A stunningly accurate prenatal test is fast replacing traditional screening for Down syndrome, but the transition has been a bumpy one
For nearly a decade, Yuk Ming Dennis Lo had no idea that he was quite literally pouring the discovery of his career down the drain.
He had been trying to find traces of fetal DNA in the mother’s bloodstream, a breakthrough that would enable noninvasive genetic testing during pregnancy for the first time.
But he was looking for fetal DNA in the wrong place. While hunting for fetal cells in the mother’s blood, Lo’s research team from the Chinese University of Hong Kong were routinely disposing the plasma.
It was only after reading two papers in Nature Medicine that reported finding cell free tumour DNA floating around in the bloodstream of cancer patients that Lo thought to check the mother’s plasma. Eureka!
He found that around 10% of all the cell free DNA in the mother’s blood came from the fetus. The evidence was stark; some pregnant women had traces of Y chromosome in their blood sample. The chromosome could not have come from anywhere other than a male fetus.
So startling was that discovery that Lo likened it to finding a car’s engine somewhere other than under the bonnet.
A paper published in 1997 outlined several exciting prospects. It predicted that as little as 10µl maternal plasma could be used to screen for chromosomal aneuploidies (such as Down syndrome) and sex-linked disorders.
The placenta was later confirmed as the source of the DNA; as fetal cells underwent apoptosis (programmed cell death) on the outer layer of the placenta, they released their DNA into the mother’s veins.
It would take a further two decades of persistence to translate the initial finding into a workable screening test. The researchers had to wait for the Human Genome Project to be completed before they could start building the technology in earnest.
The price of DNA sequencing also needed to drop before the test could go mainstream.
Eventually, in 2011, the first noninvasive prenatal testing (NIPT) was launched in the US at a cost of over $2000. That figure has dropped to a few hundred dollars today.
“Many people regard it as the most rapidly adopted genomic test ever,” says Lo, who is considered the father of NIPT and a professor of chemical pathology in Hong Kong.
“In China alone, four million NIPTs were done in 2017, which is amazing,” he told The Medical Republic. “And so globally now you’re looking at a few million tests per year.”
The Hong Kong government will offer NIPT as a publicly funded program this year. Some European countries are following suit.
NIPT was first introduced in Australia in 2012. It is currently used by the majority of pregnant women despite costing $450, which is not Medicare rebatable.
Researchers rarely live to see their discoveries change the world. Lo, who made Nature’s list of the top 20 most prolific inventors last year, is among the lucky few.
“In a way, I am surprised how quickly it has been used,” he says. “Initially we thought it would be taken up in a more-gentle way, but it has been a very rapid transformation.”
For Lo, it’s the enormous satisfaction that comes with translating his thoughts and dreams into reality that motivates him.
“It is almost like a movie director having a story they are trying to make into a movie,” he says. “I like to see that process.”
WHAT’S CHANGED?
Fewer pregnant women receive a false positive result for Down syndrome using NIPT compared with traditional screening methods.
This has dramatically reduced the number of unnecessary diagnostic tests, such as chorionic villus sampling (CVS) or amniocentesis, which carry around a 1% risk of miscarriage.
The number of invasive prenatal tests has halved during the first two years of NIPT in Australia, with further gains expected.
Thanks to NIPT, early pregnancy is no longer the black box it once was. Couples can find out the sex of the fetus as early as 10 weeks’ gestation, as well as the risk of Edwards syndrome (trisomy 18), Patau syndrome (trisomy 13), Turner syndrome (XO), Klinefelter syndrome (XXY), triple X (XXX), and, of course, Down syndrome (trisomy 21).
“They’re getting answers earlier,” says Lucinda Freeman, genetic counsellor at Royal North Shore Hospital. “For couples that’s so important.”
NIPT is much more accurate than the Medicare-funded first trimester screening (CFTS).
CFTS calculates Down syndrome risk by combining a blood test for marker proteins, a nuchal translucency ultrasound, maternal age, weight, gestation and an examination of the fetus’ nasal bone. The test is usually conducted by specialists between 9+0 and 13+6 weeks gestation.
