Human Reproduction Update

Human Reproduction Update Advance Access originally published online on October 22, 2008
Human Reproduction Update 2009 15(1):139-151; doi:10.1093/humupd/dmn047

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The use of cell-free fetal nucleic acids in maternal blood for non-invasive prenatal diagnosis

Caroline F. Wright¹ and Hilary Burton

PHG Foundation Strangeways Research Laboratory, 2 Worts Causeway, Cambridge CB1 8RN, UK

¹ Correspondence address. Tel: +44-1223-740200; Fax: +44-1223-740892; E-mail: caroline.wright{at}phgfoundation.org

	Abstract

TOP Abstract Introduction Materials and Methods Conclusion Funding References

 
BACKGROUND: Cell-free fetal nucleic acids (cffNA) can be detected in the maternal circulation during pregnancy, potentially offering an excellent method for early non-invasive prenatal diagnosis (NIPD) of the genetic status of a fetus. Using molecular techniques, fetal DNA and RNA can be detected from 5 weeks gestation and are rapidly cleared from the circulation following birth.

METHODS: We searched PubMed systematically using keywords free fetal DNA and NIPD. Reference lists from relevant papers were also searched to ensure comprehensive coverage of the area.

RESULTS: Cell-free fetal DNA comprises only 3–6% of the total circulating cell-free DNA, therefore diagnoses are primarily limited to those caused by paternally inherited sequences as well as conditions that can be inferred by the unique gene expression patterns in the fetus and placenta. Broadly, the potential applications of this technology fall into two categories: first, high genetic risk families with inheritable monogenic diseases, including sex determination in cases at risk of X-linked diseases and detection of specific paternally inherited single gene disorders; and second, routine antenatal care offered to all pregnant women, including prenatal screening/diagnosis for aneuploidy, particularly Down syndrome (DS), and diagnosis of Rhesus factor status in RhD negative women. Already sex determination and Rhesus factor diagnosis are nearing translation into clinical practice for high-risk individuals.

CONCLUSIONS: The analysis of cffNA may allow NIPD for a variety of genetic conditions and may in future form part of national antenatal screening programmes for DS and other common genetic disorders.

Key words: cell-free fetal DNA (cffDNA) / non-invasive prenatal diagnosis (NIPD) / cell-free fetal nucleic acids / prenatal diagnosis / genetic diagnosis

	Introduction

TOP Abstract Introduction Materials and Methods Conclusion Funding References

 
Prenatal screening and diagnosis are routinely offered in antenatal care and are considered to be important in allowing women to make informed choices about the continuation of pregnancies affected by genetic conditions. Prenatal testing is now part of established obstetric practice in many countries, although currently genetic abnormality accounts for only 1% of terminations in the UK (Human Genetics Commission, 2004).

Prenatal testing falls into two categories: first, prenatal ‘screening’ is offered to all pregnant women as part of routine antenatal care to determine if their baby is at significant risk of having a particular disorder, such as Down syndrome (DS) or sickle cell disease; and second, in cases deemed to be high risk, prenatal ‘diagnosis’ is offered, which aims to provide a definitive diagnosis of a particular disorder the baby might have. Diagnostic testing currently requires removal of a sample of fetal cells directly from the uterus for genetic analysis, using either chorionic villus sampling (CVS) between 11 and 14 weeks gestation or amniocentesis after 15 weeks. However, these invasive procedures carry a risk of miscarriage of around 1% (Mujezinovic and Alfirevic, 2007).

Although this approach to obtaining fetal DNA currently provides the gold standard test for prenatal diagnosis, many women decide not to undergo invasive testing, either because it is unpleasant and carries a small but significant risk of miscarriage or because they would not terminate the pregnancy irrespective of the results. Of the 700 000 pregnant women per year in the UK, around 30 000 amniocentesis and 8000 CVS tests were conducted in the period 2002–2003 (Human Genetics Commission, 2004), resulting in an estimated 460 miscarriages of potentially healthy fetuses.

A reliable and convenient method for non-invasive prenatal diagnosis (NIPD) has long been sought to reduce this risk of miscarriage and allow earlier testing. Although some work has investigated using fetal cells obtained from the cervical mucus (Fejgin et al., 2001; Mantzaris et al., 2005), most research has focused on strategies for detecting genetic elements from the fetus present in the maternal circulation.

	Materials and Methods

TOP Abstract Introduction Materials and Methods Conclusion Funding References

 
We searched PubMed up to August 2008 for relevant papers in English using the key words free fetal DNA and NIPD. Reference lists from the papers were also searched.

Cell-free fetal nucleic acids

In contrast to popular belief that the placenta forms an impermeable barrier between mother and child, there is bidirectional traffic between the fetus and the mother during pregnancy (Lo et al., 1996). Multiple studies have shown that both intact fetal cells and cell-free fetal nucleic acids (cffNA) cross the placenta and circulate in the maternal bloodstream.

Intact fetal cells circulating in maternal blood present an attractive target for NIPD, particularly for the diagnosis of fetal sex and chromosomal abnormalities by simple karyotyping. Although the existence of fetal cells in maternal blood has been known for more than a century, isolation of intact fetal nucleated red blood cells for the purpose of prenatal diagnosis was not achieved until 1990 (Bianchi et al., 1990). Since then, the isolation and detection of fetal cells from maternal blood has been extensively investigated (Bianchi, 1999; Jackson, 2003), and various methods of fetal cell enrichment developed, with varying degrees of success (Sekizawa et al., 2007). However, results to date have been disappointing, due to the scarcity of intact fetal cells in the maternal circulation (around one cell per ml of maternal blood) (Bianchi et al., 1997), low efficiency of enrichment (Bianchi et al., 1997) and difficulties with chromosomal analysis associated with abnormally dense nuclei in some cells (Babochkina et al., 2005). Although certain fetal cells (specifically nucleated red blood cells) have a relatively short lifespan in maternal blood (Lurie and Mamet, 2000), other fetal cell types can persist in the maternal circulation for decades following pregnancy (Bianchi et al., 1996), potentially causing false-positive results in subsequent pregnancies. Therefore, although research into sophisticated cell sorting techniques is ongoing, the majority of recent research has focused on cell-free fetal DNA (cffDNA) in the maternal blood, of which there is significantly more present (by a factor of almost 1000) (Lo et al., 1998b).

