CN114480609A - Method for identifying chromosome insertion translocation carrying embryo and normal embryo - Google Patents
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Abstract
The invention relates to a method for identifying a chromosome insertion translocation carrying embryo and a normal embryo, belongs to the fields of genetic diagnosis and human assisted reproduction, and particularly relates to a pre-implantation detection technology (PGT). According to the method, family haplotype linkage analysis is carried out on a patient with balanced insertion and translocation of the chromosome, a mate thereof, an embryo inseminated in vitro, a translocation carrier relative or a fetal tissue with abnormal insertion segment or a chromosome of an embryo with abnormal insertion segment, so that the embryo with inserted translocation of the chromosome and the embryo with normal chromosome can be rapidly, simply and accurately distinguished, and the chromosome aneuploidy of the embryo is screened at the same time, thereby realizing timely detection of a diseased embryo before embryo transplantation, transplantation of a non-diseased embryo, prevention of birth of a defect infant and realization of timely blocking genetic transmission of inserted translocation of the chromosome to a next generation before embryo transplantation. Promoting the development and progress of human assisted reproductive technology to a certain extent.
Description
Technical Field
The invention belongs to the field of gene diagnosis and human assisted reproduction, and particularly relates to a pre-embryo implantation detection technology (PGT), which is a core family haplotype linkage analysis method capable of identifying whether an embryo carries parent insertion translocation chromosome genetic information.
Background
An insertion is one of chromosomal translocations, also known as insertion translocations, and is one of the structural variations of chromosomes. Common insertion translocations typically involve three breakpoints, where a single chromosomal fragment is released between two breakpoints and this fragment is subsequently inserted into the third breakpoint, resulting in a single chromosomal fragment being inserted into one chromosome and the other. The chromosome carrying the insert, called the receptor, has a breakpoint; the chromosome that provides the insert, called the donor, has two breakpoints. When chromosomal insertion occurs, the chromosomal segment can be inserted in the same direction, with the relative orientation to the centromere unchanged, i.e., the original banding sequence is maintained, which is referred to as forward insertion; insertion can also be in the opposite direction to the centromere, i.e., with the order reversed, known as inverted insertion. According to the size of the insertion segment, the insertion and translocation can be divided into invisible insertion and translocation and macroscopic insertion and translocation, wherein the insertion and translocation segment is small, the common cytogenetics chromosome karyotype analysis cannot detect the insertion and translocation segment, and a complex genetic detection technology such as a fluorescence in situ hybridization technology, a single molecule sequencing technology and the like is needed, but the situation is generally non-pathogenic, the occurrence rate in the population is high, and the fertility of the person is not influenced. The latter insertion translocation segment is generally larger, can be diagnosed by cytogenetics chromosome karyotype analysis, has lower incidence, but can affect fertility, and can generate infertility, recurrent abortion or abnormal fetus fertility.
The reason is that when the germ cells undergo meiosis, many different types of gametes are formed. When the insert is small, homologous chromosomes can be independently paired in a bivalent form, and as two bivalent bodies independent of each other are normally separated, four types of gametes are formed, wherein the proportion of each gamete is 1:1:1:1, two gametes are balanced and have normal amount of genetic material (one is balanced insert carriers and the other is normal), the other two gametes are unbalanced, one gametes can generate partial trisomy of the insert and the other gametes can generate partial monomer of the insert, and the two unbalanced gametes can cause embryonic dysplasia after fertilization and development into embryos and comprise infertility, recurrent abortion or abnormal fetus fertility chromosomes and the like. When the insert is larger, homologous chromosomes can exist in the form of quadrivalence and recombination within the insert can easily occur, in which case gametes are formed more complex, the proportion of balanced gametes is lower and clinical symptoms are more evident. Studies have shown that inserts that undergo homologous recombination have an average length of about 1.5% of the haploid chromosomal length, while inserts that do not undergo homologous recombination are generally less than 1% of the haploid chromosomal length.
The carriers of chromosome insertion and translocation usually turn to assisted reproduction technology, and the pregnancies are assisted by the Preimplantation genetics detection (PGT) technology, and the PGT technology can screen the euploid embryos without partial trisomy or monosome of chromosome insertion fragments in the carriers of chromosome insertion and translocation, so that the clinical pregnancy rate is improved. At present, the technologies for realizing PGT at home and abroad mainly include microarray-comparative genome hybridization (array-CGH), microarray single nucleotide polymorphism (SNP-array), Next Generation Sequencing (NGS) technology and the like. However, these PGT detection techniques commonly used in clinic can only detect single or triple copies of the insertion of the embryo, and there is no report on identifying the chromosome insertion translocation carrying embryo and the normal embryo, that is, the existing techniques can not accurately detect the balanced chromosome structure variation, and can not further diagnose whether the embryo of the insertion translocation patient is the chromosome insertion translocation carrying embryo or the normal embryo, and the probability of 50% of all the chromosome number normal whole-time somatic embryos is the chromosome insertion translocation carrying embryo. If the bearing fetus is still a carrier of chromosomal insertion translocations, it may subsequently face the same fertility problem, giving the patient both couples and even the entire family a great potential mental strain.
Disclosure of Invention
Aiming at the defects in the prior art, the invention establishes a method for performing pre-embryo implantation haplotype analysis (PGT) based on a whole-genome large-scale SNP genotyping result, and can distinguish an embryo carrying an inserted translocation chromosome from an embryo carrying a normal chromosome in time before embryo transplantation of in vitro fertilization, so that the embryo with the completely normal chromosome is preferentially transplanted, and the chromosome inserted translocation inheritance is blocked in time for the next generation.
The invention provides a family haplotype construction method for identifying chromosome insertion translocation carrying embryos and normal embryos, which comprises the following steps:
1) genotyping of the sample: inserting chromosome into translocation patients, carrying out large-scale SNP genotype detection on both couples and at least one patient relative; the couple and the relatives of the couple are called as reference samples; preferably, the patient relative may be selected from the patient's parents, children, siblings, or couples who have chromosomal insertion translocations, abortions, induction of labor, or live births; more preferably, the patient relatives prefer patient parents, and may also be other relatives; preferably, the patient's relatives may be selected from relatives also carrying chromosomal insertion translocations, and may also be selected from normal chromosomal relatives;
2) determining the SNPs site of the information: the selection criteria of the information SNPs are as follows:
a) SNP sites that are heterozygous in a chromosomal insertion translocation patient, homozygous in its partner, and also homozygous in the patient's relatives when the patient's parents, children or siblings are used as a reference sample; selecting information SNPs covering the insertion translocation breakpoint at the chromosome 3, the insertion translocation chromosome and the normal homologous chromosome corresponding to the insertion translocation breakpoint, wherein at least 1 information SNP is selected in each Mb range of the chromosome; in the region covering the breaking point, the information SNPs are selected from the range of 1-5Mb upstream and downstream of the breaking point;
b) when the aborted, induced or live fetus or embryo with abnormal copy number of the insert (monomer or trisomy) is used as a reference sample, the SNP locus which is heterozygous in a chromosome insertion translocation patient and homozygous in a mate is not required for a fetus or embryo sample, and the SNP locus covered in the insert is not used; selecting information SNPs covering the insertion translocation breakpoint of the receptor chromosome, the receptor chromosome and a normal homologous chromosome corresponding to the receptor chromosome, wherein at least 1 information SNP is selected in each Mb range of the chromosome; in the region covering the breaking point, the information SNPs are selected from the range of 1-5Mb upstream and downstream of the breaking point; selecting information SNPs on the donor chromosome (excluding the insert) and a normal homologous chromosome corresponding to the donor chromosome, which cover the two insertion translocation breakpoints of the donor chromosome, wherein at least 1 information SNP is selected in each Mb range; in the region covering the breaking point, the information SNPs are selected from the range of 1-5Mb upstream and downstream of the breaking point;
3) constructing a family haplotype: gathering the information SNPs sites determined by a) or b) in the step 2), obtaining the haplotypes of the whole chromosomes, which cover three break regions and are inserted into the translocation chromosome (acceptor and donor) and the homologous chromosomes thereof, by the family linkage analysis, and gathering the haplotypes of different chromosomes is called as the family haplotypes.
In a second aspect, the present invention also provides a construction system for identifying a family haplotype of a chromosome insertion translocation carrying embryo and a normal embryo, the system comprising software capable of computing processing sample data, and hardware for carrying the software, further comprising:
1) the system also comprises hardware for storing genotyping data for carrying out large-scale SNP genotype detection on both couples of a patient with chromosome insertion and translocation and at least one patient relative; the couple and the relatives of the couple are called as reference samples; preferably, the patient relative may be selected from the patient's parents, children, siblings, or couples who have chromosomal insertion translocations, abortions, induction of labor, or live births; more preferably, the patient relatives prefer patient parents, and may also be other relatives; preferably, the patient's relatives may be selected from relatives also carrying chromosomal insertion translocations, and may also be selected from normal chromosomal relatives;
2) determining the SNPs site of the information: the selection criteria of the information SNPs are as follows: a) SNP sites that are heterozygous in a chromosomal insertion translocation patient, homozygous in its partner, and also homozygous in the patient's relatives when the patient's parents, children or siblings are used as a reference sample; selecting information SNPs covering the insertion translocation breakpoint at the chromosome 3, the insertion translocation chromosome and the normal homologous chromosome corresponding to the insertion translocation breakpoint, wherein at least 1 information SNP is selected in each Mb range of the chromosome; in the region covering the breaking point, the information SNPs are selected from the range of 1-5Mb upstream and downstream of the breaking point;
b) when the aborted, induced or live fetus or embryo with abnormal copy number of the insert (monomer or trisomy) is used as a reference sample, the SNP locus which is heterozygous in a chromosome insertion translocation patient and homozygous in a mate is not required for a fetus or embryo sample, and the SNP locus covered in the insert is not used; selecting information SNPs covering the insertion translocation breakpoint of the receptor chromosome, the receptor chromosome and a normal homologous chromosome corresponding to the receptor chromosome, wherein at least 1 information SNP is selected in each Mb range of the chromosome; in the region covering the breaking point, the information SNPs are selected from the range of 1-5Mb upstream and downstream of the breaking point; selecting information SNPs on the donor chromosome (excluding the insert) and a normal homologous chromosome corresponding to the donor chromosome, which cover the two insertion translocation breakpoints of the donor chromosome, wherein at least 1 information SNP is selected in each Mb range; in the region covering the breaking point, the information SNPs are selected from the range of 1-5Mb upstream and downstream of the breaking point;
3) constructing a family haplotype: gathering the information SNPs sites determined by a) or b) in the step 2), obtaining the haplotypes of the whole chromosomes, which cover three break regions and are inserted into the translocation chromosome (acceptor and donor) and the homologous chromosomes thereof, by the family linkage analysis, and gathering the haplotypes of different chromosomes is called as the family haplotypes.
