Author: David Bennett-Orphan diseases/UK specialist
Non-Invasive Prenatal Genetic Testing

Non-Invasive Prenatal Genetic Testing

Non-Invasive Prenatal Genetic Testing Date:09 August 2016
Non-Invasive Prenatal genetic Testing (NIPT) is a relatively new tool for identifying fetal abnormalities resulting from chromosomal/genetic errors. This article describes the current and potential applications, and issues and concerns expressed by healthcare professionals concerning the use of this test, including its commercial provision to the public.

Current methods to assess fetal anomolies
The use of non-invasive testing in early pregnancy (first and second trimesters) is routine, with ultrasonography and maternal blood markers, known as the Combined test, representing the current standard of care. The decision parents face following receipt of the test results can be the most difficult they are ever likely to deal with. Understanding the meaning of the result provided is crucial to their decision making.

The Combined Test is a screen to detect high risk pregnancies.The false positive rate is up to 7%. To confirm a diagnosis, invasive methods to directly assess fetal tissue, amniocentesis and chorionic villus sampling (CVS) are used. However, these procedures carry a small (0.5%) risk of miscarriage.  Women who refuse prenatal screening cite the risk to their pregnancy as a reason.

NIPT – a new option
The obvious need is for a non-invasive method which directly assesses fetal tissue, is accurate, and produces few false positives. The breakthrough technique, NIPT, has been developed to meet this need. NIPT is a relatively new tool available to clinical practice (2011). It has not been validated beyond a population at high risk of fetal aneuploidies (Note that NIPT is also called NIP Screening).

Scientific basis of NIPT
NIPT is based on the presence of fetal DNA (cell-free fetal DNA; cffDNA) in the maternal circulation (via the placenta). This cell-free placental DNA can represent up to 20% of the cell-free DNA in the maternal circulation. NIPT involves the analysis of cffDNA by sequencing DNA in the maternal blood. Computational analysis permits the mapping of all sequences to maternal and fetal chromosomes. During pregnancy cffDNA accumulates in the maternal circulation and is relatively stable. The reduced cost of DNA sequencing is making NIPT feasible as a prenatal test in clinical practice. NIPT is advised from 10 weeks gestation.

Compared to an accuracy rate of 84-90% with the Combined Test, an accuracy of 99% is quoted for NIPT when used for Down Syndrome. The low false positive rate of 1 – 3% in conditions evaluated so far (see table below) has made the technique attractive.

According to Lucy Jenkins (Director of North East Thames Genetics Laboratory, London, UK and the first NHS laboratory in the UK with the capability to undertake NIPT testing): ‘The important point about NIPT is that this is still a screening rather than a diagnostic test for fetal aneuploidies. If a woman receives a positive result from NIPT, providers should advise an invasive test to confirm the risk’. The fetal DNA under test is derived from the placenta. Usually this will have the same karyotype as the fetus, both being derived from the same zygote. In unusual but well documented cases, notably Confined Placental Mosacism, placental and fetal karyotype can vary, with the cytogenetic abnormality being confined to the placenta. Lucy Jenkins cautions that it is also possible that placental tissue is ‘normal’ in the case of a baby affected by aneuploidy such as Downs or Turner Syndrome and so give rise to false negative results.

NIPD for single gene disorders
When used for identifying single gene mutation, non-invasive prenatal genetic testing is definitive, and the term NIPD is correct. The accuracy of these tests means that confirmation via an invasive procedure is not indicated. At the moment, conditions for which NIPD can be used are limited (see table). The number of conditions to be covered by NIPD is likely to grow. In addition to paternal dominant conditions, recessive conditions can be usefully tested when there is a possibility of the mother and father carrying different mutations (e.g. cystic fibrosis).
NIPD has different utility depending on geographical region, or the level of healthcare resources available. For example, thalassemia or sickle cell disease are more prevalent in low and middle income countries. Local authorities may choose to target these disorders.

