CN113106155A - Application of single nucleotide polymorphism rs925368 in screening of leprosy patients - Google Patents

Application of single nucleotide polymorphism rs925368 in screening of leprosy patients Download PDF

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CN113106155A
CN113106155A CN202110600172.2A CN202110600172A CN113106155A CN 113106155 A CN113106155 A CN 113106155A CN 202110600172 A CN202110600172 A CN 202110600172A CN 113106155 A CN113106155 A CN 113106155A
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张福仁
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Dermatology Hospital Affiliated To Shandong First Medical University
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Abstract

The invention discloses an application of single nucleotide polymorphism rs925368 in screening of leprosy patients. The technical scheme protected by the invention is the application of a substance for detecting the polymorphism or genotype of rs925368 in a human genome in preparing products for screening leprosy patients and the application of a substance for detecting the polymorphism or genotype of rs925368 in a human genome in preparing products for detecting single nucleotide polymorphism related to leprosy. The substance for detecting the polymorphism or the genotype of the rs925368 can be combined with other substances (such as substances for detecting other single nucleotide polymorphisms or genotypes related to leprosy) to prepare a product for screening leprosy patients.

Description

Application of single nucleotide polymorphism rs925368 in screening of leprosy patients
Technical Field
The invention relates to the application of single nucleotide polymorphism rs925368 in screening leprosy patients in the field of biotechnology.
Background
Leprosy, also known as Hansen's disease, is an infectious disease caused by mycobacterium leprosum (m.leprae) that primarily invades the skin and peripheral nerves. Despite the effective treatment currently available, leprosy remains a major cause of teratogenicity and of social problems in some countries, particularly developing countries. To understand the genetic basis of leprosy susceptibility, the genome-wide association analysis (GWAS) of leprosy locates 17 common variant sites, revealing the role of innate immunity and adaptive immunity in leprosy. However, these common risk variant sites are mostly located in non-coding regions and can only partially account for the risk of leprosy. Currently, there is no systematic study of variations in protein coding regions, particularly low frequency variations and rare variations. These variations have been shown to be associated with genetic susceptibility to some diseases.
Disclosure of Invention
The invention aims to solve the technical problem of how to screen leprosy patients and detect leprosy susceptibility.
In order to solve the technical problem, the invention firstly provides any one of the following purposes:
A1) the application of the substance for detecting the polymorphism (namely allele) or genotype of rs925368 in the human genome in preparing and screening products for leprosy patients;
A2) the application of the substance for detecting the polymorphism (namely allele) or genotype of rs925368 in human genome in preparing the product for detecting leprosy susceptibility;
A3) the application of the substance for detecting the polymorphism (namely allele) or genotype of rs925368 in human genome in preparing products for detecting the single nucleotide polymorphism related to leprosy;
A4) the application of the substance for detecting the polymorphism (namely allele) or genotype of rs925368 in human genome in the preparation of products for identifying or assisting in identifying the single nucleotide polymorphism related to leprosy;
B1) the application of the polymorphism (namely allele) or genotype of rs925368 in the human genome in the preparation of products for screening leprosy patients;
B2) the application of the polymorphism (namely allele) or genotype of rs925368 in human genome in the preparation of products for detecting leprosy susceptibility;
B3) the application of the substance for detecting the polymorphism (namely allele) or genotype of rs925368 in the human genome in screening leprosy patients;
B4) the application of the substance for detecting the polymorphism (namely allele) or genotype of rs925368 in human genome in detecting leprosy susceptibility;
B5) the use of a substance for detecting the polymorphism (i.e., allele) or genotype of rs925368 in the human genome for detecting the single nucleotide polymorphism related to leprosy;
B6) the use of a substance for detecting the polymorphism (i.e. allele) or genotype of rs925368 in the human genome for identifying or assisting in identifying the single nucleotide polymorphism related to leprosy;
B7) the use of a polymorphism (i.e. allele) or genotype of rs925368 in the human genome for screening leprosy patients;
B8) use of a polymorphism (i.e. allele) or genotype of rs925368 in the human genome for detecting leprosy susceptibility.
rs925368 is a SNP site of a biallelic polymorphism on human chromosome 12q24.11, belonging to the common mutation (MAF 42%), located in the exon of the GIT2 gene, which is a switch (C/T, G/a on its complementary strand). The rs925368 genotype is CC, CT or TT. CC is homozygote with rs925368 locus as C, TT is homozygote with rs925368 locus as T, and CT is heterozygote with rs925368 locus as C and T. The polymorphism (i.e. allele) or genotype for detecting rs925368 in the human genome can be specifically the nucleotide type for detecting rs 925368.