In a third aspect, the present invention provides a system for identifying a chromosomal insertion translocation carrying embryo and a normal embryo, the system comprising software capable of computing processing sample data, and hardware for carrying the software, comprising:
1) the system also comprises hardware for storing genotyping data for carrying out large-scale SNP genotype detection on couples of chromosome insertion translocation patients, at least one patient relative and couples in vitro fertilization embryos of the chromosome insertion translocation patients; the couple and the relatives of the couple are called as reference samples; preferably, the patient relative may be selected from the patient's parents, children, siblings, or couples who have chromosomal insertion translocations, abortions, induction of labor, or live births; more preferably, the patient relatives prefer patient parents, and may also be other relatives; the in vitro fertilized embryo of a couple of a patient is called a pending sample;
2) the software determines the sites of the information SNPs according to the following rules: the selection criteria of the information SNPs are as follows:
a) SNP sites that are heterozygous in a chromosomal insertion translocation patient, homozygous in its partner, and also homozygous in the patient's relatives when the patient's parents, children or siblings are used as a reference sample; selecting information SNPs covering the insertion translocation breakpoint at the chromosome 3, the insertion translocation chromosome and the normal homologous chromosome corresponding to the insertion translocation breakpoint, wherein at least 1 information SNP is selected in each Mb range of the chromosome; in the region covering the breaking point, the information SNPs are selected from the range of 1-5Mb upstream and downstream of the breaking point;
b) when the aborted, induced or live fetus or embryo with abnormal copy number of the insert (monomer or trisomy) is used as a reference sample, the SNP locus which is heterozygous in a chromosome insertion translocation patient and homozygous in a mate is not required for a fetus or embryo sample, and the SNP locus covered in the insert is not used; selecting information SNPs covering the insertion translocation breakpoint of the receptor chromosome, the receptor chromosome and a normal homologous chromosome corresponding to the receptor chromosome, wherein at least 1 information SNP is selected in each Mb range of the chromosome; in the region covering the breaking point, the information SNPs are selected from the range of 1-5Mb upstream and downstream of the breaking point; selecting information SNPs on the donor chromosome (excluding the insert) and a normal homologous chromosome corresponding to the donor chromosome, which cover the two insertion translocation breakpoints of the donor chromosome, wherein at least 1 information SNP is selected in each Mb range; in the region covering the breaking point, the information SNPs are selected from the range of 1-5Mb upstream and downstream of the breaking point;
3) the software compares the chromosome haplotype information (gene analysis data) in a sample to be determined with the haplotype information (genotyping data) of a reference sample in a family haplotype, focuses on the haplotype information of the upper and lower reaches of the insertion breakpoint region (three positions) in the family haplotype, and judges whether the homologous recombination occurs in the chromosome insertion breakpoint region or not through the haplotype of the whole chromosome:
i) when referring to relatives with the same insertion translocation as the carrier:
a. if the chromosome of the sample to be determined is not recombined in a region corresponding to the chromosome insertion translocation breakpoint in the family haplotype, carrying an embryo for insertion translocation when the haplotype information of the region of the sample to be determined is consistent with the haplotype information of a chromosome insertion translocation patient in the reference sample and the haplotype information of a relative having the same insertion translocation with the insertion translocation patient; when the haplotype information of the region of the undetermined sample is consistent with that of the chromosome insertion translocation patient in the reference sample, but is inconsistent with that of the relatives with the same insertion translocation of the insertion translocation patient, the embryo is a completely normal embryo with chromosomes;
b. if homologous recombination occurs in the chromosome of the sample to be determined corresponding to the region of the chromosome insertion translocation breakpoint in the family haplotype, the judgment standard of the insertion translocation carrier or the chromosome completely normal embryo is opposite to i) a;
ii) when referring to a normal chromosome relative:
a. if the region of the chromosome of the sample to be determined, which corresponds to the chromosome insertion translocation breakpoint in the family haplotype, is not recombined, when the haplotype information of the region of the sample to be determined is consistent with the haplotype information of the chromosome insertion translocation patient in the reference sample and the haplotype information of the normal relatives of the chromosome, the embryo is the embryo with the completely normal chromosome; when the haplotype information of the region of the undetermined sample is consistent with the haplotype information of the chromosome insertion translocation patient in the reference sample but is not consistent with the haplotype information of the normal relatives of the chromosome, the insertion translocation carries the embryo;
b. if homologous recombination occurs in the region of the chromosome of the sample to be determined, which corresponds to the chromosome insertion translocation breakpoint in the family haplotype, the judgment standard of the embryo carrying the insertion translocation or completely normal chromosome is opposite to ii) a;
iii) when reference is made to a fetal tissue or embryo sample with a missing copy number of the inserted translocation fragment:
a. if the chromosome of the sample to be determined is not recombined in a region corresponding to the chromosome insertion translocation breakpoint in the family haplotype, when the haplotype information of the region of the sample to be determined is consistent with the haplotype information of the chromosome insertion translocation patient in the reference sample and the chromosome insertion translocation patient in the fetal tissue or the chromosome breakpoint region of the donor of the embryo sample, but is not consistent with the haplotype information of the acceptor chromosome, the sample to be determined carries the embryo for insertion translocation; when the haplotype information of the region of the undetermined sample is consistent with the haplotype information of the chromosome insertion translocation patient in the reference sample and the receptor chromosome of the fetal tissue or the embryo sample but is not consistent with the haplotype information of the donor chromosome, the embryo with completely normal chromosome is obtained;
b. if homologous recombination occurs in the region of the chromosome of the sample to be determined, which corresponds to the chromosome insertion translocation breakpoint in the family haplotype, the judgment standard of the insertion translocation carried or the chromosome complete normal embryo is opposite to the criterion of iii) a;
iv) when reference is made to a fetal tissue or embryonic sample in which the copy number of the translocated fragment inserted is repeated:
a. if the chromosome of the sample to be determined is not recombined in a region corresponding to the chromosome insertion translocation breakpoint in the family haplotype, when the haplotype information of the region of the sample to be determined is consistent with the haplotype information of the chromosome insertion translocation patient in the reference sample and the chromosome insertion translocation patient in the fetal tissue or the donor chromosome breakpoint region of the embryo sample, but is not consistent with the acceptor chromosome haplotype information, the embryo with the completely normal chromosome is obtained; when the haplotype information of the region of the undetermined sample is consistent with the haplotype information of the chromosome insertion translocation patient in the reference sample and the receptor chromosome of the fetal tissue or the embryo sample but is not consistent with the haplotype information of the donor chromosome, carrying an embryo for insertion translocation;
b. if homologous recombination occurs in the region of the chromosome of the sample to be determined, which corresponds to the chromosome insertion breakpoint in the family haplotype, the judgment standard of the embryo with insertion translocation or completely normal chromosome is opposite to iv) a;
the chromosome carrying the insert, called the recipient chromosome, and the chromosome providing the insert, called the donor chromosome.
In some embodiments of the present invention, in the first to third aspects above, the large scale SNP genotyping detection described in step 1) covers 23 pairs of chromosomes; the large-scale SNP genotype detection method preferably selects a gene chip and gene sequencing.
In some embodiments of the present invention, in the first to third aspects, the sample for detecting chromosomal insertion translocation between a patient's couple, parent, daughter, or sibling is derived from somatic cells, preferably peripheral blood; the fetal sample can be derived from skin tissue, villus tissue of aborted or induced fetus, or from peripheral blood tissue of live-born diseased fetus.
In some embodiments of the present invention, in the first to third aspects, the chromosomal insertion translocation breakpoint region is covered in step 2) a), and the information SNPs are selected from a range of 2 to 4Mb upstream and downstream of the chromosomal translocation breakpoint, preferably from a range of 2Mb upstream and downstream of the chromosomal insertion translocation breakpoint; covering the recipient chromosome insertion translocation breakpoint region and the donor chromosome two insertion translocation breakpoints to the insert outer region in the step 2) b), wherein the information SNPs are selected from the range of 2-4Mb, preferably from the range of 2Mb, from the upstream and downstream of the recipient chromosome insertion translocation breakpoint region and from the donor chromosome two insertion translocation breakpoints to the insert outer region.
In some embodiments of the present invention, in the first to third aspects, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 information SNPs per Mb of chromosome in step 2) are selected.
In some embodiments of the present invention, in the first to third aspects, the embryo is developed to 3-7 days, 1-10 cells are obtained from biopsy as the sample for detecting the in vitro fertilized embryo in step 1); preferably cells derived from an embryonic blastomere biopsy or blastocyst trophectoderm biopsy.
In some embodiments of the present invention, in the first to third aspects, the cells obtained by biopsy are lysed and whole genome amplification is performed; the whole genome amplification method may be selected from the MDA method, the MALBAC method, or other whole genome amplification methods.
The method has the advantages that the embryo carried by chromosome insertion translocation and the embryo carried by chromosome normal can be accurately identified before embryo transplantation by a PGT technology, the 'chromosome normal' is preferentially transplanted, namely the embryo carrying insertion translocation is not carried, the genetic transmission of the chromosome insertion to the next generation is timely blocked before the embryo transplantation, and the chromosome number abnormality and the structural variation in the embryo are detected, so that the method not only solves the birth problem of a patient per se, but also eliminates the potential birth problem of the next generation, and has important clinical significance. The method is simple and convenient to apply, saves time, is efficient, can accurately identify the chromosome insertion translocation carried embryo and the chromosome normal embryo strategy, is a problem to be solved in clinic, and realizes accurate medical treatment.
Drawings
FIG. 1 is a diagram showing the pattern of insertion and translocation
FIG. 2 is a family chart of case 1
FIG. 3 shows the result of detecting abnormal copy number of inserted fragment of aborted fetal tissue in case 1 patient
FIG. 4 is the results of haplotype analysis of receptor chromosome region across breakpoint for case 1 family reference samples and biopsied embryos
FIG. 5 is the result of haplotype analysis of the donor chromosome breakpoint outer region of case 1 family reference samples and biopsy embryos
FIG. 6 shows the result of karyotype detection of umbilical cord blood chromosomes of newborn after successful delivery of embryo transplantation in case 1
FIG. 7 shows the result of the detection of the variation in the umbilical cord blood chromosome copy number of newborn after successful delivery of embryo transplantation in case 1
Detailed Description
Example 1: reference sample collection for patients and patient parents
3 chromosome balance insertion translocation carrying families which receive auxiliary reproduction are recruited, and the candidates are from Shanghai collection inheritance and sterility diagnosis and treatment center of the subsidiary obstetrics and gynecology hospital of the university of Compound denier. Every family needs to sign a written informed consent, and the research scheme is approved by the ethical committee of human subjects in Shanghai Collection of love genetics and sterility diagnosis and treatment center of the obstetrics and gynecology department of the university of Compound denier.
