Maternal plasma bisulfite DNA sequencing: tomorrow starts today.
Maternal Plasma DNA Bisulfite Sequencing
The report in this issue of Clinical Chemistry by Lun, Chiu, and coworkers of the Centre for Research into Circulating Fetal Nucleic Acids at the Chinese University of Hong Kong is an excellent example of this process (2). The investigators used genomewide bisulfite DNA sequencing of maternal plasma to comprehensively assess the epigenomic profiles of both fetal and maternal epigenomes noninvasively, serially, and on a genomewide scale. As a proof of principle, the authors correctly detected trisomy 21 by assessing the methylation density of chromosome 21 and correctly deduced the imprinting status of 4 selected loci: H19  [H19, imprinted maternally expressed transcript (non-protein coding)], KCNQ1OT1 [KCNQ1 opposite strand/antisense transcript 1 (non-protein coding)], MEST (mesoderm specific transcript), and GNAS (GNAS complex locus).
To Boldly Go Where No One Has Gone Before
This report makes you eager to explore roads previously less traveled or considered dead ends, roads that now appear to be going someplace interesting. The routes include developing applications with both short- and long-term impacts and addressing important questions related to various disciplines. The following examples illustrate this process.
PRESYMPTOMATIC MONITORING OF FETAL WELL-BEING
Of the approximately 2 million loci for which the mother was homozygous, Lun, Chiu, and coworkers found that DNA sequencing of maternal plasma demonstrated the presence of a nonmaternal (i.e., fetal) allele in about 5.5% of cases. These fetal-specific reads covered 218 010 and 263 611 CpG sites on the autosomes for first- and third-trimester plasma samples, respectively. This finding means that serial analysis allows monitoring, even during individual pregnancies, of the gestational-dependent increase in methylation for about 45 000 placenta-specific CpG sites. The biological and clinical implications are manifold. Given the correlation of CpG status with gene expression, placenta-specific CpG profiling could generate information about the expression and changes in genes otherwise inaccessible or poorly accessible by other means (e.g., RNA sequencing of maternal plasma). This approach can be used to monitor both normal and abnormal changes in CpG profiles as a function of gestational age, differentiation stage, genetic variation, and/or pregnancy complications with a placental origin or such disorders as preeclampsia, HELLP  (hemolysis, increased liver enzymes and low platelets) syndrome, and intrauterine growth restriction. For example, the STOX1 (storkhead box 1) gene, which is associated with the familial form of preeclampsia in the Netherlands (and experimentally confirmed in a transgenic mouse model), is mainly expressed by subtypes of trophoblast cells that contribute little, if anything, to the maternal plasma RNA pool (3, 4). This low level of expression prevents or complicates STOX1 RNA profiling if one wants to assess the risk of women carrying the susceptibility allele. An alternative, the use of maternal-plasma bisulfite DNA sequencing for CpG profiling of the differentially methylated region in intron 1 of the STOX1 gene (5), along with the large set of effector genes regulated by this master control gene, could permit presymptomatic first-trimester profiling of fetal well-being in women at risk for developing preeclampsia.
EPIGENOMIC PROFILING ESI DISCORDANT MONOZYGOTIC PAROUS TWIN SISTERS
Twin studies have shown that monozygotic twin sisters are frequently, if not always, discordant with respect to preeclampsia and/or HELLP. In such cases, only one of the twins develops a pregnancy complicated by preeclampsia and/or HELLP (6). Such outcomes are usually explained by differences in the paternal genetic contribution (the parous sisters have different partners) and/or the effects of external factors. With this human model, bisulfite DNA sequencing of maternal plasma would aEow an evaluation of both components. The elegant combinatorial approach of Lun, Chiu, and coworkers allows simultaneous assessment of the differential or additional effects of both the paternal allele and external influences, while excluding (by design) the effect of normal genetic variation (i.e., other than epigenetic).
RESETTING OF EPIGENOMIC MEMORY LEADING TO INCREASED RISK FOR CARDIOVASCULAR DISEASES
The physiological demands of pregnancy on the maternal cardiovascular system and the need to establish a connection between the maternal and fetal vascular systems can catapult a woman into a metabolic syndrome that predisposes her to vascular endothelial dysfunction and atherosclerosis in adult life. In a prospective study of 3416 women, the association of preeclampsia with the calculated 10-year risk of cardiovascular disease (CVD) had, according to the Framingham prediction score, an odds ratio of 1.31 (95% Cl, 1.11-1.53) (7).
Long-term persisting changes in the absence of de novo mutations are by definition epigenetic. In other words, when preeclampsia or HELLP with permanent changes in the nature and number of epigenomic marks (mostly 5-methylcytosine) resets the maternal and/or fetal epigenome, the changed epigenetic memory will lead to persistent changes in the transcription of specific genes (given the location of the epigenomic marks in regulatory regions, such as CpG islands) and thereby lead to an increased CVD risk for both the mother and child. Therefore, CpG profiling of both fetal and maternal differentially methylated regions by bisulfite DNA sequencing of maternal plasma could be informative, not only for the presymptomatic monitoring of fetal well-being during pregnancy but also for the long-term risk for CVD of both mother and child. Exploring these persisting changes at the time they occur will permit the design of truly effective therapies.
