A method for the detection of chromosomal aneuploidies

Method of detecting chromosome aneuploidy

The present invention relates to pregnancy detection, and particularly relates to a method for detecting chromosome aneuploidy.

Chromosomal aneuploidy is an important cause of fetal malformations before and after birth. The assessment of chromosomal aneuploidy is usually related to the study of fetal viability and the diagnosis before birth. Methods to detect and describe chromosomal aneuploidy include metaphase mitotic chromosome karyotype analysis, fluorescence in situ hybridization (FISH) (Homer, J. et al.,<i>Prenat Diagn</i> 23: 566-571 (2003)), quantitative fluorescent polymerase chain reaction (PCR) (Mann, K.<i>Methods Mol Med</i> 92: 141-156 (2004)), gene dose PCR (Zimmermann, B. et al.,<i>Clin Chem</i> 48: 362-363 (2002) and array-based comparative genomic hybridization (CGH) (Hu, DGet al.,<i>Mol Hum Reprod</i>(2004))。

Fetal chromosomal aneuploidy is a known important cause of fetal death and causes 50% of spontaneous abortions in the first trimester of pregnancy (Chitty, L.<i>Br Med Bull</i> 54: 839-856 (1998)). The survival of the fetus is particularly related to certain types of chromosomal aneuploidy. Trisomy 21 or Downs syndrome is the most common autosomal aneuploidy, and one in 800 surviving babies born with Downs syndrome (Hook, EB<i>Lancet</i> 2:169-172 (1981)). Trisomy 21 is the main reason for couples to choose prenatal check-ups. At present, the exact examination of fetal chromosome 21 trisomy and other chromosomal aneuploidy is mainly through invasive methods such as amniocentesis and chorionic villus sampling (CVS) to obtain fetal genetic material for genetic analysis. Due to the invasive nature of the method, this method has a certain risk of spontaneous abortion. Other non-invasive methods classify pregnancy based on the risk of a fetus with trisomy 21. Invasive examinations are only recommended when the substantial risk of the fetus during pregnancy is greater than the risk of miscarriage caused by invasive diagnostic methods. Current risk classification strategies include maternal age analysis, maternal serum biochemical markers and fetal ultrasound characteristics analysis (Nicolaides, KH et al.,<i>Prenat Diagn</i> 22：308-315(2002))。

In order to obtain better sensitivity and specificity, various combinations of markers and methods have been evaluated (Wald, NJet al.,<i>Prenat Diagn</i> 17: 821-829 (1997)), including three inspections and four inspections (Wald, NJet al.,<i>Lancet</i> 361:835-836 (2003)), comprehensive testing (Wald, NJet al.,<i>N Engl J Med</i> 341:461-467 (1999)) and screening in the first trimester of pregnancy (Wapner, R. et al.,<i>N Engl J Med</i> 349: 1405-1413 (2003)). The currently used serum biochemical markers include α-fetoprotein, unconjugated estriol, total or free β-human chorionic gonadotropin, inhibin-A, pregnancy-associated plasma protein-A (PAPP-A) ). For pregnancies with a high risk of miscarriage indicated by these test features, amniotic fluid diagnosis or CVS is finally recommended.

Recently, the discovery of circulating extracellular free fetal nucleic acid in maternal plasma provides an alternative source of fetal genetic material samples required for non-invasive examination (Lo, YMD et al.,<i>Lancet</i><b> 350</b>: 485-487 (1997); Poon, LLM et al.,<i>Clin Chem</i><b> 46</b>: 1832-1834 (2000)). In addition, it has been shown that the concentration of circulating fetal DNA in the plasma of women with trisomy 21 fetuses is significantly higher than the concentration of circulating fetal DNA in the plasma of women with euploid fetuses (Lo, YMD et al.,<i>Clin Chem</i><b> 45</b>:1747-1751(1999); Zhong,XYet al.,<i>Prenat Diagn</i><b> 20</b>: 795-798 (2000)). Recently, circulating fetal RNA has been shown to be a promising and gender-independent fetal nucleic acid marker in maternal plasma (Ng, EKO et al.,<i>Clin Chem</i><b> 49</b>: 727-731 (2003); Ng, EKO et al.,<i>Proc Natl Acad Sci USA</i><b> 100</b>: 4748-4753 (2003)). Therefore, for pregnancy risk classification, the quantification of circulating fetal nucleic acid can be used as another effective prenatal detection marker.

It has been shown that mRNA transcripts of placental manifestations can be detected in maternal plasma, such as those encoding human placental prolactin (hPL), human chorionic gonadotropin β subunit (β hCG) (Ng, EKO et al.,<i>Clin Chem</i><b> 49</b>: 727-731 (2003)), corticotropin releasing hormone (CRH) (Ng, EKO<i>et al.,Proc Natl Acad Sci USA</i><b> 100</b>: 4748-4753 (2003b)), tissue factor pathway inhibitor (TFPI2), KiSS-1 metastasis inhibitor (KISS1) and placenta specific 1 (PLAC1) (Tsui, NBY et al.,<i>J Med Genet</i><b> 41</b>: 461-7 (2004)). It has been known that these mRNA types derived from the placenta are pregnancy-specific (Ng, EKO et al.,<i>Proc Natl Acad Sci USA</i><b> 100</b>: 4748-4753 (2003); Tsui, NBY et al.,<i>J Med Genet</i><b> 41</b>: 461-7 (2004)). In particular, there have been reports about the abnormal increase in the concentration of CRH mRNA in maternal plasma in preeclamptic pregnancy (Ng, EKO et al.,<i>Clin Chem</i><b> 49</b>: 727-731 (2003)). Because these placental markers are pregnancy-specific but gender and polymorphism independent, they are useful in non-invasive prenatal examinations for all pregnancies.

Chromosome aneuploidy changes the dose of genes located on the aneuploidy chromosomes. The modified gene dosage can be expressed by the abnormal allele ratio of the gene. The abnormal allele ratio is represented by the abnormal ratio of allele polymorphisms in the RNA transcripts of the locus located on the aneuploid chromosome. An example of such a polymorphism is a single nucleotide polymorphism (SNP), in which when the chromosomal aneuploidy involved occurs, the ratio of SNP alleles is abnormal. Therefore, a reference range of the RNA-SNP ratio of a normal pregnancy can be established, and when a deviation from the reference range is observed, it can be determined that the fetus has chromosome 21 trisomy. Compared with conventional cytogenetic analysis methods, the technology proposed by the present invention does not require pre-culture of fetal cells, thus shortening the analysis time. In addition, maternal blood samples can be obtained through non-invasive methods, thus reducing the potential harm that may be caused to the fetus and the mother.

In one embodiment, the present invention provides an improved method for detecting the chromosomes of the fetus in the pregnant woman by comparing the allele ratio of the fetus's RNA transcript in the pregnant woman with the corresponding ratio of the pregnant woman with a chromosomal normal fetus. abnormal. In this method, the use of the ratio greatly improves the sensitivity of detecting fetal chromosomal abnormalities, especially when compared with the total concentration of only a certain allele or all RNA transcripts.

The first step of the method involves determining the allele ratio of the RNA transcripts of the fetus in the pregnant woman. This is accomplished by obtaining a biological sample containing RNA from the pregnant woman, wherein the biological sample containing RNA contains fetal RNA. The alleles are then distinguished from RNA transcribed from at least one locus on at least one chromosome of interest, and then the allele ratio of the RNA transcript is determined. The second step involves comparing the allele ratio obtained from the pregnant woman with the allele ratio of a standard control, which refers to a corresponding biological sample obtained from a pregnant woman with a chromosomal normal fetus The average allele ratio of fetus, wherein, compared with the standard control, the increase or decrease of the allele ratio indicates that the risk of fetal chromosomal abnormality is increased.

In some embodiments, the present invention provides a method that can be used on one of chromosomes 21, 18, and 13 trisomy. In another embodiment, the chromosomal abnormality is trisomy 21. In another embodiment, the chromosomal abnormality is chromosome 13 trisomy. In another embodiment, the chromosomal abnormality is chromosome 18 trisomy. In another embodiment, the chromosomal abnormalities involve X chromosomes and Y chromosomes.

In another embodiment, the present invention provides a method, wherein the biological sample obtained from the step of obtaining a biological sample containing RNA includes maternal blood, maternal plasma or serum, amniotic fluid, chorionic villus samples, derived from pre-implantation embryos Living tissue materials, fetal nucleated cells or fetal cell fragments isolated from maternal blood, maternal urine, maternal saliva, womens reproductive tract cleansing and abdominal puncture samples. In another embodiment, the biological sample is maternal blood. In another embodiment, the biological sample is a chorionic villus sample. In another embodiment, the biological sample contains cellular components and cell fragments from maternal blood.

In another embodiment, the present invention provides a method wherein the fetal RNA obtained from the step of obtaining a biological sample containing RNA is derived from the placenta.

In another embodiment, the present invention provides a method, wherein the step of identifying the RNA allele involves reverse transcription polymerase chain reaction (RT-PCR).

In another embodiment, the present invention provides a method, wherein the steps of identifying the RNA alleles and/or determining the ratio of different alleles are accomplished by using the following methods, such as primer extension reaction, mass spectrometry, using at least Hybridization of one probe, hybridization using at least one fluorescently labeled probe, direct sequencing, selection and sequencing, and electrophoresis.

In some embodiments, the present invention provides a method, wherein the allele step involves identifying RNA alleles, determining the ratio of different alleles, and comparing the ratio of the previous step with a standard through sequence variation Compare. In another embodiment, the sequence variation refers to a single nucleotide polymorphism (SNP). In another embodiment, the sequence variation refers to an insertion/deletion polymorphism. In another embodiment, sequence variation refers to a simple tandem repeat polymorphism.

In another embodiment, the present invention provides a method, wherein the RNA is transcribed from a chromosome selected from the group consisting of chromosome 21, chromosome 18, chromosome 13, X chromosome and Y chromosome. In another embodiment, the RNA is transcribed from chromosome 18. In another embodiment, the RNA is transcribed from chromosome 13.

In another embodiment, the present invention provides a method wherein the level of expression of the RNA in the placenta is twice or more higher than the level of expression in maternal blood. In another embodiment, the expression level of the RNA in the placenta is 5 times or more higher than the expression level in the maternal blood. In another embodiment, the expression level of the RNA in the placenta is 10 times or more higher than the expression level in the maternal blood.

In another embodiment, the present invention provides a method, wherein the RNA is mRNA. In another embodiment, the present invention provides a method, wherein the RNA is selected from collagen VI α 1 (<i>COL6A1</i>), superoxide dismutase 1 (<i>SOD1</i>), collagen VI α 2(<i>COL6A2</i>), mitochondrial ATP synthase O subunit (<i>ATP5O</i>), BTG family member 3 (<i>BTG 3</i>), a disintegrin-like and metalloproteinase 1 (reprolysin type) containing thrombospondin type 1 motif (<i>ADAMTS1</i>), β-position APP-Clease 2 (<i>BACE2</i>), intersectin1(<i>ITSN1</i>), amyloid β (A4) precursor protein (<i>APP</i>), ATP synthase, H⁺Transport, mitochondrial F0 complex, subunit F6(<i>ATP5J</i>), Down syndrome discriminant region gene 5 (<i>DSCR5</i>), placenta specific 4 (<i>PIAC4</i>), speculated protein BC005107 (<i>LOC90625</i>), ribosomal protein L17 (<i>RPL17</i>), Serine protease inhibitor branch B (ovalbumin) component 2 (<i>SERPINB2</i>), Collagen IV α 2(<i>COL4A2</i>) Is transcribed from at least one locus. In another embodiment, the RNA is transcribed from a locus that contains a single nucleotide polymorphism. In another embodiment, the RNA is transcribed from collagen VI α 1 (<i>COL6A1</i>) And collagen VI α 2(<i>COL6A2</i>) At least one locus. In another embodiment, the RNA transcribed from the SNP is^Arg850_Hisor^Ser932_Serof<i>COL6A1</i>Locus. In another embodiment, the RNA transcribed from the SNP is^Val728_Valof<i>COL6A2</i>Locus.

In another embodiment, the present invention provides a method, wherein said RNA is transcribed from placenta-specific 4 (<i>PLAC4</i>) Locus. In another embodiment, the RNA is transcribed from<i>PLAC4</i>Any different transcripts of genes, such as AF269287, AK027868, AK092431, BC093685, BC101615, BC101617, L13197, NM_182832 and LOC191585. In another embodiment, the RNA is transcribed from a single nucleotide polymorphism or an insertion-deletion polymorphism<i>PLAC4</i>Gene. These polymorphisms can be selected from rs3804026, rs4818219, rs9977003, rs7844, rs9015, rs13643, rs9305729, rs9305730, rs5019195, rs5019194, rs5844069, rs1049904, rs16998089, rs12482116, rs11909439, rs7278659, rs12106409, rs2106183584, rs12106401 rs8130833, rs10222145 and rs9981478, or others located in<i>PLAC4</i>Other polymorphisms within the locus, such as<i>PLAC4</i>-41471145 and<i>PLAC4</i>-41476236。

In some embodiments, the invention provides a method, wherein the woman is in the first trimester of pregnancy. In another embodiment, the woman is in the third or last trimester of pregnancy.

In another embodiment, the present invention provides a method, wherein if the allele ratio of the fetal RNA transcript of a pregnant woman is higher or lower than 1 standard deviation of the standard control, then the step of comparing represents the chromosomal abnormality of the fetus Increased risk. In another embodiment, if the allele ratio of the fetal RNA transcripts of the pregnant woman is higher or lower than 2 standard deviations of the standard control, then the step of comparing represents an increased risk of the fetal chromosomal abnormality. In some embodiments, if the allele ratio of the fetal RNA transcript of the pregnant woman is higher or lower than 3 standard deviations of the standard control, then the step of comparing represents an increased risk of the fetal chromosomal abnormality.

In another embodiment, the present invention provides a kit for detecting chromosomal abnormalities in a pregnant woman's fetus. One component of the set is the primer used to amplify the region of interest. Another component of the set is a standard control representing the average proportion of alleles in the corresponding biological samples obtained from pregnant women with chromosomal normal fetuses. In another embodiment, the third component of the kit is a hybridization probe that distinguishes different alleles of each RNA type.

