Non-invasive prenatal testing method capable of simultaneously diagnosing aneuploidies of autosomal chromosomes and sex chromosomes

Abstract

NIPTeR (an open-source R package that enables fast NIPT analysis), which is the most widely used non-invasive diagnosis method in the prior art, can only analyze trisomy in autosomes, and has the limitation of being unable to diagnose aneuploidies in sex chromosomes. Conversely, the present invention relates to a non-invasive prenatal testing method capable of simultaneously diagnosing numerical abnormalities (aneuploidy) of both autosomal chromosomes and sex chromosomes. The method according to the present invention has the effect of providing the same level of accuracy as amniocentesis, which is a confirmatory test used to verify NIPT, while addressing the limitation of NIPTeR (an open-source R package that enables fast NIPT analysis) of being unable to diagnose sex chromosome aneuploidies, and is thus expected to be widely used in the field of prenatal diagnosis.

Description

A noninvasive prenatal testing method capable of simultaneously diagnosing aneuploidy of autosomes and sex chromosomes

[0001] The present invention relates to a non-invasive prenatal testing method capable of simultaneously diagnosing aneuploidy of autosomes and sex chromosomes.

[0002] In modern times, the risk of developing genetic diseases in the fetus is rapidly increasing due to the aging population of those who marry and give birth. Early detection of genetic abnormalities in the fetus early in pregnancy allows for prompt medical intervention to ensure safety while minimizing suffering for both the mother and the fetus. Therefore, early diagnosis of genetic abnormalities in the fetus is crucial. Genetic abnormalities that can occur in the fetus include translocations, deletions, duplications, insertions, and numerical abnormalities (e.g., trisomy). These chromosomal abnormalities can cause structural and functional abnormalities throughout the fetus's body. Numerical genetic abnormalities, in particular, are known to occur more frequently with increasing maternal age. Therefore, the need to assess and diagnose fetal diseases before birth is growing, a practice known as prenatal diagnosis.

[0003] Prenatal diagnosis methods can be broadly divided into invasive and noninvasive methods. Examples of invasive diagnostic methods include chorionic villi sampling (CVS), performed between 10 and 12 weeks of pregnancy; amniocentesis, which analyzes fetal chromosomes by measuring the concentration of AFP in amniotic fluid using an immunoassay between 15 and 20 weeks of pregnancy; and cordocentesis, which extracts fetal blood directly from the umbilical cord under ultrasound guidance between 18 and 20 weeks of pregnancy. However, these invasive diagnostic methods can shock the fetus during the examination, causing miscarriage, disease, or malformation. Therefore, noninvasive diagnostic methods are mainly used recently. Among the noninvasive diagnostic methods, the most actively used method is NIPTeR (an open-source R Package that enables fast NIPT (Non-invasive prenatal testing) analysis). This is a trisomy analysis algorithm developed by Lennart F. Johansson's research team in the Netherlands in 2017. It has been used as a tool to accurately analyze trisomies of chromosomes 13, 18, and 21. However, this method can only analyze trisomies in autosomes, and has the limitation of not being able to diagnose aneuploidy in sex chromosomes.

[0004] Accordingly, the present invention was conceived to address the aforementioned problems in conventional technology, and relates to a noninvasive prenatal testing method capable of simultaneously diagnosing numerical abnormalities (aneuploidy) of autosomes and sex chromosomes. The method of the present invention is safe, simple, and highly accurate for both pregnant women and fetuses, and is therefore expected to find significant use in the field of prenatal diagnosis.

[0005] The present invention has been devised to solve the above-mentioned problems in conventional technology, and relates to a non-invasive prenatal testing method capable of simultaneously diagnosing aneuploidy of autosomes and sex chromosomes.

[0006] In one aspect, the present invention provides a non-invasive prenatal testing method capable of simultaneously diagnosing aneuploidy of autosomes and sex chromosomes.

[0007] As another aspect of the present invention, a non-invasive prenatal testing device capable of simultaneously diagnosing aneuploidy of autosomes and sex chromosomes is provided.

[0008] As another aspect of the present invention, a method for providing information on diagnosing chromosomal aneuploidy of a fetus capable of simultaneously diagnosing aneuploidy of autosomes and sex chromosomes is provided.

[0009] However, the technical problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by those skilled in the art from the description below.

[0010] Hereinafter, various embodiments described herein will be described with reference to the drawings. In the following description, various specific details, such as specific configurations, compositions, and processes, are set forth to provide a thorough understanding of the present invention. However, certain embodiments may be practiced without one or more of these specific details, or in conjunction with other known methods and configurations. In other instances, well-known processes and manufacturing techniques have not been described in specific detail so as not to unnecessarily obscure the present invention. Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, configuration, composition, or characteristic described in connection with the embodiment is included in one or more embodiments of the present invention. Thus, the appearances of "in one embodiment" or "an embodiment" in various places throughout this specification do not necessarily refer to the same embodiment of the present invention. Additionally, the particular features, configurations, compositions, or characteristics may be combined in any suitable manner in one or more embodiments.

[0011] Unless otherwise specifically defined in the specification, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs.

[0012] Throughout the specification, whenever a part is said to "include" a component, this does not mean that it excludes other components, but rather that it may include other components, unless otherwise stated.

[0013] In the present invention, 'prenatal diagnosis' refers to diagnosing congenital abnormalities in an embryo or fetus before birth, and is also called prenatal diagnosis or intrauterine diagnosis. For the purpose of early detection of congenital abnormalities related to development and maternal protection, tests are mainly performed using fetuses or shed material from fetuses as samples, using cytogenetic analysis, biochemical analysis, and imaging methods. Techniques include invasive methods (amniocentesis, chorionic villus sampling, fetal blood collection, fetal skin biopsy, fetal liver biopsy, etc.) and non-invasive methods (ultrasound, collection of fetal cells from maternal blood, etc.).

