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New tools for an old quest

Carl P. Weiner, MD, MBA

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Due in great part to the more than 20 years of ground-breaking work by Lo et al,1 the long-awaited noninvasive

renatal diagnosis of trisomy 21 is almost at hand, perhapsacking only a few technical enhancements to reduce or elimi-ate a small false error rate to be widely applicable. Even morexciting is the potential to examine the whole fetal genome inearch of disorders not caused by aneuploidy.2

Decades in the making, the testing of cell-free fetal deoxyri-bonucleic acid (DNA) is disruptive technology that challengesthe billion-dollar-a-year market for aneuploidy screening anddiagnosis. However, the range of clinical applications remainsmodest by definition: DNA-based disease, and as such, cell-freeplasma DNA testing may be vulnerable to non-DNA methodsthat can identify both the most common types of clinicallyrelevant fetal aneuploidy plus have the potential to identifynon-DNA based diseases. The present report by Bahado-Singhand colleagues applying metabolomics to the diagnosis of tri-somy 21 is a first effort and may prove an example of suchtechnology.

The investigative team sought unique metabolic markers inmaternal sera from a cohort of 90 pregnancies at 12 weeksenriched with 30 proven fetuses with trisomy 21. The researchteam used nuclear magnetic resonance (NMR) spectroscopy.Perhaps because NMR spectroscopy is relatively insensitive,only 40 metabolites were identified in both euploid and tri-somy 21 pregnancies, and only 3 of the 40 metabolites werepredictably altered in pregnancies with fetal trisomy 21: 3-hy-droxybutyrate, 3-hydroxyisovalerate, and 2-hydroxybutyrate.Each of the metabolites was significantly increased in affectedpregnancies.

By searching the Human Metabolomics Database, the inves-tigators suggested potential explanations for the impact of tri-somy 21. First, they noted that 3-hydroxybutyrate is both anenergy source and a substrate for the synthesis of phospholip-ids and sphingolipids required for brain growth and myelina-tion. Myelination is delayed in the brain of trisomy 21 fetusescompared with controls3 and decreased myelination often as-sociated with mental retardation. Thus, the identified increasein 3-hydroxybutyrate could reflect decreased utilization by thetrisomy 21 fetus.

From the Department of Obstetrics and Gynecology and Center for theDevelopmental Origins of Adult Health and Disease, University of KansasSchool of Medicine, Kansas City, KS.

The author reports no conflict of interest.

0002-9378/free© 2013 Mosby, Inc. All rights reserved.http://dx.doi.org/10.1016/j.ajog.2012.12.028

See related article, page 371

Second, 3-hydroxyisovalerate is a marker of biotinidase de-ficiency, and biotin deficiency can be associated with findingssimilar to those of trisomy 21 including hypotonia, learningdisability, seizures, and brain atrophy. The effect of trisomy 21on biotin biosynthesis does not appear to have been directlyexamined, except from the standpoint of nutritional intake.4

Lastly, 2-hydroxybutyrate is typically increased under con-ditions of oxidative stress during which it is a byproduct ofglutathione synthesis. One of the key enzymes in that pathway,cystathione �-synthase is overexpressed in the brains of tri-somy 21 patients, and oxidative stress is considered a likelycause of neurotoxicity in trisomy 21.5 However, we note glu-athione S-transferase activity and reduced glutathione itselfre dramatically reduced in children with trisomy 21.6 It isossible that as the tissue of the affected child ages, it loses thebility to quench oxygen-free radicals.

The finding that a panel of metabolic markers is alteredithin the maternal serum metabolome of pregnancies with a

risomy 21 fetus is not, as the authors noted, surprising. Afterll, trisomy 21 is specifically associated with altered trophoblastctivity as reflected in the levels of numerous placental proteinsound in the maternal serum.7 However, trisomy 21 is also

associated with widespread abnormalities of transcription,8

suggesting that the impact of trisomy 21 on physiology is farwider than the function of genes located on the number 21chromosome. From that perspective, it is somewhat surprisingthe investigators did not identify, with the technology used, anyunique metabolites associated with trisomy 21. It is possible amore sensitive method, such as gas chromatography massspectroscopy, might yield a different result. Time will tell.

