KR20200028153A - Non-invasive prenatal testing method using cell free dna fragment derived maternal blood - Google Patents

Abstract

The present invention relates to a non-invasive prenatal test method using cell-free DNA fragments derived from maternal blood. Specifically, the present invention relates to a non-invasive prenatal test method for determining chromosomal aneuploidy of a fetus or maternal mosaicism based on the difference in length between DNA fragments of the mother and the fetus in the cell-free DNA fragment derived from the maternal blood. According to the present invention, the method can be useful for the non-invasive prenatal test since the chromosomal aneuploidy of the fetus and the maternal mosaicism can be more accurately determined using a z-score calculated from each of DNA fragments by isolating cell-free DNA fragments derived from the mother or the fetus from whole cell-free DNA fragments isolated from the mother blood depending on the length of the DNA fragment.

Description

Non-invasive prenatal testing method using maternal blood-derived cell-free DNA fragments {NON-INVASIVE PRENATAL TESTING METHOD USING CELL FREE DNA FRAGMENT DERIVED MATERNAL BLOOD}

The present invention relates to a non-invasive prenatal test method using maternal blood-derived cell-free DNA fragments, specifically separating maternal blood-derived cell-free DNA fragments and fetal-derived cell-free DNA fragments based on differences in the DNA fragment lengths of the mother and fetus And a non-invasive prenatal screening method for determining chromosome aneuploidy or maternal maternalism of the fetus based on the z-score for each.

One of the important efforts in human medical research is the discovery of genetic malformations underlying underlying adverse health outcomes. Prenatal examination for the detection of such genetic abnormalities determines and diagnoses the presence or absence of a fetus before birth through chromosome aneuploidy of the fetus.

In general, pregnant women over the age of 35, pregnant women with a hereditary disease or congenital malformation, or pregnant women with multiple births, are classified as high-risk pregnant women. Recently, the number of high-risk pregnant women tends to increase steadily, which is attributed to the increased average age of women. When classified as a high-risk pregnant woman, precautions are required for prenatal care and the need for prenatal examination is raised for the safety of the mother and fetus.

Prenatal testing is divided into invasive prenatal testing and non-invasive prenatal testing (NIPT). Invasive prenatal tests include amniocentesis, chorionic villi sampling, and cordocentesis. However, the invasive prenatal test may cause an abortion, disease, or malformation by impacting the fetus during the test process, and recently, a non-invasive prenatal test method has been developed to overcome such problems.

On the other hand, a general non-invasive prenatal test method distinguishes normal and aneuploidy chromosomes by calculating z-scores through a normalization process that divides the number of sequencing fragments arranged on each chromosome by the total number of sequencing fragments. However, it is difficult to accurately determine if the fetal fraction (FF) is low or if DNA derived from the mother is amplified or deleted.

Recently, a massively parallel signature sequencing (MPSS) method, which is a next generation sequencing (NGS) technology, has been introduced, and cell-free DNA (cfDNA) present in maternal blood has been introduced. As cell-free fetal DNA (cffDNA) was discovered, a non-invasive prenatal test method using the same was developed. However, since the amount of cffDNA of the fetus present in the mother's blood is relatively small, a method of generating and discriminating a large number of NGS reads is generally used, and thus economic problems are emerging. In addition, there is a need to develop a more accurate and sensitive test method free of diagnostic errors in fetal malformations, such as false positive (FP) or false negative (FN).

In this regard, Korean Patent Publication No. 10-2017-0036648 discloses the average number of leads of a specific chromosome and the chromosome to determine whether it is aneuploidy, in order to determine whether aneuploidy is chromosome from DNA sequence information obtained from a blood sample of a mother By comparing the average number of leads of other chromosomes except for removing the deviation between experiments, and using the ratio of the weighted averaged number of chromosomal leads as the coefficient of variation (CV) value, the reliability and specificity of the result are improved to improve the false positive probability. It provides a non-invasive method for analyzing fetal chromosomes.

Accordingly, the present inventors were studying to develop an accurate non-invasive prenatal test method, and the whole cell-free DNA fragment isolated from the mother blood was separated into a mother- or fetal-derived cell-free DNA fragment depending on the length of the DNA fragment, and these The present invention was completed by developing an analysis method capable of more accurately determining the chromosome aneuploidy or maternal maternalism of the fetus using the z-score calculated from each.

