DNA Fetus
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Cell-free (cf)DNA based non-invasive prenatal testing (NIPT) hit a milestone last August when the American College of Obstetricians and Gynecologists (ACOG) and the Society for Maternal-Fetal Medicine recommended it should be offered to all pregnant women regardless of their age or risk status. Since being introduced commercially 10 years ago, its accuracy for detecting common trisomies has improved enormously, and it has now become a standard of care in many countries around the world.

“The biggest change over the last couple of years has been the implementation of NIPT as standard for the diagnosis of Down’s, Edwards’ and Patau syndrome. That’s a game changer, because it means that an awful lot of invasive testing is avoided,” Zofia Miedzybrodzka, a clinician and professor of medical genetics at the University of Aberdeen in the U.K., told Clinical Omics.

Researchers now want to find out how NIPT can be extended to increase the number of mutations it can detect and work is underway to combine testing for fetal abnormalities with searching for biomarkers of maternal health that could indicate if a woman is at risk of developing pregnancy complications such as pre-eclampsia.

Since the advent of cheaper and more accessible genetic testing, carrier screening options have exploded with many companies developing tests in this space, ranging from basic panels to ‘preconception screens’ that test for a wide variety of genetic variants that increase risk for rare and sometimes common diseases.

In an attempt to help bring clarity to offerings of ‘expanded carrier testing’ from many providers, the American College of Medical Genetics and Genomics (ACMG) published a guidance document this summer that classified carrier testing into four recommended tiers. The document suggests that all prospective parents should be offered at least ‘tier 3’ testing comprising testing for variants in a set of 113 genes, but how this guidance will translate into clinical practice is yet to be seen.

women's health illustration
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Cheaper, more extensive NIPT options

CfDNA based NIPT has now become a ‘gold standard’ test for all pregnant women, instead of only those over 35 years old and, since the ACOG announcement last August, it is also being reimbursed by insurance companies for all pregnant women.

But these tests still have limitations. Most of only include the three main trisomies—Down’s, Edwards’ and Patau syndrome, or trisomies 21, 18 and 13—and those affecting the X and Y chromosome. Many are based on next-generation sequencing (NGS) technology and can be costly and time consuming, sometimes taking up to three weeks to return a result.

Adtiya Rajagopal
Adtiya Rajagopal, ChromaCode

“There is a pressing need to enable NIPT tests to be more scalable and affordable to labs and patients than existing solutions predominantly based on NGS,” Adtiya Rajagopal, founder and chief technology officer of ChromaCode, told Clinical Omics.

ChromaCode is based in California and developed high-definition (HD)PCR technology that can produce 10 times the content using standard PCR machines and has a NIPT test in development. “This approach enables laboratories to deliver content on the much more scalable and affordable PCR platforms that, to date, could only have been done using expensive, and slow next-generation sequencing,” explained Rajagopal. It is also quicker. “With HDPCR, we could, in principle, get a result back in 1 day instead of 14 to 21 days for complex genomic applications that NGS would take.”

Also based in California, Natera has long been a leader in the NIPT field and has the only available SNP-based NIPT test called Panorama. In February this year it announced results from the SMART study, the largest prospective NIPT registry study of its kind with over 20,000 participants, and also launched a new version of Panorama that uses artificial intelligence (AI) to improve accuracy.

Sheetal Parmar, Natera
Sheetal Parmar, Natera

“Panorama AI combines AI with Natera’s proprietary SNP-based methodology to give highly accurate results on difficult-to-call cases,” Sheetal Parmar, VP of Medical Affairs at Natera, told Clinical Omics.  “Importantly, we maintained industry-leading accuracy while significantly lowering ‘no-call’ rates. In addition, we, improved accuracy for 22q11.2 deletion detection.”

While most NIPTs are based on testing the blood for cfDNA fragments from the fetus, single-cell based NIPTs are also in development, although the technology is still at an early stage.

“I don’t see that as being a real game changer in the near future,” said Nathan Slotnick, an experienced obstetric geneticist now working as a medical advisor for San Francisco-headquartered medical genetics company Invitae, which has both NIPT and carrier tests on the market. “We’re talking about one in a trillion cells here and to be able to efficiently separate them out is problematic right now. So, there may be applications, but not at this point.”

Clarifying expanded carrier testing options

Carrier screening as a concept and practice started 50 years ago and was largely focused on diseases that were prevalent in specific ethnic communities such as screening for Tay–Sachs disease in the Ashkenazi Jewish population or sickle cell disease in African Americans. Since then, a more pan-ethnic approach has been adopted by providers.

“When you look at carrier testing frequencies over the last couple decades, you’ll find that using ethnic definitions is really pretty much non-productive,” Slotnick said.

Since the completion of the Human Genome Project and the revolution in NGS development, carrier testing has changed significantly with many tests launched onto the market by different companies and labs in recent years. The sheer diversity of choices has created confusion amongst both prospective parents and clinicians working in this area, prompting the launch of new recommendations by the ACMG this summer.

The advisory document suggests abandoning the term ‘expanded carrier screening’ and instead splits carrier testing into four tiers. Tier 1 is relatively basic and involves cystic fibrosis and spinal muscular atrophy screening, as well as risk-based screening dependent on family background. Tier 2 screens for conditions that are severe or moderate in phenotype and a carrier frequency of at least 1/100 in the population. Tier 3 involves screening for conditions with a carrier frequency of 1/200 or greater and is now recommended for all pregnant women. Tier 4 is the most intensive and screens for less common conditions than Tier 3. There is no lower limit, but the recommendations suggest Tier 4 screening should be only carried out if warranted by family history. As part of the Tier 3 recommendation, the ACMG suggested a panel of 113 genes, both autosomal recessive and X-linked, should be tested for as standard, but they concede that this may take time to roll out across the U.S.