Although the vast majority of human DNA is located inside cells, the presence of small amounts of extracellular DNA in the circulation of both healthy and diseased subjects was discovered in 1947 (Mandel and Metais, 1948). Although its biological source and potential function remain uncertain (Stroun et al., 2000), it is thought to be a product of apoptosis (programmed cell death), resulting in fragmentation and ejection of chromosomal DNA from the cell (Jahr et al., 2001). An increased concentration of circulating cell-free DNA in the serum of cancer patients was reported in 1977 (Leon et al., 1977), but it was not until 1997 that the presence of fetal DNA in the maternal circulation was demonstrated by Lo et al. (1997).

Fetal DNA originates from apoptotic placenta cells (trophoblasts) derived from the embryo (Tjoa et al., 2006; Alberry et al., 2007) and comprises around 3–6% of the total cell-free DNA in maternal circulation during early and late pregnancy, respectively (Lo et al., 1998b) (the other 94–97% being maternal cell-free DNA). Unlike cellular DNA, circulating cffDNA consists predominantly of short DNA fragments rather than whole chromosomes, of which 80% are <193 base-pairs in length (Chan et al., 2004). Fetal DNA can be detected from the 4th week of gestation (Illanes et al., 2007), though only reliably from 7 weeks, and the concentration increases with gestational age—from the equivalent of 16 fetal genomes per millilitre of maternal blood in the first trimester to 80 in the third trimester (Birch et al., 2005)—with a sharp peak during the last 8 weeks of pregnancy (Lo et al., 1998b; Birch et al., 2005). In contrast to fetal cells, cffDNA is rapidly cleared from the maternal circulation with a half life of 16 min and is undetectable 2 h after delivery (Lo et al., 1999c). Although a study in 2002 indicated that cffDNA could be detected in maternal plasma decades after pregnancy (Invernizzi et al., 2002), this finding has not been repeated and has been attributed to contamination with fetal cell DNA due to the particular extraction method used (Tomaiuolo et al., 2007). Note that cffDNA may also be detectable for several days following termination of a pregnancy (Wataganara et al., 2004a).

Additionally, cell-free fetal mRNA (cffRNA) has also been detected in the maternal circulation in 2000 (Poon et al., 2000), derived from genes that are uniquely actively expressed in the placenta. Although the majority of work to date has focused on cffDNA, both types of cffNA, i.e. DNA and RNA, could potentially be used for the NIPD of specific genetic characteristics of the fetus.

Detection of cffDNA

Distinguishing—or ideally isolating—fetally derived cell-free DNA in an overwhelming background of maternal cell-free DNA is a significant technical challenge. There are a number of general problems associated with detecting cffDNA in the maternal circulation:

1. the concentration of all cell-free DNA in blood is relatively low;
   
2. the total amount of cell-free DNA varies between individuals;
   
3. cffDNA molecules are outnumbered 20:1 by maternal cell-free DNA molecules;
   
4. the fetus inherits half its genome from the mother.
   

A number of methods have therefore been developed to address these problems. Initially, the cell-free DNA must be separated from the rest of the blood. Once a sample of maternal blood has been taken, the plasma (or serum) fraction is separated from cellular matter by centrifugation. This is followed by isolation and purification of all cell-free DNA. The yield of cell-free DNA varies with different handling procedures (Chiu et al., 2001; Randon et al., 2003) and is a limiting factor for fetal genotyping from maternal blood. Although methodologies currently vary, a recent workshop was held to evaluate a number of different protocols and has subsequently initiated a standardization process for extraction of cffDNA from maternal plasma (Legler et al., 2007).

Once the cell-free DNA fragments have been purified, small differences between the fetal and maternal DNA sequences are exploited in order to make a specific fetal diagnosis. To date, the majority of studies have focused on the detection of paternally inherited sequences that are entirely absent from the maternal genotype, such as those on the Y chromosome of male fetuses. This target is particularly attractive as it comprises a large portion of DNA which is not otherwise present in women. Variable regions of repeated DNA (short tandem repeats or STRs) can be used to identify paternally inherited sequences, by comparing the number of repeats present with the paternal and maternal sequences. Paternal alleles on the autosomal chromosomes can also be detected, if they are known to be absent in the maternal genome, but this requires detailed sequence knowledge of the paternal genotype of interest, as well as detection methods that can distinguish DNA sequences that might only differ by a single nucleotide. Importantly, all these methods rely upon the fetus inheriting a uniquely paternal sequence that is conveniently located for a particular diagnosis.

The most common technique currently used for detection and identification of specific cffDNA sequences is polymerase chain reaction (PCR). A number of different types of PCR have been explored, of which the most popular is real-time quantitative PCR (Traeger-Synodinos, 2006), as it combines high sensitivity with a closed detection system, thereby minimizing the risk of contamination. Nested PCR (Al-Yatama et al., 2001) and pyrophosphorolysis-activated polymerization PCR (Boon et al., 2007) have also been investigated in this context. Higher precision of measurement may be possible through the application of digital PCR, which allows the exact number of original template DNA molecules to be counted (Fan and Quake, 2007; Lo et al., 2007a). Another sensitive technique that has recently been applied to the identification of cffDNA is mass spectrometry (Ding et al., 2004), in which the precise mass of each DNA fragment is analysed to determine the genetic sequence, and hence detect fetal-specific alleles that differ from the maternal sequence by as little as a single base.

As with all tests, the accuracy and reliability of detection can be significantly improved by increasing the signal-to-noise ratio. There are two alternative methods specifically aimed at increasing the proportion of fetal DNA relative to maternal DNA in the sample:

1. Selective enrichment of fetal DNA, based on a difference in the average physical length of fetal and maternal DNA fragments, which can be exploited to increase the relative amount of cffDNA. Fetally derived DNA fragments are generally smaller than those that are maternally derived, being predominantly <313 base-pairs in length (Chan et al., 2004). Therefore, by using standard size fractionation to select only DNA fragments <300 base-pairs, circulating cffDNA can be enriched such that it comprises around 70% of the total cell-free DNA (Li et al., 2004b), prior to detection and identification by either PCR (Li et al., 2005) or mass spectrometry (Li et al., 2006).
   
2. Suppression of maternal DNA by the addition of formaldehyde (Dhallan et al., 2004), a chemical that is thought to stabilize intact cells, thereby inhibiting further release of maternal DNA into the sample and increasing the relative proportion of fetal DNA. However, the use of formaldehyde for this purpose is controversial, as the relative enrichment of fetal DNA from this process is highly irregular; reports suggest a wide range of results, varying from 5% to 96% of the total cell-free DNA (Benachi et al., 2005), and the repeatability of the published protocol has been called into question by several studies (Chinnapapagari et al., 2005; Chung et al., 2005).
   

