KR20190102810A - Fetal gender determination method through non-invasive prenatal test - Google Patents

Abstract

The present invention relates to a method for determining the sex of a fetus through non-invasive prenatal diagnosis. More particularly, the present invention relates to a method for determining the sex of a fetus through non-invasive prenatal diagnosis, using a non-invasive prenatal diagnosis method for sequencing a male-specific region of the Y chromosome (MSY), which is a region present only on the Y chromosome, from a biological sample of a mother. Therefore, the present invention provides the method which allows users to determine the sex of a fetus through non-invasive prenatal diagnosis with higher accuracy than before.

Description

Fetal gender determination through non-invasive prenatal diagnosis {FETFET GENDER DETERMINATION METHOD THROUGH NON-INVASIVE PRENATAL TEST}

The present invention relates to a method for determining the sex of a fetus through non-invasive prenatal diagnosis, and more particularly, by sequencing the MSY region, which is a region present only on the Y chromosome, from a mother's biological sample using a non-invasive prenatal diagnosis method. The present invention relates to a method of determining the sex of a fetus with higher accuracy than before.

Prenatal diagnosis can be divided into invasive and non-invasive diagnosis. Examples of invasive diagnostics include chorionic villi sampling (CVS) performed between 10 and 12 weeks of gestation, and fetal chromosomes by measuring the concentration of AFP in amniotic fluid using immunoassay between 15 and 20 weeks of gestation. Amniocentesis to analyze, cordocentesis to extract fetal blood directly from the umbilical cord under ultrasound guidance between 18 and 20 weeks of gestation.

However, these invasive diagnostic methods may shock the fetus during the test, causing miscarriage, disease or malformation. Methods based on fetal material acquisition by amniocentesis or chorionic villus sampling may be invasive, and even by skilled clinicians may pose a negligible risk for pregnancy. In practice, these invasive diagnostic methods are generally used when the maternal age is high or when there is a marker that increases the likelihood of Down syndrome fetal pregnancy through pre-screening through biochemical tests or ultrasonography. Noninvasive diagnostic methods are being developed to overcome these problems.

The discovery of cell-free fetal DNA (cffDNA) in cell-free DNA (cfDNA) in maternal serum provided a powerful tool for developing non-invasive prenatal diagnosis. cffDNA enters maternal blood during apoptosis of placental cells and is a short fragment of about 80 to 200 bp that is detected in maternal blood starting at 5-7 weeks of gestation. It is an effective way to exclude. Although it is easy to distinguish sex from whole cells according to technological development, it is still difficult to distinguish sex from cfDNA (cell-free DNA) in pregnant women, and cell-free fetal, the fraction of fetus in maternal cfDNA. DNA; cffDNA) is more difficult when less than 10%.

Massively parallel sequencing techniques such as Next Generation Sequencing (NGS) allow the analysis of all nucleic acids present in a sample, making it very useful for the analysis of cffDNA with very low concentrations of the desired sample. useful.

In recent years, the technology has been upgraded and Non-Invasive Prenatal Test (NIPT) technology is being performed based on next generation sequencing (NGS) (Bianchi DW et al., (2014) DNA sequencing versus standard prenatal aneuploidy screening N Engl J Med. 370: 799808., Sparks AB et al., (2012) Noninvasive prenatal detection and selective analysis of cell-free DNA obtained from maternal blood: evaluation for trisomy 21 and trisomy 18. Am J Obstet Gynecol. 206. : 319.e19., Ehrich M et al., (2011) Noninvasive detection of fetal trisomy 21 by sequencing of DNA in maternal blood: a study in a clinical setting.Am J Obstet Gynecol. 204: 205.e111.), Illumina Fast and efficient clinic analytical testing is performed using two platforms, Ion Torrent and Ion Torrent. Initially, the sensitivity and specificity of NIPT analysis using the Illumina platform was around 98% (Lau TK et al., (2014) Non-invasive prenatal testing for fetal chromosomal abnormalities by low). -coverage wholegenome sequencing of maternal plasma DNA: review of 1982 consecutive cases in a single center.Ultrasound Obstet Gynecol 43: 254264., Ashoor G et al., (2012) Chromosome-selective sequencing of maternal plasma cell-free DNA for firsttrimester detection of trisomy 21 and trisomy 18. Am J Obstet Gynecol. 206: 322.e15.), recently having sensitivity and specificity of more than 99%.

