Cell-free Fetal Nucleic Acid Identifier Markers in Maternal Circulation.

From the discovery of cell-free fetal (cff)-DNA in 1997 so far, many studies have been performed on various aspects of cff-nucleic acid. It is undoubted that currently, invasive prenatal diagnosis progresses to the noninvasive test. However, there are many problems. One of the most challenging issues in this field is differentiation and detection of the small amount of cff-nucleic acid in maternal plasma. Many markers and methods have been used for this purpose. This review makes an attempt to review and compare the studies in the field. Six identifier markers including Y-specific sequence, polymorphisms, epigenetic difference, DNA size difference, fetal mRNA, and microRNA as well as the advantages and disadvantages of each marker are discussed. This review provides a relatively perfect set on cff-nucleic acid biomarkers in various physiological and pathological status of pregnancy, helping to review and compare the prior obtained results, and improving designation in future studies.

Introduction

Prenatal diagnosis is a critical issue of gynecological practice. To perform a genetic test in this field, it is necessary to obtain placental or fetal material. This material, currently, is achieved through invasive procedures such as chorionic villus sampling or amniocentesis. These invasive procedures carry 1% risk of miscarriage, and other maternal and fetal complications have been reported to relate to these invasive procedures.[1] In 1997, the presence of cell-free fetal DNA (cff-DNA) in the circulation of pregnant women was reported.[2] Fragmented cff-DNA originates primarily from apoptotic syncytiotrophoblasts.[3] It is demonstrated now that maternal plasma and serum contain cff-DNA, but the mean fractional concentration in plasma is higher than serum 3.4–6.2% and 0.13–1.0%, respectively depending on the gestational age.[4] It is detectable as early as 18 days following embryo transfer in in vitro fertilization pregnancy and is soon cleared from maternal circulation after delivery, with a mean half-life of 16 min, so it is a suitable source for noninvasive prenatal diagnosis (NIPD) in pregnancy.[56] Following these reports, a new area of research in the diagnostic field was opened; however, there are two problems with cff-DNA for the development of the noninvasive diagnostic test, the small proportion of cff-DNA in maternal plasma as little as ~19%, and coexistence with maternal DNA.[7] The presence of cff-DNA in maternal circulation is accepted universally, and although via the discovery of cff-DNA many potentially clinical applications have been assessed such as fetal rhesus D status, sex-linked disorders, monogenic disorders, aneuploidies, and many pregnancy complications such as preeclampsia and preterm labor,[891011121314] but confirming the presence of cff-DNA in maternal plasma extracts is still a challenge in diagnostic tests. Researchers have applied many methods to differentiate the fetal-derived sequences from that of mother. This study makes an approach to cff-nucleic acids identifier markers applied by researchers of the field including Y-specific sequence, polymorphisms, epigenetic difference, DNA size difference, fetal mRNA, and microRNA (miRNA) as well as the advantages and disadvantages of each marker.

Y-Chromosomal Sequences

One of the most common targets used for the detection of cff-DNA in maternal circulation has been Y-specific sequences. If one can detect Y-specific sequences in plasma of a pregnant woman, it means that the plasma contains a male fetus cf-DNA in detectable level. These Y-specific sequences can be quantified as an index of cff-DNA in maternal circulation because they are genetic sequences of the fetus not in the maternal genome. Thus, it can be used for the evaluation of cff-DNA extraction method and detection systems. The first report of cff-DNA in maternal plasma used Y-specific sequences.[2] Since then, many groups have validated the initial finding and used Y-specific sequences to differentiate the fetal-derived sequences from that of the mother as the primary step for confirmation of cff-DNA in NIPD. The other application of prenatal sex determination is in pregnant women carriers of an X-linked disease, because identification of a male fetus indicates hemizygosity for the X chromosome and potential disease; therefore, invasive testing can be avoided in female bearing pregnancy. For some endocrine disease such as congenital adrenal hyperplasia, prenatal diagnosis can help to prevent the disease via antenatal treatment.[15] Researchers have performed fetal sex determination using various sex-specific markers through a broad range of gestational age. The most reported study on fetal sex determination has used single copy SRY gene or the multicopy DYS14 marker sequence of the TSPY gene on Y chromosome. The studies on sex determination which used sex-specific markers and their outcomes are summarized in Table 1.[1516171819] Almost all studies have used polymerase chain reaction (PCR) or real-time PCR as detecting methods. All in all, the accuracy of tests increases in higher gestational age and in the use of real-time PCR than conventional PCR.[16] Many groups have used the combination or ratio of markers and obtained highly reliable results than the single marker.[1518] In addition to some advantages, use of Y-specific sequences as cff-DNA identifier markers is associated with the number of limitations. Hence, this method is applicable only in male-bearing pregnancies, and a negative test can imply either the fetus is female, or the cff-DNA is below the detection limit of the experiment.[20]