Republic of Korea Patent Publication No. 10-2017-0036648

An object of the present invention is to provide a method and apparatus for non-invasive prenatal testing using a cell-free DNA fragment derived from maternal blood.

In order to achieve the above object, the present invention comprises the steps of producing a full sequence fragment from cell-free DNA extracted from the mother's blood; Arranging the whole nucleotide sequence fragments produced at homologous positions of a human reference genome sequence; The entire sequence fragment is divided into fetal-derived sequence fragments and maternal-derived sequence fragments along the length, and z-score (z-) for each of the entire sequence fragment, fetal-derived sequence fragment, and maternal-derived sequence fragments. calculating a score); And comparing the z-scores of each of the calculated nucleotide sequence fragments, fetal derived nucleotide sequence fragments, and maternal derived nucleotide sequence fragments to determine maternal capisism or fetal chromosomal aneuploidies. to provide.

In addition, the present invention is a production unit for producing a full sequence fragment from cell-free DNA extracted from the mother's blood; An alignment portion that aligns the produced entire sequence fragment at a homology position of a human reference genome sequence; The entire sequence fragment is divided into fetal-derived sequence fragments and maternal-derived sequence fragments along the length, and z-score (z-) for each of the entire sequence fragment, fetal-derived sequence fragment, and maternal-derived sequence fragments. a calculating unit for calculating a score); And a non-invasive prenatal test apparatus comprising a determination unit for determining the maternal moxisism or chromosome aneuploidy of the fetus by comparing the z-scores of each of the calculated nucleotide sequence fragments, fetal derived nucleotide sequences, and maternal derived nucleotide sequences. to provide.

In the method according to the present invention, the whole cell-free DNA fragment isolated from the mother blood is separated into a mother- or fetal-derived cell-free DNA fragment depending on the length of the DNA fragment, and the chromosome of the fetus using the z-score calculated from each of them. Since it is possible to more accurately judge aneuploidy or maternal maternalism, it can be useful for non-invasive prenatal examination.

1 is a graph confirming the length of fragments obtained from whole cell-free DNA isolated from pregnant or non-pregnant women (non-pregnancy.avar: average value of non-pregnant women, pregnancy.avar: average value of pregnant women).
FIG. 2 shows three chromosomes present in chromosome 21 of the mother using z-scores for the number of fragments obtained from whole acellular DNA, maternal-derived acellular DNA or fetal-derived acellular DNA isolated from maternal blood of a pregnant woman. It is a diagram showing the result of confirming the chromosomes as a heatmap (Original: original data, Fetus: fetal data, Mom: maternal data).
FIG. 3 shows the chromosome 21 present in chromosome 21 using a z-score for the number of fragments obtained from whole acellular DNA, maternal-derived acellular DNA or fetal-derived acellular DNA isolated from maternal blood of a pregnant woman. It is a diagram showing the result of confirming the chromosomes as a heatmap (Original: original data, Fetus: fetal data, Mom: maternal data).
FIG. 4 divides the average value of z-scores for the number of fragments obtained from whole acellular DNA, maternal-derived acellular DNA, or fetal-derived acellular DNA separated from maternal blood into cases determined by trisomy or maternal maternalism It is a graph (Original: original data, Fetus: fetal data, Mom: maternal data).
Figure 5 is a monochromosome present on the X chromosome of a fetus using z-scores for the number of fragments obtained from whole acellular DNA, maternal-derived acellular DNA or fetal-derived acellular DNA isolated from the maternal blood of a pregnant woman It is a figure showing the result of confirming with a heat map (Original: original data, Fetus: fetus-derived data, Mom: maternal-derived data).
Figure 6 is a monochromosome present on the X chromosome of the mother using a z-score for the number of fragments obtained from the whole cell-free DNA, maternal-derived acellular DNA or fetal-derived acellular DNA isolated from the maternal blood of a pregnant woman It is a diagram showing the result of confirming with a heat map (Original: original data, Fetus: fetal derived data, Mom: maternal derived data).
Figure 7 is divided by the case that the average value of the z-score for the number of fragments obtained from the whole cell-free DNA, maternal-derived cell-free DNA or fetal-derived cell-free DNA isolated from the maternal blood divided into monochromosomal or maternal capisism It is a graph (Original: original data, Fetus: fetal data, Mom: maternal data).