From 3 months to 6 months in 2021 in 2020, 3 families have a history of recurrent spontaneous abortion or a history of birth of fetuses with chromosomal abnormalities, and couples carried by chromosomal insertion translocation are hereinafter referred to as "patients" for short, and "patient spouses" for short. 10ml of peripheral blood of each pair of a patient couple and a patient relative (the patient relative can be selected from parents, children, brothers and sisters of the patient, or the couple with chromosome balance insertion translocation who aborts, induces or live-birth fetuses, fetal tissue can be derived from skin tissue, villus tissue of the aborted or induced-birth fetuses, and peripheral blood tissue of the live-birth diseased fetuses, and the patient relative preferably selects parents and can be other relatives) is extracted at the same time of recruitment. Using a part of peripheral blood for lymphocyte culture and carrying out karyotype analysis; and extracting DNA from the other part of peripheral blood according to a conventional mode in the field, and preparing for typing of the subsequent SNPs. If the couple has the abortion tissue with abnormal copy number of the past insertion fragment or the offspring with abnormal copy number of the survival insertion fragment, the abortion tissue or the peripheral blood tissue of the offspring can be collected to extract DNA for the subsequent SNPs typing.
(1) 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 plug of culture flask, injecting 30-40 drops of whole blood into 10ml of culture medium, shaking up, 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).
All the steps need to be operated aseptically
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 karyotype is the same as their mother, the patient's insertion translocation is inherited from the mother; as with the father, the patient's insertion translocation is inherited from the father. If the patient's parent's peripheral blood chromosome karyotype does not develop the same insertion translocation as the patient, the patient's insertion translocation is a new issue. 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.
For translocation karyotypes from families 1-3, see Table 1. Fig. 2 shows a family map of family No. 1.
TABLE 1.1-3 insertion of lines translocation karyotype table
| Family serial number | Number of chromosomes | The person who carries the medicine | Receptor chromosomes | Donor chromosome | Insertion of translocated chromosomal karyotypes |
| 1 | 46 | XX | Chromosome 1 | Chromosome 2 | ins(1；2)(q21.3；q31.1q32.2) |
| 2 | 46 | XX | Chromosome 4 | Chromosome 5 | ins(4；5)(q21；q14.1q23.1) |
| 3 | 46 | XX | Chromosome 9 | |
ins(9；18)(q32；q12.3q21.2) |
(2) Detection of insert copy number abnormalities (monomer or trisomy) using Illumina microarray gene chip for CNV detection method as follows:
1. preparation before experiment
1) Personal protection: and (4) changing the experimental clothes, wearing the hat, the mask and the shoe covers to enter the laboratory according to the relevant program contents of the safety protection of the laboratory.
2) Ultraviolet disinfection: and (3) opening an ultraviolet lamp in the ultra-clean workbench, sterilizing for 30min, closing the ultraviolet lamp, opening a fan, and ventilating for 10 min.
3) Printing related materials: gene chip operation record table, gene chip sample register table, etc.
4) Preparation of a kit: except for the reagents matched with the gene chip, 0.1M NaOH, isopropanol, formamide, 0.5M EDTA, deionized water and absolute ethyl alcohol are also required to be prepared, the operation needs three days to be completed, and the frozen reagent to be used before the experiment is placed at room temperature for melting.
5) Preparing consumables: preparing a 96-deep-hole plate, two matched rubber sleeves, filter element suction heads of various specifications, a sample adding groove, a small yellow plate and the like.
6) Sample preparation: taking out the patient couple and the relatives sample from a refrigerator at the temperature of-20 ℃, checking whether the sample number is consistent with the number on the record sheet, after the check is finished, placing the sample according to the sample loading sequence of the record sheet after the sample is instantly separated by vortex.
2. Formulating MSA3
1) Melting MA1, MA2 and MSM at normal temperature, mixing by reversing, and mixing by 0.1M NaOH.
2) Taking out the sample from a refrigerator at the temperature of-20 ℃, placing the sample on a small yellow plate, placing the sample on a room temperature, naturally melting the sample, performing vortex instantaneous separation, and placing the sample according to a recording list and a sample loading sequence.
3) And selecting N sampling holes on a 96-deep-hole plate according to the number N of the samples, and marking.
4) The hybridization oven was opened in advance and the temperature was adjusted to 37 ℃.
5) mu.L of MA1 was pipetted into selected wells of a 96-well plate using a line gun.
6) A4. mu.L sample of DNA product was pipetted into the corresponding well.
7) The sample wells were filled with 4. mu.L of 0.1M NaOH by pipetting with a line gun, covered with a rubber sleeve, shaken at 1600rpm for 1min, centrifuged at 280Xg for 1min and allowed to stand at room temperature for 10 min.
8) mu.L of MA2 was pipetted into the sample wells using a line gun.
9) Using a row gun to suck 38 mu L of MSM, adding the MSM into a sample hole, covering a rubber sleeve, oscillating at 1600rpm for 1min, centrifuging at 280Xg for 1min, and then incubating at 37 ℃ for 20-24h in a hybridization furnace.
3. Fragmentation of MSA3
1) Melting FMS at normal temperature, and mixing by reversing after melting.
2) The metal bath was opened in advance and the temperature was adjusted to 37 ℃.
3) The 96 deep well plate was removed from the hybridization oven, 25. mu.L of FMS was pipetted into the sample wells using a row gun, covered with a gum cover, shaken at 1600rpm for 1min, centrifuged at 280Xg for 1min and then metal bathed for 1 h.
4. Precipitated MSA3
1) PM1 was removed and prepared in isopropanol.
2) The refrigerated centrifuge was turned on in advance and pre-cooled at a set temperature of 4 ℃.
3) The 96 deep well plate was removed from the metal bath, 50. mu.L of PM1 was pipetted into the sample wells using a row gun, covered with a rubber sleeve, shaken at 1600rpm for 1min, incubated in the metal bath at 37 ℃ for 5min and centrifuged at 280Xg for 1 min.
4) Sucking 155 μ L of isopropanol with a line gun, adding into the sample well, covering with a new rubber sleeve, turning 10 times, and placing in a refrigerator at 4 deg.C for 30 min.
5) Centrifuging a 96-deep-hole plate at 4 ℃ and 3000Xg for 20min, then knocking the liquid dry, suspending and inverting the liquid on a super clean bench, and air-drying for 1 h.
5. Resuspending MSA3
1) RA1 was removed in advance for thawing.
2) The temperature of the hybridization furnace is adjusted to 48 ℃, the aluminum sealing film instrument is opened in advance, and the metal bath is adjusted to 95 ℃ in advance.
3) Sucking 23 μ L of RA1 with a row gun, adding into the sample well, sealing with aluminum film, incubating in a hybridization furnace at 48 deg.C for 1h at 1800rpm after incubation, shaking for 1min, centrifuging for a short time (less than or equal to 280Xg), incubating in a metal bath at 95 deg.C for 20min, standing at room temperature for 30min, and centrifuging for a short time (less than or equal to 280 Xg).
6. Chip hybridization
1) 450. mu.L of PB2 was added to each of the upper and lower wells of the chip box.
2) And (3) mounting the chip to a chip box, enabling the bar code end to be close to the chip box, and sucking a 15 mu L sample to a chip hole corresponding to the record sheet.
3) Carefully cover the chip box, place it into the hybridization oven smoothly, open the shaking mode, incubate at 48 ℃ for 16-20 h.
7. Cleaning chip
1) The chip cartridge was taken out of the hybridization oven and left to cool at room temperature for 25 min.
2) 200 microliter PB1 was added to each of the two glass plates, the chip was removed, the seal film was carefully removed, and the first glass plate was pulled up and down for 1min and then the second glass plate was placed and pulled up and down for 1 min.
8. Chip dyeing
1) The air bath was turned on in advance and the instrument was set at about 45.75 c, subject to a thermometer measuring the instrument surface temperature of 44 c (the set temperature was slightly greater than the actual temperature due to heat loss).
2) Adding 330mL of absolute ethyl alcohol into an XC4 reagent bottle one day in advance, vigorously shaking for 15s, standing at room temperature, vigorously shaking again before use, and standing.
3) The method comprises the following steps of: deionized water: 0.5M EDTA 95% formamide was prepared at a ratio of 47.5:2.4: 0.1.
4) Placing RA1, XC1, XC2, TEM, STM and ATM at normal temperature for melting, and taking XC3 and PB1 as normal temperature reagents.
5) 150mL of PB1 was added to the chip mounting box, a chip assembly was formed in the order of holder + chip + membrane + slide + iron holder, the bar code end was close to itself, the non-bar code end of the membrane was cut off, the bar code end was up, and placed in an air bath at 44 ℃.
6) Wells were formed between the chip and the slide, and 150 μ L of RA1 was added to the wells every 30s, and repeated 6 times.
7) Add 450. mu.L of XC1 and incubate for 10 min.
8) Add 450. mu.L of XC2 and incubate for 10 min.
9) Add 200. mu.L TEM and incubate for 15 min.
10) Add 450. mu.L 95% formamide, incubate for 1min, add 450. mu.L 95% formamide again, incubate for 6 min.
11) And adjusting the temperature of the air bath to the actual surface temperature to be the labeling temperature of the STM reagent tube.
12) Add 450. mu.L of XC3, incubate for 1min, add 450. mu.L of XC3 again, incubate for 20 min.
13) The air bath was adjusted to about 32.0 ℃.
14) And when the temperature of the air bath is reduced to the labeling temperature of the STM reagent tube, adding 250 mu L of STM, and incubating for 10 min.
15) Add 450. mu.L of XC3, incubate for 1min, add 450. mu.L of XC3 again, incubate for 5 min.
16) Add 250. mu.L of ATM and incubate for 10 min.
17) Add 450. mu.L of XC3, incubate for 1min, add 450. mu.L of XC3 again, incubate for 5 min.
18) Add 250. mu.L of STM and incubate for 10 min.
19) Add 450. mu.L of XC3, incubate for 1min, add 450. mu.L of XC3 again, incubate for 5 min.
20) Add 250. mu.L of ATM and incubate for 10 min.
21) Add 450. mu.L of XC3, incubate for 1min, add 450. mu.L of XC3 again, incubate for 5 min.
22) Add 250. mu.L of STM and incubate for 10 min.
