CN114480667A

CN114480667A - Method for detecting fetal chromosome balance structural variation through pregnant woman peripheral blood free DNA

Info

Publication number: CN114480667A
Application number: CN202210073440.4A
Authority: CN
Inventors: 张硕; 徐丛剑
Original assignee: Obstetrics and Gynecology Hospital of Fudan University
Current assignee: Obstetrics and Gynecology Hospital of Fudan University
Priority date: 2022-01-21
Filing date: 2022-01-21
Publication date: 2022-05-13
Also published as: WO2023138131A1

Abstract

The invention discloses a method for detecting fetal chromosome balance structural variation through pregnant woman peripheral blood free DNA, which comprises the steps of constructing a core family haplotype model and detecting the fetal structural variation. By collecting the genealogy of the couples carrying the structural rearrangement variation of the chromosome, carrying out SNP genotyping on the genealogy, determining the haplotypes of the structural rearrangement chromosome and the structural normal chromosome, and constructing a whole genome haplotype model. Capturing and sequencing free DNA of peripheral blood of the pregnant woman, calculating and analyzing the genotype and the haplotype of the fetus in the free DNA according to a hidden Markov model, and predicting the structural rearrangement of the chromosome of the embryo by analyzing whether the fetus carries the haplotype near the structural rearrangement chromosome breaking point region. The invention provides the detection of fetal balance chromosome structural abnormality through the peripheral blood cfDNA of the pregnant woman for the first time, and provides important guidance for preventing and diagnosing the chromosome disease.

Description

Method for detecting fetal chromosome balance structural variation through pregnant woman peripheral blood free DNA

Technical Field

The invention belongs to the field of genetic diagnosis of chromosome variation, and particularly relates to a method for detecting fetal chromosome balance structural variation through pregnant woman peripheral blood free DNA.

Background

Genetic diseases are important causes of birth defects and fertility disorders, and pose serious threats to human health and life. Clinically common genetic diseases include monogenic and chromosomal disorders, including chromosomal numerical and structural abnormalities. For the prevention of birth in genetically-ill infants, invasive prenatal diagnosis remains 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 of infants can be evaluated based on cell-free total DNA (cffDNA) in the peripheral blood plasma of pregnant women. The prior NIPT technology has a very high detection rate for common aneuploidy, has already matured for monogenic diseases, and is gradually applied to clinic. But still has the defects that the prior NIPT technology can not detect the structural variation of the balanced chromosome, and no relevant research reports are found at home and abroad. For carriers with chromosome structural abnormality, only chromosome aneuploidy or deletion/duplication of large fragments can be detected through NIPT, and for detecting whether fetuses carry balanced chromosome structural abnormality, diagnosis can be carried out only through invasive prenatal diagnosis amniotic fluid puncture at present. Because invasive prenatal diagnosis has certain abortion and infection risks, many pregnant women refuse to accept invasive examination, and some pregnant women have invasive prenatal diagnosis contraindications, such as preposed placenta, oligohydramnios, infectious diseases and the like. Therefore, there is a clinical need to develop an NIPT technique suitable for patients with chromosomal abnormality, which can detect chromosomal aneuploidy and balanced chromosomal abnormality of the fetus at the same time, and reduce the risk of fetal abortion or infection caused by invasive prenatal diagnosis. The technology can be suitable for structural distortion of different breakpoint positions, independent design for different chromosome structural distortion is not needed, universality is achieved, guidance is provided for noninvasive prenatal detection of chromosome structural abnormality carriers, and great clinical significance is achieved.

The chromosome structural abnormality is also called chromosome rearrangement, and refers to chromosome aberration generated by chromosome or chromosome monomer through a breaking-rearrangement or interchange mechanism, mainly including chromosome translocation, inversion, deletion/duplication and the like, and the most common chromosome balance translocation in the clinical field of obstetrics and gynecology is an important reason for poor pregnancy fatality such as infertility, recurrent abortion, fetal developmental malformation and congenital birth defects. The chromosome balance translocation refers to the structural change of chromosomes caused by the simultaneous breakage of two chromosomes and the missplicing exchange, including reciprocal translocation and Robertsonian translocation. The total incidence rate in the population is about 0.27%, the incidence rate in the patients with infertility is about 1.1%, and the incidence rate in the patients with recurrent spontaneous abortion is higher and can be as high as 4.08%.

The carriers of chromosome balance translocation usually have no obvious clinical symptoms before the birth age, and usually show repeated abortion or infertility at the birth age, and have low fertility. The reason is that the germ cells of the carriers can generate a large amount of unbalanced chromosome abnormal gametes, embryos formed after the unbalanced gametes are fertilized have implantation failure, spontaneous abortion, birth defects of newborns and the like due to the chromosome abnormality, in addition, the risk of premature ovarian failure of women is increased, and males usually show oligoasthenospermia. Therefore, the balanced translocation of the chromosome seriously affects the fertility health of human beings and is an important cause of birth defects of newborns.

For carriers with chromosomal aberrations, pregnancy is generally required to have invasive prenatal diagnosis of the fetus, including chorionic puncture during the early pregnancy, amniotic puncture during the middle pregnancy, and umbilical cord blood puncture during the late pregnancy. The chromosome karyotype analysis is carried out on the fetus through prenatal diagnosis genetics examination, including the traditional karyotype and molecular karyotype, the traditional karyotype has lower resolution, generally, the diagnosis can be carried out only by fragments of more than 5Mb or 10Mb, compared with the molecular karyotype, the accuracy of the molecular karyotype is higher, the latent unbalanced translocation of small fragments can be diagnosed, the resolution is about 100kb, the currently commonly used molecular karyotype detection technology comprises a main microarray-comparative genome hybridization technology, a microarray single nucleotide polymorphism, a next generation sequencing technology and the like, but the technologies can not detect balanced structural distortion.

