WO2023138131A1 - Method for detecting fetal balanced chromosome structure variation by means of cell-free dna in peripheral blood of pregnant woman - Google Patents

Abstract

Provided is a method for detecting a fetal balanced chromosome structure variation by means of cell-free DNA (cfDNA) in peripheral blood of a pregnant woman, comprising construction of a nuclear family haplotype model and fetal structure variation detection. A nuclear family of a couple carrying a chromosome structure rearrangement variation, SNP genotyping is performed on the family, a haplotype of a chromosome having a rearranged structure and a haplotype of a chromosome having a normal structure are defined, and a whole-genome haplotype model is constructed. Capture sequencing is performed on cfDNA in the peripheral blood of the pregnant woman, a fetal genotype and haplotype in cfDNA are calculated and analyzed according to a hidden Markov model, and embryo chromosome structure rearrangement is predicted by analyzing whether a fetus carries a haplotype near a breakpoint area of the chromosome having a rearranged structure. It is the first time to perform detection of a fetal balanced chromosome structure abnormality by means of cfDNA in the peripheral blood of the pregnant woman, and important guidance is provided for chromosome disease prevention and diagnosis.

Description

A method for detecting balanced structural variation of fetal chromosomes by free DNA in peripheral blood of pregnant women technical field

The invention belongs to the field of genetic diagnosis of chromosomal variation, in particular to a method for detecting balanced structural variation of fetal chromosomes through free DNA in the peripheral blood of pregnant women.

Background technique

Genetic diseases are an important cause of birth defects and fertility disorders, and pose a serious threat to human health and life. Clinically common genetic diseases include single gene diseases and chromosomal diseases, and chromosomal diseases include abnormal chromosome number and abnormal chromosome structure. For preventing the birth of children with genetic diseases, invasive prenatal diagnosis is still the clinical gold standard, but there is a certain risk of abortion infection. In recent years, with the rapid development of non-invasive prenatal testing (NIPT), the risk of genetic diseases in children can be assessed based on the fetal free DNA (cell-free fetal DNA, cffDNA) in the peripheral blood plasma of pregnant women. The existing NIPT technology has a very high detection rate for common aneuploidies, and it has also begun to mature for single-gene diseases and is gradually being applied clinically. However, there are still deficiencies. Existing NIPT technology cannot detect balanced chromosome structural variation, and there are no relevant research reports at home and abroad. For carriers of chromosomal structural abnormalities, NIPT can only detect chromosomal aneuploidy or large deletions/duplications. To detect whether a fetus carries a balanced chromosomal structural abnormality, it can only be diagnosed through invasive prenatal amniocentesis. Because invasive prenatal diagnosis has a certain risk of miscarriage and infection, many pregnant women refuse to accept invasive prenatal diagnosis. In addition, some pregnant women have contraindications to invasive prenatal diagnosis, such as placenta previa, oligohydramnios, and infectious diseases. Therefore, clinically, it is necessary to develop a NIPT technology suitable for patients with chromosomal structural abnormalities, which can simultaneously detect fetal chromosomal aneuploidy and balanced chromosomal structural abnormalities, and reduce the risk of fetal miscarriage or infection caused by invasive prenatal diagnosis. Moreover, this technology can be applied to structural aberrations at different breakpoint positions, and there is no need to design separately for different chromosomal structural aberrations. It is universal and provides guidance for non-invasive prenatal testing of carriers of chromosomal structural abnormalities, which has great clinical significance.

Chromosomal structural abnormalities, also known as chromosomal rearrangements, refer to chromosomal aberrations caused by the break-replacement or exchange mechanism of chromosomes or chromatids, mainly including chromosomal translocations, inversions, deletions/duplications, etc. In the field of obstetrics and gynecology, the most common chromosomal balanced translocations are important causes of adverse pregnancy outcomes such as infertility, recurrent miscarriages, fetal developmental deformities, and congenital birth defects. Chromosomal balanced translocation refers to the change of chromosome structure caused by two chromosomes breaking at the same time and missplicing and exchange, including reciprocal translocation and Robertsonian translocation. The overall incidence rate in the population is about 0.27%, the incidence rate in infertility patients is about 1.1%, and the incidence rate in patients with repeated spontaneous abortion is higher, up to 4.08%.

Carriers of balanced chromosomal translocations usually have no obvious clinical symptoms before childbearing age, and usually manifest as recurrent miscarriage or infertility and low fertility at childbearing age. The reason is that the germ cells of the carrier will produce a large number of unbalanced chromosomal abnormal gametes. The embryos formed after fertilization of these unbalanced gametes will have implantation failure, spontaneous abortion, and birth defects due to chromosomal abnormalities. In addition, women have an increased risk of premature ovarian failure, and men usually show oligoasthenospermia. Therefore, balanced chromosomal translocations seriously affect human reproductive health and are an important cause of birth defects in newborns.

For carriers of chromosomal structural aberrations, invasive prenatal diagnosis of the fetus is generally required after pregnancy, including chorionic villus puncture in the first trimester, amniocentesis in the second trimester, and umbilical cord blood aspiration in the third trimester. Carry out chromosomal karyotype analysis on the fetus through prenatal diagnostic genetic examination, including traditional cell karyotype and molecular karyotype. The resolution of traditional karyotype is low. Generally, fragments larger than 5Mb or 10Mb can be diagnosed. In contrast, molecular karyotype is more accurate and can diagnose occult unbalanced translocations of small fragments. distortion.

In 1997, Dennis Lo et al. detected the specific SRY gene on the Y chromosome in the peripheral blood of pregnant women pregnant with male fetuses by fluorescent quantitative PCR technology, and confirmed for the first time that there was a small amount of fetal-derived free DNA fragments in the maternal peripheral blood, which opened the era of NIPT detection in the peripheral blood of pregnant women. Fetal-derived DNA fragments are considered to come from placental trophoblast cells or naturally exfoliated fetal cells. They can be detected in the peripheral blood of pregnant women at 7 weeks of pregnancy. The length of maternal-derived DNA fragments in plasma is concentrated at about 166 bp, and the length of fetal-derived DNA fragments is shorter and more concentrated at 143 bp. In addition, maternal-derived and fetal-derived DNA fragments tend to break at different chromosome position coordinates. According to these characteristics, cell-free DNA from the fetus and cell-free DNA from the mother can be distinguished in the peripheral blood of pregnant women.

At present, NIPT technology has been widely used in the detection of chromosomal aneuploidy and some microdeletion and microduplication syndromes. It has very high sensitivity and specificity. Studies have shown that compared with serological screening methods, NIPT has greatly improved the detection rate and screening efficiency. The detection rate of 21-trisomy syndrome is over 98-99%, and the detection rate of 18-trisomy and 13-trisomy syndrome is over 95%. Later, thanks to the development of next-generation sequencing technology, NIPT for some single-gene genetic diseases became a research hotspot. A common technique is to use a quantitative method to directly detect the mutant allele of the pathogenic site in the cffDNA in the peripheral blood of pregnant women. Another common method is to use SNP linkage analysis, that is, the method of haplotype analysis for detection. By constructing the haplotype of pregnant women and their spouses, the direct detection of the mutation site is converted into an indirect analysis of a haplotype at the site to improve detection sensitivity. This method has a wide range of applications and is not affected by the mutation type. Unfortunately, there is no NIPT technology for the detection of chromosome balance structural aberrations so far.

