American Association for Clinical Chemistry
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April 2011 Clinical Laboratory News: New Paradigms in Carrier and Prenatal Screening

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April 2011: Volume 37, Number 4

New Paradigms in Carrier and Prenatal Screening
How Soon Will They be in Use Clinically?

By Genna Rollins

Since the Human Genome Project closed shop in 2003 some 2 years ahead of schedule with the crowning achievements of having sequenced 99% of human DNA to 99.99% accuracy and mapping 3.7 million human single nucleotide polymorphisms (SNPs), the hunt has been on to find practical applications for this treasure trove of information. So far, with some notable exceptions, little has been put to use in routine patient care. But that may be changing, according to experts. Two recent studies involving carrier and prenatal screening set the scientific community abuzz and opened promising avenues for genetic testing.

One effort, led by Stephen Kingsmore, MB, ChB, DSc, reported development of a preconception carrier test that can simultaneously screen for 448 severe recessive disorders, based on target enrichment, massively parallel (next generation) sequencing, and powerful bioinformatics (Sci Transl Med 2011;3:65ra4). The other, a validation study under the direction of Y. M. Dennis Lo, DM, DPhil, reported successfully using multiplexed, massively parallel sequencing of fetal DNA in maternal plasma to detect trisomy 21 (BMJ 2011:345:c7401). These findings, taken together with efforts by other research teams, constitute a dramatic down payment on the promise of molecular diagnostics and present a fundamentally new paradigm for genetic testing, according to Laird Jackson, MD, professor of obstetrics and gynecology and medical genetics at Drexel University in Philadelphia.

“For those who are disappointed that the Human Genome Project 10 years on has led to little beneficial impact on human disease, these applications should be a welcome sign of return on the investment. Current progress in genetic testing technology offers to merge the concepts of carrier testing, pregnancy screening, and prenatal diagnosis for genetic disease into one manageable continuum,” he predicted.

Ready for Prime Time?

Perhaps the most intriguing aspect of these efforts is that both investigative teams plan to make their tests available in some fashion and at reasonable costs by the end of this year. However, both the researchers and other experts cautioned that there are many miles to go and hurdles to cross before the tests gain widespread adoption. “We’ve seen the future in that tests of this kind are likely to emerge over time as the mainstay, and the days of our looking at one gene and one test at a time are numbered,” said Bruce Korf, MD, PhD, professor and chair of genetics at the University of Alabama at Birmingham. “What isn’t so clear is when these advances will be ready for routine use. There are questions of cost-effectiveness, technical, clinical utility, ethical, legal, and social issues that have to be addressed before these technologies can be reliably deployed in a clinical context.” Korf also is president of the American College of Medical Genetics.

Parents’ Sorrow Prompts Research

Kingsmore’s efforts started with anguished parents. Craig Benson, president and CEO of Rules-Based Medicine, and his wife, Charlotte, learned in 2008 that their daughter, Christiane, just 5 years old, had Batten disease, a fatal neurodegenerative illness. That year, the Bensons established the Beyond Batten Disease Foundation and approached Kingsmore about the possibility of developing a carrier screening test that could identify not only Batten disease but also other rare and debilitating autosomal recessive disorders. Kingsmore and his team, then at the National Center for Genome Resources (NCGR) in Santa Fe, took up the challenge.

Their first step was to critically assess molecular diagnostic technology. “The idea of carrier screening is nothing new, but most carrier screens are offered using targeted mutation testing. So we looked at the platforms available for the detection of individual variants like SNPs and compared them to sequencing in terms of cost and effectiveness. Our conclusion was that a fixed content system like a custom DNA chip wouldn’t be something that could keep pace as we found new mutations, unless we redesigned the chip every few months. So we decided a sequencing platform would be the best,” explained Callum Bell, PhD, program lead at NCGR and lead author of the team’s study.

