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Innovations: Prenatal diagnosis: progress through plasma nucleic acids
Author: Y. M. Dennis Lo
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"sequences in the plasma of women carrying male fetuses 2 . This discovery offered excit- ing new avenues for non-invasive prenatal diagnosis. Today, the clinical potential of this strategy has to some extent been realized, particularly with respect to the prenatal assessment of fetal rhesus D blood group status 3 . Here we discuss the current knowledge regarding the trafficking of fetal nucleic acids to the maternal circulation, and advances towards the elusive goal of non-invasive prenatal diagnosis. Biology of circulating fetal DNA After the surprising discovery of cell-free fetal DNA in maternal plasma, much had to be learnt about its nature. Circulating fetal DNA consists predominantly of short DNA fragments, of which 80% are less than 200 bp 4 . Furthermore, fetal DNA represents only a subfraction (on average, 3?6%) of the INNOVATION Prenatal diagnosis: progress through plasma nucleic acids Y. M. Dennis Lo and Rossa W. K. Chiu Abstract | Over the past 40 years, much effort has been spent on developing non- invasive prenatal diagnostic methods. Since 1997, the progress of this field has been accelerated by the unexpected finding of extracellular fetal nucleic acids in maternal plasma. These developments have been translated into many novel genetic, epigenetic and gene-expression markers, and are expected to have a fundamental impact on the future practice of prenatal diagnosis. Prenatal diagnosis is an indispensable part of modern obstetrics care. Fetal genetic diseases that are targeted by most current pro- grammes include chromosomal aneuploidies and several single-gene disorders. Although many non-invasive screening approaches are available, the definitive prenatal diagnosis of these disorders requires the analysis of fetal genetic material, which is obtained through invasive procedures such as amniocentesis and chorionic villus sampling. Because these procedures are associated with a small, but not immaterial, risk of fetal miscarriage, many researchers, over several decades, have searched for non-invasive alternatives to sample for fetal genetic material. One such approach is the isolation of fetal nucleated cells that have trespassed into the maternal circulation (BOX 1). However, such cells are extremely rare and require specialized isola- tion processes, prompting investigators to look for alternative approaches. Since the 1940s, extracellular DNA has been reported to be present in the plasma of human subjects. In particular, it was demonstrated in the 1990s that a proportion of this extracellular plasma DNA in some cancer patients contains tumour-derived genetic signatures 1 . In 1997, Lo et al. rea- soned by analogy that extracellular DNA of fetal origin might similarly be detectable in the plasma of pregnant women 2 . The first demonstration of the presence of cell-free fetal DNA in the plasma of pregnant women was the detection of Y-chromosomal DNA cell-free DNA that is present in maternal plasma 5 . The earliest gestational age at which circulating fetal DNA is detectable in maternal plasma is day 18 after embryo transfer by assisted reproduction 6 . Fetal DNA concentrations seem to increase as gestation progresses 5 ; following delivery, fetal DNA is cleared extremely rapidly from maternal plasma, with a mean half-life of about 16 minutes 7 . It has been suggested that the renal system is one of the mechanisms of clearance of plasma DNA, as fetal DNA has been reported by some 8 , but not all 9 , investi- gators to be present in the urine of pregnant women. The consensus in the literature is that there is no long-term persistence of fetal DNA in maternal plasma 10,11 , with the exception of an isolated report by Invernizzi et al. 12 . Their data might be explained by the persistence of some fetal nucleated cells for extended periods following delivery 13 . It has also been suggested that these cells could have contaminated a proportion of the plasma samples used in that study due to a subopti- mal centrifugation step for the separation of cells from the plasma 11 . Many lines of evidence indicate that the placenta is the predominant source of fetal DNA in maternal plasma. This evidence includes the presence of placental genetic signatures in maternal plasma in cases with confined placental chromosomal abnormalities 14 , the demonstration of placental epigenetic signatures in maternal plasma DNA 15 , and the presence of circulating fetal DNA after the placenta has been formed but before the fetal circulatory system has been established 6 . Apoptosis and necrosis, which are normal cell turnover events in the placenta, have Box 1 | Fetal nucleated cells in maternal blood The search for fetal nucleated cells in maternal peripheral blood started in the late 1960s. Multiple fetal nucleated cell populations have been reported in maternal peripheral blood, including fetal nucleated red blood cells 54 , lymphocytes 55 and trophoblasts 56 . Many methods have been attempted for their isolation and detection, including fluorescence-activated cell sorting 54,55 , magnetic-activated cell sorting 57 , microdissection 58 and size separation 56 . However, the extreme rarity of these cells, of the order of about one cell in each millilitre of maternal blood 59 , is a major obstacle to their robust detection. In this regard, a landmark large-scale study, the National Institute of Child Health and Development Fetal Cell Isolation Study, reported a sensitivity for male fetal cell detection of 41.4% and a false-positive rate of 11.1% (REF. 57). This group of investigators concluded that technological advances are needed before clinical application of fetal cell analysis can be achieved. NATURE REVIEWS | GENETICS VOLUME 8 | JANUARY 2007 | 71 PERSPECTIVES been shown in a placental explant model to be responsible for fetal DNA release 16 . The association between fetal DNA release and placental cell death might also partly explain the elevation in circulating fetal DNA that has been reported in several pregnancy-associated disorders, such as pre-eclampsia 17,18 . Clinical applications Fetal DNA in maternal plasma can be used to obtain valuable information about the fetus or pregnancy, either for prenatal diagnosis or monitoring (TABLE 1). These applications can be divided into two groups: those that require the qualitative analysis of circulating fetal DNA, and those that require its quantitative assessment. These analyses typically start with the separation of plasma from the cellular components of maternal blood, followed by the extrac- tion of DNA from the plasma fraction 19,20 . For both of these types of application, it is important to bear in mind that fetal DNA only represents a minor fraction of the DNA that is present in maternal plasma 5 . Therefore, the analytical method must detect this subpopulation of cell-free DNA with high sensitivity, and differentiate the target fetal DNA from the overwhelming background of maternal DNA. In practice, the easiest way to achieve this is by targeting chromosomes, genes or genetic polymor- phisms and mutations that the fetus has inherited from the father, and that are either absent or different in the mother. Qualitative assessments. Clinical informa- tion from such analysis can result in either the positive identification of, or the exclu- sion of, a genetic trait or disease (FIG. 1). At the chromosomal level, the most obvious applications are those that detect sequences from the Y chromosome of male fetuses in maternal plasma. These tests are close to 100% accurate, and are useful for the pre- natal risk stratification of pregnancies that involve mothers who carry mutations for sex-linked diseases such as haemophilia A 21 . Fetal gender determination is also clinically useful for the prenatal management of congenital adrenal hyperplasia, as the virili- zation of the genitalia that is associated with this disease is only a problem for female fetuses 22 . At the genetic level, the most important procedure is, perhaps, the detec- tion of the rhesus blood group antigen D (RHD) gene from a RHD-positive fetus in the plasma of RHD-negative mothers (15% of the Caucasian population) 23 . These moth- ers, when carrying a RHD-positive fetus, might have rhesus D incompatibility, a condi- tion that requires further management. As this test is close to 100% accurate, it has now been introduced as a routine test in a number of centres 3 . The first polymorphisms or mutations to be detected from fetal DNA were those associated with multiple nucleotide differ- ences between the fetal and maternal DNA, including microsatellite polymorphisms 24 , trinucleotide repeat expansion (for example, within the gene that causes myotonic dys- trophy) 25 , and mutations caused by multiple base deletion (for example, the 4 bp deletion in codons 41 and 42 of the haemoglobin-? (HBB) gene, which causes ?-thalassaemia) 26 . The technical challenge of distinguishing between fetal and maternal circulating DNA down to a single nucleotide has recently been overcome by the development of a single-allele base extension reaction protocol for mass spectrometry analysis 27 . This extends the plasma DNA approach to the non-invasive prenatal diagnosis of many single-gene disorders. For genetic diseases that are caused by more than one mutation, and in which the father and mother carry different mutations, this approach can allow direct detection of the unique paternally inherited mutations (FIG. 1). For situations in which the father and mother carry the same mutation, this method can be used to trace the inheritance of the mutant (or normal) chromosome by the fetus through the analysis of linked SNPs 27 (FIG. 1). Table 1 | Potential clinical applications of analysing fetal nucleic acids in maternal plasma Approach Analytical principles Marker Clinical applications Refs Fetal DNA analysis for diagnosis or exclusion of fetal genetic disorders Qualitative detection of fetal-specific sequence (for example, Y chromosome, RHD, paternal-specific disease-associated alleles) in maternal plasma by real-time, fluorescent, nested or conventional PCR, and mass- spectrometry based methods, such as single-allele base extension reaction. Fetal gender Sex-linked disorders, congenital adrenal hyperplasia 21,22 Fetal RHD Early identification of pregnancies with rhesus D incompatibility, risk stratification of pregnancies requiring treatment with RHD immunoglobulin 3,23 Fetal HLA haplotype HLA-linked diseases; for example, congenital adrenal hyperplasia 60 Paternally derived mutations ?