Table 1

Examples of studies used Y- chromosome sequences for detection of cff-DNA in maternal plasma

Paternally Inherited Polymorphic Markers

The other way for differentiating fetal-derived sequences from the mother is the use of paternally inherited polymorphic markers. Some studies have used a panel of number of biallelic highly polymorphic markers to confirm the presence of fetal DNA in maternal plasma.[2122] In some studies, the use of these polymorphic markers helps to avoid invasive procedure in more than 60% of cases.[21] Other groups have developed a detection method for determination of the presence of either free fetal DNA or fetal cells in maternal circulation using polymorphic short tandem repeats (STR), or a panel of single nucleotide polymorphisms (SNP); the data were summarized in Table 2.[232425] The most important advantage of these markers is that they are gender independent and can potentially ascertain the presence of fetal DNA in female bearing pregnancies. However, the use of paternally inherited polymorphism is associated with many disadvantages and requires prior data on the polymorphic status of the parent; therefore, it is labor intensive and could apply only to individuals who own polymorphisms, and due to obtaining an appropriated number of informative alleles, a large number of polymorphisms need to be used.[2026]

Table 2

Examples of studies used polymorphic markers for detection of cff-DNA in maternal plasma

Epigenetic Markers

The epigenetic markers can differentiate cff-DNA independently of the gender or polymorphic status of the parents and fetus. Epigenetic modifications are molecular events that affect gene expression without changing in DNA sequences; they are stable through cell division. The best studied epigenetic event is DNA methylation which refers to the addition of methyl group to cytosine residues in DNA sequence; when this occurs in promoters of genes, gene expression may be switched off.[2728] The specific DNA methylation signatures of tumoral DNA were detected in 1999 for the first time, and the possibility of using them as noninvasive biomarkers was examined.[293031] After such developments, various studies were performed to detect cff-DNA from maternal plasma.[3233] Detection of cff-DNA from maternal plasma using epigenetic markers has been performed based on two patterns. The first pattern is an imprinted locus, in which the DNA methylation patterns are inherited in a parent-of-origin-specific manner. The methylation status of this locus can distinguish between the fetus and the mother. In 2002, using this method for the first time, the stretch of DNA that a fetus has inherited from the mother was detected.[32] However, this method is so complicated to be used in routine tests as a fetal marker.[20] Therefore, the second pattern, placenta-specific methylation pattern has been explored. Many groups have demonstrated that the methylation pattern of human placenta is different from other somatic tissues.[3435] Now, it is believed that cff-DNA in maternal plasma is derived from syncytiotrophoblasts, and maternal free DNA is derived from hematopoietic cells.[336] Therefore, these two fractions of free DNA in maternal plasma can be distinguished based on their different methylation status in a single genomic locus. The first successful study was in 2005, based on the different methylation patterns of maspin gene in the mother and fetus.[33] This was the first universal fetal marker which could be used in all pregnancies independent of gender or polymorphic status, and this is the main advantage of this epigenetic pattern. Since then, many studies were developed based on this pattern.[373839] In general, the detection of epigenetic markers in maternal plasma requires two steps; the first is differentiation of methylated and unmethylated sequences using bisulfate modification or differential cleavage by restriction enzymes. The second is identification or quantification of fetal-specific methylation pattern via sequencing, methylation-specific PCR, or real-time PCR.[20] Many studies have used bisulfate treating method; however, the main disadvantage of this technique is that bisulfate treating is proved to degrade the huge amount of DNA template, so not suitable for cff-DNA, which is low in maternal plasma.[40] If there are recognition sites of methylation-sensitive restriction enzymes within the differentially methylated region of placenta genome, this can be the basis of cff-DNA identification in maternal plasma.[3841] Compared with bisulfate treating, this digestion-based method brings less damage to the cell-free DNA in plasma, therefore, is more appropriate for cff-DNA.[20] Many applications for epigenetic markers have been reported in NIPD including (1) to indicate the presence of fetal DNA or as quantitative markers to quantify the amount of fetal DNA in maternal plasma, (2) to indicate the quantitative aberrations of cff-DNA in some disorders for example, in preeclampsia, (3) reports have demonstrated the feasibility of using fetal epigenetic markers for prenatal diagnosis of aneuploidy.[3337] Another application of epigenetic factors is cff-DNA enrichment. It is now accepted that the amount of cff-DNA in maternal plasma is very little and coexists with maternal-free DNA. These two problems are the major challenges for the use of cff-DNA in the diagnostic field. Hence, researchers need an enrichment method to solve these problems, which means a selective amplification of cff-DNA to overcome scarcity and a huge amount of maternal background. One group has developed a method for preferentially amplification of cff-DNA in maternal plasma using the first universal epigenetic marker for fetal DNA, maspin [Table 3].[3341] The other disadvantage of using epigenetic markers is that these assays are multiprocedure and relatively labor intensive.

