CN116287193B - SNP locus for evaluating effect of adalimumab on psoriasis treatment, kit and application thereof - Google Patents

SNP locus for evaluating effect of adalimumab on psoriasis treatment, kit and application thereof Download PDF

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CN116287193B
CN116287193B CN202310077706.7A CN202310077706A CN116287193B CN 116287193 B CN116287193 B CN 116287193B CN 202310077706 A CN202310077706 A CN 202310077706A CN 116287193 B CN116287193 B CN 116287193B
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崔勇
左先波
许静凯
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China Japan Friendship Hospital
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Abstract

The invention provides a group of gene mutation sites for evaluating the effect of adalimumab on treating psoriasis, and a kit and application thereof, and belongs to the technical field of biological medicines. The invention screens and obtains a group of specific gene mutation sites based on genome DNA data of people suffering from psoriasis in Chinese Han nationality, and provides a method for guiding psoriasis patients to treat adalimumab and evaluating the safety and effectiveness of adalimumab in treating psoriasis according to the gene mutation sites.

Description

SNP locus for evaluating effect of adalimumab on psoriasis treatment, kit and application thereof
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a set of SNP loci for evaluating the effect of adalimumab on treating psoriasis, a kit and application thereof.
Background
Psoriasis is commonly called as psoriasis, and is a common chronic, recurrent, inflammatory and systemic disease. The disease course is chronic, easy to recur, and has no cure method, and the life quality of patients is seriously affected.
Adalimumab is a fully human IgG1 antibody to TNF- α, which was first approved by the FDA in 2002 for the treatment of moderate to severe active rheumatoid arthritis, was approved by the FDA in 2005 for psoriatic arthritis, and was approved by the FDA in 2008 for moderate to severe plaque psoriasis. The primary pharmacological effect of adalimumab is to bind to free TNF- α, block interactions with cell surface TNF- α receptors, and thereby modulate TNF- α induction/modulation responses.
Patients carrying different mutations show different response rates in the treatment of adalimumab during the treatment of psoriasis with adalimumab. In order to realize accurate treatment, the patient is enabled to walk less during treatment, and the economic burden of the patient is further reduced. Through public database search, a plurality of clinical experimental studies analyze the response of adalimumab drugs carrying different variations, but lack systematic evaluation, and have lower guiding significance for psoriasis patients. And the population targeted by the researches is mainly European and American population, and has not been clearly guided in Chinese population and Asia population. In addition, when a part of psoriasis patients are treated by adalimumab, serious side effects occur, even life is endangered, so that the indication that the patient is treated by the adalimumab is solved, the accurate treatment of the psoriasis can be realized, and meanwhile, the side effects of the patient can be greatly reduced.
Although adalimumab can significantly improve the skin damage condition of patients with psoriasis and improve the survival quality of the patients, patients carrying different mutations show different response rates in the treatment of adalimumab in the process of treating psoriasis by the adalimumab. Thus, there is a need to develop a set of variant sites for assessing or guiding adalimumab drug treatment.
Disclosure of Invention
Therefore, the invention aims to provide an SNP locus for evaluating the effect of adalimumab on treating psoriasis, which is used as a drug action target and a drug toxicity related locus, and can evaluate whether the adalimumab can be used for treating psoriasis and also evaluate the toxic and side effects of the adalimumab on psoriasis patients.
The invention also provides a kit for detecting the gene mutation site for evaluating the effect of adalimumab on treating psoriasis, and provides a detection tool for guiding a psoriasis patient to treat the adalimumab or evaluating the safety and effectiveness of the adalimumab on treating the psoriasis.
