孕妇血液中含有胎儿的全部基因组序列-----英文

DOI: 10.1126/scitranslmed.3001720 , 61ra91 (2010);2 Sci Transl Med , et al.Y. M. Dennis Lo Mutational Profile of the Fetus Maternal Plasma DNA Sequencing Reveals the Genome-Wide Genetic and http://stm.sciencemag.org/content/2/61/61ra91.full.html can be found at: and other services, including high-resolution figures,A complete electronic version of this article http://stm.sciencemag.org/content/suppl/2010/12/06/2.61.61ra91.DC1.html can be found in the online version of this article at: Supplementary Material http://stm.sciencemag.org/content/2/61/61ra91.full.html#ref-list-1 , 18 of which can be accessed free:cites 37 articlesThis article http://www.sciencemag.org/about/permissions.dtl in whole or in part can be found at: article permission to reproduce this of this article or about obtaining reprintsInformation about obtaining is a registered trademark of AAAS. Science Translational Medicinerights reserved. The title NW, Washington, DC 20005. Copyright 2010 by the American Association for the Advancement of Science; all last week in December, by the American Association for the Advancement of Science, 1200 New York Avenue (print ISSN 1946-6234; online ISSN 1946-6242) is published weekly, except theScience Translational Medicine o n D ec em be r 8 , 2 01 0 st m .s ci en ce m ag .o rg D ow nl oa de d fro m PRENATAL D IAGNOS I S Maternal Plasma DNA Sequencing Genome-Wide Genetic and Mutati of the Fetus . K I te e e ho A m . a molecular basis of this observation is not known. Better understand- allel sequencing to study the genomic sequence and size distribution ping ism (SNP) genotyping extracted from paternal S sample, with the Af- 0 system (table S1). The s (Fig. 1). We defined and mother were both Category 2 SNPs were both homozygous, but for the same allele. Category 3 SNPs were those in which the father which both the father end, on DNA extracted ), equivalent to an av- e, were aligned to the e (Hg18 NCBI.36). For amily, the fetus was an obligate heterozygote. The fetal SNP allele inherited from the father R E S EARCH ART I C L E o n D ec em be r 8 , 2 01 0 st m .s ci en ce m ag .o rg D ow nl oa de d fro m Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, New Territories, Hong Kong SAR, China. 4Sequenom Inc., San Diego, CA 92121–1331, USA. wide genetic map of a fetus from the maternal plasma DNA sequences and from information about the paternal genotype and maternal haplotype. were those in which the father was homozygo heterozygous. Category 5 SNPs were those in and the mother were heterozygous. Sequencing of plasma DNA We performed PE sequencing, 50 bp for each from maternal plasma. Reads (3.931 billion erage of 65-fold coverage of a human genom non–repeat-masked reference human genom each of the 45,392 category 1 SNPs in this f 1Centre for Research into Circulating Fetal Nucleic Acids, Li Ka Shing Institute of Health Sciences, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, New Territories, Hong Kong SAR, China. 2Department of Chemical Pathology, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, New Territories, Hong Kong SAR, China. 3Department of Obstetrics and Gynaecology, The of fetal DNA in maternal plasma. We further constructed a genome- was heterozygous and the mother was homozygous. Category 4 SNPs us and the mother was ing of this size difference might allow one to develop methods for the selective enrichment of fetal DNA from maternal plasma. It is also not known whether the entire fetal genome is represented in maternal plasma. Complete representation might make it possible to deduce a whole-genome genetic map, or even the entire genomic sequence, of a fetus noninvasively. However, this is a technically challenging task because most (about 90%) of the DNA in maternal plasma is derived from the mother, and the DNA molecules in plasma are short fragments (8). Here, we have used paired-end (PE) massively par- Single-nucleotide polymorphism genoty Genome-wide single-nucleotide polymorph for ~900,000 SNPs was performed for DNA and maternal buffy coat samples, and the CV fymetrix Genome-Wide Human SNP Array 6. SNPs were classified into different categorie category 1 SNPs as those for which the father homozygous, but for a different allele each. those in which the father and mother were consistently reported to be shorter than maternal DNA (8), but the gestation. A portion of the CVS DNA was stored for the study. Y. M. Dennis Lo,1,2* K. C. Allen Chan,1,2 Hao Sun,1,2 Eric Z Fiona M. F. Lun,1,2 Yama W. Zheng,1,2 Tak Y. Leung,3 Tze Charles R. Cantor,4 Rossa W. K. Chiu1,2 (Published 8 December 2010; Volume 2 Issue 61 61ra91) Cell-free fetal DNA is present in the plasma of pregnant women. primarily maternally derived DNA fragments. We sequenced a ma genomic coverage. We showed that the entire fetal and maternal g ma at a constant relative proportion. Plasma DNA molecules show miniscent of