Hereinafter, the present invention will be described in detail.

The present invention comprises the steps of producing a full sequence fragment from cell-free DNA extracted from the mother's blood; Arranging the whole nucleotide sequence fragments produced at homologous positions of a human reference genome sequence; The entire sequencing fragment is divided into fetal-derived sequencing fragments and maternal-derived sequencing fragments along the length, and z-scores (z-) for each of the whole sequencing fragments, fetal-derived sequencing fragments and maternal-derived sequencing fragments. calculating a score); And comparing the z-scores of each of the calculated nucleotide sequence fragments, fetal derived nucleotide sequence fragments, and maternal derived nucleotide sequence fragments to determine maternal capisism or fetal chromosomal aneuploidies. to provide.

In one aspect, the non-invasive prenatal test method according to the present invention provides a step of producing a full sequence fragment from cell-free DNA extracted from maternal blood.

As used herein, the term "non-invasive prenatal testing" refers to a test method capable of identifying chromosomal abnormalities in a fetus without invasive surgical procedures, unlike prenatal testing methods such as amniotic fluid testing or chorionic membrane testing. it means. The non-invasive prenatal test method may be performed through sequencing of cell-free fetal DNA present in the mother's blood.

In the method according to the invention, the blood may be serum or plasma, specifically plasma.

As used herein, the term "cell-free DNA (cfDNA)" refers to a piece of DNA that does not exist in the nucleus of a cell and is floating in the blood. Currently, the onset of cancer, metastasis, relapse, anti-cancer agent reactivity, etc. It is used for prediction. The cfDNA can be isolated from blood by methods known in the art. The isolated cfDNA can be produced as a sequence fragment by using a large-scale parallel sequencing method. At this time, the produced sequence fragment may be 1 to 10 million.

The term, "massively parallel signature sequencing (MPSS)" is a next generation sequencing (NGS) method, which produces DNA, RNA and other nucleotide sequences as fragments and amplifies them. It refers to a method of sequencing that rapidly reads large amounts of genomic information by reading several fragments simultaneously and combining the sequence data thus obtained using bioinformatics.

The large-scale parallel sequencing may be performed according to a method well known in the art, and the method may be appropriately modified by a skilled person in some cases. Specifically, for large-scale parallel sequencing, 454 platform (Margulies et al., Nature, 2005, 437: 376-380), Illumina Genome Analyzer, Illumina HiSeq2000, HisSeq2500, MiSeq, NextSeq500, Life Tech Ion PGM, Ion Proton, Ion S5, Ion S5, Ion S5XL, SOLiD (Applied Biosystems), Helicos True Single Molecule DNA sequencing technology (Harris et al., Science, 2008, 320: 106-109), single molecule from Pacific Biosciences or It can be performed by real-time (SMART ™) technology.

In this case, the sequencing may be performed by a paired-end sequencing or a single-read sequencing method, specifically, a paired-end sequencing method. You can.

In another aspect, the non-invasive prenatal test method according to the present invention provides a step of aligning the produced entire sequence fragment at the homologous position of the human reference genomic sequence.

The human reference genomic sequence is a sequence stored in a nucleotide sequence database, which is known as a human genomic sequence, and can be used to compare sequences of nucleotide sequence fragments produced by the method according to the present invention. The human reference genomic sequence may be a known sequence such as build 37 (build 37, GRCh37), hg18, hg19, hg38, etc., and may be hg18 according to an embodiment of the present invention.

In this step, it is possible to remove the overlapping sequencing fragments while arranging the entire sequencing fragment at the homology position of the human reference genome sequence. In addition, at this time, in the process of amplifying the sequence of a fragment, forward and reverse sequences can be used in subsequent steps by taking only data derived from the same sequence fragment.