The invention provides a group of genomic variation sites for evaluating the effect of adalimumab on treating psoriasis, which comprises the following SNP molecular markers: rs1799724, rs2233945, rs510432, rs2071303, rs2145623, rs1800629, rs7234029, rs3218253, rs2032582, rs1135216, rs10280623, rs10497346, rs10248420;
the rs1799724 is a polymorphic site T/C present at position 31542482 of chromosome 6 on the basis of genomic version GRCh37 (hg 19);
the rs2233945 is a polymorphic site A/C existing at 31107361 of chromosome 6 on the basis of genome version GRCh37 (hg 19);
the rs510432 is a polymorphic site T/C existing at 106774030 th position of chromosome 6 on the basis of genome version GRCh37 (hg 19);
the rs2071303 is a polymorphic site T/C existing at 26091336 th position of chromosome 6 on the basis of genome version GRCh37 (hg 19);
the rs2145623 is a polymorphic site G/C existing at 35839236 th chromosome 14 on the basis of a genome version GRCh37 (hg 19);
the rs1800629 is based on the polymorphic site A/G existing at 31543031 position of chromosome 6 of the genome version GRCh37 (hg 19);
the rs7234029 is a polymorphic site G/A existing at 12877060 of chromosome 18 on the basis of genome version GRCh37 (hg 19);
the rs3218253 is a polymorphic site A/G existing at 37544810 of chromosome 22 on the basis of genome version GRCh37 (hg 19);
the polymorphic site A/C of the 87160618 locus of chromosome 7 of rs2032582 on the basis of the genome version GRCh37 (hg 19);
the rs1135216 is a polymorphic site C/T existing at 32814975 th position of chromosome 6 on the basis of genome version GRCh37 (hg 19);
the rs10280623 is a polymorphic site C/T existing at 87202544 th chromosome 7 on the basis of a genome version GRCh37 (hg 19);
the rs10497346 is a polymorphic site C/T existing at 169771196 th chromosome 2 on the basis of a genome version GRCh37 (hg 19);
the rs10248420 is the polymorphic site G/A present at position 87164986 of chromosome 7 on the basis of genomic version GRCh37 (hg 19).
Preferably, the rs1799724, rs2233945, rs510432, rs2071303, rs2145623, rs1800629, rs7234029, rs3218253, rs2032582, rs1135216, rs10280623, rs10497346 and rs10248420 are the polymorphic site at position 220 in the DNA fragment shown in the nucleotide sequences SEQ ID NO. 1 to SEQ ID NO. 10 and the polymorphic site at position 451 in the DNA fragment shown in the nucleotide sequences SEQ ID NO. 11 to SEQ ID NO. 13.
The invention provides a primer group for amplifying the gene mutation site, wherein the primer group for amplifying rs1799724 comprises a forward primer with a nucleotide sequence shown as SEQ ID NO. 14 and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 15;
the amplified rs2233945 comprises a forward primer with a nucleotide sequence shown as SEQ ID NO. 16 and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 17;
the amplified rs510432 comprises a forward primer with a nucleotide sequence shown as SEQ ID NO. 18 and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 19;
the amplified rs2071303 comprises a forward primer with a nucleotide sequence shown as SEQ ID NO. 20 and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 21;
amplification rs2145623 comprises a forward primer with a nucleotide sequence shown as SEQ ID NO. 22 and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 23;
amplification rs1800629 comprises a forward primer with a nucleotide sequence shown as SEQ ID NO. 24 and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 25;
amplification rs7234029 comprises a forward primer with a nucleotide sequence shown as SEQ ID NO. 26 and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 27;
amplification rs3218253 includes a forward primer having a nucleotide sequence shown as SEQ ID NO. 28 and a reverse primer having a nucleotide sequence shown as SEQ ID NO. 29;
the amplified rs2032582 comprises a forward primer with a nucleotide sequence shown as SEQ ID NO. 30 and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 31;
the amplified rs1135216 comprises a forward primer with a nucleotide sequence shown as SEQ ID NO. 32 and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 33;
the amplified rs10280623 comprises a forward primer with a nucleotide sequence shown as SEQ ID NO. 34 and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 35;
the amplified rs10497346 comprises a forward primer with a nucleotide sequence shown as SEQ ID NO. 36 and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 37;
amplification rs10248420 includes a forward primer having a nucleotide sequence shown as SEQ ID NO. 38 and a reverse primer having a nucleotide sequence shown as SEQ ID NO. 39.
The invention provides a kit for detecting the genetic variation locus, which comprises the primer group and a PCR amplification buffer solution.
The invention provides application of the gene mutation site or the primer group in preparation of a kit for evaluating safety and effectiveness of adalimumab in treating psoriasis.