I. Definition

In this specification, the term "chromosomal abnormality" usually refers to a state of chromosomal abnormality, in which the number of chromosomes is not an integral multiple of the normal haploid number: it is usually either with an extra chromosome or a chromosome is missing. "Chromosomal abnormality" can sometimes mean that one or more chromosomal parts are not an integer multiple of the normal haploid number. The cause is, for example, a chromosomal translocation. A common chromosomal abnormality is chromosomal aneuploidy. The common form of chromosomal aneuploidy is trisomy, which has an extra chromosome. For example, trisomy of chromosome 18 is a chromosomal abnormality, that is, the third chromosome 18 is found in the cell, and the patient with the third chromosome 21 found in the cell has trisomy 21. Chromosome translocations (such as the translocation of chromosomes 21 and 14, which results in part of chromosome 14 being replaced by additional chromosome 21) can cause part of chromosome 21 trisomy.

In this specification, the term "biological sample containing RNA" refers to a biological sample containing ribonucleic acid (RNA) (for example, as described below). RNA refers to a polynucleotide having a sequence corresponding to at least a part of the preselected site of the human genome. RNA in this specification includes mRNA, ribosomal mRNA and small RNA. RNA can be a protein coding sequence such as mRNA, or a non-coding sequence such as ribosomal RNA, small RNA or other transcription sequences with no detailed defined function. mRNA is transcribed from the DNA of genes, and proteins can be translated from it through the action of ribosomes. Ribosomal RNA (rRNA) is a non-coding RNA that cannot be translated into protein. Small RNAs (miRNAs) are a subtype of "small molecule RNAs" that are distinguished by their source rather than their function. Small RNAs are less than 30 nucleotides in length and are transcribed from DNA but not translated into protein. Those skilled in the art will understand that other types of RNA are also useful in the present invention.

In this specification, the terms "fetus", "placental origin" and "placental manifestation" refer to the source of a certain type of RNA that can be detected in biological samples derived from pregnant women, such as blood. For example, fetal RNA types are transcribed from fetal DNA sequences. The placental origin or placental expression RNA is also a type of fetal RNA. Those skilled in the art should understand that other types of fetal RNA are also useful in the present invention. Placental-derived or placental-expressed RNA is fetal RNA transcribed in the placenta.

In this specification, the term "identifying RNA alleles transcribed from at least one locus on at least one chromosome of interest" refers to detecting specific RNA alleles transcribed from a specific locus on the chromosome And quantitative. There are many ways to detect and quantify alleles, including the use of hybridization probes and real-time quantitative polymerase chain reaction (QRT-PCR). Other methods include the use of mass spectrometry (MS), electrophoresis, pyrosequencing, primer extension microarrays, wafers, and sequencing.

In this specification, the term "allele ratio" refers to the ratio of one allele population to another allele population in a biological sample. In some cases, it is possible to have three alleles at a specific location in a trisomy fetus. In this case, "allele ratio" refers to the ratio of any one allele population to another allele population, or the ratio of any one allele population to the other two allele populations.

In this specification, the term "standard control" refers to those suitable for use in the method of the present invention, in order to compare a specific locus, such as<i>COL6A1</i>，<i>SOD1</i>，<i>COL6A2</i>，<i>ATP5O</i>，<i>BTG3</i>，<i>ADAMTS1</i>，<i>BACE2</i>，<i>ITSN1</i>，<i>APP</i>，<i>ATP5J</i>，<i>DSCR5</i>，<i>PLAC4</i>，<i>LOC90625</i>，<i>RPL17</i>，<i>SERPINB2 or COL4A2</i>The allele ratio of the transcribed RNA-SNP is measured on the sample. Such a sample contains the ratio of known RNA-SNP alleles transcribed from a specific locus, which can accurately reflect the average ratio of such RNA-SNP alleles in pregnant women with chromosomal normal fetuses . For those skilled in the art, the standard control can also represent an average ratio, a median ratio, or other useful ratios. The measurement of the standard control will be described in detail later.

In this specification, the term "pregnant woman" refers to a group of pregnant women with normal chromosomal fetuses. Taking into account certain characteristics, such as the ratio of RNA alleles transcribed from the site of interest, it can represent a randomly selected one. Group of women with fetuses with normal chromosomes. The group selected in this way should contain a sufficient number of women so that the average, average, median or other mathematical relationship of the RNA allele ratios transcribed from the sites of interest of these women can be reasonably accurate Sex reflects the ratio of RNA alleles in the general population of healthy pregnant women with normal fetuses. The mothers who are at risk of fetal chromosomal aneuploidy can be screened out in the first trimester of pregnancy, that is, about 13 weeks of gestation. Such mothers can also be screened out in the third or last trimester of pregnancy. The third trimester of pregnancy refers to the period from 14 weeks to 27 weeks of pregnancy. The last trimester of pregnancy refers to the period from the 28th week of pregnancy to the end of pregnancy, which is about 40 weeks. In addition, the preferred age of pregnancy depends on the RNA marker used in the test.

In this specification, the term "normal chromosomes" means that the number of chromosomes is an integer multiple of the number of normal haploids, such as 2 times the number of chromosomes in a single coat, and the number of each chromosome is the same (except for sex chromosomes, if there are two different males) The sex chromosomes, X and Y, each have only one copy).

In this specification, the term "increase or decrease in the standard control ratio" refers to a positive or negative change compared to the standard control ratio. The increase is preferably at least 10%, more preferably at least 50%, and most preferably at least 100%. Similarly, the reduction is preferably at least 10%, more preferably at least 50%, and most preferably at least 90%. In addition, the increase or decrease of the ratio may have at least one standard deviation from the standard control ratio. The increase or decrease can be at least 2 standard deviations from the standard control ratio. The increase or decrease can be at least 3 standard deviations from the standard control.

In this specification, the term "single nucleotide polymorphism (SNP)" refers to a nucleic acid sequence variation involving a single nucleotide change. The SNPs useful in the present invention include SNPs that appear in the corresponding RNA transcripts transcribed from the locus of interest, the locus including collagen VI α 1 (<i>COL6A1</i>), superoxide dismutase 1 (<i>SOD1</i>), collagen VI α 2(<i>COL6A2</i>), mitochondrial ATP synthase O subunit (<i>ATP5O</i>), BTG family member 3 (<i>BTG3</i>), a disaggregin-like and metalloproteinase I containing thrombospondin type 1 motif (<i>ADAMTS1</i>), β-position APP cleavage enzyme 2 (<i>BACE2</i>), intersectin 1(<i>ITSN1</i>), amyloid β (A4) precursor protein (<i>APP</i>), ATP synthase, H⁺Transport, mitochondrial F0 complex, subunit F6(<i>ATP5J</i>), Down syndrome discriminant region gene 5 (<i>DSCR5</i>), placenta specific 4 (<i>PLAC4</i>), speculated protein BC005107 (<i>LOC90625</i>), ribosomal protein L17 (<i>RPL17</i>), Serine protease inhibitor branch B (ovalbumin) member 2 (<i>SERPINB2</i>) And collagen IV α 2(<i>COL4A2</i>). Such SNPs are called RNA-SNPs because they involve a single nucleotide change on RNA. The alleles of the RNA-SNPs can thus be used in the present invention to determine the ratio of RNA-SNP alleles in the biological samples of pregnant women, and compare the ratios with those obtained from each biological sample of pregnant women with healthy fetuses. To compare. Those skilled in the art should understand that other markers and insertion/deletion polymorphisms are equally useful in the present invention.

In this specification, the term "maternal blood" refers to a blood sample or product from a pregnant woman or a woman who is tested for pregnancy. The term also includes whole blood or any blood component. "Maternal blood" includes plasma and serum. Maternal blood samples that are essentially free of cells are called "cell-free" and generally do not contain platelets.

In this specification, the term "cell components or cell fragments" refers to the part of cells remaining in a biological sample, including platelets, apoptotic bodies and syncytiotrophoblast microparticles.

II. The method of the present invention

The present invention develops a method for genetic determination of fetal chromosomal aneuploidy using the pregnancy specificity of fetal expression transcripts, and thus establishes a non-invasive diagnostic method. In one embodiment, the fetal manifestation transcript is manifested in the placenta. In particular, the present invention can detect single nucleotide polymorphisms (SNPs) on RNA transcripts with tissue-specific expression patterns encoded by genes on aneuploid chromosomes. Other polymorphisms such as insertion/deletion polymorphisms and simple tandem repeat polymorphisms can also be detected by the method of the present invention. The status of the locus is determined by analyzing the ratio of informative SNPs transcribed from the RNA of the locus of interest. In short, the present invention compares the allele ratios between the site-specific polymorphic sites on the tissue-specific RNA transcripts of aneuploid fetuses and euploid fetuses.

Therefore, the present invention can be applied to the prenatal diagnosis of chromosome 21 trisomy, which involves the analysis of informative SNPs on the RNA transcripts transcribed from the chromosome 21 locus with placental tissue expression. Then, the ratio of the informational SNPs of the placental expression RNA transcripts in the maternal blood is compared to determine the trisomy of fetal chromosome 21. The fetal specificity of the marker in maternal blood is manifested by placental tissue, and the aneuploidy state is determined by the abnormal ratio of information SNPs in the RNA transcript.

Based on the analysis of the placental expression RNA-SNPs encoded by the locus on chromosome 21, the method of the present invention can be used for genetic determination of fetal chromosome 21 trisomy and its prenatal diagnosis. The RNA-SNP alleles can be identified by, for example, real-time quantitative polymerase chain reaction (QRT-PCR) analysis, and the ratio between the gene doses specifying the locus can be determined. In other embodiments, the RNA-SNP alleles can be identified by primer extension followed by mass spectrometry (MS).

A. Chromosomal abnormalities detectable using the method of the present invention

The present invention provides methods for determining chromosomal aneuploidy such as trisomy 21. This method can also detect other fetal aneuploidies, such as those that appear on chromosomes 18, 13, X and Y or involve these chromosomes. When analyzing maternal blood, this method can non-invasively detect fetal chromosomal aneuploidy. The use of the RNA-SNP detection goes beyond the scope of detecting chromosomal abnormalities and extends to detecting other genetic variations of the fetus, such as paternal genetic polymorphisms and mutations.

B. Useful biological samples for detecting fetal chromosomal abnormalities in the present invention

The first step in implementing the present invention is to obtain biological samples from pregnant women who are suitable for being tested by the method of the present invention, or women who are pregnant or not. As mentioned above, the suitable gestational age will also vary depending on the detected conditions and sometimes the different RNA markers used.

The biological sample may be maternal blood, including maternal plasma or serum. In some cases, the biological sample is acellular. In other environments, the biological sample contains cellular components or cell debris in the maternal plasma. Other biological samples include amniotic fluid, chorionic villus samples, live tissues derived from pre-implantation embryos, maternal urine, maternal saliva, fetal cavity puncture samples, infant nucleation cells or infant cell fragments, or from pregnant womens reproductive tract cleaning Samples of objects.

When the biological sample is maternal blood, blood is collected from women according to standard procedures used in general hospitals or clinics. Collect an appropriate amount of peripheral blood, such as 3-20ml, and store it in accordance with standard procedures before subsequent processing.

The biological sample useful in the present invention contains fetal RNA. The fetal RNA is transcribed from chromosomes 21, 18, 13, X and Y chromosomes. In addition, the RNA expressed in the placenta can be 2 times, 5 times, 10 times or even higher than the expression of maternal blood or different parts of blood components.

Fetal RNA usually comes from any tissue at the root of the fetus, including but not limited to the placenta, and it can also be mRNA. Various methods can be used to extract RNA from biological samples. There are many ways to extract RNA from biological samples. Common RNA preparation methods can be used (e.g. Sambrook and Russell,<i>Molecular Cloning: A Laboratory Manual</i> 3d ed., "Molecular Selection: Laboratory Manual" 3rd Edition, 2001); a variety of commercially available reagents or kits, such as Trizol reagent (Invitrogen, Carlsbad, California), Oligotex Direct mRNA Kits (Qiagen, Valencia , California), RNeasy Mini Kits (Qiagen, Hilden, Germany) and PolyATtract^® Series 9600^TM(Promega, Madison, Wisconsin) to obtain RNA from womens blood samples. These methods can also be combined to extract RNA. The samples should be handled carefully during the amplification and quantification steps, treated with DNase from start to finish, and set appropriate negative controls to eliminate DNA in RNA preparation.

C. Methods of identifying RNA transcription alleles

The alleles of RNA transcripts can be identified by a series of methods, including PCR, mass spectrometry (MS), gel electrophoresis, pyrophosphate sequencing, primer extension analysis, chip, sequencing and the use of one or more fluorescent probes Hybridization.

1. Method for identifying RNA-SNP alleles based on PCR technology

Once RNA is extracted from the biological sample, the RNA of interest, such as<i>COL6A1</i>，<i>SOD1</i>，<i>COL6A2</i>，<i>ATP5O</i>，<i>BTG3</i>，<i>ADAMTS1</i>，<i>BACE2</i>，<i>ITSN1</i>，<i>APP</i>，<i>ATP5J</i>，<i>DSCR5</i>，<i>PLAC4</i>，<i>LOC90625</i>，<i>RPL17</i>，<i>SERPINB2</i>or<i>COL4A2</i>The amount of each specific SNP allele is analyzed.

Variants of the genes selected in the present invention are still useful. E.g,<i>PLAC4</i>Useful variants of genes include those with GenBank accession numbers AF269287, AK027868, AK092431, BC093685, BC101615, BC101617, L13197, NM_182832 and LOC191585. Transcribed from<i>PLAC4</i>The RNA of the genetic seat of the gene contains one or more single nucleotide polymorphisms or insertion-deletion polymorphisms. Transcribed from<i>PLAC4</i>Gene polymorphisms include the following SNPs database ((dbSNP) www.ncbi.nlm.nih.gov/SNP/)) accession number (with human genome based on UCSC genome browser (genome.ucsc.edu/) May 2004 (hg17) compiled<i>PLAC4</i>Equivalent) identified polymorphism: rs3804026 (<i>PLAC4</i>-41469163), rs4818219(<i>PLAC4</i>-41469764), rs9977003(<i>PLAC4</i>-41470591), rs7844(<i>PLAC4</i>-41470699), rs9015(<i>PLAC4</i>-41470877), rs13643(<i>PLAC4</i>-41471296), rs9305729(<i>PLAC4</i>-41472272), rs9305730(<i>PLAC4</i>-41472277), rs5019195(<i>PLAC4</i>-41473295), rs5019194(<i>PLAC4</i>-41473302), rs5844069(<i>PLAC4</i>-41473306), rs1049904(<i>PLAC4</i>-41473392), rs16998089 (<i>PLAC4</i>-41473496), rs12482116(<i>PLAC4</i>-41475590), rs11909439 (<i>PLAC4</i>-41475912), rs7278659(<i>PLAC4</i>-41476875), rs12106409(<i>PLAC4</i>-41477273), rs12106395(<i>PLAC4</i>-41477340), rs12106401(<i>PLAC4</i>-41477425), rs12106434(<i>PLAC4</i>-41477486), rs2183584(<i>PLAC4</i>-41477956), rs3949725(<i>PLAC4</i>-41478283), rs8130833(<i>PLAC4</i>-41478755), rs10222145(<i>PLAC4</i>-41480512) and rs9981478 (<i>PLAC4</i>-41480564), or located in<i>PLAC4</i>-41471145 and<i>PLAC4</i>-41476236<i>PLAC4</i>Other polymorphisms in the locus, based on the UCSC Genome Browser (genome.ucsc.edu/), the human genome in May 2004 (hg17) compilation, are located on the 41471145 and 41476236 nucleotide equivalents of chromosome 21, respectively. Those skilled in the art should also understand<i>PLAC4</i>Other polymorphisms in are equally useful in the present invention.