In the prenatal diagnosis of genetic diseases, it is of great significance to find a low-cost, low-risk, high-sensitivity, and high-accuracy non-invasive prenatal diagnosis technology for the detection of chromosome balance structural aberrations.

Contents of the invention

Aiming at the gaps in the above-mentioned prior art, the present invention establishes a new NIPT technology based on targeted capture combined with Bayesian HMM analysis. Firstly, the patient’s core family is collected, genome-wide targeted capture single nucleotide polymorphism (SNP) allelic typing is carried out, effective information loci are defined to construct the core family haplotype model, and normal and structural variant chromosome haplotypes in the family are clarified; at the same time, the maternal peripheral blood free DNA (cfDNA) is targeted capture and sequenced, and the fetal haplotype is estimated through HMM analysis. The fetal haplotype is used to determine whether it carries chromosomal structural variation. The invention can not only detect the aneuploidy of the fetus, but also detect the variation of the balanced chromosome structure, and the technology is universal for different chromosome structure variations.

Concrete technical scheme of the present invention is as follows:

The first aspect of the present invention provides a method for constructing a nuclear family genome-wide haplotype model for identifying fetal chromosomal abnormalities, comprising the following steps:

(1) Sample genotyping: large-scale SNP genotyping of the following objects:

a. Both spouses of chromosomal rearrangement carriers;

b. At least one relative of the carrier: including the parents of the carrier, offspring of the carrier and other relatives;

Among them, the relatives of the carrier can be relatives who have the same chromosomal structural rearrangement as the carrier, or relatives with normal chromosomes;

When the relatives of the carrier are selected from the carrier's parents or other relatives, one of the carrier's parents carries the same chromosome structure rearrangement as the carrier;

The above b is referred to as the reference sample;

(2) Determine the effective information SNPs site:

a. When one of the carrier's parents or other relatives is used as the reference sample, the SNP sites that are heterozygous in the chromosome structure rearrangement carrier, homozygous in the carrier's spouse, and homozygous in the reference sample are valid information SNPs;

b. When the offspring of the carrier are used as the reference sample, the SNP sites that are heterozygous in the chromosome structure rearrangement carrier and homozygous in the carrier spouse are valid information SNPs;

(3) Construct the genome-wide haplotype model of the core family: collect the effective information SNPs identified in step (2) to conduct family haplotype linkage analysis, obtain the genome-wide haplotype of the core family, clarify the haplotype of the structurally rearranged chromosome and the haplotype of the normal chromosome, and determine the genome-wide haplotype of the parent.

The second aspect of the present invention provides a system for constructing a core family genome-wide haplotype model for identifying fetal chromosomal abnormalities, the system includes software for processing sample data and hardware for carrying the above-mentioned software,

(1) The system also includes hardware that stores reference samples and genotyping data of large-scale SNP genotype detection of both carrier couples; the reference sample is at least one carrier relative: including parents of carriers, offspring of carriers and other relatives; wherein, the relatives of the carrier can be relatives with the same chromosome structure rearrangement as the carrier, or relatives with normal chromosomes; when the carrier relatives are selected from the carrier's parents or other relatives, one of the carrier's parents carries the same chromosome structure rearrangement as the carrier;

(2) The software determines the effective information SNPs site according to the following rules:

a. When one of the carrier's parents or other relatives is used as the reference sample, the SNP sites that are heterozygous in the chromosome structure rearrangement carrier, homozygous in the carrier's spouse, and homozygous in the reference sample are valid information SNPs;

b. When the offspring of the carrier are used as the reference sample, the SNP sites that are heterozygous in the chromosome structure rearrangement carrier and homozygous in the carrier spouse are valid information SNPs;

(3) The software constructs the whole-genome haplotype model of the core family according to the following principles: the reference samples determined in step (2) and the effective information SNPs loci of the carrier couple are collected for family haplotype linkage analysis, the whole-genome haplotype of the core family is obtained, the haplotype of the structurally rearranged chromosome and the haplotype of the normal chromosome are clarified, and the whole-genome haplotype of the parent is determined.

The third aspect of the present invention provides a method for identifying fetal chromosomal abnormalities through free DNA in peripheral blood of pregnant women, comprising the following steps:

S1: Construction of the genome-wide haplotype model of the core family

(1) Sample genotyping: large-scale SNP genotyping of the following objects:

a. Both spouses of chromosomal rearrangement carriers;

b. At least one relative of the carrier: including the parents of the carrier, offspring of the carrier and other relatives;

Among them, the relatives of the carrier can be relatives who have the same chromosomal structure rearrangement as the carrier, and can also be relatives with normal chromosomes;

When the relatives of the carrier are selected from the carrier's parents or other relatives, one of the carrier's parents carries the same chromosome structure rearrangement as the carrier;

The above b is referred to as the reference sample;

(2) Determine the effective information SNPs site:

a. When one of the carrier's parents or other relatives is used as the reference sample, the SNP sites that are heterozygous in the chromosome structure rearrangement carrier, homozygous in the carrier's spouse, and homozygous in the reference sample are valid information SNPs;

b. When the offspring of the carrier are used as the reference sample, the SNP sites that are heterozygous in the chromosome structure rearrangement carrier and homozygous in the carrier spouse are valid information SNPs;

(3) Construct the whole-genome haplotype model of the core family: collect the effective information SNPs sites determined in step (2) to carry out family haplotype linkage analysis, obtain the whole-genome haplotype of the core family, clarify the haplotype of the structurally rearranged chromosome and the haplotype of the normal chromosome, and determine the whole-genome haplotype of the parent;

S2: Predictive Analysis of Fetal Genotypes and Haplotypes

SNP allelic typing was carried out on the cfDNA of pregnant women's peripheral blood by targeted capture sequencing method, and the fetal genotype and haplotype in cfDNA of pregnant women's peripheral blood were estimated and analyzed according to the hidden Markov statistical model;

S3: Identification of Chromosomal Structural Abnormalities

According to the haplotypes of the parents and the fetus, by analyzing the haplotype of the fetal chromosomal structure rearrangement breakpoint region and whether homologous recombination has occurred in this region, the fetal chromosomal structure variation is detected. Relatives as reference samples include carrier parents, carrier offspring and other relatives:

1) When the relatives of the carrier who carry the chromosomal structural rearrangement are used as the reference sample:

a. If there is no recombination in the fetal chromosomal structure rearrangement breakpoint area, when the haplotype information of the fetal chromosomal structure rearrangement breakpoint area is consistent with the haplotype information of the reference sample, it is diagnosed as a chromosomal structure rearrangement carrier fetus;

b. If homologous recombination occurs in the fetal chromosome breakpoint region, the judgment standard is opposite to a;