In 1997, Dennis Lo et al detected specific SRY gene on Y chromosome in the peripheral blood of pregnant woman carrying male fetus by fluorescent quantitative PCR technology, and confirmed that there is a small amount of fetus-derived free DNA fragment in the peripheral blood of mother for the first time, opening the age of using the peripheral blood of pregnant woman for NIPT detection. The fetal-derived DNA fragments are considered to be derived from placenta trophoblast cells or naturally-exfoliated fetal cells, can be detected in the peripheral blood of the pregnant woman at 7 weeks of pregnancy, and have the concentration increased along with the increase of the gestational week, short half life and no longer detectable after 2 hours of delivery of the pregnant woman. The length of the maternal-derived DNA fragments in the plasma is concentrated to about 166bp, the fetal-derived DNA fragments are more concentrated to 143bp, and the maternal-derived and fetal-derived DNA fragments tend to be broken at different chromosome position coordinates, so that the free DNA derived from the fetus in the peripheral blood of the pregnant woman can be distinguished from the free DNA derived from the mother according to the characteristics.

At present, the NIPT technology is widely applied to the detection of chromosome aneuploidy and a few microdeletion and microdropism syndromes, and has very high sensitivity and specificity, researches show that compared with a serological screening method, the NIPT technology has greatly improved detection rate and screening efficiency, the detection rate of 21-trisomy syndrome is more than 98-99%, the detection rate of 18-trisomy and 13-trisomy syndromes is more than 95%, and good evaluation is obtained in the aspect of preventing and controlling birth defects of newborns. Since then, owing to the development of the next-generation sequencing technology, NIPT against partial monogenetic genetic diseases became a hot point of research. A common technique is to directly detect mutant alleles of pathogenic sites in cffDNA in the peripheral blood of pregnant women using a quantitative method. The other common method is to utilize SNP linkage analysis, namely a haplotype analysis method to carry out detection, and convert the direct detection of a mutation site into indirect analysis of a haplotype of the site to improve the detection sensitivity by constructing the haplotype of the pregnant woman and the mate thereof. Unfortunately, no NIPT technique for detection of chromosomal balance structural aberrations has emerged to date.

In prenatal diagnosis of genetic diseases, it is of great significance to find a noninvasive prenatal diagnosis technology aiming at chromosome balance structural aberration detection with low cost, low risk, high sensitivity and high accuracy.

Disclosure of Invention

Aiming at the blank existing in the prior art, the invention establishes a new NIPT technology based on the analysis of target capture combined with Bayesian HMM. Firstly, collecting a patient core family, carrying out whole genome targeted capture Single Nucleotide Polymorphism (SNP) allele typing, defining effective information sites to construct a core family haplotype model, and determining normal and structural variation chromosome haplotypes in the family; meanwhile, the method is used for carrying out targeted capture sequencing on free DNA (cfDNA) in peripheral blood of the pregnant woman, conjecturing a fetal haplotype through HMM analysis, determining whether the fetal haplotype carries chromosome structure variation or not through the fetal haplotype, and analyzing fetal chromosome aneuploidy. The invention can not only detect the aneuploidy of the fetus, but also detect the structural variation of the balanced chromosome, and the technology has universality to different structural variations of the chromosome.

The specific technical scheme of the invention is as follows:

the invention provides a method for constructing a core family genome-wide haplotype model for identifying structural abnormality of a chromosome of a fetus in a first aspect, which comprises the following steps:

(1) genotyping of the sample: the following subjects were subjected to large-scale SNP genotype detection:

a. both couples having carriers of chromosomal rearrangement;

b. at least one carrier relative: including carrier parents, carrier offspring and other relatives;

wherein, the carrier relatives can be relatives with the same chromosome structural rearrangement as the carrier, and can also be relatives with normal chromosomes;

when the carrier relative is selected from the carrier parent or other relative, the carrier parent carries the same chromosome structure rearrangement as the carrier;

b is referred to as a reference sample;

(2) determining the effective information SNPs:

a. when a carrier parent or other relatives are used as a reference sample, the carrier is heterozygous in the chromosome structure rearrangement carrier, homozygous in the carrier mate, and SNP sites which are also homozygous in the reference sample are effective information SNPs sites;

b. when carrier filial generation is taken as a reference sample, the carrier is heterozygous in the chromosome structure rearrangement carrier, and SNP loci which are homozygous in the carrier partner are effective information SNPs loci;

(3) constructing a core family whole genome haplotype model: and (3) performing family haplotype linkage analysis on the SNPs sites with the effective information determined in the step (2) to obtain a core family whole genome haplotype, determining the haplotype of structurally rearranged chromosomes and the haplotype of normal chromosomes, and determining the whole genome haplotype of parents.