The dbSNP accession number only refers to the SNP position where the SNP genome sequence exists. The range of the SNP sequence can be selected from the upstream or downstream part of the transcribed genome sequence.

Other RNA-SNPs used in the present invention include the human genome on chromosome 21 compiled in May 2004 (hg17) based on the UCSC genome browser (genome.ucsc.edu/)<i>COL6A1</i>(<i>COL6A1</i>-46247817)^Arg850_His，<i>COL6A1</i>(<i>COL6A1</i>-46248064)^Ser932_Serand<i>COL6A2</i>(<i>COL6A2</i>-46370341)^Val728_Val。

In most cases, it is desirable to use a variety of nucleic acid amplification methods known in the art (listed above and described in detail below) to amplify the target sequence. In particular, nucleic acid amplification refers to the use of enzymes to synthesize a nucleotide replicon containing a sequence complementary to the amplified nucleotide sequence. The nucleic acid amplification method is particularly effective when the content of the target sequence in the sample is very low. By amplifying the target sequence and detecting the synthesis of the replicon, the sensitivity of the analysis can be greatly improved, because the lower content of the target sequence at the beginning of the analysis can ensure that the target sequence in the test sample is derived from the organism of interest Or virus.

The methods used for polynucleotide amplification such as polymerase chain reaction (PCR), ligation amplification reaction (or ligase chain reaction (LCR)), and amplification methods are based on the application of Q-β replicase. Other useful methods include strand substitution amplification (SDA), thermophilic SDA, nucleotide sequence-based amplification (3SR or NASBA), and transcription-related amplification (TAA). These methods are well known in the art and widely used. Reagents and instruments for PCR can be purchased.

Before the amplification step, it is usually necessary to synthesize a DNA replica (cDNA) from the RNA transcript of interest. This step can be accomplished by reverse transcription, a separate step or in a homologous reverse transcription polymerase chain reaction (RT-PCR) of a modified polymerase chain reaction that amplifies RNA. Suitable methods for nucleic acid PCR amplification can be found in Romero and Rotbart "Diagnostic Molecular Biology: Principles and Applications" (<i>Diagnostic Molecular Biology: Principles and Applications</i>)pp.401-406; Persing et al.,eds.,Mayo Foundation,Rochester,MN,1993; Egger et al.,<i>J.Clin.Microbiol.</i><b>33</b>: 1442-1447, 1995; and U.S. Patent No. 5,075,212.

The PCR method is well known in the art and therefore need not be described here. For a review of PCR methods, experimental methods and primer design principles, please refer to Innis et al.'s "PCR Experimental Methods: Methods and Application Guide" (<i>PCR Protocols: A Guide to Methods and Applications</i>), Academic Press, Inc. NY, 1990. You can also obtain PCR reagents and procedures from retailers such as Roche Molecular Systems.

Generally, thermostable enzymes are used to implement PCR in an automated process. In this process, the temperature of the reaction mixture is usually automatically circulated between the denaturation zone, the primer annealing zone, and the extension reaction zone. Machines suitable for this purpose are commercially available.

The primer used to amplify the RNA transcript is preferably complementary to or specifically hybridizes to the sequence flanking the target site. The polynucleotide sequence obtained by amplification can be directly sequenced. Alternatively, the amplified products can be colonized before sequencing. Another possibility is to design primers to overlap with the SNP site when performing allele-specific PCR operations. Allele-specific PCR can identify RNA-SNP alleles because only the correct hybridization primers can be amplified. The PCR primer refers to the transcription from the site of interest in polymerase chain reaction (PCR), such as<i>COL6A1, SOD1, COL6A2, ATP5O, BTG3, ADAMTS1, PACE2, ITSN1, APP, ATP5J, DSCR5, PLAC4, LOC90625, RPL17, SERPINB2</i>or<i>COL4A2</i>The RNA accounting sequence is amplified by oligonucleotides.

Although PCR amplification of the target RNA-SNP allele is usually performed in the present invention, it should be understood in the art that any known method can be used to amplify RNA in a blood sample, such as ligase chain reaction (LCR). ), transcription-mediated amplification, self-sustaining sequence replication or nucleotide sequence-based amplification (NASBA), each method can provide sufficient amplification. The recently developed branch-DNA technology can also be used to amplify the RNA-labeled signal in the blood. The branch-DNA (b-DNA) signal amplification used to directly quantify the nucleic acid sequence of clinical samples can be found in Nolte,<i>Adv.Clin.Chem.</i><b>33</b>：201-235,1998。

2. Other methods to identify RNA-SNP alleles

Other standard techniques known to those skilled in the art can also be used to determine the type of RNA of interest. Although the amplification step usually precedes the determination step, amplification may not be required in the method of the present invention. For example, whether it is performed before or after the amplification step, size separation (such as gel electrophoresis) can be used to identify the type of RNA of interest. After electrophoresis on a well-known agarose or polyacrylamide gel and labeling with ethidium bromide (see, for example, the aforementioned Sambrook and Russell, "Molecular Cloning: Laboratory Manual" 3rd edition, Molecular Cloning: A Laboratory Manual 3d ed., 2001), the presence of the same band as the standard control can indicate the presence of target RNA, and the amount of the band can be compared with the standard according to the intensity of the band. Optionally, based on the intensity of the signal generated by the probe, it can be<i>COL6A1, SOD1, COL6A2, ATP5O, BTG3, ADAMTS1, BACE2, ITSN1, APP, ATP5J, DSCR5, PLAC4, LOC90625, RPL17, SERPINB2</i>or<i>COL4A2</i>RNA-specific oligonucleotide probes transcribed from the locus to detect the presence of these types of RNA, and the comparison with the standard indicates the amount of the RNA.

Sequence-specific probe hybridization is a well-known method for detecting specific nucleic acids under conditions containing other nucleic acid types. Under sufficiently stringent hybridization conditions, the probe specifically hybridizes to only substantially complementary sequences. The stringency of the hybridization conditions can be relaxed to allow different amounts of sequence mismatches.

There are many hybridization methods in the art, including liquid phase, solid phase or mixed phase hybridization analysis methods. The following article provides an overview of various hybrid analysis methods: Singer<i>et al.,Biotechniques</i>(Biotechnology)<b>4</b>: 230,1986; Haase <i>et al.</i>,<i>Methods in Virology</i>(Methods of Virology), pp.189-226,1984; Wilkinson,<i>In situ Hybridization</i>(In situ hybridization), Wilkinson ed., IRL Press, Oxford University Press, Oxford; and Hames and Higgins eds.,<i>Nucleic Acid Hybridization: A Practical Approach</i>(Nucleic Acid Hybridization: Operation Method), IRL Press, 1987.

The hybridization complex can be measured according to a known method, which is not the focus of the present invention. Any method commonly used to determine the presence of hybrid nucleic acids can be used to label nucleic acid probes that can specifically hybridize to the target nucleic acid, that is, the RNA type of interest or the amplified DNA. The common measurement method is³H,¹²⁵I,³⁵S,¹⁴C or³²P and other labeled probes are subjected to autoradiography. According to the ease of synthesis, stability and half-life of the selected isotope, there is a research preference for the choice of radioisotope. Other labels include labeling of compounds (such as biotin and digoxigenin) that can bind to fluorescent groups, chemiluminescent agents, and enzyme-labeled antiligands or antibodies. Alternatively, the probe may be directly conjugated to a label such as a fluorescent group, chemiluminescent agent, or enzyme. The choice of the label depends on the required sensitivity, ease of conjugation with the probe, stability requirements and available equipment.

The probes and primers necessary for the practice of the present invention can be synthesized and labeled using well-known techniques. Can be based on the first by Beaucage and Caruthers,<i>Tetrahedron Letts.</i>,<b>22</b>: 1859-1862, the solid-phase phosphatidite triester method described in 1981, such as Needham-VanDevanter <i>et al.</i>,<i>NucleicAcids Res.</i><b>12</b>: 6159-6168, 1984, using an automatic synthesizer to chemically synthesize oligonucleotides used as probes and primers. Such as Pearson and Regnier,<i>J.Chrom.</i>,<b>255</b>:137-149, 1983, the oligonucleotides can be purified by natural acrylamide gel electrophoresis or anion exchange high performance liquid chromatography (HPLC).

Other useful methods for identifying RNA transcription alleles include direct sequencing, such as pyrophosphate sequencing. The process of pyrophosphate sequencing involves a primer-template complex in which one of the four deoxynucleoside triphosphates is added at a time. When the deoxynucleoside triphosphate is combined with the DNA polymerase, a light signal is emitted. The intensity of the generated light signal is proportional to the number of bases added. Infer the downstream sequence from this.

The primer extension reaction is also useful in the present invention. The method of primer extension reaction to identify SNP alleles is as follows. The method is completed by joining deoxynucleotides and/or dideoxynucleotides to primer extension primers, which in turn hybridize with parts adjacent to the SNP site . The primer is extended by polymerase. The SNP of the primer extension can be physically measured by mass spectrometry or identified by a labeling part such as biotin. Since the SNP site can only be extended by complementary deoxynucleotides and/or dideoxynucleotides that are bound by a specific label or produce a product with a specific molecular weight, the SNP alleles can be identified.

Mass spectrometry can detect polynucleotides, such as PCR replicons or primer extension products. By comparing the molecular weight of the detected signal with the expected molecular weight of the target polynucleotide, the sequence of the polynucleotide that appears can be verified. The relative signal intensity of a specific polynucleotide sequence indicates the relative number of specific alleles, so the allele ratio can be directly calculated from the obtained data. For the method of mass spectrometry analysis of genotype, please refer to Pusch et al.,<i>fharmacogenomics</i><b> 3</b>：537-548,2002。

D. Determine the ratio of RNA-SNP alleles

Generally speaking, determining the ratio of RNA-SNP alleles involves calculating the relative number of each RNA-SNP allele in the sample, and dividing the measured value of one RNA-SNP allele by the other RNA-SNP alleles The value of the gene. Using a PCR-based detection system can divide one allele from other alleles based on the PCR-related label strength, primer extension, or hybridization reaction product. Other methods for detecting the ratio of RNA-SNP alleles include comparing the abundance of the sequencing product and the number of cloned sequences for each allele. The RNA-SNP ratio can also be determined by comparing the mass spectrometric signal intensities of the alleles by mass spectrometry.

Alternatively, the RNA-SNP ratio can also be determined by the difference in the fluorescence intensity (Rn) accumulated for each allele in the reaction, or the PCR reaction for each allele accumulated to the number of PCR cycles required for the fluorescence intensity of the valve ( Ct) to determine. ΔCt and ΔΔRn reflect the RNA-SNP allele ratio because these values are proportional to the logarithm of the RNA allele value. As a result, the difference in the initial cycle value (ΔCt) or accumulated fluorescence intensity (ΔΔRn) of each allele reflects the SNP ratio of RNA transcripts. For example, allele A can be detected with FAM (6-carboxyfluorescein) label, while allele B can be detected with fluorescent probes such as VIC (Applied Biosystems). Calculate the Ct and Rn values of alleles A and B. The difference between the two Ct values can be determined by the valve cycle value, and the ΔCt value is generated. The difference between the two Rn values can be determined by the accumulated fluorescence intensity and produce the Rn value. Since Ct and Rn are logarithmically related to the abundance of PCR products, the Ct and Rn values of each RNA-SNP are calculated, and these differences (Ct and Rn values) reflect the two RNA alleles The ratio of RNA-SNP between genes.

E. Compare the RNA-SNP ratio with a standard control

Once the allelic ratio of the subject is determined, the ratio is compared with a standard control to detect the presence of fetal aneuploidy. When compared with the known value determined by the control sample, whether the RNA-SNP ratio is higher or lower than the control, it indicates the presence of fetal aneuploidy. For example, when compared with the known value determined by the control sample, regardless of the ΔCt value, ΔΔRn value, label or mass spectrum intensity ratio between the SNP alleles is higher or lower than the standard control, Both indicate the presence of fetal aneuploidy.

In order to establish a standard control, first select a group of healthy pregnant women with healthy fetuses. These women should have a similar gestational age, and the gestational age should be within an appropriate pregnancy stage for detection of conditions such as fetal chromosomal aneuploidy using the method of the present invention. Similarly, samples from a group of healthy non-pregnant women can be used to establish standard controls. The health status of selected pregnant women and fetuses can be confirmed by routine methods, including cytogenetic analysis or fetal genetic analysis of the biological samples obtained by the above methods. The standard control can be measured before the detection of fetal aneuploidy.

In addition, the selection group of healthy pregnant women with healthy fetuses must have a reasonable size so that the code calculated from the group<i>COL6A1, SOD1, COL6A2, ATP5O, BTG3, ADAMTS1, BACE2, ITSN1, APP, ATP5J, DSCR5, PLAC4, LOC90625, RPL17, SERPINB2</i>or<i>COL4A2</i>The average, average, or median ratio of RNA-SNP alleles can reasonably represent the normal, average, or median value from the total population of healthy women pregnant with healthy fetuses. In some instances, the group contains at least 10 women.

Once an average value is established from the ratio of RNA-SNP alleles based on the value of each individual woman in the selected group, this value can be used as a criterion for the type of RNA. Any blood sample containing similar proportions of the same type of RNA can be used as a standard control. It is also possible to manually prepare an established average ratio of the same type of RNA, containing the code<i>COL6A1, SOD1, COL6A2, ATP5O, BTG3, ADAMTS1, BACE2, ITSN1, APP, ATP5J, DSCR5, PLAC4, LOC90625, RPL17, SERINB2</i>or<i>COL4A2</i>RNA solution and use it as a standard control.