-thalassaemia, haemoglobin E disease, cystic fibrosis, congenital adrenal hyperplasia, myotonic dystrophy, Huntington disease 25-27, 31,60 Fetal DNA analysis for assessment or monitoring of pregnancy- associated disorders Quantification of fetal DNA concentration in maternal plasma by real-time quantitative or competitive PCR, and comparison with gestational age-matched normal pregnancies. Fetal conditions Chromosomal aneuploidy, intrauterine growth retardation, fetomaternal haemorrhage 29,61 Other pregnancy complications Pre-eclampsia, pre-term labour, ectopic pregnancy, invasive placentation, hyperemesis gravidarum, polyhydramnios 17,18,28, 62,63 Placental RNA analysis Quantification of placentally expressed transcripts by real-time quantitative reverse-transcriptase PCR. CGB Gestational trophoblastic disease 49 CRH Pre-eclampsia 48 Transcript quantification; RNA-SNP allelic ratio determination Chromosome- 21 encoded transcripts Fetal trisomy 21 44,50 Epigenetic analysis Real-time quantitative methylation- specific PCR; epigenetic allelic ratio determination Hypomethylated SERPINB5 Pre-eclampsia, fetal trisomy 18 15 CGB, chorionic gonadotropin, beta polypeptide; CRH, corticotropin releasing hormone; HLA, human leukocyte antigen; RHD, rhesus blood group, D antigen. PERSPECTIVES 72 | JANUARY 2007 | VOLUME 8 www.nature.com/reviews/genetics c d abQuantitative assessments. Abnormally high levels of circulating fetal DNA occur in several pregnancy-associated disorders, including pre-eclampsia 17,18 , pre-term labour 28 , and fetal chromosomal aneu- ploidies 29,30 (TABLE 1). Therefore, there have been suggestions that measuring the overall amount of circulating fetal DNA might be used as a general screening tool for preg- nancy-associated disorders 18 . However, most groups have used loci on the Y chromosome as markers for circulating fetal DNA, limiting this approach to pregnancies that involve male fetuses. Therefore, as discussed below, one active area of research is the search for fetal DNA markers that can be used irrespective of the gender and genetic poly- morphisms of the fetus. Another limitation is the significant overlap in circulating fetal DNA levels between normal and complicated pregnancies 17,29,30 , which has reduced the sen- sitivity and specificity of this type of screening strategy. One possible solution is to search for fetal nucleic-acid markers that are specifically associated with a particular pathology; for example, fetal RNA (see below). Recent advances In this section we highlight a number of recent developments. It is expected that sig- nificant developments and discussion in these areas will take place over the next few years. Fetal DNA enrichment. Many of the analyti- cal and diagnostic limitations of fetal DNA in maternal plasma stem from the relative concentration of fetal DNA to the over- whelming background of maternal DNA, of the order of 1:20 or less 5 . The fractional concentration of fetal DNA could theoreti- cally be increased by one of two approaches: through the selective enrichment of fetal DNA, or by suppressing the maternal DNA background. It has recently been demon- strated that the size of fragments of fetal DNA in maternal plasma is generally smaller than that of the background maternal DNA 4 . This size difference allows a degree of fetal DNA enrichment, by harvesting the DNA molecules of smaller molecular weight from the plasma following gel electrophoresis 31 . The main limitations of this approach are that the degree of enrichment that is achieved is only moderate, and the manipu- lations involved in the electrophoretic and subsequent DNA isolation steps are susceptible to exogenous contamination. The second concern could potentially be overcome by the development of devices which allow these steps to be carried out in an enclosed environment. To suppress the background maternal DNA, it is necessary to explore its possible sources. As this is relatively difficult to study directly in pregnant women due to the lack of markers for differentiating between DNA that originates from different tissues, Lui et al. studied individuals who had undergone successful sex-mismatched bone-marrow transplantation 32 : in these individuals, the haematopoietic and non-haemat- opoietic systems were of different gender. Interestingly, it was found that the ?gender? of the plasma DNA switched towards that of the donor, indicating that most of the plasma DNA (median ~60% in male-to-female bone marrow transplantation 32 ) was of haemat- opoietic origin. The sources of the non-haematopoietic plasma DNA are not completely documented, but studies on patients following sex-mismatched heart, liver and kidney transplantations indicate that these organs contribute only a minor percentage of non-haematopoietic DNA in plasma 33 . If these findings can be extrapolated to pregnant women, then the haematopoietic cells should also be the main contributor to the background maternal DNA. Haematopoietic DNA might be released in vivo before the blood is sampled and/or as a consequence of blood sampling, resulting Figure 1 | Principles of circulating fetal DNA analysis for prenatal diagnosis of single-gene disorders. a | Release of fetal DNA into the maternal