[0014] Genetic prenatal diagnosis is performed when one or more parents are suspected of having a chromosomal abnormality or a carrier of a serious genetic disease, a history of abnormal delivery, advanced maternal age, a chromosomal abnormality requiring further confirmation by ultrasound, a single-gene disease, or an inborn error of metabolism. For karyotyping, amniocentesis is required for safety reasons, and blood samples are typically collected between 15 and 18 weeks of pregnancy. For DNA diagnosis, chorionic villus sampling is also performed, collected transvaginally or transperitoneally between 9 and 11 weeks of pregnancy. Fetal blood samples are collected transperitoneally from the umbilicus after 16 weeks of pregnancy. For cytogenetic diagnosis, karyotyping using fluorescence in situ hybridization (FISH) or fetal cell culture is performed, and for molecular genetic diagnosis, direct diagnosis using southern blotting, polymerase chain reaction (PCR), and indirect diagnosis using restriction fragment length polymorphism (RFLP) and PCR-RFLP are performed.

[0015] In the present invention, 'abnormal chromosome number', also called aneuploidy, refers to a state in which the number of chromosomes per cell in a cell, organism or lineage is not an integer multiple of the basic number, but is 1 or more or less than the integer multiple, that is, a state in which the genome has an incomplete structure, and a cell or organism in such a state is called an aneuploid. In general, a case in which the number of chromosomes is more than an integer multiple of the basic number is called hyperchromasia, and a case in which the number of chromosomes is less than the integer multiple of the basic number is called hypochromasia. In particular, in the case of diploidy, a case in which two homologous chromosomes of a pair are missing is called 0somy, a case in which one is missing and the other is present is called monosomy, and a case in which one extra chromosome exists in addition to a pair of homologous chromosomes is called trisomy. Representative autosomal aneuploidies include, but are not limited to, Down syndrome, which is trisomy 21, Edward syndrome, which is trisomy 18, which causes developmental disorders during pregnancy, and Patau syndrome, which is trisomy 13, which causes severe malformations. Cat-cry syndrome, which produces a characteristic meowing sound, is caused by partial deletion of the short arm of chromosome 5. Representative sex chromosome aneuploidy is Klinefelter syndrome, which is XXY and is mainly characterized by testicular dysfunction. There is also Turner syndrome, which has one X chromosome (XO) and underdeveloped ovaries, XXX females with three X chromosomes, and YY syndrome, which is XYY (YY male).

[0016] In one aspect, the present invention provides a non-invasive prenatal testing method capable of simultaneously diagnosing aneuploidy of autosomes and sex chromosomes.

[0017] The non-invasive prenatal examination method of the present invention is a method that can be performed by a computing device.

[0018] In the present invention, the method is improved from NIPTeR (v1.0.2), a conventional non-invasive prenatal diagnosis method. The NIPTeR method is a standard recommended by the Ministry of Food and Drug Safety (Ministry of Food and Drug Safety, Republic of Korea, Laws / Data > Law Information > Civil Complainant Guide > Registration Numbers: Guide-0926-01 and Guide-0926-02). The definitions of terms in the NIPTeR method and the method of the present invention, which is an improvement thereof, are as follows.

[0019] Read: This refers to the base pair information for the amount of analysis generated from a DNA or cDNA fragment included in a sequencing library, and is the output data or sequence (a fragment of a base sequence) from base sequencing.

[0020] Read length: This refers to the length of the base sequence of a read. It also refers to the average length of the entire read.

[0021] Mapping: Used in the same sense as alignment, it refers to the step of estimating the original location of the read in the genome by comparing it with the reference sequence.

[0022] Depth of coverage: The average number of reads per base position in a specific region. Typically, units of “x” are used (e.g., 5x, 10x, 20x, 100x).

[0023] Strand bias: This refers to a phenomenon in which the directionality of reads is biased due to one of the two DNA strands having a higher sequencing efficiency than the other strand or for other reasons.

[0024] GC content: This refers to the ratio of guanine (G) to cytosine (C) among all bases in the target region. It is known that a higher GC content leads to higher DNA density and less denaturation.

[0025] GC bias: GC bias refers to a decrease in read depth in regions with high or low GC content. Causes of this GC bias include enrichment PCR bias.

[0026] BAM file (Binary Alignment / Map format file): A binary version of the SAM file (Sequence Alignment / Map format file), this is a file format that contains the alignment results of the reads to the reference genome.

[0027] SAM file (Sequence Alignment / Map format file): A tab-delimited text file containing information on how sequences are mapped to specific locations in a reference genome. It consists of a header and an alignment section. The header section includes information such as version, alignment status, and length, while the alignment section contains sequence information and its quality.

[0028] Bin file (also known as binary file): A binary file is a computer file format other than a text file, containing data encoded in binary format for computer storage and processing purposes. It is often used interchangeably with "non-text file."

[0029] FASTQ: A file containing the ID, base sequence, and corresponding quality indicators for each read. It is typically the starting point for NGS BI pipelines and is a de facto standard text-based file format.

[0030] The above NIPTeR method specifically consists of the following steps.