An increase in the proportion of RNA-SNP that is at least 10% higher than the average of the control sample indicates an increased probability of having a fetus with a chromosomal abnormality. In some cases, the ratio of RNA-SNP has increased by at least 50%. In other cases, the ratio of RNA-SNP has increased by at least 100%. In other cases, an RNA-SNP ratio of at least 10% lower than the average of the control sample indicates an increased probability of having a fetus with a chromosomal abnormality. In some cases, the reduction is at least 50% or 90%.

A ratio of RNA-SNP that is at least one standard deviation above or below the average of the control sample indicates an increased probability of having a fetus with a chromosomal abnormality. The ratio of RNA-SNP that is at least two standard deviations higher or lower than the average of the control sample also indicates an increased probability of having a fetus with a chromosomal abnormality. A ratio of RNA-SNP that is at least three standard deviations higher or lower than the average of the control sample indicates an increased probability of having a fetus with a chromosomal abnormality. In some cases, the ratio of RNA-SNP within one standard deviation above or below the average of the control sample also indicates an increased probability of having a fetus with a chromosomal abnormality.

F. A kit for detecting chromosomal abnormalities in the fetus of a pregnant woman

The present invention also provides a kit for detecting chromosomal abnormalities of a fetus pregnant by a pregnant woman. The set of the present invention includes primers for amplifying the target region. The primers used in the set of the present invention to distinguish alleles have been described above. The primer may be marker-specific or function in a non-specific manner.

The other component of the set of the present invention is a standard control representing the average ratio of RNA alleles of pregnant women with chromosomal normal fetuses. The standard control is determined using the same method as described above for detecting chromosomal abnormalities in the fetus of pregnant women.

The kit of the present invention also contains hybridization probes for identifying different alleles of each RNA. The different alleles of each RNA can be single nucleotide polymorphisms, insertion/deletion polymorphisms, or simple tandem repeat polymorphisms. The hybridization probes used in the kit of the present invention are the same hybridization probes used in the aforementioned method for detecting chromosomal abnormalities in the fetus of a pregnant woman. The hybridization probe can be radioactive, fluorescently labeled, chemiluminescent or enzymatic. Oligonucleotides used as probes can be used according to known techniques (Beaucage and Caruthers,<i>Tetrahedron Letts.</i>, 22: 1859-1862, 1981, using an automatic synthesizer, according to Needham-VanDevanter et al.,<i>Nucleic Acids Res.</i>12: 6159-6168, 1984). The probes of the present invention can be specific or non-specific. Those skilled in the art should understand that there are other methods that can be used in the set of the present invention to distinguish the alleles of each RNA, such as the method described above.

The kit of the present invention may also contain other ingredients understood by those skilled in the art that can be used in the kit of the present invention.

The following examples are provided for illustrative purposes only and not for restrictive purposes. It should be easily understood by those skilled in the art that basically similar results can be obtained by changing and modifying various non-critical parameters.

III. Examples

The method of the present invention is used to detect the chromosomal abnormality of an individual who is heterozygous at a specific locus, that is, to detect whether the individual has an abnormal number of chromosomes at the specific locus, for example, 2 chromosomes are normal, and 3 chromosomes are normal. The chromosomes are abnormal. In the case of pregnancy, when the biological sample is directly collected from a pregnant woman, the individual whose chromosomal aneuploidy is detected is the fetus pregnant by the pregnant woman. In an embodiment where this method is used for prenatal diagnosis, the extra copy of the gene in the placental tissue is manifested together with its corresponding normal gene pair. As a result of the additional duplication of this gene, the ratio of the RNA alleles in the placenta deviates from the allele ratio of the normal placenta. The RNA transcripts are then released into the maternal blood, and their relative abundance reflects the expression of placental genes. Accordingly, the ratio of RNA alleles detected from the blood of pregnant women with chromosomal abnormal fetuses or their constituents (such as plasma) differs from the ratio of pregnant women with chromosomal euploid fetuses.

The use of RNA transcripts to detect chromosomal abnormalities requires identification of fetal-specific markers with detectable levels of expression in maternal blood, identification of transcription regions with high placental expression markers, and determination of the transcripts in maternal blood The ability to determine that the transcript is pregnancy-specific, determine the allele ratio of the transcript, and thereby determine the presence or absence of chromosomal abnormalities.

The following examples give examples of identifying useful markers and transcripts, detecting the transcripts and confirming that the transcripts are pregnancy-specific, and quantifying the transcripts and determining the ratio of RNA alleles. Methods of detecting chromosomal abnormalities. Those skilled in the art should understand that there are other methods and techniques that can also be applied to the present invention.

Example 1: Identification of SNP for detection of fetal triploid chromosome 21

Identifying the SNP used to detect the chromosome 21 triploid of the fetus requires identifying the type of RNA that is present in the fetal cell and that it has a detectable concentration in the biological sample being analyzed.

Identify fetal-specific transcripts with high levels of placental expression

Through microarray analysis of each tissue sample, gene expression profiles of five cases of chorionic villus samples (CVS) in the first trimester were obtained. When the placental expression transcript was identified from the circulating RNA molecules of maternal plasma, the gene expression profile of maternal whole blood (especially maternal hematopoietic cells) was obtained and compared with the corresponding gene expression profile of placental tissue. By comparing with the corresponding whole blood samples in all five comparisons, transcripts with increased levels of expression in CVS samples were screened to identify placental expression transcripts in early pregnancy.

Sample processing and RNA extraction. Five samples of placenta tissue in the first trimester were collected from pregnant womens CVS before the therapeutic termination of pregnancy. The fetal karyotype proved to be normal in all samples. Placental tissue samples are stored in RNAlater immediately after collection^TM(Ambion^®, Austin, TX) and placed at -80°C for RNA extraction. Collect 6 milliliters of maternal peripheral blood while collecting tissue samples and save it in PAXgene^TMBlood RNA test tube (PreAnalytiX, Hombrechtikon, Switzerland). Total RNA from placenta tissue was extracted with Trizol kit (Invitrogen, Carlsbad, California), and purified with RNeasy mini kit (Qiagen, Hilden, Germany), and the operation was in accordance with the instructions. Peripheral blood fluid total RNA use PAXgene according to instructions^TMBlood RNA kit (PreAnalytiX, Hombrechtikon, Switzerland) extraction, which also includes DNase treatment (RNase-free DNase combination, Qiagen, Hilden, Germany).

High-density oligonucleotide arrays were used to analyze gene expression. For each sample, label 10 micrograms of extracted RNA according to the instructions and use GeneChip^®The human genome U133A was crossed with U133B Arrays (Affymetrix, Santa Clara, California). After hybridization, each array is washed and placed in GeneChip^®Stain in Fluidics Station 400 (Affymetrix). The wafer was scanned with GeneArray scanner (Affymetrix) and GeneChip^® Microarray Suite 5.0 (Affymetrix) for analysis. The transcripts derived from chromosome 21, which are mainly expressed in placental tissue rather than the blood around the mother, were selected. Among the 7226 gene transcripts expressed in CVS samples, 1245 transcripts expressed higher levels in CVS than maternal peripheral blood samples. Among these transcripts, 13 placental expression genes are located on chromosome 21 (Table 1).

<tables><img file="TWI367259B_D0001.tif" /></tables>

Identify available SNPs and calculate allele ratios

Some single nucleotide polymorphisms (SNPs) have been identified from public databases. These SNPs are located in the transcriptional region of the locus with high placental tissue expression on chromosome 21. The allele ratio of each SNP is calculated by the Chinese and Caucasian populations. The SNP with high heterozygosity is the target SNP.

Among the 13 genes located on chromosome 21 (Table 1), which are mainly expressed in the placenta, 4 genes have the highest level of placental expression, namely collagen VI α 1 (COL6A1), superoxide dismutase 1 (SOD1), Collagen VI α 2 (COL6A2), mitochondrial ATP synthase O subunit (ATP5O), selected these genes for further analysis. SNPs located in the exons of these genes were selected to study mRNA polymorphisms. The PCR primers flanking the corresponding SNPs (Table 2) were used to directly sequence the DNA of 10 Chinese and 10 Caucasian white blood cells to determine the proportion of each allele of these coding polymorphisms. Sequencing was done with BigDye Terminator Cycle Sequencing v1.1 (Applied Biosystems, Foster City, California) and a 3100 DNA analyzer (Applied Biosystems).

<tables><img file="TWI367259B_D0002.tif" /></tables>

The allelic ratios of the transcribed SNPs are shown in Table 3. SNPs with a heterozygosity of at least 30% in one of the two populations are considered informative and can be selected as targets for further analysis and development.

Table 3. Pairs of unrelated individuals in 10 Chinese and 10 Caucasians<i>COL6A1,SOD1,COL6A2</i>and<i>ATP5O</i>Genotype analysis of encoding SNP<tables><img file="TWI367259B_D0003.tif" /></tables>

Identify the detectability and pregnancy specificity of the selected transcript

Using real-time quantitative RT-PCR, the four transcripts with placental expression on chromosome 21 can be detected in maternal plasma. The RT-PCR is performed by amplifying the non-polymorphic regions of these transcripts. Compared with non-pregnant women, the concentration of these transcripts present in the plasma of pregnant women is particularly high. In addition, the concentration of these transcripts present in the plasma of pregnant women decreases sharply with the birth of the child. Therefore, the placenta is the main source of these mRNA transcripts in maternal plasma.

Sample collection and processing. Whole blood samples were collected from 6 non-pregnant women and 10 pregnant women in the first trimester. Peripheral blood samples were collected from five pregnant women in the third trimester before and 24 hours after birth. Collect 12 ml of blood sample in EDTA test tube and centrifuge at 1600 xg for 10 minutes at 4°C, then carefully transfer the plasma to an empty polypropylene test tube. The plasma sample was centrifuged at 16000 xg for 10 minutes at 4°C, and the supernatant was collected in a new polypropylene test tube. The reaction to extract RNA from maternal plasma is as follows: Mix 4 ml of Trizol LS reagent (Invitrogen, Carlsbad, California) and 0.8 ml of chloroform (Ng, EKO et al., Clin Chem 48: 1212-1217 (2002)) with 3.2 ml of plasma , The mixed solution was centrifuged at 12000 xg for 15 minutes at 4°C, the aqueous layer was transferred to a new test tube, and an equal volume of 70% ethanol was added. The mixture was then treated with an RNeasy mini-column (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Total RNA was eluted with 60μl of RNase-free water and stored at -80°C. DNase treatment is used to remove any contaminated DNA (RNase-Free DNase Set, Qiagen, Hilden, Germany).

Instant QRT-PCR development. Develop QRT-PCR to improve collagen VI α 1(<i>COL6A1</i>), superoxide dismutase 1 (<i>SOD1</i>), collagen VI α 2(<i>COL6A2</i>), mitochondrial ATP synthase O subunit (<i>ATP5O</i>) MRNA is measured. The primer sequence and TaqMan^®Minor groove binding (MGB) fluorescent probes (Applied Biosystems, Foster City, California, USA) are shown in Table 4. The calibration curve is made with serial dilutions of single-stranded synthetic DNA oligonucleotides purified by high performance liquid chromatography specific to the corresponding amplicon, and the concentration ranges from 1 X 10⁶Copy to 10 copies. according to<i>COL6A1</i>,<i>SOD1</i>,<i>COL6A2</i>and<i>APT5O</i>The synthesized DNA oligonucleotide sequence is shown in Table 4. The absolute concentration of all transcripts is expressed as the ratio of the number of copies to the number of milliliters of plasma.

<tables><img file="TWI367259B_D0004.tif" /></tables><tables><img file="TWI367259B_D0005.tif" /></tables>

The QRT-PCR reaction was performed in a 25 μl system according to the manufacturer's guide (EZ rTth RNA PCR Kit Applied Biosystems). The QRT-PCR reaction was completed on a combined thermal cycler and fluorescence detector (ABI Prism 7700, Applied Biosystems). For all four transcripts, the concentrations of PCR primers (Proligo) and fluorescent probes (Applied Biosystems) were 300 nM and 100 nM, respectively. 5 μl of the extracted plasma RNA was used for amplification. Multiple negative water blanks are included in each analysis.

Used to<i>COL6A1,SOD1,COL6A2</i>and<i>ATP5O</i>The temperature setting for the analysis is as follows: the reaction is kept at 50°C for 2 minutes to allow the uracil N-glycosidase contained therein to act, and then reverse transcription is performed at 60°C for 30 minutes. After 5 minutes of denaturation at 95°C, perform denaturation at 92°C for 15 seconds and 57°C (<i>COL6A1</i>and<i>ATP5O</i>) Or 56°C(<i>COL6A2</i>) Or 59°C(<i>SOD1</i>) 40 cycles of annealing/extension for 1 minute.

The placenta shows that the transcript of chromosome 21 can be detected in maternal plasma and is pregnancy-specific. Non-pregnant women, pregnant women in the first trimester and last trimester<i>COL6A1</i>The median plasma mRNA concentrations were 0 copies/ml, 0 copies/ml and 72.6 copies/ml (Figure 2A; for<i>SOD1</i>They are 25.3 copies/ml, 53.0 copies/ml and 155.6 copies/ml (Figure 2B);<i>COL6A2</i>Is 0.8 copy/ml, 2.1 copy/ml and 8.8 copy/ml (Figure 2C);<i>ATP5O</i>The values are 6.2 copies/ml, 88.2 copies/ml and 126.4 copies/ml (Figure 2D). For all four transcripts, their plasma concentrations in the third trimester pregnant women were significantly higher than those in the non-pregnant group (Mann-Whitney Rank Sum test, for<i>COL6A1</i>,<i>SOD1</i>,<i>COL6A2</i>and<i>ATP5O</i> mRNA <i>P</i>&lt;0.05). In addition, in the pre-delivery plasma samples,<i>COL6A1</i>,<i>SOD1</i>,<i>COL6A2</i>and<i>ATP5O</i> The median mRNA concentrations were 72.6 replicates/ml, 155.6 replicates/ml, 8.8 replicates/ml and 126.4 replicates/ml (Figure 3A corresponds to<i>COL6A1</i> mRNA, corresponding to Figure 3B<i>SOD1</i> mRNA, corresponding to Figure 3C<i>COL6A2</i> mRNA and 3D image correspondence<i>ATP5O</i> mRNA). And for plasma samples after childbirth,<i>COL6A1</i>,<i>SOD1</i>,<i>COL6A2</i>and<i>ATP5O</i> The median mRNA concentrations were 0 copies/ml, 56.2 copies/ml, 0.8 copies/ml and 56.2 copies/ml, respectively.

