Review Gynecol Obstet Invest 2014;77:73–77 DOI: 10.1159/000355693
Received: June 6, 2013 Accepted: September 17, 2013 Published online: October 26, 2013
Noninvasive Prenatal Diagnosis Using Next-Generation Sequencing Liang Xu b Rui Shi a a
Department of Obstetrics and Gynecology, and b Research Center for Translational Medicine, Shanghai East Hospital, Tongji University School of Medicine, Shanghai, China
Abstract Nowadays, prenatal diagnosis is necessary for pregnant women. For the parents who are expecting a child, the genetic test may provide the information whether they are carrying rare gene mutations and whether they are at risk of passing them onto their offspring. However, the ultimate determination of genetic diseases often requires invasive procedures such as amniocentesis and chorionic villus sampling, which may cause fetal miscarriage. A noninvasive type of prenatal diagnosis needs to be developed in clinical practice to dispel safety concerns. In this paper, we will review the technical advancement of using maternal circulating nucleic acids as the sample in noninvasive studies, and highlight the utilization of next-generation sequencing in the screening of genetic diseases. © 2013 S. Karger AG, Basel
There are more than 3,000 mendelian disorders [1] accounting for about 20% of deaths in infancy [2]. Besides the routine ultrasound examination concerning fetal de© 2013 S. Karger AG, Basel 0378–7346/13/0772–0073$38.00/0 E-Mail [email protected] www.karger.com/goi
velopment, for those who are at high risk of genetic disease, prenatal diagnosis usually consists of diagnosis of chromosomal aneuploidies and several single-gene disorders. Current prenatal screening of pregnant women considers only a few specific diseases such as trisomy 21, the genetic cause of Down syndrome. Although there are many noninvasive screening approaches available, an invasive approach is still the gold standard for prenatal diagnosis of genetic disorders. The risk of complications related to abortion is as high as 0.5% [3, 4], and the miscarriage rate after chorionic villus sampling is as high as 6.8% [5]. An ideal prenatal genetic diagnostic screening should be precise and noninvasive to meet the clinical demand.
Characteristics of Circulating Fetal DNA
In the plasma of pregnant women, DNA molecules are mainly short DNA fragments. The DNA size in pregnant women is significantly larger than that in nonpregnant women [6]. Since the study by Lo et al. [7] in 1997 revealed that cell-free fetal DNA exists in maternal plasma, much of fetal DNA nature has been uncovered. Fetal DNA can be traced in maternal plasma as early as day 18 after the embryo transfer [8]. As gestation progresses, the amount of fetal DNA increases, and usually reaches 3–6% Rui Shi, MD Department of Obstetrics and Gynecology Shanghai East Hospital, Tongji University School of Medicine No. 150, Ji Mo Road, Shanghai 200120 (China) E-Mail shezzle @ 126.com
Downloaded by: UCSF Library & CKM 169.230.243.252 - 12/11/2014 1:18:14 AM
Key Words Noninvasive prenatal diagnosis · Next-generation sequencing · Circulating fetal DNA
of total cell-free DNA on average [7]. The origin of this fetal DNA, as many documents indicate, derives from the maternal placenta [9, 10]. The apoptosis and necrosis of placental cells contribute to fetal DNA release. Fetal DNA molecules are shorter than maternal ones, most of which are less than 300 bp [6]. The circulating fetal DNA cannot persist for a long time, for its concentration dramatically decreases postpartum [11–13].
group. They detected β-thalassemia mutations using single-allele base extension reaction and mass spectrometry (MS) [26]. In this method, a set of well-designed primers is applied to specifically amplify mutant fetal allele, but not to the maternal allele (father is the carrier of the mutation), so the following mass spectrum assay is able to easily identify the presence of a mutant fetal allele. The PCR-MS strategy is also applicable to detect fetal trisomy 21 by determining the ratio between single nucleotide polymorphism (SNP) alleles in PLAC4 mRNA [27].