In another aspect, in the non-invasive prenatal test method according to the present invention, the entire sequence fragments arranged above are divided into fetal-derived sequence fragments and maternal-derived sequence fragments along the length, and the entire sequence fragment and fetal-derived sequence fragments And calculating z-scores for each base fragment derived from the parent.

The fetal-derived nucleotide sequence fragment and the parent-derived nucleotide sequence fragment may be divided according to the length of the sequence fragment. In this case, the fetal-derived sequence fragment may be 150 bp or less, and the maternal-derived sequence fragment may be 185 bp or more. By dividing the nucleotide sequence fragment derived from the fetus or mother according to the length of the nucleotide sequence fragment, the result derived by the non-invasive prenatal test method according to the present invention can be used to determine whether the maternal capisism or fetal chromosomal aneuploidy.

The z-score may be calculated by a method well known in the art, or may be calculated by an appropriately modified method in some cases. In the test method according to the present invention, the z-score can be calculated using the number of DNA fragments of the chromosome to check chromosome aneuploidy in the aligned base sequence. The chromosome may be an autosomal or sex chromosome. The autosomal may be any one or more selected from the group consisting of chromosomes 1 to 22 in humans, and the sex chromosome may be an X or Y chromosome in humans.

Specifically, the z-score can be calculated by Equation 1 below:

[Equation 1]

(Where i is chromosome 1 to 22, X chromosome or Y chromosome,

reference is a normal sample group,

target means the parent sample to be tested).

In another aspect, the non-invasive prenatal test method according to the present invention provides maternal capisism or fetal chromosomal aneuploidies from z-scores for the whole sequencing fragment, fetal derived sequencing fragment and maternal derived sequencing fragment calculated above. Provides steps for judging.

As used herein, the term "maternal mosaicism" refers to a case in which the chromosome of a fetus is normal, but the maternal has an abnormal karyotype, and the non-invasive prenatal test determines that chromosome aneuploidy appears. That is, the non-invasive prenatal test method according to the present invention can improve the accuracy of results by reducing false-positive errors caused by maternal maternalism.

As used herein, the term "chromosomal aneuploidy" means that the number of chromosomes differs from the number of normal chromosomes (2), i.e., 0, 1, 3 or more chromosomes are present. . This chromosome aneuploidy can be used to determine a rare genetic disorder in the fetus.

In the test method according to the present invention, maternal maternalism or chromosome aneuploidy of the fetus can be determined by:

i) if the z-score for the fetal sequence fragment is greater than the z-score for the entire sequence fragment, the fetus is determined to be a trisomy;

ii) if the z-score for the nucleotide sequence fragment derived from the parent is greater than the z-score for the entire sequencing fragment, then it is determined that the parent is a mother isism;

iii) If the z-score for the fetal sequence fragment is smaller than the z-score for the entire sequence fragment, it is determined that the fetus is a monostain; or

iv) If the z-score for the nucleotide sequence fragment derived from the parent is smaller than the z-score for the entire sequencing fragment, then it is determined that the maternal capsism.

That is, maternal maternalism or chromosome aneuploidy of the fetus can be judged by:

i) If the value obtained by subtracting the z-score for the entire sequence fragment from the z-score for the fetal sequence fragment is greater than 0, it is determined that the fetus is a trisomy;

ii) If the value obtained by subtracting the z-score for the entire sequencing fragment from the z-score for the nucleotide sequence fragment derived from the parent is greater than 0, it is determined that it is maternal capisism;

iii) If the value obtained by subtracting the z-score for the entire sequence fragment from the z-score for the fetal sequence fragment is less than 0, it is determined that the fetus is a monostain; or

iv) If the value obtained by subtracting the z-score for the entire sequencing fragment from the z-score for the nucleotide sequence fragment derived from the parent is less than 0, it is determined that it is maternal capisism.

Specifically, fetal chromosomal aneuploidies can be judged by:

i) The z-score for the fetal sequence fragment is greater than the z-score for the entire sequence fragment,

ii) If the z-score for the nucleotide sequence fragment derived from the parent is smaller than the z-score for the entire sequencing fragment, the fetus is considered to be a trisomy.