NIPT is more than 99% accurate (with a 0.2% false positive rate), while CFTS is only around 90% accurate (with a 5% false positive rate).
“[CFTS] is not a particularly accurate test,” says Professor Graeme Suthers, a pathologist and the head of Sonic Genetics. “With CFTS, we have to do about 20 amniocenteses for every pregnancy that really does have Down syndrome.”
Prior to NIPT, pregnant women were unable to find out information about sex chromosome aneuploidies without undergoing amniocentesis or CVS.
Knowing the risk for chromosomal conditions earlier gives women more options if they decide to have an invasive diagnostic procedure and potentially terminate the pregnancy, says Ms Freeman.
In the past, women and couples would also have to wait for the ultrasound at 11 to 13+6 weeks to know the sex of their future child.
Pregnant women who choose NIPT are still given an ultrasound as it can detect a larger range of abnormalities – including neural tube defects and non-genetic abnormalities.
TIPS AND TRICKS
GPs have now begun to supersede specialists as the main providers of Down syndrome screening because NIPT can be offered so early in pregnancy.
“Most of the NIPTs would go through a GP now,” says Ms Freeman.
NIPT is also one of the first technologies to push GPs into the realm of genetics – an area where GPs often admit to feeling out of their comfort zone.
“We have seen an incredible shift in the management antenatal risk of chromosome abnormalities, and it is not surprising that there have been some growing pains in this space,” says Professor Suthers.
Education programs for GPs emphasise that not all pregnant women are suitable for NIPT.
For example, women with a weight above 120 kg are more likely to return a failed result, as their blood sample may not contain enough fetal DNA to perform a test. (The amount of fetal DNA in the mother’s bloodstream decreases with maternal weight.)
“It’s been a very steep education curve,” says maternal fetal medicine specialist, Rosalie Grivell from Flinders University.
Around half of women presenting for pregnancy care in Australia are overweight or obese, so GPs offering NIPT need to be aware of this particular test limitation, says Associate Professor Grivell. It is possible for pregnant women with obesity to attempt an NIPT, but there are other options available, she says.
Some pathology labs claim to be able to complete the NIPT with lower amounts of fetal DNA, namely those labs that use whole genome sequencing. However, the research establishing this superiority has been criticised for its reliance on computer simulations. Labs that use single nucleotide polymorphism methods require at least 4% fetal DNA in the blood sample.
Pregnant women with a very high risk of Down syndrome, such as those with a 1-in-10 risk based on CFTS, may be best served by an invasive test rather than an NIPT, says Associate Professor Grivell.
A common misconception about NIPT for both healthcare professionals and patients is that it is a diagnostic rather than a screening test. One pathology lab, Genea, has even changed the name to “NIPS” to avoid the confusion. (“S” stands for “screening test”)
Equally problematic is GPs forgetting to mention the other genetic disorders that NIPT tests for, says Ms Freeman.
When a NIPT comes back with a positive result for sex aneuploidies, such as Turner syndrome and Klinefelter syndrome, patients are very often angry or confused because they did not know that was a possible outcome, she says.
The genetic counselling around these disorders is challenging because the NIPT is not as accurate and these conditions have a variable, and less well-known, impact on the child and the family.
Couples in this “difficult and distressing” situation often say they would have preferred not to know about their baby’s risk for these other genetic conditions, according to Ms Freeman.
While it is possible to request an NIPT without screening for these extra genetic conditions from some labs, couples that wish to know their child’s sex will always be informed about the risk of sex aneuploidies, she says.
“The difficulty is that finding out those other results is tied up with finding out the sex of the baby,” says Ms Freeman.
Before ordering an NIPT, genetic counsellors will generally highlight the possibility that women and couples may find out something unexpected and ethically challenging.
While it would be wonderful for GPs to enlighten patients about these possibilities, Ms Freeman recognises that these conversations take a lot of time. At the very least, however, GPs can add a few more sentences to their consultations, and explain that the NIPT could unearth some unnerving results – and offer to provide more information, she says.