Susan Klugman, president-elect of ACMG
According to Susan Klugman, president-elect of ACMG, it is working to provide laboratory standards and guidelines for NIPT and carrier testing that will aid in broader use of these tests.

“The ACMG has convened a group of experts to work on a companion document for laboratory standards and guidelines,” explained Susan Klugman, president-elect of the ACMG. “This will help laboratories to be able to set up platforms for all the disorders in Tier 3. Testing for many of the disorders is quite complex and uses different genetic testing technologies.”

Outside of the U.S. it seems likely that Tier 1 and 2 may remain the norm in many places. For example, in countries with a public healthcare system, such as the NHS in the U.K., there is currently a lack of funding for more extensive carrier testing unless prospective parents choose to buy a private or direct-to-consumer test.

Most carrier tests understandably focus on risks for passing on monogenic or rare diseases, but newcomer Orchid Health is taking its ‘preconception test’ into the common, complex disease arena and are using polygenic risk scores to predict a couple’s risk of having a child with conditions such as breast cancer, Alzheimer’s disease, or type 1 diabetes.

As onset of many such conditions is influenced by the environment, the accuracy of polygenic risk scores for predicting the chances of having a child with one of these conditions is questionable. Polygenic risk scores are also susceptible to ethnicity bias as many currently use scores based on predominantly White European-ancestry source populations.

Combining testing for maximum benefit for mother and baby

Sema4 is an AI-driven genomic and clinical data intelligence company. It is taking a holistic, all-round approach to reproductive health and in September launched Sema4 Elements, which comprises various elements including carrier screening, NIPT and newborn screening.

“The Sema4 testing portfolio is powered by advanced sequencing technologies, utilizing an NGS platform that includes low pass genome sequencing to power molecular ancestry for personalized residual risk determination,” explained Lisa Edelmann, chief diagnostics officer at Sema4.

“It also incorporates several accessory technologies to boost detection rates for genes that are not amenable to NGS, including genotyping, Sanger sequencing, enzyme analysis, capillary electrophoresis, and multiplex-ligation dependent probe amplification.”

The company is also involved in research into reproductive health including the Better Understanding the Metamorphosis of Pregnancy (BUMP) study being run by nonprofit 4YouandMe. This study began in February and aims to recruit 1,000 women to investigate a range of different passive and active measures.

There is increasing evidence that technology similar to that used for NIPT could also be used to detect women at risk of prenatal or birth complications. For example, using cell free DNA in the blood as a measure of preeclampsia risk.

Nathan Slotnick, Invitae
Nathan Slotnick, Invitae

“One of the projects that I’m working on right now is trying to find whether currently available prenatal pregnancy tests, like NIPT, can be used to identify patients at risk for other obstetric conditions, preterm labor, preeclampsia, other issues to do with placentation,” said Slotnick.

“If I can define in a carrier test that a woman is at risk for developing a cardiomyopathy related to a pregnancy… then I know that that patient will be followed more closely and can have a much better outcome, not just for the pregnancy, but also for the rest of her life.”

This kind of maternal testing is something several companies in addition to Invitae are looking into, such as Natera. Other companies have a focus of testing for pregnancy complications, such as Nx Prenatal, which is developing an exosome-based blood test to predict pre-eclampsia and preterm birth, and Sera Prognostics, which is developing a biomarker blood test to predict preterm birth.

What’s next for reproductive genetic testing?

With a fast-moving field like genetic testing, it can be hard to predict the future, but it seems that NIPT is here to stay. Experts predict it will only become more accurate and, as with Natera’s Panorama AI test, cover more pathogenic variants in greater detail.

As with other areas of medicine, AI and bioinformatics will likely have a greater role to play, which can already be glimpsed with Natera’s Panorama AI and other similar tests in development.

“The ability of companies to aggregate data and the big public data sets are helping a lot, but also the ability of companies that do clinical bioinformatics,” said Miedzybrodzka.

“The slicker they’ve got, the more work we can do with the scientists we have, because a lot more stuff is automatically filtered. One of the big debates that we still have to have in the U.K. is about automatically feeding our variant interpretations into databases like ClinVar. If we had the informatics systems that allowed us to do that, it would help worldwide the interpretation of data and save us all a lot of work.”

The extent and nature of some types of carrier screening can be confusing and concerning, but the new measures set out by the ACMG are a step in the right direction towards better communication and fair prioritization of such tests.

With testing now spanning across the reproductive journey, including areas such as fertility and in vitro fertilization, many companies and researchers are keen to link up the processes. The aim seems to be to create a set of connected tests that begin at the carrier stage and end with newborn testing with a view to maximizing overall maternal and fetal health.

“We predict that prenatal screening will continue to expand so that even more information about the health of the pregnancy can be obtained from a single sample of blood. For example, that could mean detection of additional genetic conditions or possibly identification of adverse pregnancy outcomes, such as preeclampsia or pre-term birth,” says Parmar.

“Another area that I think we’ll be focusing on is the ability to identify causes of unexplained stillbirth and early neonatal death,” adds Slotnick. “Current society recommendations include some rather specific ideas about how these situations should be examined, but they’re not done as well as they probably should be. I think that we can get for many, many families some useful information so that they can prepare for the next pregnancy and help make it to a good outcome.”

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