Universal fetal markers

A major area of current research is aimed at finding universal fetal-specific markers that could be used either as diagnostic tests in their own right or to confirm and quantify the presence of fetal DNA independent of sex or other specific diagnostic tests. Universal fetal markers could be used alongside clinically relevant diagnostic tests as a positive control for the presence of cffDNA, in order to highlight false-negative results either caused by low levels of circulating DNA below the detection limit of the test or problems with the DNA extraction process.

One method under investigation is the detection of specific DNA sequences located on the autosomal chromosomes that can be shown to be paternally inherited, including:

1. Single nucleotide polymorphisms (SNPs), or point mutations, which differ between the maternal and paternal genomes but may not be directly linked to a specific disease. Although there are a number of studies confirming that this method of detection is possible (Li et al., 2006; Dhallan et al., 2007), it relies upon selective enrichment of the cffDNA followed by analysis by a highly sensitive technique such as mass spectrometry, as the maternal and fetal genotypes in question only differ by a single base-pair making them challenging to distinguish. It also relies upon finding SNPs which differ between the maternal and paternal (and therefore fetal) genomes.
   
2. Polymorphic segments of DNA that vary between the maternal and paternal genomes, such as STR sequences. Because of the highly variable nature of STRs, most people will possess two alleles (versions) of each—one inherited from each parent—with a different number of repeats. Therefore, the paternally inherited fetal STR sequence will differ in the number of repeats from the maternal sequence. Amplification of these STR sequences will therefore result in two major products corresponding to the maternal alleles (and the maternally inherited fetal allele) and one minor product corresponding to the paternally inherited fetal allele. This technique was first reported in 2000 (Pertl et al., 2000) and has subsequently been developed using simultaneous detection of multiple different STR regions using real-time PCR (Liu et al., 2007). A similar technique has been applied to insertion-deletion alleles (Page-Christiaens et al., 2006). However, the technique has yet to be optimized for clinical diagnostic use and the sensitivity and specificity have not been established.
   

Another method for identifying fetal DNA makes use of differences in gene activation between the mother and growing fetus. There are several biological mechanisms that can be exploited for this purpose:

1. Epigenetic modifications, specifically DNA methylation of certain genes, which differs between cells of the mother versus the growing fetus—e.g. genes that are important for growth and development may be methylated (silenced) in adults, but unmethylated (active) in the developing fetus. In these cases, fetal alleles can be distinguished from maternal alleles by analysing differences in DNA modification patterns, using either methylation sensitive PCR or bisulphate sequencing (Poon et al., 2002). Recent work has revealed that the promoter region of two tumour suppressor genes—maspin (Chim et al., 2005) and RASSF1A (Chan et al., 2006b)—are differentially methylated in the placenta (and DNA from cells derived therein) relative to maternal cells, providing the first truly universal markers for fetal DNA.
   
2. Detection of mRNA derived from genes that are uniquely active in the placenta or fetus. The presence of male fetal RNA in the maternal circulation was first demonstrated in 2000 using RNA derived from the Y chromosome of male fetuses (Poon et al., 2000) originating from the placenta (Ng et al., 2003). Subsequently, fetal transcriptomics has been used to identify numerous fetal/placental RNA species from the autosomal chromosomes, which are unique to the fetus and detectable in maternal plasma (Oudejans et al., 2003; Go et al., 2004; Tsui et al., 2004; Tsui and Lo, 2006). Like cffDNA, cffRNA is detectable within the maternal circulation early on in the first trimester and is rapidly cleared following birth, with a half-life of 14 min (Chiu et al., 2006). Since the expression of certain genes is unique to pregnancy, detection of placental/fetal RNA is an extremely promising avenue for research, as it is relatively easy to isolate completely from background maternal RNA.
   
3. Detection of proteins derived from genes that are uniquely expressed in the placenta or fetus. Placentally expressed genes may result in potentially diagnostic fetal proteins in the maternal bloodstream (Avent et al., 2008). However, the study of fetal proteomics is currently only just beginning, with the primary aim of improving the panel of serum markers used in screening for DS, and is outside the scope of this review.
   

Further work is needed to select and refine the best method for universal detection of fetal nucleic acids from the maternal circulation.

Clinical applications

Owing to the difficulties with isolating cffDNA from the maternal circulation, its diagnostic potential at present is limited to known differences between fetal and maternal genomes. It is important to emphasize that complete fetal genotyping is not conceivable using cffDNA in the maternal circulation and that the genetic information derived from cffDNA is entirely restricted to the specific DNA sequence (or chromosome) detected. However, given modern high-throughput technologies, such as DNA microarrays (Ge et al., 2006), thousands of different sequences could potentially be detected simultaneously if required for multiple diagnoses.

There are a number of quite discrete clinical applications of cffNA analysis in prenatal screening and/or diagnosis, based on distinct and detectable differences between fetal and maternal genomes:

1. sex determination—by detecting cffDNA sequences on the Y chromosome;
   
2. single gene disorders—by detecting a paternally inherited allele in cffDNA;
   
3. pregnancy-related disorders—by detecting either the presence of a working copy of the Rhesus gene or an elevation in the absolute concentration of cffDNA;
   
4. aneuploidy—by detecting an abnormal concentration of a particular chromosome, potentially using cffRNA specific to the fetus and chromosome of interest.
   

Clinically, these applications separate into diagnosis of fetal sex and certain single gene disorders by medical specialists in high-risk families, and screening of pregnant women for Rhesus factor and DS/aneuploidy as part of routine antenatal care. A summary of the cell-free nucleic acid sequences that have been detected from maternal plasma or serum for the purposes of NIPD in each of these categories is given in Table I.

View this table: [in this window] [in a new window]   Table I Summary of nucleic acid sequences that have been detected in maternal blood for the purposes of NIPD (see text for details and references)

 
There are a number of technical and clinical obstacles to achieving high diagnostic accuracy in any of these applications. False negatives can be the result of failure to extract or detect sufficient material, due to individual variability in the amount of total cell-free DNA and the small proportion of fetal versus maternal cell-free DNA; false positives can be the result of either technical issues, such as contamination, or clinical abnormalities such as the presence of a non-identical vanishing twin. Therefore, extensive clinical trials will be required for each application to evaluate both the analytical and clinical validity before this technique could be used reliably in a clinical setting. The clinical utility of the technique and its socio-ethical implications will vary enormously with the application, and a full evaluation will be needed in order to ensure feasibility of delivery, equity of access and value to patients.