In addition, fetal chromosomal aneuploidies can be judged by:

i) The z-score for the fetal sequence fragment is smaller than the z-score for the entire sequence fragment,

ii) If the z-score for the nucleotide sequence fragment derived from the parent is greater than the z-score for the entire sequencing fragment, the fetus is considered to be a monostain.

The test method according to the present invention may determine an autosomal abnormality, an abnormal sex chromosome, or an deletion of an autosomal. For example, the autosomal abnormality may be a case where the number of chromosomes is 3 or more, specifically 3. Diseases that may appear as autosomal abnormalities may be Down's syndrome, Edward's syndrome, Fatau syndrome, trisomy 9, trisomy 16 or trisomy 22. Meanwhile, the sex chromosome abnormality may be an X or Y chromosome added or deleted. Diseases that may appear as sex chromosomal abnormalities may be specifically Turner's syndrome, TripleX syndrome, Klinefelter's syndrome, or Jacob's syndrome. In addition, the autosomal deletion may be a disease in which a part of the autosomal deletion may occur. Diseases that may appear as autosomal deletions include, specifically, mononuclear chromosome deletion syndrome, 2q33.1 deletion syndrome, Walf-Hirschhorn syndrome, cat crying syndrome, Williams syndrome, Jacobson syndrome, Prader-Willi / Angelman syndrome, or D. George syndrome.

In addition, the present invention is a production unit for producing a full sequence fragment from cell-free DNA extracted from the mother's blood; An alignment portion that aligns the produced entire sequence fragment at a homology position of a human reference genome sequence; The entire sequencing fragment is divided into fetal-derived sequencing fragments and maternal-derived sequencing fragments along the length, and z-scores (z-) for each of the whole sequencing fragments, fetal-derived sequencing fragments and maternal-derived sequencing fragments. a calculating unit for calculating a score); And a non-invasive prenatal test apparatus comprising a determination unit for determining the maternal moxisism or chromosome aneuploidy of the fetus by comparing the z-scores of each of the calculated nucleotide sequence fragments, fetal derived nucleotide sequences, and maternal derived nucleotide sequences. to provide.

The non-invasive prenatal test apparatus may have the same characteristics as the non-invasive prenatal test method described above. As an example, the sequencing fragment may be produced by a large-scale parallel sequencing method, and the produced sequencing fragment may be divided into fetal or maternal sequencing fragments according to its length. In addition, chromosome aneuploidy of a maternal moxisism or a fetus can be determined by the following using z-scores calculated from each of the entire sequencing fragment, fetal-derived sequencing fragment and maternal-derived sequencing fragment:

i) if the z-score for the fetal sequence fragment is greater than the z-score for the entire sequence fragment, the fetus is determined to be a trisomy;

ii) if the z-score for the nucleotide sequence fragment derived from the parent is greater than the z-score for the entire sequencing fragment, then it is determined that the parent is a mother isism;

iii) If the z-score for the fetal sequence fragment is smaller than the z-score for the entire sequence fragment, it is determined that the fetus is a monostain; or

iv) If the z-score for the nucleotide sequence fragment derived from the parent is smaller than the z-score for the entire sequencing fragment, then it is determined that the maternal capsism.

The non-invasive prenatal test apparatus according to the present invention may determine an autosomal abnormality, an abnormal sex chromosome, or a deletion of a chromosome. Specifically, the autosomal abnormality may cause Down syndrome, Edward's syndrome, Fatau syndrome, trisomy 9, trisomy 16 or trisomy 22, and the sex chromosome abnormality is Turner syndrome, triple X Can cause syndrome, Klinefelter syndrome or Jacobs syndrome, wherein the autosomal deletion is the number 1 monochromosomal deletion syndrome, 2q33.1 deletion syndrome, Walf-Hirschhorn syndrome, cat crying syndrome, Williams syndrome, Jacobson syndrome, Prader- May cause Willie / Angelman syndrome or DiGeorge syndrome.

Hereinafter, the present invention will be described in detail by examples. However, the following examples are only to illustrate the present invention, the present invention is not limited by these. Any thing that has substantially the same configuration and the same operation and effect as the technical idea described in the claims of the present invention is included in the technical scope of the present invention.