nuclease-cleaved nucleosomes, with the fetal DNA s peak relative to a 143-bp peak, when compared with maternal DN map and determined the mutational status of the fetus from the information about the paternal genotype and maternal haplotype genome-wide scanning to diagnose fetal genetic disorders prenat INTRODUCTION During pregnancy, a median of 10% of the DNA in the plasma of pregnant women is fetally derived (1, 2), offering opportunities for noninvasive prenatal diagnosis (3). Thus far, detection of paternally inherited traits [for example, sex (4) and rhesus D blood group status (5)] and fetal chromosomal aneuploidies (6, 7) is the main appli- cation. Yet, little is known about the physical and biological charac- teristics of fetal DNA in maternal plasma. Circulating fetal DNA is *To whom correspondence should be addressed. E-mail: loym@cuhk.edu.hk www.Scien Reveals the onal Profile Chen,1,2 Peiyong Jiang,1,2 . Lau,3 t consists of short DNA fragments among rnal plasma DNA sample at up to 65-fold nomes were represented in maternal plas- d a predictable fragmentation pattern re- wing a reduction in a 166–base pair (bp) . We constructed a genome-wide genetic aternal plasma DNA sequences and from Our study suggests the feasibility of using lly in a noninvasive way. RESULTS Clinical case We recruited a pregnant couple attending an obstetrics clinic for the prenatal diagnosis of b-thalassemia. The father was a carrier of the -CTTT 4–base pair (bp) deletion of codons 41/42, and the pregnant mother was a carrier of the A→G mutation at nucleotide −28 of the HBB gene (9). Blood samples were taken from the father and from the mother before chorionic villus sampling (CVS) at 12 weeks of should be readily detected as a unique sequence in maternal plasma ceTranslationalMedicine.org 8 December 2010 Vol 2 Issue 61 61ra91 1 pr h ec to 6 o t ve a o m na ze N m in R E S EARCH ART I C L E o n D ec em be r 8 , 2 01 0 st m .s ci en ce m ag .o rg D ow nl oa de d fro m ternal DNA was largely constant across t data have suggested that the GC bias aff total DNA in maternal plasma is likely related to the sequencing platform used ( indication of the differential representation ent GC content. Our data therefore suggest fetal and maternal genomes is relatively e High-resolution plasma DNA size an The size of each sequenced plasma DNA from the genome coordinates of the ends of the fetal and total sequences were determ (Fig. 2C) and individually for each chromoso dant total sequences (predominantly mater The most significant difference in the si fetal and the total DNA was that fetal D the 166-bp peak (Fig. 2C) and a relative pro The latter likely corresponded to the trimm where p is the number of sequenced reads of the fetal-specific allele (the A allele for the category 1 SNP in Fig. 1) and q is the read count of the other allele, which is shared by the maternal and fetal genomes (the C allele for the category 1 SNP in Fig. 1). The values of f determined for every chromosome were highly consist- ent (Table 1). The depth of coverage of fetal and maternal sequences (in 1-Mb windows) across the genome is plotted in Fig. 2B. It correlated with the GC content of each genomic window (fig. S2). The number of fetal sequences as a propor- tion of the total sequences in each win- dow was consistent with the fractional fetal DNA concentration determined on a 1). These data indicated that the relative and could be used for studying the dis- tribution of fetal DNA sequences across the genome in maternal plasma. Figure 2A shows the number of times the paternally inherited fetal alleles for the category 1 SNPs were observed in ma- ternal plasma as the depth of sequencing increased. With data from 3.931 billion reads, a fetal allele was observed at least once for 93.94% of these SNPs (table S2). These results were consistent with Poisson distribution predictions assuming that the whole fetal genome was evenly distributed in maternal plasma (fig. S1). The fractional fetal DNA concentration in the maternal plasma, f, can be calcu- lated from the sequencing data: f ¼ 2p pþ q ment from a nucleosome to its core particle of ~146 bp (12). From www.Scien chromosomal level (Table oportion of fetal and ma- e entire genome. Previous ting the measurement of be an analytical artifact , 7, 10, 11), rather than an f DNA molecules of differ- hat the distribution of the n in maternal plasma. lysis molecule can be deduced f the PE reads. The sizes ined for the whole genome e (fig. S3). The most abun- l) were 166 bp in length. distribution between the A exhibited a reduction in inence of the 143-bp peak. g of a ~20-bp linker frag- ~143 bp and below, the distributions of both fetal and total DNA demonstrated a 10-bp periodicity reminiscent of nuclease-cleaved nu- cleosomes (12). These data suggest that plasma DNA fragments are derived from the enzymatic processing of DNA from apoptotic cells. In