Diagnosis Using Circulating Fetal Nucleic Acids
74
Gynecol Obstet Invest 2014;77:73–77 DOI: 10.1159/000355693
Sequencing Circulating Fetal DNA
PCR assay combined with MS permits the chance of gene polymorphism detection, but it seems difficult to cover multiple genes or SNP alleles, so researchers attempt to directly sequence the maternal plasma DNA in order to acquire multiplex detection of the fetal genome noninvasively. Fetal DNA represents a small fraction in maternal plasma compared to predominant maternal DNA; therefore the sequencing detection should be sensitive enough to capture the ‘dim’ fetal DNA. By means of massively parallel genomic sequencing, trisomy 21, one of the most common genetic disorders featured in three 21 chromosomes, is successfully detected [28]. In this method, sequences that only match just one location in human genome are counted and classified to each chromosome. Individual chromosome has its own counting ratio. Due to the excessive chromosome 21, trisomy 21 is diagnosed by a high ‘dose’ of 21 unique counting ratio compared with the corresponding euploid reference sample. Similar methods have extended to other aneuploidy defect detections such as trisomy 13 and 18 [29].
Construct Fetal Genome Using Cell-Free DNA
Although fetal aneuploidy could be detected only with maternal circulating DNA using massively parallel sequencing [28], other mendelian diseases that severely affect the fetus remain unsolved. It seems impossible to cover hundreds of single-gene disorders merely with a low-precision strategy. Comparably, next-generation sequencing could provide higher throughput and sensitivity, offering the opportunity to reconstruct fetal genome noninvasively (table 1). Bell et al. [30] have attempted to apply this tool in preconception carrier screening, and they scanned for 448 severe recessive childhood diseases with just one detection. Xu /Shi
Downloaded by: UCSF Library & CKM 169.230.243.252 - 12/11/2014 1:18:14 AM
As the content of fetal nucleic acids can reach 13% of total DNA in maternal cell-free plasma [14], this gives the opportunity to detect information about the fetal genome. The simplest idea is to examine sex-linked diseases. Congenital adrenal hyperplasia, for instance, is a problem of virilization of the genitalia related only to female fetuses [15]. If genes specifically residing in Y chromosome sequences are not detected in maternal circulating DNA, the fetus is not at risk of suffering this disorder [16]. Theoretically, any Y chromosome-specific sequence such as Sox14 or Tbx3 [14] could be a sex determination marker in clinical detection, and generally it can reach 95.4% sensitivity and 98.6% specificity [17]. Similarly, the ones that do not exist in the mother’s genome but are carried by the fetus can also be used for analyzing genetic disease. Rhesus D (RHD) belongs to this category. It encodes rhesus blood group antigen D in RHD-positive fetus and will induce hemolysis of RHD incompatibility in RHD-negative mothers. Such qualitative assessments of maternal and fetal DNA in a specific genome region are convenient and reliable. The procedure of standard PCR is easy to perform, the accuracy is satisfactory (close to 100%) [18] and adapted to the analysis of other fetal genetic disorders such as myotonic dystrophy and β-thalassemia [19–21]. Although previous studies have shown that quantitative analysis of mRNA in maternal plasma using RT-PCR could monitor disease condition and progress [22–24], or even detect chromosome 21-encoded mRNA [25], it is not feasible and applicable in clinics because it is difficult to make a determined value (or relative multiples) to define a disease. PCR-based qualitative analysis can only define a small portion of genetic disorders. More single-gene disorders often involve gene polymorphisms or mutations, so the screening of genetic diseases demands the detection to the extent of the single base level, which requires improvement of detection technology and combination of multiple tools. This has been achieved by Dennis Lo’s
Color version available online
Fig. 1. Fetal genome in maternal circulat-
ing DNA. A human somatic cell contains two complete haploid sets. During meiosis, the two chromosomes of the mother or father are recombined and randomly assorted to the offspring. As a result, three haplotypes occur in maternal plasma: haplotype that the fetus inherited from the mother (red), haplotype that the fetus inherited from the father (green), and the maternal haplotype that is not transmitted to the fetus (yellow). Colors refer to the online version only.