2) When the relatives of the carrier who do not carry the chromosomal structural rearrangement are used as the reference sample:

a. If there is no recombination in the fetal chromosomal structure rearrangement breakpoint area, when the haplotype information of the fetal chromosomal structure rearrangement breakpoint area is consistent with the haplotype information of the reference sample, it is diagnosed as a non-chromosomal structure rearrangement carrier fetus, that is, a fetus with a normal chromatid karyotype; when they are inconsistent, it is diagnosed as a chromosomal structure rearrangement carrier fetus.

b. If homologous recombination occurs in the fetal chromosome breakpoint region, the judgment standard is opposite to a;

Also includes S4: Detection of fetal chromosomal aneuploidy

Using low-depth whole-genome high-throughput sequencing, the cfDNA sequencing reads data are compared with normal controls to detect common aneuploidy and large segment copy number variation of chromosomes, and determine whether there is aneuploidy in cffDNA in pregnant women's plasma, as well as large segmental deletions and duplications related to chromosomal structural variation.

The fourth aspect of the present invention provides a system for identifying fetal chromosomal abnormalities through free DNA in peripheral blood of pregnant women, the system includes software for processing sample data and hardware for carrying the above software,

S1: the system also includes hardware that stores reference samples and genotyping data of large-scale SNP genotyping detection in the peripheral blood of pregnant women; the reference sample is at least one carrier relative: including carrier parents, carrier offspring and other relatives; wherein, the carrier relatives can be relatives with the same chromosome structure rearrangement as the carrier, or relatives with normal chromosomes; when the carrier relatives are selected from the carrier's parents or other relatives, one of the carrier's parents carries the same chromosome structure rearrangement as the carrier;

S2: The software constructs the whole genome haplotype model of the core family according to the following rules:

(1) Determine effective information SNPs:

a. When one of the carrier's parents or other relatives is used as the reference sample, the SNP sites that are heterozygous in the chromosome structure rearrangement carrier, homozygous in the carrier's spouse, and homozygous in the reference sample are valid information SNPs;

b. When the offspring of the carrier are used as the reference sample, the SNP sites that are heterozygous in the chromosome structure rearrangement carrier and homozygous in the carrier spouse are valid information SNPs;

(2) Collect the effective information SNPs loci determined in step (1) to carry out family haplotype linkage analysis, obtain the whole genome haplotype of the core family, clarify the haplotype of structurally rearranged chromosomes and the haplotype of normal chromosomes, and determine the whole genome haplotype of the parents;

S3: The software predicts and analyzes the fetal genotype and haplotype according to the following rules

SNP allelic typing was carried out on the cfDNA of pregnant women's peripheral blood by targeted capture sequencing method, and the fetal genotype and haplotype in cfDNA of pregnant women's peripheral blood were estimated and analyzed according to the hidden Markov statistical model;

S4: The software identifies abnormalities in chromosome structure according to the following rules

According to the haplotypes of the parents and the fetus, by analyzing the haplotype of the fetal chromosomal structure rearrangement breakpoint region and whether homologous recombination has occurred in this region, the fetal chromosomal structure variation is detected. Relatives as reference samples include carrier parents, carrier offspring and other relatives:

1) When the relatives of the carrier who carry the chromosomal structural rearrangement are used as the reference sample:

a. If there is no recombination in the fetal chromosomal structure rearrangement breakpoint area, when the haplotype information of the fetal chromosomal structure rearrangement breakpoint area is consistent with the haplotype information of the reference sample, it is diagnosed as a chromosomal structure rearrangement carrier fetus;

b. If homologous recombination occurs in the fetal chromosome breakpoint region, the judgment standard is opposite to a;

2) When the relatives of the carrier who do not carry the chromosomal structural rearrangement are used as the reference sample:

a. If there is no recombination in the fetal chromosomal structure rearrangement breakpoint area, when the haplotype information of the fetal chromosomal structure rearrangement breakpoint area is consistent with the haplotype information of the reference sample, it is diagnosed as a non-chromosomal structure rearrangement carrier fetus, that is, a fetus with a normal chromatid karyotype; when they are inconsistent, it is diagnosed as a chromosomal structure rearrangement carrier fetus.

b. If homologous recombination occurs in the fetal chromosome breakpoint region, the judgment standard is opposite to a;

S4: The software detects fetal chromosomal aneuploidy according to the following rules

Using low-depth whole-genome high-throughput sequencing, the cfDNA sequencing reads data are compared with normal controls to detect common aneuploidy and large segment copy number variation of chromosomes, and determine whether there is aneuploidy in cffDNA in pregnant women's plasma, as well as large segmental deletions and duplications related to chromosomal structural variation.

In the above technical solution of the present invention, the chromosomal structural abnormality is chromosome balance structural variation. Preferably, the chromosome balanced structural variation includes chromosome balanced translocation and inversion.

In the above technical solution of the present invention, the SNP genotype detection of the carrier couple and the carrier relatives is performed on the gDNA capture sequencing of the peripheral blood of the carrier couple and the carrier relatives.

In the above technical solution of the present invention, the design principle of the peripheral blood cfDNA capture probe is as follows: query the population genome database, select the minimum allele frequency according to the frequency of SNP sites, especially in East Asian populations, between 0.3-0.7, and the interval between adjacent sites is 300-500Kb, evenly distributed in the genome, and verified by haploview, the linkage disequilibrium R2 between the sites must be greater than 0.8.

In the above technical solution of the present invention, no less than two effective SNP sites are required when analyzing the haplotype of the fetal chromosomal structure rearrangement breakpoint region and whether homologous recombination occurs in this region.

The beneficial effects of the present invention are:

(1) The present invention proposes for the first time the detection of fetal chromosomal abnormality through cfDNA in the peripheral blood of pregnant women, which provides important guidance for the prevention and diagnosis of chromosomal diseases and fills in the international technical gap.

(2) The present invention is innovatively based on the core family, combines the core family haplotype model with Bayesian HMM analysis, and accurately detects the fetal chromosome structural variation through indirect haplotype linkage analysis, avoiding the problem of directly detecting the breakpoint.

(3) The present invention is a comprehensive NIPT universal technology platform, which can complete the detection of fetal chromosomal aneuploidy and structural abnormality through one detection, and solve the problem that the existing NIPT technology cannot detect balanced chromosome structural variation. Moreover, the SNP genetic marker sites designed in the present invention have high-throughput characteristics, and are applicable to all types of chromosome structure rearrangement carriers.

Description of drawings

Figure 1 is a hypothetical diagram for the establishment of non-invasive detection of fetal chromosomal aberrations using plasma cell-free DNA of pregnant women in nuclear families, HMM: hidden Markov model;

Fig. 2 is technical path of the present invention;

Figure 3 is the karyotype diagram of the carrier of case 3;

Figure 4 is the haplotype map of fetal translocation chromosome in case 3.