23) Add 450. mu.L of XC3, incubate for 1min, add 450. mu.L of XC3 again, incubate for 5 min.
24) Adding 310mL of PB1 into the plastic dish in advance, taking down all chips, closing the air bath, detaching the iron clamping pieces by a file, recycling the iron clamping pieces, the glass slides and the support, discarding the diaphragm, placing the chips on a rack in the plastic dish with the bar code end upward, lifting up and down ten times, and standing for 5 min.
25) Adding 310mL of XC4 into the other plastic dish in advance, placing the rack containing the chips into the plastic dish, lifting and pulling up and down for ten times, and standing for 5 min.
26) And (3) clamping the bar code end of the chip by using a pair of tweezers, sequentially clamping the chips, carefully draining redundant liquid on the edge of the plastic dish, horizontally placing the chip on a chip rack, carefully placing the chip rack into a vacuum suction device, pumping for about 50-55min until the surface of the chip is XC4, and taking out the chip.
9. Gene chip scanning
1) And downloading the DAMP File corresponding to each chip on Decode File Client Support software provided by Illumina in advance according to the ID number of the chip, and putting the DAMP File into a corresponding path of a computer connected with the iScan scanner.
2) Opening an iScan scanner and an iScan Control Software system on a computer, clicking 'Start' to automatically open an engine room after the instruments are automatically initialized, placing a chip on a chip groove on a metal platform of the engine room, pressing buttons on two sides of the metal platform to adjust the position of the chip until the chip is completely clamped in the groove, clicking 'Next', waiting for the scanner to Scan a chip bar code, selecting an Input Path as a folder for placing a chip DAMP file after scanning is finished, selecting an Output Path as a folder for data Output, and clicking 'Scan' to Scan.
3) After the scanning is finished, clicking 'Done', pressing a button on the scanner to open the cabin, pressing buttons on two sides of the metal table to take out the chips, putting the chips into a packaging box originally containing the chips, storing the chips at normal temperature, and pressing the button on the scanner to close the cabin.
4) Copying the IDAT file of each chip in a data output folder, and closing the iScan Control Software system and the iScan scanner.
10. Gene chip data quality control
By carrying out software analysis on original gene chip data, the Genome Studio provides a call rate value representing the proportion of detected SNP sites to the total number of SNP sites, the higher the value is, the better the data quality is, and the highest value is 100%; karyo Studio can provide LogR dev values, which are a fluctuating indicator of CNV homogeneity, with higher values, poorer homogeneity and more difficult CNV analysis.
11. Gene chip analysis principle and process
CNV was calculated mainly by two indices Log R Ratio (LRR) which is the ratio between observed and expected fluorescence intensity and B Allel Frequency (BAF) which is the ratio of Infinium assigned B allele, with values of BAF for AA, BB and AB tending to 0, 1 and 0.5, respectively, in normal samples, and if the BAF value for a locus approaches 0.66, this indicates the occurrence of duplication (ABB).
By comparing the observed LRR and BAF values, preliminary copy number estimates were calculated for each site. L is0Being the possibility of a homozygous deletion, L1Possibility of being haploid, L2Possibility of being diploid, L3Possibility of being a triploid, L4The probability of tetraploids is calculated as follows:
L0＝LDD
L1＝LA+LB
L2＝LAA+LAB+LBB
L3＝LAAA+LAAB+LABB+LBBB
L4＝LAAAA+LAAAB+LABBB+LBBBB
wherein the probability L of a given genotype AAB is calculatedAABThe parameters of table 1 and the following standard values can be used:
CNV calculated 14 genotype parameters, genotype AABB was not modeled, as this represents two independent repetitive events, which rarely occur in nature. (CN-copy number, DD-homozygous deletion, SD-standard deviation).
First, a preliminary copy number estimate (X) is calculated, X being the average of 5 copy models, weighted by their respective likelihoods:
then find the maximum | Z using a sliding window approachijThe maximum value is found using the following formula, where n is the number of loci on the chromosome.
Si＝X1+...+Xi,1≤i≤n
Next, a copy number is assigned to the region between each successive breakpoint, and the L at each position in the region is calculated0，L1， L2，L3，L4. For each assumed copy number K (0-4), L for each KKThe values are added, the K with the highest sum is the copy number of the region, and a confidence score is assigned to evaluate the region with the copy number not being 2. FIG. 3 shows the result of detecting abnormal copy number of inserted fragment in aborted fetal tissues (reference sample) of family 1 patients.
Example 2: in vitro fertilization, blastocyst biopsy and Whole Genome Amplification (WGA)
1. In vitro fertilization
In Vitro Fertilization (IVF) of the recruited families, the in vitro fertilization method following the methods routine in the art; maternal/paternal age, phenotype, ovulation outcome, fertilized egg count, and blastocyst count for final biopsy for these families are listed in table 2. Through the in vitro fertilization, 31 blastula are obtained from 3 families for subsequent biopsy and haplotype analysis research, 2 ovulation induction cycles are carried out on family 1, and one ovulation induction cycle is carried out on families 2 and 3.
TABLE 2.1-3 family basic cases and in vitro fertilization cases
1Case 1 family is the number of oocytes obtained for 2 ovulation cycles, MII, number of fertilized embryos, number of D3 embryos and number of D5/D6 blastocyst biopsies
2. Blastocyst biopsy and whole genome amplification
The embryos at blastocyst stage were taken and 3 to 10 cells were removed from trophectoderm on day 5 or 6 of embryonic development. The biopsy cells were lysed in PCR tubes containing alkaline denaturing buffer (KOH). Whole Genome Amplification (WGA) was then performed by multiple replacement amplification (MDA) method. Isothermal DNA amplification (Repli-g single cell whole genome amplification kit, QIAGEN GmbH, Hilden, Germany) was performed with phi 29DNA polymerase according to the method described in the kit instructions, and the specific procedure was as follows:
(1) preparation before experiment
1) Personal protection: and (4) changing the experimental clothes, wearing the hat, the mask and the shoe covers to enter the laboratory according to the relevant program contents of the safety protection of the laboratory.
2) Ultraviolet disinfection: and (3) opening an ultraviolet lamp in the ultra-clean workbench, sterilizing for 30min, closing the ultraviolet lamp, opening a fan, and ventilating for 10 min.
3) Printing related materials: experimental operation record sheet, bar code, etc.
4) Preparing an instrument: and (4) checking whether the equipment is normal or not, wherein the equipment comprises a vortex mixer, a palm three-tube centrifugal machine, a PCR instrument and the like.
5) Preparation of a kit: preparing a single cell whole genome amplification reagent (MDA method), checking whether all components in the kit are complete and intact within the effective period, and putting the reagent on an ice box in advance for later use.
6) Preparing consumables: comprises filter element suction heads with various specifications, an inlet EP pipe with 0.2mL, an inlet EP pipe with 1.5mL, a double-sided board and an ice box.
7) Sample preparation: and (5) verifying whether the name and the number of the sample are consistent, and placing the sample on an ice box for standby after the name and the number are consistent.
8) Preparation of positive control: a positive control was prepared, which was a 2.5 μ L sample containing 5-10 cells (ensuring that there were always cells in the tube).
(2) Single cell whole genome amplification by MDA method
1) Two 0.2mL inlet EP tubes were prepared and 2.5. mu.L and 4. mu.L of water were added as volume control tubes for use.
2) And centrifuging the sample for 1min, checking the volume of the liquid, comparing the volume of the liquid with a control tube, photographing and storing the comparison result, recording the comparison result in a book, and making the sample volume traceable.
3) Buffer D2 was prepared (the volume of Buffer D2 given in the table below was sufficient for 12 reactions and could be stored at-20 ℃ without complete exhaustion of one experiment, but the storage time could not exceed 3 months).
| Components | Volume of |
| DTT，1M | 3μL |
| Reconstituted Buffer DLB | 33μL |
| Total volume | 36μL |
4) Add 3. mu.L Buffer D2 to each sample tube, mix the tube walls evenly by flicking and centrifuge briefly to collect at the bottom of the tube, and place the centrifuged sample tube into the PCR ice box. Note that: ensure that the liquid in the tube is not adhered to the tube wall, and avoid blowing by using a liquid transfer device, and avoid the cell sample from being adhered to the suction head of the liquid transfer device.
5) The PCR instrument was turned on and the PCR program was set as follows: 10min at 65 ℃; hold (hot lid temperature: 105 ℃ C.).
6) And (3) placing the sample tube in a PCR instrument for carrying out a cracking reaction, quickly adding 3 mu L of Stop Solution after the PCR reaction is finished, flicking the tube wall, uniformly mixing and centrifuging for a short time. The sample was placed on an ice box before the next reaction mixture was ready.
7) Preparing a reaction mixed solution:
| components | Volume of |
| H2O sc | 9μL |
| REPLI-g sc Reaction Buffer | 29μL |
| REPLI-g sc DNA Polymerase | 2μL |
| Total volume | 40μL |
8) Immediately, 40. mu.L of the reaction mixture was added to the prepared 10. mu.L of DNA sample (step 4.2.7), thoroughly shaken and mixed, and collected by brief centrifugation.
9) Putting the sample into a PCR instrument for single cell amplification reaction, wherein the PCR program is as follows: 59min59s 8cycles at 30 ℃; 3min at 65 ℃; hold at 4 ℃.
10) The amplification product was stored at-20 ℃ for future use.
(3) Determination of concentration of amplification product
1) Quantification with Qubit: and (4) performing concentration determination after the MDA amplification product is diluted 10 times without nuclease water.
2) Qualified requirements of concentration quality inspection: all samples were tested at concentrations > 300 ng/. mu.L. If the concentration is lower than the range, subsequent experiments are not carried out, and the reason is analyzed.
(4) Quality inspection of MDA amplification product
1) Preparation before experiment
a) Personal protection: according to the contents of the relevant procedures of safety protection of the laboratory, the laboratory clothes, the cap, the mask and the shoe cover are replaced to enter the laboratory.
b) Ultraviolet disinfection: and (3) turning on an ultraviolet lamp in the ultra-clean workbench, sterilizing for 30min, turning off the ultraviolet lamp, turning on a fan, blowing for 3 levels, and ventilating for 10 min.
c) Printing related materials: experimental operation record sheet, bar code, etc.
d) Preparing an instrument: and (4) checking whether the equipment is normal or not, wherein the equipment comprises a palm three-tube centrifugal machine, a micro-bench centrifugal machine, a qPCR instrument and the like.
e) Preparation of reagents: comprises qPCR reagent and nuclease-free water, checks whether each component in the kit is complete and intact within the effective period, and places the kit on an ice box for dissolving for later use.
f) Preparing consumables: the special suction head comprises filter element suction heads with various specifications, 1.5mL inlet EP pipes, 2mL inlet EP pipes, eight connecting pipes and pipe covers special for QPCR, a double-sided plate and an ice box.
g) Sample preparation: and (5) verifying whether the name and the number of the sample are consistent, and placing the sample on an ice box for standby after the name and the number are consistent.