In a second aspect, the invention provides a construction system for identifying a core family genome-wide haplotype model of a fetal chromosomal structural abnormality, the system comprising software for processing sample data and hardware for carrying the software,

(1) the system further comprises hardware storing genotyping data for large scale SNP genotyping of both the reference sample and the carrier couple; the reference sample is at least one carrier relative: including ranking carrier parents, carrier offspring and other relatives; wherein, the carrier relatives can be relatives with the same chromosome structural rearrangement as the carrier, and can also be relatives with normal chromosomes; when the carrier relative is selected from the carrier parent or other relative, the carrier parent carries the same chromosome structure rearrangement as the carrier;

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

(3) the software constructed a core pedigree genome-wide haplotype model according to the following principles: and (3) performing family haplotype linkage analysis on the SNPs sites of the effective information of the reference sample determined in the step (2) and the effective information of the carrier couple to obtain a core family whole genome haplotype, determining the haplotype of structurally rearranged chromosomes and the haplotype of normal chromosomes, and determining the whole genome haplotype of the parent.

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

s1: construction of core family genome-wide haplotype model

a. both couples having carriers of chromosomal rearrangement;

b is referred to as a reference sample;

(2) determining the effective information SNPs:

(3) constructing a core family whole genome haplotype model: performing family haplotype linkage analysis on the SNPs sites of the effective information determined in the step (2) to obtain a core family whole genome haplotype, determining the haplotype of structurally rearranged chromosomes and the haplotype of normal chromosomes, and determining the whole genome haplotype of parents;

s2: predictive analysis of fetal genotypes and haplotypes

Carrying out SNP allele typing on the pregnant woman peripheral blood cfDNA by a target capture sequencing method, and carrying out calculation analysis on the fetal genotype and the haplotype in the pregnant woman peripheral blood cfDNA according to a hidden Markov statistical model;

s3: identification of chromosomal structural abnormalities

Detecting the structural variation of the fetus chromosome by analyzing the haplotype of the rearrangement breakpoint region of the chromosome structure of the fetus and whether homologous recombination occurs in the region according to the haplotype of the parent and the fetus, wherein the relatives serving as reference samples comprise parents of carriers, offspring of carriers and other relatives:

1) when referring to the relatives of the carrier carrying the rearrangement of the chromosome structure:

a. if the fetal chromosome structure rearrangement breakpoint region is not recombined, when the haplotype information of the fetal chromosome structure rearrangement breakpoint region is consistent with the haplotype information of the reference sample, diagnosing that the fetal chromosome structure rearrangement carries the fetus; when the two are inconsistent, the non-chromosome structural rearrangement is diagnosed as carrying the fetus, namely, the chromosome karyotype normal fetus is stained;

b. if the fetal chromosome breakpoint region is subjected to homologous recombination, the judgment standard is opposite to a;

2) when the relatives of the carriers not carrying the chromosomal structural rearrangement are taken as the reference samples:

a. if the fetal chromosome structure rearrangement breakpoint region is not recombined, when the haplotype information of the fetal chromosome structure rearrangement breakpoint region is consistent with the haplotype information of the reference sample, diagnosing that the non-chromosome structure rearrangement carries a fetus, namely, dyeing a normal fetus with a haplotype karyotype; when there is no match, the chromosomal rearrangement is diagnosed as carrying a fetus.

further comprising S4: fetal chromosomal aneuploidy detection

And (3) comparing cfDNA sequencing reads data with normal control by utilizing low-depth whole genome high-throughput sequencing to detect common aneuploidy and large fragment copy number variation of the chromosome, and judging whether the cffDNA in the plasma of the pregnant woman has aneuploidy and large fragment deletion and repetition related to chromosome structure variation.

In a fourth aspect, the invention provides a system for identifying fetal chromosomal structural abnormalities in pregnant woman's peripheral blood free DNA, the system comprising software for processing sample data and hardware for carrying the software,

s1: the system also comprises hardware for storing genotyping data of the large-scale SNP genotype detection of the reference sample and the peripheral blood of the pregnant woman to be detected; 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 structural rearrangement as the carrier, and can also be relatives with normal chromosomes; when the carrier relative is selected from the carrier parent or other relative, the carrier parent carries the same chromosome structure rearrangement as the carrier;

s2: the software constructs a core family genome-wide haplotype model according to the following rules:

(1) determining the effective information SNPs:

(2) performing family haplotype linkage analysis on the SNPs sites of the effective information determined in the step (1) to obtain a core family whole genome haplotype, determining the haplotype of structurally rearranged chromosomes and the haplotype of normal chromosomes, and determining the whole genome haplotype of parents;

s3: the software predictively analyzes fetal genotype and haplotype according to the following rules

s4: the software identifies chromosomal structural abnormalities according to the following rules

a. if the fetal chromosome structure rearrangement breakpoint region is not recombined, when the haplotype information of the fetal chromosome structure rearrangement breakpoint region is consistent with the haplotype information of the reference sample, diagnosing that the fetal chromosome structure rearrangement carries the fetus; when the two are inconsistent, the non-chromosome structural rearrangement is diagnosed as carrying a fetus, namely, a normal fetus with a chromatoplasmosis;

a. if the fetal chromosome structure rearrangement breakpoint region is not recombined, when the haplotype information of the fetal chromosome structure rearrangement breakpoint region is consistent with the haplotype information of the reference sample, diagnosing that the non-chromosome structure rearrangement carries a fetus, namely, dyeing a normal fetus with a haplotype karyotype; when there is disagreement, it is diagnosed that the chromosomal rearrangement carries a fetus.

s4: the software detects fetal chromosomal aneuploidies according to the following rules

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

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

In the above technical scheme of the present invention, the design principle of the peripheral blood cfDNA capture probe is as follows: querying a population genome database, selecting the minimum allele frequency between 0.3 and 0.7 according to the SNP locus frequency, particularly the frequency in east Asia population, uniformly distributing the minimum allele frequency in the genome with the interval between adjacent loci of 300-500Kb, and verifying by haploview that the linkage disequilibrium R2 between loci is more than 0.8.