A subsequent literature review identified 448 autosomal recessive disorders that cause severe illness or death in childhood. The investigators then quickly concluded that their efforts would be better spent on targeted gene capture rather than genome-wide sequencing. “Knowing that about 85 percent of mutations are found in coding regions and are either single nucleotide variants or small insertions or deletions, we looked for methods for sequencing solely that fraction of the genome that codes for those genes,” said Bell. After considering several platforms, they ultimately went with the Agilent SureSelect and targeted 7,717 regions from 437 genes. The researchers then developed a powerful bioinformatics disease tree to assess the clinical utility of their method. (See Table,below) They found that 93% of nucleotides had at least 20x coverage, with 95% sensitivity and 100% specificity for substitution, insertion/deletion, splicing, gross deletion mutations, and SNPs. On average, each person tested carried 2.8 severe recessive mutations.

The Pathway to Carrier Screening

Researchers Callum Bell, Stephen Kingsmore, and their colleagues combined target enrichment, massively parallel sequencing, and powerful bioinformatics analysis to develop a test that screens for 448 autosomal recessive disorders. This flowchart provides an overview of the steps involved in performing the test.

Click here for chart

Legend: Blue boxes–Quality assessment and control; Green boxes–Lab or bioinformatics process; Red box–Report production

Courtesy Darrell Dinwiddie, PhD, Children’s Mercy Hospital, Kansas City, Mo.

For Further Information:

Bell CJ, Dinwiddie DL, Miller NA, Hateley SL, et al. Carrier Testing for Severe Childhood Recessive Diseases by Next Generation Sequencing. Sci Transl Med 2011;3:65ra4.

Chiu RWK, Akolekar R, Zheng YWL, Leung TY, et al. Non-invasive prenatal assessment of trisomy 21 by multiplexed maternal plasma DNA sequencing: large scale validity study. BMJ;2011:342:c7401.

Chiu RWK, Chan KCA, Gao Y, Lau VYM, et al. Noninvasive prenatal diagnosis of fetal chromosomal aneuploidy by massively parallel genomic sequencing of DNA in maternal plasma. NAS;2008;105:20458–63.

Chiu RWK, Lo YMD. Non-invasive prenatal diagnosis by fetal nucleic acid analysis in maternal plasma: the coming of age. Semin Fetal Neonatal Med 2011;16:88–93.

Ehrich M, Deciu C, Zwiefelhofer T, Tynan J, et al. Noninvasive detection of fetal trisomy 21 by sequencing of DNA in maternal blood: a study in a clinical setting. Am J Obstet Gynecol 2011;205.e1–11.

Lo YMD, Chan KCA, Sun H, Chen EZ, et al. Maternal plasma DNA sequencing reveals the genome-wide genetic and mutational profile of the fetus. Sci Transl Med. 2010;61:61ra91.

Lo, YMD, Chiu RWK. Noninvasive approaches to prenatal diagnosis of hemoglobinopathies using fetal DNA in maternal plasma. Hematol Oncol Clin N Am 2010;24:1179–1186.

Tsui NBY, Kadir RA, Chan KCA, Chi C, et al. Noninvasive prenatal diagnosis of hemophilia by microfluidics digital PCR analysis of maternal plasma DNA. Blood doi:10.1182/blood-2010-10-310789.

Zimmermann BG, Grill S, Holzgreve W, Zhong XY, et al. Digital PCR: a powerful new tool for noninvasive prenatal diagnosis? Prenat Diagn 2008;28:1087–93.


A Diagnostic Tool, Too

With this proof-of-concept completed, Kingsmore’s team now has its sights set on offering, by the end of this year and for limited clinical use, a beefed-up version of the test that will identify about 600 conditions. Kingsmore, who relocated to Children’s Mercy Hospital in Kansas City, Mo. at the end of 2010 and is now director of that institution’s Center for Pediatric Genomic Medicine, hopes to make the test available at Children’s under research protocols and CLIA regulations. “Our initial target group is for pediatric diagnosis, not carrier screening. It would be for children who are sick and have a presentation that makes their physician think they may have one of these recessive illnesses,” he explained.

Kingsmore favors this approach as a way to learn how best to report results for the groundbreaking diagnostic, and as a means of launching it without having to address a plethora of thornier issues related to using it in preconception carrier screening. “We’ll have a set of physicians who can give us instant feedback on whether the reporting mechanisms are understandable,” he said. “Also, the pre-test probability of a positive result will be quite different and the symptomatology will give a clinical-pathological correlation. That’ll be immensely useful in validating the clinical utility of the test before we do carrier screening.”