2) Primer preparation
When the quality inspection primers are designed, the completeness of the single-cell amplification product and the difference of the amplification efficiency of different amplicons are considered, so that the amplification products are distributed on different chromosomes as much as possible, and the lengths of the amplification products are different.
The specific primers correspond to the DNA sequences of the amplification products as follows:
MQ1：
GCCTGCCCAGACCTAGTAATTGCTGTAGTCTTTTCCTTTTGGATTCCTAGCAGTGGATGCTTGAAT ACAAATACCAAGTGGGCTCCAGCCAGTGAGGGTAATAGTGTACAGCAGTAAACTGTCTGTTAGCTGAT GCTTACATTTCAGACAAATTGAGGAGGTTGTCAAAGGCATTAGCTTCTATCCTTTCCAGGGAATCAATC TGAGAGATTTCACTAGGGAAGAGAGGAGGGGGAAAAAGAGAAAGAAACATGAAAGAAATGACCGT GTCTGCATGATTATGAGCTATGTGTGTGACCCAACAACTGGGGACACCACTGTGTCCCCCTGAAAAGA ACAAAGATGTGCAAGTTGTCCCCAAATCATACAGTGCTAACATCTCCCTTTAGGAGCACTGATGGGCA MQ2：
TTTCGACATGGAGGCCACTGGCTTGCCCTTCTCCCAGCCCAAGGTCACGGAGCTGTGCCTGCTG GCTGTCCACAGATGTGCCCTGGAGAGCCCCCCCACCTCTCAGGGGCCACCTCCCACAGTTCCTCCAC CACCGCGTGTGGTAGACAAGCTCTCCCTGTGTGTGGCTCCGGGGAAGGCCTGCAGCCCTGCAGCCAG CGAGATCACAGGTCTGAGCACAGCTGTGCTGGCAGCGCATGGGCGTCAATGTTTTGATGACAACCTG GCCAACCTGCTCCTAGCCTTCCTGCGGCGCCAGCCACAGCCCTGGTGCCTGGTGGCACACAATGGTG ACCGCTACGACTTCCCCCTGCTCCAAGCAGAGCTGGCTATGCTGGGCCTCACCAGTGCTCTGGATGGT GCCTTCTGTGTGGATAGCATCACTGCGCTGAAGGCCCTGGAGCGAGCAAGCAGCCCCTCAGAACACG GCCCAAGGAAGAGCTATAGCCTAGGCAGCATCTACACTCGCCTGTATGGGCAGTCCCCTCCAGACTCG CACACGGCTGAGGGTGATGTCCTGGCCCTGCTCAGCATCTGTCAGTGGAGACCACAGGCCCTGCTGC GGTGGGTGGATGCTCACGCCAGGCCTTTCGGCACCATCAGGCCCATGTATGGGGTCACAGCCTCTGCT AGGACCAAGCCAAGACCATCTGCTGTCACAACCACTGCACACCTGGCCACAACCAGGAACACTAGT CCCAGCCT
MQ3：
GGGTTGTCCCCTGGATATAGGAGGGGTGAGGTGGGTCAGAGGGTGCCGGGAGGGGCCTGGATCA GCACTTCAAAAGGTGGGTGCCCGCCACCCAGGGGTGGCCCCAAGGAGAGGAGAGGAGCTGGCCATG TGCAGAGATGGGGCCCTGTGATGCAGACACACCTCGGTACCAGTTGCGCAGGGAGGTCATGACGAAG AAGCGGATGGCCTGGTTCGAGCCCTGCTTCAGGACAGTGGCTGTGAGGCCCTGGTACGTCCCCTTCA GCCCTGCGGGAAGGCAGGCACGGGGTTACCCTGCAGCCTCTCAGGCCCCGGTTGGGAATTGGTGTGT GTGGGGGGTGGGTGTTGCACAAAGCCCAAGGCAGGATGGGAGGTGCAGGCCCTTCCCACCCTGGCC CAGGTCCCTGAGCCCTATCAGGAAGGTCGAGTGGCTCACCTTGTTCCCGCACAATCTCCCTAACCCCG TGGAAGAATCCTCTGTACTTGGGGTTTGGGGAGGTCTGGTCGTGGATGAACTT
MQ4：
TCAAGTTTCCTCTCACCTTTATTCTACTTCCTCATTAACAAGTCATCGGTTTGATACATTAATCATG GCAAAAAAATCACAGAGGAAGCACTAATCTGTCATTTTTTTCTGTGACCTGCAGACTCTTTGACACCC GGTACAGCATCCGGGAGAATGGGCAGCTCCTCACCATCCTGAGTGTGGAAGACAGTGATGATGGC
MQ5：
GCGATCGATGCAGTGGACATGGTACTCATGGGAACAAGGTAGTTTACGAAGTTTGTTGCCTTCTG TATATTCTGTAATGCAAACACTACAGGTTTTTAATGCATCATTTTCACCAAAACTTCTCATTGCCAAGTT GTCAATCTGTTCTTTGGTGAGTCCTCTAGGTTGGTCATCATCATCCTCATTTAAGAGGAAAAACTGAGC CAGGCTAAGGAAGGGCAAAGAGCCACTTTCATCAAATGTGACTGGGGCCCTATGTCGACCCTCTCGC CTGGCACCTGATGAGCCTGATGATGAGCTTCCTTCATTACTGCCTTCAAATAAATCTGAGCTAGTTTCT GAACTTTCACCACCGGAACTGGAACTAGGACTGGAACTGGAACTTGAACTGGAACTGGAACTCGAA CTGGAACTGGAACTCGAACTGGAACCAGAACTACTACCACCACCAGAACCTCCTCTTCCACTCCGTG ACTCTGCCCTTTCCATATTTCGATTTGAGACTGAGCCAGTAGGCTCTGAGTCGCTATCACTGTACATAA AATAGCTTAACTCACCAAAACCTGTCATTATCTGCCTTAACATGGTCTGAATTGCAACAGATGTAGTCT CACTTAAACCAGTATTTAAGATTCTACGAATGGGAATTCTGATGGTACT
MQ6：
CTCAAGACGTGGAGGCATCTGGGCTTAGCTGAGGTCACACTACTAGTGGGACCTACAGAAAAAC CCAGGTGTGTCACCAGCAGGGCGGGCACCATCTCTGCAGCCGACACCAGCTTCCCCAACCTCCAGAC AGGAGAAAGCATGAGGGTCCCCACTGAAATTCTGGCTCACAGAGTCACCTCCTATGCTGTGGCTTTTT TCAATCCCAGAGTTTGTCCAGCGAGGCAAAGACCTGGTCACGGCGTCTCTGGCTCACCAGGTGGAGG GAACGGCAAAACTCACGCTGGCCCAAGAGGAGGAACAGAGAAGCTTCCTGGCTGAGGCCCAGCCG ACTGCTGACCCGGAAAAGTTTCTCGAGGTGACTCACATCCCCAGCCTCTGCACATGTGGGTGAGCCA GTTGTAGCTCTGTTCCCGTGACTGAGCACGGGACGCCGGAGGTATTCATCAGGCATGAGGTTATCTGC CTACTTCCCATGTGTCAG
a) centrifuging 8 pairs of primer dry powder at 12,000rpm for 1min, adding nuclease-free water to dilute to 100 μ M mother solution, and storing at-20 deg.C for a long time; when in use, a small amount of mother liquor is diluted to 2 mu M of working solution, and the working solution is stored at the temperature of minus 20 ℃ for later use.
b) And (3) primer selection: generally, all 8 pairs or part of primers are used for MDA amplification products to carry out quality inspection verification, the verification coverage is comprehensive, and the reliability is high.
3) Template preparation
a) MDA amplification product: and diluting the MDA amplification product which meets the requirements after quantification by 10 times by using nuclease-free water to serve as a QPCR reaction template.
b) Positive quality control: mixed human peripheral blood or human cell line genomic DNA at a concentration ≈ 50 ng/. mu.L.
c) Negative quality control: nuclease-free water.
4) Configuring a PCR reaction system
The following table is the qPCR reaction system.
| Name of reagent | Single tube system (20ul) | Remarks to note |
| KAPASYBR Mix 2× | 10μL | Mix containing SYBR fluorescent dye |
| Template DNA | 2μL | Note: MDA product needs to be diluted by 10 times |
| Primer F, 2. mu.M | 3μL | |
| Primer R, 2. mu.M | 3μL | |
| High Rox | 0.4μL | Note: selecting according to different instruments |
| ddH2O | 1.6μL |
5) PCR cycling parameters are shown in the following Table
6) Analysis of results
I) Judging and labeling:
a. positive results: the melting peak of the amplification product is single and sharp, and the expected range of the Tm value is fixed; the amplification curve is typically sigmoidal and the typical Ct value is ≦ 28 (unable to > 30).
That is, if 4 conditions (single sharp melting peak, fixed Tm value range, S-shaped curve, Ct value less than or equal to 28) are satisfied simultaneously, the product is positive; if the conditions (especially Ct value and melting peak pattern) are not satisfied, the sample is negative or weakly positive.
b. Negative results: the amplification product has a plurality of melting peaks or no single sharp main peak, and the Tm value is not in the expected range; the amplification curve is not typically sigmoidal, with a typical Ct value > 30.
That is, if any one of the above 4 conditions exists, the test strip is negative or weakly positive; in particular, Ct values >30 must be negative, multiple melting peaks and no main peak must be negative.
II) flow of results analysis:
a. according to the principle of 'judgment standard', positive quality control and negative quality control are analyzed firstly, and the positive quality control result is ensured to be positive, and the negative quality control result is ensured to be negative. If the negative and positive quality control results are incorrect, it indicates improper operation or the quality of the reagent is problematic, and the reason needs to be eliminated and re-verified.
b. And analyzing the MDA amplification products after the positive and negative quality control results are correct, comparing the positive and negative results according to the 'judgment standard' principle, and analyzing each MDA amplification product one by one.
c. And (5) sorting and summarizing analysis results, and keeping quality inspection records.