Table 1. Prenatal detection using circulating fetal nucleic acids in maternal plasma
qPCR SRY gene, RHD gene, β-globin gene mutation
Potential risk congenital adrenal hyperplasia [15, 16, 35], rhesus D incompatibility [36], β-thalassemia [20, 21], myotonic dystrophy [19] preeclampsia [23, 24]
SRY mRNA, CRH mRNA PCR + MS Disease-specific DNA sequence (deletion or mutation) SNP of PLAC4 mRNA MPGS 36 bp of each plasma DNA molecule NGS 160 × average target coverage, 93% of nucleotides >20 × coverage 50 × coverage of haploid genome, >100 × coverage of exome ∼30 × coverage of genomic DNA
448 severe recessive childhood diseases [30] whole genomic analysis [31] whole genomic analysis [32]
The rationale that the second sequencing aiding noninvasive prenatal diagnosis is based on these premises is as follows: (1) blocks of DNA, which are long stretches or large fragments of genetic substance, are transmitted from parents to their offspring; (2) SNP, which is a single DNA base pair position with two different alleles of the mother’s genome, could be utilized to determine the mother’s two haplotypes, and (3) sequencing reads should be numerous to permit quantitative analysis at specific genomic regions.
Fan et al. [31] have shown a good example utilizing maternal blood sample to reconstruct the whole fetal genome. In their study, a sample of peripheral blood was obtained and separated into two parts: the cell part and the cell-free part. The former was to extract the mother’s DNA to directly determine maternal haplotypes. In this step, an SNP-linked DNA block is induced to discriminate alleles in the mother’s two haplotypes. Obviously, longer reads of sequencing permit longer segment mapping to the genome and consequently more reliable re-
Noninvasive Prenatal Diagnosis Using Next-Generation Sequencing
Gynecol Obstet Invest 2014;77:73–77 DOI: 10.1159/000355693
β-thalassemia [26] trisomy 21 [27] trisomy 21 [28, 37, 38], trisomy 18, and trisomy 13 [29]
75
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Approach and target
Color version available online
Fig. 2. Deducing fetal genome with mater-
nal peripheral blood. Full-length haplotypes of the mother’s genome are obtained from maternal peripheral blood and determined by SNP analysis. Fetal haplotypes are deduced from maternal plasma DNA, which is a mixture of maternal and fetal DNA. During the course of fetal genome analysis, deep sequencing of maternal circulating DNA is required to count the specific fetal alleles.
76
Gynecol Obstet Invest 2014;77:73–77 DOI: 10.1159/000355693
Noninvasive prenatal diagnosis has long been considered an ideal examination method not only for the pregnant woman but also her family. The new-generation sequencing technology opens up a possibility of screening hundreds of disease-susceptible genes once at the cost of only several milliliters of blood. No one can deny that it is a great progress technically, but it also means more caution and responsibility [34]. The more comprehensive the genetic information about the fetus, the stricter the standard of informing the patient, and we should make every effort to avoid any abuse related to decrypting the ‘ATCG’ of the little person we are expecting to welcome.
Acknowledgements This work was supported by a grant from National Natural Science Fund (81100124).
Disclosure Statement The authors declare no conflict of interest.
Xu /Shi
Downloaded by: UCSF Library & CKM 169.230.243.252 - 12/11/2014 1:18:14 AM
sults. The part of cell-free plasma contains circulating DNA whose features we have mentioned above. When sequencing this kind of DNA, each allele displays four portions in which two are from the mother and two from the fetus. As one haplotype of the fetus is inherited from the father and the other from the mother, indeed three types of haplotype exist in the cell-free DNA mixture (fig. 1). Because we could determine maternal whole genome sequences (two haplotypes) depending on the sequencing of the cell part DNA extracted from the mother’s peripheral blood, when the percentage of fetal DNA is clear (usually determined by male specific allele), we can easily deduce fetal SNP linkage alleles. Stringing these SNP blocks together manifests the construction of the fetal chromosome (fig. 2). Of note, deeper sequencing should be performed to provide enough data to count the SNPs at individual alleles. Testing the father’s DNA, even though this is not mandatory, would be an additional reference in determining the paternal haplotype inherited by the fetus [32]. One should keep in mind that this road map, in spite of the accurate forecast of fetal genetic secrets, is constructed based on three premises. As a matter of fact, the average de novo mutation rate is 1.20 × 10–8 per nucleotide per generation [33], and the father’s age contributes to the diversity in the mutation rate of SNPs; thus, the father’s genetic information should be added to enhance the analysis of the fetal genome.