circulation. Before release, the numbers of paternally (red) and maternally inherited (green) DNA molecules are equal. The block arrow denotes the release of fetal DNA from the placenta into the maternal circulation. b | In maternal plasma, fetal DNA molecules circulate among a background of maternal DNA sequences. Because the fetal DNA allele that the fetus inherits from its mother (green) is genetically identical to the background maternal sequences (green), the allele that the fetus inherits from its father (red) is the most readily distinguishable fetal-specific sequence in maternal plasma. c | Non-invasive pre- natal assessment of the fetal genotype could be made on the basis of the positive detection of a molecular feature (yellow star) that is unique to the paternally inherited fetal allele. Examples of unique molecular features include the Y chromosome, RHD blood antigen (when the mother is RHD-negative) and paternally inherited mutations or polymorphisms. d | Alternatively, non-invasive prenatal assessment could be made on the basis of excluding the inheritance of a molecular feature (yellow star) that is unique to the paternally inherited fetal allele. For example, non-invasive pre- natal assessment of an autosomal recessive disease could be achieved by excluding the fetal inheritance of the paternal mutation if the mother and father each carry a different mutation. If the couple both carry an identical mutation, the disease could be excluded in the fetus through the detection of a polymorphism that is uniquely linked to the paternal wild-type allele. PERSPECTIVES NATURE REVIEWS | GENETICS VOLUME 8 | JANUARY 2007 | 73 from the lysis of maternal nucleated blood cells during venesection or in transit in the blood-collection tubes. Dhallan et al. therefore reasoned that, if the maternal blood cells were stabilized through fixation with formaldehyde, the in vitro release of DNA from the maternal blood cells could be minimized 34 . However, several groups were unable to reproduce the data that Dhallan et al. presented to support this line of reasoning 35,36 . One possible explanation for their results could be the use of an imprecise method to determine fetal DNA concentration, which would yield frac- tional fetal DNA concentrations in some samples that are much higher than the actual values, therefore overestimating the effect, if any, of the formaldehyde treatment 35 . Fetal epigenetic markers. As fetal DNA is surrounded by a majority background of maternal DNA, it has generally been assumed that it would be technically chal- lenging to detect a DNA sequence that the fetus has inherited from its mother. To challenge this assumption, Poon et al. studied an imprinted region between the insulin-like growth factor 2 (IGF2) and H19 genes, which is methylated if inherited from the father and unmethylated if inher- ited from the mother 37 . Poon et al. rea- soned that, in a pregnant woman who has inherited a copy of this imprinted region from her father (so it is methylated in the cells of the pregnant woman), and who has passed this copy on to her fetus (so it is unmethylated in the cells of the fetus), the methylation status of the fetal and maternal DNA in maternal plasma should be distinguishable from one another 37 . This has led to the first detection of maternally inherited fetal DNA sequences in maternal plasma, and has shown that epigenetic markers might allow one to overcome the limitations of genetic markers in non-invasive prenatal diagnosis. Another limitation of fetal genetic markers for the detection of fetal DNA in maternal plasma is that no single marker is applicable to all pregnancies. For example, the use of Y-chromosomal markers is only applicable to women who are carrying male fetuses. By contrast, Chim et al. have recently demonstrated that SERPINB5 (encoding maspin) is hypomethylated in the placenta and hypermethylated in maternal blood cells 15 . So, hypomethylated SERPINB5 sequences can be used as a universal marker for fetal DNA in maternal plasma, irrespec- tive of the gender and polymorphisms of the fetus 15 . This marker was used to detect the elevation of the concentrations of circulating fetal DNA in pre-eclampsia 15 . The main limitation of this marker is the need to carry out bisulfite treatment to con- vert the fetal-specific epigenetic signature into sequence differences that can then be detected by methods such as PCR. Bisulfite conversion is labour-intensive and inevita- bly destroys a large proportion of the treated DNA 38 . It would therefore be desirable to search for placental-specific (that is, fetal-specific) epigenetic signatures that are opposite to those exhibited by SERPINB5; that is, hypermethylated in the placenta, but hypomethylated in maternal blood cells. If found, such epigenetic markers could be detected with the help of methylation-sensitive restriction enzymes, which can be used to cut the hypomethyl- ated maternal sequences, but will leave the hypermethylated fetal sequences intact for amplification and detection. Plasma RNA markers. The presence of fetal RNA in maternal plasma was first demonstrated using transcripts from the Y chromosome 39 . This discovery was surprising in view of the known lability of RNA. It has subsequently been dem- onstrated that the unexpected stability of