Example 3: SNP genotyping and haplotyping (haplotypes) analysis
1. SNP genotype detection
The Illumina SNP microarray is used for SNP genotype detection, each chip contains nearly 70 ten thousand SNPs, and 23 pairs of chromosomes can be fully covered. 31 samples obtained by blastocyst biopsy and whole genome amplification in example 2 were grouped according to family, 3 whole genome amplification samples of the family, the couple and the relatives of the patient were grouped into one group, and microarray SNP genotype detection and analysis were performed, the grouping being shown in Table 3. The specific experimental method of the gene chip is as described in example 1, and will not be described repeatedly.
TABLE 3.1-3 family SNP array Experimental groups
Note: f represents a female, M represents a male
2. Embryo chromosome aneuploidy and insert copy number variation detection
The principle and process of detecting embryo chromosome aneuploidy and insert copy number variation by the SNP gene chip are the same as those described in example 1, and will not be described repeatedly herein.
3. Haplotype analysis
After obtaining all SNPs information detected by the chip, a family haplotype is constructed and used for identifying an insertion translocation carrying embryo and a normal embryo. The specific operation method comprises the following steps:
A. constructing a family haplotype:
1) genotyping of the sample: SNP genotyping is carried out on both couples and relatives of the patients (in the embodiment, aborted fetus tissues and embryos (blastocysts) with abnormal insertion copy number) of the patients; the couple and the relatives of the couple are called as reference samples;
2) determining the SNPs site of the information: the selection criteria of the information SNPs are as follows: the carrier is heterozygous in the chromosome insertion translocation carrier, is homozygous in the mate thereof and has no requirement on the sample of the relatives of the patient; the SNP sites covered in the insert were not used; selecting information SNPs covering the insertion translocation breakpoint of the receptor chromosome, the receptor chromosome and a normal homologous chromosome corresponding to the receptor chromosome, wherein at least 1 information SNP is selected in each Mb range of the chromosome; in the region covering the breaking point, the information SNPs are selected from the range of 1-5Mb upstream and downstream of the breaking point; selecting information SNPs on the donor chromosome (excluding the insert) and a normal homologous chromosome corresponding to the donor chromosome, which cover the two insertion translocation breakpoints of the donor chromosome, wherein at least 1 information SNP is selected in each Mb range; in the region covering the breaking point, the information SNPs are selected from the range of 1-5Mb upstream and downstream of the breaking point;
3) constructing a family haplotype: gathering the information SNPs sites determined in the step 2), obtaining the haplotype of the whole chromosome covering three fracture regions and inserted into the whole chromosome (acceptor and donor) of the translocation chromosome and the homologous chromosome thereof by family linkage analysis, and gathering the haplotype of different chromosomes is called as family haplotype.
B. Method for identifying chromosomal insertion translocations carrying embryos and normal embryos:
SNP genotyping is carried out on in vitro fertilized embryos (blastocysts) of couples of patients, which are called undetermined samples. Comparing the haplotype information of the chromosome in the sample to be determined with the haplotype information of the reference sample, focusing on the haplotype information of the upstream and downstream of the insertion breakpoint region (three positions) in the family haplotype, and judging whether the homologous recombination occurs in the chromosome insertion breakpoint region or not through the haplotype of the whole chromosome:
i) when referring to relatives with the same insertion translocation as the carrier:
a. if the chromosome of the sample to be determined is not recombined in a region corresponding to the chromosome insertion translocation breakpoint in the family haplotype, carrying an embryo for insertion translocation when the haplotype information of the region of the sample to be determined is consistent with the haplotype information of a chromosome insertion translocation patient in the reference sample and the haplotype information of a relative having the same insertion translocation with the insertion translocation patient; when the haplotype information of the region of the undetermined sample is consistent with that of the chromosome insertion translocation patient in the reference sample, but is inconsistent with that of the relatives with the same insertion translocation of the insertion translocation patient, the embryo is a completely normal embryo with chromosomes;
b. if homologous recombination occurs in the chromosome of the sample to be determined corresponding to the region of the chromosome insertion translocation breakpoint in the family haplotype, the judgment standard of the insertion translocation carrier or the chromosome completely normal embryo is opposite to i) a;
ii) when referring to a normal chromosome relative:
a. if the region of the chromosome of the sample to be determined, which corresponds to the chromosome insertion translocation breakpoint in the family haplotype, is not recombined, when the haplotype information of the region of the sample to be determined is consistent with the haplotype information of the chromosome insertion translocation patient in the reference sample and the haplotype information of the normal relatives of the chromosome, the embryo is the embryo with the completely normal chromosome; when the haplotype information of the region of the undetermined sample is consistent with the haplotype information of the chromosome insertion translocation patient in the reference sample but is not consistent with the haplotype information of the normal relatives of the chromosome, the insertion translocation carries the embryo;
b. if homologous recombination occurs in the region of the chromosome of the sample to be determined corresponding to the chromosomal insertion translocation breakpoint in the pedigree haplotype, the criteria for judging an embryo carrying an insertion translocation or having a completely normal chromosome are the opposite of those in ii) a.
iii) when reference is made to a fetal tissue or embryo sample with a missing copy number of the inserted translocation fragment:
a. if the chromosome of the sample to be determined is not recombined in a region corresponding to the chromosome insertion translocation breakpoint in the family haplotype, when the haplotype information of the region of the sample to be determined is consistent with the haplotype information of the chromosome insertion translocation patient in the reference sample and the chromosome insertion translocation patient in the fetal tissue or the chromosome breakpoint region of the donor of the embryo sample, but is not consistent with the haplotype information of the acceptor chromosome, the sample to be determined carries the embryo for insertion translocation; when the haplotype information of the region of the undetermined sample is consistent with the haplotype information of the chromosome insertion translocation patient in the reference sample and the receptor chromosome of the fetal tissue or the embryo sample but is not consistent with the haplotype information of the donor chromosome, the embryo with completely normal chromosome is obtained;
b. if homologous recombination occurs in the region of the chromosome of the sample to be determined, which corresponds to the chromosome insertion translocation breakpoint in the family haplotype, the judgment standard of the insertion translocation carried or the chromosome complete normal embryo is opposite to the criterion of iii) a;
iv) when reference is made to a fetal tissue or embryonic sample in which the copy number of the translocated fragment inserted is repeated:
a. if the chromosome of the sample to be determined is not recombined in a region corresponding to the chromosome insertion translocation breakpoint in the family haplotype, when the haplotype information of the region of the sample to be determined is consistent with the haplotype information of the chromosome insertion translocation patient in the reference sample and the chromosome insertion translocation patient in the fetal tissue or the donor chromosome breakpoint region of the embryo sample, but is not consistent with the acceptor chromosome haplotype information, the embryo with the completely normal chromosome is obtained; when the haplotype information of the region of the undetermined sample is consistent with the haplotype information of the chromosome insertion translocation patient in the reference sample and the receptor chromosome of the fetal tissue or the embryo sample but is not consistent with the haplotype information of the donor chromosome, carrying an embryo for insertion translocation;
b. if homologous recombination occurs in the region of the chromosome of the sample to be determined, which corresponds to the chromosome insertion breakpoint in the family haplotype, the judgment standard of the embryo with insertion translocation or completely normal chromosome is opposite to iv) a;
4. chromosome aneuploidy and insertion translocation prediction result of No. 1-3 family embryo
Through analysis, 31 embryos to be transplanted of the family No. 1-3 are diagnosed, 16 are chromosome insertion translocation carryovers or normal chromosome euploids, 10 are insertion translocation copy number monomer or trisomy variation (5 of the chromosome insertion translocation copy number monomer or trisomy variation (other chromosome aneuploidy abnormality), and 5 show variation unrelated to insertion translocation. Analyzing the 16 examples of euploid embryos by using fetal tissues or fetuses with abnormal insertion translocation copy number as references, and predicting each embryo by the distinguishing method described in the Biii) or the Biv), wherein the prediction results are consistent: 8 are embryos carried by insertion translocations, the remaining 8 chromosomes are perfectly normal, i.e. do not carry insertion translocations. And the predicted results of different insertion translocation breakpoint regions of each embryo have consistency. The information of the test results of family embryos of 1-2 is shown in Table 4, FIG. 4 shows the results of haplotype analysis of the receptor chromosome region across the breakpoint of the family reference sample of case 1 and the biopsy embryo, and FIG. 5 shows the results of haplotype analysis of the donor chromosome region outside the breakpoint of the family reference sample of case 1 and the biopsy embryo.
TABLE 4.1-3 family embryos Using the test results of the patent of the invention
Example 4: effect verification of embryo transplantation and embryo insertion translocation screening method
1. Embryo transfer
Embryos not carrying insertion translocations (family 1, 2-1 embryos and family 2, 1 embryos) referred to in table 4 of example 3 above were used for embryo transfer according to methods conventional in the art. After 2 frozen blastocysts are thawed, the blastocysts survive, after 2 transplantation cycles, the family 1 is successfully fertilized after the first transplantation, and the family 2 is successfully implanted after the transplantation, but the case is biochemical pregnancy through follow-up visits.
2. Collecting fetal umbilical cord blood for analyzing and verifying cell chromosome karyotype during amniotic fluid puncture or pregnancy in middle gestation period
After the embryo is successfully transplanted, the prediction accuracy of the method for identifying the chromosome insertion translocation carrying embryo and the normal embryo is verified by carrying out amniotic fluid puncture in the middle term of pregnancy or collecting the fetal umbilical cord blood karyotype analysis in the gestation period.
(1) 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) Preparation method of umbilical cord blood chromosome
The preparation of cord blood chromosomes was the same as the preparation of peripheral blood chromosomes in example 1 and will not be repeated here.
(3) Detection of aneuploidy and insert copy number variation in amniotic fluid cells or umbilical cord blood
The Illumina microarray gene chip was used for aneuploidy and insert copy number variation detection as described in example 1, and the description thereof is not repeated. Or a high-throughput sequencing CNV-seq detection method is used, and the specific experimental method is carried out according to the instruction, which is not described in detail herein. Table 5 shows specific information on the predicted results obtained using the method for identifying a chromosomal insertion translocation-carrying embryo and a normal embryo according to the present invention in embryos that have been successfully transplanted in case 1 pedigree and the results of genetic test verification of umbilical cord blood cells of newborns. Fig. 6 shows the karyotype detection results of umbilical cord blood chromosomes of the newborn after successful delivery of family embryo transplantation of case 1, and fig. 7 shows the detection results of the variation of the number of copies of umbilical cord blood chromosomes of the newborn, including chromosomal aneuploidy and insert monomer/trisomy.
TABLE 5 comparison of PGT embryo assay results with the post-transplantation neonatal umbilical cord blood cytogenetic validation results
As can be seen from Table 5, the results of the family haplotype prediction and the newborn umbilical cord blood cell genetic analysis are completely consistent after verification, and the method for identifying the chromosome insertion translocation carrying embryo and the normal embryo is proved to be accurate and reliable and can be practically applied to the clinical auxiliary reproduction.