Detailed ways

The present invention puts forward the hypothesis that "the genetic haplotype of the core family can represent the karyotype, and then detect the chromosome structure aberration of the fetus", to establish a new NIPT technology for the detection of the chromosome structure aberration. The technology mainly includes two parts: the construction of the core family haplotype model and the detection of fetal structural variation. In the first part, by collecting families of couples carrying chromosome structural rearrangement variants, the families include carrier couples and carrier relatives (carrier relatives give priority to carrier parents and carrier offspring, and other relatives are also preferred), and use targeted capture sequencing to perform SNP genotyping on core family samples, and combine bioinformatics analysis methods to clarify the haplotypes of structurally rearranged chromosomes and structurally normal chromosomes, and construct a genome-wide haplotype model. Because the model includes genome-wide haplotypes, haplotypes at any chromosomal position can theoretically be obtained, with generalizability to different structural variants. In the second part, SNP allelic typing was also performed on the cell-free DNA in the peripheral blood of pregnant women by the targeted capture sequencing method, and the fetal genotype and haplotype in the cell-free DNA were calculated and analyzed according to the hidden Markov model (HMM). In addition, the chromosome aneuploidy of the fetus is detected at the same time through the sequencing data, so as to achieve the purpose of simultaneously completing the detection of chromosome number and structural abnormalities in one detection.

The hypotheses of non-invasive detection of fetal chromosomal structural aberrations were established based on the nuclear family and the free DNA in the peripheral blood of pregnant women, as shown in Figure 1. The schematic diagram shows that by combining targeted capture sequencing and bioinformatics analysis, the haplotypes of the core family are constructed to clarify the haplotypes of the couple. After deep sequencing of the free DNA in the peripheral blood of pregnant women, the fetal haplotypes are calculated according to the HMM model analysis, and then the fetal karyotypes are deduced according to the fetal chromosomal haplotypes, so as to realize the non-invasive detection of fetal balanced chromosome structural variation. Hap0 and Hap1 indicate that the fetus has inherited the same and different haplotypes from the proband at this position, respectively. The technical route is shown in Figure 2. For reference samples, parents and offspring of carriers are given priority. When the reference sample is selected from the carrier's parents or other relatives, one of the carrier's parents is required to carry the same chromosomal rearrangement as the carrier. The specific plan is as follows:

(1) Core family collection, DNA extraction and capture sequencing

To collect eligible patient families, one of the couples is required to be a carrier of chromosomal rearrangement. At the same time, the peripheral blood of the parents of the carrier is drawn, and the source of the chromosomal rearrangement of the case is determined according to the test results of the parents, that is, which parent is inherited from. Collect peripheral blood samples from both the couple and the parents of the carrier to construct basic family information and lay the foundation for the construction of an analysis model. Cell-free DNA was extracted from peripheral blood of core families and peripheral blood of pregnant women, and targeted capture sequencing was performed after library construction.

The above-mentioned principles for the design of targeted capture probes for cfDNA in the peripheral blood of pregnant women are: query the population genome database, and according to the frequency of SNP sites, especially in East Asian populations, select the minimum allele frequency between 0.3-0.7, the interval between adjacent sites is about 300-500Kb, evenly distributed in the genome, and verified by haploview, the linkage disequilibrium R2 between the sites must be greater than 0.8.

(2) Construct a family genome-wide haplotype model to determine the parental genome-wide haplotype

Perform genome-wide SNP typing on family samples, define effective information SNP selection criteria, perform haplotype linkage analysis on effective information SNP genetic markers, construct a family-wide genome haplotype model, and use the haplotype model to clarify the haplotypes of structurally rearranged chromosomes and structurally normal chromosomes, and determine the genome-wide haplotypes of the parents.

The selection criteria for valid information SNPs are as follows: a. When one of the parents or other relatives of the carrier is used as the reference sample, the effective information SNP requirements are heterozygous in the carrier and homozygous in the spouse of the carrier and the reference sample; b. When the offspring of the carrier is used as the reference sample, the valid information SNP requirements are heterozygous in the carrier and homozygous in the spouse of the carrier.

(3) Cell-free DNA in the peripheral blood of pregnant women to detect the haplotype of the whole genome of the fetus

SNP allelic typing was carried out on the cfDNA of pregnant women's peripheral blood by targeted capture sequencing method, combined with parental haplotype, fetal free concentration and sequencing error rate, according to the binomial distribution model and the Hidden Markov statistical model (HMM) based on Bayesian analysis, the fetal genotype and haplotype in cfDNA were predicted and analyzed. Relatives with the same chromosomal structure rearrangement as the carrier and relatives with normal chromosomes were used as references to explore the consistency of fetal haplotypes constructed under different reference samples.

(4) Fetal chromosomal rearrangement detection

Association of haplotypes at chromosomal breakpoint regions with whole chromosome haplotypes: Homologous recombination occurs between homologous chromosomes during meiosis, interfering with haplotype outcomes. Focus on analyzing the relationship between the haplotype at the breakpoint of chromosome structure rearrangement and the haplotype of the entire chromosome, and analyze the homologous recombination at the breakpoint.

Consistency of haplotypes at the two breakpoint positions of chromosome structural rearrangement: theoretically, the haplotypes at the two breakpoint positions should be structurally rearranged haplotypes or normal chromosome haplotypes at the same time.

According to the haplotypes of the parents and the fetus, the fetal chromosomal structure variation is detected by analyzing the haplotype of the fetal chromosomal structure rearrangement breakpoint region and whether homologous recombination has occurred in this region.

1) When referring to the relatives of the carrier who carry the chromosomal structural rearrangement:

a. If there is no recombination in the fetal chromosomal structure rearrangement breakpoint region, when the haplotype information in the fetal chromosomal structure rearrangement breakpoint region is consistent with the haplotype information in the reference sample, it is diagnosed as a chromosomal structure rearrangement carrier fetus. When they are inconsistent, the fetus is diagnosed as a fetus with a non-chromosomal structural rearrangement, that is, a fetus with a normal chromatid karyotype.

b. If homologous recombination occurs in the region of the fetal chromosome breakpoint, the judgment standard is the opposite of a.

2) When referring to the relatives of the carrier who do not carry the chromosomal structural rearrangement:

a. If there is no recombination in the fetal chromosomal structure rearrangement breakpoint region, when the haplotype information in the fetal chromosomal structure rearrangement breakpoint region is consistent with the haplotype information in the reference sample, it is diagnosed as a non-chromosomal rearrangement carrier fetus, that is, a fetus with a normal chromatid karyotype. When they are inconsistent, it is diagnosed as carrying a fetus with chromosomal rearrangement.

b. If homologous recombination occurs in the region of the fetal chromosome breakpoint, the judgment standard is the opposite of a.

After the detection and analysis model is analyzed, the fetus is diagnosed. For fetuses that do not carry the haplotype of chromosome structure rearrangement, the diagnosis is normal chromosome structure; for fetuses carrying the haplotype of chromosome structure rearrangement, the diagnosis is for chromosome structure rearrangement; for fetuses with aneuploidy or chromosome structure imbalance, the fetus is directly diagnosed as chromosomal abnormality.