[0031] [1] Measurement of fetal fraction

[0032] A key factor determining the reliability of NIPT testing for aneuploidy is the proportion of fetal DNA in maternal plasma, which serves as the evaluation criteria for assessing whether to proceed with prenatal testing. For read counting methods, test accuracy is influenced by fetal fraction and sequencing depth, so evaluation criteria must be clearly established. Generally, a fetal fraction of 4% or higher is considered reliable for reliable results. This is the basis for securing reliable data on fetal fraction and coverage depth.

[0033] [2] Elimination of GC bias

[0034] To remove the influence of GC bias, a three-step process is used to correct for sequencing bias caused by varying GC contents on each chromosome using LOESS regression analysis.

[0035] (1) LOESS regression analysis

[0036] All chromosomes are divided into bins of a specific size, and the number of reads for each bin and the GC contents within each bin are calculated. After filtering out bins containing reference genome sequences with unreadable sequences, the fit prediction value (URloess) of each bin is calculated using the GC contents of the corresponding bin according to the unique read count (UR) of each bin through LOESS regression analysis. e(UR) is the expected value for the unique read count within each bin, and after correcting by the overall mean of each bin, the number of reads for each chromosome is added using the corrected number of reads in each bin for the corresponding chromosome.

[0037] (2) Normalization

[0038] The second step is to correct for differences between samples sequenced simultaneously. First, corrected unique reads for each sample and chromosome are obtained from each base sequence analysis. Then, the corrected unique reads for each chromosome due to differences between samples are calculated. Then, the read ratio for each chromosome in each sample is calculated.

[0039] (3) Linear regression model

[0040] Because the GC content distributions vary between different sequencing runs, the correlation between reads and GC content is used to calculate the read ratio and statistical significance using a linear regression model. The predicted read ratio is then applied to a normal distribution.

[0041] [3] Detection of trisomy

[0042] Because the chromosomal distribution of reads can vary between sequences or even within the same sequence, which can lead to unclear results in the identification of chromosomal aneuploidies, the number of reads mapped to the target chromosome must be corrected by the number of reads mapped to the reference genome. Trisomy of all or specific chromosomes can be identified using standard Z-scores and normalized chromosome values ​​derived from the reference genome.

[0043] The non-invasive prenatal testing method of the present invention capable of diagnosing aneuploidy of autosomes and sex chromosomes simultaneously is an improvement of the NIPTeR method, and improves upon the limitation of NIPTeR, which cannot diagnose aneuploidy of sex chromosomes.

[0044] To this end, the method of the present invention comprises the steps of (a) performing whole genome sequencing on cell-free fetal DNA obtained from a biological sample isolated from the mother; (b) calculating the number of reads for each chromosome from a BAM file generated as a result of the sequencing and dividing the reads into binary files of 30,000 bp to 5,000,000 bp in size and correcting them; (c) correcting GC bias using local regression analysis on the corrected bin values; (d) re-correcting the variance difference between the control group and the sample to be analyzed using a Chi-Squared Test; and, (e) calculating a Z-score value.

[0045] In the case where the method of the present invention seeks to confirm the aneuploidy of the sex chromosome, the BAM file in the step (b) obtains the number of reads for each chromosome and calculates the number of reads for 1,000,000 bp to 5,000,000 bp, 1,000,000 bp to 4,000,000 bp, 1,000,000 bp to 3,000,000 bp, 2,000,000 bp to 5,000,000 bp, 2,000,000 bp to 4,000,000 bp, 2,000,000 bp to 3,000,000 bp, 3,000,000 bp to 5,000,000 bp, or 3,000,000 bp to It may be corrected by dividing it into a binary file of 4,000,000bp, preferably 3,000,000bp in size.

[0046] In the method of the present invention, the Z-score value may be the Z-score of the X chromosome (Z-score(X)), the Z-score of the Y chromosome (Z-score(Y)), the predicted Z-score of the X chromosome (Z-score(X')), and the ratio (R) of Z-score(X) and Z-score(X'). In this case, the Z-score(X), Z-score(Y), Z-score(X'), and R values ​​may be calculated by the following [Mathematical Formula 1] to [Mathematical Formula 4].

[0047] [Mathematical Formula 1]

[0048]

[0049] [Equation 2]

[0050]

[0051] [Equation 3]

[0052]

[0053] [Equation 4]

[0054]

[0055] In the above mathematical expression 3, m may be -0.01 to 1, -0.1 to 1, -0.5 to 1, -0.6 to -0.9, or -0.7 to -0.9, preferably -0.8 to -0.9, and b may be -10 -10 -10 inland -17 , -10 -10 -10 inland -15 , -10 -11 -10 inland -16 , -10 -12 -10 inland -17 , or -10 -12 -10 inland -15 , preferably -10 -13 -10 inland -15 It could be.

[0056] In addition, the method of the present invention interprets the sex chromosome of the fetus as aneuploid XO when the Z-score(Y) value is less than 3 or the Z-score(X) value is greater than 3, or interprets the sex chromosome of the fetus as normal XX when the Z-score(Y) value is less than 3 or the absolute value of Z-socre(X) is less than 3, or interprets the sex chromosome of the fetus as aneuploid XXX when the Z-score(Y) value is less than 3 and the absolute value of Z-score(X) is greater than 3, or interprets the sex chromosome of the fetus as aneuploid XXY when the Z-score(Y) value is greater than 3 or the Z-score(X) value is less than -3 and the Z-score(X) value is greater than 3. If the Z-score(X') value is greater than the value, the sex chromosome of the fetus may be interpreted as aneuploid XYY, or if the Z-score(Y) value is greater than 3 and R ∈ {-0.8, 0.8}, the sex chromosome of the fetus may be interpreted as normal XY.

