in fetal medicine. So far, NIPD has been clinically most successful in the testing for fetal sex and Rhesus blood group. Detection of fetal sex by the identification of the Y chromosome was the first clinically applicable NIPT technique to be developed [85]. Identification of fetal sex is useful for couples at risk of

Summary

CffDNA based screening for the common autosomal aneuploidies is undoubtedly the most superior screening method for trisomy 21 to date, with unprecedented sensitivity and specificity. However, the implementation of NIPT into clinical practice has been challenging for the medical profession due to the speed of introduction, the cost of this novel technology, and our nascent understanding of the biology of cfDNA in maternal plasma. It is now established that NIPT for trisomies 21, 18 and 13 is an

Conflict of interest

None.

Practice Points

  • NIPT is the most sensitive and specific screening test for trisomy 21

  • NIPT for trisomy 21, 18 and 13 is suitable for use in low and high risk populations

  • the positive predictive value of NIPT is lower when used in low risk populations

  • routine screening for sex chromosome abnormalities and microdeletions is not recommended by most professional societies

  • “high risk” NIPT results require confirmatory diagnostic testing with amniocentesis or CVS

  • women with a fetal abnormality

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