(5) Detection of fetal chromosomal aneuploidy

Using low-depth whole-genome high-throughput sequencing, the cfDNA sequencing reads data are compared with normal controls to detect common aneuploidy and large segment copy number variation of chromosomes, and determine whether there is aneuploidy in cffDNA in pregnant women's plasma, as well as large segmental deletions and duplications related to chromosomal structural variation.

(6) Follow up the results of fetal prenatal diagnosis and pregnancy outcome

According to the results of fetal chromosomal rearrangement and aneuploidy detection, follow-up the results of amniotic fluid cell chromosome karyotype of fetuses in the second trimester, compare with the results of the detection and analysis model, and analyze the effectiveness of this technical method. The fetus was followed up to birth, and the birth status was recorded.

Based on the above-mentioned model detection system, the present invention preliminarily included 6 case families meeting the requirements in the early stage, all of which successfully detected the chromosome number and structural results of the fetus. Carry out prenatal genetic diagnosis to the fetus by amniocentesis, the result shows that fetal amniotic fluid cell or umbilical cord blood diagnosis result is completely consistent with the NIPT detection technology of the present invention to the detection result of embryo at the early stage, currently there are 5 fetuses born, growth and development are all normal.

In the above technical solution of the present invention, attention should be paid to the selection of the size of the haplotype analysis region: when analyzing the breakpoint region, sufficient genetic markers must be used for linkage analysis of the structural rearrangement breakpoint region, generally requiring no less than 2 effective SNP sites; when analyzing the haplotype of the entire chromosome, attention should be paid to identifying the homologous recombination that occurs during the meiotic classification of germ cells to avoid misdiagnosis due to homologous recombination. At the same time, pay attention to the uniformity of the distribution of genetic markers to ensure that there are effective genetic markers in the analyzed area.

The present invention will be described in detail below in conjunction with specific embodiments. Unless otherwise specified, the technical means used in the present invention are conventional technical means that can be mastered by those skilled in the art. The embodiments of the present invention are not intended as specific limitations on the implementation of the present invention.

Example 1: Collection of both spouses and reference samples of chromosomal structural variation carriers

Six couples carrying chromosomal structural variants, including balanced chromosome translocation and chromosome inversion, were recruited. The selected candidates were all from the Jiai Reproductive Center of the Obstetrics and Gynecology Hospital of Fudan University. The research protocol was approved by the Human Subjects Ethics Committee of the Obstetrics and Gynecology Hospital of Fudan University.

All couples have a history of recurrent spontaneous abortion or chromosomal abnormalities. One of the couples carrying balanced chromosome translocations or inversions is referred to as "patient" and the other as "patient's spouse" in the following text. At the same time of recruitment, draw about 10ml of peripheral blood from each patient couple and relatives of patients (parents and children of patients are preferred, other relatives are also acceptable). A part of the peripheral blood was used for lymphocyte culture and karyotype analysis; the other part of the peripheral blood was extracted according to the conventional methods in the art for subsequent sequencing.

Peripheral blood chromosome preparation method is as follows:

1. Cell culture

1). Blood collection: disinfect the skin with alcohol, collect blood from the cubital vein, pass the injection needle directly through the rubber stopper of the culture bottle, inject 30-40 drops of whole blood into 10ml of culture medium, shake gently and place in a 37°C incubator for cultivation.

2). Cultivation: the time is 68 hours. During the culture period, shake gently regularly to make the cells fully contact with the culture medium.

3). Colchicine treatment: 2-4 hours before terminating the culture, add colchicine to the culture solution (2 drops with a No. 5 needle tip of a 1 ml syringe, so that the final concentration is 0.07 μg/ml).

All the above steps need to be performed aseptically.

2. Chromosome preparation

1). Collect cells: transfer all the cultures into a clean centrifuge tube, centrifuge at 1000rpm for 8-10 minutes, and discard the supernatant.

2). Hypotonic treatment: Add 8ml of hypotonic solution pre-warmed at 37°C to a graduated centrifuge tube, mix well with a dropper, and place in a constant temperature water bath at 37°C for 15-25 minutes.

3). Pre-fixation: After hypotonicity, add 0.5ml fixative solution, mix gently and then centrifuge at 1000rpm for 8-10 minutes.

4). One fixation: Discard the supernatant, add 5ml of fixative solution, mix gently, and let stand for 20 minutes. Centrifuge at 1000rpm and discard the supernatant.

5). Two fixes, three fixes: the same fix.

6). Preparation of suspension: After discarding the supernatant, add an appropriate amount of fixative to make a cell suspension depending on the number of cells.

7). Drop sheet: absorb the cell suspension from 10-20cm high and drop it on a dry and clean glass slide, blow it gently, and air dry.

8). Dyeing: 1:10Giemsa dyeing for 5-10 minutes, rinse with fine water to remove excess dyeing solution, and air dry.

9). Microscopic examination: look for well-dispersed and moderately stained cleavage phases under a low-magnification microscope, observe and count the chromosome morphology under an oil microscope.

If the patient's peripheral blood karyotype is the same as that of the mother, the patient's balanced translocation is inherited from the mother; if the patient's peripheral blood karyotype is the same as that of the father, the patient's balanced translocation is inherited from the father. When the patient's parents are unable to take blood (such as death) or do not agree to take blood, the patient's siblings or other relatives can also take peripheral blood for karyotype analysis, and it can also be used as a reference sample when constructing the haplotype of the family.

The translocation karyotypes of families 1-6 are shown in Table 1. Figure 3 shows the peripheral blood karyotype of family No. 3.

Table 1. Chromosome karyotype of families No. 1-6

family number	Carrier Karyotype	Carrier paternal karyotype	Carrier mother's karyotype
1	46,XX,t(3;6)(p10;p10)	46,XY	46,XX,t(3;6)(p10;p10)
2	46,XY,t(1;12)(q21;p11)	46,XY,t(1;12)(q21;p11)	46,XX
3	46,XX,t(1;20)(q25;p11.2)	46,XY	46,XX,t(1;20)(q25;p11.2)
4	45,XY,rob(13;14)(q10;q10)	45,XY,rob(13;14)(q10;q10)	46,XX
5	45,XX,rob(13;14)(q10;q10)	46,XY	45,XX,rob(13;14)(q10;q10)
6	46,XY,inv(7)(p21q21)	46,XY,inv(7)(p21q21)	46,XX

Example 2: Construction of the haplotypes of the husband and wife and the fetus

1. Genomic DNA extraction from peripheral blood

Preliminary preparation: 1.5ml EP tube, consumables such as water bath, centrifuge, shaker, etc., well marked, the protease provided in the kit, AW1 and AW2 are added with corresponding volume of absolute ethanol before use. If BufferAL precipitates, warm at 56°C and shake gently to dissolve. Before using BufferAW1 and BufferAW2 for the first time, you need to add the corresponding volume of absolute ethanol according to the label on the reagent bottle to make it a working solution. Turn on the water bath and set at 56 °C.