In the above use, the substance for detecting the polymorphism or genotype of rs925368 in the human genome may be a PCR primer and/or a single-base extension primer for amplifying a genomic DNA fragment including rs 925368.
In the above use, the ratio of individuals of said CC and said CT genotypes in a population of leprosy patients is higher than the ratio of the corresponding genotypes in a normal population, respectively. The product may comprise the PCR primer and/or the single base extension primer.
In the application, the leprosy can be leprosy of Chinese people.
In order to solve the technical problems, the invention also provides a product containing a substance for detecting the polymorphism or genotype of rs925368 in the human genome.
The product containing the substance for detecting the polymorphism or the genotype of rs925368 in the human genome, provided by the invention, is any one of the products a) to d):
a) products that detect single nucleotide polymorphisms (i.e., alleles) or genotypes associated with leprosy;
b) identifying or aiding in identifying products of single nucleotide polymorphisms (i.e., alleles) or genotypes associated with leprosy;
c) screening products of leprosy patients;
d) and detecting the leprosy susceptibility product.
In the above product, the substance for detecting the polymorphism or genotype of rs925368 in the human genome may be a PCR primer and/or a single base extension primer for amplifying a genomic DNA fragment including rs 925368.
In the product, the leprosy can be leprosy of Chinese people.
In order to solve the technical problem, the invention also provides the following methods of M1) or M2):
m1) a method of screening for leprosy, comprising: detecting the genotype of the rs925368 locus in the genome of a to-be-detected object, wherein if the genotype of the rs925368 locus is a CC genotype, the to-be-detected object is or is a candidate for a leprosy patient; if the genotype of the rs925368 locus is a CT genotype, the object to be detected is or is a candidate for a leprosy patient; if the genotype of the rs925368 locus is the TT genotype, the object to be detected is or is a candidate of a non-leprosy patient;
m2), a method for detecting leprosy susceptibility, comprising: detecting the genotype of the rs925368 locus in the genome of a to-be-detected object, wherein if the genotype of the rs925368 locus is a CC genotype, the to-be-detected object is susceptible or candidate susceptible leprosy; if the genotype of the rs925368 locus is a CT genotype, the object to be detected is susceptible or candidate susceptible leprosy; if the genotype of the rs925368 locus is TT genotype, the object to be detected is not susceptible or candidate is not susceptible to leprosy.
In the method, the leprosy can be leprosy of Chinese people.
In the method, the detection of the genotype of the rs925368 locus in the genome of the object to be detected can be performed by using the substance for detecting the polymorphism or the genotype of the rs925368 locus.
Experiments prove that the risk allele of rs925368 is C, and the proportion of the allele in a leprosy patient population is 38.77 percent higher than that of the allele in a normal healthy population. The P value of rs925368 is 9.18 × 10-17And the relative risk of rs925368 is 1.44, indicating that rs925368 is a single nucleotide polymorphism related to leprosy. Of the three genotypes of rs925368, the proportion of individuals with CC genotype and CT genotype in the leprosy patient population is respectively higher than that of individuals with corresponding genotype in the normal human population, and the proportion of individuals with TT genotype in the leprosy patient population is lower than that of individuals with TT genotypeThe proportion of individuals of that genotype in the normal human population.
In practical application, the substance for detecting the polymorphism (allele) or the genotype of rs925368 and other substances (such as substances for detecting other single nucleotide polymorphism or the genotype related to leprosy) can be combined together to prepare a product for screening leprosy patients.