Preferably, the method for evaluating the safety and effectiveness of adalimumab in treating psoriasis comprises the following steps:
extracting genome DNA of psoriasis patients;
using the extracted genome DNA as a template, and carrying out PCR amplification by using the primer group to obtain an amplification product, and sequencing to obtain a sequencing result of a genetic variation locus;
obtaining genotyping of the genetic variation site of the psoriasis patient based on the sequencing result of the genetic variation site;
marking each SNP locus according to the genotyping of the genetic variation locus of the psoriasis patient by contrasting a marking table, and carrying out the marking to obtain a total score in a formula I and a formula II;
total score = score of rs1799724 x 0.64+ score of rs2233945 x 0.56+ score of rs510432 x 0.83+ score of rs2071303 x 1.20+ score of rs2145623 x 0.84+ score of rs1800629 x 0.70+ score of rs7234029 x 1.16+ score of rs3218253 x 1.23 formula I
Total score = score of rs2032582 x 1.16+score of rs1135216 x 0.78+score of rs10280623 x 0.62+score of rs10497346 x 0.66+score of rs10248420 x 0.87 formula II
The validity is evaluated according to the total score calculated according to formula I, and the safety is evaluated according to the total score calculated according to formula II:
when the psoriasis patients with the total score of more than 0 are effectively and safely treated by adalimumab, the higher the score is, the better the curative effect is; patients with psoriasis having a total score of less than 0 are not recommended to be treated with adalimumab and may have side effects after use.
The invention provides application of the gene mutation site or the primer group in preparing a kit for guiding a psoriasis patient to treat adalimumab.
Preferably, the method of directing treatment of adalimumab in a psoriasis patient comprises the steps of:
extracting genome DNA of psoriasis patients;
using the extracted genome DNA as a template, and carrying out PCR amplification by using the primer group to obtain an amplification product, and sequencing to obtain a sequencing result of a genetic variation locus;
obtaining genotyping of the genetic variation site of the psoriasis patient based on the sequencing result of the genetic variation site;
marking each SNP locus according to the genotyping of the genetic variation locus of the psoriasis patient by contrasting a marking table, and taking the SNP locus into a formula III to obtain a total score;
total score = rs1799724 score x 0.64+rs2233945 score x 0.56+rs510432 score x 0.83+rs2071303 score x 1.20+rs2145623 score x 0.84+rs1800629 score x 0.70+rs7234029 score x 1.16+rs3218253 score x 1.23+rs2032582 score x 1.16+rs1135216 score x 0.78+rs10280623 score x 0.62+rs10497346 score x 0.66+rs10248420 score x 0.87 formula III
Determining the dosing regimen of adalimumab based on the total score:
patients with psoriasis when the total score is greater than 0 are suitable for treatment with adalimumab; patients with psoriasis who have a total score of less than 0 are not recommended to be treated with adalimumab.
Preferably, the scoring table is as follows:
the invention provides a group of gene mutation sites for evaluating the effect of adalimumab on treating psoriasis, which comprises the following SNP molecular markers: rs1799724, rs2233945, rs510432, rs2071303, rs2145623, rs1800629, rs7234029, rs3218253, rs2032582, rs1135216, rs10280623, rs10497346, rs10248420. The invention is based on genome variation data of psoriasis of Chinese Han people, defines the action site of adalimumab based on the obtained genome variation data, can be applied to the effectiveness and safety of adalimumab in treating psoriasis, and is used for guiding the administration scheme of the adalimumab. Meanwhile, the kit relates to 13 genome variation sites, has wider detection range and higher sensitivity, and can provide effectiveness and safety guidance for psoriasis patients when treating with adalimumab more comprehensively.
Detailed Description
The invention provides a group of gene mutation sites for evaluating the effect of adalimumab on treating psoriasis, which comprises the following SNP molecular markers: rs1799724, rs2233945, rs510432, rs2071303, rs2145623, rs1800629, rs7234029, rs3218253, rs2032582, rs1135216, rs10280623, rs10497346, rs10248420; the rs1799724 is a polymorphic site T/C present at position 31542482 of chromosome 6 on the basis of genomic version GRCh37 (hg 19); the rs2233945 is a polymorphic site A/C existing at 31107361 of chromosome 6 on the basis of genome version GRCh37 (hg 19); the rs510432 is a polymorphic site T/C existing at 106774030 th position of chromosome 6 on the basis of genome version GRCh37 (hg 19); the rs2071303 is a polymorphic site T/C existing at 26091336 th position of chromosome 6 on the basis of genome version GRCh37 (hg 19); the rs2145623 is a polymorphic site G/C existing at 35839236 th chromosome 14 on the basis of a genome version GRCh37 (hg 19); the rs1800629 is based on the polymorphic site A/G existing at 31543031 position of chromosome 6 of the genome version GRCh37 (hg 19); the rs7234029 is a polymorphic site G/A existing at 12877060 of chromosome 18 on the basis of genome version GRCh37 (hg 19); the rs3218253 is a polymorphic site A/G existing at 37544810 of chromosome 22 on the basis of genome version GRCh37 (hg 19); the polymorphic site A/C of the 87160618 locus of chromosome 7 of rs2032582 on the basis of the genome version GRCh37 (hg 19); the rs1135216 is a polymorphic site C/T existing at 32814975 th position of chromosome 6 on the basis of genome version GRCh37 (hg 19); the rs10280623 is a polymorphic site C/T existing at 87202544 th chromosome 7 on the basis of a genome version GRCh37 (hg 19); the rs10497346 is a polymorphic site C/T existing at 169771196 th chromosome 2 on the basis of a genome version GRCh37 (hg 19); the rs10248420 is the polymorphic site G/A present at position 87164986 of chromosome 7 on the basis of genomic version GRCh37 (hg 19).
