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

A chromosome and fetal technology, applied in the field of genetic diagnosis of chromosome variation, can solve problems such as no detection of NIPT technology

Pending Publication Date: 2022-05-13
THE OBSTETRICS & GYNECOLOGY HOSPITAL OF FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Unfortunately, so far there is no NIPT technology for the detection of chromosome balance structural aberrations

Method used

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  • Method for detecting balance structure variation of fetal chromosome through free DNA of peripheral blood of pregnant woman
  • Method for detecting balance structure variation of fetal chromosome through free DNA of peripheral blood of pregnant woman
  • Method for detecting balance structure variation of fetal chromosome through free DNA of peripheral blood of pregnant woman

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0111] Example 1: Collection of both spouses and reference samples of chromosomal structural variation carriers

[0112] 6 couples carrying chromosomal structural variants, including balanced chromosomal translocation and chromosomal inversion, were recruited from Jiai Reproductive Center of Obstetrics and Gynecology Hospital Affiliated to Fudan University. Ethics committee approval.

[0113] All couples have a history of recurrent spontaneous abortion or chromosomal abnormalities. One of the couples carrying balanced chromosome translocations or inversions is referred to as "patient" and the other as "patient's spouse" in the following text. At the same time of recruitment, draw about 10ml of peripheral blood from each patient couple and relatives of patients (parents and children of patients are preferred, other relatives are also acceptable). A part of the peripheral blood was used for lymphocyte culture and karyotype analysis; the other part of the peripheral blood was ex...

Embodiment 2

[0134] Example 2: Construction of the haplotypes of the husband and wife and the fetus

[0135] 1. Genomic DNA extraction from peripheral blood

[0136] Preliminary preparations: 1.5ml EP tube, water bath, centrifuge, shaker and other consumables, well marked, protease provided in the kit, AW1 and AW2 add corresponding volume of absolute ethanol before use. If BufferAL precipitates, warm at 56°C and shake gently to dissolve. Before using BufferAW1 and BufferAW2 for the first time, you need to add the corresponding volume of absolute ethanol according to the label on the reagent bottle to make it a working solution. Turn on the water bath and set at 56 °C.

[0137] The experimental steps are as follows:

[0138] (1) Add proteinase K 20μl, anticoagulant blood 200μl, buffer AL 200μl, vortex and incubate at 56°C for 10 minutes.

[0139] (2) Add 200 μl of absolute ethanol, vortex and mix to make a homogeneous solution.

[0140] (3) Transfer the mixed solution to the DNeasy Min...

Embodiment 3

[0157] Example 3: Screening of Fetal Chromosomal Structural Variations

[0158] According to the haplotype of the fetus, the karyotype of the fetus is predicted. The prediction principles are:

[0159] When the relatives of the carrier who carry the chromosomal rearrangement are used as the reference sample:

[0160] a. If there is no recombination in the breakpoint region of fetal chromosomal structure rearrangement

[0161] When the haplotype information of the fetal chromosomal structure rearrangement breakpoint region is consistent with the haplotype information of the reference sample, it is diagnosed as a chromosomal rearrangement carrier fetus; when they are inconsistent, it is diagnosed as a non-chromosomal structure rearrangement carrier fetus, that is, Chromosomally normal fetus.

[0162] b. If homologous recombination occurs in the region of the fetal chromosome breakpoint, the judgment standard is the opposite of a.

[0163] Table 3 shows the prediction results...

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Abstract

The invention discloses a method for detecting fetal chromosome balance structure variation through pregnant woman peripheral blood free DNA. The method comprises construction of a core family haplotype model and fetal structure variation detection. The method comprises the following steps: collecting chromosome structure rearrangement variation family carrying Fuffles, carrying out SNP (Single Nucleotide Polymorphism) genotyping on the family, defining haplotypes of structure rearrangement chromosomes and structure normal chromosomes, and constructing a whole genome haplotype model. Free DNA of peripheral blood of a pregnant woman is captured and sequenced, the genotype and haplotype of a fetus in the free DNA are calculated and analyzed according to a hidden Markov model, and embryo chromosome structure rearrangement is predicted by analyzing whether the fetus carries the haplotype near a structure rearrangement chromosome breaking point area or not. According to the invention, detection of fetal balance chromosome structure abnormity through the maternal peripheral blood cfDNA is provided for the first time, and important guidance is provided for prevention and diagnosis of chromosome diseases.

Description

technical field [0001] The invention belongs to the field of genetic diagnosis of chromosomal variation, in particular to a method for detecting balanced structural variation of fetal chromosomes through free DNA in the peripheral blood of pregnant women. Background technique [0002] Genetic diseases are an important cause of birth defects and fertility disorders, and pose a serious threat to human health and life. Clinically common genetic diseases include single gene diseases and chromosomal diseases, and chromosomal diseases include abnormal chromosome number and abnormal chromosome structure. For preventing the birth of children with genetic diseases, invasive prenatal diagnosis is still the clinical gold standard, but there is a certain risk of abortion infection. In recent years, with the rapid development of non-invasive prenatal testing (NIPT), the risk of genetic diseases in children can be assessed based on the fetal free DNA (cell-free fetal DNA, cffDNA) in the ...

Claims

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Application Information

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IPC IPC(8): C12Q1/6888C12Q1/6869G16B20/20G16B30/10G16B50/00
CPCC12Q1/6888C12Q1/6869G16B20/20G16B30/10G16B50/00C12Q2600/156C12Q2535/122C12Q2537/165
Inventor 张硕徐丛剑
Owner THE OBSTETRICS & GYNECOLOGY HOSPITAL OF FUDAN UNIV
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