References
Noninvasive Prenatal Diagnosis Using Next-Generation Sequencing
14 Nygren AO, Dean J, Jensen TJ, et al: Quantification of fetal DNA by use of methylationbased DNA discrimination. Clin Chem 2010; 56:1627–1635. 15 Rijnders RJ, der Schoot CE v, Bossers B, de Vroede MA, Christiaens GC: Fetal sex determination from maternal plasma in pregnancies at risk for congenital adrenal hyperplasia. Obstet Gynecol 2001;98:374–378. 16 Chiu RW, Lau TK, Cheung PT, Gong ZQ, Leung TN, Lo YM: Noninvasive prenatal exclusion of congenital adrenal hyperplasia by maternal plasma analysis: a feasibility study. Clin Chem 2002;48:778–780. 17 Devaney SA, Palomaki GE, Scott JA, Bianchi DW: Noninvasive fetal sex determination using cell-free fetal DNA: a systematic review and meta-analysis. JAMA 2011;306:627–636. 18 Finning KM, Martin PG, Soothill PW, Avent ND: Prediction of fetal D status from maternal plasma: introduction of a new noninvasive fetal RHD genotyping service. Transfusion (Paris) 2002;42:1079–1085. 19 Amicucci P, Gennarelli M, Novelli G, Dallapiccola B: Prenatal diagnosis of myotonic dystrophy using fetal DNA obtained from maternal plasma. Clin Chem 2000; 46: 301– 302. 20 Chiu RW, Lau TK, Leung TN, Chow KC, Chui DH, Lo YM: Prenatal exclusion of beta thalassaemia major by examination of maternal plasma. Lancet 2002;360:998–1000. 21 Li Y, Di NE, Vitucci A, Zimmermann B, Holzgreve W, Hahn S: Detection of paternally inherited fetal point mutations for beta-thalassemia using size-fractionated cell-free DNA in maternal plasma. JAMA 2005; 293: 843– 849. 22 Levine RJ, Qian C, Leshane ES, et al: Twostage elevation of cell-free fetal DNA in maternal sera before onset of preeclampsia. Am J Obstet Gynecol 2004;190:707–713. 23 Lo YM, Leung TN, Tein MS, et al: Quantitative abnormalities of fetal DNA in maternal serum in preeclampsia. Clin Chem 1999; 45: 184–188. 24 Ng EK, Leung TN, Tsui NB, et al: The concentration of circulating corticotropin-releasing hormone mRNA in maternal plasma is increased in preeclampsia. Clin Chem 2003;49: 727–731.
25 Oudejans CB, Go AT, Visser A, et al: Detection of chromosome 21-encoded mRNA of placental origin in maternal plasma. Clin Chem 2003;49:1445–1449. 26 Ding C, Chiu RW, Lau TK, et al: MS analysis of single-nucleotide differences in circulating nucleic acids: application to noninvasive prenatal diagnosis. Proc Natl Acad Sci USA 2004; 101:10762–10767. 27 Lo YM, Tsui NB, Chiu RW, et al: Plasma placental RNA allelic ratio permits noninvasive prenatal chromosomal aneuploidy detection. Nat Med 2007;13:218–223. 28 Chiu RW, Chan KC, Gao Y, et al: Noninvasive prenatal diagnosis of fetal chromosomal aneuploidy by massively parallel genomic sequencing of DNA in maternal plasma. Proc Natl Acad Sci USA 2008;105:20458–20463. 29 Chen EZ, Chiu RW, Sun H, et al: Noninvasive prenatal diagnosis of fetal trisomy 18 and trisomy 13 by maternal plasma DNA sequencing. PLoS One 2011;6:e21791. 30 Bell CJ, Dinwiddie DL, Miller NA, et al: Carrier testing for severe childhood recessive diseases by next-generation sequencing. Sci Transl Med 2011;3:65ra4. 31 Fan HC, Gu W, Wang J, Blumenfeld YJ, ElSayed YY, Quake SR: Non-invasive prenatal measurement of the fetal genome. Nature 2012;487:320–324. 