contrast, size analysis of reads that mapped to the non–histone-bound mitochondrial genome did not show this nucleosomal pattern (Fig. 2C). These results provide a molecular explanation for the previously reported size differences between fetal and maternal DNA using Y chromo- some and selected polymorphic genetic markers (8, 13, 14), and show that such size differences exist across the entire genome. General principles for constructing a fetal genetic map After having demonstrated that the entire fetal genome was evenly rep- resented in maternal plasma, we attempted to construct a genome-wide genetic map of the fetus. Maternal plasma DNAmolecules are short frag- ments and the fetal sequences are in the minority. Here, we used the genetic structure of the parental genomes as scaffolds for assembling the fetal genetic map from the maternal plasma DNA sequences. The map resolution depends on the known resolution of the parental genomes. First, we used the category 2 SNPs (Fig. 1), in which the father and father can be regarded as category 3. Category 4 allows the inheritance status of the maternal haplotype to be studied. One application is the tracking of fetal inheritance of a haplotype block close to a mutation carried by the mother. Here, noninvasive fetal genomic analysis was carried out for a family undergoing prenatal diagnosis for b-thalassemia. Asterisk denotes that information on the ma- ternal haplotype is required for the RHDO analysis. Category 5 SNPs were not analyzed in this study, but might be useful for the prenatal diagnosis of autosomal recessive disorders with consanguineous parents or genetic diseases with a strong founder effect. Fig. 1. Noninvasive fetal genomic analysis from maternal plasma DNA. Parental SNP combinations can be grouped into five categories. Categories 1, 2, and 3 allow the basic parameters for maternal plasma DNA sequencing to be established, including the percentage coverage of the fetal genome, fractional concentration of fetal DNA, and sequencing error rate. Category 3 also allows the fetal in- heritance status of SNP alleles unique to the father to be studied. Mutations uniquely carried by the mother were both homozygous for the same allele, to estimate the error ceTranslationalMedicine.org 8 December 2010 Vol 2 Issue 61 61ra91 2 3% The th Supp n .55% le g in ied uirem on s as s NPs rent rna inhe . To ad i Ps w oth ch o bein 40 to 100 reads per SNP), and fetal-specific read sequencing depth (blue; range, 1 to 8 reads ve . S gr s w conc ana . 7 11.49 R E S EARCH ART I C L E o n D ec em be r 8 , 2 01 0 st m .s ci en ce m ag .o rg D ow nl oa de d fro m per SNP). (C) Size distribution of fetal DNA (blue curve), total DNA (red cur drial DNA (green broken curve). Numbers denote the DNA size at the peaks tions of the structural organization of a nucleosome are shown above the right, DNA double helix wound around a nucleosomal core unit with the cleavage shown; a nucleosome core unit with ~146 bp of DNA (red tape) and a nucleosomal core unit with an intact ~20-bp linker sequence. www.Scien ), and mitochon- chematic illustra- aph. From left to ites for nuclease ound around it; 21 10.87 22 11.19 X 11.10 Whole genome 11.43 ceTranslationalMedi 8 11.53 9 11.51 10 11.36 11 11.51 12 11.41 13 11.47 14 11.38 15 11.07 16 11.08 17 11.17 18 11.60 19 11.55 20 11.33 Fig. 2. Sequencing of fetal and total DNA in maternal plasma. (A) Depth of coverage of fetal-specific SNP alleles versus the number of sequenced reads. (B) Sequencing depth and GC content across the whole genome. Chromosome ideograms (outer ring) are oriented pter-qter in a clockwise direction (centromeres are shown in yellow). Other tracks (from outside to inside): GC content (green; range, 30 to 55%), total sequencing depth (red; range, rate of plasma DNA sequencing. For the 500,457 category 2 SNPs in this family, the fetus would be homozygous for the alleles concerned. The sequencing error rate was expressed as the number of reads with an un- expected allele as a proportion of all reads covering the category 2 SNP loci andwas 0.30 seen in 4.04% of seen more than o allele seen in at 4 11.49 5 11.66 6 11.43 2 3 cine.org 8 December 2010 11.57 11.59 1 11.57 Chromosome Fetal DNA concentration (%) different chromosomes calculated based on the lysis of category 1 SNPs for Table 1. Fractional entrations of fetal DNA had a 50% chance of g inherited by the fetus. erozygous and the m one of the alleles. Ea er was homozygous for f the two paternal alleles 129,835 category 3 SN here the father was het- allele that the fetus h nherited, we studied the in a stepwise fashion determine the paternal of the fetus We deduced the fetal ritance from each parent Deducing the pate l inheritance homozygous for diffe alleles (table S2). 