Example 1. Confirmation of cell free DNA (cfDNA) obtained from pregnant women and non-pregnant women

1-1. Isolation of cfDNA from blood

To compare the fragment length of cfDNA obtained from pregnant and non-pregnant women, blood from pregnant or non-pregnant women was first collected.

Specifically, 10 ml of blood was taken from the subject and placed in a cell-free DNA blood collection tube of VanGenes, which was centrifuged for 15 minutes at 1,900 g at room temperature. After centrifugation, the supernatant was transferred to a 1.5 ml tube, which was again centrifuged for 15 minutes at 16,000 g at room temperature to obtain the supernatant again. CfDNA was isolated from 2 ml of the supernatant (plasma) obtained above using the QIAsymphony DSP Virus / Pathogen Midi Kit (Qiagen).

1-2. Check the length of the cfDNA fragment

In Example 1-1. 100 ng of cfDNA isolated from pregnant or non-pregnant women was prepared for each fragment library set through 75-cycle paired-end sequencing using Illumina NextSeq500. Did. Fragments prepared using the BWA (version 0.7.10) program were then aligned to the homologous position of the human reference genomic sequence (hg19). At this time, the overlapped nucleotide sequence fragments were removed using a Picard (version 1.81) program. Among the fragments arranged, only the data obtained when the short sequences arranged in the forward and reverse directions were derived from the same sequential fragment (this is properly called paired read).

The number of sequencing fragments arranged on each chromosome was calculated using the obtained bam file and the SAMtools (version 0.1.18) program, and the distribution of the length of each fragment was graphically shown in FIG. 1.

As shown in FIG. 1, sequencing fragments having a length of 160 bp or less were present in a higher proportion in cfDNA isolated from pregnant women than in non-pregnant women. On the other hand, pregnant women with many short-length fragments of 160 bp or less had relatively low fragments with lengths of 160 bp or more compared to non-pregnant women (FIG. 1). That is, it can be seen from the above that the length of the cfDNA fragment of the pregnant woman is short by cffDNA contained in the cfDNA of the pregnant woman.

Example 2. Isolation of cfDNA from maternal blood

In order to diagnose chromosomal aneuploidy of the fetus through a non-invasive prenatal test, cfDNA was first isolated from maternal blood in the following manner. At this time, blood was collected from 630 pregnant women, of which 195 had a trisomy aneuploidy on chromosome 21. cfDNA was isolated by conditions and methods as described in Example 1-1., except that blood was obtained from pregnant women.

Example 3. Arrangement of cfDNA fragments and calculation of the number of sequencing fragments

Using the 100 ng of cfDNA isolated in Example 2, cfDNA fragments were arranged under the same conditions and methods as described in Example 1-2., And the number of sequence fragments arranged was calculated. At this time, the calculated data was stored as original data. On the other hand, using a bam file and a SAMtools (version 0.1.18) program, only sequences with a sequence length shorter than 150 bp were taken to calculate the number of fragments and stored as fetal derived data. In addition, the number of fragments was calculated by taking only sequences with a length greater than 185 bp, and stored as maternal-derived data. At this time, the base sequence having a length of 150 bp to 185 bp is a mixed region not suitable for distinguishing a fetus from a mother, and was not used to analyze data derived from a fetus or mother.

Experimental Example 1. Trisomy 21 analysis of chromosome 21

Trisomy of chromosome 21 was analyzed using z-score calculated using original data, fetal derived data and maternal derived data calculated in Example 3. The general z-score was calculated using Equation 1 below for a specific chromosome using a blood sample of a mother whose chromosome aneuploidy was not found as a normal sample group, and the analysis algorithm was performed by referring to a known literature. (Hyuk Jung Kwon et al., Open Journal of Genetics, 2017, 7, p. 1-8).

[Equation 1]

(Where i is chromosome 1 to 22, X chromosome or Y chromosome,

reference is a normal sample group,

target means the parent sample to be tested)

In the result obtained from the above, if the value obtained by subtracting the z-score calculated from the original data from the z-score calculated from the fetal derived data exceeds 0, it is determined that the fetus has a trisomy, and the z-calculated from the maternal derived data. When the value obtained by subtracting the z-score calculated from the original data in the score exceeds 0, it was determined as maternal mosaicism. The results are shown in FIG. 1 by creating a heatmap graph. At this time, one column of the chromosome section 21 represents each z-score value, which is the basis for determining aneuploidy in comparison with a threshold value.