[0057] In the case where the method of the present invention seeks to confirm aneuploidy of an autosome, the BAM file in step (b) may be a file that calculates the number of reads for each chromosome and divides it into a binary file of 30,000 bp to 70,000 bp, 30,000 bp to 60,000 bp, 30,000 bp to 50,000 bp, 40,000 bp to 70,000 bp, 40,000 bp to 60,000 bp, 40,000 bp to 50,000 bp, 50,000 bp to 70,000 bp, or 50,000 bp to 60,000 bp, preferably 50,000 bp in size, for correction.

[0058] At this time, the method may be to calculate the Z-score value by linear regression analysis, and the method may be to additionally calculate the NCV (Normalized Chromosome Value) and RBZ (Regression based Z-score) values, and if the Z-score value exceeds 4, the NCV value exceeds 3, or the RBZ value exceeds 3, the autosomes of the fetus may be interpreted as trisomy aneuploidy. At this time, in the method, the trisomy aneuploidy may be Down syndrome, Edwards syndrome, or Patau syndrome.

[0059] In another aspect of the present invention, the present invention provides a non-invasive prenatal testing device capable of simultaneously diagnosing aneuploidy of autosomes and sex chromosomes.

[0060] The non-invasive prenatal testing device of the present invention is a device including a computing device.

[0061] The non-invasive prenatal testing device of the present invention is a device that utilizes the non-invasive prenatal testing method of the present invention described above, which can simultaneously diagnose aneuploidy of autosomes and sex chromosomes. The term "use" herein refers to a device including a computer that programs the device.

[0062] To this end, the device of the present invention comprises a first module for whole genome sequencing of fetal cell-free DNA (cfDNA) obtained from a biological sample isolated from the mother; a second module for calculating the number of reads for each chromosome from a BAM file generated as a result of the sequencing and dividing the reads into binary files of 30,000 bp to 5,000,000 bp in size and correcting them; a third module for correcting GC bias using local regression analysis of the corrected bin values; a fourth module for re-correcting the variance difference between the control group and the target sample using a chi-square test; and a fifth module for calculating a Z-score value.

[0063] In the case where the device of the present invention is a device for confirming the aneuploidy of a sex chromosome, the BAM file in step (b) obtains the number of reads for each chromosome and converts the number of reads to 1,000,000 bp to 5,000,000 bp, 1,000,000 bp to 4,000,000 bp, 1,000,000 bp to 3,000,000 bp, 2,000,000 bp to 5,000,000 bp, 2,000,000 bp to 4,000,000 bp, 2,000,000 bp to 3,000,000 bp, 3,000,000 bp to 5,000,000 bp, or 3,000,000 bp to It may be corrected by dividing it into a binary file of 4,000,000bp, preferably 3,000,000bp in size.

[0064] In the device of the present invention, the Z-score value may be the Z-score of the X chromosome (Z-score(X)), the Z-score of the Y chromosome (Z-score(Y)), the predicted Z-score of the X chromosome (Z-score(X')), and the ratio (R) of Z-score(X) and Z-score(X'). In this case, the Z-score(X), Z-score(Y), Z-score(X'), and R values ​​may be calculated by the following [Mathematical Formula 1] to [Mathematical Formula 4].

[0065] [Mathematical Formula 1]

[0066]

[0067] [Equation 2]

[0068]

[0069] [Equation 3]

[0070]

[0071] [Equation 4]

[0072]

[0073] In the above mathematical expression 3, m may be -0.01 to 1, -0.1 to 1, -0.5 to 1, -0.6 to -0.9, or -0.7 to -0.9, preferably -0.8 to -0.9, and b may be -10 -10 -10 inland -17 , -10 -10 -10 inland -15 , -10 -11 -10 inland -16 , -10 -12 -10 inland -17 , or -10 -12 -10 inland -15 , preferably -10 -13 -10 inland -15 It could be.

[0074] In addition, the device of the present invention interprets the sex chromosome of the fetus as aneuploidy XO when the Z-score(Y) value is less than 3 or the Z-score(X) value is greater than 3, or interprets the sex chromosome of the fetus as normal XX when the Z-score(Y) value is less than 3 or the absolute value of Z-socre(X) is less than 3, or interprets the sex chromosome of the fetus as aneuploidy XXX when the Z-score(Y) value is less than 3 and the absolute value of Z-score(X) is greater than 3, or interprets the sex chromosome of the fetus as aneuploidy XXY when the Z-score(Y) value is greater than 3 or the Z-score(X) value is less than -3 and the Z-score(X) value is greater than 3. It may further include a sixth module that interprets the sex chromosome of the fetus as aneuploid XYY if the Z-score(X') value is greater than 3, or interprets the sex chromosome of the fetus as normal XY if the Z-score(Y) value is greater than 3 and R ∈ {-0.8, 0.8}.

[0075] In the case where the device of the present invention is a device for confirming aneuploidy of an autosome, the BAM file in step (b) may be a file that calculates the number of reads for each chromosome and divides it into a binary file of 30,000 bp to 70,000 bp, 30,000 bp to 60,000 bp, 30,000 bp to 50,000 bp, 40,000 bp to 70,000 bp, 40,000 bp to 60,000 bp, 40,000 bp to 50,000 bp, 50,000 bp to 70,000 bp, or 50,000 bp to 60,000 bp, preferably 50,000 bp in size, for correction.

[0076] At this time, the device may calculate the Z-score value by linear regression analysis, and the device may further include a seventh module that calculates NCV (Normalized Chromosome Value) and RBZ (Regression based Z-score) values, and the device may further include an eighth module that interprets the autosomes of the fetus as trisomy aneuploidy when the Z-score value is greater than 4, the NCV value is greater than 3, or the RBZ value is greater than 3. At this time, in the device, the trisomy aneuploidy may be Down syndrome, Edwards syndrome, or Patau syndrome.