However, Kingsmore acknowledged that interest in the technology is keen and whether he will be able to hold the throttle on its commercialization remains to be seen. “We’re in dialogue with a lot of different parties now,” he said. “Our goal is to have implementation of the test occur in the non-profit arena if possible. We think it should be broadly available at cost, and we’re pushing it in that direction, but others see it as a great commercial opportunity. In the end, there probably will be a number of places where these kind of technologies get rolled out.”

A Lengthy Discovery Process

Lo’s findings are the culmination of a long scientific odyssey dating to 1997, when he was the first to demonstrate that cell-free fetal DNA is in the blood of pregnant women. This discovery opened a new pathway for noninvasive prenatal diagnostics, albeit one fraught with challenges. “A lot of people were not that optimistic then because without a membrane surrounding fetal nucleic acid—which occupies about 10 percent of the mother’s plasma—you can’t use conventional methods like fluorescence in situ hybridization to count the number of chromosomes,” recalled Lo, who is professor of chemical pathology and Li Ka Shing professor of medicine at The Chinese University of Hong Kong. He will be speaking on aneuploidy detection during pregnancy by non-invasive fetal DNA measurement at the 2011 AACC Annual Meeting in Atlanta.

“Initially our approach was to look at sequences that were only present in the baby and absent in the mother, such as the Y chromosome, which the baby has inherited from its father. Those are easy—if you detect them, you know you’re looking at fetal DNA,” he said. This approach eventually led to development of molecular tests in use today, for fetal sex determination and RhD genotyping in RhD-negative mothers, in which positive results indicate male and RhD-positive babies, respectively.

Not content with these advances, Lo’s team pursued methods to improve on the tests, not only to boost their specificity, but also ultimately to be able to detect fetal chromosomal aneuploidies, trisomy 21 being one of the most common. One approach was to exploit DNA methylation changes that differ between placental and maternal blood cells, while another involved identifying placental-derived mRNA. However, both these methods have downsides, according to Lo. “Because they’re based on SNPs, you can only use them in the part of the population who has the required configuration of the SNPs. That means you’d need a combination of those polymorphisms to cover a large population,” he explained.

This led Lo’s group to search for still better ways of detecting fetal aneuploidies independent of polymorphisms, and in 2007, they reported using digital PCR to count single molecules. “We showed that for every two-fold decrease in fetal DNA proportion you need to count four times as many fetal molecules. But if you count molecules one at a time, which we did using digital PCR, you can reach that kind of precision,” Lo explained. “So for a 10 percent fetal DNA proportion, you’d probably need to count tens of thousands of molecules, and of course, conducting tens of thousands of PCRs would be very time-consuming.”

Luckily, massively parallel sequencing technology was just starting to enter the mainstream at that time, and Lo used it to crack the nut he had been working on for years. “With next generation sequencing you can literally analyze millions of molecules, and we showed that you can use it to detect trisomy 21,” he said. In that report, Lo described the potential of next generation sequencing (PNAS 2008;105:20458-63). It’s “such a powerful tool for quantifying the relative genomic representation of plasma DNA molecules that only an amount corresponding to just a representative fraction of the human genome would need to be sequenced,” he wrote.

That proof of principle report involved just 28 cases, so the team’s next hurdle was to validate the findings in a larger study population, and that they accomplished, with 753 subjects, as described in the British Medical Journal report. The investigators compared 2- and 8-plex sequencing strategies, and found the former more precise, with 100% sensitivity and 97.9% specificity (See Table, below).

Noninvasive Detection of Fetal Trisomy 21

Researchers massively sequenced maternal plasma DNA using two multiplex protocols to detect fetal trisomy 21. The true detection rate of the 2-plex protocol correctly identified 86 of 86 cases tested.