Wherein, the substance for detecting the polymorphism or genotype of rs925368 in human genome can be a reagent and/or an instrument required for determining the polymorphism or genotype of rs925368 by at least one of the following methods: DNA sequencing, restriction enzyme fragment length polymorphism, single-strand conformation polymorphism, denaturing high performance liquid chromatography, SNP chip, TaqMan probe technology and Sequenom MassArray technology. Wherein, the reagents and/or instruments required for determining the polymorphism or genotype of rs925368 by utilizing the Sequenom MassArray technology comprise PCR primer pairs, extension primers based on single-base extension reaction, phosphatase, resin, chips, MALDI-TOF (matrix-assisted laser desorption/ionization-time of flight mass spectrometry) and/or other reagents and instruments required by the Sequenom MassArray technology; the reagent and/or instrument required for determining the polymorphism or genotype of the rs925368 by using the TaqMan probe technology comprises a TaqMan probe, a PCR primer pair, a quantitative PCR instrument, a genotyping module and/or other reagents required by the TaqMan probe technology; the SNP chip includes a chip based on nucleic acid hybridization reaction, a chip based on single base extension reaction, a chip based on allele-specific primer extension reaction, a chip based on "one-step" reaction, a chip based on primer ligation reaction, a chip based on restriction enzyme reaction, a chip based on protein DNA binding reaction and/or a chip based on fluorescent molecule DNA binding reaction. In one embodiment of the present invention, an Infinium Human outer Beadchip chip from Illumina is utilized.
The product can be a reagent or a kit, and can also be a system consisting of the reagent or the kit and an instrument, such as a system consisting of a primer and a DNA sequencer, a system consisting of a PCR reagent and a DNA sequencer, a system consisting of a TaqMan probe, a PCR primer pair, a quantitative PCR instrument and a module for genotyping and other reagents required by TaqMan probe technology, a system consisting of a probe, a PCR primer pair and other reagents and instruments required by Ligase Detection Reaction (LDR), and a system consisting of a PCR primer pair, a single base extension primer, a chip, a PCR instrument, a module for genotyping and/or other reagents and instruments required by Sequenom MassArray technology.
Amplifying a genome DNA fragment including rs925368 by using a PCR primer, carrying out single base extension reaction by using an obtained PCR amplification product as a template and a single base extension primer, detecting the sequence of the obtained extension product, and determining the polymorphism (namely allele) and the genotype of the rs 925368. The PCR primer has no special requirements on the sequence as long as the genomic DNA fragment including rs925368 can be amplified. The extension primer can be designed according to the upstream of rs925368 in the human genome (excluding the SNP site), and the last 1 nucleotide of the extension primer corresponds to the first 1 nucleotide of rs925368 in the human genome. The extension primer can also be designed according to the downstream of rs925368 in the human genome (excluding the SNP site), and the 1 st nucleotide of the extension primer corresponds to the last 1 st nucleotide of rs925368 in the human genome.
In the invention, the PCR primer can consist of rs925368-F and rs 925368-R;
rs925368-F is any one of the following single-stranded DNA from a1) to a 4):
a1) single-stranded DNA shown in a sequence 4 in a sequence table;
a2) single-stranded DNA obtained by adding one or several nucleotides to the 5 'end and/or 3' end of a 1);
a3) a single-stranded DNA having 85% or more identity to the single-stranded DNA defined in a1) or a 2);
a4) single-stranded DNA which hybridizes with the single-stranded DNA defined in a1) or a2) under stringent conditions;
the rs925368-R is any one of the following single-stranded DNA from b1) to b 4):
b1) single-stranded DNA shown in a sequence 5 in a sequence table;
b2) single-stranded DNA obtained by adding one or several nucleotides to the 5 'end and/or 3' end of b 1);
b3) a single-stranded DNA having 85% or more identity to the single-stranded DNA defined in b1) or b 2);
b4) a single-stranded DNA which hybridizes with the single-stranded DNA defined in b1) or b2) under stringent conditions.
The single-base extension primer (rs925368-E) can be any one single-stranded DNA of the following c1) to c 4):
c1) single-stranded DNA shown in sequence 6 in the sequence table;
c2) single-stranded DNA obtained by adding one or several nucleotides to the 5 'end and/or 3' end of c 1);
c3) a single-stranded DNA having 85% or more identity to the single-stranded DNA defined in c1) or c 2);
c4) single-stranded DNA which hybridizes under stringent conditions with the single-stranded DNA defined in c1) or c 2).
a2) The single-stranded DNA obtained by adding one or more nucleotides to the 5 'end and/or the 3' end of a1) is the single-stranded DNA obtained by adding one to ten nucleotides to the 5 'end and/or the 3' end of the single-stranded DNA shown in the sequence 4. b2) The single-stranded DNA obtained by adding one or more nucleotides to the 5 'end and/or the 3' end of b1) is the single-stranded DNA obtained by adding one to ten nucleotides to the 5 'end and/or the 3' end of the single-stranded DNA shown in the sequence 5. c2) The single-stranded DNA obtained by adding one or more nucleotides to the 5 'end and/or the 3' end of c1) is the single-stranded DNA obtained by adding one to ten nucleotides to the 5 'end and/or the 3' end of the single-stranded DNA shown in sequence 6.