Fetal sex

Owing to the relative ease with which the Y chromosome of a male fetus can be distinguished from maternal DNA, the most common clinical application of NIPD using cffDNA to date is fetal sex determination, predominantly in cases where a male fetus is at risk of a sex-linked disease, such as haemophilia or Duchenne muscular dystrophy. Although each disease is individually relatively rare, it has been estimated that their cumulative incidence is around 5 in 10 000 live births (Baird et al., 1988). Determination of fetal sex non-invasively could reduce the number of invasive diagnoses required for each specific disease by half, with the particular advantage of sparing most female fetuses from unnecessary invasive diagnostic testing. Sex determination is also important in cases where development of external genitalia is ambiguous and in some endocrine disorders, such as congenital adrenal hyperplasia, where there is masculinization of the female fetus, which is preventable with antenatal treatment.

The majority of studies using cffDNA for sex determination use real-time PCR to detect genes on the Y chromosome of male fetuses; most detect the sex-determining region Y (SRY), although a number of other Y chromosome specific sequences present in multiple copies per male genome have also been investigated [particularly DYS (Honda et al., 2002; Zimmermann et al., 2005; Deng et al., 2006), DYZ (Honda et al., 2001) and DAZ (Stanghellini et al., 2006)]. This method is potentially prone to false-negative results, as female fetuses are not detected directly but inferred by a negative result for the Y chromosome, which could also be caused by undetectable levels of cffDNA. A number of studies have tried to address this issue by testing the total amount of cell-free DNA extracted, detection of the paternally inherited X chromosome (Tang et al., 1999; Zhu et al., 2005) or by simultaneously testing for autosomal universal fetal markers as a positive control for the presence of cffDNA (Chim et al., 2005; Liu et al., 2007). In contrast, false-positive tests can be significantly minimized by controlling for contamination with non-fetal male DNA.

There are multiple studies which describe the use of cffDNA to determine fetal sex, including several large studies specifically looking at test accuracy. Although in principle NIPD of sex using cffDNA is applicable to any sex-linked disease, to date it has been published within the context of haemophilia, Duchenne muscular dystrophy, X-linked mental retardation, adrenoleukodystrophy, Alport’s syndrome, X-linked severe immunodeficiency, retinitis pigmentosa, X-linked hydrocephalus, anhidrotic ectodermal dysplasia, Hunter’s syndrome, Menke’s syndrome and Lesch–Nyhan syndrome (Costa et al., 2002), as well as some endocrine disorders including congenital adrenal hyperplasia (Chitty et al., 2007). Two representative studies include an Italian study published in 2005 in which the SRY sequence was detected (Galbiati et al., 2005) and a Japanese study published in 2001 in which the DYS14 sequence was detected (Sekizawa et al., 2001). In the former study, the accuracy of fetal sex prediction (i.e. the proportion of cases in which fetal sex was correctly predicted) was 99.4%, 97.8% and 100% in the first, second and third trimesters, respectively, and SRY only failed to be detected in 7 out of 246 male pregnancies (Galbiati et al., 2005). The latter study focused on fetal sex determination at 7–16 weeks gestation and achieved a test sensitivity of 97.2% and a specificity of 100%, with a positive predictive value of 100% and a negative predictive value of 97.5% (Sekizawa et al., 2001). Within the UK testing laboratories, 97.6% accuracy was achieved in tests performed at 7 weeks gestation or more between March 2006 and April 2007 (Chitty et al., 2007).

It has recently been shown that some false positives are due to the presence of a vanishing (male) twin (L. Chitty, personal communication), though this is only expected to cause a false positive result in around 0.3–0.7% of cases (Gail Norbury, personal communication). In order to reduce this problem, it has been suggested that all cffDNA testing for fetal sex should be accompanied by an ultrasound scan, which could be done early in pregnancy, as loss of the twin usually occurs in the first 7 weeks (Landy and Keith, 1998). The identification of a vanishing twin on ultrasound could make the results of a cffDNA test ambiguous in around 1 in 200 women. In those women with a vanishing twin, but a negative test result (0.66% * 0.25% = 0.17%) due to both the fetus and vanishing twin being female, the technique is still applicable. Therefore, only a positive result is ambiguous (0.66% * 0.75% = 0.5% of the cases), accounting for around 1 in 200 women.

The accuracy of this technique is continually improving, as laboratories hone and refine DNA extraction and detection, the limits of testing are being defined and the clinical aetiology of false results are being discovered. A recent summary of 23 selected studies, most of which use the real-time PCR analysis of the SRY gene, highlighted that most achieved specificities of 100% (Avent and Chitty, 2006), which suggests that although false-positive results are preventable, false-negative results due to failure to detect the Y chromosome sequence may still be problematic. Together, these studies confirm that sex determination from cffDNA is not only feasible, but also more accurate than using ultrasound in the first trimester (Avent and Chitty, 2006).

Single gene disorders

Thousands of human diseases are caused by mutations in just a single gene, and it has been estimated that their combined occurrence is around 3.6 in 1000 live births (Baird et al., 1988). Prenatal diagnosis of single gene disorders using invasive techniques is an accepted part of clinical practice and performed through a clinical geneticist when there is a family history of a particular disease. However, the detection of fetal point mutations from cffDNA is extremely technically challenging, due to the predominance of very similar maternal DNA sequences.

First, despite selective enrichment of fetal DNA prior to sequence detection, detection of fetal point mutations on the autosomal chromosomes is severely hindered by the predominance of maternal DNA sequences. Furthermore, the enrichment of cffDNA means that it is impossible to calculate the dosage (i.e. number) of any homozygous mutations. Therefore, diagnosis remains limited to the detection of alleles not already present in the mother. Second, detection of large-scale mutations (e.g. disorders caused by expansion, insertion or duplication of sequences) is restricted to sequences <300 base-pairs in length, due to the fragmented nature of ffDNA (Norbury and Norbury, 2008). In addition, like testing for fetal sex, using cffDNA to test for paternally inherited single gene disorders should ideally be coupled with an ultrasound scan to avoid false-positive results caused by a vanishing (affected) twin.

Diagnosis of dominant diseases that are paternally inherited (or occur de novo as a result of spontaneous mutations arising during oocyte or sperm formation) is possible due to the absence of the disease causing allele in the maternal genome. To date, the use of cffDNA for NIPD has been published for this following dominant single gene disorders in at least one pregnancy.

• Huntington’s disease, a dominant adult-onset neurological disease, caused by an expansion in the number of copies (>36) of a three base-pair repeated sequence in the HD gene on chromosome 4 which is thought to play an important role in nerve cells. A paternally inherited expansion of 37 repeats has been detected using cffDNA (Gonzalez-Gonzalez et al., 2003).
  