True Detection Rate
False Positive
2-plex sequencing protocol
100% (86/86)*
2.1% (3/146)**
8-plex sequencing protocol
79.1% (68/86)*
1.1% (6/571)***

*Among 86 trisomy 21 cases; **Among 146 non-trisomy 21 cases;***Among 571 non-trisomy 21 cases

Source: Adapted from BMJ 2011:345:c7401

A Substitute for Amniocentesis?

Lo plans to make the test available for use in clinical practice in Hong Kong by the end of this year. However, many details have yet to be worked out, including the cost of the test, although Lo indicated that he expects it will be competitive with amniocentesis. “We’re talking about five percent of pregnant women who deliver in Hong Kong–about 3,000 each year–who would go for an invasive prenatal test, and we’re proposing that they be channeled into this sequencing test,” he explained. “It would be introduced as a lab-developed test, and initially, I think it would be a good idea to run it in parallel with what clinicians normally do. This will help them get used to what this test is like even though the bioinformatics are complicated.”

Sequenom, headquartered in San Diego, Calif., has licensed technologies from Lo, and recently reported using massively parallel DNA sequencing to detect fetal trisomy 21 in 449 maternal blood samples, with 100% sensitivity and 99.7% specificity (Am J Obstet Gynecol 2011;204:205.e1–11). The company launched a clinical validation study for its SensiGene Trisomy 21 test in December 2010.

The Counseling Bottleneck

As appealing as the prospects for both tests may be, experts agreed that simply having the technologies available will not be the magic wand that suddenly disseminates them. “Although the whole package is fairly complex, the actual sequencing is simple. The tricky bit relates to physician interface on the front end so there’s appropriate ordering and pre-test genetic counseling, and on the back end, interpreting results and delivering them in a format that physicians will understand,” said Kingsmore. “All of this has to be provided in the context of genetic counseling, and that will tax the entire healthcare industry because there aren’t enough genetic counselors or clinical geneticists to handle all of this. It’s a real bottleneck.”

Kingsmore’s point resonated with several experts, including Jackson, who emphasized that even physicians misinterpret currently available prenatal tests. “There are huge segments of the professional population who think a positive serum screening test for Down syndrome means the mother has a Down syndrome fetus. But there’s only at most a five percent risk that that’s true,” he observed. “It’s not an easy concept to get across to most people, and you can put all the fine print on the report that you want, but the part that’s read is that it’s positive for Down syndrome.”

The Genotype-Phenotype Disconnect

Given the intricacies of genetic counseling, Korf cautioned that these new approaches will only ratchet up the complexity factor. “We’re moving quickly to the point where we can know a lot more at the genotype level than we know how to interpret phenotypically,” he said. “We’ve been in a somewhat insulated position historically where we test for things like thalassemia, which we know a lot about and can give parents who are carriers good information about what to expect. But as we start moving beyond those well-characterized situations, we’ll start finding things where we’re not so certain what they mean.”

Medical ethicist Lainie Friedman Ross, MD, PhD, already sees signs of this genotype-phenotype disconnect. In the Bell-Kingsmore study, for example, “the authors included a broad range of conditions which many of us wouldn’t consider that severe. For instance, our clinic has detected women with two abnormal genes for Type 1 Gaucher disease, meaning they have the disease, and have never known it. So the authors’ approach of basically doing carrier screening for any autosomal or exome conditions without considering that they manifest in a continuum was frustrating to me,” she said. Ross is Carolyn and Matthew Bucksbaum professor of clinical ethics and professor of pediatrics, medicine, and surgery at the University of Chicago.

She also questioned exactly how Kingsmore’s test would be implemented in carrier screening, particularly if it were used in a general, let’s-see-what-pops-up strategy. “There’s nothing in the classic medical ethics literature that justifies broad consent, meaning we’re looking for any pathogenic genes and if we find them, we can then discuss the range of severity of symptoms, time-of-onset, and options the patient may have. So this notion that we’ll test for lots of conditions and discuss any that come up positive afterwards doesn’t seem like we’re approaching the problem ethically from an informed consent and patient empowerment perspective,” Ross contended.