[0077] In another aspect of the present invention, the present invention provides a method for diagnosing a chromosomal aneuploidy in a fetus, or a method for providing information for the diagnosis.

[0078] The above method of the present invention is a method that can be performed by a computing device.

[0079] The method of the present invention is characterized by including a non-invasive prenatal testing method capable of simultaneously diagnosing aneuploidy of autosomes and sex chromosomes of the present invention described above.

[0080] To this end, the method of the present invention comprises the steps of (a) performing whole genome sequencing on cell-free fetal DNA obtained from a biological sample isolated from the mother; (b) calculating the number of reads for each chromosome from a BAM file generated as a result of the sequencing and dividing the reads into binary files of 30,000 bp to 5,000,000 bp in size and correcting them; (c) correcting GC bias using local regression analysis on the corrected bin values; (d) re-correcting the variance difference between the control group and the sample to be analyzed using a Chi-Squared Test; and, (e) calculating a Z-score value.

[0081] In the case where the method of the present invention seeks to confirm the aneuploidy of the sex chromosome, the BAM file in the step (b) obtains the number of reads for each chromosome and calculates the number of reads for 1,000,000 bp to 5,000,000 bp, 1,000,000 bp to 4,000,000 bp, 1,000,000 bp to 3,000,000 bp, 2,000,000 bp to 5,000,000 bp, 2,000,000 bp to 4,000,000 bp, 2,000,000 bp to 3,000,000 bp, 3,000,000 bp to 5,000,000 bp, or 3,000,000 bp to It may be corrected by dividing it into a binary file of 4,000,000bp, preferably 3,000,000bp in size.

[0082] In the method of the present invention, the Z-score value may be the Z-score of the X chromosome (Z-score(X)), the Z-score of the Y chromosome (Z-score(Y)), the predicted Z-score of the X chromosome (Z-score(X')), and the ratio (R) of Z-score(X) and Z-score(X'). In this case, the Z-score(X), Z-score(Y), Z-score(X'), and R values ​​may be calculated by the following [Mathematical Formula 1] to [Mathematical Formula 4].

[0083] [Mathematical Formula 1]

[0084]

[0085] [Equation 2]

[0086]

[0087] [Equation 3]

[0088]

[0089] [Equation 4]

[0090]

[0091] In the above mathematical expression 3, m may be -0.01 to 1, -0.1 to 1, -0.5 to 1, -0.6 to -0.9, or -0.7 to -0.9, preferably -0.8 to -0.9, and b may be -10 -10 -10 inland -17 , -10 -10 -10 inland -15 , -10 -11 -10 inland -16 , -10 -12 -10 inland -17 , or -10 -12 -10 inland -15 , preferably -10 -13 -10 inland -15 It could be.

[0092] In addition, the method of the present invention interprets the sex chromosome of the fetus as aneuploid XO when the Z-score(Y) value is less than 3 or the Z-score(X) value is greater than 3, or interprets the sex chromosome of the fetus as normal XX when the Z-score(Y) value is less than 3 or the absolute value of Z-socre(X) is less than 3, or interprets the sex chromosome of the fetus as aneuploid XXX when the Z-score(Y) value is less than 3 and the absolute value of Z-score(X) is greater than 3, or interprets the sex chromosome of the fetus as aneuploid XXY when the Z-score(Y) value is greater than 3 or the Z-score(X) value is less than -3 and the Z-score(X) value is greater than 3. If the Z-score(X') value is greater than the value, the sex chromosome of the fetus may be interpreted as aneuploid XYY, or if the Z-score(Y) value is greater than 3 and R ∈ {-0.8, 0.8}, the sex chromosome of the fetus may be interpreted as normal XY.

[0093] In the case where the method of the present invention seeks to confirm aneuploidy of an autosome, the BAM file in step (b) may be a file that calculates the number of reads for each chromosome and divides it into a binary file of 30,000 bp to 70,000 bp, 30,000 bp to 60,000 bp, 30,000 bp to 50,000 bp, 40,000 bp to 70,000 bp, 40,000 bp to 60,000 bp, 40,000 bp to 50,000 bp, 50,000 bp to 70,000 bp, or 50,000 bp to 60,000 bp, preferably 50,000 bp in size, for correction.

[0094] At this time, the method may be to calculate the Z-score value by linear regression analysis, and the method may be to additionally calculate the NCV (Normalized Chromosome Value) and RBZ (Regression based Z-score) values, and if the Z-score value exceeds 4, the NCV value exceeds 3, or the RBZ value exceeds 3, the autosomes of the fetus may be interpreted as trisomy aneuploidy. At this time, in the method, the trisomy aneuploidy may be Down syndrome, Edwards syndrome, or Patau syndrome.

[0095] Hereinafter, the present invention will be described in detail based on examples.

[0096] The prenatal testing method of the present invention is a non-invasive prenatal testing method capable of simultaneously diagnosing numerical abnormalities (aneuploidy) of autosomes and sex chromosomes, and has the effect of improving the limitations of NIPTeR (an open-source R Package that enables fast analysis), which cannot diagnose aneuploidy of sex chromosomes, while providing the same accuracy as the evaluation results of amniotic fluid examination, which is a confirmatory NIPT test.