Example 2: Determination of chromosome 21 trisomy of fetuses pregnant by pregnant women by real-time quantitative RT-PCR

The two SNPs with the highest polymorphic ratio were used as target SNPs for allele-specific QRT-PCR. Design a discriminative hybridization probe to identify the different alleles of each SNP. The results of real-time PCR and direct sequencing to obtain the same genotype confirmed the allelic specificity of the probe. These probes are then combined with the QRT-PCR reaction, and the corresponding expression levels of the different alleles of each SNP in the placental tissue are first measured. RNA was extracted from normal CVS, normal expiring placenta, and placenta of pregnant women with trisomy 21, and determined by allele-specific QRT-PCR. Since the allele ratio of each SNP is basically different in pregnancy with and without chromosome 21 trisomy, maternal plasma can be used to determine the fetal chromosome 21 trisomy.

Sample collection and processing. Placental tissue samples were collected in the form of chorionic villus sampling (CVS) from 13 pregnant women in the first trimester and 20 pregnant women in the last trimester before the therapeutic termination of pregnancy or immediately after the cesarean section. Collect the placental tissue samples of seven chromosome 21 trimester pregnancies at the same time. All tissues are handled as described above.

Development of allele-specific QRT-PCR. Two coding SNPs with separate functions with the highest polymorphism ratio were selected. These two SNPs were rs1053312 (dbSNP sequence number) and rs2839114 of the COL6A1 and COL6A2 genes, respectively. An allele-specific QRT-PCR reaction was established to determine the relative concentration of two alleles for each SNP. In order to detect alleles, each QRT-PCR reaction contains two fluorescent MGB probes specific for one allele. These two probes are labeled with FAM (6-carboxyfluorescein) and VIC fluorescent dyes. The primer and probe sequences of allele-specific QRT-PCR are shown in Table 5. The results of real-time PCR and direct sequencing to obtain the same genotype confirmed the allelic specificity of the probe.

<tables><img file="TWI367259B_D0006.tif" /></tables>

Set up a QRT-PCR reaction for allele detection and corresponding quantification according to the previous steps. The PCR primers (Proligo) and fluorescent probes (Applied Biosystems) used in the reaction are at 450nM and 100nM, and the amplification template is 17ng. Placental RNA samples. Each QRT-PCR reaction is performed in Applied Biosystems 7700 Sequence Detector. The thermal cycle conditions are as described above, and the annealing/extension temperature is 59°C. Only heterozygous samples are analyzed.

Statistical Analysis. Statistical analysis was performed using Sigma Stat 2.03 software (SPSS).

Relative quantification of chromosome 21 encoding RNA-SNP. The relative values of these two alleles are calculated using the difference in the number of valve cycles (Ct) or the difference in accumulated fluorescence intensity (Rn), the calculation formula is as follows: Ct=Ct_FAM-Ct_VIC

Rn=Rn_FAM-Rn_VIC

Where Ct_FAMAnd Ct_VICIt is the valve cycle number of allele A (detected with FAM-labeled probe) and allele B (detected with VIC-labeled probe). Rn_FAMAnd Rn_VICThe accumulated fluorescence intensity values of allele A and allele B. The above values are calculated using SDS v1.9 software (Applied Biosystems). The Ct value and Rn value are logarithmically related to the PCR product abundance, so the difference between the Ct value and Rn value of each RNA-SNP reflects the RNA-SNP ratio of these two alleles.

For SNP rs1053312<i>(COL6A1)</i>In terms of allele-specific QRT-PCR detection, it was found to be heterozygous in 1 CVS, 6 normal pregnancy placenta and 3 chromosome 21 trisomy placenta samples. As shown in Figure 4, the ΔCt values of the three chromosome 21 trisomy placenta samples deviated from the ΔCt values of the normal pregnancy CVS and the mature placenta.

For SNP rs2839114<i>(COL6A2)</i>In terms of allele-specific QRT-PCR detection, 8 CVS, 13 mature placenta of normal pregnancy and 3 chromosome 21 trisomy placenta samples showed heterozygosity. As shown in Figure 5A, the Ct values of the three chromosome 21 trisomy placenta samples deviated from the Ct value of the mature placenta of normal pregnancy, and two of the chromosome 21 trisomy placenta samples showed higher Ct Value, while the other has a lower ΔCt value. The deviation of the C value of chromosome 21 trisomy can be reduced to higher or lower than the normal pregnancy range, depending on the excess of this allele. Similar results were obtained with the Rn value (Figure 5B).

Example 3: Using primer extension and mass spectrometry to determine the trisomy 21 of the fetus pregnant by a pregnant woman

The two SNPs with the highest polymorphism rate (<i>COL6A1</i>Rs1053320 and<i>COL6A2</i>Rs2839114) was selected for further analysis. The primer extension reaction can determine the SNP genotype of placental tissue samples. The sample is processed with mass spectrometry to distinguish different RNA-SNP alleles, and the relative expression level of RNA-SNP alleles is detected to calculate the allele ratio. Since the difference in the ratio of each SNP allele detected from pregnancy with and without trisomy 21 is sufficiently high, maternal plasma can be used to determine trisomy 21 of the fetus.

Placental tissue collection and processing. A sample of placental tissue at the middle trimester of trimester was collected from pregnant women with trisomy 21 fetuses before the therapeutic termination of pregnancy. Immediately after the birth of the fetus with normal chromosomes, placental tissue samples were collected in the last trimester. These placental tissue samples were divided into two parts, one of which was placed in RNAlater immediately after collection^TM(Ambion^®, Austin, Texas) and stored at -80°C until RNA extraction, while the other aliquot was immediately stored at -80°C until DNA extraction. According to the manufacturer's guidelines, the QIAamp small kit is used to extract DNA from placental tissue. Total RNA extraction from placental tissues was performed using Trizol Reagent (Invitrogen, Carlsbad, California) for extraction and RNeasy small kit (Qiagen, Hilden, Germany) for purification according to the manufacturer's guidelines.

Placental DNA and RNA amplification. For RNA amplification, 450ng of placental RNA was reverse transcribed with random hexamers (TheromScript, Invitrogen) according to the manufacturer's guidelines. In each PCR reaction (AmpliTaq Gold, Applied Biosystems), cDNA corresponding to 50 ng of total RNA or 25 ng of DNA was used. The primer sequences for amplifying each SNP site on COL6A1 and COL6A2 are shown in Table 6. The final concentration of primers in 25μl PCR system is 200nM. The PCR reaction conditions were: denaturation at 95°C for 10 minutes, then denaturation at 94°C for 20 seconds, annealing at 56°C for 30 seconds, extension at 72°C for 1 minute, and finally incubation at 72°C for 3 minutes.

<tables><img file="TWI367259B_D0007.tif" /></tables>

SNP was determined by primer extension reaction. The primer extension reaction was designed to determine the SNP genotype of placental tissue samples. The placental RNA-SNP genotype was compared with the genotype obtained from placental DNA. Placental DNA and RNA PCR products were treated with shrimp alkaline phosphatase (Sequenom, San Diego, USA) at 37°C for 40 minutes, followed by treatment at 85°C for 5 minutes to remove excess dNTPs. The mixture of primer extension primer and 2',3'-dideoxynucleoside triphosphate (ddNTP) and dNTP is added to the processed PCR product. Designed test<i>COL6A1</i>and<i>COL6A2</i>The primer sequence of the primer extension of the selected SNP site is shown in Table 7. MassARRAY according to Sequenom standard^TM Homogeneous MassEXTEND^TMThe (hME) protocol performs a base extension reaction. The base extension conditions were as follows: denaturation at 94°C for 2 minutes, followed by denaturation at 94°C for 5 seconds, annealing at 52°C for 5 seconds, and extension at 72°C for 5 seconds for 75 cycles. The sequence of the primer extension product of each SNP allele is shown in Table 7. The final product of the extension was desalted by SpectroCLEAN resin (Sequenom) in the reaction buffer. Use a SpectroPoint (Sequenom) micro distributor to distribute about 10 nL of the reaction solution to the 384-format SpectroCHIP (Sequenom). A bench-top MALDI-TOF mass spectrometer was used to obtain the data. The expected molecular weights of all relevant peaks were calculated before analysis (Table 7) and identified by mass spectrometry. The SNP genotype is determined by scoring the presence or absence of the mass spectrum signal corresponding to a specific SNP allele.

<tables><img file="TWI367259B_D0008.tif" /></tables>

Determination of SNP ratio. Automatically input mass spectrometry data into SpectroTYPER (Sequenom) database for automatic analysis. The ratio of the peak frequency of the quality signal of the SNP in the two SNP alleles of the fetal heterozygote was determined. The SNP ratios of normal chromosome and trisomy 21 fetuses were compared. The ratio of SNP in placental DNA of trisomy 21 to that of normal fetus<i>COL6A1</i>(Figure 6A) and<i>COL6A2</i>(Figure 7A) The SNP ratio is deviated. Compared with normal fetuses, whether the SNP ratio of trisomy 21 fetuses decreases or decreases depends on the SNP genotype of trisomy 21. The SNP ratio of the placental tissue SNP is in the trisomy 21 fetus<i>COL6A1</i>(Figure 6B) and<i>COL6A2</i>(Figure 7B) RNA transcripts also show deviations from normal fetuses.

Example 4: Non-invasive prenatal detection of trisomy 21 fetuses by primer extension and mass spectrometry analysis using circulating fetal RNA in maternal plasma

In order to show that other SNPs can also be used to detect chromosomal abnormalities in the fetuses of pregnant women, we are specific to the placenta 4(<i>PLAC4</i>) (Table 19) was studied. The primer extension reaction analysis can determine the RNA-SNP genotypes of the placental tissue and maternal plasma samples. The primer extension products were analyzed by mass spectrometry to quantify the ratio of RNA-SNP alleles. Fetuses with and without trisomy 21<i>PLAC4</i> The difference in the ratio of alleles of SNP is high enough so that maternal plasma can be used to detect trisomy 21 of the fetus.

Placenta specific 4 (<i>PLAC4</i>) Identification of available coding SNPs in genes and determination of allele ratios

Identification of SNP. Placenta specific 4 (<i>PLAC4</i>) Genes are highly expressed in the placenta, but lower in white blood cells.<i>PLAC4</i>The gene is located in a key region of Down syndrome on chromosome 21.<i>PLAC4</i>The gene sequence is listed in Table 19, spanning 41469028-41480585 nucleotides on chromosome 21, this sequence is based on the compilation of the human genome on the UCSC genome browser (genome.ucsc.edu/) in May 2004 (hg17) . Listed in Table 19<i>PLAC4</i>The gene sequence contains all known and predicted<i>PLAC4</i> RNA splicing variants, their GenBank sequence numbers are: AF269287, AK027868, AK092431, BC093685, BC101615, BC101617, L13197, NM_182832 and LOC191585. Correct<i>PLAC4</i>The exons/transcription regions of genes are sequenced to identify polymorphic SNPs. Placental DNA samples extracted from 10 unrelated Chinese pregnant women were used for direct sequencing. The DNA was first amplified by PCR with 20ng of genomic DNA. Sequencing was performed with BigDye Terminator Cycle Sequencing v1.1 (Applied Biosystems, Foster City, California) and Model 3100 DNA analyzer (Applied Biosystems).

Determination of allele frequency.<i>PLAC4</i>Four coding SNPs in the gene transcription region were detected to have polymorphisms in the Chinese population (Table 8). These four SNPs are located at the nucleotide positions 41470591, 41471145, 41476236 and 41478755 of the human genome compiled in May 2004 (hg17) on the UCSC genome browser (genome.ucsc.edu/). The dbSNP sequence numbers of the SNPs located at nucleotide positions 41470591 and 41478755 on chromosome 21 are rs9977003 and rs8130833, respectively. Based on the nucleotide position of chromosome 21 compiled by the human genome on the UCSC genome browser (genome.ucsc.edu/) in May 2004 (hg17), the remaining two new SNPs were named<i>PLAC4</i>-41471145 and<i>PLAC4</i>-41476236. Examples of their allelic ratios are shown in Table 9. Among the four SNPs, rs8130833 has the highest polymorphism, so it is analyzed in depth.

<tables><img file="TWI367259B_D0009.tif" /></tables>

<tables><img file="TWI367259B_D0010.tif" /></tables>

Application of SNP labeling in maternal plasma<i>PLAC4</i> RNA allele ratio

Sample collection and processing. Placental tissue samples of the first trimester and the middle trimester were collected from seven pregnant women with trisomy 21. Chorionic villus sampling (CVS) was used to collect placental tissue from 26 pregnant women with normal karyotype fetuses. Placental tissue samples are stored in RNAlater immediately after collection^TM(Ambion^®, Austin, Texas) and placed at -80°C until RNA extraction. During the first trimester of pregnancy, peripheral blood was collected from 43 pregnant women with multiple fetuses and 5 pregnant women with a trisomy 21 fetus. The plasma samples were collected as described in Example 1.

According to the product instructions, the Trizol kit (Qiagen, Hilden, Germany) was used to extract total RNA from placental tissue. RNA is extracted from 1.6 to 3.2 ml maternal plasma samples. Add 3ml of Trizol LS reagent and 0.8ml of chloroform to each milliliter of plasma. The mixture is 12,000 x at 4°C<i>g</i>Centrifuge for 15 minutes. Collect the aqueous layer after centrifugation. 538 microliters of absolute ethanol was added to each 1 ml of the liquid phase layer. The mixed solution was treated with an RNeasy mini-column (Qiagen, Hilden, Germany) according to the product instructions. Each column was eluted with 48μl RNase-free water to obtain total RNA. The RNA finally eluted from the two columns is combined. Then DNase treatment was performed to remove DNA contamination (Invitrogen, Carlsbad, California, USA).

Reverse transcription and PCR amplification. According to the product description (ThermoScript, Invitrogen, Carlsbad, California, USA), reverse transcription was performed on 1.25 μg placental RNA or 48 μl plasma RNA using gene-specific primers (sequences shown in Table 10) in a 40 or 100 μl reaction system, respectively.