In the invention, the gene mutation site is obtained based on genome screening of psoriasis patients of Chinese Han people. The locus of the polymorphism at the 220 th position in the DNA fragment shown in the nucleotide sequences SEQ ID NO: 1-SEQ ID NO:10 and the locus of the polymorphism at the 451 th position in the DNA fragment shown in the nucleotide sequences SEQ ID NO: 11-SEQ ID NO:13 are preferable for the rs1799724, rs2233945, rs510432, rs2071303, rs2145623, rs1800629, rs7234029, rs3218253, rs2032582, rs1135216, rs10280623, rs10497346 and rs10248420.
The invention provides a primer group for amplifying the gene mutation site, wherein the primer group for amplifying rs1799724 comprises a forward primer with a nucleotide sequence shown as SEQ ID NO. 14 and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 15; the amplified rs2233945 comprises a forward primer with a nucleotide sequence shown as SEQ ID NO. 16 and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 17; the amplified rs510432 comprises a forward primer with a nucleotide sequence shown as SEQ ID NO. 18 and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 19; the amplified rs2071303 comprises a forward primer with a nucleotide sequence shown as SEQ ID NO. 20 and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 21; amplification rs2145623 comprises a forward primer with a nucleotide sequence shown as SEQ ID NO. 22 and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 23; amplification rs1800629 comprises a forward primer with a nucleotide sequence shown as SEQ ID NO. 24 and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 25; amplification rs7234029 comprises a forward primer with a nucleotide sequence shown as SEQ ID NO. 26 and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 27; amplification rs3218253 includes a forward primer having a nucleotide sequence shown as SEQ ID NO. 28 and a reverse primer having a nucleotide sequence shown as SEQ ID NO. 29; the amplified rs2032582 comprises a forward primer with a nucleotide sequence shown as SEQ ID NO. 30 and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 31; the amplified rs1135216 comprises a forward primer with a nucleotide sequence shown as SEQ ID NO. 32 and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 33; the amplified rs10280623 comprises a forward primer with a nucleotide sequence shown as SEQ ID NO. 34 and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 35; the amplified rs10497346 comprises a forward primer with a nucleotide sequence shown as SEQ ID NO. 36 and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 37; amplification rs10248420 includes a forward primer having a nucleotide sequence shown as SEQ ID NO. 38 and a reverse primer having a nucleotide sequence shown as SEQ ID NO. 39.
The source of the primer set is not particularly limited in the present invention, and sources of primers well known in the art may be used. In the embodiment of the invention, the primer set delegates Metpeta and company synthesis.
The invention provides a kit for detecting the genetic variation locus, which comprises the primer group and a PCR amplification buffer solution.
The source of the PCR amplification Buffer is not particularly limited, and the PCR amplification Buffer well known in the art may be used, and in the present embodiment, the PCR amplification Buffer is a2 XPCR Buffer (containing 1.25U/. Mu.l DNA Polymerase,4mM Mg 2+ ，800μM dNTP)。
The invention provides application of the gene mutation site or the primer group in preparation of a kit for evaluating safety and effectiveness of adalimumab in treating psoriasis.