The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "identity" includes a nucleotide sequence having 85% or more, or 90% or more, or 95% or more identity to the nucleotide sequence shown in SEQ ID No. 4, SEQ ID No. 5 or SEQ ID No. 6 of the present invention. Identity can be assessed visually or by computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to assess the identity between related sequences.
The stringent conditions are hybridization and washing of the membrane 2 times, 5min each, at 68 ℃ in a solution of 2 XSSC, 0.1% SDS, and 2 times, 15min each, at 68 ℃ in a solution of 0.5 XSSC, 0.1% SDS; alternatively, hybridization was carried out at 65 ℃ in a solution of 0.1 XSSPE (or 0.1 XSSC), 0.1% SDS, and the membrane was washed.
The above-mentioned identity of 85% or more may be 85%, 90% or 95% or more.
In embodiments of the invention, rs925368 uses the SEQUENOM genotyping platform, i.e.
Figure BDA0003092451540000051
Molecular weight array technique, Sequenom
Figure BDA0003092451540000052
The detection process combines a multiple PCR technology, a MassARRAY iPLEX single base extension technology and a matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF) mass spectrometry technology to carry out typing detection. The DNA template containing the SNP site area is amplified through a PCR technology, and then a single base extension reaction is carried out with the PCR product by using a specific extension primer. Because the different bases of the polymorphic sites lead to the difference of the molecular weights of the extended products due to the different terminal bases of the extended products, the base difference caused by SNP polymorphism is reflected by the difference of the molecular weights, the size of the molecular weight of the extended products is detected by a matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) mass spectrometry technology, and SNP typing detection is carried out by judging the difference of the molecular weights by applying special analysis software.
The invention discovers that rs925368 is the single nucleotide polymorphism related to leprosy in a sample (4817 leprosy patients and 12132 healthy people) from Chinese population. The substance for detecting the polymorphism (i.e. allele) or the genotype of the rs925368 and other substances (such as substances for detecting other single nucleotide polymorphism (i.e. allele) or the genotype related to the leprosy) can be combined together to prepare the product for screening leprosy patients.
Drawings
FIG. 1 shows the results of meta analysis of all samples (4817 cases and 12132 healthy controls) using a fixed effect model.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents, instruments and the like used in the following examples are commercially available unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged. In the following examples, unless otherwise specified, the 1 st position of each nucleotide sequence in the sequence listing is the 5 'terminal nucleotide of the corresponding DNA, and the last position is the 3' terminal nucleotide of the corresponding DNA.
Examples 1, rs13259978, rs925368, rs671 and rs75680863 are single nucleotide polymorphisms associated with leprosy
The method comprises the following steps: the inventors carried out a two-stage genome-wide association analysis study of protein-encoding regions for 4817 leprosy patients and 12132 normal controls in the chinese population. First, the SNP sites (MAF > 0.1%) of 40,491 coding regions of 1,648 leprosy patients and 2,318 normal controls were analyzed by exon chip (first stage). The 34 candidate SNP sites were then validated in the northern china population (3,169 leprosy patients and 9,814 normal controls) (second stage).
Study object
The study subjects for the exon chip discovery phase (first phase) were 1,670 leprosy patients and 2,321 normal controls in northern china. Validation phase (second phase) 39 mutation sites were validated in an additional 3,169 leprosy patients and 9,814 normal controls in northern china. The leprosy patients and the normal controls were all Chinese Han population and were geographically matched. Clinical information for the subjects is shown in table 1.
TABLE 1 study object information
Figure BDA0003092451540000061
Wherein, the selection criteria of the leprosy patient are that 2 or more than 2 of the following 4 patients are met, or the diagnosis is established by the 3 rd patient: 1. skin lesions with sensory disturbance and anhidrosis, or areas with numbness; 2. peripheral nerve involvement, manifested as a massive nerve trunk with corresponding dysfunction; 3. detecting leprosy bacillus by a skin lesion tissue section or a tissue fluid smear; 4. characteristic changes are seen pathologically.