For each PCR amplification reaction, the template is 40 μl placental cDNA or 100 μl maternal plasma cDNA, and the reaction system is 80 μl or 200 μl, respectively. Each reaction contains 0.6X HotStar<i>Taq</i> PCR buffer, 0.9mM MgCl₂(Qiagen), dATP, dGTP and dCTP each 25μM, 50μM dUTP (Applied Biosystems), forward and reverse primers each 200nM (Integrated DNA Technologies), and 0.02U/μl HotStar <i>Taq</i>Polymerase (Qiagen). The reaction conditions were: denaturation at 95°C for 7 minutes, then denaturation at 95°C for 40 seconds, annealing at 56°C for 1 minute, extension at 72°C for 1 minute, and finally holding at 72°C for 3 minutes.

<tables><img file="TWI367259B_D0011.tif" /></tables>

The primer extension reaction was used for SNP identification and allele ratio quantification. The primer extension reaction was the same as in Example 3. The PCR product was first treated with shrimp alkaline phosphatase (Sequenom, San Diego, USA). Combine 4μl base extension mixture containing 771nM extension primer (Integrated DNA Technologies), 1.15U Thermosequenase (Sequenom) and 64μM ddATP, ddCTP, ddTTP and dGTP (Sequenom, San Diego, USA) with 5μl water and 5μl PCR The products are mixed. The heating process is: 94°C, 2 minutes, and then enters 100 cycles of 94°C, 5 seconds, 52°C, 5 seconds, and 72°C, 5 seconds. The sequence and molecular weight of the extension primer and extension product of each SNP allele are shown in Table 11. As described in Example 3, the molecular weight of the final extension product was determined with a MALDI-TOF mass spectrometer. The ratio of the peak area area of the primer extension product, which represents the two SNP alleles of the fetus with heterozygous SNP, is determined.

<tables><img file="TWI367259B_D0012.tif" /></tables>

Development of real-time QRT-PCR system

developed<i>PLAC4</i> QRT-PCR system of mRNA to evaluate chromosome 21 trisomy and maternal plasma of normal pregnancy<i>PLAC4</i> Whether there is a quantitative difference in mRNA concentration. The primer sequence (Integrated DNA Technologies, Coralville, IA), TaqMan^®The minor groove binding (MGB) fluorescent probe sequence (Applied Biosystems, Foster City, California, USA) and the calibration control sequence (Proligo, Singapore) are shown in Table 12.

<tables><img file="TWI367259B_D0013.tif" /></tables>

According to the manufacturer's guide (EZ r<i>Tth</i> RNA PCR kit (Applied Biosystems) performs QRT-PCR reaction in 25μl system. The QRT-PCR reaction in ABI PRISM^® Performed in 7900HT (Applied Biosystems, Foster City, California, USA). PCR primers and fluorescent probes were used at concentrations of 400 nM and 100 nM, respectively. 5 μl of the extracted RNA was used for amplification. The thermal cycling process is: 50°C, 2 minutes, then reverse transcription at 60°C for 30 minutes, then 95°C denaturation for 5 minutes, and enter 45 cycles of 95°C, 15 seconds and 60°C, 1 minute.

Measured in placenta and maternal plasma samples collected from pregnant women with trisomy 21 and normal fetuses<i>PLAC4</i> Differences in the ratio of RNA alleles

SNP rs8130833 was used to compare the allele ratios of RNA transcripts in the placenta of chromosomal normal fetuses and trisomy fetuses on chromosome 21. By dividing the relative value of allele G (heavier allele, that is, the extension product of this allele shows higher quality on the mass spectrometer) by allele A (lower quality allele, That is, the relative value of the extension product of the allele showing lower quality on the mass spectrometer) calculates the allele ratio. As shown in Figure 8, all chromosome 21 trisomy samples exhibited significantly different allele ratios than normal samples. The allele ratios of trisomy samples of chromosome 21 can be divided into two groups. Trisomy samples with additional G alleles exhibited allele ratios above the normal range, while samples with additional A alleles exhibited allele ratios below the normal range.

SNP rs8130833 was also used to compare the ratio of RNA transcript alleles in maternal plasma of chromosomal normal fetuses and trisomic fetuses on chromosome 21. Except for one, all other trisomy samples showed deviations from the normal sample allele ratio (Figure 9). The data can indicate the use of polymorphic markers such as SNP to analyze the allele ratio of circulating fetal-specific transcripts in maternal plasma to non-invasively detect fetal chromosomal aneuploidy.

In the placenta<i>PLAC4</i> RNA and maternal plasma circulating<i>PLAC4</i> The SNP rs8130833 allele ratio between RNAs was found to be positively correlated (Figure 10) (Pearson correlation,<i>P</i>&lt;0.05). This finding further proves that the placenta is releasing<i>PLAC4</i> A major source of RNA to maternal plasma.

Chromosome integer multiples and chromosome 21 trisomy circulates in pregnancy<i>PLAC4</i> mRNA comparison

Cycle between normal chromosome and trisomic pregnancy on chromosome 21<i>PLAC4</i> The mRNA concentration was compared. Plasma samples were collected from 29 pregnant women with euploid fetuses and 5 pregnant women with trisomy 21 fetuses in the first trimester and middle trimester. As mentioned earlier, the real-time one-step RT-PCR was used to determine the content of plasma samples<i>PLAC4</i> mRNA concentration. As shown in Figure 11, the plasma samples of all trisomy<i>PLAC4</i> mRNA. The median values of trisomy 21 and normal pregnancy were 5581 copies/ml and 4836 copies/ml, respectively. Plasma between normal chromosome and trisomy 21 pregnancy<i>PLAC4</i> There was no statistically significant difference in mRNA concentration. This shows that only the maternal plasma<i>PLAC4</i> mRNA quantification cannot assess the presence of trisomy 21 fetuses.

Example 5: Detecting chromosome 18 trisomy by primer extension and mass spectrometry using fetal expression RNA in the placenta.

In order to show that other genes can be used to detect other chromosomal abnormalities, the serine protease inhibitor branch B (ovalbumin) member 2 (<i>SERPINB2</i>) (GenBank serial number: NM_002575) The ability to detect trisomy of chromosome 18 was studied. The primer extension response analysis can determine the SNP genotype of the placental tissue sample. The sample is processed by mass spectrometry to distinguish different RNA-SNP alleles, and the relative expression level of RNA-SNP alleles is determined to calculate the allele ratio. Because of the difference between the pregnancy with and without chromosome 18 trisomy<i>SERPINB2</i> The difference in the ratio of SNP alleles is high enough so that placental RNA samples can be used to determine trisomy 18 fetuses.

Determination of the allele ratio of serine protease inhibitor branch B (ovalbumin) member 2 in the placenta

The serine protease inhibitor branch B (ovalbumin) member 2 (SERPINB2) was selected.<i>SERPINB2</i>The gene is located on chromosome 18. Identified from a public database located in<i>SERPINB2</i>Polymorphic SNPs in the coding region of the gene (Table 13) and analyzed.

<tables><img file="TWI367259B_D0014.tif" /></tables>

Sample collection and processing. Placental tissue samples in the first trimester and the middle trimester were collected from 4 pregnant women with trisomy fetuses on chromosome 18. The placental tissues of 8 pregnant women with normal chromosomes were also collected by chorionic villus sampling (CVS). Save the sample in RNAlater immediately after collection^TM(Ambion^®, Austin, TX) and placed at -80°C until RNA extraction. According to the product instructions, the Trizol kit (Qiagen, Hilden, Germany) was used to extract total RNA from placental tissue. The extracted RNA was then subjected to DNase treatment to remove DNA contamination (Invitrogen, Carlsbad, California, USA).

Reverse transcription and PCR amplification. According to the product instructions (ThermoScript, Invitrogen, Carlsbad, California, USA), 1.25 μg of placental RNA was reverse transcribed in a 20 μl reaction system using gene-specific primers (sequences shown in Table 14).

For each PCR amplification reaction, the template is 20 μl placental cDNA, and the reaction system is 40 μl. Each reaction contains 0.6X HotStar<i>Taq</i> PCR buffer, 0.9mM MgCl₂(Qiagen), 25μM dATP, dGTP and dCTP each, 50μM dUTP (Applied Biosystems), forward and reverse primers each 200nM (Integrated DNA Technologies), and 0.02U/μ HotStar <i>Taq</i>Polymerase (Qiagen). The PCR primer sequence is shown in Table 14. The reaction conditions were: denaturation at 95°C for 7 minutes, then denaturation at 95°C for 40 seconds, annealing at 56°C for 1 minute, extension at 72°C for 1 minute, and finally holding at 72°C for 3 minutes.

<tables><img file="TWI367259B_D0015.tif" /></tables>

SNP was determined by primer extension reaction. The primer extension reaction was the same as in Example 3. The PCR product was first treated with shrimp alkaline phosphatase (Sequenom, San Diego, USA). Combine 4μl base extension mixture containing 771nM extension primer (Integrated DNA Technologies), 1.15U Thermosequenase (Sequenom) and 64μM ddATP, ddCTP, ddTTP and dGTP (Sequenom, San Diego, USA) with 5μl water and 5μl PCR The products are mixed. The heating process is: 94°C, 2 minutes, and then enter 100 cycles of 94°C, 5 seconds, 52°C, 5 seconds, and 72°C, 5 seconds. The sequence and molecular weight of the extension primer and extension product of each SNP allele are shown in Table 15. As described in Example 3, the molecular weight of the final extension product was determined with a MALDI-TOF mass spectrometer. The ratio of the peak area area of the primer extension product, which represents the two SNP alleles of the fetus with heterozygous SNP, is determined.

<tables><img file="TWI367259B_D0016.tif" /></tables>

Trisomy 18 in the placenta<i>SBRPINB2</i>The deviation of the transcript allele ratio from that of the normal placenta

For fetuses with normal chromosomes and trisomy 18<i>SERPINB2</i> The SNP ratio of mRNA was compared. The allele ratio is calculated by dividing the relative value of allele G (high molecular weight gene) by the relative value of allele A (low molecular weight gene). As shown in Figure 12, the allele ratios of all trisomy samples of chromosome 18 are significantly different from those of normal samples. Divide the allele ratios of the trisomy samples of chromosome 18 into two groups. Trisomy samples with additional G alleles exhibited allele ratios above the normal range, while samples with additional A alleles exhibited allele ratios below the normal range.

Example 6: Determination of chromosome 13 trisomy using fetal expression RNA in placenta by primer extension and mass spectrometry analysis

In order to show that other genes can be used to detect other chromosomal abnormalities, the collagen VI α 2(<i>COL4A2</i>) (GenBank serial number: X05610) The ability to detect trisomy of chromosome 13 was studied. The primer extension response analysis can determine the SNP genotype of the placental tissue sample. The sample is processed with mass spectrometry analysis to distinguish different RNA-SNP alleles, and the relative expression level of RNA-SNP alleles is determined to calculate the allele ratio. Because of the difference between pregnancies with and without trisomy 13<i>SERPINB2</i> The difference in the ratio of SNP alleles is high enough that placental RNA samples can be used to determine trisomy 13 fetuses.

Measure the SNP ratio of collagen IV α 2mRNA in the placenta

Select collagen VI α 2(<i>COL4A2</i>)。<i>COL4A2</i>The gene is located on chromosome 13. Identified from a public database located in<i>COL4A2</i>Polymorphic SNPs in the coding region of the gene (Table 16) and analyzed.

<tables><img file="TWI367259B_D0017.tif" /></tables>

Sample collection and processing. The first trimester and middle trimester plateau tissue samples were collected from 3 pregnant women with trisomy fetuses on chromosome 13. The placental tissues of 7 pregnant women with normal chromosomal fetuses were also collected by chorionic villus sampling (CVS). Save the sample in RNAlater immediately after collection^TM(Ambion^®, Austin, TX) and placed at -80°C until RNA extraction. According to the product instructions, the Trizol kit (Qiagen, Hilden, Germany) was used to extract total RNA from placental tissue. The extracted RNA was then subjected to DNase treatment to remove DNA contamination (Invitrogen, Carlsbad, California, USA).

Reverse transcription and PCR amplification. According to the product instructions (ThermoScript, Invitrogen, Carlsbad, California, USA), 1.25 μg of placental RNA was reverse transcribed in a 40 μl reaction system using gene-specific primers (sequence shown in Table 17).

For the PCR amplification reaction, the template is 40 μl placental cDNA, and the reaction system is 80 μl. Each reaction contains 0.6X HotStar<i>Taq</i> PCR buffer, 0.9mM MgCl₂(Qiagen), each 25μM dATP, dGTP and dCTP, 50μM dUTP (Applied Biosystems), forward and reverse primers each 200nM (Integrated DNA Technologies), and 0.02U/μl HotStar <i>Taq</i>Polymerase (Qiagen). The PCR primer sequence is shown in Table 17. The reaction conditions were: denaturation at 95°C for 7 minutes, then denaturation at 95°C for 40 seconds, annealing at 56°C for 1 minute, extension at 72°C for 1 minute, and finally holding at 72°C for 3 minutes.

<tables><img file="TWI367259B_D0018.tif" /></tables>

SNP was determined by primer extension reaction and the allele ratio was quantified. The primer extension reaction was the same as in Example 3. The PCR product was first treated with shrimp alkaline phosphatase (Sequenom, San Diego, USA). 4μl base extension mixture containing 771nM extension primer (Integrated DNA Technologies), 1.15U Thermosequenase (Sequenom) and 64μM ddATP, ddCTP, ddTTP and dGTP (Sequenom, San Diego, USA) with 5μl water and 5μl PCR product mix. The heating process is: 94°C, 2 minutes, and then enter 100 cycles of 94°C, 5 seconds, 52°C, 5 seconds, and 72°C, 5 seconds. The sequence and molecular weight of the extension primer and extension product of each SNP allele are shown in Table 18. As described in Example 3, the molecular weight of the final extension product was determined with a MALDI-TOF mass spectrometer. The ratio of the peak area area of the primer extension product, which represents the two SNP alleles of the fetus with heterozygous SNP, is determined.

<tables><img file="TWI367259B_D0019.tif" /></tables>The extension primer anneals the reverse strand of the sequence. Bold letters indicate the residues of dNTPs and ddNTPs added to the extension primer

Trisomy 13 in the placenta<i>SERPINB2</i>The deviation of the transcript allele ratio from that of the normal placenta

For fetuses with normal chromosomes and trisomy 13<i>COL4A2</i>The SNP ratio of mRNA was compared. The allele ratio is calculated by dividing the relative value of allele G (high molecular weight gene) by the relative value of allele A (low molecular weight gene). As shown in Figure 13, the allele ratios of all trisomy samples of chromosome 13 are significantly different from those of normal samples. Divide the allele ratios of chromosome 13 trisomy samples into two groups. Trisomy samples with additional G alleles exhibited allele ratios above the normal range, while samples with additional A alleles exhibited allele ratios below the normal range.