In the present invention, the method for evaluating the safety and effectiveness of adalimumab in treating psoriasis preferably comprises the steps of:
extracting genome DNA of psoriasis patients;
using the extracted genome DNA as a template, and carrying out PCR amplification by using the primer group to obtain an amplification product, and sequencing to obtain a sequencing result of a genetic variation locus;
obtaining genotyping of the genetic variation site of the psoriasis patient based on the sequencing result of the genetic variation site;
marking each SNP locus according to the genotyping of the genetic variation locus of the psoriasis patient by contrasting a marking table, and carrying out the marking to obtain a total score in a formula I and a formula II;
total score = score of rs1799724 x 0.64+ score of rs2233945 x 0.56+ score of rs510432 x 0.83+ score of rs2071303 x 1.20+ score of rs2145623 x 0.84+ score of rs1800629 x 0.70+ score of rs7234029 x 1.16+ score of rs3218253 x 1.23 formula I
Total score = score of rs2032582 x 1.16+score of rs1135216 x 0.78+score of rs10280623 x 0.62+score of rs10497346 x 0.66+score of rs10248420 x 0.87 formula II
The validity is evaluated according to the total score calculated according to formula I, and the safety is evaluated according to the total score calculated according to formula II:
when the psoriasis patients with the total score of more than 0 are effectively and safely treated by adalimumab, the higher the score is, the better the curative effect is; patients with psoriasis having a total score of less than 0 are not recommended to be treated with adalimumab and may have side effects after use.
The method for extracting genomic DNA from a psoriasis patient according to the present invention is not particularly limited, and methods for extracting genomic DNA from a human sample known in the art may be employed. In embodiments of the invention, extraction of genomic DNA is preferably accomplished using commercial kits.
In the present invention, the reaction system for PCR amplification is preferably a 20. Mu.l system. The reaction program of the PCR amplification is preferably 94 ℃ pre-denaturation for 3min; denaturation at 94℃for 30s, annealing at 60℃for 30s, elongation at 72℃for 30s,35 cycles; stop at 72℃for 5min.
The sequencing is preferably accomplished using the Sanger sequencing method.
In the present invention, the scoring table is as follows:
wherein, the non-mutated base represents a reference base of normal population, 0 in the result represents that the base is not mutated, 1 represents that the base is mutated, 0/0 represents that none of the alleles at the locus is mutated, 0/1 or 1/0 represents that one of the alleles at the locus is mutated, and 1/1 represents that double mutation is generated at the alleles at the locus. For example, the rs1799724 locus is given a score of 0 (0/0) if the detection result is T/T, a score of 1 (1/0) if the detection result is T/C, a score of 1 (0/1) if the detection result is C/T, a score of 2 (1/1) if the detection result is C/C, and the same as the scoring method for other SNP loci.
In the invention, 8 SNP loci of rs1799724, rs2233945, rs510432, rs2071303, rs2145623, rs1800629, rs7234029 and rs3218253 are used for evaluating the effectiveness of adalimumab, and rs2032582, rs1135216, rs10280623, rs10497346 and rs10248420 are used for evaluating the toxicity of adalimumab.
The invention provides application of the gene mutation site or the primer group in preparing a kit for guiding a psoriasis patient to treat adalimumab.
In the present invention, the method of directing the treatment of adalimumab in a psoriasis patient preferably comprises the steps of: extracting genome DNA of psoriasis patients;
using the extracted genome DNA as a template, and carrying out PCR amplification by using the primer group to obtain an amplification product, and sequencing to obtain a sequencing result of a genetic variation locus;
obtaining genotyping of the genetic variation site of the psoriasis patient based on the sequencing result of the genetic variation site;
marking each SNP locus according to the genotyping of the genetic variation locus of the psoriasis patient by contrasting the marking table, and taking the SNP locus into a formula III to obtain a total score;
total score = rs1799724 score x 0.64+rs2233945 score x 0.56+rs510432 score x 0.83+rs2071303 score x 1.20+rs2145623 score x 0.84+rs1800629 score x 0.70+rs7234029 score x 1.16+rs3218253 score x 1.23+rs2032582 score x 1.16+rs1135216 score x 0.78+rs10280623 score x 0.62+rs10497346 score x 0.66+rs10248420 score x 0.87 formula III
Determining the dosing regimen of adalimumab based on the total score:
patients with psoriasis when the total score is greater than 0 are suitable for treatment with adalimumab; patients with psoriasis who have a total score of less than 0 are not recommended to be treated with adalimumab.
Experiments of the invention prove that psoriasis patients carrying different genetic variation sites show different response levels to adalimumab treatment, wherein 20 psoriasis patients (PAS 4-PAS 23) calculate total scores by adopting the method of the invention, the total score is more than 1, after a few months of adalimumab administration treatment, only PS8 patients are treated, the psoriasis is not improved, PS4, PS5, PS7 and PS9 are obviously improved after 6 months of treatment, the PASI scores are respectively reduced by 90.34%, 76.27%, 85.03% and 94.91%, and the psoriasis skin damage area of PS9 patients with high scores is reduced to the greatest. The method for guiding adalimumab to treat psoriasis is accurate and reliable, and can be suitable for clinical guiding drug application.