32 Kitzman JO, Snyder MW, Ventura M, et al: Noninvasive whole-genome sequencing of a human fetus. Sci Transl Med 2012;4:137ra76. 33 Kong A, Frigge ML, Masson G, et al: Rate of de novo mutations and the importance of father’s age to disease risk. Nature 2012; 488: 471–475. 34 Goldstein DB: Growth of genome screening needs debate. Nature 2011;476:27–28. 35 Costa JM, Benachi A, Gautier E: New strategy for prenatal diagnosis of X-linked disorders. N Engl J Med 2002;346:1502. 36 Lo YM, Hjelm NM, Fidler C, et al: Prenatal diagnosis of fetal RhD status by molecular analysis of maternal plasma. N Engl J Med 1998;339:1734–1738. 37 Ehrich M, Deciu C, Zwiefelhofer T, et al: Noninvasive detection of fetal trisomy 21 by sequencing of DNA in maternal blood: a study in a clinical setting. Am J Obstet Gynecol 2011;204:205.e1–e11. 38 Chiu RW, Akolekar R, Zheng YW, et al: Noninvasive prenatal assessment of trisomy 21 by multiplexed maternal plasma DNA sequencing: large scale validity study. BMJ 2011;342: c7401.
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1 Antonarakis SE, Beckmann JS: Mendelian disorders deserve more attention. Nat Rev Genet 2006;7:277–282. 2 Costa T, Scriver CR, Childs B: The effect of Mendelian disease on human health: a measurement. Am J Med Genet 1985;21:231–242. 3 Tabor A, Philip J, Madsen M, Bang J, Obel EB, Norgaard-Pedersen B: Randomised controlled trial of genetic amniocentesis in 4606 low-risk women. Lancet 1986;1:1287–1293. 4 American College of Obstetricians and Gynecologists: ACOG Practice Bulletin No. 88, December 2007. Invasive prenatal testing for aneuploidy. Obstet Gynecol 2007; 110: 1459– 1467. 5 Jauniaux E, Pahal GS, Rodeck CH: What invasive procedure to use in early pregnancy. Baillieres Best Pract Res Clin Obstet Gynaecol 2000;14:651–662. 6 Chan KC, Zhang J, Hui AB, et al: Size distributions of maternal and fetal DNA in maternal plasma. Clin Chem 2004;50:88–92. 7 Lo YM, Corbetta N, Chamberlain PF, et al: Presence of fetal DNA in maternal plasma and serum. Lancet 1997;350:485–487. 8 Guibert J, Benachi A, Grebille AG, Ernault P, Zorn JR, Costa JM: Kinetics of SRY gene appearance in maternal serum: detection by real time PCR in early pregnancy after assisted reproductive technique. Hum Reprod 2003; 18: 1733–1736. 9 Chim SS, Tong YK, Chiu RW, et al: Detection of the placental epigenetic signature of the maspin gene in maternal plasma. Proc Natl Acad Sci USA 2005;102:14753–14758. 10 Masuzaki H, Miura K, Yoshiura KI, Yoshimura S, Niikawa N, Ishimaru T: Detection of cell free placental DNA in maternal plasma: direct evidence from three cases of confined placental mosaicism. J Med Genet 2004; 41: 289–292. 11 Lo YM, Zhang J, Leung TN, Lau TK, Chang AM, Hjelm NM: Rapid clearance of fetal DNA from maternal plasma. Am J Hum Genet 1999;64:218–224. 12 Rijnders RJ, Christiaens GC, Soussan AA, van der Schoot CE: Cell-free fetal DNA is not present in plasma of nonpregnant mothers. Clin Chem 2004; 50: 679–681, author reply 681. 13 Smid M, Galbiati S, Vassallo A, et al: No evidence of fetal DNA persistence in maternal plasma after pregnancy. Hum Genet 2003; 112:617–618.