Table 3

Examples of studies used epigenetic markers for cff-DNA in maternal plasma

Differential Characteristic

Fragmentation of cff-DNA in maternal plasma has been proved by many groups. It is proved that the major amount of cff-DNA is shorter than 300 bp, and maternal-free DNA molecules are obviously longer.[4243] Although this differential characteristic is not a marker, but it has been successfully recruited by some groups as a basis of enrichment method in size separation manner on agarose gel electrophoresis. Although this method is prone to contamination, it has been solved by strict anticontamination measures used at all stages of sample preparation, and the contamination has been avoided.[444546] Many studies have used various methods as an enrichment method for improving the results of subsequent diagnostic tests, but some are too complex, expensive in fee, and labor intensive to be applied in clinical practice [Table 4].[414445474849505152] Enrichment method based on the size separation on agarose gel is easy to perform, not expensive, and available in almost all laboratories [Figure 1].

Table 4

Summarization of cff-DNA enrichment methods applied by researchers

Figure 1

Schematic diagram representative various enrichment methods for cff-DNA. *Single Allele Base Extension Reaction

Fetal mRNA

After the discovery of fetal DNA in maternal plasma, many studies confirmed the presence of fetal DNA in maternal plasma and tried to use the fetal-free DNA in many diagnostic destinations mentioned above. However, the presence of fetal RNA was not known till 2000, and one group for the first time, using the two-step reverse transcription (RT)-PCR assay, demonstrated the presence of Y chromosome-specific zinc finger protein mRNA which is a fetal-derived, male-specific mRNA in plasma of pregnant women carrying male fetuses.[53] The study also showed that the detection rate of plasma fetal RNA is lower than that of plasma fetal DNA. It is possible because of degradation nature of the fetal RNA in maternal blood. Although some groups have demonstrated that the placenta-mRNA is stable in maternal plasma, possibly it is because of the presence of microparticles that protect them from degradation. This study followed other studies which demonstrated the detectability of RNA of human placental lactogen (hPL) and the β-subunit of human chorionic gonadotropin (β-hCG) mRNA in pregnancy, using placental-specific mRNA analysis.[5354] Many groups have examined the possibility of the use of fetal mRNA in maternal plasma as a marker for pregnancy complications such as fetal-maternal hemorrhage or preeclampsia. Due to significant increase in fetal CRH mRNA concentration in preeclamptic pregnancy rather than healthy pregnancy and rapidly clearance through 2 h postpartum, it is suggested that maternal plasma CRH mRNA might be a new molecular marker for preeclampsia.[5556] In 2007, Lo et al., using PLAC4 mRNA-SNP on chromosome 21, determined the allelic ratio of interest SNP in heterozygote fetus and diagnosed trisomy 21 with comparable sensitivity and specificity to many other multimarker screening strategies. An additional advantage of this strategy is insensitivity to the gestational age at which sampling is performed unlike the current strategy which uses serum biochemical markers; thus, the clinical use of this strategy is relatively simple. The main drawbacks of this method are that this method is polymorphism-dependent and usable only for heterozygote fetus. PLAC4 SNP (rs8130833) has a heterozygosity rate of 45% in the studied population. Therefore, it has a low population coverage; this can be compensated by combining several SNPs of genes transcribed from chromosome 21.[57] Some studies have attempted to systematically identify placental tissue gene expression profiling using oligonucleotide microarrays followed by real-time quantitative RT-PCR for the detection of identified expressed gene in maternal plasma. In this study, six genes could be identified: hPL, β-hCG, CRH, TFPI2, KISS1, and PLAC1, and their transcript are detected in maternal plasma [Table 5].[58] Plasma fetal RNA analysis has a number of advantages: First, these markers can provide valuable data on gene expression patterns of fetal tissues. For example, complicated pregnancies such as preeclampsia have been confirmed associated with abnormal gene expression. Thus, with the development of further RNA markers, RNA analysis of maternal plasma may lead to the noninvasive monitoring of the gene expression of an unborn fetus in many physiological and pathological statuses. The second is gender- and polymorphism-independent unlike previously described markers. Third, the use of these markers are relatively simple and inexpensive involving mRNA extraction, and RT-PCR analysis can be used for many plasma RNA markers unlike other markers such as epigenetic markers which need multi or damaging procedures such as bisulfate conversion.[54]