The following describes in detail a set of genetic variation sites for evaluating the effect of adalimumab on psoriasis, and the kit and use thereof, provided by the present invention, with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
Gene mutation site for evaluating effect of adalimumab on psoriasis treatment and amplification primers thereof
Sequencing analysis of genomic DNA was performed on 10,000 chinese ethnic psoriasis and 20,000 control populations using a ASA (Asian Screening Array) whole genome chip commonly used in GWAS studies, followed by a major analysis of genomic variation sites associated with adalimumab, with the differential site results shown in table 1:
table 1: genomic variation analysis of psoriasis in Chinese Han population
Note that: CHR represents chromosome number, SNP represents SNP site name, BP represents physical position, allele represents Allele, f_a represents Allele frequency, f_u represents Allele frequency, CHISQ represents chi-square test, P represents P value, OR represents dominance ratio, SE represents standard error. The positions of the SNP sites in Table 1 are shown in SEQ ID NO: 1-SEQ ID NO: position 220 in 13.
Amplification primers were designed upstream and downstream based on the SNP sites in Table 1, and the resulting primer sequences are shown in Table 2.
TABLE 2 primer sequences for detecting mutation sites of genes
Example 2
Method for evaluating effectiveness and safety of adalimumab in treating psoriasis and guiding drug administration based on gene mutation sites and amplification primers thereof in example 1
1. Extracting 2.0ml of peripheral blood of a psoriasis patient by using an EDTA anticoagulation tube, and extracting genome DNA by using a commercialized kit;
2. performing PCR amplification on the genomic DNA of a patient by using the primers designed in the embodiment 1, and performing genotype detection on the obtained PCR amplification product by using a Sanger sequencing platform after gel cutting and recovery;
wherein the reaction conditions for PCR amplification were 20. Mu.l of the system. The reaction program of the PCR amplification is preferably 94 ℃ pre-denaturation for 3min; denaturation at 94℃for 30s, annealing at 60℃for 30s, elongation at 72℃for 30s,35 cycles; stop at 72℃for 5min.
3. Obtaining the genotyping of the psoriasis patient, and scoring the psoriasis patient against a scoring table (see table 3);
table 3 scoring table
Note that: refAllele is a reference base of normal population, 0 in Result is a base which is not mutated, and 1 is a base which is mutated.
4. According to the formula I and the formula II, calculating the effectiveness and toxicity scores of the psoriasis patient on the treatment with adalimumab, wherein SNP loci shown by sequence numbers 1-8 are used for evaluating the effectiveness, SNP loci shown by sequence numbers 9-14 are used for evaluating the toxicity, the scoring method is that the psoriasis patient with the score being more than 0 is effective on the treatment with adalimumab, and the higher the score is, the better the curative effect is; patients with a score of less than 0 do not recommend treatment with adalimumab, and side effects may occur after use;
total score = score of rs1799724 x 0.64+ score of rs2233945 x 0.56+ score of rs510432 x 0.83+ score of rs2071303 x 1.20+ score of rs2145623 x 0.84+ score of rs1800629 x 0.70+ score of rs7234029 x 1.16+ score of rs3218253 x 1.23 formula I
Total score = score of rs2032582 x 1.16+score of rs1135216 x 0.78+score of rs10280623 x 0.62+score of rs10497346 x 0.66+score of rs10248420 x 0.87 formula II
5. Providing advice to utilize adalimumab treatment in terms of fractional psoriasis patients;
calculating the total score of the psoriasis patient according to a formula III, and giving a suggestion whether the psoriasis patient is suitable for adalimumab treatment or not according to the total score result, wherein the specific method is that the psoriasis patient with the score being more than 0 is effectively treated by the adalimumab, and the higher the score is, the better the curative effect is; psoriasis patients with a score of less than 0 are not recommended to be treated with adalimumab and may have side effects after use.
Total score = rs1799724 score x 0.64+rs2233945 score x 0.56+rs510432 score x 0.83+rs2071303 score x 1.20+rs2145623 score x 0.84+rs1800629 score x 0.70+rs7234029 score x 1.16+rs3218253 score x 1.23+rs2032582 score x 1.16+rs1135216 score x 0.78+rs10280623 score x 0.62+rs10497346 score x 0.66+rs10248420 score x 0.87 formula III
Example 3
Guidance and assessment of whether 20 psoriatic patients are suitable for adalimumab treatment using the assessment method of the invention
20 medically confirmed psoriasis patients (PSA 4 to PSA 23) were genotyped using the method of example 2 and scored for medication according to the scoring method.