Received: June 6, 2013 Accepted: September 17, 2013 Published online: October 26, 2013
Noninvasive Prenatal Diagnosis Using Next-Generation Sequencing Liang Xu b Rui Shi a a
Department of Obstetrics and Gynecology, and b Research Center for Translational Medicine, Shanghai East Hospital, Tongji University School of Medicine, Shanghai, China
Abstract Nowadays, prenatal diagnosis is necessary for pregnant women. For the parents who are expecting a child, the genetic test may provide the information whether they are carrying rare gene mutations and whether they are at risk of passing them onto their offspring. However, the ultimate determination of genetic diseases often requires invasive procedures such as amniocentesis and chorionic villus sampling, which may cause fetal miscarriage. A noninvasive type of prenatal diagnosis needs to be developed in clinical practice to dispel safety concerns. In this paper, we will review the technical advancement of using maternal circulating nucleic acids as the sample in noninvasive studies, and highlight the utilization of next-generation sequencing in the screening of genetic diseases. © 2013 S. Karger AG, Basel
There are more than 3,000 mendelian disorders [1] accounting for about 20% of deaths in infancy [2]. Besides the routine ultrasound examination concerning fetal de© 2013 S. Karger AG, Basel 0378–7346/13/0772–0073$38.00/0 E-Mail [email protected] www.karger.com/goi
velopment, for those who are at high risk of genetic disease, prenatal diagnosis usually consists of diagnosis of chromosomal aneuploidies and several single-gene disorders. Current prenatal screening of pregnant women considers only a few specific diseases such as trisomy 21, the genetic cause of Down syndrome. Although there are many noninvasive screening approaches available, an invasive approach is still the gold standard for prenatal diagnosis of genetic disorders. The risk of complications related to abortion is as high as 0.5% [3, 4], and the miscarriage rate after chorionic villus sampling is as high as 6.8% [5]. An ideal prenatal genetic diagnostic screening should be precise and noninvasive to meet the clinical demand.
Characteristics of Circulating Fetal DNA
In the plasma of pregnant women, DNA molecules are mainly short DNA fragments. The DNA size in pregnant women is significantly larger than that in nonpregnant women [6]. Since the study by Lo et al. [7] in 1997 revealed that cell-free fetal DNA exists in maternal plasma, much of fetal DNA nature has been uncovered. Fetal DNA can be traced in maternal plasma as early as day 18 after the embryo transfer [8]. As gestation progresses, the amount of fetal DNA increases, and usually reaches 3–6% Rui Shi, MD Department of Obstetrics and Gynecology Shanghai East Hospital, Tongji University School of Medicine No. 150, Ji Mo Road, Shanghai 200120 (China) E-Mail shezzle @ 126.com
Downloaded by: UCSF Library & CKM 169.230.243.252 - 12/11/2014 1:18:14 AM
Key Words Noninvasive prenatal diagnosis · Next-generation sequencing · Circulating fetal DNA
of total cell-free DNA on average [7]. The origin of this fetal DNA, as many documents indicate, derives from the maternal placenta [9, 10]. The apoptosis and necrosis of placental cells contribute to fetal DNA release. Fetal DNA molecules are shorter than maternal ones, most of which are less than 300 bp [6]. The circulating fetal DNA cannot persist for a long time, for its concentration dramatically decreases postpartum [11–13].
group. They detected β-thalassemia mutations using single-allele base extension reaction and mass spectrometry (MS) [26]. In this method, a set of well-designed primers is applied to specifically amplify mutant fetal allele, but not to the maternal allele (father is the carrier of the mutation), so the following mass spectrum assay is able to easily identify the presence of a mutant fetal allele. The PCR-MS strategy is also applicable to detect fetal trisomy 21 by determining the ratio between single nucleotide polymorphism (SNP) alleles in PLAC4 mRNA [27].
Diagnosis Using Circulating Fetal Nucleic Acids
74
Gynecol Obstet Invest 2014;77:73–77 DOI: 10.1159/000355693
Sequencing Circulating Fetal DNA
PCR assay combined with MS permits the chance of gene polymorphism detection, but it seems difficult to cover multiple genes or SNP alleles, so researchers attempt to directly sequence the maternal plasma DNA in order to acquire multiplex detection of the fetal genome noninvasively. Fetal DNA represents a small fraction in maternal plasma compared to predominant maternal DNA; therefore the sequencing detection should be sensitive enough to capture the ‘dim’ fetal DNA. By means of massively parallel genomic sequencing, trisomy 21, one of the most common genetic disorders featured in three 21 chromosomes, is successfully detected [28]. In this method, sequences that only match just one location in human genome are counted and classified to each chromosome. Individual chromosome has its own counting ratio. Due to the excessive chromosome 21, trisomy 21 is diagnosed by a high ‘dose’ of 21 unique counting ratio compared with the corresponding euploid reference sample. Similar methods have extended to other aneuploidy defect detections such as trisomy 13 and 18 [29].