[0097] Figure 1 is a diagram illustrating a method for analyzing trisomy and sex chromosome aneuploidy of an autosome according to one embodiment of the present invention. In Figure 1, the 'Chi-squared based variation feduction' route is a method capable of analyzing trisomy of an autosome using the conventionally known NIPTeR (v1.0.2) method, and the 'Calculate chromosome fraction' route represents a method capable of analyzing sex chromosome aneuploidy newly developed in the present invention.

[0098] In conventional clinical testing methods (amniocentesis and Harmony), 401 previously reported negative samples were sequenced using WGS and analyzed using an analysis algorithm developed by our research team to assess specificity. As a result, the algorithm developed in this invention confirmed negative results for both trisomy and sex chromosome aneuploidy in 401 negative samples, demonstrating its potential as a viable replacement for existing testing methods.

[0099] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples, in accordance with the gist of the present invention.

[0100] Throughout the specification, whenever a part is said to "include" a component, this does not mean that it excludes other components, but rather that it may include other components, unless otherwise stated.

[0101] Example.

[0102] [Development of Analysis Algorithm]

[0103] 1. Detection of chromosomal numerical abnormalities

[0104] In order to detect chromosomal numerical abnormalities, changes in the copy number of the entire chromosome were detected using the WGS (Whole Genome Sequencing) method, thereby detecting abnormal chromosomes. In the present invention, after sequencing was performed using the WGS library kit (IDT; USA), the calculated copy number value was converted into a z-score to detect abnormal trisomy and sex chromosome aneuploidy in autosomes.

[0105] 2. Analysis of chromosomal numerical abnormalities

[0106] 2-1. WGS using maternal plasma

[0107] Maternal whole blood (8 cc) was collected in a cell-free DNA-dedicated tube (Roche cfDNA collection tube; Roche) and centrifuged at 1600 xg for 12 minutes at 4℃ to separate plasma. Cell-free DNA (cfDNA) was obtained from the separated plasma (2 cc) using a MagNa96 automated extraction reagent kit (MagNA Pure cfNA Buffer Set; Roche). The obtained cfDNA fragments were prepared at a level of 1–10 ng and then end-repaired. Specifically, the sample DNA underwent end prep to attach dA-Tailing, and adapters were attached to both ends of the sequence using an enzymatic method. Afterwards, impurities were removed using beads, and the DNA with the attached adapters was amplified through PCR amplification, and impurities were removed once more to secure the final library. This was sequenced using the Illumina NextSeq 550 NGS system. The sequence file produced by sequencing was analyzed for chromosomal aneuploidy through the same procedure as SCA detection in Figure 1.

[0108] 2-2. Analysis of autosomal trisomy and sex chromosome aneuploidy

[0109] For the analysis of autosomal and sex chromosome aneuploidy, NIPTeR (v1.0.2) was used as the basic framework as shown in Fig. 1. NIPTeR uses Z-score, Normalized Chromosome Value (NCV), and Regression Based Z-score (RBZ) as the final calculation formulas for the analysis of autosomal trisomy. To this end, in [Step 1], the number of sequence reads is calculated for each chromosome from the bam files of the input files of the control group and the sample to be analyzed, and this is divided into bins of 50,000 bp in size and corrected. In [Step 2], the bias of the GC content is corrected for the calculated bin values ​​using LOESS analysis. In [Step 3], the difference in variance between the control group and the sample to be analyzed is corrected again using the chi-square test for the corrected bin values. Based on the Z-score, NCV (Normalized Chromosome Value), and RBZ (Regression based Z-score) values ​​calculated through this, cases where Z-score > 4, NCV and RBZ > 3 are determined as trisomy aneuploidy of autosomes. The scripts for [Step 1] to [Step 3] above are shown in Table 1 below.

[0110]

[0111] At the same time, in the present invention, the same bam file used for autosomal analysis was used for aneuploidy analysis of sex chromosomes after correcting the total read count to 3,000,000 using bbmap (v39.06). The final calculation formula for sex chromosome aneuploidy analysis was the Z-score of the X chromosome (Z-score(X)), the Z-score of the Y chromosome (Z-score(Y)), the predicted Z-score of the X chromosome (Z-score(X')), and the ratio (R) of Z-score(X) and Z-score(X'). The definitions (calculation formulas) of the above factors are shown in Table 2 below.

[0112]

[0113] For sex chromosome aneuploidy analysis, the bin values ​​of NIPTeR (v1.0.2) [Step 2] derived from data of normal control clinical specimens (n=158) were used. Afterwards, the sex chromosome read counts of the control and target samples were obtained using the corrected bin values, and then the Z-score (X) and Z-score (Y) of the target sample were calculated using the mean and standard deviation values ​​of the sex chromosome reads of the control group.

[0114] Meanwhile, female fetuses (XX) have more X chromosomes than male fetuses (XY), and in male fetuses, the Y chromosome and X chromosome each appear as a single chromosome. Therefore, a linear regression equation can be obtained by modeling the relationship between the X chromosome and the Y chromosome through linear regression analysis on the X chromosome and the Y chromosome. In the present invention, the Z-score (X) calculated from 158 clinical specimens used as normal controls and the linear regression equation derived through linear regression analysis of the Z-score (Y) were used to calculate the Z-score (X') to analyze sex chromosome aneuploidy. The Z-score (X') calculation equation is shown in Table 3.

[0115]

[0116] The m and b values ​​of the regression equation in Table 3 above were calculated and derived from clinical samples (n=158) of the normal control group (normal maternal blood), and m=-0.8667, b=-10 -14 The value of was obtained. The Z-score(X') obtained through this serves as a reference value used to determine sex chromosome aneuploidy, and the R value expressed through the ratio with the Z-score(X) serves as a reference value used to determine gender. The criteria used to determine sex chromosome aneuploidy are shown in Table 4 below.