Inclusion criteria for normal controls (normal healthy persons): no history of leprosy and no family history of leprosy (including primary, secondary and tertiary relatives of leprosy patients); no history of other infectious diseases; there was no history of other autoimmune diseases, systemic diseases and family history.
The study was approved by the ethical review committee of the institute of dermatologic disease control, Shandong province, academy of medicine and sciences, Shandong province.
Typing and quality control
An Infinium Human outer Bead Chip of Illumina corporation is selected to classify 1,670 leprosy patients and 2,321 normal controls, and the total number of chips is 27,089 (in 1,998 Chinese samples, 27,089 samples are identified by whole Exome sequencing>Present in 1 sample) non-synonymous coding region variants. Exclusion of non-coding region variation and MAF<Variation of 0.1%, and association analysis of intron and intergenic regions on exon chip and rs13259978, rs925368, rs671, rs79259738 and rs 75680863. In the verification stage, sequence MassARRAY system (Agena Bioscience), QuantStaudio is selectedTMTyping studies were performed with 12K Flex Real-Time PCR (Applied Biosystems) and 7900HT fast Real-Time PCR systems (Applied Biosystems). The quality control filtration criteria were as follows: eliminating variation and detection rate of uncertain population<90% and does not comply with Hardy-Weinberg equilibrium law (P)<1×10-3) And the detection rate of the sample<95% of the cases.
Statistical analysis
In the first stage, correlation analysis between phenotype and single variant genotype was performed by GMMAT-v 0.7. In the second stage, MAF is corrected by a linear regression model in PLINK>Correlation analysis was performed on 1% of SNPs using the exact profile of Fisher's in PLINKRate method for MAF<1% of SNPs were subjected to correlation analysis. Using a fixed Effect model (on MAF by META-v7.0 software)>1% of SNPs by inverse variance method for MAF<1% of the SNPs were analyzed using the z-statistic combination method). And (4) performing meta analysis on the combined data of the discovery stage and the verification stage. Simultaneously conform to p<5×10-8The variation of (a) is a statistically significant variation.
Exome discovery phase analysis
273,028 variant sites were typed for 1,670 leprosy patients and 2,321 normal controls. All samples were confirmed by principal component analysis to be of chinese ancestry and found to have a better match of genotypes in leprosy patients and normal controls. After a series of samples and mutation sites filtered, a total of 46,373 coding region mutation sites with MAF > 0.1% were found and analyzed in 1,648 leprosy patients and 2,318 healthy controls.
Full exon analysis revealed strong associations within the MHC region. After removing all the variant sites within the MHC region, the Q-Q plots of the remaining SNP sites fit a zero distribution, indicating that the likelihood of exon association analysis results due to population confounders is minimized (lambda value 0.99). Meanwhile, Q-Q plot analysis was performed for SNP sites with different MAF thresholds (MAF > 0.5%, MAF > 1% and MAF > 5%), consistent with the above results.
Through research, 17 variation sites (P) are found<1.0×10-3) Found to have an association with leprosy.
Verification phase analysis
The 17 newly discovered mutation sites were typed in 3,169 leprosy patients and 9,814 healthy controls in the northern region of china. There were 12 mutation sites that could be successfully typed, and 4 sites were statistically significant in meta analysis during the discovery and validation stages (P)<5×10-8) And without genetic heterogeneity, these 4 sites were: SLC7a2 gene rs13259978(chr8:17396415) (P ═ 1.74 × 10-8OR 1.28), GIT2 gene rs925368(chr12:110390979:) (P9.18 × 10)-17OR 1.44), ALDH2 gene rs671(chr12:112241766) (P2.0 × 10-20,OR＝1.35) TCN2 gene rs75680863(chr22:31007023) (P ═ 8.37 × 10-21OR ═ 0.74) (table 3).