plasma RNA molecules might be due to their association with particulate matter 40 , which might protect them against plasma RNase digestion 41 . The realization that the placenta is the main source of circulating fetal RNA 42 has greatly increased the number of molecular markers that can be developed for prenatal diagnosis and monitoring. The development of a microarray-based method for systematically identifying gene tran- scripts that are expressed in the placenta but absent in maternal blood cells has further increased the pace of plasma-RNA marker development 43 . To date, over ten placental mRNA species have been shown to be specific to pregnancy and detectable in maternal plasma 42?45 . Circulating placental mRNA is partially degraded, with a preponderance of 5? frag- ments 46 . This indicates that assays that are designed towards the 5? end of a transcript are likely to be more sensitive than those that are designed towards the 3? end. Circulating placental mRNA is associated with particles 42 , and it has been suggested that such particles might be related to syncytiotrophoblast microparticles, which are thought to be shed by the placenta, especially in pathological conditions such as pre-eclampsia 47 . The main advantages of the plasma RNA approach include its potential disease specificity and broad population coverage, irrespective of fetal gender and polymorphisms. In this regard, quantitative aberrations of selected placen- tal mRNA species have been observed in various pregnancy-associated disorders, including corticotropin releasing hormone (CRH) mRNA in pre-eclampsia 48 and cho- rionic gonadotropin ?-polypeptide (CGB) mRNA in gestational trophoblastic disease 49 . Glossary Amniocentesis A clinical procedure that involves the insertion of a needle through the womb to sample the amniotic fluid that bathes the fetus. Chorionic villus sampling A clinical procedure that is carried out to obtain a biopsy of the placenta during early pregnancy. Confined placental chromosomal abnormalities Chromosomal abnormalities that are restricted in distribution to the placenta, so are not found throughout all cells of the fetus. Fetomaternal haemorrhage Bleeding from a fetal source into the maternal circulation. Gestational trophoblastic disease The tumorous growth of fetal tissues in a maternal host. Hyperemesis gravidarum Severe vomiting in pregnancy. Mass spectrometry An analytical technique that detects and identifies small molecules, for example, peptide fragments and oligonucleotides, on the basis of their molecular mass and charge. Polyhydramnios An excess of amniotic fluid. Pre-eclampsia A clinical condition that presents during the second half of pregnancy; hallmark manifestations are hypertension, oedema and proteinuria. Rhesus D incompatibility When a woman with rhesus D antigen (RHD)- negative blood group is pregnant with a RHD-positive fetus, immune destruction of the fetal red blood cells can occur if the maternal immune system has previously been sensitized by the RHD antigen. Single-allele base extension reaction An analytical protocol that involves an extension reaction with an oligonucleotide primer that targets the detection of a specific polymorphic allele or a mutation. Syncytiotrophoblast microparticles Subcellular particles derived from syncytiotrophoblasts, which are the outermost layer of cells that cover the floating villi of the placenta. PERSPECTIVES 74 | JANUARY 2007 | VOLUME 8 www.nature.com/reviews/genetics MassMass Int ensity Int ensity 1:1 2:1 21 21 18 18 Placenta Maternal plasma Maternal blood cells a Euploid fetusb c Trisomy Chromosomal aneuploidy detection. The detection of fetal chromosomal aneuplodies using plasma nucleic acids represents a considerable challenge in the field. First, fetal nucleic acids only represent a minor fraction of the nucleic acids that are present in the plasma of a pregnant woman. Second, the dissociation of circulating nucleic acids from intact cells makes the elucidation of chromosome dosage difficult. One solution to the first problem is to focus on a subfrac- tion of the nucleic acids in maternal plasma that is almost completely fetal-specific, thereby minimising the interference from the background maternal nucleic acids (FIG. 2). One such subfraction is mRNA that is expressed only in the placenta 42 . To provide the aneuploidy information, the placentally expressed mRNA species should be expressed by the chromosome that is involved in the aneuploidy; for example, chromosome 21 in Down syndrome 44 . Using this strategy, Lo et al. have recently demonstrated that transcripts from placenta specific 4 (PLAC4), a gene that lies on chro- mosome 21, are detectable in the plasma of pregnant women 50 . To address the problem of obtaining chromosome-dosage informa- tion, Lo et al. reasoned that, in a euploid fetus that is heterozygous for a SNP within the coding region of PLAC4, the ratio of the two alleles should be 1:1, whereas that for a fetus with trisomy 21 should be 1:2 or 2:1 (REF. 50). Using this approach, trisomy 21 was detected non-invasively with a sensitiv- ity of 90% (9 cases were detected out of the 10 trisomy-21 cases that were tested) and a specificity of 96% (54 cases were excluded out of the 56 euploid cases that were tested) in informative cases 50 . These figures, which