In the technical scheme of the invention, when analyzing the haplotype of the rearrangement breakpoint region of the fetal chromosome structure and whether the region has homologous recombination, not less than 2 effective SNP sites are required.

The invention has the beneficial effects that:

(1) the invention provides the detection of fetal chromosome structural abnormality through the peripheral blood cfDNA of the pregnant woman for the first time, provides important guidance for prevention and diagnosis of chromosome diseases and fills the blank of international technology.

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

(3) The invention is a comprehensive NIPT general technical platform, can complete the detection of fetal chromosome aneuploidy and structural abnormality through one-time detection, and solves the problem that the existing NIPT technology can not detect balanced chromosome structural variation. The SNP genetic marker locus designed by the invention has high flux characteristic and is suitable for all types of chromosome structure rearrangement carriers.

Drawings

FIG. 1 is a diagram of noninvasive fetal chromosome structural aberration detection hypothesis established by maternal plasma free DNA of a core family, HMM, hidden Markov model;

FIG. 2 is a technical scheme of the present invention;

FIG. 3 is a karyotype chart of case 3 carriers;

FIG. 4 is a haplotype map of fetal translocation chromosomes in case 3.

Detailed Description

The invention provides a new NIPT technology aiming at chromosome structure aberration detection by proposing a hypothesis that a core family genetic haplotype can represent a karyotype and further carry out chromosome structure aberration detection on a fetus. The technology mainly comprises two parts: constructing a core family haplotype model and detecting the structural variation of the fetus. In the first part, a complete genome haplotype model is constructed by collecting a pedigree carrying couples through chromosome structure rearrangement variation, wherein the pedigree comprises carrier couples and carrier relatives (the carrier relatives preferentially consider carrier parents and carrier offspring and can also be other relatives), carrying out SNP genotyping on a core pedigree sample by adopting a targeted capture sequencing method, and determining haplotypes of a structure rearrangement chromosome and a structure normal chromosome by combining a bioinformatics analysis method. Since the model includes a genome-wide haplotype, haplotypes for any chromosomal location are theoretically available, with versatility for different structural variations. In the second section, SNP allelic typing is also performed on maternal peripheral blood free DNA by a targeted capture sequencing method, fetal genotype and haplotype in free DNA are computationally analyzed according to a Hidden Markov Model (HMM), and fetal chromosomal structural rearrangement is detected by analyzing whether the fetus carries a haplotype near the point of chromosomal disruption of the structural rearrangement. In addition, the chromosome aneuploidy of the fetus is detected simultaneously through sequencing data, and the purpose of simultaneously detecting the chromosome number and structural abnormality by one-time detection is realized.

The hypothesis of noninvasive detection of fetal chromosomal structural aberration established based on core families and maternal peripheral blood free DNA is shown in fig. 1. The schematic diagram shows that the haplotypes of two couples of the couple are determined by combining target capture sequencing and bioinformatics analysis to build the haplotypes of a core family, the haplotypes of a fetus are calculated according to HMM model analysis after the peripheral blood free DNA of a pregnant woman is deeply sequenced, and the karyotype of the fetus is deduced according to the chromosome haplotypes of the fetus, so that the noninvasive detection of the balanced chromosome structural variation of the fetus is realized. Hap0 and Hap1 indicate that the fetus inherits the same and different haplotype at this location as the proband, respectively. Technical route as shown in fig. 2, the carrier parent and carrier child are prioritized with reference to the sample. When the reference sample is selected from the carrier's parent or other relatives, the carrier is required to carry the same chromosomal rearrangements as the carrier on the parent side. The specific scheme is as follows:

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

The families of patients meeting the conditions are collected, and the husband and the wife are required to be carriers of the chromosome structure rearrangement. Meanwhile, peripheral blood of parents of the carrier is extracted, and the source of the chromosome structure rearrangement of the case is determined according to the detection result of the parents, namely the case is determined to be inherited from any one of the parents. Collecting peripheral blood samples of parents of couples and carriers, constructing basic family information and laying a foundation for constructing an analysis model. Extracting free DNA of the peripheral blood of the core family and the peripheral blood of the pregnant woman, and performing targeted capture sequencing after library construction.

The principle of the design of the pregnant woman peripheral blood cfDNA target capture probe is as follows: querying a population genome database, selecting the minimum allele frequency between 0.3 and 0.7 according to the SNP locus frequency, particularly the frequency in east Asia population, uniformly distributing the minimum allele frequency in the genome with the interval between adjacent loci of about 300-500Kb, and verifying by haploview that the linkage disequilibrium R2 between loci is more than 0.8.

(2) Constructing a family genome-wide haplotype model to determine a parent genome-wide haplotype

Carrying out whole genome SNP typing on a family sample, defining an effective information SNP selection standard, carrying out haplotype linkage analysis on an effective information SNP genetic marker, constructing a family whole genome haplotype model, determining the haplotype of structurally rearranged chromosome and the haplotype of structurally normal chromosome through the haplotype model, and determining the whole genome haplotype of a parent.

The effective information SNP selection criteria are as follows: a. when a carrier parent side or other relatives are used as a reference sample, the effective information SNP requirement simultaneously meets the requirement that the carrier is heterozygous and the carrier is homozygous in a carrier spouse and the reference sample; b. when the carrier offspring is used as a reference sample, the effective information SNP requirement simultaneously meets the requirements of being heterozygous in a carrier and homozygous in a carrier mate.