Current applications of NIPT and NIPD
The following table summarises some of the main documented applications for NIPT/NIPD.  The technology is used in over 60 countries with uptake in the US being rapid.
Application Purpose Considerations
Down syndrome (screening test). Note that commercial tests are available for several other aneuploidies (including 13, 16, 18, 21, 22, X, and Y) so that most common chromosome and sex chromosome aneuploidies can be detected. Assess level of chromosome 21 sequences. A raised ratio of these sequences compared to those from other chromosomes suggests trisomy in the fetus. A positive test result is not diagnostic; an invasive test is needed for confirmation of the NIPT result.
Fetal sex determination
Risk of X-linked conditions, notably Duchenne Muscular dystrophy, and those at risk of Congenital Adrenal Hyperplasia. If fetus is male then invasive testing is indicated.  In CAH the insight from NIPT can guide use of dexamethasone treatment (that is, discontinue if fetus is male).
Fetal Rhesus D typing in RhD-negative mothers
RhD- women who have been sensitized, have a history of haemolytic disease of the newborn or have elevated levels of anti-D antibodies in pregnancy.
In the case of a RhD+ fetus close monitoring during pregnancy can guide clinical intervention.
Determining fetal RHD type early during pregnancy (from 11 weeks) can target anti-D prophylaxis.
Single gene disorders Test for paternal dominant genes or de novo mutations.
Currently available for
FGFR3-related skeletal dysplasias (e.g. achondroplasia, thanatophoric dysplasia) and Apert  / craniosynostosis syndromes (FGFR2 alterations).
Not applicable when mother has the affected mutation.
Relative Haplotype Dosage Analysis for autosomal recessive conditions (e.g. Cystic Fibrosis, Congenital Adrenal Hyperplasia). Can be applied irrespective of parental mutations. Currently technically challenging and expensive so yet to reach mainstream practice.

For the majority of pregnancies not associated with a special risk factor, general screening rather than NIPT is advised due to the higher rate of false positives in a general obstetric population. Other ‘contra-indications’ include pregnancy via donor eggs and surrogacy.

Use of NIPT in practice
The uptake of NIPT on a global basis is variable. The options available are to use NIPT: The use of this test within the UK public health service (NHS) is being clarified. Based on the RAPID NIPT Evaluation Study, as part of an evaluation process NIPT will be offered to women with a high risk screening result for Down’s, Edward’s and Patau’s syndrome (greater than 1:150). In the event of a positive NIPT result, an invasive test will be advised.

For those mothers with a concern about their pregnancy but who may not qualify for an NHS- funded NIPT, there is the option provided by private clinics. This has raised the concern that unless mothers are professionally counseled and advised, a positive NIPT may be considered diagnostic of a fetal abnormality rather than indicating a need to undertake an invasive test.

Whether working in the public or private sector however, healthcare professionals require training in order that parents are given full balanced information on the interpretation of NIPT. In the context of a ‘diagnostic rush’, for example created by the prospect of newborn screening for an ever increasing number of genetic conditions, availability of  the required professional expertise cannot always be assumed (see Down’s Syndrome Society concern).
Down’s Syndrome Society concern
There is concern that NIPT could be viewed as a diagnostic test and not only in mothers at high risk. Without access to expert advice from their healthcare professional, parents might not be able to make an informed and balanced decision on their pregnancy. According to Sheila Heslam (Services Director):  ‘Given the huge number of pregnancies in the UK every week, the resources to provide full advice prior to testing are not available.  Even if NIPT proves to be comparable or superior to the traditional combined test, there is a need for sufficient and adequately trained healthcare professionals. Simply pushing the new test without putting in place the right support could be detrimental to antenatal care.’

The plummeting cost of genome sequencing is enabling the development of new and powerful options to screen patients and diagnose conditions. The pace of development is placing a strain on healthcare systems around the world.  NIPT is a new technology with significant consequences for budgets, clinician training, patient education and the organization of healthcare provision. The development of evidence-based guidelines to support the responsible application of NIPT follows the gradual pace of scientific process.  The concern is that the uptake by the public of new tests will progress at a faster rate.

Further reading
D K Kalousek and M Vekemans. Confined placental mosaicism. J Med Genet. 1996 Jul; 33(7): 529–533.

Allyse M,  Minear M A,  Berson E,  Sridhar S,  Rote M, Hung A, and Chandrasekharan S. Non-invasive prenatal testing: a review of international implementation and challenges. Int J Womens Health. 2015; 7: 113–126.
Great Ormond Street Hospital for Children NHS Foundation Trust. Introduction to NIPD / NIPT: A guide for patients and healthcare professionals. Accessed 17/7/16 

Great Ormond Street Hospital launches new safer, more accurate test for Down’s Syndrome. 27 March 2015. Accessed 17/7/16
Prenatal cell-free DNA screening. Mayo Clinic. Accessed 17/7/16 

WHO Fact sheet N°308. Sickle-cell disease and other haemoglobin disorders. January 2011. Accessed 17/7/16

Lucy Jenkins
is the Director of the Regional Genetics Laboratories and Head of Service for Molecular Genetics. The Molecular Genetics Laboratory, along with Cytogenetics, forms the North East Thames Regional Genetics Laboratories. Great Ormond Street Hospital for children, NHS Trust.
Sheila Haslem is Services Director at Down's Syndrome Association