were achieved using only a single marker, are comparable to those that are currently possible only with multi-marker strategies. The false-positive and false-negative results of this ?RNA-SNP? approach are seen in cases with a relatively low absolute PLAC4 mRNA concentration in maternal plasma. It is possible that the improvement in plasma processing and RNA extraction methods from maternal plasma will improve the total yield, and therefore the amount of PLAC4 mRNA that is available for this kind of analysis, further reducing the chance of misdiagnosis. The main limitation of the RNA-SNP approach is the need to find informative SNPs in the target RNA transcripts. For example, the first PLAC4 SNP that was used for this approach has a heterozygosity rate of only ~0.45 (REF. 50). There are a number of other transcribed SNPs in PLAC4, and at least two of these seem to hold promise for RNA-SNP analysis (unpublished observa- tions). Further increase in informativeness can potentially be achieved by exploring other placentally expressed genes, and their associated SNPs, on chromosome 21. A similar strategy might be applicable to the other trisomies that are important in prena- tal diagnosis, trisomy 18 and trisomy 13. As an alternative to using placental mRNA to focus on the fetal-specific subfraction of circulating nucleic acids in maternal plasma, it is also theoretically possible to analyse circulating DNA that bears a fetal-specific epigenetic signature (FIG. 2). Analogous to the RNA-SNP strategy, the locus that bears the fetal-specific DNA- methylation signature should be on the Figure 2 | Principles of circulating fetal DNA and RNA analysis for the prenatal detection of trisomies. a | In maternal plasma, most of the background maternal nucleic-acid sequences are derived from maternal blood cells; however, evidence suggests that the fetal-derived sequences originate from the placenta. Therefore, prenatal diagnosis of fetal trisomies could be achieved through the detection of placenta-specific DNA or RNA that is derived from the chromosome of interest. The placental specificity of the molecules could be ascertained by DNA-methylation dif- ferences between the placenta and maternal blood cells, as occurs in genes such as SERPINB5 15,51 (green double-stranded molecules), which is hypomethylated (open circles) in placental cells and hypermethylated (filled circles) in maternal cells. Alternatively, genes that are expressed by the placenta but not by maternal blood cells, such as PLAC4 mRNA (orange single-stranded molecules), could be targeted 50 . SERPINB5 and PLAC4 are encoded by chromosomes 18 and 21, respectively. Grey squares and triangles denote base-pair differences, which are indicative of different parental origin. b | Chromosome-dosage information could be obtained by assessing the ratio of heterozy- gous alleles of polymorphic markers within the placenta-derived nucleic-acid molecules. For a biallelic polymorphism, we would expect an equal proportion of the two alleles in a heterozygous euploid fetus, whereas an additional dose of one of the alleles is expected for a trisomic fetus. c | Therefore, the allelic ratio of the placenta-specific sequence would be 1:1 for a euploid fetus but 2:1 for a trisomic fetus, which could be identified by methods such as mass spectrometry 50,51 . PERSPECTIVES NATURE REVIEWS | GENETICS VOLUME 8 | JANUARY 2007 | 75 chromosome that is involved in the chro- mosomal aneuploidy. Chromosome-dosage information can then be obtained by SNP allelic ratio analysis. The theoretical basis of this ?epigenetic allelic ratio? approach is illus- trated in FIG. 2 (REF. 51). SERPINB5 (REF. 15), discussed above, is on chromosome 18, and non-invasive prenatal detection of trisomy 18 using the epigenetic allelic ratio approach with this gene has recently been achieved 51 . This strategy also has potential applications in the detection of other trisomies, provided that chromosome-specific and fetal-specific DNA-methylation signatures can be found. With the Human Epigenome Project (see online links), it is possible that such infor- mation will be forthcoming over the next few years. Conclusions In the nine years since the first discovery of cell-free fetal nucleic acids in maternal plasma 2 , these molecules have provided many useful new markers for non-invasive prenatal diagnosis and monitoring. In particular, prenatal RHD genotyping from maternal plasma is now routinely carried out in a number of centres 3 . The recent demon- stration that circulating fetal cell-free nucleic acids can be used for the direct detection of trisomy 21 in the fetus 50 will fuel further interest in this area, although much work is needed to replicate these data in a large- scale, multi-centre setting. Furthermore, the impact of this development on existing prenatal screening programmes and the cur- rent repertoire of tests, including ultrasound scanning and maternal serum biochemical screening 52 , will need to be assessed and discussed. On the cost issue, judging from the current platforms of plasma nucleic- acid-based testing, this methodology should compare favourably with existing invasive methods of prenatal diagnosis, especially when the relatively expensive clinicians? time involvement is factored in. The molecular characterization of plasma