(3) Pregnant woman peripheral blood free DNA detection fetus whole genome haplotype

SNP allelic typing is carried out on the peripheral blood cfDNA of the pregnant woman through a target capture sequencing method, and prediction analysis is carried out on the fetal genotype and the haplotype in the cfDNA according to a binomial distribution Model and a Hidden Markov statistical Model (HMM) based on Bayesian analysis by combining a parental haplotype, fetal free concentration and a sequencing error rate. The concordance of fetal haplotypes constructed under different reference samples was investigated using as references relatives with the same chromosomal structural rearrangement as the carrier and relatives with normal chromosomes, respectively.

(4) Fetal chromosomal structural rearrangement detection

Association between chromosomal breakpoint region haplotype and whole chromosome haplotype: homologous recombination occurs between homologous chromosomes during meiosis, interfering with haplotype outcomes. The relation between the haplotype at the position of the rearrangement breakpoint of the chromosome structure and the haplotype of the whole chromosome is mainly analyzed, and the homologous recombination condition at the position of the breakpoint is analyzed.

Identity of haplotypes at the two breakpoint positions of a chromosomal structural rearrangement: theoretically, the haplotypes for both breakpoint locations should be both structurally rearranged haplotypes or both normal chromosomal haplotypes.

According to the haplotype of the parent and the fetus, the structural variation of the chromosome of the fetus is detected by analyzing the haplotype of the rearrangement breakpoint region of the chromosome structure of the fetus and whether the homologous recombination occurs in the region.

1) When referring to the carrier's relatives carrying a rearrangement of the chromosomal structure:

a. if the fetal chromosomal structural rearrangement breakpoint region is not recombined, when the haplotype information of the fetal chromosomal structural rearrangement breakpoint region is consistent with the haplotype information of the reference sample, the fetal chromosomal structural rearrangement is diagnosed to carry the fetus. When they are not consistent, it is diagnosed that the non-chromosomal structural rearrangement carries a fetus, i.e., a normal fetus with a chromatoplastic karyotype.

b. If homologous recombination occurs in the fetal chromosomal breakpoint region, the criterion is reversed from a.

2) When referring to the non-chromosomal structural rearrangement-bearing relatives of the carrier:

a. if the fetal chromosome structure rearrangement breakpoint region is not recombined, when the haplotype information of the fetal chromosome structure rearrangement breakpoint region is consistent with the haplotype information of the reference sample, the non-chromosomal structure rearrangement carrying fetus is diagnosed, namely the chromosome karyotype normal fetus is stained. When there is no match, the chromosomal rearrangement is diagnosed as carrying a fetus.

And diagnosing the fetus after the analysis is carried out through the detection analysis model. Diagnosing that the chromosome structure is normal for the fetus which does not carry the chromosome structure rearrangement haplotype; for a fetus carrying a chromosomal structural rearrangement haplotype, a chromosomal structural rearrangement is diagnosed; for aneuploid or chromosome structure imbalance fetuses, the chromosome abnormality fetuses are directly diagnosed.

(5) Fetal chromosomal aneuploidy detection

(6) Follow-up prenatal diagnosis result and pregnancy outcome of fetus

And (3) according to the chromosome structure rearrangement and aneuploidy detection results of the fetus, follow-up visiting the chromosome karyotype result of the amniotic fluid cell of the fetus in the middle of gestation, comparing the result with the detection analysis model result, and analyzing the effectiveness of the technical method. Follow-up the fetus to birth and record the birth condition.

Based on the model detection system, the invention preliminarily incorporates 6 cases families meeting the requirements in the prior period, and successfully detects the chromosome number and structure results of the fetus. The prenatal genetic diagnosis of the fetus is carried out through amniotic fluid puncture, and the result shows that the diagnosis result of amniotic fluid cells or umbilical cord blood of the fetus is completely consistent with the detection result of the early-stage NIPT detection technology of the invention on the embryo, and at present, 5 fetuses are born and all grow normally.

In the above technical solution of the present invention, attention is paid to the selection of the size of the haplotype analysis region: when analyzing the breakpoint region, it is generally required that not less than 2 effective SNP sites are provided for sufficient genetic markers for linkage analysis of the structurally rearranged breakpoint region; when analyzing the whole chromosome haplotype, attention is paid to identifying the homologous recombination condition in the germ cell reduction classification process, and misdiagnosis caused by homologous recombination is avoided. Meanwhile, the uniformity of the distribution of the genetic markers is noticed, and the effective genetic markers exist in the analyzed region.

The present invention will be described in detail with reference to specific examples. Unless otherwise specified, the technical means used in the present invention are conventional technical means that can be grasped by those skilled in the art. The examples are not intended as specific limitations on the practice of the invention.

Example 1: collection of reference samples from couples and couples who are carriers of chromosomal structural variation

Couples carried by 6 chromosome structural variations, including chromosome balance translocation and chromosome inversion, were recruited, and were obtained from the college reproductive center of the subsidiary obstetrics and gynecology hospital of the university of double denier, and the study protocol was approved by the ethical committee of human subjects in the obstetrics and gynecology hospital of the university of double denier.

All couples have a history of recurrent spontaneous abortion or chromosomal abnormality, and couples carried with chromosomal balance translocation or inversion are hereinafter referred to as "patients" for one party and "patient couples" for the other party. At the same time of recruitment, about 10ml of peripheral blood of each pair of patient couples and patient relatives (the priority of the patient parents and children, and other relatives can be considered) is extracted. Using a part of peripheral blood for lymphocyte culture and carrying out karyotype analysis; another portion of the peripheral blood is subjected to DNA extraction in a manner conventional in the art for subsequent sequencing.