Although for a clear understanding of the present invention, the above has been described in detail by way of illustrations and examples, those skilled in the art should still understand that certain changes and modifications can be made within the scope of the appended patent application of the present invention. In addition, each reference mentioned in the present invention is fully cited as a reference in this application, just as if each document is individually cited as a reference.

<tables><img file="TWI367259B_D0020.tif" /></tables><tables><img file="TWI367259B_D0021.tif" /></tables><tables><img file="TWI367259B_D0022.tif" /></tables><tables><img file="TWI367259B_D0023.tif" /></tables><tables><img file="TWI367259B_D0024.tif" /></tables>Underscore: Corresponds to the data from the NCBI database (NM_182832)<i>PLAC4</i> mRNA reference sequence¹Nucleotide counterparts on chromosome 21 based on the compilation of the human genome on the UCSC genome browser (genome.ucsc.edu/) in May 2004 (hg17)

<tables><img file="TWI367259B_D0025.tif" /></tables><tables><img file="TWI367259B_D0026.tif" /></tables><tables><img file="TWI367259B_D0027.tif" /></tables>

Figure 1 shows the analysis of maternal blood, serum or plasma by relative quantification of alleles of the placental expression transcript located on chromosome 21. The strategy of a certain embodiment is used to perform non-trisomy on fetal chromosome 21. Schematic diagram of invasive identification. (A) Normal and trisomy 21 fetuses possessing a heterozygous SNP locus transcribed on chromosome 21. Trisomy 21 fetuses have an extra copy of this gene. "A" and "G" respectively represent the alleles of the transcribed SNP. (B) The gene is expressed in placental tissue, and the expression of the encoded SNPs indicates that the obtained transcripts are allelic. Due to the performance of the extra copy of the gene, the ratio of the two RNA alleles in the placenta of trisomy 21 should deviate from the ratio of the control. (C) The RNA transcripts are released into the maternal blood, and their relative abundance can reflect the expression profile of the placental genes. Therefore, the ratio of the two RNA alleles in the maternal blood, serum, and plasma of pregnancy with trisomy 21 should deviate from the ratio in normal pregnancy.

Figure 2 shows a box plot of the concentration of placental-derived mRNA transcripts in maternal plasma of non-pregnant women, women in the first trimester of pregnancy, and women in the third trimester of pregnancy. (A)<i>COL6A1</i> mRNA. (B)<i>SOD1</i> mRNA. (C)<i>COL6A2</i> mRNA and (D) ATP5O mRNA. Control, non-pregnant woman; 1^stThree months, women in the first trimester of pregnancy; 3^rdWomen in the third trimester of pregnancy. The line inside the box represents the median. The box represents 25^thAnd 75^thThe interval between percentage points. Error bars indicate 10^thAnd 90^thThe interval between percentage points. Solid dots indicate 10^thAnd 90^thData points outside of the hundreds of divisions.

Figure 3 shows the clearance of placental-derived mRNA transcripts from maternal plasma after delivery. (A), (B), (C) and (D) are in maternal plasma before delivery and 24 hours after delivery, respectively<i>COL6A1</i>、<i>SOD1</i>、<i>COL6A</i>2 and<i>ATP5O</i> The concentration of mRNA. Each line represents a pair of plasma samples obtained from a subject.

Figure 4 shows two of the placental tissues<i>COL6A1</i>The ratio of alleles of the SNP rs1053312 transcript of the gene. The relative amount between the RNA alleles is determined by allele-specific real-time quantitative RT-PCR. Analyzed from normal CVS (<i>CVS</i>), the placenta of normal term pregnancy (<i>PLN</i>) And chromosome 21 trisomy placenta (<i>T21</i>) RNA. Number of cycles through the valve (<i>Ct</i>) To calculate the ratio.

Figure 5 shows two of the placental tissues measured by allele-specific real-time quantitative RT-PCR<i>COL6A2</i>The ratio of alleles of the gene SNP rs2839114 transcript. Analyzed from normal CVS (<i>CVS</i>), the placenta of normal term pregnancy (<i>PLN</i>) And chromosome 21 trisomy placenta (<i>T21</i>) RNA. Number of cycles through (A) valve (<i>Ct</i>) Difference or (B) Difference in fluorescence intensity (ΔΔRn) obtained in the last PCR cycle to calculate the ratio.

Figure 6 shows the allele frequencies of SNPrs1053320 (COL6A1) in the placental tissues of heterozygous normal fetuses and trisomy fetuses with chromosome 21 determined by primer extension and subsequent mass spectrometry. (A) Placental DNA. (B) Placental RNA.

Figure 7 shows the allele frequencies of SNP rs2839114 (COL6A2) in the placental tissues of heterozygous normal fetuses and trisomy 21 fetuses determined by primer extension and subsequent mass spectrometry. (A) Placental DNA. (B) Placental RNA.

Figure 8 shows the placenta of a pregnant woman with a normal fetus and a trisomy fetus on chromosome 21<i>PLAC4</i>The proportion of alleles in the transcript. The allele ratio was determined by primer extension and subsequent mass spectrometry analysis using SNP rs8130833.

Figure 9 shows a plasma sample of a pregnant woman with a normal fetus and a fetus with trisomy on chromosome 21<i>PLAC4</i>The allele ratio of the transcript.

Figure 10 shows the placenta and maternal plasma samples<i>PLAC4</i>Allelic ratio correlation of transcripts.

Figure 11 shows the maternal plasma of chromosome 21 trisomy pregnancy and control pregnancy<i>PLAC4</i> Comparison of mRNA concentration. The box represents 25^thAnd 75^thThe interval between percentage points. Error bars indicate 10^thAnd 90^thThe interval between percentage points. Solid dots indicate 10^thAnd 90^thData points outside of the hundreds of divisions.

Figure 12 shows the placenta of a pregnant woman with a normal fetus and a trisomy fetus on chromosome 18<i>SERPINB2</i>The allele ratio of the transcript. The allele ratio was determined by primer extension and subsequent mass spectrometry analysis using SNP rs6098.

Figure 13 shows the placenta of a pregnant woman with a normal fetus and a trisomy fetus on chromosome 13<i>COL4A2</i>The allele ratio of the transcript. The allele ratio was determined by primer extension and subsequent mass spectrometry analysis using rs7990383.

&lt;110&gt; 香港中文大學(The Chinese University of Hong Kong)

&lt;120&gt; 檢測染色體非整倍性的方法

&lt;150&gt; US 60/663,173

&lt;151&gt; 2005-03-18

&lt;160&gt; 77

&lt;170&gt; PatentIn version 3.3

&lt;210&gt; 1

&lt;211&gt; 19

&lt;212&gt; DNA

&lt;213&gt; COL6A1直接定序的正向引子

&lt;400&gt; 1 <img file="TWI367259B_D0028.tif" />

&lt;210&gt; 2

&lt;211&gt; 20

&lt;212&gt; DNA

&lt;213&gt; COL6A1直接定序的正向引子

&lt;400&gt; 2 <img file="TWI367259B_D0029.tif" />

&lt;210&gt; 3

&lt;211&gt; 20

&lt;212&gt; DNA

&lt;213&gt; COL6A1直接定序的反向引子

&lt;400&gt; 3 <img file="TWI367259B_D0030.tif" />

&lt;210&gt; 4

&lt;211&gt; 20

&lt;212&gt; DNA

&lt;213&gt; COL6A1直接定序的反向引子

&lt;400&gt; 4 <img file="TWI367259B_D0031.tif" />

&lt;210&gt; 5

&lt;211&gt; 20

&lt;212&gt; DNA

&lt;213&gt; SOD1直接定序的正向引子

&lt;400&gt; 5 <img file="TWI367259B_D0032.tif" />

&lt;210&gt; 6

&lt;211&gt; 20

&lt;212&gt; DNA

&lt;213&gt; SOD1直接定序的正向引子

&lt;400&gt; 6 <img file="TWI367259B_D0033.tif" />

&lt;210&gt; 7

&lt;211&gt; 20

&lt;212&gt; DNA

&lt;213&gt; SOD1直接定序的反向引子

&lt;400&gt; 7 <img file="TWI367259B_D0034.tif" />

&lt;210&gt; 8

&lt;211&gt; 22

&lt;212&gt; DNA

&lt;213&gt; SOD1直接定序的反向引子

&lt;400&gt; 8 <img file="TWI367259B_D0035.tif" />

&lt;210&gt; 9

&lt;211&gt; 20

&lt;212&gt; DNA

&lt;213&gt; COL6A2直接定序的正向引子

&lt;400&gt; 9 <img file="TWI367259B_D0036.tif" />

&lt;210&gt; 10

&lt;211&gt; 20

&lt;212&gt; DNA

&lt;213&gt; COL6A2直接定序的正向引子

&lt;400&gt; 10 <img file="TWI367259B_D0037.tif" />

&lt;210&gt; 11

&lt;211&gt; 20

&lt;212&gt; DNA

&lt;213&gt; COL6A2直接定序的正向引子

&lt;400&gt; 11 <img file="TWI367259B_D0038.tif" />

&lt;210&gt; 12

&lt;211&gt; 20

&lt;212&gt; DNA

&lt;213&gt; COL6A2直接定序的反向引子

&lt;400&gt; 12 <img file="TWI367259B_D0039.tif" />

&lt;210&gt; 13

&lt;211&gt; 20

&lt;212&gt; DNA

&lt;213&gt; COL6A2直接定序的反向引子

&lt;400&gt; 13 <img file="TWI367259B_D0040.tif" />

&lt;210&gt; 14

&lt;211&gt; 20

&lt;212&gt; DNA

&lt;213&gt; COL6A2直接定序的反向引子

&lt;400&gt; 14 <img file="TWI367259B_D0041.tif" />

&lt;210&gt; 15

&lt;211&gt; 20

&lt;212&gt; DNA

&lt;213&gt; ATP5O直接定序的正向引子

&lt;400&gt; 15 <img file="TWI367259B_D0042.tif" />

&lt;210&gt; 16

&lt;211&gt; 20

&lt;212&gt; DNA

&lt;213&gt; ATP5O直接定序的反向引子

&lt;400&gt; 16 <img file="TWI367259B_D0043.tif" />

&lt;210&gt; 17

&lt;211&gt; 23

&lt;212&gt; DNA

&lt;213&gt; COL6A1即時QRT-PCR測定的正向引子

&lt;400&gt; 17 <img file="TWI367259B_D0044.tif" />

&lt;210&gt; 18

&lt;211&gt; 18

&lt;212&gt; DNA

&lt;213&gt; COL6A1即時QRT-PCR測定的反向引子

&lt;400&gt; 18 <img file="TWI367259B_D0045.tif" />

&lt;210&gt; 19

&lt;211&gt; 16

&lt;212&gt; DNA

&lt;213&gt; COL6A1即時QRT-PCR測定的探針

&lt;400&gt; 19 <img file="TWI367259B_D0046.tif" />

&lt;210&gt; 20

&lt;211&gt; 90

&lt;212&gt; DNA

&lt;213&gt; COL6A1即時QRT-PCR測定的標準曲線的序列

&lt;400&gt; 20 <img file="TWI367259B_D0047.tif" />

&lt;210&gt; 21

&lt;211&gt; 19

&lt;212&gt; DNA

&lt;213&gt; SOD1即時QRT-PCR測定的正向引子

&lt;400&gt; 21 <img file="TWI367259B_D0048.tif" />

&lt;210&gt; 22

&lt;211&gt; 18

&lt;212&gt; DNA

&lt;213&gt; SOD1即時QRT-PCR測定的反向引子

&lt;400&gt; 22 <img file="TWI367259B_D0049.tif" />

&lt;210&gt; 23

&lt;211&gt; 21

&lt;212&gt; DNA

&lt;213&gt; SOD1即時QRT-PCR測定的探針

&lt;400&gt; 23 <img file="TWI367259B_D0050.tif" />

&lt;210&gt; 24

&lt;211&gt; 65

&lt;212&gt; DNA

&lt;213&gt; SOD1即時QRT-PCR測定的標準曲線的序列

&lt;400&gt; 24 <img file="TWI367259B_D0051.tif" />

&lt;210&gt; 25

&lt;211&gt; 21

&lt;212&gt; DNA

&lt;213&gt; COL6A2即時QRT-PCR測定的正向引子

&lt;400&gt; 25 <img file="TWI367259B_D0052.tif" />

&lt;210&gt; 26

&lt;211&gt; 19

&lt;212&gt; DNA

&lt;213&gt; COL6A2即時QRT-PCR測定的反向引子

&lt;400&gt; 26 <img file="TWI367259B_D0053.tif" />

&lt;210&gt; 27

&lt;211&gt; 15

&lt;212&gt; DNA

&lt;213&gt; COL6A2即時QRT-PCR測定的探針

&lt;400&gt; 27 <img file="TWI367259B_D0054.tif" />

&lt;210&gt; 28

&lt;211&gt; 61

&lt;212&gt; DNA

&lt;213&gt; COL6A2即時QRT-PCR測定的標準曲線的序列

&lt;400&gt; 28 <img file="TWI367259B_D0055.tif" />

&lt;210&gt; 29

&lt;211&gt; 21

&lt;212&gt; DNA

&lt;213&gt; ATP5O即時QRT-PCR測定的正向引子

&lt;400&gt; 29 <img file="TWI367259B_D0056.tif" />

&lt;210&gt; 30

&lt;211&gt; 21

&lt;212&gt; DNA

&lt;213&gt; ATP5O即時QRT-PCR測定的反向引子

&lt;400&gt; 30 <img file="TWI367259B_D0057.tif" />

&lt;210&gt; 31

&lt;211&gt; 15

&lt;212&gt; DNA

&lt;213&gt; ATP5O即時QRT-PCR測定的探針

&lt;400&gt; 31 <img file="TWI367259B_D0058.tif" />

&lt;210&gt; 32

&lt;211&gt; 65

&lt;212&gt; DNA

&lt;213&gt; ATP5O即時QRT-PCR測定的標準曲線的序列

&lt;400&gt; 32 <img file="TWI367259B_D0059.tif" />

&lt;210&gt; 33

&lt;211&gt; 20

&lt;212&gt; DNA

&lt;213&gt; COL6A1 RNA-SNPs等位基因特異即時QRT-PCR測定的正向引子

&lt;400&gt; 33 <img file="TWI367259B_D0060.tif" />

&lt;210&gt; 34

&lt;211&gt; 15

&lt;212&gt; DNA

&lt;213&gt; COL6A1 RNA-SNPs等位基因特異即時QRT-PCR測定的反向引子

&lt;400&gt; 34 <img file="TWI367259B_D0061.tif" />

&lt;210&gt; 35

&lt;211&gt; 14

&lt;212&gt; DNA

&lt;213&gt; COL6A1 RNA-SNPs等位基因特異即時QRT-PCR測定的探針(等位基因G)