The experimental steps are as follows:

(1) Add proteinase K 20μl, anticoagulant blood 200μl, bufferAL 200μl, vortex and incubate at 56°C for 10 minutes.

(2) Add 200 μl of absolute ethanol, vortex and mix to make a homogeneous solution.

(3) Transfer the mixed solution to the DNeasy Mini spin column and centrifuge at 6000x g (8000rpm) for 1 minute. Replace collection tube.

(4) Transfer the DNeasy Mini spin column to a new collection tube, add 500μl BufferAW1, and centrifuge at 6000xg (8000rpm) for 1 minute. Replace collection tube.

(5) Transfer the DNeasy Mini spin column to a new collection tube, add 500μl Buffer AW2□, centrifuge at 20,000xg (14,000rpm) for 3 minutes, and then centrifuge for 1 minute. Replace collection tube. At this step, it is necessary to pay attention to the complete drying of the membrane surface of DNeasy Mini spin column. If there is still ethanol remaining, it will interfere with the next experimental reaction. If the DNeasy Mini spin column touches the liquid surface in the collection tube during the replacement of the collection tube, aspirate the liquid in the collection tube and centrifuge at 20,000x g (14,000rpm) for one minute.

(6) Transfer DNeasy Mini spin column to a clean 1.5ml or 2ml centrifuge tube, add 30-100AE. Dissolve at room temperature for 3 minutes and centrifuge at 6000xg (8000rpm) for 1 minute.

2. Extraction of free DNA from peripheral blood of pregnant women

The initial volume of plasma samples from pregnant women was 1.8mL, cfDNA was extracted using Laifeng Free DNA Extraction Kit (Shanghai Laifeng Biotechnology Co., Ltd., DK607), and the final elution volume was 60uL. All experimental operations were performed according to the kit instructions.

3. Capture sequencing

(1) Next generation sequencing library preparation

The initial amount of gDNA genome-wide library construction was 400ng, and Hieff NGS Fast-Pace DNA Fragmentation Reagent and Hieff NGS Fast-Pace DNA Ligation Module kits (Hieff Biotechnology (Shanghai) Co., Ltd., 12609ES96, 12607ES96) were used to construct the library, and the final library volume was 25uL. The initial volume of cfDNA genome-wide library construction was 50ul, and the library was constructed using Hieff NGS MaxUp II DNA Library Prep Kit for Illumina Universal DNA Library Prep Kit (Hieff Biotechnology (Shanghai) Co., Ltd., 12200ES08), and the final library volume was 25uL. All experimental operations were performed according to the kit instructions.

(2) Probe hybridization capture

The Twist Standard Hybridization and Wash Kit (Twist Bioscience, USA) was used to hybridize and capture the established genome-wide library. The initial hybridization amount of the gDNA library was 200ng, the initial hybridization amount of the cfDNA library was 300ng, and the hybridization time was 16 hours. All experimental operations were performed according to the kit instructions.

(3) Next generation sequencing

The capture library was sequenced by MGISEQ-T7, with 1000Mb of on-machine data for each gDNA library and 4000Mb for each cfDNA library, and the sequencing mode was PE150.

4. The haplotype construction of the couple and the fetus

Capture and sequence the genotype of the SNP site in the family, and define the effective information SNP selection criteria. The cases of the embodiments of the present invention all use the parent who has the same structural variation as the carrier as the reference sample, and the SNP sites that are heterozygous in the chromosome structure rearrangement carrier, homozygous in the carrier spouse, and also homozygous in the reference sample are valid information SNPs. The haplotype linkage analysis of the effective genetic markers in the nuclear family was carried out, and the haplotypes of the structurally abnormal chromosomes and the haplotypes of the normal chromosomes were clarified through the haplotype linkage analysis, and the haplotype model of the core family was constructed to clarify the haplotypes of the parents. Then combined with the parental haplotype, fetal free concentration and sequencing error rate, the fetal genotype and haplotype in the peripheral blood cfDNA of pregnant women were estimated and analyzed according to the hidden Markov statistical model. The principle hypothesis diagram is shown in Figure 2. Table 2 shows the haplotypes of the breakpoint region of fetal structural variation in families 1-6. Fig. 4 is the haplotype diagram of fetal translocation chromosome in case 3,

Table 2. Haplotypes in the breakpoint region of fetal structural variation in families 1-6

family number	Carrier and reference karyotype	Breakpoint-1 domain haplotype	Breakpoint-2 domain haplotype
1	46,XX,t(3;6)(p10;p10)	normal haplotype	normal haplotype
2	46,XY,t(1;12)(q21;p11)	structural variant carrier haplotype	structural variant carrier haplotype
3	46,XX,t(1;20)(q25;p11.2)	normal haplotype	normal haplotype
4	45,XY,rob(13;14)(q10;q10)	normal haplotype	normal haplotype
5	45,XX,rob(13;14)(q10;q10)	normal haplotype	normal haplotype
6	46,XY,inv(7)(p21q21)	normal haplotype	normal haplotype

Example 3: Screening of Fetal Chromosomal Structural Variations

According to the haplotype of the fetus, the karyotype of the fetus is predicted. The prediction principles are:

When the relatives of the carrier who carry the chromosomal rearrangement are used as the reference sample:

a. If there is no recombination in the breakpoint region of fetal chromosomal structure rearrangement

When the haplotype information of the fetal chromosomal structure rearrangement breakpoint region is consistent with the haplotype information of the reference sample, the fetus is diagnosed as a chromosomal structure rearrangement carrier fetus;

b. If homologous recombination occurs in the region of the fetal chromosome breakpoint, the judgment standard is the opposite of a.

Table 3 shows the prediction results of fetal chromosome karyotype in families 1-6.

Table 3. Prediction results of fetal chromosome karyotype in No. 1-6 families

family number	Prediction of fetal karyotype by this technology
1	46,XN
2	46,XN,t(1;12)(q21;p11)
3	46,XN
4	46,XN
5	46,XN
6	46,XN

Remarks: N stands for one sex chromosome

Example 4: Verification of the effectiveness of screening methods for fetal chromosomal structural variation

1. Amniocentesis in the second trimester for cytogenetic analysis and verification

The accuracy of the screening method for detecting fetal chromosomal structural variation of the present invention is verified by comparing with the conventional amniotic fluid karyotype in the second trimester.

Amniotic fluid cell chromosome preparation method (in situ method)

A. Cell culture

1). Transfer the amniotic fluid (about 20ml) to a sterile centrifuge tube and centrifuge at 1000rpm for 10 minutes;

2). Remove the supernatant and use it for other analysis, keep about 0.5-1ml of the cell suspension, and mix it with the culture medium to about 2-2.5ml;

3). Divide the cell suspension into 2 to 4 Chromslide dishes;

4). After culturing for 24/48 hours, add about 2.5ml amniotic fluid culture medium to each Chromslide culture dish;

5). After the 5th to 6th day of culture, observe the growth of the cells and replace with a new medium;

6). After 1-2 days, observe the growth of the cells. If the number of cell clones is sufficient, add colchicine to the culture dish and harvest the cells. The treatment time is determined according to the concentration of the colchicine solution.