The preparation method of the peripheral blood chromosome comprises the following steps:

1. cell culture

1) Blood sampling: disinfecting skin with alcohol, collecting blood from elbow vein, making injection needle directly pass through rubber stopper of culture flask, injecting 30-40 drops of whole blood into 10ml of culture medium, shaking, and culturing in 37 deg.C incubator.

2) Culturing: the time period required was 68 hours. During the culture period, the cells are shaken up periodically to make the cells fully contact with the culture medium.

3) Colchicine treatment: 2-4 hours before terminating the culture, colchicine was added to the culture (2 drops were added dropwise with 1ml syringe No. 5 needle tip to a final concentration of 0.07. mu.g/ml).

The above steps all need aseptic operation.

2. Chromosome preparation

1) Collecting cells: the whole culture was transferred to a clean centrifuge tube, centrifuged at 1000rpm for 8-10 minutes, and the supernatant was discarded.

2) Hypotonic treatment: adding 8ml of low-permeability liquid with the pre-temperature of 37 ℃ into a graduated centrifuge tube, uniformly mixing by using a dropper, and performing low-permeability for 15-25 minutes in a constant-temperature water bath with the temperature of 37 ℃.

3) Pre-fixing: after hypotonic, 0.5ml of stationary liquid is added, mixed gently and centrifuged at 1000rpm for 8-10 minutes.

4) A fixing: the supernatant was discarded, 5ml of the fixative was added, gently mixed, and allowed to stand for 20 minutes. Centrifuge at 1000rpm and discard the supernatant.

5) Fixing II and fixing III: the same is fixed.

6) Preparing a suspension: after the supernatant is discarded, a proper amount of stationary liquid is added according to the number of cells to prepare cell suspension.

7) Dropping sheet: the cell suspension is sucked from the height of 10-20cm and dropped on a dry and clean glass slide, and the glass slide is lightly blown to disperse and is air-dried.

8) Dyeing: dyeing for 5-10 minutes by 1:10Giemsa, washing off excessive dye liquor by fine water, and air-drying.

9) Microscopic examination: and (5) searching for a split phase with good dispersion and moderate dyeing under a low power microscope, and observing the form of the chromosome under an oil microscope and counting.

If the patient's peripheral blood cell karyotype is the same as their mother, the patient's balanced translocation is inherited from the maternal side; as with the father, the patient's balanced translocation is inherited from the father. When parents of a patient cannot take blood (such as vanish) or do not agree to take blood, brothers and sisters or other relatives of the patient can also take peripheral blood to perform karyotype analysis and can also be used as a reference sample in constructing family haplotypes.

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

TABLE 1.1-6 family karyotype

Family serial number	Carrier karyotype of chromosome	Carrier paternal karyotype	Carrier mother chromosome 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 monomer type of both sexes and fetus

1. Peripheral blood genome DNA extraction

Early preparation: 1.5ml of EP tube, water bath, centrifuge, oscillator and other equipment consumables are marked, and the protease provided by the kit, AW1 and AW2 are added with corresponding volume of absolute ethyl alcohol before use. If precipitation of Bufferal occurs, it can be dissolved by warming at 56 ℃ and gently shaking. Before the first use of BufferAW1 and BufferAW2, absolute ethyl alcohol with corresponding volume is added according to the label on a reagent bottle to form a working solution. The water bath was opened and set at 56 ℃.

The experimental procedure was as follows:

(1) 20 μ l of proteinase K, 200 μ l of anticoagulated blood and 200 μ l of buffer AL are added in sequence, vortexed and mixed, and incubated at 56 ℃ for 10 minutes.

(2) Add 200. mu.l of absolute ethanol and mix by vortexing to form a homogeneous solution.

(3) The mixed solution was transferred to DNeasy Mini spin column and centrifuged at 6000x g (8000rpm) for 1 min. The collection tube is replaced.

(4) DNeasy Mini spin column was transferred to a new collection tube and centrifuged for 1 min by adding 500. mu.l Buffer AW1,6000 x g (8000 rpm). The collection tube is replaced.

(5) DNeasy Mini spin column was transferred to a new collection tube and centrifuged for 3 minutes by adding 500. mu.l Buffer AW2, 20,000x g (14,000rpm) followed by 1 minute. The collection tube is replaced. This step requires attention to the complete drying of the membrane surface of the DNeasy Mini spin column. If ethanol still remains, the ethanol will interfere with the next experimental reaction. If the DNeasy Mini spin column has contact with the liquid surface in the header during replacement of the header, the header is aspirated and centrifuged at 20,000x g (14,000rpm) for one minute.

(6) The DNeasy Mini spin column was transferred to a clean 1.5ml or 2ml centrifuge tube and 30-100 AE added. Dissolve for 3 minutes at room temperature and centrifuge at 6000Xg (8000rpm) for 1 minute.

2. Pregnant woman peripheral blood free DNA extraction

The initial volume of the pregnant woman plasma sample was 1.8mL, cfDNA was extracted using a leuer free DNA extraction kit (shanghai leuer biotech ltd, DK607), and the final elution volume was 60 uL. All experimental procedures were performed according to kit instructions.