In general, females have two X chromosomes, and males have X and Y chromosomes. In mammals, the presence of Y chromosomes determines the sex. Without Y chromosomes, the gonads are differentiated into ovaries and femininity. This develops. The Y chromosome is about 60 Mb in size, derived from the X chromosome, and the X chromosome and the Y chromosome have homologous regions called pseudoautosomal regions (PAR1, PAR2), and the PAR1, PAR2 on the Y chromosome. The region excluding the region may be regarded as an MSY (male-specific region of the Y gene) region that exists only on the Y chromosome.

Y chromosome can be amplified and observed by real-time PCR from fetal DNA extracted from maternal plasma. By real-time PCR, the Y chromosome region is amplified and observed when the mother has a male fetus, but the Y chromosome region is not amplified when the mother has a female fetus.

In a conventional experiment, the DYS-14 region of Y chromosome in maternal plasma was amplified and observed by real-time PCR, and the accuracy was 92.6% ( Prenat Diagn , 26 (13), 1219-1223). When DAZ (male infertility gene), which is not amplified by real-time PCR, was observed, the accuracy was 97.3% compared with the actual newborn infants ( Acta Medica Iranica , 51 (4), 209-214).

An object of the present invention is to provide a method that can determine the sex of the fetus with a higher accuracy than conventional by analyzing the cell-free DNA obtained from the mother's biological sample using a non-invasive prenatal diagnostic method through the next generation sequencing .

It is another object of the present invention to provide an MSY region capable of discriminating the sex of a fetus through a non-invasive prenatal diagnosis method.

The present invention to solve the above problems is

(A) extracting cfDNA from the mother's biological sample to perform Next Generation Sequencing;

(B) aligning the sequenced reads into a reference genome database;

(C) UR (y)% (Unique Reads y%) of reads unique to the region of MSY for extracting the uniquely arranged reads in the MSY region of the Y chromosome from the summarized sequence and for determining fetal gender relative to the total reads Calculating;

(D) simultaneously calculating the calculated UR (y)% data to determine a cut-off value; And

(E) discriminating a male fetus when UR (y)% of the MSY region for determining fetal gender is greater than a reference value, and determining a female fetus when smaller than a reference value; It provides a method for determining the sex of the fetus through non-invasive prenatal diagnosis comprising a.

In the method for determining sex of the fetus through non-invasive prenatal diagnosis according to the present invention, the step (A) is

(a-1) separating the plasma by centrifuging the collected blood;

(a-2) extracting cfDNA from the separated plasma;

(a-3) preparing a library using the extracted cfDNA; And

(a-4) After pooling the prepared library, it is characterized in that it comprises the step of deciphering the nucleotide sequence using the NGS equipment.

In the method of gender determination of the fetus through non-invasive prenatal diagnosis according to the present invention, the biological sample is characterized in that any one of blood, plasma, serum, urine or saliva. Biological samples are obtained from the mother and can be extracted from the mother's plasma, serum, urine or saliva in addition to the blood as any sample containing one or more nucleic acid molecules of interest.

In the method for determining sex of a fetus through non-invasive prenatal diagnosis according to the present invention, the next generation sequencing method is, for example, 454 platform (Margulies, et al., Nature (2005) 437: 376 ~ 380), Illumina Genome Analyzer (Or Solexa ™ platform), Illumina HiSeq2000, HisSeq2500, MiSeq, NextSeq500, Life Tech Ion PGM, Ion Proton, Ion S5, Ion S5XL, or Applied Biosystems (SOLiD) or Helicos True Single Molecule DNA Sequencing Technology (Harris, et al., Science (2008) 320: 106-109), single molecule of Pacific Biosciences, and / or real time (SMRT ™) technology, and the like.   

In the method of gender determination of the fetus through non-invasive prenatal diagnosis according to the present invention, the step of extracting cfDNA from the mother's biological sample and performing the next generation sequencing, a large number of parallel DNA sequencing genomic DNA After dividing into many pieces, it is possible to obtain sequencing data of each piece, and to include all analysis methods for deciphering the genome by combining the sequencing data of the pieces using various bioinformatics techniques. When determining the sex of the fetus from cfDNA of pregnant women through Next Generation Sequencing (NGS) technology such as NextSeq 550Dx and Ion Proton, it is generally judged by the Y chromosome mapping rate.

In the method of gender determination of the fetus through non-invasive prenatal diagnosis according to the present invention, it is preferable to perform the next generation sequencing by dividing the 50kb bin size.