81.06% of category 1 S , where both parents were the fetal allele detecti inherited fetal allele w ensitivity. The paternally een at least twice in only specific alleles, the req ent of two reads reduced However, when appl to the detection of fetal- ast two reads, resultin a specificity of 99.45%. e category 2 SNP loci. ce to be scored, only 0 ose that an allele must be of such SNPs had a false (99,467/32,828,899). se unexpected alleles were Vol 2 Issue 61 61ra91 3 e rn lo d e es re co te al y pl I a ba R E S EARCH ART I C L E o n D ec em be r 8 , 2 01 0 st m .s ci en ce m ag .o rg D ow nl oa de d fro m Fig. 3. Relative haplotype dosage (RHDO) analysis. (A) In type a SNPs, pa the maternal alleles on Hap I. In type b SNPs, paternal alleles are identic Hap II. If the fetus inherits Hap I from the mother, it is homozygous for t type b SNPs. (B) For type a SNPs, Hap I is overrepresented in maternal there is no significant difference between the cumulative counts for Hap the fetus in this case inherits Hap II from the father, the sequential pro duces the inheritance of Hap I from the mother. www.Scien in the study that the approach can detect “artificial” maternal meiotic recombina- tions. If RHDO is used clinically, the maternal haplotype can be deduced with- out any fetal information by comparison with genotype information for other family members. Figure 3 shows the RHDO process. The two maternal haplotypes are Hap I rnal alleles are identical to to the maternal alleles on pe a and heterozygous for asma. (C) For type b SNPs, nd Hap II SNPs. Given that bility ratio test (SPRT) de- ceTranslationalMedicine.org 8 differences in genotyping and sequencing al-specific alleles observed once or twice ci, respectively, were false positives. Given ata indicated that the fetus inherited the r as the homozygous maternal allele in for fetal allele detection using the one- 95.51 and 99.42%, respectively (table nsistent with the category 2 SNP results. Deducing the maternal inheritance of the fetus Formaternal inheritance, we analyzed the category 4 SNPs (Fig. 1), where themother was heterozygous and the father was homozygous, and asked whether a slight allelic imbalance was present in maternal plasma. An imbalance would indicate that the fetus was homozygous for one mater- nal allele. This analysis could, in principle, be carried out for each SNP with locus- specific approaches such as digital poly- merase chain reaction (PCR) (15).However, for genome-wide random sequencing, the depth of coverage needed and hence the costs would be prohibitive for clinical use. Using nearby SNP alleles on the same maternal chromosome as a haplotype, wedeveloped anewapproach todetermine whether there was a relative haplotype dosage (RHDO) imbalance in maternal plasma. Because of meiotic recombina- tion, the final maternally derived hap- lotype inherited by the fetus is a mosaic of the two original maternal haplotypes. Using RHDO analysis, the combination of alleles inherited by the fetus from its mother can then be deduced as a series of inheritance blocks. The resolution for detecting this depends on the number and distribution of genetic markers known for the mother’s genome. In this proof-of-concept study, we de- duced the maternal haplotype information needed for RHDO analysis with genotype information obtained from microarray analysis of the CVS. This precluded the direct observation of maternal meiotic recombinations, but we do show later 53,070 loci. The CVS genotype data indicated that the fetus inherited the paternal-specific alleles in 65,018 category 3 SNPs. Such paternally inherited fetal alleles were observed at least once in 61,049 (that is, 93.90%) and at least twice in 52,697 (that is, 81.05%) loci, in good agree- ment with the category 1 SNP values (table S2). If we assume that the that the CVS genotyping same allele from the fath 64,817 loci, the specificiti and two-read criteria we S2). These error rates are The paternal-specific allele (as illustrated by the C allele in SNP category 3; Fig. 1) was detected at least once among the sequenced reads covering 63,962 category 3 loci and at least twice covering genotyping was perfect, th meant that sequenced pate in 2913 and 373 category 3 and Hap II (Fig. 3A). Hap I is the actual December 2010 Vol 2 Issue 61 61ra91 4 ap rn ou et ). n c ( ex yz o a fi R E S EARCH ART I C L E o n D ec em be r 8 , 2 01 0 st m .s ci en ce m ag .o rg D ow nl oa de d fro m Fig. 4. SPRT classification. (A and B) SPRT classification process for RHDO type b SNPs in a region close to the pter of chromosome 1. The classi direction from the telomeric end to the centromere. See also Tables 2 an www.Scien RHDO classifications for the type a and b SNPs, respectively (0.6 and 1.2% of these classifications). With the current sequencing coverage, the mean sizes of type a and b classification segments were 659,000 and 768,000 bp, respectively. The presence of two meiotic recombina- tions within such distances would be un- likely (19). Therefore, we proposed to accept a s