Wherein rs13259978, rs925368, rs671 and rs75680863 are typed by Sequenom MassARRAY system (Agena Bioscience) and QuantStudioTM12K Flex Real-Time PCR (Applied Biosystems) and 7900HT fast Real-Time PCR systems (Applied Biosystems) by the following specific methods:
typing of rs13259978, rs925368, rs671, rs75680863
The 4 SNPs rs13259978, rs925368, rs671 and rs75680863 are applied to a SEQUENOM genotyping platform
Figure BDA0003092451540000082
Molecular weight array technique, Sequenom
Figure BDA0003092451540000083
The detection process combines a multiple PCR technology, a MassARRAY iPLEX single base extension technology and a matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF) mass spectrometry technology to carry out typing detection. The DNA template containing the SNP site region is amplified by the PCR technology, and then a single base extension reaction is carried out by using a specific extension primer and a PCR product, wherein the PCR amplification and the extension primer of each site are shown in Table 2. Because the different bases of the polymorphic sites lead to the difference of the molecular weights of the extended products due to the different terminal bases of the extended products, the base difference caused by SNP polymorphism is reflected by the difference of the molecular weights, the size of the molecular weight of the extended products is detected by a matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) mass spectrometry technology, and SNP typing detection is carried out by judging the difference of the molecular weights by applying special analysis software.
TABLE 2, 4 SNP typing primers
Figure BDA0003092451540000081
The operation steps are as follows:
the 4 SNPs were verified using the Sequenom MassArray (San Diego, USA) platform using approximately 15ng DNA per sample. Firstly, extracting genomic DNA of peripheral blood, standardizing, amplifying genomic DNA fragments including SNP sites by sample DNA through multiple PCR reaction, extending specific single strand of the SNP sites of amplification products, desalting the extension products and transferring the extension products to a 384-hole chip. Allele detection was performed by mass spectrometry (MALDI-TOF MS), and the results were analyzed using Sequenom MassARRAY typing software.
TABLE 3 SNP and leprosy correlation analysis results
Figure BDA0003092451540000091
Note: in the context of Table 3, the following examples are,
Figure BDA0003092451540000094
minor/major allele;
Figure BDA0003092451540000095
means that the results are from a fixed effect model;
OR, OR is calculated according to the rank as the gene;
f _ a, minor allele frequency in case; f _ U, minor allele frequency in control.
TABLE 4 number of individuals of each genotype for SNP in leprosy patients and normal population
Figure BDA0003092451540000092
TABLE 5 frequency of different genotypes in leprosy patients and Normal population
Figure BDA0003092451540000093
Note: in tables 4 and 5, in the genotype of rs13259978, A1A1 represents CC, A1A2 represents CG, A2a2 represents GG;
in the genotype of rs925368, A1A1 represents CC, A1A2 represents CT, and A2A2 represents TT;
rs671 genotype, A1A1 represents AA, A1A2 represents AG, A2A2 represents GG;
in the genotype of rs75680863, A1A1 represents TT, A1A2 represents TA, and A2A2 represents AA.
In table 4, less than 4817 leprosy patients and less than 12132 normal controls were present for each SNP, because partial individual typing was unsuccessful during the typing process, and the data analysis in tables 5 and 6 was performed on the basis of table 4.
The genotypes and frequencies of the 4 SNPs in leprosy patients and normal controls are shown in tables 4 and 5. The results show that: of the three genotypes of rs13259978, the proportion of individuals with CC and CG genotypes in the leprosy patient population is respectively higher than that of individuals with the corresponding genotypes in the normal human population, and the proportion of individuals with GG genotypes in the leprosy patient population is lower than that of individuals with the genotypes in the normal human population;
of the three genotypes of rs925368, the proportion of individuals with CC and CT genotypes in the leprosy patient population is respectively higher than that of individuals with the corresponding genotypes in the normal human population, and the proportion of individuals with TT genotypes in the leprosy patient population is lower than that of the individuals with the genotypes in the normal human population;
of the three genotypes of rs671, the proportion of the individuals with AA and AG genotypes in the leprosy patient population is respectively higher than that of the individuals with the corresponding genotypes in the normal human population, and the proportion of the individuals with GG genotypes in the leprosy patient population is lower than that of the individuals with the genotypes in the normal human population;
of the three genotypes of rs75680863, the proportion of the individuals of AA genotype in the leprosy patient population is higher than that of the individuals of the genotype in the normal human population, and the proportion of the individuals of TT and TA genotypes in the leprosy patient population is respectively lower than that of the individuals of the corresponding genotype in the normal human population.