Construct Fetal Genome Using Cell-Free DNA
Although fetal aneuploidy could be detected only with maternal circulating DNA using massively parallel sequencing [28], other mendelian diseases that severely affect the fetus remain unsolved. It seems impossible to cover hundreds of single-gene disorders merely with a low-precision strategy. Comparably, next-generation sequencing could provide higher throughput and sensitivity, offering the opportunity to reconstruct fetal genome noninvasively (table 1). Bell et al. [30] have attempted to apply this tool in preconception carrier screening, and they scanned for 448 severe recessive childhood diseases with just one detection. Xu /Shi
Downloaded by: UCSF Library & CKM 169.230.243.252 - 12/11/2014 1:18:14 AM
As the content of fetal nucleic acids can reach 13% of total DNA in maternal cell-free plasma [14], this gives the opportunity to detect information about the fetal genome. The simplest idea is to examine sex-linked diseases. Congenital adrenal hyperplasia, for instance, is a problem of virilization of the genitalia related only to female fetuses [15]. If genes specifically residing in Y chromosome sequences are not detected in maternal circulating DNA, the fetus is not at risk of suffering this disorder [16]. Theoretically, any Y chromosome-specific sequence such as Sox14 or Tbx3 [14] could be a sex determination marker in clinical detection, and generally it can reach 95.4% sensitivity and 98.6% specificity [17]. Similarly, the ones that do not exist in the mother’s genome but are carried by the fetus can also be used for analyzing genetic disease. Rhesus D (RHD) belongs to this category. It encodes rhesus blood group antigen D in RHD-positive fetus and will induce hemolysis of RHD incompatibility in RHD-negative mothers. Such qualitative assessments of maternal and fetal DNA in a specific genome region are convenient and reliable. The procedure of standard PCR is easy to perform, the accuracy is satisfactory (close to 100%) [18] and adapted to the analysis of other fetal genetic disorders such as myotonic dystrophy and β-thalassemia [19–21]. Although previous studies have shown that quantitative analysis of mRNA in maternal plasma using RT-PCR could monitor disease condition and progress [22–24], or even detect chromosome 21-encoded mRNA [25], it is not feasible and applicable in clinics because it is difficult to make a determined value (or relative multiples) to define a disease. PCR-based qualitative analysis can only define a small portion of genetic disorders. More single-gene disorders often involve gene polymorphisms or mutations, so the screening of genetic diseases demands the detection to the extent of the single base level, which requires improvement of detection technology and combination of multiple tools. This has been achieved by Dennis Lo’s
Color version available online
Fig. 1. Fetal genome in maternal circulat-
ing DNA. A human somatic cell contains two complete haploid sets. During meiosis, the two chromosomes of the mother or father are recombined and randomly assorted to the offspring. As a result, three haplotypes occur in maternal plasma: haplotype that the fetus inherited from the mother (red), haplotype that the fetus inherited from the father (green), and the maternal haplotype that is not transmitted to the fetus (yellow). Colors refer to the online version only.