In the method for determining sex of a fetus through non-invasive prenatal diagnosis according to the present invention, the next generation sequencing is performed by an Ion Torrent's Ion proton system.

In the method of gender determination of the fetus through non-invasive prenatal diagnosis according to the present invention, the next generation sequencing is performed by NextSeq 550Dx of Illumina (Illumina).

In the method of gender determination of the fetus through non-invasive prenatal diagnosis according to the present invention, (B) sequencing the reads sequenced by platform for performing the next generation sequencing in a reference genome database (alignment) ) Step is to determine the location by comparing and aligning the base sequence of the DNA fragments (reference genome database) and the base sequence of the DNA fragments obtained from the mother.

In the method for determining the sex of a fetus through non-invasive prenatal diagnosis according to the present invention, after sequenced reads for each platform for performing the next generation sequencing in a reference genome database, Extracting reads uniquely arranged in the MSY region of the Y chromosome for each platform and calculating UR (y)% (Unique Reads y%) of reads unique to a specific region relative to the entire reads.

In the method for determining the sex of a fetus through non-invasive prenatal diagnosis according to the present invention, (C) a region of MSY for extracting a lead arranged uniquely in the MSY region of the Y chromosome and for determining fetal sex relative to the total lead In calculating UR (y)% of unique reads at UR (y)%, UR (y)% refers to the percentage of reads mapped uniquely to the MSY region of the Y chromosome relative to the entire gene reads.

In the method for discriminating the fetus of a fetus through non-invasive prenatal diagnosis according to the present invention, the region of MSY for determining fetal gender means a predetermined region suitable for discriminating fetal gender among a plurality of regions appropriately divided by the MSY region. Bar, Illumina's NextSeq 550Dx indicates 50 areas of Table 1 below, and Ion Torrent's Ion proton system points to 31 areas of Table 2 below.

In the present invention, (C) extracting reads uniquely arranged in the MSY region of the Y chromosome from the arranged sequence, and calculating UR (y)% (Unique Reads y%) of reads unique to a specific region compared to the entire reads. In the step, the MSY region of Y chromosome is divided into 50 kb bins, and the number of unique leads is counted in 31 regions (regions 1 to 31 in Table 1) suitable for the ion proton system. Provide gender discrimination methods.

region Ion proton system One chrY: 2800001-2850000 2 chrY: 6850001-6900000 3 chrY: 6900001-6950000 4 chrY: 7250001-7300000 5 chrY: 7550001-7600000 6 chrY: 7850001-7900000 7 chrY: 7900001-7950000 8 chrY: 8350001-8400000 9 chrY: 8600001-8650000 10 chrY: 8850001-8900000 11 chrY: 9850001-9900000 12 chrY: 14400001-14450000 13 chrY: 15550001-15600000 14 chrY: 15800001-15850000 15 chrY: 16400001-16450000 16 chrY: 16850001-16900000 17 chrY: 17100001-17150000 18 chrY: 17500001-17550000 19 chrY: 17750001-17800000 20 chrY: 17850001-17900000 21 chrY: 18150001-18200000 22 chrY: 18650001-18700000 23 chrY: 18850001-18900000 24 chrY: 19150001-19200000 25 chrY: 19500001-19550000 26 chrY: 20800001-20850000 27 chrY: 21650001-21700000 28 chrY: 22050001-22100000 29 chrY: 22700001-22750000 30 chrY: 22750001-22800000 31 chrY: 22800001-22850000

region NextSeq 550Dx 32 chrY: 2700001-2750000 33 chrY: 6850001-6900000 34 chrY: 6950001-7000000 35 chrY: 7500001-7550000 36 chrY: 7600001-7650000 37 chrY: 7650001-7700000 38 chrY: 7750001-7800000 39 chrY: 7800001-7850000 40 chrY: 7850001-7900000 41 chrY: 7900001-7950000 42 chrY: 7950001-8000000 43 chrY: 8250001-8300000 44 chrY: 8350001-8400000 45 chrY: 8400001-8450000 46 chrY: 8500001-8550000 47 chrY: 8550001-8600000 48 chrY: 8600001-8650000 49 chrY: 8750001-8800000 50 chrY: 8850001-8900000 51 chrY: 9050001-9100000 52 chrY: 9100001-9150000 53 chrY: 9850001-9900000 54 chrY: 14850001-14900000 55 chrY: 15000001-15050000 56 chrY: 15500001-15550000 57 chrY: 16150001-16200000 58 chrY: 16250001-16300000 59 chrY: 16550001-16600000 60 chrY: 16750001-16800000 61 chrY: 16800001-16850000 62 chrY: 16900001-16950000 63 chrY: 17350001-17400000 64 chrY: 17400001-17450000 65 chrY: 17550001-17600000 66 chrY: 18150001-18200000 67 chrY: 18550001-18600000 68 chrY: 18800001-18850000 69 chrY: 19450001-19500000 70 chrY: 21200001-21250000 71 chrY: 21300001-21350000 72 chrY: 22200001-22250000 73 chrY: 22250001-22300000 74 chrY: 22450001-22500000 75 chrY: 22900001-22950000 76 chrY: 23350001-23400000 77 chrY: 23400001-23450000 78 chrY: 23450001-23500000 79 chrY: 23550001-23600000 80 chrY: 23600001-23650000 81 chrY: 23850001-23900000