• Achondroplasia, a dominant form of dwarfism, caused by point mutations in the FGFR3 gene on chromosome 4 which is involved in cartilage formation. One specific point mutation which accounts for more than 98% of the cases has been detected using cffDNA (Saito et al., 2000; Li et al., 2004a, 2007a).
  
• Myotonic dystrophy, a dominant adult-onset form of muscular dystrophy (muscle wasting disease), caused by an expansion in the number of copies (50–5000) of a three base-pair repeated sequence in the DMPK gene on chromosome 19 involved in skeletal muscle. A paternally inherited expansion of 70 repeats has been detected using cffDNA (Amicucci et al., 2000).
  

Autosomal recessive diseases are significantly harder to diagnose, as there is currently no way to distinguish between identical maternal and paternal alleles. Therefore, cffDNA can only be used to determine the carrier status of the fetus in compound heterozygotes, i.e. through detection of a paternally inherited disease allele in cases where multiple disease alleles are known and the maternal and paternal inherited allele differ. This information could be used to reduce the number of invasive diagnoses required, either by increasing the risk of a diseased fetus (from one in four to one in two), or by determining that the fetus has not inherited the paternal disease allele and therefore cannot have the disease.

To date, fetal carrier status has been determined using cffDNA in the following recessive single gene disorders in at least one pregnancy.

• Cystic fibrosis, a recessive, progressive disabling disease mainly of the lungs caused by various mutations in the CFTR gene on chromosome 4 which encodes an ion channel important for mucus production. Since over 1000 mutations are linked with cystic fibrosis, specific paternally inherited point mutations not present in the maternal genome have been detected using cffDNA (Gonzalez-Gonzalez et al., 2002; Nasis et al., 2004).
  
• Haemoglobinopathy, a group of recessive blood disorders with various geographic distributions, caused by various mutations in the globin genes on chromosome 11 resulting in changes to either the structure or production of haemoglobin, leading to chronic anaemia (Johnston, 2005). Specifically, cffDNA has been used to detect paternally inherited mutations that cause β-thalassemia (Chiu et al., 2002b; Fucharoen et al., 2003), both in maternal carriers of a different β-thalassemia mutation or the sickle cell mutation (leading to sickle β-thalassemia disease), and Hb Lepore (Lazaros et al., 2006). Although the most common haemoglobinopathy, sickle cell anaemia, has been diagnosed prenatally using DNA extracted from fetal cells in the maternal circulation (Cheung et al., 1996), because the disease is caused by two identical copies of a single point mutation, it is not yet possible to use cffDNA for diagnosis.
  
• Congenital adrenal hyperplasia (21-hydroxylase deficiency), a group of recessive conditions resulting in increased production of androgens (male sex hormones), caused by various mutations in the CYP21 gene on chromosome 6. One strategy for NIPD of 21-hydroxylase deficiency is the detection of a paternally derived normal allele in high-risk pregnancies by using polymorphic STRs associated with the gene (Chiu et al., 2002a). An alternative strategy also exists as a result of the fact that, unusually for an autosomal disease, the excess of androgens has no effect on male fetuses but may cause sterility in female fetuses. Therefore, fetal sex determination using cffDNA can be used to stratify pregnancies at risk of the disease (Rijnders et al., 2001; Bartha et al., 2003), followed by prenatal treatment of female fetuses with steroid hormones.
  

The list of single-gene disorders for which cffDNA could potentially be used for prenatal diagnosis is substantially longer than the list of diseases presented here, to which the technique has already been applied. However, it is believed that the proportion of recessive diseases caused by compound heterozygosity is low, and it should be noted that the technique is currently not applicable to the most common disease to be diagnosed through invasive prenatal testing, namely sickle cell anaemia, which is caused by a single homozygous point mutation.

Pregnancy-related disorders

There are a number of pregnancy-related disorders that can be inferred by using cffDNA as a marker for fetal–maternal well-being. These can be broadly subdivided into two categories, both in terms of the underlying pathology and the proposed diagnostic method:

1. fetal Rhesus blood group, by detection of a fetal Rhesus antigen gene;
   
2. abnormal formation and functioning of the placenta, causing elevation of the cffDNA concentration, which could be used as a diagnostic marker.
   

Rhesus blood group
The Rhesus blood group system refers to the five main Rhesus antigens (C, c, D, E and e) on the surface of red blood cells. The RhD antigen, or Rhesus factor (RhD), is by far the most common and is usually indicated by a positive or negative suffix to the ABO blood type. Whether a person is RhD positive or negative is determined genetically by the dominant RHD gene. If fetal cells carrying paternally inherited RhD antigens enter the maternal circulation as a result of fetal–maternal bleeding (e.g. during birth or amniocentesis), in most cases, an RhD negative mother will produce an immune response against the fetus. These maternal antibodies can pass through the placenta and initiate the destruction of RhD positive red blood cells, putting the fetus at risk of haemolytic disease of the newborn (HDN), a potentially fatal disease characterized by anaemia. This is usually not a problem during the first pregnancy, as sufficient intact fetal blood cells are rarely exchanged with the maternal circulation. However, at birth, there is a significant exchange of blood, resulting in the production of antibodies that can persist and cross the placenta in later pregnancies, worsening the chance of severe haemolytic disease with each successive RhD positive pregnancy.

The RhD antigen is present in over 80% of the Caucasian population (although this figure varies across ethnic groups) (Wagner et al., 1995; Daniels, 2005) and accounts for the majority of cases of maternal immunization (Avent and Reid, 2000). Currently, this immune response is largely treated prophylactically by maternal injection of anti-D antibodies, administered during the third trimester and immediately following birth, which bind and neutralize fetal RhD antigens. Prior to the introduction of anti-D prophylaxis, HDN due to RhD incompatibility affected around 5% of children born to RhD negative women in Caucasian populations; currently the figure is closer to 0.5% (Pilgrim et al., 2007). However, it has been estimated that in the Caucasian population, 40% of the RhD negative women receive unnecessary antenatal anti-D prophylactically while carrying an RhD negative child (Van der Schoot et al., 2006).

Minimizing the prophylactic use of anti-D is considered to be desirable as it is a theoretical source of infection since it is harvested from blood donors. Although the determination of fetal RhD status can be achieved by amniocentesis or CVS, both these methods themselves carry a risk, not only of miscarriage, but also of maternal sensitization to RhD due to mixing of fetal and maternal blood. Non-invasive prenatal genetic determination of fetal RhD status in RhD negative mothers could provide a solution to this problem and was first shown to be feasible in 1998 (Lo et al., 1998a), since when it has been extensively developed and widely applied.