As shown in FIG. 2, the fragment obtained from a pregnant woman cfDNA having a trisomy on chromosome 21 had a high z-score value in chromosome 21 (horizontal reference) (FIG. 2).

On the other hand, as shown in Fig. 3, the z-score did not appear clearly enough to determine the chromosome aneuploidy of the fetus in the chromosome section 21 (horizontal reference) of the original data. However, when this was confirmed by dividing it with maternal or fetal derived data, it was confirmed that the z-score was significantly higher in chromosome section 21 (horizontal basis) of fetal derived data, but not high in maternal derived data (FIG. 3). . Therefore, it was confirmed from this that the fetus had aneuploidy on chromosome 21.

Thus, as described above, the z-scores of the original data obtained from each pregnant woman's blood, maternal or fetal data are identified, and divided into cases determined by fetal trisomy or maternal maternalism to create a graph for z-scores. It is shown in Figure 4 by.

As shown in FIG. 4, the z-score in all cases determined as trisomy or maternal maternalism showed effective values. When it was judged as maternal maternalism, the z-score difference was not shown in the original data and the fetus-derived data, but the z-score of the maternal-derived data was significantly higher (blue line). On the other hand, when it was judged to be a trisomy of the fetus, the z-score of fetal derived data was significantly higher (red line).

Experimental Example 2. Analysis of monochromosomes (monosomy X) of sex chromosomes

First, monochromosomes of the X chromosome, which is a sex chromosome, were analyzed using z-score calculated using original data, fetal derived data, and maternal derived data calculated in Example 3. In the results obtained from the above, if the value obtained by subtracting the z-score calculated from the original data from the z-score calculated from the fetal derived data is less than 0, it was determined that the X chromosome of the fetus is a monosomy and calculated from the maternal derived data. If the value obtained by subtracting the z-score calculated from the original data from the z-score is less than 0, it was determined as maternal maternalism. The results were plotted in a heat map graph and shown in FIGS. 5 and 6.

As shown in Fig. 5, the fragment obtained from a single pregnant woman cfDNA having a monosomy on the X chromosome of the fetus had a low z-score value in the X chromosome section (horizontal reference). That is, the results indicated that the X chromosome of the fetus was a monosomy. In addition, as a result of comparative analysis by dividing the original data into fetal or maternal-derived data, by showing a lower z-score in fetal-derived data than in maternal-derived data, it was confirmed that the result is an aneuploidy chromosome appearing in the fetus (FIG. 5). .

On the other hand, as shown in Figure 6, the fragment obtained from one pregnant woman cfDNA having a single chromosome on the X chromosome of the mother has a z-score value in the X chromosome section (horizontal reference), more from the maternal-derived data compared to the fetal-derived data By showing low, it was confirmed that the fetus has a normal chromosome (Fig. 6).

In addition, as described above, the z-score of the original data obtained from each pregnant woman's blood, maternal or fetal data is identified, and divided into cases determined by the fetal monosomy or maternal maternalism to create a graph for the z-score. It is shown in Fig. 7.

As shown in FIG. 7, the z-score in all cases determined as monosomy or maternal maternalism showed effective values. When it was judged as maternal maternalism, the z-score difference was not shown in the original data and fetal data, but the z-score of the maternal data was significantly lower (blue line). On the other hand, when it was judged to be a monosomy of the fetus, the z-score of fetal derived data was significantly lower (red line).

Therefore, in conclusion, the method according to the present invention can be applied to determine abnormalities or deletions of autosomal and sex chromosomes such as chromosomes 9, 13, and 18 as well as chromosome 21.