In addition, the sex determination method of the fetus through non-invasive prenatal diagnosis according to the present invention is divided into 50kb bin from the MSY region of the Y chromosome from the next-generation sequencing results 50 regions suitable for NextSeq 550Dx (region 32 to Table 2 above) 81) is used to measure the number of unique leads and to determine the sex of the fetus through non-invasive prenatal diagnosis to determine the sex of the fetus.

In the method of gender determination of the fetus through non-invasive prenatal diagnosis according to the present invention, (D) by calculating the UR (y)% data calculated for each platform at the same time, the reference value in the step of setting a cut-off value (cut-off value) refers to the numerical value used to determine the sex of the fetus due to the difference in lead values between boys and girls. .

In the non-invasive prenatal diagnosis according to the present invention, the fetal gender determination method, when the mapping rate of the Y chromosome is lower than the cut-off value is classified as a female fetus, if higher than the reference value to be classified as a male fetus. The Journal of Maternal-Fetal and Neonatal Medicine, 25 (8): 1370-1374.

Fetal gender determination method through non-invasive prenatal diagnosis according to the present invention reduces the risk factors for the fetus by non-invasive prenatal diagnosis, compared with the lead value in the MSY region of Y chromosome found only in male fetus by sequencing by NGS method The sex of the fetus can be determined with higher accuracy.

1 shows a data set obtained by dividing a chrY chromosome by 50 kb bin using a sample having a gender grasp in advance.
FIG. 2 illustrates a dataset in which a region in which a number of leads mapped in a male sample is less than 2 and a region in which a number of leads mapped in a female sample are greater than 3 are removed.
Figure 3 shows the number of leads mapped in 50 regions suitable for NextSeq 550Dx in the MSY region of Y chromosome.
4 and 5 illustrate datasets in which only regions where a large number of leads are mapped in a male sample and a region where the number of leads which are generally mapped in a female sample are selected are selected.
FIG. 6 shows that more than 50 males appear and less than 20 females appear when adding the number of mapped leads in 50 regions suitable for NextSeq 550Dx.
FIG. 7 (A) shows that when 31 areas (blue boxes) of the Ion Proton dataset are used by the Ion Proton system, it is more advantageous for sex discrimination of the fetus.
FIG. 7 (B) shows that the use of 50 red boxes of the NextSeq dataset by NextSeq 550Dx is more advantageous for fetal sex discrimination.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein and the experimental methods described below are well known and commonly used in the art.

Example 1 Extraction of Gender Distinctable Regions

After dividing the MSY region of the chrY chromosome by 50 kb bins using a dataset already known by Illumina's NextSeq 550Dx (FIG. 1), the number of reads mapped was less than 2 Regions with more than 3 leads in the regions and girl samples were removed (FIGS. 2 and 3).

Only regions with a large number of mapped leads in the male sample and regions with a small number of leads mapped overall in the female sample were reselected.

As a result, when the total number of mapped leads in 50 regions (regions 32 to 81 in Table 1) of the MSY regions were summed, it could be seen that 50 males and 20 female samples were less than 20 (FIG. 4, FIG. 4). 5, Figure 6).

In 50 of the MSY areas, there was a large difference in the number of leads between boys and girls, thus distinguishing the sex of the fetuses.

Example 2 Maternal cfDNA Extraction and Sequencing

Blood was collected from the mother, and plasma was separated by centrifugation. More than 30ng of cfDNA (cell-free DNA) was extracted from the separated plasma.

After the library was prepared using the extracted cfDNA, the adapter was combined (ligation) and the library was pooled to decode the base sequence.