The variety of methods available for aneuploidy screeningand its diagnosis has dramatically increased over the past sev-eral decades, so it is reasonable to consider their respective rolesin health care as we enter the era of value medicine. Pricingbecomes the driving variable when tests have comparableperformance.9

Presently, universal risk assessment by combining maternalage with multiple biochemical and ultrasound markers pro-vides a patient-specific risk with very high sensitivity and pos-itive predictive values and a low screen-positive rate (eg, a 95%detection rate with a 2.5% screen-positive rate using a risk cut-off of 1 in 100).

Such screening performance is possible when either biochemi-cal testing is performed in all pregnancies or by using a contin-gency approach in which the first stage of screening includes ma-ternal age, fetal nuchal translucency, and either tricuspid orductus venosus flow, with biochemical testing reserved for thoseat intermediate risk (about 20% of the total).10 Those womenidentified as high risk are then offered definitive testing; thesmaller the number of women classed as high risk, the smaller the

number of invasive procedures required.

MAY 2013 American Journal of Obstetrics & Gynecology 339

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Editorials www.AJOG.org

In an attempt to further reduce the number of invasive pro-cedures, cell-free plasma DNA diagnosis of trisomies 21, 18,and 13 is increasingly accepted by health care plans for use inhigh-risk but not low-risk women because of the low but realrisk of a false-positive result.11 In addition, up to 4.5% of spec-mens submitted for cell-free DNA study may prove unusable,urther delaying diagnosis.12

Should test sensitivity either be 100% or closely approach it(as it appears for trisomy 21), the need for invasive testing isessentially eliminated when the cell-free DNA test is negative,although the need for a positive test remains. Unfortunately,some companies have priced their tests to match or be slightlyless than the present maximum total cost of testing an individ-ual woman (cost of screening plus the cost of invasive fetaltesting). Thus, widespread use of cell-free DNA testing willincrease the overall cost of care from a societal perspective untilthe tests achieve the high-performance characteristics re-quired. In that instance, societal costs would decline because allwomen could be tested noninvasively with an expectation of asecure diagnosis, eliminating the cost of screening.

Returning then to the present example and speculating on anenhanced metabolomic approach would reveal several diseasespecific panels of markers diagnostic for trisomies 13, 18, and21 plus the major sex chromosome abnormalities such as XO,XXY, and XYY. Let us also assume that when the methodologyis scaled up for commercial use, a test can be delivered for aquarter to a third of the patient cost of cell-free plasma DNAtesting. Because these chromosome abnormalities constitute80-90% of those identified by amniocentesis or chorionic villussampling13 and about 90% of the remaining fetuses would be

iscovered to have an abnormality on detailed ultrasoundcanning, the incremental diagnostic information provided byell-free plasma DNA testing might not represent added valueor the vast majority of women.

One could also foresee the development of a clinical para-igm in which a noninvasive diagnosis using metabolomics ishe first testing modality offered to all pregnant women andell-free plasma DNA reserved for those in whom either theetabolomic panels are not informative for diagnosis or are

ormal but associated with an abnormal ultrasound. The costdvantages of metabolomics would be multiplied if metabolo-ic markers for other pregnancy diseases such as preeclampsia

nd preterm birth could be identified and included with mod-st additional cost. This is not possible yet, but in 5 years per-aps it could be.Other technologies have the potential to empower the same

aradigm hypothesized for metabolomics. For example, adultsith trisomy 21 express multiple transcription errors at the

xon level.8 It may be that panels of cell-free plasma RNAsriginating from the placenta of the fetus with trisomy 21 cane used for diagnosis. In addition, there are already multipletudies suggesting the maternal plasma transcriptome may be

ltered spontaneous preterm birth,14 preeclampsia, and/or 2

340 American Journal of Obstetrics & Gynecology MAY 2013

rowth restriction.15-16 Thus, it is reasonable to foresee theevelopment of a panel of cell-free plasma RNA markers usingigh-throughput, low-cost PCR that at 10-12 weeks consti-uted a pregnancy wellness examination providing accurateredictions of which women carried an abnormal fetus or wereestined to develop preeclampsia or experience spontaneousreterm birth. It seems unlikely new tests such as metabolo-ics for disorders of pregnancy that complement cell-freeNA will be long in coming. f

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