Table 5

Examples of studies used mRNA markers in maternal plasma

MicroRNA

MicroRNAs (miRNAs) are small noncoding RNAs about 20–24 nucleotides long. They are critical factors in regulating cellular gene expression, suppressing the translation of protein-coding genes at the posttranscriptional level.[59] MicroRNAs are critical in cell development, proliferation, communication, and tissue differentiation. They are involved in regulating pregnancies.[60] The miRNA expression patterns change in various pathological and physiological statuses including pregnancy. The placental development has many critical processes such as differentiation, migration, invasion, angiogenesis, proliferation, and apoptosis.[616263] It has been shown that miRNAs regulate placental development and functions through these processes.[646566] MicroRNAs are released from placental syncytiotrophoblasts into the maternal circulation via exosomal particles which are small vesicles secreted by many cells. Circulating miRNAs are complex with circulating ribonucleoprotein and high-density lipoproteins. This complex form of miRNAs converts them to stable forms by protection from digestion by RNase.[6768] If miRNA biomarkers for noninvasive diagnosis in maternal circulation is to be used, first miRNA expression profile both in normal controls and patients need to be determined using microarray or next generation sequencing. Subsequently, the under-or over-regulated miRNAs are quantified by real-time PCR to validate the repeatability of the results. Candidate miRNAs can then be tested to determine their changes in maternal serum or plasma. As an alternative strategy, we can directly examine specific miRNAs with known expression patterns that are associated with the pregnancy complication of interest.[69] Due to the role of miRNAs in placental development, the aberrant expression pattern of placental miRNAs has been detected in maternal circulation with pregnancy complications or human placental diseases such as preeclampsia,[70] intrauterine growth restriction (IUGR),[71] and miscarriage.[72] Although some complications, such as preeclampsia have been well studied, the other complications such as IUGR and miscarriage, the role of these potential biomarkers need to be confirmed by further investigations. Consideration of these data including the role of miRNA in critical steps of placenta developing, founding these genetic materials in maternal circulation in a stable form, possessing specific expression patterns in various maternal and fetal status, and importantly, the demonstration of the fact that many of these miRNAs are detectable in maternal plasma during pregnancy but undetectable in postdelivery plasma which confirm their placenta origin,[69] suggest that these regulating factors can be used as a potential biomarkers in NIPD. Although these potential markers (like mRNA) have a number of advantages such as independency on gender and polymorphism and relative simple detection assay, the clinical application of these markers need many requirements such as standardization in all steps of assay including sampling, miRNA isolation, and quantification. The obtained results should be confirmed by further analytical studies and appropriate normalization methods.[73]

Conclusion

Nowadays, it is confirmed that cff-nucleic acids are an excellent potential genetic resource for clinical NIPD due to riskless sampling, low-cost detecting test in comparison to current methods such as karyotyping and fluorescent in situ hybridization using invasive sampling and other appropriate characteristic such as detectability in early pregnancy.[15654] However, due to the scarcity of cff-nucleic acid and mixing with maternal background, all stages including sampling, plasma preparation, nucleic acid extraction, cff-nucleic acid differentiation, and detection strategies need standardization before definite clinical usage. The most challenging step is differentiation and detection of cff-nucleic acid in maternal plasma. Many studies on various aspects of cff-nucleic acid have been performed, but regarding method standardization and improvement in performance and subsequent results, the prior obtained results need to be reviewed and compared. This study provided a relatively comprehensive source on cff-nucleic acid markers and the advantages and disadvantages for interested investigators. Due to potential application of cff-nucleic acid in diagnostic field, this data collection can help to develop these diagnostic tests [Figure 2].

Figure 2

Schematic diagram representative various cff-nucleic acid identifier markers. *Those alleles present on the paternal genome and absent from the maternal genome could potentially serve as a marker to confirm the presence of fetal DNA

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Conflicts of interest

There are no conflicts of interest.