[0117]

[0118] [Analysis Algorithm Evaluation]

[0119] 1. Evaluation of the analytical performance of the developed algorithm

[0120] In order to evaluate the non-invasive prenatal testing method (algorithm) capable of simultaneously diagnosing aneuploidy of autosomes and sex chromosomes developed in the present invention, positive standard materials (NA04965; Down syndrome, NA02732; Edwards' syndrome, NA03330; Patau syndrome and, NA04626(XXX), NA03102(XXY), NA09326(XYY), NA01176(XO); Sex chromosome aneuploidy) verified by Coriell Institute (USA) were purchased and analytical performance evaluation was performed using the algorithm of the present invention. Each standard material was tested five times to confirm the accuracy of trisomy analysis and sex chromosome aneuploidy analysis results, and trisomy in autosomes was tested according to the standard recommended by the Ministry of Food and Drug Safety (Ministry of Food and Drug Safety, Republic of Korea, Laws / Data > Law Information > Civil Complainant Guide > Registration Number: Guide-0926-01, and Guide-0926-02). It was classified as positive when both z-score > 4 and NCV > 3 were satisfied, and additionally when RBZ > 3 was satisfied.

[0121] As a result of analyzing trisomy using the algorithm of the present invention in positive reference materials of autosomes, trisomy was confirmed in chromosome 21 for NA04965 material, in chromosome 18 for NA02732 material, and in chromosome 13 for NA03330 material. Each reference material showed negative results in other areas. In addition, in the continuous sex chromosome aneuploidy analysis, XXY was confirmed for NA04965 material, XY for NA02732 material, and XY for NA03330 material, which were confirmed to be the same sex as the origin specimen of each reference material. As a result of analyzing trisomy in positive reference materials of sex chromosomes, trisomy was not confirmed in autosomes for any of materials NA04626, NA03102, NA09326, or NA01176. In addition, in the serial sex chromosome aneuploidy analysis, the NA04626 material was confirmed to be XXX, the NA03102 material was confirmed to be XXY, the NA09326 material was confirmed to be XYY, and the NA03330 material was confirmed to be XO, which were confirmed to be the same sex as the origin specimen of each standard material. The results of the aneuploidy analysis performance evaluation of the developed algorithm using the above positive standard materials are shown in Table 5 below.

[0122]

[0123] 2. Evaluation of analysis results

[0124] 2-1. Clinical performance evaluation of the developed algorithm

[0125] In conventional clinical testing methods (amniocentesis and Harmony), 401 previously reported negative samples were sequenced using WGS and analyzed using an analysis algorithm developed by our research team to assess specificity. As a result, the algorithm developed in this invention confirmed negative results for both trisomy and sex chromosome aneuploidy in 401 negative samples, demonstrating its potential as a viable replacement for existing testing methods.

[0126] 2-2. Comparison of Results between the Developed Algorithm and Conventional Testing Methods

[0127] T21, T13, XXX, and XXY specimens that were previously reported as positive in clinical tests (amniocentesis and harmony) were sequenced using WGS and analyzed using the analysis algorithm developed by our research team to evaluate their clinical performance. As a result, 7 positive specimens for Trisomy 21, 3 positive specimens for XXX, and 1 positive specimen for XXY, in which both amniocentesis and harmony tests were performed, all obtained test results identical to the results of the previous tests. Meanwhile, the positive samples (Clin_T13_3, Clin_T13_4) for which only amniocentesis, a confirmatory NIPT test, was performed also showed equivalent results using the algorithm of the present invention. However, among the samples for which both amniocentesis and Harmony testing were performed, some results (blue shaded areas) that were inconsistent with the Harmony testing were confirmed in T13 (Clin_T13_1, Clin_T13_2) and SCA samples (Clin_XXX_3, Clin_XXY_2). This is shown in Table 6 below.

[0128]

[0129] In summary, the developed algorithm of the present invention was confirmed to produce identical results to those of amniocentesis, a confirmatory NIPT test (100% accuracy). Meanwhile, some samples showed inconsistent results with the Harmony test (IVD certified by the Ministry of Food and Drug Safety), a screening NIPT test. However, the algorithm of the present invention was verified for analytical performance using validated reference materials (Table 5), and additional clinical performance evaluations confirmed identical results to those of amniocentesis, a confirmatory NIPT test (Table 6). Therefore, the analytical accuracy of the developed algorithm is evaluated as verified, and it is expected to be a sufficient replacement for existing testing methods.

[0130] While specific aspects of the present invention have been described in detail above, it should be apparent to those skilled in the art that these specific descriptions are merely preferred embodiments and do not limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

[0131] The present invention relates to a noninvasive prenatal testing method capable of simultaneously diagnosing numerical abnormalities (aneuploidy) of autosomes and sex chromosomes. The method of the present invention is safe and simple for both the pregnant woman and the fetus, and is highly accurate, so it is expected to be widely utilized in the field of prenatal diagnosis.

Claims

1. (a) A step of whole genome sequencing cell-free DNA of a fetus obtained from a biological sample isolated from the mother; (b) A step of calculating the number of reads for each chromosome from the BAM file generated as a result of sequencing and dividing it into binary files of 30,000 bp to 5,000,000 bp in size to correct it; (c) a step of correcting GC bias using local regression analysis of the corrected bin values; (d) a step of re-correcting the difference in variance between the control group and the sample to be analyzed using a chi-square test; and, (e) a non-invasive prenatal screening method comprising a step of calculating a Z-score value.