3. Capture sequencing

(1) Second Generation sequencing library preparation

The gDNA whole genome library was prepared in an initial amount of 400ng, using the Hieff NGS Fast-Pace DNA Fragmentation Reagent and the Hieff NGS Fast-Pace DNA Ligation Module kit (assist Saint Biotechnology (Shanghai) GmbH, 12609ES96, 12607ES96), and a final library volume of 25 uL. The cfDNA whole genome Library was initially pooled at 50uL, pooled using the assist holy Hieff NGS MaxUp II DNA Library Prep Kit for Illumina all-purpose DNA pooling Kit (assist holy Biotechnology (Shanghai) Co., Ltd., 12200ES08), and the final Library volume was 25 uL. All experimental procedures were performed according to kit instructions.

(2) Probe hybridization capture

The constructed whole genome library was subjected to Hybridization capture using Twist Standard Hybridization and Wash Kit (Twist Bioscience, USA), with an initial amount of gDNA library Hybridization of 200ng, an initial amount of cfDNA library Hybridization of 300ng, and a Hybridization time of 16 hours, all experimental procedures were performed according to the Kit instructions.

(3) Second generation sequencing

The capture libraries were sequenced by Chiense-made MGISEQ-T7, with 1000Mb of machine data per gDNA library, 4000Mb of machine data per cfDNA library, and sequencing mode PE 150.

4. Construction of female and male parturients and fetal haplotype

In all cases of the present invention, the parents having the same structural variation as the carrier are used as the reference sample, and the SNP sites that are heterozygous in the carrier with the chromosomal structural rearrangement, homozygous in the carrier's mate, and homozygous in the reference sample are the sites of the SNPs as the effective information. Performing family haplotype linkage analysis on the core family effective genetic marker, determining the haplotype of the structurally abnormal chromosome and the haplotype of the normal chromosome through haplotype linkage analysis, constructing a core family haplotype model, and determining the haplotype of the parent. And then, calculating and analyzing the fetal genotype and the haplotype in the peripheral blood cfDNA of the pregnant woman according to a hidden Markov statistical model by combining the parental haplotype, the fetal free concentration and the sequencing error rate. The principle hypothesis diagram is shown in fig. 2. The haplotypes for the structural variant breakpoint region of family Nos. 1-6 are shown in Table 2. FIG. 4 is a haplotype of fetal translocation chromosomes in case 3,

TABLE 2.1-6 haplotype of the structural variation breakpoint region of family fetus

Family serial number	Carrier and reference karyotype	Haplotype of breakpoint-1 region	Haplotype of breakpoint-2 region
				1	46,XX,t(3；6)(p10；p10)	Normal haplotype	Normal haplotype
2	46,XY,t(1；12)(q21；p11)	Structural variation carrying haplotype	Structural variation carrying 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: fetal chromosomal structural variation screening

And (4) predicting the fetal karyotype according to the fetal haplotype. The prediction principle is as follows:

when referring to the relatives of the carrier carrying the rearrangement of the chromosome structure:

a. if the fetal chromosomal rearrangement breakpoint region is not recombined

When the haplotype information of the fetal chromosome structure rearrangement breakpoint region is consistent with the haplotype information of the reference sample, diagnosing that the chromosome structure rearrangement carries a fetus; when they are not consistent, it is diagnosed that the non-chromosomal structural rearrangement carries a fetus, i.e., a normal fetus with a chromatoplastic karyotype.

Table 3 shows the results of karyotype prediction for fetuses from family 1-6.

TABLE 3.1-6 family fetus karyotype prediction results

Family serial number	The technology can predict the chromosome karyotype of fetus
		1	46,XN
2	46,XN,t(1；12)(q21；p11)
		3	46,XN
4	46,XN
		5	46,XN
6	46,XN

Remarking: n represents a sex chromosome

Example 4: validity verification of fetal chromosome structure variation screening method

1. Cytogenetic analysis and verification by amniotic fluid puncture in the middle of gestation

The accuracy of the screening method for detecting the structural variation of the chromosome of the fetus is verified by comparing with the conventional amniotic fluid karyotype in the middle of pregnancy.

Preparation method of amniotic fluid cell chromosome (in situ method)

A. Cell culture

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

2) removing the supernatant for other analysis, keeping about 0.5-1 ml of cell suspension, and uniformly mixing the cell suspension with a culture medium to about 2-2.5 ml;

3) bisecting the cell suspension into 2-4 Chromslide culture dishes;

4) after 24/48 hours of culture, add approximately 2.5ml of amniotic fluid medium to each Chromslide dish;

5) observing the growth condition of the cells after 5-6 days of culture, and replacing a new culture medium;

6) observing the growth condition of the cells after 1-2 days, if the clone number of the cells is enough, adding colchicine into a culture dish, harvesting the cells, and determining the treatment time according to the concentration of the colza water solution.

B. Chromosome preparation

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

2) adding 3-4 ml of hypotonic solution into each culture dish, and treating for 10 minutes at room temperature;

3) directly adding 0.5-0.7 ml of fixing solution into the hypotonic solution, and treating for 5 minutes at room temperature;

4) removing the supernatant, adding 3-4 ml of fresh stationary liquid, and treating at room temperature;

5) repeating the fourth step for 1-2 times;

6) removing the fixing solution, and performing chromosome dispersion process in a Maxchrome chromosome disperser (setting appropriate parameters);

7) after drying, the slides were aged and banding was evident.

2. Cytogenetic analysis and verification of umbilical cord blood of newborn

The specific method steps are as in example 1 for the preparation of the peripheral blood chromosome, and are not described in detail here.