“You can’t prevent people from getting upset when a positive result comes back,” says Ms Freeman. “But you can make sure that the patients actually want to do a test and aren’t just doing it because they have been told that it’s available.”
Ms Freeman says genetic counsellors will see patients urgently if the fetus is identified as being at high risk of a genetic disorder.
“The saddest thing is to think that there would be a woman or a couple who have a result saying there is a high risk and then they just sit on that for a week and go onto Google. We would be very responsive to seeing people because there is a lot of misinformation out there.”
Much of the process of counselling pregnant women about Down syndrome screening will remain unchanged for GPs, says Professor Suthers. “I don’t want doctors to think that this is a radical different genomic pathway that means that they need to understand gene sequencing. They don’t.”
WHICH TEST?
In 2015, a stunt by two Boston-based specialists raised serious concerns about NIPT quality control.
The doctors sent blood samples from two non-pregnant women to American NIPT providers. If the labs were actually checking the amount of fetal DNA in each sample, as they should, they would report a failed test result for these dud samples, the specialists reasoned.
Alarmingly, three out five labs declared the fetus genetically normal, despite the sample containing no fetal DNA and the women providing the blood sample being emphatically not pregnant.
The incident revealed a heated debate raging in the NIPT pathology community worldwide.
About half of labs do not report fetal fraction (the amount of fetal DNA in the blood sample), according to a survey presented at the European Society of Human Genetics annual meeting last year.
“I thought that was striking, that there was a 50-50 split,” says Professor Suthers. Normally, pathologists are in almost total agreement over quality control matters, with a few outliers. “But here we have a divide down the middle,” he says.
NIPT detects genetic abnormalities by measuring changes in the concentration of various chromosomes in the plasma.
An abnormal pregnancy will elevate the concentration of DNA from chromosomes 21, 13 or 18, X or Y in the mother’s blood, whereas a normal pregnancy will not change the concentrations. To avoid a false negative result, it is important that labs check that there is enough fetal DNA in the sample to draw the conclusion that there has been no elevation in the concentration of these chromosomes. Fetal DNA levels could be suppressed by exercise, infection, inflammation, and some common drugs.
Sonic Genetics, which reports the fetal fraction for every sample, finds that around 3% of collections do not yield a result. The lab offers recollection and re-analysis at no additional charge. Two-thirds of recollections allow for NIPT, meaning that overall around 99% of patients get a result.
There are only a handful of accredited providers for NIPT in Australia. All of the labs contacted by The Medical Republic (Sonic Genetics, Genomic Diagnostics and Genea) confirmed that they measured the fetal fraction.
The best labs also investigate any false negatives to determine whether the failure is due to a biological or technical issue, says Professor Suthers.
In 1% of Down syndrome cases, the placenta will have a different genetic makeup to the baby, a phenomenon called confined placental mosaicism. With each investigation, Sonic Genetics has confirmed that the false negatives were due to this biological quirk rather than a flaw in the testing.
TO INFINITY AND BEYOND
NIPT is nowhere near reaching its full potential, says Lo.
The first wave of NIPT created more accurate Down syndrome screening. Trisomy 13 and 18 were quickly added to the test, followed by the sex chromosome aneuploidies.
“More recently, people are adding in something called sub-chromosomal aberrations, that is changes that are less than one chromosome in length,” says Lo. “So, this is the current wave.”
Lo predicts that the next wave will focus on single gene diseases.
“In the longer term, we have also demonstrated that you can detect the whole genome of the baby using this method,” he says.
“And last year we showed that you can even detect a class of mutation called de novo mutations. So, that is mutation which our parents don’t have and which was actually introduced when our parents created the sperm and the egg.”
These mutations are typically very difficult to detect. Lo’s group have already published a paper showing that NIPT can pick up a de novo BRAF mutation that can cause craniofaciocutaneous syndrome in the second trimester.
As for Down syndrome, has NIPT affected the number of children born with this condition globally?
“I don’t have that data,” says Lo. “But the primary goal of developing NIPT is really to save the children who are normal who are unnecessarily harmed by conventional invasive testing.”
