Method for detecting fetal thalassemia pathogenic gene and kit

Abstract

The invention discloses a method for detecting a fetal thalassemia pathogenic gene, which comprises the following steps of: (1) screening SNP sites, wherein the SNP sites are used for designing a primer pool of the thalassemia gene in an amplification genome and for capturing the probe of the thalassemia gene of the free DNA in the plasma of a pregnant woman; (2) extracting the free DNA in the plasma of the pregnant woman and the whole blood genomic DNA of the father, the mother and a born sibling and constructing a corresponding DNA library, and carrying out template preparation and enrichment; (3) sequencing the free DNA and the whole blood genomic DNA library in step (2); (4) constructing the haploid genotype of the SNP sites on the thalassemia gene, combining the sequencing informationof the free DNA and the whole blood genomic DNA library, analyzing the genetic condition of the parent source and the genetic condition of the parent source, so as to determine the corresponding genotype of the SNP sites of the fetal. According to the method, target area capture and high-throughput sequencing technology are used, so that the noninvasive antepartum detection of the thalassemia isrealized; the required sample amount is small; on the basis of detecting the mutation of the parent source, the method can realize the detection of the gene mutation of the maternal source of the fetal.

Description

technical field

[0001] The invention relates to the technical field of gene detection, in particular to a method and a kit for detecting fetal thalassemia-causing genes. Background technique

[0002] Thalassemia (referred to as thalassemia) is one of the high-incidence genetic diseases in the world. It is due to the mutation or deletion of human α, β-globin gene, which leads to the imbalance of α, β-globin peptide chain synthesis rate, which causes hemolysis. sexual anemia. The two common types of thalassemia are α-thalassemia and β-thalassemia, α-thalassemia-related genes are HBA2 and HBA1, and β-thalassemia-related genes are HBB. Thalassemia is concentrated in tropical and subtropical regions, mostly in Mediterranean countries, followed by the Middle East, India, Pakistan, Southeast Asia, southern China and North Africa. The United States is an immigrant country, and its incidence is also relatively high. The provinces south of the Yangtze River in my country are high-in...

Examples

Embodiment 1

[0044] Construction of embodiment 1 haplotype (i.e. haploid genotype)

[0045] (1) Thalassemia primer and probe design

[0046] Design region for α-thalassemia (chr16:60022-2233661): downstream (10Kb) + HBA gene + upstream (10Kb), and design region for β-thalassemia (chr11:3236690-7177304): downstream (10Kb) + HBB gene + upstream (10Kp), and then use the dbSNP database to screen out 195 α-thalassemia SNP sites and 275 β-thalassemia SNP sites. Among them, the SNP site screening conditions: the SNP site has MAF>=20% at the same time in the Thousand Genomes Chinese Southerners and Northerners database; the 100bp sequence above and below the SNP site is a specific region and has no homology on the genome; and 45 %<GC<70% SNP sites do not have 3 consecutive identical bases. According to the SNP sites screened above, the amplification primer pair was designed through the Ampliseq Designer website (https: / / www.ampliseq.com / login / login.action), and the Agilent SureDesign website (ht...

Embodiment 3

[0072] Embodiment 3 maternal genetic situation analysis

[0073] Based on the analysis of paternal inheritance, the analysis of maternal inheritance was carried out. Select the locus (MN) whose genotype is homozygous father (MM) and heterozygous mother (MN), and determine whether the maternal pathogenic haplotype is inherited according to RHDO-SPRT, wherein the SPRT curve is calculated according to the following formula:

[0074]

[0075] in,

[0076] Upper boundary and Lower boundary refer to the upper bound and the lower bound respectively. In this detection method, the inventor defines haplotype 1 as the upper bound, and defines haplotype 2 as the lower bound. Use the SPRT algorithm to calculate the sum of the sum of the variation frequencies of the selected sites corresponding to the depth. If the screened loci are all between the upper and lower bounds, then it is impossible to tell which haplotype the fetus has inherited from the mother; if one or more of the acc...