B. Chromosome Preparation

1). Tilt the Chromslide cell culture dish to completely remove the culture medium;

2). Add 3-4ml hypotonic solution to each Petri dish and treat at room temperature for 10 minutes;

3). Add 0.5-0.7ml of fixative solution directly to the hypotonic solution, and treat at room temperature for 5 minutes;

4). Remove the supernatant, add 3-4ml of fresh fixative, and treat at room temperature;

5). Repeat the fourth step 1 to 2 times;

6). Remove the fixative, and carry out the chromosome dispersion process in the Maxchrome chromosome dispersion instrument (setting appropriate parameters);

7). After the slide is dried, it is aged and banded.

2. Cytogenetic analysis and verification of neonatal umbilical cord blood

The specific method steps are as the peripheral blood chromosome preparation in Example 1, and will not be described in detail here.

Table 4 shows the karyotype results of the fetal cells of families No. 1-6, and the specific information verified by comparison with the results of the fetal karyotype detected by the technical method of the present invention.

Table 4. Comparison of the karyotype results of fetal cells in families 1-6 with the results of this technique

family number	fetal cell karyotype	The fetal chromosome karyotype predicted by the technology of the present invention	Is it consistent
1	46,XN	46,XN	unanimous
2	46,XN,t(1;12)(q21;p11)	46,XN,t(1;12)(q21;p11)	unanimous
3	46,XN	46,XN	unanimous
4	46,XN	46,XN	unanimous
5	46,XN	46,XN	unanimous
6	46,XN	46,XN	unanimous

Remarks: N stands for one sex chromosome

As can be seen from Table 4, after verification, the prediction results of family haplotypes are completely consistent with the genetic analysis results of fetal amniotic fluid or umbilical cord blood cells, which proves that the sensitivity and specificity of a method for detecting fetal chromosome balance structural variation through free DNA in the peripheral blood of pregnant women according to the present invention are 100%.

Apparently, the above-mentioned embodiments are only examples for clear description, rather than limiting the implementation. For those of ordinary skill in the art, other changes or changes in different forms can be made on the basis of the above description. It is not necessary and impossible to exhaustively list all the implementation manners here. And the obvious changes or changes derived therefrom are still within the scope of protection of the present invention.

Claims (9)

1. A method for constructing a core family genome-wide haplotype model for identifying fetal chromosomal abnormalities, characterized in that it comprises the following steps:

   (1) Sample genotyping: large-scale SNP genotyping of the following objects:

   a. Both spouses of chromosomal rearrangement carriers;

   b. At least one relative of the carrier: including the parents of the carrier, offspring of the carrier and other relatives;

   Among them, the relatives of the carrier can be relatives who have the same chromosomal structural rearrangement as the carrier, or relatives with normal chromosomes;

   When the relatives of the carrier are selected from the carrier's parents or other relatives, one of the carrier's parents carries the same chromosome structure rearrangement as the carrier;

   The above b is referred to as the reference sample;

   (2) Determine the effective information SNPs site:

   a. When one of the carrier's parents or other relatives is used as the reference sample, the SNP sites that are heterozygous in the chromosome structure rearrangement carrier, homozygous in the carrier's spouse, and homozygous in the reference sample are valid information SNPs;

   b. When the offspring of the carrier are used as the reference sample, the SNP sites that are heterozygous in the chromosome structure rearrangement carrier and homozygous in the carrier spouse are valid information SNPs;

   (3) Construct the genome-wide haplotype model of the core family: collect the effective information SNPs identified in step (2) to conduct family haplotype linkage analysis, obtain the genome-wide haplotype of the core family, clarify the haplotype of the structurally rearranged chromosome and the haplotype of the normal chromosome, and determine the genome-wide haplotype of the parent.
2. A system for constructing a core family whole-genome haplotype model for identifying fetal chromosomal abnormalities, the system includes software for processing sample data and hardware for carrying the above-mentioned software, characterized in that,

   (1) The system also includes hardware that stores reference samples and genotyping data of large-scale SNP genotype detection of both carrier couples; the reference sample is at least one carrier relative: including parents of carriers, offspring of carriers and other relatives; wherein, the relatives of the carrier can be relatives with the same chromosome structure rearrangement as the carrier, or relatives with normal chromosomes; when the carrier relatives are selected from the carrier's parents or other relatives, one of the carrier's parents carries the same chromosome structure rearrangement as the carrier;

   (2) The software determines the effective information SNPs site according to the following rules:

   a. When one of the carrier's parents or other relatives is used as the reference sample, the SNP sites that are heterozygous in the chromosome structure rearrangement carrier, homozygous in the carrier's spouse, and homozygous in the reference sample are valid information SNPs;

   b. When the offspring of the carrier are used as the reference sample, the SNP sites that are heterozygous in the chromosome structure rearrangement carrier and homozygous in the carrier spouse are valid information SNPs;

   (3) The software constructs the whole-genome haplotype model of the core family according to the following principles: the reference samples determined in step (2) and the effective information SNPs loci of the carrier couple are collected for family haplotype linkage analysis, the whole-genome haplotype of the core family is obtained, the haplotype of the structurally rearranged chromosome and the haplotype of the normal chromosome are clarified, and the whole-genome haplotype of the parent is determined.
3. A method for identifying fetal chromosomal abnormalities through free DNA in the peripheral blood of pregnant women, characterized in that it comprises the following steps:

   S1: Construction of the genome-wide haplotype model of the core family

   (1) Sample genotyping: large-scale SNP genotyping of the following objects:

   a. Both spouses of chromosomal rearrangement carriers;

   b. At least one relative of the carrier: including the parents of the carrier, offspring of the carrier and other relatives;

   Among them, the relatives of the carrier can be relatives who have the same chromosomal structural rearrangement as the carrier, or relatives with normal chromosomes;

   When the relatives of the carrier are selected from the carrier's parents or other relatives, one of the carrier's parents carries the same chromosome structure rearrangement as the carrier;

   The above b is referred to as the reference sample;

   (2) Determine the effective information SNPs site:

   a. When one of the carrier's parents or other relatives is used as the reference sample, the SNP sites that are heterozygous in the chromosome structure rearrangement carrier, homozygous in the carrier's spouse, and homozygous in the reference sample are valid information SNPs;

   b. When the offspring of the carrier are used as the reference sample, the SNP sites that are heterozygous in the chromosome structure rearrangement carrier and homozygous in the carrier spouse are valid information SNPs;

   (3) Construct the whole-genome haplotype model of the core family: collect the effective information SNPs sites determined in step (2) to carry out family haplotype linkage analysis, obtain the whole-genome haplotype of the core family, clarify the haplotype of the structurally rearranged chromosome and the haplotype of the normal chromosome, and determine the whole-genome haplotype of the parent;