nucleic acids is still in its infancy. Currently, we have only basic information about the size distribution and concentrations of circulating fetal nucleic acids. Little or no information is available regarding possible complexing of these circulating nucleic acids with other biomolecules (for example, proteins and lipids) and the nature and biophysical properties of the particles that associate with circulating fetal RNA. Apart from the intrinsic biological interest of this information, such knowledge might also allow one to develop better methods for enriching fetal nucleic acids from maternal plasma. Such enrichment could simplify analytical procedures and further improve diagnostic accuracy. Like many other rapidly developing areas of research, the social, ethical and regula- tory discussions tend to lag behind the technological progress, although this will hopefully be rectified as we enter the sec- ond decade of the field. These issues were recently highlighted by several companies in their marketing of non-invasive molecular tests of fetal gender, which they claim are broadly based on the fetal nucleic acids in maternal blood 53 . Considering the recent progress in plasma nucleic acids, we could be entering the most exciting period of non-invasive prenatal molecular diagnosis so far. We expect that, over the next few years, we will witness the influence of these new developments on many fundamental aspects of the practice of prenatal diagnosis, making prenatal testing safer for the millions of pregnant women worldwide. We also await with anticipation the unravelling of the release and clearance mechanisms and functional implications of plasma nucleic acids. Y.M. Dennis Lo and Rossa W.K. Chiu are at the Li Ka Shing Institute of Health Sciences and the Department of Chemical Pathology, The Chinese University of Hong Kong, Prince of Wales Hospital, 30-32 Ngan Shing Street, Shatin, New Territories, Hong Kong SAR, China. Correspondence to Y.M.D.L. e-mail: loym@cuhk.edu.hk doi:10.1038/nrg1982 Published online 5 December 2006 1. Chen, X. Q. et al. Microsatellite alterations in plasma DNA of small cell lung cancer patients. Nature Med. 2, 1033?1035 (1996). 2. Lo, Y. M. D. et al. Presence of fetal DNA in maternal plasma and serum. Lancet 350, 485?487 (1997). 3. Finning, K. 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Lui, Y. Y. N. et al. Origin of plasma cell-free DNA after solid organ transplantation. Clin. Chem. 49, 495?496 (2003). 34. Dhallan, R. et al. Methods to increase the percentage of free fetal DNA recovered from the maternal circulation. JAMA 291, 1114?1119 (2004). 35. Chung, G. T. et al. Lack of dramatic enrichment of fetal DNA in maternal plasma by formaldehyde treatment. Clin. Chem. 51, 655?658 (2005). 36. Chinnapapagari, S. K., Holzgreve, W., Lapaire, O., Zimmermann, B. & Hahn, S. Treatment of maternal blood samples with formaldehyde does not alter the proportion of circulatory fetal nucleic acids (DNA and mRNA) in maternal plasma. Clin. Chem. 51, 652?655 (2005). PERSPECTIVES 76 | JANUARY 2007 | VOLUME 8 www.nature.com/reviews/genetics 37. Poon, L. L. M., Leung, T. N., Lau, T. K., Chow, K. C. & Lo, Y. M. D. Differential DNA methylation between fetus and mother as a strategy for detecting fetal DNA in maternal plasma. Clin. Chem. 48, 35?41 (2002). 38. Grunau, C., Clark, S. 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Detection of chromosome 21-encoded mRNA of placental origin in maternal plasma. Clin. Chem. 49, 1445?1449 (2003). 45. Go, A. T. et al. Detection of placental transcription factor mRNA in maternal plasma. Clin. Chem. 50, 1413?1414 (2004). 46. Wong, B. C. K. et al. Circulating placental RNA in maternal plasma is associated with a preponderance of 5? mRNA fragments: implications for noninvasive prenatal diagnosis and monitoring. Clin. Chem. 51, 1786?1795 (2005). 47. Gupta, A. K. et al. Detection of fetal DNA and RNA in placenta-derived syncytiotrophoblast microparticles generated in vitro. Clin. Chem. 50, 2187?2190 (2004). 48. Ng, E. K. O. et al. The concentration of circulating corticotropin-releasing hormone mRNA in maternal plasma is increased in preeclampsia. Clin. Chem. 49, 727?731 (2003). 49. Masuzaki, H. et al. Clinical applications of plasma circulating mRNA analysis in cases of gestational trophoblastic disease. Clin. Chem. 51, 1261?1263 (2005). 50. Lo, Y. M. D. et al. Plasma placental RNA allelic ratio permits noninvasive prenatal chromosomal aneuploidy detection. Nature Med. (in the press). 51. Tong, Y. K. et al. Noninvasive prenatal detection of fetal trisomy 18 by epigenetic allelic ratio analysis in maternal plasma: theoretical and empirical considerations. Clin. Chem. 13 Oct 2006 (doi:10.1373/clinchem.2006.076851). 52. Malone, F. D. et al. First-trimester or second-trimester screening, or both, for Down?s syndrome. N. Engl. J. Med. 353, 2001?2011 (2005). 53. Bianchi, D. W. At-home fetal DNA gender testing: caveat emptor. Obstet. Gynecol. 107, 216?218 (2006). 54. Bianchi, D. W., Flint, A. F., Pizzimenti, M. F., Knoll, J. H. & Latt, S. A. Isolation of fetal DNA from nucleated erythrocytes in maternal blood. Proc. Natl Acad. Sci. USA 87, 3279?3283 (1990). 55. Herzenberg, L. A., Bianchi, D. W., Schroder, J., Cann, H. M. & Iverson, G. M. Fetal cells in the blood of pregnant women: detection and enrichment by fluorescence-activated cell sorting. Proc. Natl Acad. Sci. USA 76, 1453?1455 (1979). 