Example 3 Fetal Gender Determination by Proton Dataset

Ion proton system was tested according to Ion Torrent's protocol.

Proton datasets were tested using a total of 894 samples, and when the MSY region was divided into 50 kb bins, the female fetus was able to distinguish sex from 31 regions with little mapping (regions 1 to 31 in Table 1). .

Example 4 Determination of Fetal Gender by NextSeq Dataset

Experiments were performed according to the protocol of NextSeq 550Dx from Illumina.

The NextSeq dataset was able to distinguish sex in 50 regions with little mapping in female fetus by dividing the MSY region into 50 kb bins, using a total of 734 samples (regions 32 to 81 in Table 2). .

In FIG. 3, experimental results of eight samples are shown. When the fetus was male, all 50 reads had a read count of 3 or more, and when the fetus was female, all 50 had a read count of 2 or less.

Comparative Example Comparison with Existing Area

A total of 1628 samples were used for non-invasive prenatal diagnosis according to the present invention to determine the sex of the fetus. As a comparative example, fractionation when sequencing the entire MSY region and repeated sequencing and centromere regions on the Y chromosome In the remaining portions, the analysis was performed by four methods of fractionation by measuring the number of leads, and the results are shown in FIG. 7.

In FIG. 7, the green box represents the number of reads in the entire MSR region, and the yellow box represents the number of reads in the remaining portions except the repeated sequencing on the Y chromosome and the centromere region.

As shown in FIG. 7A, when 31 areas (blue boxes) of the Ion Proton dataset are used instead of 50 red boxes of the NextSeq dataset, the read values of the female fetus are lowered according to the Ion Proton dataset. As this becomes easier, it is more advantageous to use 31 regions of the Ion Proton dataset. As shown in FIG. 7A, the green box and the yellow box, which are the most common methods according to the Ion Proton dataset, indicate that it is difficult to distinguish a male fetus from a female fetus.

In FIG. 7B, when the 50 red areas of the NextSeq dataset are used by the NextSeq dataset, the lead value of the female fetus is lower than that of the 31 blue areas of the Ion Proton dataset. It is easier to distinguish, indicating that using 50 regions of the NextSeq dataset is more advantageous.

FIG. 7 (B) shows that the green box and the yellow box do not become the sex classification of the fetus even when using the NextSeq dataset.

Claims (8)

(A) extracting cfDNA from the mother's biological sample to perform Next Generation Sequencing;
(B) aligning the sequenced reads into a reference genome database;
(C) UR (y)% (Unique Reads y%) of the only reads in the region of MSY for extracting the uniquely arranged reads in the MSY region of the Y chromosome from the arranged sequence and determining fetal sex relative to the total reads. Calculating;
(D) determining a cut-off value from the calculated UR (y)% data; And
(E) determining if the UR (y)% is greater than the reference value as a male fetus and if less than the reference value as a female fetus; Containing
Fetal Gender Discrimination by Noninvasive Prenatal Diagnosis
The method of claim 1,
The biological sample is any one of blood, plasma, serum, urine or saliva
Fetal Gender Discrimination by Noninvasive Prenatal Diagnosis
The method of claim 1,
Step (A) is
(a-1) separating the plasma by centrifuging the collected blood;
(a-2) extracting cfDNA from the separated plasma;
(a-3) preparing a library using the extracted cfDNA; And
(a-4) pooling the prepared library, and then using the NGS equipment to read the sequence
Fetal Gender Discrimination by Noninvasive Prenatal Diagnosis
The method of claim 1,
Step (A) is to divide the next generation sequencing by 50kb bin size
Fetal Gender Discrimination by Noninvasive Prenatal Diagnosis
The method of claim 1,
The next generation sequencing is performed by the Ion Torrent's Ion proton system
Fetal Gender Discrimination by Noninvasive Prenatal Diagnosis
The method of claim 5,
When the next-generation sequencing is performed by the Ion Torrent's Ion proton system, the MSY regions for determining fetal gender are 31 areas below.
Fetal Gender Discrimination by Noninvasive Prenatal Diagnosis

The method of claim 1
The next generation sequencing is carried out by NextSeq 550Dx of Illumina Co., Ltd.
Fetal Gender Discrimination by Noninvasive Prenatal Diagnosis
The method of claim 7, wherein
When the next generation sequencing is performed by Illumina NextSeq 550Dx, MSY regions for determining fetal gender are the following 50 regions.
Fetal Gender Discrimination by Noninvasive Prenatal Diagnosis

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