Were NIPD to be offered to all RhD negative pregnant women, a very high-throughput platform would be required; for example, there are 100 000 RhD negative pregnancies annually in the UK alone (Pilgrim et al., 2007). The majority of NIPD studies of Rhesus factor using cffDNA use a quantitative real-time PCR (Geifman-Holzman et al., 2006) to identify the presence of the RHD gene in RhD negative mothers. However, research is ongoing into using mass-spectroscopy, which is inherently a much higher throughput technique, but requires significant capital investment as most laboratories do not currently own a mass spectrometer.

Since the RHD gene is deleted in the majority of RhD negative Caucasians, a paternally derived fetal RHD gene can be specifically amplified and detected with high levels of accuracy. Usually, fetal SRY is also amplified to ensure that the assay is working correctly; however, this only works in male pregnancies and inclusion of a universal fetal marker would clearly be desirable.

Numerous and extensive studies have been published regarding the determination of fetal RhD status from cffDNA. A recent meta-analysis published in 2006 was performed on 37 English-language publications including 44 protocols for non-invasive fetal RHD genotyping from maternal blood (including using fetal cellular DNA as well as cffDNA) (Geifman-Holzman et al., 2006). This study found that the overall test sensitivity and specificity were 95.4% and 98.6%, respectively. cffDNA extracted from either maternal serum or plasma gave the most accurate diagnosis of fetal RHD status (96.1% and 96.5%, respectively), with the highest diagnostic accuracy in the first trimester (Geifman-Holzman et al., 2006). Problematic false negatives were attributed to failure to detect the presence of fetal DNA, which can be addressed by concurrently utilizing other methods for fetal DNA detection as an internal control (e.g. SRY in male pregnancies); false positives were considered to be less critical as the administration of needless anti-D therapy is usually harmless and has been the mainstay of management of this condition in the absence of cffDNA technologies. There were 16 studies that reported 100% diagnostic accuracy in fetal RHD genotyping.

More recently, a number of large studies have been published reporting extremely high levels of accuracy (Table II), including a 4-year prospective study of 563 pregnant women in Belgium, which achieved 99.8% accuracy in RhD genotyping (with a single false-positive result in an organ transplant recipient), which led to modified management of RhD negative pregnant women (Minon et al., 2008). The largest study to date is a British study of 1997 previously sensitised RhD negative women at high risk of HDN, which achieved 95.7% accuracy, with only 1% false results and the rest being inconclusive (Finning et al., 2008). The authors state that if these results had been applied as a guide to treatment, only 2% of the women would have received anti-RhD unnecessarily, compared with 38% without the genotyping.

View this table: [in this window] [in a new window]   Table II Summary of recent large-scale studies of NIPD of fetal RhD status in RhD-negative pregnant women

 
The Rhesus system is highly diverse involving multiple similar genes with around 50 known variants (Avent, 1999). While in most RhD negative Caucasians, there is complete deletion of the RHD gene, in other populations such as Asians, Japanese and black Africans, the negative phenotype is associated with numerous smaller genetic (Minon et al., 2008) variations including point mutations and small DNA insertions (Avent and Reid, 2000). In such cases, a serologically RhD negative mother will test positive for the RHD gene. This problem has previously been addressed by including tests for the most common RHD polymorphisms (Singleton et al., 2000), as well as concurrent maternal RHD genotyping in phenotypically RhD negative women, to determine the presence of non-functional RHD genes (Grootkerk-Tax et al., 2006). Since test sensitivity must be high, as a false-negative result due to low concentrations of cffDNA could result in lack of potentially life saving treatment, knowledge of the ethnic group of both parents may be helpful in the selection of appropriate genotyping tests. As a result, the cffDNA test from the International Blood Group Reference Laboratory in the UK includes a test for the RHD gene, which is the most common African variant, accounting for 66% of phenotypically RhD negative black Africans (Finning et al., 2008).

Other rarer blood cell antigens can also cause HDN in mothers who are negative for the corresponding antigen, in a manner analogous to RhD maternal immunity. The presence of these antigens in the fetus could be determined prenatally using cffDNA, again reducing the unnecessary use of prophylactic antibodies. An initial study has recently been published using cffDNA in maternal plasma to diagnose the presence of blood cell antigens K (Kell), Rhesus C, c and E as well as ABO blood group in mothers whose blood cells lack the respective antigen (Finning et al., 2007; Meng et al., 2007).

Abnormal placentation
Various problems associated with placental growth and development result in altered levels of cffDNA. Elevated concentrations of cffDNA have been specifically detected in numerous pregnancy- related disorders associated with problems in the placenta (Bischoff et al., 2005; Hahn et al., 2005; Bauer et al., 2006), of which the most well-described is preeclampsia.

Preeclampsia is a serious condition that occurs in around 5–10% of pregnancies, typically any time from 20 weeks gestation until 6 weeks after the birth, characterized by high blood pressure and the presence of protein in the urine. Preeclampsia is the leading cause of premature birth and the most common dangerous complication of pregnancy. If allowed to progress undetected and develop into eclampsia, it is life-threatening to both the mother and child. Although preeclampsia stems from a defective placenta, the underlying cause is unknown and it has no known cure apart from ending the pregnancy. As preeclampsia is usually asymptomatic in the earlier stages, diagnosis depends upon blood pressure and urine measurements. Since it was first reported in 1999 (Lo et al., 1999b), numerous studies have shown conclusively that the level of cffDNA (usually measuring Y chromosome DNA of male pregnancies) is elevated by 2–3-fold before the onset of preeclampsia and 2–14-fold during preeclampsia (Hahn et al., 2005).

In addition to preeclampsia, a number of other pregnancy-related disorders have been linked to an elevated concentration of cffDNA. These include preterm labour, hyperemesis gravidarum (severe morning sickness), invasive placentation (in which the placenta contacts the maternal bloodstream), intrauterine growth restriction, feto-maternal haemorrhage and polyhydramnios (too much amniotic fluid).

It has been proposed that cffDNA could be useful as an additional screening variable to predict preeclampsia in low-risk asymptomatic patients (Farina et al., 2004). However, the absolute level of circulating cffDNA fluctuates over short periods throughout pregnancy (Hahn et al., 2001) and varies with both ethnicity (Gerovassili et al., 2006) and maternal weight (Wataganara et al., 2004b), raising important questions about the diagnostic utility of adding cffDNA concentration to the current panel of biomarkers.