Embodiment 4

[0078] Example 4 Test results of 5 thalassemia families

[0079] (1) Thalassemia test results

[0080] Families 1-5 were detected by the method of Example 2, and the haplotype construction results of families 1-5 are shown in Table 3-7. According to Mendel's law of inheritance, combined with family sample phenotype and known mutation type information, the pathogenic mutation is associated with the haplotype (for example: the father's genotype is MN, the mother's genotype is MN, and the child's genotype is NN (M is the disease-causing locus, N is the normal locus. M and N represent the bases A, T, C, G), and the child has inherited the father's haplotype 1 and the mother's haplotype 1 respectively, then The father's pathogenic locus M is on haplotype 2, and the mother's pathogenic locus M is on haplotype 2). Table 8 shows the results of the association of pathogenic loci in the father and mother of families 1-5 with haplotypes.

[0081] Table 3 Haplotype construction results...

technical field

[0001] The invention relates to the technical field of gene detection, in particular to a method and a kit for detecting fetal thalassemia-causing genes. Background technique

[0002] Thalassemia (referred to as thalassemia) is one of the high-incidence genetic diseases in the world. It is due to the mutation or deletion of human α, β-globin gene, which leads to the imbalance of α, β-globin peptide chain synthesis rate, which causes hemolysis. sexual anemia. The two common types of thalassemia are α-thalassemia and β-thalassemia, α-thalassemia-related genes are HBA2 and HBA1, and β-thalassemia-related genes are HBB. Thalassemia is concentrated in tropical and subtropical regions, mostly in Mediterranean countries, followed by the Middle East, India, Pakistan, Southeast Asia, southern China and North Africa. The United States is an immigrant country, and its incidence is also relatively high. The provinces south of the Yangtze River in my country are high-in...

Examples

Embodiment 1

[0044] Construction of embodiment 1 haplotype (i.e. haploid genotype)

[0045] (1) Thalassemia primer and probe design

[0046] Design region for α-thalassemia (chr16:60022-2233661): downstream (10Kb) + HBA gene + upstream (10Kb), and design region for β-thalassemia (chr11:3236690-7177304): downstream (10Kb) + HBB gene + upstream (10Kp), and then use the dbSNP database to screen out 195 α-thalassemia SNP sites and 275 β-thalassemia SNP sites. Among them, the SNP site screening conditions: the SNP site has MAF>=20% at the same time in the Thousand Genomes Chinese Southerners and Northerners database; the 100bp sequence above and below the SNP site is a specific region and has no homology on the genome; and 45 %<GC<70% SNP sites do not have 3 consecutive identical bases. According to the SNP sites screened above, the amplification primer pair was designed through the Ampliseq Designer website (https: / / www.ampliseq.com / login / login.action), and the Agilent SureDesign website (ht...

Embodiment 3

[0072] Embodiment 3 maternal genetic situation analysis

[0073] Based on the analysis of paternal inheritance, the analysis of maternal inheritance was carried out. Select the locus (MN) whose genotype is homozygous father (MM) and heterozygous mother (MN), and determine whether the maternal pathogenic haplotype is inherited according to RHDO-SPRT, wherein the SPRT curve is calculated according to the following formula:

[0074]

[0075] in,

[0076] Upper boundary and Lower boundary refer to the upper bound and the lower bound respectively. In this detection method, the inventor defines haplotype 1 as the upper bound, and defines haplotype 2 as the lower bound. Use the SPRT algorithm to calculate the sum of the sum of the variation frequencies of the selected sites corresponding to the depth. If the screened loci are all between the upper and lower bounds, then it is impossible to tell which haplotype the fetus has inherited from the mother; if one or more of the acc...

Embodiment 4

[0078] Example 4 Test results of 5 thalassemia families

[0079] (1) Thalassemia test results

[0080] Families 1-5 were detected by the method of Example 2, and the haplotype construction results of families 1-5 are shown in Table 3-7. According to Mendel's law of inheritance, combined with family sample phenotype and known mutation type information, the pathogenic mutation is associated with the haplotype (for example: the father's genotype is MN, the mother's genotype is MN, and the child's genotype is NN (M is the disease-causing locus, N is the normal locus. M and N represent the bases A, T, C, G), and the child has inherited the father's haplotype 1 and the mother's haplotype 1 respectively, then The father's pathogenic locus M is on haplotype 2, and the mother's pathogenic locus M is on haplotype 2). Table 8 shows the results of the association of pathogenic loci in the father and mother of families 1-5 with haplotypes.

[0081] Table 3 Haplotype construction results...

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