2. In paragraph 1, A method wherein the above Z-score values ​​are the Z-score of the X chromosome (Z-score(X)), the Z-score of the Y chromosome (Z-score(Y)), the predicted Z-score of the X chromosome (Z-score(X')), and the ratio (R) of Z-score(X) and Z-score(X').

3. In paragraph 2, A method in which the above Z-score(X), Z-score(Y), Z-score(X'), and R values ​​are calculated using the following [Mathematical Formula 1] to [Mathematical Formula 4]. [Mathematical Formula 1] [Equation 2] [Equation 3] [Equation 4] 4. In paragraph 3, The above method, If the Z-score (Y) value is less than 3 or the Z-score (X) value is greater than 3, the sex chromosome of the fetus is interpreted as aneuploid XO; If the Z-score (Y) value is less than 3 or the absolute value of Z-socre (X) is less than 3, the sex chromosome of the fetus is interpreted as normal XX; If the Z-score (Y) value is less than 3 and the absolute value of the Z-score (X) is greater than 3, the sex chromosome of the fetus is interpreted as aneuploid XXX; If the Z-score(Y) value exceeds 3, or if the absolute value of Z-score(X) is less than 3 and the Z-score(X) value is greater than the Z-score(X') value, the sex chromosome of the fetus is interpreted as aneuploid XXY; If the Z-score(Y) value is greater than 3, or if the Z-score(X) value is less than -3 and the Z-score(X) value is greater than the Z-score(X') value, the sex chromosome of the fetus is interpreted as aneuploid XYY; or, A method for interpreting the sex chromosome of the fetus as normal XY when the Z-score (Y) value is greater than 3 and R ∈ {-0.8, 0.8}.

5. In paragraph 1, A method in which the above Z-score value is calculated using linear regression analysis.

6. In paragraph 5, The above method, A method for additionally calculating NCV (Normalized Chromosome Value) and RBZ (Regression based Z-score) values.

7. In paragraph 6, The above method, A method for interpreting the autosomes of the fetus as trisomy aneuploidy when the Z-score value exceeds 4, the NCV value exceeds 3, or the RBZ value exceeds 3.

8. In paragraph 7, A method wherein the trisomy is Down syndrome, Edward syndrome or Patau syndrome.

9. A first module for whole genome sequencing of cell-free fetal DNA obtained from a biological sample isolated from the mother; A second module that calculates the number of reads for each chromosome from the BAM file generated as a result of sequencing and divides it into a binary file of 30,000 bp to 5,000,000 bp in size for correction; A third module that corrects GC bias by local regression analysis of the corrected bin values; A fourth module that recalibrates the difference in variance between the control group and the analysis sample using a chi-square test; and A noninvasive prenatal screening device comprising a fifth module for calculating a Z-score value.

10. In paragraph 9, A device wherein the above Z-score values ​​are the Z-score of the X chromosome (Z-score(X)), the Z-score of the Y chromosome (Z-score(Y)), the predicted Z-score of the X chromosome (Z-score(X')), and the ratio (R) of Z-score(X) and Z-score(X').

11. In paragraph 10, A device wherein the above Z-score(X), Z-score(Y), Z-score(X'), and R values ​​are calculated using the following [Mathematical Formula 1] to [Mathematical Formula 4]. [Mathematical Formula 1] [Equation 2] [Equation 3] [Equation 4] 12. In paragraph 11, The above device, If the Z-score (Y) value is less than 3 or the Z-score (X) value is greater than 3, the sex chromosome of the fetus is interpreted as aneuploid XO; If the Z-score (Y) value is less than 3 or the absolute value of Z-socre (X) is less than 3, the sex chromosome of the fetus is interpreted as normal XX; If the Z-score (Y) value is less than 3 and the absolute value of the Z-score (X) is greater than 3, the sex chromosome of the fetus is interpreted as aneuploid XXX; If the Z-score(Y) value exceeds 3, or if the absolute value of Z-score(X) is less than 3 and the Z-score(X) value is greater than the Z-score(X') value, the sex chromosome of the fetus is interpreted as aneuploid XXY; If the Z-score(Y) value is greater than 3, or if the Z-score(X) value is less than -3 and the Z-score(X) value is greater than the Z-score(X') value, the sex chromosome of the fetus is interpreted as aneuploid XYY; or, A device further comprising a sixth module for interpreting the sex chromosome of the fetus as normal XY if the Z-score(Y) value is greater than 3 and R ∈ {-0.8, 0.8}.

13. In paragraph 9, A device wherein the above Z-score value is calculated by linear regression analysis.

14. In paragraph 13, The above device, A device further comprising a seventh module for calculating NCV (Normalized Chromosome Value) and RBZ (Regression based Z-score) values.

15. In paragraph 14, The above device, A device further comprising an eighth module for interpreting the fetus' autosomes as trisomy aneuploidy when the Z-score value exceeds 4, the NCV value exceeds 3, or the RBZ value exceeds 3.

16. In paragraph 15, A device wherein the above trisomy is Down syndrome, Edward syndrome or Patau syndrome. 17.(a) A step of whole genome sequencing cell-free DNA of a fetus obtained from a biological sample isolated from the mother; (b) A step of calculating the number of reads for each chromosome from the BAM file generated as a result of sequencing and dividing it into binary files of 30,000 bp to 5,000,000 bp in size to correct it; (c) a step of correcting GC bias using local regression analysis of the corrected bin values; (d) a step of re-correcting the difference in variance between the control group and the sample to be analyzed using a chi-square test; and, (e) a method for providing information on the diagnosis of chromosomal aneuploidy of a fetus, comprising the step of calculating a Z-score value.

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