TABLE 6 allele frequency (%) of SNP in leprosy patient population and variation from control
Figure BDA0003092451540000101
And (3) calculating the difference P value of the gene frequencies in the leprosy patient population and the normal human population by using a two-classification logistic regression model (log-additive model), and determining whether the SNP has significance, wherein the genotype is counted by adopting an additive model. As a result, it was found that the gene frequencies of the respective alleles of rs13259978, rs925368, rs671 and rs75680863 were significantly different in both the normal human population and the leprosy patient population.
As can be seen from table 6, the risk allele of rs13259978 is C, which is increased by 28.63% in leprosy patients compared to normal controls; the risk allele of rs925368 is C, which is increased by 38.77% in leprosy patients compared to normal controls; the risk allele of rs671 is A, which is increased by 26.26% in leprosy patients compared with normal control; the risk allele of rs75680863 is a, which is increased by 5.88% in leprosy patients compared to normal controls.
The experimental result shows that the polymorphism, or genotype, or allele frequency of rs13259978, rs925368, rs671 and rs75680863 can be used for screening leprosy patients.
<110> Shandong first medical university Hospital affiliated with skin disease (Shandong province research institute for skin disease prevention and treatment, Shandong province Hospital for skin disease)
Application of <120> single nucleotide polymorphism rs925368 in screening of leprosy patients
<160> 12
<170> PatentIn version 3.5
<210> 1
<211> 30
<212> DNA
<213> Artificial sequence
<400> 1
acgttggatg ggtttattgg aacacctgcc 30
<210> 2
<211> 30
<212> DNA
<213> Artificial sequence
<400> 2
acgttggatg tatatcagta tcccaggccc 30
<210> 3
<211> 16
<212> DNA
<213> Artificial sequence
<400> 3
gccccaccgg tttgcc 16
<210> 4
<211> 30
<212> DNA
<213> Artificial sequence
<400> 4
acgttggatg tctgtgtctt cgtctgatgc 30
<210> 5
<211> 30
<212> DNA
<213> Artificial sequence
<400> 5
acgttggatg catcaataac cagcacagcg 30
<210> 6
<211> 22
<212> DNA
<213> Artificial sequence
<400> 6
gggtcgttga gagtcaagac aa 22
<210> 7
<211> 30
<212> DNA
<213> Artificial sequence
<400> 7
acgttggatg caggtcccac actcacagtt 30
<210> 8
<211> 30
<212> DNA
<213> Artificial sequence
<400> 8
acgttggatg agttgggcga gtacgggctg 30
<210> 9
<211> 19
<212> DNA
<213> Artificial sequence
<400> 9
cactcacagt tttcacttc 19
<210> 10
<211> 29
<212> DNA
<213> Artificial sequence
<400> 10
cctggagcac ttgaacccca gcatctatg 29
<210> 11
<211> 25
<212> DNA
<213> Artificial sequence
<400> 11
gtggtgacag gcccaaacta gtacc 25
<210> 12
<211> 20
<212> DNA
<213> Artificial sequence
<400> 12
cctctacctg cacagcctca 20

Claims (10)

1. The application of the substance for detecting the polymorphism or genotype of rs925368 in human genome in preparing and screening products for leprosy patients.
2. The application of the substance for detecting the polymorphism or genotype of rs925368 in human genome in preparing products for detecting leprosy susceptibility.
3. The application of the substance for detecting the polymorphism or genotype of rs925368 in human genome in preparing products for detecting the single nucleotide polymorphism related to leprosy.
4. The application of the substance for detecting the polymorphism or genotype of rs925368 in human genome in the preparation of products for identifying or assisting in identifying the single nucleotide polymorphism related to leprosy.
5. Any of the following applications:
B1) the application of the polymorphism or genotype of rs925368 in the human genome in the preparation of products for screening leprosy patients;
B2) the application of the polymorphism or genotype of rs925368 in human genome in the preparation of products for detecting leprosy susceptibility;
B3) the application of the substance for detecting the polymorphism or genotype of rs925368 in the human genome in screening leprosy patients;
B4) the application of the substance for detecting the polymorphism or genotype of rs925368 in human genome in detecting leprosy susceptibility;
B5) the application of the substance for detecting the polymorphism or genotype of rs925368 in human genome in detecting the single nucleotide polymorphism related to leprosy;
B6) the application of the substance for detecting the polymorphism or genotype of rs925368 in human genome in identifying or assisting in identifying the single nucleotide polymorphism related to leprosy;
B7) the application of the polymorphism or genotype of rs925368 in the human genome in screening leprosy patients;
B8) the application of the polymorphism or genotype of rs925368 in human genome in detecting leprosy susceptibility.