The results are shown in Table 4.
Wherein the score of all other patients except PSA7 patient was greater than 1 and the score of PSA14 was highest. Based on this, 20 psoriasis patients were given a recommendation to treat with adalimumab, with a dosing regimen of 40mg subcutaneous injection, once every two weeks. Patients were scored for PASI (psoriasis area and severity index) before adalimumab treatment and 6 months of treatment, respectively. Psoriasis efficacy criterion: the PASI score before and after treatment was recorded according to the psoriasis lesion area and severity index (psoriasis area and severity index, PASI) score criteria, and efficacy determination was made according to the rate of decrease in PASI score (see formula IV).
PASI score decrease (%) = (pre-treatment PASI score-post-treatment PASI score)/pre-treatment PASI score x 100% formula IV
The results show that the adverse reactions such as headache, pruritus, transaminase rise and the like appear in the PSA7 patients after the administration, the patients stop being treated by adalimumab after one month after the administration, and the other 19 patients are treated for 6 months, the psoriasis skin lesions of the PSA8, PSA14 and PSA21 patients with obviously improved psoriasis skin lesions and high scores are basically resolved and healed.
Table 5 PASI scoring results after treatment with adalimumab in 20 psoriasis patients
Patient numbering PASI-0 PASI-6
PSA4 52.8 5.4
PSA5 31.6 7.5
PSA6 33.4 5
PSA7 13.9 -
PSA8 39.3 1
PSA9 33.3 3
PSA10 35.6 3
PSA11 25.8 2
PSA12 50 7
PSA13 25.1 3
PSA14 23.9 0
PSA15 14.1 1.5
PSA16 38.2 3
PSA17 20 2
PSA18 22.7 2
PSA19 40.6 4.5
PSA20 26.9 3
PSA21 23 1
PSA22 19 2
PSA23 34.3 4
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
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Claims (1)

1. The application of a group of SNP molecular markers or a primer group for amplifying the SNP molecular markers in the preparation of a kit for guiding a psoriasis patient to treat adalimumab is characterized in that the psoriasis patient is a Chinese Han people psoriasis patient;
the SNP molecular marker consists of the following SNP molecular markers: rs1799724, rs2233945, rs510432, rs2071303, rs2145623, rs1800629, rs7234029, rs3218253, rs2032582, rs1135216, rs10280623, rs10497346 and rs10248420;
the rs1799724 is a polymorphic site T/C present at position 31542482 of chromosome 6 on the basis of genomic version GRCh37 (hg 19);
the rs2233945 is a polymorphic site A/C existing at 31107361 of chromosome 6 on the basis of genome version GRCh37 (hg 19);
the rs510432 is a polymorphic site T/C existing at 106774030 th position of chromosome 6 on the basis of genome version GRCh37 (hg 19);
the rs2071303 is a polymorphic site T/C existing at 26091336 th position of chromosome 6 on the basis of genome version GRCh37 (hg 19);
the rs2145623 is a polymorphic site G/C existing at 35839236 th chromosome 14 on the basis of a genome version GRCh37 (hg 19);
the rs1800629 is based on the polymorphic site A/G existing at 31543031 position of chromosome 6 of the genome version GRCh37 (hg 19);
the rs7234029 is a polymorphic site G/A existing at 12877060 of chromosome 18 on the basis of genome version GRCh37 (hg 19);
the rs3218253 is a polymorphic site A/G existing at 37544810 of chromosome 22 on the basis of genome version GRCh37 (hg 19);
the polymorphic site A/C of the 87160618 locus of chromosome 7 of rs2032582 on the basis of the genome version GRCh37 (hg 19);
the rs1135216 is a polymorphic site C/T existing at 32814975 th position of chromosome 6 on the basis of genome version GRCh37 (hg 19);
the rs10280623 is a polymorphic site C/T existing at 87202544 th chromosome 7 on the basis of a genome version GRCh37 (hg 19);
the rs10497346 is a polymorphic site C/T existing at 169771196 th chromosome 2 on the basis of a genome version GRCh37 (hg 19);
the rs10248420 is a polymorphic site G/A existing at 87164986 th chromosome 7 on the basis of a genome version GRCh37 (hg 19);