SEQUENCE LISTING
<110> affiliated obstetrical and gynecological hospital of the university of Compound Dan; shanghai Jiai inheritance and sterility diagnosis and treatment center Co., Ltd
<120> a method for discriminating a chromosome insertion translocation carrying embryo from a normal embryo
<130> 1
<160> 6
<170> PatentIn version 3.3
<210> 1
<211> 404
<212> DNA
<213> Artificial
<220>
<223> MQ1
<400> 1
gcctgcccag acctagtaat tgctgtagtc ttttcctttt ggattcctag cagtggatgc 60
ttgaatacaa ataccaagtg ggctccagcc agtgagggta atagtgtaca gcagtaaact 120
gtctgttagc tgatgcttac atttcagaca aattgaggag gttgtcaaag gcattagctt 180
ctatcctttc cagggaatca atctgagaga tttcactagg gaagagagga gggggaaaaa 240
gagaaagaaa catgaaagaa atgaccgtgt ctgcatgatt atgagctatg tgtgtgaccc 300
aacaactggg gacaccactg tgtccccctg aaaagaacaa agatgtgcaa gttgtcccca 360
aatcatacag tgctaacatc tccctttagg agcactgatg ggca 404
<210> 2
<211> 744
<212> DNA
<213> Artificial
<220>
<223> MQ2
<400> 2
tttcgacatg gaggccactg gcttgccctt ctcccagccc aaggtcacgg agctgtgcct 60
gctggctgtc cacagatgtg ccctggagag cccccccacc tctcaggggc cacctcccac 120
agttcctcca ccaccgcgtg tggtagacaa gctctccctg tgtgtggctc cggggaaggc 180
ctgcagccct gcagccagcg agatcacagg tctgagcaca gctgtgctgg cagcgcatgg 240
gcgtcaatgt tttgatgaca acctggccaa cctgctccta gccttcctgc ggcgccagcc 300
acagccctgg tgcctggtgg cacacaatgg tgaccgctac gacttccccc tgctccaagc 360
agagctggct atgctgggcc tcaccagtgc tctggatggt gccttctgtg tggatagcat 420
cactgcgctg aaggccctgg agcgagcaag cagcccctca gaacacggcc caaggaagag 480
ctatagccta ggcagcatct acactcgcct gtatgggcag tcccctccag actcgcacac 540
ggctgagggt gatgtcctgg ccctgctcag catctgtcag tggagaccac aggccctgct 600
gcggtgggtg gatgctcacg ccaggccttt cggcaccatc aggcccatgt atggggtcac 660
agcctctgct aggaccaagc caagaccatc tgctgtcaca accactgcac acctggccac 720
aaccaggaac actagtccca gcct 744
<210> 3
<211> 518
<212> DNA
<213> Artificial
<220>
<223> MQ3
<400> 3
gggttgtccc ctggatatag gaggggtgag gtgggtcaga gggtgccggg aggggcctgg 60
atcagcactt caaaaggtgg gtgcccgcca cccaggggtg gccccaagga gaggagagga 120
gctggccatg tgcagagatg gggccctgtg atgcagacac acctcggtac cagttgcgca 180
gggaggtcat gacgaagaag cggatggcct ggttcgagcc ctgcttcagg acagtggctg 240
tgaggccctg gtacgtcccc ttcagccctg cgggaaggca ggcacggggt taccctgcag 300
cctctcaggc cccggttggg aattggtgtg tgtggggggt gggtgttgca caaagcccaa 360
ggcaggatgg gaggtgcagg cccttcccac cctggcccag gtccctgagc cctatcagga 420
aggtcgagtg gctcaccttg ttcccgcaca atctccctaa ccccgtggaa gaatcctctg 480
tacttggggt ttggggaggt ctggtcgtgg atgaactt 518
<210> 4
<211> 200
<212> DNA
<213> Artificial
<220>
<223> MQ4
<400> 4
tcaagtttcc tctcaccttt attctacttc ctcattaaca agtcatcggt ttgatacatt 60
aatcatggca aaaaaatcac agaggaagca ctaatctgtc atttttttct gtgacctgca 120
gactctttga cacccggtac agcatccggg agaatgggca gctcctcacc atcctgagtg 180
<210> 5
<211> 660
<212> DNA
<213> Artificial
<220>
<223> MQ5
<400> 5
gcgatcgatg cagtggacat ggtactcatg ggaacaaggt agtttacgaa gtttgttgcc 60
ttctgtatat tctgtaatgc aaacactaca ggtttttaat gcatcatttt caccaaaact 120
tctcattgcc aagttgtcaa tctgttcttt ggtgagtcct ctaggttggt catcatcatc 180
ctcatttaag aggaaaaact gagccaggct aaggaagggc aaagagccac tttcatcaaa 240
tgtgactggg gccctatgtc gaccctctcg cctggcacct gatgagcctg atgatgagct 300
tccttcatta ctgccttcaa ataaatctga gctagtttct gaactttcac caccggaact 360
ggaactagga ctggaactgg aacttgaact ggaactggaa ctcgaactgg aactggaact 420
cgaactggaa ccagaactac taccaccacc agaacctcct cttccactcc gtgactctgc 480
cctttccata tttcgatttg agactgagcc agtaggctct gagtcgctat cactgtacat 540
aaaatagctt aactcaccaa aacctgtcat tatctgcctt aacatggtct gaattgcaac 600
agatgtagtc tcacttaaac cagtatttaa gattctacga atgggaattc tgatggtact 660
<210> 6
<211> 484
<212> DNA
<213> Artificial
<220>
<223> MQ6
<400> 6
ctcaagacgt ggaggcatct gggcttagct gaggtcacac tactagtggg acctacagaa 60
aaacccaggt gtgtcaccag cagggcgggc accatctctg cagccgacac cagcttcccc 120
aacctccaga caggagaaag catgagggtc cccactgaaa ttctggctca cagagtcacc 180
tcctatgctg tggctttttt caatcccaga gtttgtccag cgaggcaaag acctggtcac 240
ggcgtctctg gctcaccagg tggagggaac ggcaaaactc acgctggccc aagaggagga 300
acagagaagc ttcctggctg aggcccagcc gactgctgac ccggaaaagt ttctcgaggt 360
gactcacatc cccagcctct gcacatgtgg gtgagccagt tgtagctctg ttcccgtgac 420
tgagcacggg acgccggagg tattcatcag gcatgaggtt atctgcctac ttcccatgtg 480
tcag 484
Claims (9)
1. A method of constructing a family haplotype for identifying a chromosomal insertion translocation-carrying embryo and a normal embryo, comprising the steps of:
1) genotyping of the sample: inserting chromosome into translocation patients, carrying out large-scale SNP genotype detection on both couples and at least one patient relative; the couple and the relatives of the couple are called as reference samples; preferably, the patient relative may be selected from the patient's parents, children, siblings, or couples who have chromosomal insertion translocations, abortions, induction of labor, or live births; more preferably, the patient relatives prefer patient parents, and may also be other relatives; preferably, the patient's relatives may be selected from relatives also carrying chromosomal insertion translocations, and may also be selected from normal chromosomal relatives;
2) determining the SNPs site of the information: the selection criteria of the information SNPs are as follows:
a) SNP sites that are heterozygous in a chromosomal insertion translocation patient, homozygous in its partner, and also homozygous in the patient's relatives when the patient's parents, children or siblings are used as a reference sample; selecting information SNPs covering the insertion translocation breakpoint at the chromosome 3, the insertion translocation chromosome and the normal homologous chromosome corresponding to the insertion translocation breakpoint, wherein at least 1 information SNP is selected in each Mb range of the chromosome; in the region covering the breaking point, the information SNPs are selected from the range of 1-5Mb upstream and downstream of the breaking point;
b) when the aborted, induced or live fetus or embryo with abnormal copy number (monomer or trisomy) of the insert is used as a reference sample, the SNP locus is heterozygous in a chromosome insertion translocation patient and homozygous in a mate thereof, so that the SNP locus is not required for a fetus or embryo sample, and the SNP locus covered in the insert is not used; selecting information SNPs covering the insertion translocation breakpoint of the receptor chromosome, the receptor chromosome and a normal homologous chromosome corresponding to the receptor chromosome, wherein at least 1 information SNP is selected in each Mb range of the chromosome; in the region covering the breaking point, the information SNPs are selected from the range of 1-5Mb upstream and downstream of the breaking point; selecting information SNPs on the donor chromosome (excluding the insert) and a normal homologous chromosome corresponding to the donor chromosome, which cover the two insertion translocation breakpoints of the donor chromosome, wherein at least 1 information SNP is selected in each Mb range; in the region covering the breaking point, the information SNPs are selected from the range of 1-5Mb upstream and downstream of the breaking point;
3) constructing a family haplotype: gathering the information SNPs sites determined by a) or b) in the step 2), obtaining the haplotypes of the whole chromosomes, which cover three break regions and are inserted into the translocation chromosome (acceptor and donor) and the homologous chromosomes thereof, by the family linkage analysis, and gathering the haplotypes of different chromosomes is called as the family haplotypes.
2. A construction system for identifying a family haplotype of a chromosomal insertion translocation carrying an embryo and a normal embryo, the system comprising software capable of computing processing sample data and hardware for carrying the software, characterized in that,
1) the system also comprises hardware for storing genotyping data for carrying out large-scale SNP genotype detection on both couples of a patient with chromosome insertion and translocation and at least one patient relative; the couple and the relatives of the couple are called as reference samples; preferably, the patient relative may be selected from the patient's parents, children, siblings, or couples who have chromosomal insertion translocations, abortions, induction of labor, or live births; more preferably, the patient relatives prefer patient parents, and may also be other relatives; preferably, the patient's relatives may be selected from relatives also carrying chromosomal insertion translocations, and may also be selected from normal chromosomal relatives;
2) determining the SNPs site of the information: the selection criteria of the information SNPs are as follows: a) SNP sites that are heterozygous in a chromosomal insertion translocation patient, homozygous in its partner, and also homozygous in the patient's relatives when the patient's parents, children or siblings are used as a reference sample; selecting information SNPs covering the insertion translocation breakpoint at the chromosome 3, the insertion translocation chromosome and the normal homologous chromosome corresponding to the insertion translocation breakpoint, wherein at least 1 information SNP is selected in each Mb range of the chromosome; in the region covering the breaking point, the information SNPs are selected from the range of 1-5Mb upstream and downstream of the breaking point;
b) when the aborted, induced or live fetus or embryo with abnormal copy number of the insert (monomer or trisomy) is used as a reference sample, the SNP locus which is heterozygous in a chromosome insertion translocation patient and homozygous in a mate is not required for a fetus or embryo sample, and the SNP locus covered in the insert is not used; selecting information SNPs covering the insertion translocation breakpoint of the receptor chromosome, the receptor chromosome and a normal homologous chromosome corresponding to the receptor chromosome, wherein at least 1 information SNP is selected in each Mb range of the chromosome; in the region covering the breaking point, the information SNPs are selected from the range of 1-5Mb upstream and downstream of the breaking point; selecting information SNPs on the donor chromosome (excluding the insert) and a normal homologous chromosome corresponding to the donor chromosome, which cover the two insertion translocation breakpoints of the donor chromosome, wherein at least 1 information SNP is selected in each Mb range; in the region covering the breaking point, the information SNPs are selected from the range of 1-5Mb upstream and downstream of the breaking point;
3) constructing a family haplotype: gathering the information SNPs sites determined by a) or b) in the step 2), obtaining the haplotypes of the whole chromosomes, which cover three break regions and are inserted into the translocation chromosome (acceptor and donor) and the homologous chromosomes thereof, by the family linkage analysis, and gathering the haplotypes of different chromosomes is called as the family haplotypes.