6. A product containing a substance for detecting rs925368 polymorphism or genotype in human genome, which is any one of a) to d):
a) products for detecting single nucleotide polymorphisms or genotypes associated with leprosy;
b) identifying or aiding in identifying a product of a single nucleotide polymorphism or genotype associated with leprosy;
c) screening products of leprosy patients;
d) and detecting the leprosy susceptibility product.
7. The product of claim 6, wherein: the substance for detecting the polymorphism or the genotype of the rs925368 in the human genome is a PCR primer and/or a single-base extension primer for amplifying a genome DNA segment including the rs 925368.
8. The product of claim 7, wherein: the PCR primer consists of rs925368-F and rs 925368-R;
rs925368-F is any one of the following single-stranded DNA from a1) to a 4):
a1) single-stranded DNA shown in a sequence 4 in a sequence table;
a2) single-stranded DNA obtained by adding one or several nucleotides to the 5 'end and/or 3' end of a 1);
a3) a single-stranded DNA having 85% or more identity to the single-stranded DNA defined in a1) or a 2);
a4) single-stranded DNA which hybridizes with the single-stranded DNA defined in a1) or a2) under stringent conditions;
the rs925368-R is any one of the following single-stranded DNA from b1) to b 4):
b1) single-stranded DNA shown in a sequence 5 in a sequence table;
b2) single-stranded DNA obtained by adding one or several nucleotides to the 5 'end and/or 3' end of b 1);
b3) a single-stranded DNA having 85% or more identity to the single-stranded DNA defined in b1) or b 2);
b4) a single-stranded DNA which hybridizes with the single-stranded DNA defined in b1) or b2) under stringent conditions.
9. The product according to claim 7 or 8, characterized in that: the single-base extension primer is any one single-stranded DNA of the following c1) to c 4):
c1) single-stranded DNA shown in sequence 6 in the sequence table;
c2) single-stranded DNA obtained by adding one or several nucleotides to the 5 'end and/or 3' end of c 1);
c3) a single-stranded DNA having 85% or more identity to the single-stranded DNA defined in c1) or c 2);
c4) single-stranded DNA which hybridizes under stringent conditions with the single-stranded DNA defined in c1) or c 2).
10. The following M1) or M2):
m1) a method of screening for leprosy, comprising: detecting the genotype of the rs925368 locus in the genome of a to-be-detected object, wherein if the genotype of the rs925368 locus is a CC genotype, the to-be-detected object is or is a candidate for a leprosy patient; if the genotype of the rs925368 locus is a CT genotype, the object to be detected is or is a candidate for a leprosy patient; if the genotype of the rs925368 locus is the TT genotype, the object to be detected is or is a candidate of a non-leprosy patient;
m2), a method for detecting leprosy susceptibility, comprising: detecting the genotype of the rs925368 locus in the genome of a to-be-detected object, wherein if the genotype of the rs925368 locus is a CC genotype, the to-be-detected object is susceptible or candidate susceptible leprosy; if the genotype of the rs925368 locus is a CT genotype, the object to be detected is susceptible or candidate susceptible leprosy; if the genotype of the rs925368 locus is TT genotype, the object to be detected is not susceptible or candidate is not susceptible to leprosy.
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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106399572A (en) * 2016-12-07 2017-02-15 山东省皮肤病性病防治研究所 Application of polymorphism of single nucleotide rs149308743 in screening of leprosy patients

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106399572A (en) * 2016-12-07 2017-02-15 山东省皮肤病性病防治研究所 Application of polymorphism of single nucleotide rs149308743 in screening of leprosy patients

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
ZHENZHEN WANG ET,AL.: ""Discovery of four exonic and one intergenic novel susceptibility loci for leprosy", 《CLINICAL GENETICS》 *
伊正君等: "《临床分子生物学检验技术》", 31 January 2020, 武汉：华中科技大学出版社 *

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Application publication date: 20210713