PREIMPLANTATION MONITORING OF EMBRYO COMPETENCE
As the authors of this Clinical Chemistry report have indicated, their approach could be applied to other areas of medicine in which plasma DNA analysis is of interest, such as the methylomes of cancers or the methylomes of transplanted organs. In the context of pregnancy, one can even envision their method being applicable at the preimplantation stage. Scaruffi and coworkers recently tested whether preimplantation human embryos released DNA into culture medium and whether cell-free DNA provided additional, reliable, and predictive parameters for assessing embryo competence (8). Both genomic DNA and mitochondrial DNA in spent medium were significantly correlated with fragmentation rate, one of the morphologic parameters conventionally used for assessing embryo quality, and mitochondrial DNA mirrored the blastulation potential of both fragmented and high-grade embryos. Bisulfite sequencing of the DNA released by preembryos into the culture medium could allow preimplantation monitoring of embryo competence and/or the effect of culture conditions. Given the correlation between methylation status and DNA size (2) and given the presumed homeostatic role of fragmentation in blastocyst formation (8), explorations of this sort could generate novel insights into fundamental processes of human biology, such as those underlying the earliest stages of human development.
In conclusion, the genomewide, data-driven approach of bisulfite DNA sequencing of maternal plasma described in this issue of Clinical Chemistry generates novel, exciting hypotheses that address fundamental and clinically important questions in various areas, including pregnancy at various stages (before, during, and after), oncology, and transplantation. Tomorrow has started today!
Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article.
Authors' Disclosures or Potential Conflicts of Interest: No authors declared any potential conflicts of interest.
(1.) Grossman SR, Andersen KG, Shiyakhter I, Tabrizi S, Winnicki S, Yen A, et al. Identifying recent adaptations In large-scale genomic data. Cell 2013;152: 703-13.
(2.) Lun FMF, Chiu RWK, Sun K, Leung TY, Jiang P, Chan KCA, et al. Noninvasive prenatal methylomic analysis by genomewide bisulfite sequencing of maternal plasma DNA. Clin Chem 2013;59:1583-94.
(3.) van Dijk M, Mulders J, Poutsma A, Konst AA, tachmeijer AM, Dekker GA, et al. Maternal segregation of the Dutch preeclampsla locus at 10q22 with a new member of the winged helix gene family. Nat Genet 2005;37: 514-9.
(4.) Dorldot L, Passet 8, Mehats C, Rigourd V, Rarbaux S, Ducat A, et al. Preeclampsla-like symptoms Induced In mice by fetoplacental expression of STOX1 are reversed by aspirin treatment. Hypertension 2013;61:662-8
(5.) van Dijk M, Drewlo S, Oudejans CB. Differential methylation of STOX1 in human placenta. Epigenetics 2010;6:e1001015.
(6.) Treloar SA, Cooper DW, Brennecke SP, Grehan MM, Martin NG. An Australian twin study of the genetic basis of preeclampsia and eclampsia. Am J Obstet Gynecol 2001;184:374-81.
(7.) Fraser A, Nelson SM, Macdonald-Wallis C, Cherry L, Butler E, Sattar N, et al. Associations of pregnancy complications with calculated cardiovascular disease risk and cardiovascular risk factors in middle age: the Avon Longitudinal Study of Parents and Children. Circulation 2012;125:1367-80.
(8.) Scaruffi P, Stigliani S, Venturini PL, Anserini P. DNA profiling of culture medium as a novel, non-invasive tool for embryo assessment. Hum Reprod 2013;28(Suppl 1):i1-4.
Cees B.M. Oudejans  *
 Department of Clinical Chemistry, VU University Medical Center, Amsterdam, the Netherlands.
 Human genes: H19, H19, imprinted maternally expressed transcript (non-protein coding); KCNQ10T1, KCNQ1 opposite strand/antisense transcript 1 (non-protein coding); MEST, mesoderm specific transcript; GNAS, GNAS complex locus; ST0X1, storkhead box 1.
 Nonstandard abbreviations: HELLP, hemolysis, increased liver enzymes, and low platelets (syndrome); CVD, cardiovascular disease.
* Address correspondence to this author at: Department ot Clinical Chemistry, VU University Medical Center, De Boelelaan 1117, 1081 HV Amsterdam, the Netherlands. Fax 20-4443895; e-rnaii email@example.com.
Received August 8, 2013; accepted August 12, 2013.
Previously published online at DOI: 10.1373/clinchem.2013.212787
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|Author:||Oudejans, Cees B.M.|
|Date:||Nov 1, 2013|
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