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

TABLE 4.1-6 comparison of the karyotype results of the fetal cells of family lines with the results of the assay according to this technique

Family serial number	Fetal cell karyotype	Fetal karyotypes predicted by the techniques of the invention	Whether it is consistent
				1	46,XN	46,XN	Uniformity
2	46,XN,t(1；12)(q21；p11)	46,XN,t(1；12)(q21；p11)	Uniformity
				3	46,XN	46,XN	Uniformity
4	46,XN	46,XN	Uniformity
				5	46,XN	46,XN	Uniformity
6	46,XN	46,XN	Uniformity

Remarking: n represents a sex chromosome

As can be seen from Table 4, the results of the family haplotype prediction and the fetal amniotic fluid or umbilical cord blood cell genetic analysis are completely consistent after verification, and the sensitivity and specificity of the method for detecting the fetal chromosomal balance structural variation through the maternal peripheral blood free DNA are both 100%.

It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.

Claims

1. A construction method of a core family genome-wide haplotype model for identifying fetal chromosomal structural abnormalities, comprising the following steps:

a. both couples having carriers of chromosomal rearrangement;

b is referred to as a reference sample;

(2) determining the effective information SNPs:

(3) constructing a core family whole genome haplotype model: and (3) performing family haplotype linkage analysis on the SNPs sites with the effective information determined in the step (2) to obtain a core family whole genome haplotype, determining the haplotype of structurally rearranged chromosomes and the haplotype of normal chromosomes, and determining the whole genome haplotype of the parents.

2. A construction system for identifying a core pedigree genome-wide haplotype model of fetal chromosomal abnormalities, the system comprising software for processing sample data and hardware for carrying the software,

3. A method for identifying fetal chromosomal structural abnormalities from maternal peripheral blood free DNA, comprising the steps of:

s1: construction of core family genome-wide haplotype model

a. both couples having carriers of chromosomal rearrangement;

b is referred to as a reference sample;

(2) determining the effective information SNPs:

s2: predictive analysis of fetal genotypes and haplotypes

s3: identification of chromosomal structural abnormalities

a. if the fetal chromosome structure rearrangement breakpoint region is not recombined, when the haplotype information of the fetal chromosome structure rearrangement breakpoint region is consistent with the haplotype information of the reference sample, diagnosing that the non-chromosome structure rearrangement carries a fetus, namely, dyeing a normal fetus with a haplotype karyotype; when the two are inconsistent, the chromosome structure rearrangement is diagnosed as carrying a fetus;

further comprising S4: fetal chromosomal aneuploidy detection

4. A system for identifying fetal chromosomal structural abnormalities in a pregnant woman from peripheral blood free DNA, the system comprising software for processing sample data and hardware for carrying the software,

s1: the system further comprises hardware storing genotyping data for large scale SNP genotyping of both the reference sample and the carrier couple; 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 structural rearrangement as the carrier, and can also be relatives with normal chromosomes; when the carrier relative is selected from the carrier parent or other relative, the carrier parent carries the same chromosome structure rearrangement as the carrier;

(1) determining the effective information SNPs:

a. when the carrier parent or other relatives are used as a reference sample, SNP loci which are heterozygous in a chromosome structure rearrangement carrier, homozygous in a carrier mate and also homozygous in the reference sample are effective information SNPs loci;

(2) collecting the SNPs sites of the effective information determined in the step (1) to perform family haplotype linkage analysis to obtain a core family whole genome haplotype, determining the haplotype of structurally rearranged chromosome and the haplotype of normal chromosome, and determining the whole genome haplotype of the parent;

2) when the non-chromosomal rearrangement-bearing relatives of the carrier are taken as reference samples:

a. if the fetal chromosome structure rearrangement breakpoint region is not recombined, when the haplotype information of the fetal chromosome structure rearrangement breakpoint region is consistent with the haplotype information of the reference sample, diagnosing that the non-chromosome structure rearrangement carries a fetus, namely, dyeing a normal fetus with a haplotype karyotype; when the difference is not consistent, the chromosome structure rearrangement is diagnosed as carrying the fetus;

And (3) comparing cfDNA sequencing reads data with normal control by using low-depth whole genome high-throughput sequencing to detect common aneuploidy and large-fragment copy number variation of the chromosome, and judging whether the cffDNA in the plasma of the pregnant woman has aneuploidy and large-fragment deletion and repetition related to chromosome structure variation.

5. A method or system according to any one of claims 1 to 4 wherein the chromosomal structural abnormality is a structural variation in chromosomal balance.

6. The method or system of claim 5, wherein the chromosomal balance structural variation comprises a chromosomal balance translocation and inversion.

7. The method or system according to any one of claims 1 to 4, wherein SNP genotyping of both the carrier couples and the carrier relatives is performed by performing a gDNA capture sequencing of peripheral blood of both the carrier couples and the carrier relatives.

8. The method or system according to claim 3 or 4, wherein the peripheral blood cfDNA capture probe is designed as follows: querying a population genome database, selecting the minimum allele frequency between 0.3 and 0.7 according to the SNP locus frequency, particularly the frequency in east Asia population, uniformly distributing the minimum allele frequency in the genome with the interval between adjacent loci of 300-500Kb, and verifying by haploview that the linkage disequilibrium R2 between loci is more than 0.8.

9. The method or system of claim 3 or 4, wherein no less than 2 effective SNP sites are required for analyzing the haplotype of the rearrangement breakpoint region of the fetal chromosomal structure and whether homologous recombination has occurred in the region.