56. Beroud, C. et al. Prenatal diagnosis of spinal muscular atrophy by genetic analysis of circulating fetal cells. Lancet 361, 1013?1014 (2003). 57. Bianchi, D. W. et al. Fetal gender and aneuploidy detection using fetal cells in maternal blood: analysis of NIFTY I data. National Institute of Child Health and Development Fetal Cell Isolation Study. Prenat. Diagn. 22, 609?615 (2002). 58. Cheung, M. C., Goldberg, J. D. & Kan, Y. W. Prenatal diagnosis of sickle cell anaemia and thalassaemia by analysis of fetal cells in maternal blood. Nature Genet. 14, 264?268 (1996). 59. Bianchi, D. W. et al. PCR quantitation of fetal cells in maternal blood in normal and aneuploid pregnancies. Am. J. Hum. Genet. 61, 822?829 (1997). 60. Chiu, R. W. K. et al. Noninvasive prenatal exclusion of congenital adrenal hyperplasia by maternal plasma analysis: a feasibility study. Clin. Chem. 48, 778?780 (2002). 61. Lau, T. K., Lo, K. W., Chan, L. Y. S., Leung, T. Y. & Lo, Y. M. D. Cell-free fetal deoxyribonucleic acid in maternal circulation as a marker of fetal-maternal hemorrhage in patients undergoing external cephalic version near term. Am. J. Obstet. Gynecol. 183, 712?716 (2000). 62. Lazar, L., Nagy, B., Ban, Z., Nagy, G. R. & Papp, Z. Presence of cell-free fetal DNA in plasma of women with ectopic pregnancies. Clin. Chem. 52, 1599?1601 (2006). 63. Sekizawa, A. et al. Increased cell-free fetal DNA in plasma of two women with invasive placenta. Clin. Chem. 48, 353?354 (2002). Acknowledgements The work in the authors? laboratories is supported by the Hong Kong Research Grants Council, the Innovation and Technology Fund and the Li Ka Shing Foundation. Competing interests statement The authors declare competing financial interests: see web version for details. DATABASES The following terms in this article are linked online to: Entrez Gene: http://www.ncbi.nlm.nih.gov/entrez/query. fcgi?db=gene CGB | CRH | H19 | HBB | IGF2 | PLAC4 | RHD | SERPINB5 OMIM: http://www.ncbi.nlm.nih.gov/entrez/query. fcgi?db=OMIM ?-thalassaemia | Down syndrome | haemophilia A FURTHER INFORMATION Chinese University of Hong Kong Department of Chemical Pathology: http://www.cpy.cuhk.edu.hk Human Epigenome Project: http://www.epigenome.org Access to this links box is available online. OPINION Recombination: an underappreciated factor in the evolution of plant genomes Brandon S. Gaut, Stephen I. Wright, Car�ne Rizzon, Jan Dvorak and Lorinda K. Anderson Abstract | Our knowledge of recombination rates and patterns in plants is far from being comprehensive. However, compelling evidence indicates a central role for recombination, through its influences on mutation and selection, in the evolution of plant genomes. Furthermore, recombination seems to be generally higher and more variable in plants than in animals, which could be one of the primary reasons for differences in genome lability between these two kingdoms. Much additional study of recombination in plants is needed to investigate these ideas further. The nuclear genomes of plants are remark- ably variable in terms of characteristics such as genome size, chromosome number, gene order and gene density. The grass family (Poaceae) serves as a fitting example. Since their origin ~77 million years ago 1 , the grasses have diverged to range ~55-fold in diploid genome size and at least 10-fold in diploid chromosome number 2 . Some of this genome lability can be attributed to ancient polyploid events 3 . But paleopolyploidy by itself cannot explain the structural variation that exists among the genomes of extant plants. For one thing, paleopolyploid events might be too infrequent 4 . The most recent polyploidy event in the evolutionary lineage of Arabidopsis thaliana occurred anywhere from 25 million years to 100 million years ago 5,6 , too long ago to contribute to differences in genome size and chromosomal number between A. thaliana and its closest relatives. In addition to changes in ploidy, ongoing mechanisms of gene deletion, genome rearrangement and localized gene duplica- tions that do not involve changes in ploidy undoubtedly contribute to genomic flux. In A. thaliana, for example, the proportion of genes that have been duplicated by local- ized events is comparable to that of genes that have been duplicated by polyploidy. These mechanisms might accelerate after polyploidy events occur, but also function in their absence 7 . In recent years, plant evolutionary genomicists have focused primarily on paleopolyploid events. However, one process that we believe has not received adequate attention is recombination, which generates mutations and influences the strength of natural selection. Here we high- light the increasing body of evidence that indicates that recombination has had an important role in plant genome evolution. We begin by briefly reviewing the muta- tional properties of recombination, its role in natural selection and genome-wide PERSPECTIVES NATURE REVIEWS | GENETICS VOLUME 8 | JANUARY 2007 | 77 "
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Genetics
Gene Inheritance and Transmission
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Nucleic Acid Structure and Function
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