In addition, she expressed concerns about Lo’s short-term plans to offer a non-invasive screening test for trisomy 21 only, without also including trisomy 13 and 18. “Trisomy 13 and 18 are much more severe than 21, so if you’re a pro-choice woman who’d consider abortion on the basis of trisomy, you’d want to know all three. So right now, you’d end up with amniocentesis anyway, because Lo’s test can’t promise you the other two,” she explained. Lo’s lab already is working on expanding the technology to screen for trisomy 13 and 18. “It’s slightly more technically difficult than trisomy 21 because the precision with which one can measure trisomy 13 and 18 is less exact. But I think it’s solvable by using appropriate bioinformatics,” Lo indicated.

What’s the Mutation Penetration?

Adding to the complexity of genetic counseling associated with the tests, Bell and Kingsmore found that nearly a third of mutations cited in the literature were either common polymorphisms or misannotated, casting doubts on existing notions about the prevalence of autosomal recessive diseases. “We found a lot of disease-causing mutations in the human genome mutation database that were present in multiple individuals in our set, adding up to a frequency greater than five percent, which is the cutoff people have used for calling something a SNP. After that it’s considered a common polymorphism,” Bell explained. “We also found references in the primary literature saying there was a particular mutation, but when we sequenced it by traditional PCR and Sanger sequencing, we’d confirm that those sources were wrong.”

These discrepancies, many of which have to do with the rarity of some of the diseases and the technologies available when they were annotated initially, underscore the need for a broad coalition of stakeholders, particularly laboratorians, to develop a robust mutation reference database, according to Kingsmore. “One of the very practical issues that’s coming out of this is, you can have a test with an analytical precision of near-perfect sensitivity and specificity and a beautiful area under the curve, but if you’ve referenced a database that’s wrong 20 percent of the time, then you’re not delivering the power of that assay. The only solution is to develop a clinical grade mutation database,” he noted.

Who Will Crunch the Data?

Bell, Kingsmore, and Lo also cautioned about the need for sophisticated bioinformatics to process the data output from next generation sequencing. Lo suggested labs might accomplish this using cloud computing, in which data would be uploaded via the Internet, processed by a high-powered third-party bioinformatics system, and transmitted back to the lab. However, Bell was less sanguine about that approach. “You need pretty sophisticated software engineers to take advantage of all that. Using cloud computing would free you from the capital expense of buying equipment and to some extent the personnel costs, but regardless of where the computing is done—by cloud or in a local infrastructure—you still have to have the right algorithms and programs. The cloud might provide practical advantages but the computational approach is the most important piece,” he said.

A Bright Future

Even with all the what-ifs and technical issues to be sorted through, experts endorsed the long-term prospects for the clinical utility of both tests. In the case of Lo’s test for trisomy 21, Jackson shares his view that the technology ultimately may replace nearly all amniocentesis and chorionic villus sampling tests. “Right now, there’s a very small risk with these invasive procedures, connected with expertise. They work safely and successfully in centers where they’re done every day or every week, so the mother has to commute to these specialized centers,” he explained. “If Dennis’s work evolves to the clinically applicable stage, all of that goes away and the mother will just go down and get her blood drawn where it can be appropriately stored and sent to a lab. It democratizes access to that service.”

Korf hailed the promise of Kingsmore’s test as a pediatric diagnostic tool. “I wish I could say we could see a child, figure out the differential diagnosis, order only those tests most likely to be productive and come to an answer—that’s the ideal,” he said. “But the reality is, you can’t always stage a work-up the way you’d like to, and that’s why a test like this could make a big difference in the long run. You can cut to the chase and answer a lot of questions that may have taken months or years to figure out otherwise.”

Similarly, Jackson’s sees the test’s potential in carrier screening. “It’s a significant step in the right direction for anticipatory management of the risks posed by the occurrence of genetic disorders as we reproduce,” he noted.

Looking beyond development of these two tests, Korf encouraged labs to keep abreast of fast-changing developments in this up-and-coming field. “We’ll reach a point where doing a whole genome will rival the cost of doing one gene, so that will change the paradigm in terms of how testing is done,” he observed. “Labs will have to think of new approaches for how genetic tests are performed, interpreted, and deployed. We’re talking about a generation of effort to establish how and when genetic tests are done that could be overturned in a short period of time.”