   S2: Predictive Analysis of Fetal Genotypes and Haplotypes

   SNP allelic typing was carried out on the cfDNA of pregnant women's peripheral blood by targeted capture sequencing method, and the fetal genotype and haplotype in cfDNA of pregnant women's peripheral blood were estimated and analyzed according to the hidden Markov statistical model;

   S3: Identification of chromosomal structural abnormalities

   According to the haplotypes of the parents and the fetus, by analyzing the haplotype of the fetal chromosomal structure rearrangement breakpoint region and whether homologous recombination has occurred in this region, the fetal chromosomal structure variation is detected. Relatives as reference samples include carrier parents, carrier offspring and other relatives:

   1) When the relatives of the carrier who carry the chromosomal structural rearrangement are used as the reference sample:

   a. If there is no recombination in the fetal chromosomal structure rearrangement breakpoint area, when the haplotype information of the fetal chromosomal structure rearrangement breakpoint area is consistent with the haplotype information of the reference sample, it is diagnosed as a chromosomal structure rearrangement carrier fetus;

   b. If homologous recombination occurs in the fetal chromosome breakpoint region, the judgment standard is opposite to a;

   2) When the relatives of the carrier who do not carry the chromosomal structural rearrangement are used as the reference sample:

   a. If there is no recombination in the fetal chromosomal structure rearrangement breakpoint area, when the haplotype information of the fetal chromosomal structure rearrangement breakpoint area is consistent with the haplotype information of the reference sample, it is diagnosed as a non-chromosomal structure rearrangement carrier fetus, that is, a fetus with a normal chromatid karyotype; when they are inconsistent, it is diagnosed as a chromosomal structure rearrangement carrier fetus.

   b. If homologous recombination occurs in the fetal chromosome breakpoint region, the judgment standard is opposite to a;

   Also includes S4: Detection of fetal chromosomal aneuploidy

   Using low-depth whole-genome high-throughput sequencing, the cfDNA sequencing reads data are compared with normal controls to detect common aneuploidy and large segment copy number variation of chromosomes, and determine whether there is aneuploidy in cffDNA in pregnant women's plasma, as well as large segmental deletions and duplications related to chromosomal structural variation.
4. A system for identifying fetal chromosomal abnormalities through free DNA in the peripheral blood of pregnant women, the system includes software for processing sample data and hardware for carrying the above software, characterized in that,

   S1: the system also includes hardware for storing reference samples and genotyping data of large-scale SNP genotyping of both carrier couples; the reference sample is at least one carrier relative: including carrier parents, carrier offspring and other relatives; wherein, the carrier relatives can be relatives with the same chromosome structure rearrangement as the carrier, or relatives with normal chromosomes; when the carrier relatives are selected from the carrier's parents or other relatives, one of the carrier's parents carries the same chromosome structure rearrangement as the carrier;

   S2: The software constructs the whole genome haplotype model of the core family according to the following rules:

   (1) Determine effective information SNPs:

   a. When one of the carrier's parents or other relatives is used as the reference sample, the SNP sites that are heterozygous in the chromosome structure rearrangement carrier, homozygous in the carrier's spouse, and homozygous in the reference sample are valid information SNPs;

   b. When the offspring of the carrier are used as the reference sample, the SNP sites that are heterozygous in the chromosome structure rearrangement carrier and homozygous in the carrier spouse are valid information SNPs;

   (2) Collect the effective information SNPs loci determined in step (1) to carry out family haplotype linkage analysis, obtain the whole genome haplotype of the core family, clarify the haplotype of structurally rearranged chromosomes and the haplotype of normal chromosomes, and determine the whole genome haplotype of the parents;

   S3: The software predicts and analyzes the fetal genotype and haplotype according to the following rules

   SNP allelic typing was carried out on the cfDNA of pregnant women's peripheral blood by targeted capture sequencing method, and the fetal genotype and haplotype in cfDNA of pregnant women's peripheral blood were estimated and analyzed according to the hidden Markov statistical model;

   S4: The software identifies abnormalities in chromosome structure according to the following rules

   According to the haplotypes of the parents and the fetus, by analyzing the haplotype of the fetal chromosomal structure rearrangement breakpoint region and whether homologous recombination has occurred in this region, the fetal chromosomal structure variation is detected. Relatives as reference samples include carrier parents, carrier offspring and other relatives:

   1) When the relatives of the carrier who carry the chromosomal structural rearrangement are used as the reference sample:

   a. If there is no recombination in the fetal chromosomal structure rearrangement breakpoint area, when the haplotype information of the fetal chromosomal structure rearrangement breakpoint area is consistent with the haplotype information of the reference sample, it is diagnosed as a chromosomal structure rearrangement carrier fetus;

   b. If homologous recombination occurs in the fetal chromosome breakpoint region, the judgment standard is opposite to a;

   2) When the relatives of the carrier who do not carry the chromosomal structural rearrangement are used as the reference sample:

   a. If there is no recombination in the fetal chromosomal structure rearrangement breakpoint area, when the haplotype information of the fetal chromosomal structure rearrangement breakpoint area is consistent with the haplotype information of the reference sample, it is diagnosed as a non-chromosomal structure rearrangement carrier fetus, that is, a fetus with a normal chromatid karyotype; when they are inconsistent, it is diagnosed as a chromosomal structure rearrangement carrier fetus.

   b. If homologous recombination occurs in the fetal chromosome breakpoint region, the judgment standard is opposite to a;

   S4: The software detects fetal chromosomal aneuploidy according to the following rules

   Using low-depth whole-genome high-throughput sequencing, the cfDNA sequencing reads data are compared with normal controls to detect common aneuploidy and large segment copy number variation of chromosomes, and determine whether there is aneuploidy in cffDNA in pregnant women's plasma, as well as large segmental deletions and duplications related to chromosomal structural variation.
5. The method or system according to any one of claims 1-4, wherein the structural abnormality of the chromosome is a balanced structural variation of the chromosome.
6. The method or system according to claim 5, wherein the balanced structural variation of chromosomes includes balanced translocations and inversions of chromosomes.
7. The method or system according to any one of claims 1-4, characterized in that the SNP genotype detection of the carrier couple and the carrier relatives is performed on the peripheral blood gDNA capture sequencing of the carrier couple and the carrier relatives.
8. The method or system according to claim 3 or 4, wherein the design principle of the peripheral blood cfDNA capture probe is as follows: query the population genome database, select the minimum allele frequency according to the frequency of SNP sites, especially in East Asian populations, between 0.3-0.7, and the interval between adjacent sites is 300-500Kb, evenly distributed in the genome, and verified by haploview, the linkage disequilibrium R2 between the sites must be greater than 0.8.
9. The method or system according to claim 3 or 4, characterized in that no less than 2 effective SNP sites are required when analyzing the haplotype of the region at the breakpoint of fetal chromosomal structure rearrangement and whether homologous recombination occurs in the region.

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