Claims (13)

1) producing a whole sequence fragment from cell-free DNA extracted from the mother's blood;
2) arranging the entire sequence fragment produced in step 1) at the homologous position of the human reference genomic sequence;
3) Divide the entire sequencing fragments arranged in step 2) into fetal-derived sequencing fragments and maternal-derived sequencing fragments along the length, for each of the whole sequencing fragments, fetal-derived sequencing fragments and maternal-derived sequencing fragments calculating a z-score; And
4) Comparing the z-scores of the whole sequencing fragment, the fetal sequencing fragment, and the maternal sequencing fragment, calculated in step 3), to determine the maternal maternalism or chromosome aneuploidy of the fetus. Invasive prenatal testing method.
The method of claim 1, wherein the sequencing fragment is produced by a large-scale parallel sequencing method.
According to claim 1, In the process of dividing the entire sequence fragment into fetal-derived sequence fragments and maternal-derived sequence fragments along the length, the fetal-derived sequence fragments are separated to 150 bp or less, non-invasive prenatal test method.
The method of claim 1, wherein in the process of dividing the entire sequencing fragment into fetal-derived sequencing fragments and maternal-derived sequencing fragments along the length, the maternal-derived sequencing fragments are separated by 185 bp or more, non-invasive prenatal testing method.
The method of claim 1, wherein maternal maternalism or chromosome aneuploidy of the fetus is determined by:
i) if the z-score for the fetal sequence fragment is greater than the z-score for the entire sequence fragment, the fetus is determined to be a trisomy;
ii) if the z-score for the nucleotide sequence fragment derived from the parent is greater than the z-score for the entire sequencing fragment, then it is determined that the parent is a mother isism;
iii) If the z-score for the fetal sequence fragment is smaller than the z-score for the entire sequence fragment, it is determined that the fetus is a monostain; or
iv) If the z-score for the nucleotide sequence fragment derived from the parent is smaller than the z-score for the entire sequencing fragment, then it is determined that the maternal capsism.
The method of claim 1, wherein the fetal chromosome aneuploidy is determined by:
i) The z-score for the fetal sequence fragment is greater than the z-score for the entire sequence fragment,
ii) If the z-score for the nucleotide sequence fragment derived from the parent is smaller than the z-score for the entire sequencing fragment, the fetus is considered to be a trisomy.
The method of claim 1, wherein the fetal chromosome aneuploidy is determined by:
i) The z-score for the fetal sequence fragment is smaller than the z-score for the entire sequence fragment,
ii) If the z-score for the nucleotide sequence fragment derived from the parent is greater than the z-score for the entire sequencing fragment, the fetus is considered to be a monostain.
The method of claim 1, wherein the z-score is calculated by Equation 1 below:
[Equation 1]

(Where i is chromosome 1 to 22, X chromosome or Y chromosome,
reference is a normal sample group,
target means the parent sample to be tested).
The method of claim 1, wherein the non-invasive prenatal test method determines an autosomal abnormality, a sex chromosomal abnormality, or an autosomal deletion.
The method of claim 7, wherein the autosomal abnormality causes Down syndrome, Edward syndrome, Fatau syndrome, trisomy 9, trisomy 16 or trisomy 22.
The method of claim 7, wherein the sex chromosome abnormality causes Turner syndrome, TripleX syndrome, Klinefelter syndrome or Jacobs syndrome.
The autosomal deletion of claim 7, wherein the autosomal deletion is: 1 short chromosome deletion syndrome, 2q33.1 deletion syndrome, Walf-Hirschhorn syndrome, cat crying syndrome, Williams syndrome, Jacobson syndrome, Prader-Willi / Angelman syndrome or D. Non-invasive prenatal test method that causes George syndrome.
1) a production unit that produces a whole sequence fragment from cell-free DNA extracted from the mother's blood;
2) an alignment portion for arranging the entire sequencing fragment produced in step 1) at the homology position of the human reference genome sequence;
3) Divide the entire sequencing fragments arranged in step 2) into fetal-derived sequencing fragments and maternal-derived sequencing fragments along the length, for each of the whole sequencing fragments, fetal-derived sequencing fragments and maternal-derived sequencing fragments a calculation unit for calculating a z-score; And
4) A ratio comprising a determination unit for determining maternal maternalism or chromosome aneuploidy of a fetus by comparing z-scores of the whole sequencing fragment, the fetal sequencing fragment and the maternal sequencing fragment calculated in step 3) above. Invasive prenatal testing device.

Images