3. A system for identifying a chromosomal insertion translocation carrying embryo and a normal embryo, said system comprising software capable of computing processing sample data, and hardware for carrying said software,
1) the system also comprises hardware for storing genotyping data for carrying out large-scale SNP genotype detection on couples of chromosome insertion translocation patients, at least one patient relative and couples in vitro fertilization embryos of the chromosome insertion translocation patients; the couple and the relatives of the couple are called as reference samples; preferably, the patient relative may be selected from the patient's parents, children, siblings, or couples who have chromosomal insertion translocations, abortions, induction of labor, or live births; more preferably, the patient relatives prefer patient parents, and may also be other relatives; the in vitro fertilized embryo of a couple of a patient is called a pending sample;
2) the software determines the sites of the information SNPs according to the following rules: the selection criteria of the information SNPs are as follows:
a) SNP sites that are heterozygous in a chromosomal insertion translocation patient, homozygous in its partner, and also homozygous in the patient's relatives when the patient's parents, children or siblings are used as a reference sample; selecting information SNPs covering the insertion translocation breakpoint at the chromosome 3, the insertion translocation chromosome and the normal homologous chromosome corresponding to the insertion translocation breakpoint, wherein at least 1 information SNP is selected in each Mb range of the chromosome; in the region covering the breaking point, the information SNPs are selected from the range of 1-5Mb upstream and downstream of the breaking point;
b) when the aborted, induced or live fetus or embryo with abnormal copy number (monomer or trisomy) of the insert is used as a reference sample, the SNP locus is heterozygous in a chromosome insertion translocation patient and homozygous in a mate thereof, so that the SNP locus is not required for a fetus or embryo sample, and the SNP locus covered in the insert is not used; selecting information SNPs covering the insertion translocation breakpoint of the receptor chromosome, the receptor chromosome and a normal homologous chromosome corresponding to the receptor chromosome, wherein at least 1 information SNP is selected in each Mb range of the chromosome; in the region covering the breaking point, the information SNPs are selected from the range of 1-5Mb upstream and downstream of the breaking point; selecting information SNPs on the donor chromosome (excluding the insert) and a normal homologous chromosome corresponding to the donor chromosome, which cover the two insertion translocation breakpoints of the donor chromosome, wherein at least 1 information SNP is selected in each Mb range; in the region covering the breaking point, the information SNPs are selected from the range of 1-5Mb upstream and downstream of the breaking point;
3) the software compares the chromosome haplotype information (gene analysis data) in a sample to be determined with the haplotype information (genotyping data) of a reference sample in a family haplotype, focuses on the haplotype information of the upper and lower reaches of the insertion breakpoint region (three positions) in the family haplotype, and judges whether the homologous recombination occurs in the chromosome insertion breakpoint region or not through the haplotype of the whole chromosome:
i) when referring to relatives with the same insertion translocation as the carrier:
a. if the chromosome of the sample to be determined is not recombined in a region corresponding to the chromosome insertion translocation breakpoint in the family haplotype, carrying an embryo for insertion translocation when the haplotype information of the region of the sample to be determined is consistent with the haplotype information of a chromosome insertion translocation patient in the reference sample and the haplotype information of a relative having the same insertion translocation with the insertion translocation patient; when the haplotype information of the region of the undetermined sample is consistent with that of the chromosome insertion translocation patient in the reference sample, but is inconsistent with that of the relatives with the same insertion translocation of the insertion translocation patient, the embryo is a completely normal embryo with chromosomes;
b. if homologous recombination occurs in the chromosome of the sample to be determined corresponding to the region of the chromosome insertion translocation breakpoint in the family haplotype, the judgment standard of the insertion translocation carried or the chromosome complete normal embryo is opposite to i) a;
ii) when referring to a normal chromosome relative:
a. if the region of the chromosome of the sample to be determined, which corresponds to the chromosome insertion translocation breakpoint in the family haplotype, is not recombined, when the haplotype information of the region of the sample to be determined is consistent with the haplotype information of the chromosome insertion translocation patient in the reference sample and the haplotype information of the normal relatives of the chromosome, the embryo is the embryo with the completely normal chromosome; when the haplotype information of the region of the undetermined sample is consistent with the haplotype information of the chromosome insertion translocation patient in the reference sample but is not consistent with the haplotype information of the normal relatives of the chromosome, the insertion translocation carries the embryo;
b. if homologous recombination occurs in the region of the chromosome of the sample to be determined, which corresponds to the chromosome insertion translocation breakpoint in the family haplotype, the judgment standard of the embryo carrying the insertion translocation or completely normal chromosome is opposite to ii) a;
iii) when reference is made to a fetal tissue or embryo sample with a missing copy number of the inserted translocation fragment:
a. if the chromosome of the sample to be determined is not recombined in a region corresponding to the chromosome insertion translocation breakpoint in the family haplotype, when the haplotype information of the region of the sample to be determined is consistent with the haplotype information of the chromosome insertion translocation patient in the reference sample and the chromosome insertion translocation patient in the fetal tissue or the chromosome breakpoint region of the donor of the embryo sample, but is not consistent with the haplotype information of the acceptor chromosome, the sample to be determined carries the embryo for insertion translocation; when the haplotype information of the region of the undetermined sample is consistent with the haplotype information of the chromosome insertion translocation patient in the reference sample and the receptor chromosome of the fetal tissue or the embryo sample but is not consistent with the haplotype information of the donor chromosome, the embryo with completely normal chromosome is obtained;
b. if homologous recombination occurs in the region of the chromosome of the sample to be determined, which corresponds to the chromosome insertion translocation breakpoint in the family haplotype, the judgment standard of the insertion translocation carried or the chromosome complete normal embryo is opposite to the criterion of iii) a;
iv) when reference is made to a fetal tissue or embryonic sample in which the copy number of the translocated fragment inserted is repeated:
a. if the chromosome of the sample to be determined is not recombined in a region corresponding to the chromosome insertion translocation breakpoint in the family haplotype, when the haplotype information of the region of the sample to be determined is consistent with the haplotype information of the chromosome insertion translocation patient in the reference sample and the chromosome insertion translocation patient in the fetal tissue or the donor chromosome breakpoint region of the embryo sample, but is not consistent with the acceptor chromosome haplotype information, the embryo with the completely normal chromosome is obtained; when the haplotype information of the region of the undetermined sample is consistent with the haplotype information of the chromosome insertion translocation patient in the reference sample and the receptor chromosome of the fetal tissue or the embryo sample but is not consistent with the haplotype information of the donor chromosome, carrying an embryo for insertion translocation;
b. if homologous recombination occurs in the region of the chromosome of the sample to be determined, which corresponds to the chromosome insertion breakpoint in the family haplotype, the judgment standard of the embryo with insertion translocation or completely normal chromosome is opposite to iv) a;
the chromosome carrying the insert, called the recipient chromosome, and the chromosome providing the insert, called the donor chromosome.
4. The method of any one of claims 1 to 3, wherein step 1) said large scale SNP genotyping assay covers 23 pairs of chromosomes; the large-scale SNP genotype detection method preferably selects a gene chip and gene sequencing.
5. The method according to any one of claims 1 to 3, wherein the test sample for the chromosomal insertion translocation patient, the patient's couple, parent, daughter, sibling is derived from somatic cells, preferably peripheral blood; the fetal sample can be derived from skin tissue, villus tissue of aborted or induced fetus, or from peripheral blood tissue of live-born diseased fetus.
6. The method according to any one of claims 1 to 3, wherein in step 2) a) the chromosomal insertion translocation breakpoint region is overlaid and the informative SNPs are selected from the range of 2-4Mb upstream and downstream of the chromosomal translocation breakpoint, preferably from the range of 2Mb upstream and downstream of the chromosomal insertion translocation breakpoint; covering the recipient chromosome insertion translocation breakpoint region and the donor chromosome two insertion translocation breakpoints to the insert outer region in the step 2) b), wherein the information SNPs are selected from the range of 2-4Mb, preferably from the range of 2Mb, from the upstream and downstream of the recipient chromosome insertion translocation breakpoint region and from the donor chromosome two insertion translocation breakpoints to the insert outer region.
7. The method according to any one of claims 1 to 6, wherein at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 informative SNPs per Mb of chromosome are selected in step 2).
8. The method of claim 3, wherein 1-10 cells are obtained from a biopsy as a test sample of the in vitro fertilized embryo of step 1) when the embryo develops to days 3-7; preferably cells derived from an embryonic blastomere biopsy or blastocyst trophectoderm biopsy.
9. The method of claim 8, wherein the cells obtained from the biopsy are lysed and subjected to whole genome amplification; the whole genome amplification method may be selected from the MDA method, the MALBAC method, or other whole genome amplification methods.
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| CN117925820A (en) * | 2024-01-22 | 2024-04-26 | 广州菲尔医学检验有限公司 | Method for detecting variation before embryo implantation |
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| CN115433777A (en) * | 2022-10-26 | 2022-12-06 | 北京中仪康卫医疗器械有限公司 | Integrated identification method for CNV, SV and SGD abnormalities and abnormal sources of embryos |
| CN117925820A (en) * | 2024-01-22 | 2024-04-26 | 广州菲尔医学检验有限公司 | Method for detecting variation before embryo implantation |
| CN117925820B (en) * | 2024-01-22 | 2024-08-02 | 广州菲尔医学检验有限公司 | Method for detecting variation before embryo implantation |
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