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Method of predicting gestational related diseases based on peripheral blood free DNA high-throughput sequencing

A high-throughput, peripheral blood technology, applied in biochemical equipment and methods, genomics, sequence analysis, etc., can solve the problems of clinical application limitations, easy sample degradation, and high extraction costs

Active Publication Date: 2019-12-17
SOUTHERN MEDICAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, the free fetal DNA in the peripheral blood of pregnant women has achieved certain results in the prenatal diagnosis of birth defects, but still faces many problems, such as: the free fetal DNA in the peripheral blood of pregnant women usually only accounts for 5-20% of the total maternal DNA. %, resulting in a low positive rate; the prenatal screening of fetal free DNA in the peripheral blood of pregnant women is only suitable for a small number of types of birth defects, more birth defects caused by complex factors such as polygenes, and more pregnant women during pregnancy Related diseases, not yet applicable
Although the detection of free RNA in peripheral blood can directly detect the expression level of some genes, it faces shortcomings such as easy degradation of samples, high extraction costs, and unstable quantification, and its clinical application is relatively limited.

Method used

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  • Method of predicting gestational related diseases based on peripheral blood free DNA high-throughput sequencing
  • Method of predicting gestational related diseases based on peripheral blood free DNA high-throughput sequencing
  • Method of predicting gestational related diseases based on peripheral blood free DNA high-throughput sequencing

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] 1. Experimental method

[0059] (1) Collect free peripheral blood DNA, peripheral blood leukocyte DNA and RNA from the same sample. Micrococcal Nuclease (MNase) was used to treat peripheral blood leukocyte DNA to obtain the DNA sequence bound to nucleosomes. Perform high-throughput sequencing of free peripheral blood DNA, peripheral blood leukocyte RNA, and peripheral blood leukocyte nucleosome-bound DNA, compare the sequencing results with the genome sequence map, and calculate the transcription start sites from all genes in the same sample The number of regional DNA fragments, the number of nucleosome-bound DNA fragments, and the number of RNA fragments of the gene to be tested.

[0060] This step is specifically to determine where the DNA or RNA fragment comes from on the chromosome: after DNA double-end sequencing (alternatively, single-end sequencing can also be used), the sequences at both ends can be compared with the human genome standard sequence 37.1( http: / ...

Embodiment 2

[0075] Based on the above research results of genes, the present invention uses machine learning algorithms to provide a relatively non-invasive and economical and convenient early prediction method for pregnancy-related diseases through the optimal combination of different differential genes, which can be used for pre-eclampsia, gestational diabetes, fetal growth disorders Early detection and screening of pregnancy-related diseases such as limited and macrosomia.

[0076] Specifically, a pregnancy-related disease prediction model based on high-throughput sequencing of peripheral blood cell-free DNA includes three modules:

[0077] (1) High-throughput sequencing and analysis module for free peripheral blood DNA of samples to be tested:

[0078] Perform high-throughput sequencing of free peripheral blood DNA of the sample to be tested, compare the sequencing results with the genome sequence map, and calculate the number of DNA fragments from the transcription start site region of...

Embodiment 3

[0101] Embodiment 3 detection example

[0102] 1. Example of gestational diabetes detection

[0103] Operate according to the method of Example 2. In step 2, the total number of aligned sequences of the sample is counted. In this example, sample 1 and sample 2 are 69479 and 57037, respectively. Calculate the number of DNA fragments in the region of the transcription start site of the gene to be tested in the same sample, and use Formula 1 to correct the abundance of DNA fragments. Table 3 is an example of the calculation of the abundance of DNA fragments in the region of the transcription start site of the gene to be tested in two samples:

[0104] table 3

[0105]

[0106] Use Equation 2 to calculate the risk of developing gestational diabetes mellitus. An example calculation is as follows:

[0107] Sample 1 (pre-onset sample with confirmed gestational diabetes):

[0108] logit(Y)=0.957+0.565×CC2D2B–1.060×NAT10–1.070×SIPA1–0.620×ZNF565–0.805×ZNF552–0.367×WDR35+0.559×M...

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Abstract

The invention discloses a method of predicting gestational related diseases based on pregnant woman peripheral blood free DNA high-throughput sequencing. A research discovers that the distribution condition of the pregnant woman peripheral blood free DNA in a gene transcription starting site region can reflect the physiological states of the pregnant woman and the fetus; the serum free DNA abundance based on the gene transcription starting site area has significant difference between a patient with a gestational related disease and a healthy pregnant woman, and can effectively predict the onset of the gestational related disease. A gestational related disease screening prediction model based on peripheral blood free DNA detection is constructed; the method can predict the onset of the gestational related disease before the clinical symptoms of the gestational related diseases occur, is a non-invasive, economical, convenient and accurate method for early prediction of the gestational related diseases, and has a good application prospect in development of prediction and screening products for the gestational related diseases.

Description

technical field [0001] The invention belongs to the technical field of disease detection products. More specifically, it relates to a method for predicting pregnancy-related diseases based on high-throughput sequencing of cell-free peripheral blood DNA. Background technique [0002] Diseases related to pregnancy can affect a series of physiological processes of pregnancy, harm the health of pregnant women and fetuses, and even endanger the lives of pregnant women and fetuses in severe cases. Some pregnancy-related diseases have a high incidence frequency in the population, including preeclampsia (incidence 3-8%), gestational diabetes (5-10%), fetal growth restriction (5-10%), macrosomia children (7 ~ 12%) and so on. [0003] At present, according to the clear diagnostic indicators of these pregnancy-related diseases, the diagnosis can be made when the fetus is close to maturity or has been delivered. The diagnosis time of fetal growth restriction was after the birth of th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G16B30/10G16B25/10G16B20/20G16B5/00C12Q1/6883C12Q1/6869
CPCG16B5/00G16B30/10G16B25/10G16B20/20C12Q1/6883C12Q1/6869C12Q2600/158C12Q2535/122C12Q2537/165
Inventor 杨学习韩博炜郭智伟吴英松
Owner SOUTHERN MEDICAL UNIVERSITY
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