&lt;400&gt; 35 <img file="TWI367259B_D0062.tif" />

&lt;210&gt; 36

&lt;211&gt; 14

&lt;212&gt; DNA

&lt;213&gt; COL6A1 RNA-SNPs等位基因特異即時QRT-PCR測定的探針(等位基因A)

&lt;400&gt; 36 <img file="TWI367259B_D0063.tif" />

&lt;210&gt; 37

&lt;211&gt; 17

&lt;212&gt; DNA

&lt;213&gt; COL6A2 RNA-SNPs等位基因特異即時QRT-PCR測定的正向引子

&lt;400&gt; 37 <img file="TWI367259B_D0064.tif" />

&lt;210&gt; 38

&lt;211&gt; 14

&lt;212&gt; DNA

&lt;213&gt; COL6A2 RNA-SNPs等位基因特異即時QRT-PCR測定的反向引子

&lt;400&gt; 38 <img file="TWI367259B_D0065.tif" />

&lt;210&gt; 39

&lt;211&gt; 16

&lt;212&gt; DNA

&lt;213&gt; COL6A2 RNA-SNPs等位基因特異即時QRT-PCR測定的探針(等位基因G)

&lt;400&gt; 39 <img file="TWI367259B_D0066.tif" />

&lt;210&gt; 40

&lt;211&gt; 16

&lt;212&gt; DNA

&lt;213&gt; COL6A2 RNA-SNPs等位基因特異即時QRT-PCR測定的探針(等位基因A)

&lt;400&gt; 40 <img file="TWI367259B_D0067.tif" />

&lt;210&gt; 41

&lt;211&gt; 30

&lt;212&gt; DNA

&lt;213&gt; 擴增COL6A1上SNP位點的正向引子

&lt;400&gt; 41 <img file="TWI367259B_D0068.tif" />

&lt;210&gt; 42

&lt;211&gt; 30

&lt;212&gt; DNA

&lt;213&gt; 擴增COL6A1上SNP位點的反向引子

&lt;400&gt; 42 <img file="TWI367259B_D0069.tif" />

&lt;210&gt; 43

&lt;211&gt; 30

&lt;212&gt; DNA

&lt;213&gt; 擴增COL6A2上SNP位點的正向引子

&lt;400&gt; 43 <img file="TWI367259B_D0070.tif" />

&lt;210&gt; 44

&lt;211&gt; 30

&lt;212&gt; DNA

&lt;213&gt; 擴增COL6A2上SNP位點的反向引子

&lt;400&gt; 44 <img file="TWI367259B_D0071.tif" />

&lt;210&gt; 45

&lt;211&gt; 17

&lt;212&gt; DNA

&lt;213&gt; COL6A1的rs1053320的延伸引子序列

&lt;400&gt; 45 <img file="TWI367259B_D0072.tif" />

&lt;210&gt; 46

&lt;211&gt; 18

&lt;212&gt; DNA

&lt;213&gt; COL6A1的rs1053320的等位基因T的延伸序列

&lt;400&gt; 46 <img file="TWI367259B_D0073.tif" />

&lt;210&gt; 47

&lt;211&gt; 20

&lt;212&gt; DNA

&lt;213&gt; COL6A1的rs1053320的等位基因C的延伸序列

&lt;400&gt; 47 <img file="TWI367259B_D0074.tif" />

&lt;210&gt; 48

&lt;211&gt; 20

&lt;212&gt; DNA

&lt;213&gt; COL6A2的rs2839114的延伸引子序列

&lt;400&gt; 48 <img file="TWI367259B_D0075.tif" />

&lt;210&gt; 49

&lt;211&gt; 21

&lt;212&gt; DNA

&lt;213&gt; COL6A2的rs2839114的對位基因A的延伸序列

&lt;400&gt; 49 <img file="TWI367259B_D0076.tif" />

&lt;210&gt; 50

&lt;211&gt; 23

&lt;212&gt; DNA

&lt;213&gt; COL6A2的rs2839114的對位基因C的延伸序列

&lt;400&gt; 50 <img file="TWI367259B_D0077.tif" />

&lt;210&gt; 51

&lt;211&gt; 180

&lt;212&gt; DNA

&lt;213&gt; 人(Homo sapiens)

&lt;220&gt;

&lt;221&gt; rs8130833

&lt;222&gt; (1)..(180)

&lt;223&gt; dbSNP序列號

&lt;220&gt;

&lt;221&gt; 位於PLAC4編碼區域的SNP

&lt;222&gt; (74)..(74)

&lt;223&gt; [A/G]

&lt;220&gt;

&lt;221&gt; PLAC4-41478755

&lt;222&gt; (74)..(74)

&lt;223&gt; 數字表示在基於UCSC基因組瀏覽器(genome.ucsc.edu/)人類基因組2004年5月(hg17)彙編的上SNP的位置T

&lt;400&gt; 51 <img file="TWI367259B_D0078.tif" />

&lt;210&gt; 52

&lt;211&gt; 180

&lt;212&gt; DNA

&lt;213&gt; 人(Homo sapiens)

&lt;220&gt;

&lt;221&gt; 位於PLAC4編碼區域的SNP

&lt;222&gt; (84)..(84)

&lt;223&gt; [A/G]

&lt;220&gt;

&lt;221&gt; PLAC4-41471145

&lt;222&gt; (84)..(84)

&lt;223&gt; 數字表示在基於UCSC基因組瀏覽器(genome.ucsc.edu/)人類基因組2004年5月(hg17)彙編的上SNP的位置

&lt;400&gt; 52 <img file="TWI367259B_D0079.tif" />

&lt;210&gt; 53

&lt;211&gt; 180

&lt;212&gt; DNA

&lt;213&gt; 人(Homo sapiens)

&lt;220&gt;

&lt;221&gt; rs9977003

&lt;222&gt; (1)..(180)

&lt;223&gt; dbSNP序列號

&lt;221&gt; 位於PLAC4編碼區域的SNP

&lt;222&gt; (70)..(70)

&lt;223&gt; [A/G]

&lt;220&gt;

&lt;221&gt; PLAC4-41470591

&lt;222&gt; (70)..(70)

&lt;223&gt; 數字表示在基於UCSC基因組瀏覽器(genome.ucsc.edu/)人類基因組2004年5月(hg17)彙編的上SNP的位置

&lt;400&gt; 53 <img file="TWI367259B_D0080.tif" /><img file="TWI367259B_D0081.tif" />

&lt;210&gt; 54

&lt;211&gt; 180

&lt;212&gt; DNA

&lt;213&gt; 人(Homo sapiens)

&lt;220&gt;

&lt;221&gt; 位於PLAC4編碼區域的SNP

&lt;222&gt; (75)..(75)

&lt;223&gt; [C/T]

&lt;220&gt;

&lt;221&gt; PLAC4-41476236

&lt;222&gt; (75)..(75)

&lt;223&gt; 數字表示在基於UCSC基因組瀏覽器(genome.ucsc.edu/)人類基因組2004年5月(hg17)彙編的上SNP的位置

&lt;400&gt; 54 <img file="TWI367259B_D0082.tif" />

&lt;210&gt; 55

&lt;211&gt; 25

&lt;212&gt; DNA

&lt;213&gt; 反轉錄含有SNP rs8130833序列的引子

&lt;400&gt; 55 <img file="TWI367259B_D0083.tif" />

&lt;210&gt; 56

&lt;211&gt; 30

&lt;212&gt; DNA

&lt;213&gt; PCR擴增含有SNP rs8130833序列的正向引子

&lt;400&gt; 56 <img file="TWI367259B_D0084.tif" />

&lt;210&gt; 57

&lt;211&gt; 30

&lt;212&gt; DNA

&lt;213&gt; PCR擴增含有SNP rs8130833序列的反向引子

&lt;400&gt; 57 <img file="TWI367259B_D0085.tif" />

&lt;210&gt; 58

&lt;211&gt; 18

&lt;212&gt; DNA

&lt;213&gt; SNP rs8130833未延伸引子

&lt;400&gt; 58 <img file="TWI367259B_D0086.tif" />

&lt;210&gt; 59

&lt;211&gt; 19

&lt;212&gt; DNA

&lt;213&gt; SNP rs8130833的等位基因A的延伸產物

&lt;400&gt; 59 <img file="TWI367259B_D0087.tif" />

&lt;210&gt; 60

&lt;211&gt; 20

&lt;212&gt; DNA

&lt;213&gt; SNP rs8130833的等位基因G的延伸產物

&lt;400&gt; 60 <img file="TWI367259B_D0088.tif" />

&lt;210&gt; 61

&lt;211&gt; 20

&lt;212&gt; DNA

&lt;213&gt; 對PLAC4 mRNA進行即時QRT-PCR測定的正向引子

&lt;400&gt; 61 <img file="TWI367259B_D0089.tif" />

&lt;210&gt; 62

&lt;211&gt; 22

&lt;212&gt; DNA

&lt;213&gt; 對PLAC4 mRNA進行即時QRT-PCR測定的反向引子

&lt;400&gt; 62 <img file="TWI367259B_D0090.tif" />

&lt;210&gt; 63

&lt;211&gt; 15

&lt;212&gt; DNA

&lt;213&gt; 對PLAC4 mRNA進行即時QRT-PCR測定的探針

&lt;400&gt; 63 <img file="TWI367259B_D0091.tif" />

&lt;210&gt; 64

&lt;211&gt; 74

&lt;212&gt; DNA

&lt;213&gt; 對PLAC4 mRNA進行即時QRT-PCR測定的校準序列

&lt;400&gt; 64 <img file="TWI367259B_D0092.tif" />

&lt;210&gt; 65

&lt;211&gt; 184

&lt;212&gt; DNA

&lt;213&gt; 人(Homo sapiens)

&lt;220&gt;

&lt;221&gt; 位於SERPINB2基因編碼區內的多態SNP

&lt;222&gt; (111)..(111)

&lt;223&gt; [A/G]

&lt;220&gt;

&lt;221&gt; rs6098

&lt;222&gt; (111)..(111)

&lt;223&gt; dbSNP序列號

&lt;400&gt; 65 <img file="TWI367259B_D0093.tif" />

&lt;210&gt; 66

&lt;211&gt; 30

&lt;212&gt; DNA

&lt;213&gt; 反轉錄和PCR擴增SERPINB2基因的正向引子

&lt;400&gt; 66 <img file="TWI367259B_D0094.tif" />

&lt;210&gt; 67

&lt;211&gt; 30

&lt;212&gt; DNA

&lt;213&gt; 反轉錄和PCR擴增SERPINB2基因的反向引子

&lt;400&gt; 67 <img file="TWI367259B_D0095.tif" />

&lt;210&gt; 68

&lt;211&gt; 17

&lt;212&gt; DNA

&lt;213&gt; SERPINB2基因編碼SNP的未延伸引子序列

&lt;400&gt; 68 <img file="TWI367259B_D0096.tif" />

&lt;210&gt; 69

&lt;211&gt; 18

&lt;212&gt; DNA

&lt;213&gt; SERPINB2基因編碼SNP的對位基因A的延伸產物

&lt;400&gt; 69 <img file="TWI367259B_D0097.tif" />

&lt;210&gt; 70

&lt;211&gt; 19

&lt;212&gt; DNA

&lt;213&gt; SERPINB2基因編碼SNP的對位基因G的延伸產物

&lt;400&gt; 70 <img file="TWI367259B_D0098.tif" />

&lt;210&gt; 71

&lt;211&gt; 183

&lt;212&gt; DNA

&lt;213&gt; 人(Homo sapiens)

&lt;220&gt;

&lt;221&gt; 位於COL4A2基因編碼區內的多態SNP

&lt;222&gt; (86)..(86)

&lt;223&gt; [G/A]

&lt;220&gt;

&lt;221&gt; rs7990383

&lt;222&gt; (86)..(86)

&lt;223&gt; dbSNP序列號

&lt;400&gt; 71 <img file="TWI367259B_D0099.tif" />

&lt;210&gt; 72

&lt;211&gt; 30

&lt;212&gt; DNA

&lt;213&gt; 反轉錄和PCR擴增COL4A2基因的正向引子

&lt;400&gt; 72 <img file="TWI367259B_D0100.tif" />

&lt;210&gt; 73

&lt;211&gt; 30

&lt;212&gt; DNA

&lt;213&gt; 反轉錄和PCR擴增COL4A2基因的反向引子

&lt;400&gt; 73 <img file="TWI367259B_D0101.tif" />

&lt;210&gt; 74

&lt;211&gt; 17

&lt;212&gt; DNA

&lt;213&gt; COL4A2基因編碼SNP的未延伸引子

&lt;400&gt; 74 <img file="TWI367259B_D0102.tif" />

&lt;210&gt; 75

&lt;211&gt; 18

&lt;212&gt; DNA

&lt;213&gt; COL4A2基因編碼SNP的對位基因G的延伸產物

&lt;400&gt; 75 <img file="TWI367259B_D0103.tif" />

&lt;210&gt; 76

&lt;211&gt; 20

&lt;212&gt; DNA

&lt;213&gt; COL4A2基因編碼SNP的對位基因A的延伸產物

&lt;400&gt; 76 <img file="TWI367259B_D0104.tif" />

&lt;210&gt; 77

&lt;211&gt; 11558

&lt;212&gt; DNA

&lt;213&gt; 人(Homo sapiens)

&lt;220&gt;

&lt;221&gt; PLAC4基因座的基因組序列

&lt;222&gt; (1)..(11558)

&lt;223&gt; 基於UCSC基因組瀏覽器(genome.ucsc.edu/)的人類基因組2004年5月(hg17)彙編，該PLAC4基因座跨越了21號染色體的41469028-41480585核苷酸對應物

&lt;400&gt; 77 <img file="TWI367259B_D0105.tif" /><img file="TWI367259B_D0106.tif" /><img file="TWI367259B_D0107.tif" /><img file="TWI367259B_D0108.tif" /><img file="TWI367259B_D0109.tif" /><img file="TWI367259B_D0110.tif" /><img file="TWI367259B_D0111.tif" /><img file="TWI367259B_D0112.tif" /><img file="TWI367259B_D0113.tif" />