Aneuploidy

Aneuploidy refers to a change in the number of chromosomes present inside a cell. This is often caused by non-disjunction in meiosis during gamete production. Although relatively common in developing embryos, most aneuploidies lead to spontaneous miscarriage early in pregnancy; in 1988, it was estimated that chromosomal abnormalities were present in around 1.8 per 1000 live births (Baird et al., 1988). Most of the disorders present with clinical features including physical abnormalities, sterility and learning disability caused by an increased (or decreased) gene dosage as a result of gaining (or losing) a particular chromosome. Milder symptoms are associated with mosaicism, which occurs in around 1–3% of the cases (Devlin and Morrison, 2004) when chromosomes incorrectly divide during mitosis after fertilization, resulting in a variable mixture of two types of cells in the body, some with 47 chromosomes (aneuploid) and others with the normal complement of 46 chromosomes.

Apart from those with a specific risk—due to family history, for example—prenatal testing for chromosomal abnormalities is normally conducted in two steps: screening and risk assessment of all pregnancies, followed by prenatal diagnosis of high-risk cases. The current gold standard for diagnosis of any aneuploidy is provided by invasive removal of fetal cells by CVS or amniocentesis followed by karyotyping, though more recently, molecular techniques including PCR and fluorescent in situ hybridization have been used. NIPD diagnosis is therefore desirable if it is sufficiently robust to reduce or replace the need for amniocentesis/CVS [note that either method (invasive or non-invasive) is prone to misdiagnosis in the case of mosaicism, particularly in individuals where aneuploid cells are outnumbered by normal cells]. It is also hoped that it might replace or improve current screening tests.

By far, the most common known aneuploidy compatible with life is trisomy 21 (DS). Rarely, DS can also be caused by an inherited or sporadic defect, whereby an extra copy of all or part of chromosome 21 becomes attached to another chromosome (usually 14) to form a single aberrant chromosome. DS is associated with intellectual impairment, severe learning difficulties and excess mortality caused by long-term health problems such as heart disease. The incidence of DS varies strongly with maternal age, from around 0.07% at age 20 to 1% at age 40 (UK National Screening Committee, 2006), and in recent years, screening of older women has significantly decreased the proportion of DS live births.

The current screening protocol for DS involves a blood test for numerous maternal protein markers associated with DS (maternal serum alpha-fetoprotein, unconjugated estriol, human chorionic gonadotrophin and inhibin-A) as well as a nuchal translucency ultrasound scan of the fetus. The risk score calculated for each pregnancy is heavily influenced by maternal age and only reaches a detection rate of around 85%, with a false-positive rate of 5%, by integration of multiple different tests (Reddy and Mennuti, 2006). Interestingly, a study in Denmark found that offering standard screening to all women, and ignoring maternal age, halved the number of DS births (Mayor, 2007).

Other aneuploidies with known clinical significance include Edward syndrome (trisomy 18) and Patau syndrome (trisomy 13), both of which are frequently fatal within the first few months of life. In 2005, 428 cases of Edward and 165 cases of Patau were diagnosed in England and Wales, over 90% of which were prenatally diagnosed and in more than half the pregnancies were subsequently terminated (National Down Syndrome Cytogenetic Register, 2005). Abnormalities associated with the number of sex chromosomes are also observed, including Turner syndrome (X0) and triple X syndrome (XXX) in female births and Klinefelter syndrome (XXY) and XYY syndrome in male births, each of which is associated with various phenotypes including sterility and mild reduction in intellectual skills. In principle, cffNA could be used to diagnose any of these chromosomal abnormalities prenatally.

The key distinguishing factor that groups the aneuploidies together is an increase (or decrease) in the relative amount of specific chromosomal material. For the purposes of prenatal diagnosis, detection of aneuploidy therefore requires not just detection but also accurate quantification of the DNA derived from a specific chromosome. To date, there is no consensus on the best way to detect fetal aneuploidy using fetal nucleic acids from the maternal circulation, though a number of different methods have been described in the literature.

Although some evidence suggests that total cffDNA (specifically Y chromosome DNA in male pregnancies) or even total cell-free DNA (fetal plus maternal) might be elevated 2–3-fold in certain aneuploid pregnancies (Lo et al., 1999a; Spencer et al., 2003; Wataganara et al., 2003), this variation has been attributed to different experimental protocols and a recent comprehensive study showed no significant correlation between the level of total cffDNA or total cell-free DNA and trisomies 21, 13 and 18 (Gerovassili et al., 2007). Given that levels of cffDNA vary widely (Hahn et al., 2001) and are elevated in a number of other pregnancy-related conditions, more successful diagnostic strategies are likely to focus on detection and quantification of chromosome specific markers which must necessarily be altered by aneuploidy.

Other proposed methods rely upon detecting chromosome specific SNPs that differ in the maternal and paternal genomes. By calculating a ratio between alleles present in the maternal circulation, it is possible to directly determine the number of fetal chromosomes present. However, this method is highly sensitive to the level of cffNA and requires substantial enrichment of the fetal-derived sequences. Several different techniques have been proposed to address this problem, and a proof-of-principle study using either trisomy 13, 18 or 21 carried out.

1. Simultaneous detection of hundreds of paternally inherited SNPs on the chromosome of interest, using formaldehyde to suppress the maternal cell-free DNA in maternal plasma (Dhallan et al., 2007). The results are then compared with the maternal and paternal genotypes, quantified to determine the ratio of different paternal and maternal alleles at each site and thus the number of chromosomes of interest present in the fetus determined.
   
2. Exploitation of differences in the DNA methylation pattern between placenta and maternal cells, of a gene containing an informative SNP on the chromosome of interest, in order to selectively amplify and detect unmethylated sequences only (Tong et al., 2006). For example, the maspin gene, one of the proposed universal fetal markers, is serendipitously located on chromosome 18, and multiple differentially methylated genes have also been found on chromosome 21 (Chim et al., 2008a). This technique relies upon the fetus inheriting a conveniently located paternal SNP not present in the mother. However, this method is prone to false-positive results due to incomplete digestion of the (differentially methylated) maternal DNA, and therefore may have limited application.
   