Table 1. Prenatal detection using circulating fetal nucleic acids in maternal plasma
qPCR SRY gene, RHD gene, β-globin gene mutation
Potential risk congenital adrenal hyperplasia [15, 16, 35], rhesus D incompatibility [36], β-thalassemia [20, 21], myotonic dystrophy [19] preeclampsia [23, 24]
SRY mRNA, CRH mRNA PCR + MS Disease-specific DNA sequence (deletion or mutation) SNP of PLAC4 mRNA MPGS 36 bp of each plasma DNA molecule NGS 160 × average target coverage, 93% of nucleotides >20 × coverage 50 × coverage of haploid genome, >100 × coverage of exome ∼30 × coverage of genomic DNA
448 severe recessive childhood diseases [30] whole genomic analysis [31] whole genomic analysis [32]
The rationale that the second sequencing aiding noninvasive prenatal diagnosis is based on these premises is as follows: (1) blocks of DNA, which are long stretches or large fragments of genetic substance, are transmitted from parents to their offspring; (2) SNP, which is a single DNA base pair position with two different alleles of the mother’s genome, could be utilized to determine the mother’s two haplotypes, and (3) sequencing reads should be numerous to permit quantitative analysis at specific genomic regions.
Fan et al. [31] have shown a good example utilizing maternal blood sample to reconstruct the whole fetal genome. In their study, a sample of peripheral blood was obtained and separated into two parts: the cell part and the cell-free part. The former was to extract the mother’s DNA to directly determine maternal haplotypes. In this step, an SNP-linked DNA block is induced to discriminate alleles in the mother’s two haplotypes. Obviously, longer reads of sequencing permit longer segment mapping to the genome and consequently more reliable re-
Noninvasive Prenatal Diagnosis Using Next-Generation Sequencing
Gynecol Obstet Invest 2014;77:73–77 DOI: 10.1159/000355693
β-thalassemia [26] trisomy 21 [27] trisomy 21 [28, 37, 38], trisomy 18, and trisomy 13 [29]
75
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Approach and target
Color version available online
Fig. 2. Deducing fetal genome with mater-
nal peripheral blood. Full-length haplotypes of the mother’s genome are obtained from maternal peripheral blood and determined by SNP analysis. Fetal haplotypes are deduced from maternal plasma DNA, which is a mixture of maternal and fetal DNA. During the course of fetal genome analysis, deep sequencing of maternal circulating DNA is required to count the specific fetal alleles.
76
Gynecol Obstet Invest 2014;77:73–77 DOI: 10.1159/000355693
Noninvasive prenatal diagnosis has long been considered an ideal examination method not only for the pregnant woman but also her family. The new-generation sequencing technology opens up a possibility of screening hundreds of disease-susceptible genes once at the cost of only several milliliters of blood. No one can deny that it is a great progress technically, but it also means more caution and responsibility [34]. The more comprehensive the genetic information about the fetus, the stricter the standard of informing the patient, and we should make every effort to avoid any abuse related to decrypting the ‘ATCG’ of the little person we are expecting to welcome.
Acknowledgements This work was supported by a grant from National Natural Science Fund (81100124).
Disclosure Statement The authors declare no conflict of interest.
Xu /Shi
Downloaded by: UCSF Library & CKM 169.230.243.252 - 12/11/2014 1:18:14 AM
sults. The part of cell-free plasma contains circulating DNA whose features we have mentioned above. When sequencing this kind of DNA, each allele displays four portions in which two are from the mother and two from the fetus. As one haplotype of the fetus is inherited from the father and the other from the mother, indeed three types of haplotype exist in the cell-free DNA mixture (fig. 1). Because we could determine maternal whole genome sequences (two haplotypes) depending on the sequencing of the cell part DNA extracted from the mother’s peripheral blood, when the percentage of fetal DNA is clear (usually determined by male specific allele), we can easily deduce fetal SNP linkage alleles. Stringing these SNP blocks together manifests the construction of the fetal chromosome (fig. 2). Of note, deeper sequencing should be performed to provide enough data to count the SNPs at individual alleles. Testing the father’s DNA, even though this is not mandatory, would be an additional reference in determining the paternal haplotype inherited by the fetus [32]. One should keep in mind that this road map, in spite of the accurate forecast of fetal genetic secrets, is constructed based on three premises. As a matter of fact, the average de novo mutation rate is 1.20 × 10–8 per nucleotide per generation [33], and the father’s age contributes to the diversity in the mutation rate of SNPs; thus, the father’s genetic information should be added to enhance the analysis of the fetal genome.
References
Noninvasive Prenatal Diagnosis Using Next-Generation Sequencing
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Gynecol Obstet Invest 2014;77:73–77 DOI: 10.1159/000355693
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