RU2636618C1 - Method and test system for determination of share of fetal dna in plasma of pregnant woman using high-productive sequencing methods - Google Patents

Abstract

FIELD: medicine.SUBSTANCE: invention is intended to determine the share of fetal DNA in the plasma of a pregnant woman using high-productive sequencing methods. 53 single nucleotide polymorphisms (SNP) located on 1-12 chromosomes with a heterozygote frequency of 40-50%, and specific primers to them are used. The share of fetal DNA is determined as a median of the fetal DNA share value for all informative SNPs.EFFECT: effective determination of the share of fetal DNA in the blood plasma of a pregnant woman, regardless of the sex of the fetus.4 tbl, 2 ex

Description

The present method relates to the field of molecular genetics, obstetrics. The proposed method allows to determine the proportion of fetal DNA in the blood plasma of a pregnant woman, regardless of the gender of the fetus.

The level of technology.

In the blood plasma of a pregnant woman, in addition to free circulation DNA (ssDNA) of the woman herself, there is also DNA belonging to the fetus. This is used to screen for a number of genetic abnormalities, such as changes in the number of chromosomes (aneuploidy). When conducting such studies, an important parameter affecting the sensitivity of the method is the proportion of fetal DNA among all cfDNA. In case of pregnancy with a male fetus, the representation of fragments of the Y chromosome can be used to determine the proportion of fetal DNA, which is not a technical difficulty. In the case of pregnancy with a female fetus, the determination of the proportion of fetal DNA requires additional research.

Currently, several methods are used in the world to determine the proportion of fetal DNA in the total ssDNA pool. All of them are based on the distribution of fragment counts for which their origin can be determined. For example, the use of fragments specific for the Y chromosome, which is possible only for a male fetus or regardless of gender: differently labeled cfDNA fragments, single nucleotide polymorphisms, differences in fetal and maternal DNA in length.

There is a patent (Methods for non-invasive prenatal ploidy calling US 2011/0288780 A1) that describes a procedure for determining the presence of fetal aneuploidy using about 20,000 polymorphic loci, including the step of determining the proportion of fetal DNA. However, the accuracy of estimating the fraction of fetal DNA is achieved using a significant number of studied loci.

There is a patent (Resolving genome fractions using polymorphism counts US 21012/0264121 A1) that describes the procedure for determining the DNA fraction of an impurity, including the fraction of fruit DNA, based on counting the frequency of occurrence of polymorphic alleles, including SNPs. The method also involves the use of a large number of loci.

When using single nucleotide polymorphisms to determine the proportion of fetal DNA, informative (suitable for determining the proportion of fetal DNA) will be the points at which the mother has the homozygous genotype AA, and the fetus is heterozygous AB. In this case, the percentage of fruit DNA can be defined as the double ratio of the number of readings of the B allele to all readings: The percentage of fruit DNA = 2 * B / (A + B).

The greatest probability is to get an informative version of SNPs with the same ratio of the frequencies of occurrence of both alleles. To select SNPs with an appropriate frequency of occurrence, Hapmap data from large-scale population studies (International, T. & Consortium, N. The International NarMar Project Nature 426, 789-796 (2003)) can be used.

The objective of the invention is to determine the proportion of fetal DNA in the blood plasma of a pregnant woman, regardless of the gender of the fetus.

This invention differs from those listed above by the use of a small number of SNPs with a high frequency of occurrence of heterozygotes to determine the proportion of fetal DNA and a set of primers.

Description of the invention

To determine the proportion of fetal DNA, 53 single nucleotide polymorphisms (SNPs) are used located on 1-12 chromosomes with a heterozygous frequency of 40-50%. A list of SNPs of their positions in the genome and the frequency of occurrence in the Russian population is shown in table 1. The list of primers is shown in table 2.

The method is as follows.

1. Peripheral blood sampling from a vein in a pregnant woman

2. Using sequential centrifugation or any other method adapted for this study, the plasma is separated.

3. Extracellular DNA containing maternal and fetal fractions is released from plasma.

4. Spend the amplification of DNA fragments, including SNPs.

5. Sequences (sequencing) of the obtained DNA fragments are determined using high-throughput sequencing methods.

6. For each of the SNPs, the representation of both alleles is calculated. SNPs with a fraction of less than the presented allele from 0.0075 to 0.20 and a total coverage of at least 1000 fragments are considered informative. And for each SNP, the fraction of fruit DNA is calculated by the formula: 2 * B / (A + B), where A is the proportion of the more presented allele, and B is the fraction of the less represented allele.

7. The percentage of fruit DNA is defined as the median of the percentage of fruit DNA for all informative alleles.

Implementation of the invention (example)

The results of determining the fraction of fetal DNA using SNPs were compared with the results of determining the fraction of fetal DNA from the Y chromosome.

Sample 1: the proportion of fruit DNA on the Y chromosome - 14%.

Table 3 shows the results of calculating the frequency of occurrence of alleles for each SNP. Only 13 informative SNPs. The proportion of fruit DNA, determined on the basis of sequencing of SNP sequences, 13%.

Sample 2: the proportion of fruit DNA on the Y chromosome is 11%.

Table 4 shows the results of calculating the frequency of occurrence of alleles for each SNP. A total of 17 informative SNPs. rs7696439 is excluded from consideration due to low coverage - 204 fragments. The proportion of fruit DNA, determined on the basis of sequencing of SNP sequences, 11.4%.

Claims (4)

The method for determining the fraction of fetal DNA in the blood plasma of a pregnant woman using high-throughput sequencing methods is different in that they use 53 single nucleotide polymorphisms (SNPs) located on 1-12 chromosomes with a heterozygous frequency of 40-50%, and specific primers for them:

, at the same time, for each of the SNPs, the representation of both alleles is calculated, SNPs with a fraction of less than the presented allele from 0.0075 to 0.20 and a total coverage of at least 1000 fragments are considered informative; for each informative SNP, the fraction of fetal DNA is calculated by the formula: 2 * B / (A + B), where A is the proportion of the more represented allele, and B is the fraction of the less represented allele; the fraction of fetal DNA is defined as the median of the fraction of fetal DNA for all informative SNPs.