3. Detection of uniquely placentally derived RNA, which contains an informative SNP, from the chromosome of interest (Lo et al., 2007b). Again, this technique relies upon the fetus inheriting two different SNP alleles in a region which is transcribed into RNA only by the growing fetus (Fig. 1) and therefore is not applicable to individuals who are homozygous at that position. The most promising example of this technique to date is the detection and quantification of PLAC4 RNA, expressed uniquely by the placenta and located on chromosome 21, which has been used to detect DS prenatally. Numerous other genes have been found to be uniquely expressed by the placenta during pregnancy (Go et al., 2007; Chim et al., 2008b), offering a wide variety of cffRNA targets each with potentially different SNPs. This method has great potential for high accuracy, as the RNA is uniquely expressed by the placenta/fetus, and therefore the problem of detecting it in the background of maternal RNA is minimal. Large clinical trials using this technique are currently ongoing in the USA, using SNPs that are believed to be informative in over 90% of the population.
   

Recently, an entirely different technique for detecting fetal aneuploidy was proposed using digital PCR (Lo et al., 2007a), a highly sensitive technique which uses dilution to isolate single template DNA molecules to be amplified, in order to detect very small differences in chromosome ratios. Importantly, in this method, fetally derived DNA (or RNA) is not specifically distinguished from maternal DNA; instead, the technique provides a measure of the total (i.e. fetal plus maternal) dosage of a particular chromosome relative to another reference chromosome. Although this technique requires prior enrichment of the cffDNA to achieve high accuracy, it should be applicable to all cases because it is not reliant on heterozygous SNPs.

View larger version (20K): [in this window] [in a new window] [Download PowerPoint slide]   Figure 1 Principle of calculating the fetal chromosome dosage to screen for aneuploidy, using the allelic ratio of a specific heterozygous SNP on cell-free fetal mRNA. Note that this method will only work when the SNP in question in heterozygous.

 
Most recently, high-throughput shotgun sequencing has been applied to the diagnosis of fetal aneuploidies (trisomies 13, 18 and 21) during the second and third trimesters, which does not necessitate any differentiation of fetal versus maternal DNA (Fan et al. 2008). Using simultaneous sequencing of millions of short DNA fragments, it is possible to compare the amount of sequences produced from different chromosomes to detect any small over-representation caused by aneuploidy, or potentially smaller chromosomal imbalances, in the fetus. Before a prenatal test for DS can be considered for either screening or diagnostic purposes, large trials will be needed to determine the clinical sensitivity and specificity of the method in different populations compared with the current protocol. It is not yet clear that the method will be accurate enough to replace traditional invasive diagnostic testing using karyotyping; however, even if this is the case, cffNA may still be useful in screening to risk stratify all pregnancies (regardless of maternal age) prior to offering definitive diagnosis, again depending upon its accuracy relative (or in addition) to the current method. In either case, were the test to be offered to all pregnant women, around 700 000 tests per annum could be expected in the UK alone.

Ethical, social and legal issues

The fact that NIPD requires only a small sample of blood raises numerous ethical, social and legal implications, due to the ease with which the test can be performed. Since the test can be used very early in the first trimester of pregnancy, with no risk to the mother, it is therefore likely to become much more common practice than amniocentesis. Although a detailed discussion of the issues is outside the scope of this review, it would be remiss not to highlight a few of the key factors relating specifically to the potential applications of cffNA, such as non-medical uses of the technology and implications for informed consent.

Aside from the wider debate on the ethics of abortion, one of the biggest concerns surrounding the use of cffDNA is the relative ease with which fetal sex can be determined. Although research and clinical use of NIPD of fetal sex currently only includes cases where there is a risk of an inherited disorder, sex determination could also potentially be used more widely for the purposes of family balancing or for preference of a particular sex. Indeed, there are a number of companies actively developing NIPD, including several offering prenatal fetal sex determination over the internet using cffDNA from a small finger-prick blood sample. There is considerable concern regarding this broader application of the test, in particular due to the extreme preference for male babies shown in some parts of the world such as India (George, 2006) and China (Chan et al., 2006a), and the substantial cultural variation in the value attached to informed choice in this context (van den Heuvel and Marteau, 2008).

Although currently the technology is primarily limited to the detection of paternally inherited sequences, in the longer term, the methods of measuring gene dosage may be developed and applied to detecting other genetic traits also present in the mother. Ultimately, not only causative genetic traits, but potentially also genetic variants associated with increased susceptibility to disease—such as BRCA1/2 mutations in breast cancer—may also be added to the list of mutations that could possibly be diagnosed prenatally using cffDNA. The addition of tests for increased risk of developing adult-onset diseases is significantly more ethically problematic than testing for inherited or congenital disorders, particularly as the person may never actually develop the disease. There has already been extensive discussion of this subject in the area of preimplantation genetic diagnosis and assisted reproduction (Robertson, 2003).

Finally, the possible addition of the technology to national screening programmes, combined with multiplexing technology platforms, means that multiple diseases may be tested for early and simultaneously in all pregnant women. Achieving truly informed consent may therefore be extremely difficult, potentially leading to routinisation and undue pressure upon the mother, who would perhaps rather realise her right ‘not to know’ about the genetic status of her fetus before birth (Newson, 2008), or who might realize only too late that she might have preferred not to be tested.

	Conclusion

TOP Abstract Introduction Materials and Methods Conclusion Funding References

 
cffNA can be detected in the maternal circulation during pregnancy, offering a potentially superb method for early NIPD of the genetic status of a fetus. They can be detected from 5 weeks gestation and are rapidly cleared from the circulation following birth. Currently, since the cffDNA comprises only 3–6% of the total circulating cell-free DNA, diagnoses are primarily limited to paternally inherited sequences as well as conditions that can be inferred by the unique expression of fetal RNA by the placenta.

The main advantages of using cffNA over conventional techniques of prenatal diagnosis (amniocentesis and CVS) are that the sampling method is non-invasive and therefore poses no risk to mother or child, and it can be performed early during the first trimester and would most likely be cheaper. Broadly, the potential applications fall into two categories of prenatal testing:

1. high genetic risk families, including sex determination in cases at risk of X-linked diseases and detection of specific paternally inherited single gene disorders;
   
2. routine antenatal care in all pregnancies, including aneuploidy screening, particularly trisomy 21 (DS), and diagnosis of Rhesus factor status in RhD negative women.
   

Currently, only sex determination and RhD diagnosis are nearing translation into clinical practice for high-risk individuals. In the longer term, depending upon the development of appropriate technology accompanied by robust evaluation, whether by commercial companies or state provided health services, the analysis of cffNA may form part of national antenatal screening programmes for all pregnant women.

	Funding

TOP Abstract Introduction Materials and Methods Conclusion Funding References

 
The work was funded by the Foundation for Genomics and Population Health (PHG Foundation).

	References

TOP Abstract Introduction Materials and Methods Conclusion Funding References

 

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Received on June 26, 2008; revised September 15, 2008; accepted on September 25, 2008

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