the rs1799724, rs2233945, rs510432, rs2071303, rs2145623, rs1800629, rs7234029, rs3218253, rs2032582, rs1135216, rs10280623, rs10497346 and rs10248420 are polymorphic sites at the 220 rd position in the DNA fragment shown in the nucleotide sequence SEQ ID NO. 1-SEQ ID NO. 10 and polymorphic sites at the 451 rd position in the DNA fragment shown in the nucleotide sequence SEQ ID NO. 11-SEQ ID NO. 13;
the primer consists of the following primers:
the primer pair for amplifying rs1799724 comprises a forward primer with a nucleotide sequence shown as SEQ ID NO. 14 and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 15;
the amplified rs2233945 comprises a forward primer with a nucleotide sequence shown as SEQ ID NO. 16 and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 17;
the amplified rs510432 comprises a forward primer with a nucleotide sequence shown as SEQ ID NO. 18 and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 19;
the amplified rs2071303 comprises a forward primer with a nucleotide sequence shown as SEQ ID NO. 20 and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 21;
amplification rs2145623 comprises a forward primer with a nucleotide sequence shown as SEQ ID NO. 22 and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 23;
amplification rs1800629 comprises a forward primer with a nucleotide sequence shown as SEQ ID NO. 24 and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 25;
amplification rs7234029 comprises a forward primer with a nucleotide sequence shown as SEQ ID NO. 26 and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 27;
amplification rs3218253 includes a forward primer having a nucleotide sequence shown as SEQ ID NO. 28 and a reverse primer having a nucleotide sequence shown as SEQ ID NO. 29;
the amplified rs2032582 comprises a forward primer with a nucleotide sequence shown as SEQ ID NO. 30 and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 31;
the amplified rs1135216 comprises a forward primer with a nucleotide sequence shown as SEQ ID NO. 32 and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 33;
the amplified rs10280623 comprises a forward primer with a nucleotide sequence shown as SEQ ID NO. 34 and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 35;
the amplified rs10497346 comprises a forward primer with a nucleotide sequence shown as SEQ ID NO. 36 and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 37;
amplification rs10248420 includes a forward primer having a nucleotide sequence shown as SEQ ID NO. 38 and a reverse primer having a nucleotide sequence shown as SEQ ID NO. 39;
the method of directing treatment of adalimumab in a psoriasis patient comprises the steps of:
extracting genome DNA of psoriasis patients;
taking the extracted genome DNA as a template, carrying out PCR amplification by using the primer group to obtain an amplification product, and sequencing to obtain a sequencing result of SNP molecular markers;
obtaining the genotyping of the SNP molecular marker of the psoriasis patient based on the sequencing result of the SNP molecular marker;
marking each SNP locus according to the genotyping of the SNP molecular marker of the psoriasis patient by contrasting a marking table, and taking the SNP loci into a formula III to obtain a total score;
total score = score of rs1799724 x 0.64+ score of rs2233945 x 0.56+ score of rs510432 x 0.83+ score of rs510432 x 1.20+ score of rs2145623 x 0.84+ score of rs1800629 x 0.70+ score of rs7234029 x 1.16+ score of rs3218253 x 1.23+ score of rs2032582 x 1.16+ score of rs1135216 x 0.78+ score of rs10280623 x 0.62+ score of rs10497346 x 0.66+ score of rs10248420 x 0.87 formula III
Determining the dosing regimen of adalimumab based on the total score:
patients with psoriasis when the total score is greater than 0 are suitable for treatment with adalimumab; patients with psoriasis who have a total score of less than 0 do not recommend treatment with adalimumab;
the scoring table is as follows:
SNP locus Non-mutated bases Results 1 score Results 2 score Results 3 score Results 4 score rs1799724 T 0/0 (0) 0/1(1) 1/0(1) 1/1(2) rs2233945 A 0/0 (0) 0/1(1) 1/0(1) 1/1(2) rs510432 T 0/0 (0) 0/1(1) 1/0(1) 1/1(2) rs2071303 T 0/0 (0) 0/1(1) 1/0(1) 1/1(2) rs2145623 G 0/0 (0) 0/1(1) 1/0(1) 1/1(2) rs1800629 A 0/0 (0) 0/1(1) 1/0(1) 1/1(2) rs7234029 G 0/0 (0) 0/1(1) 1/0(1) 1/1(2) rs3218253 A 0/0 (0) 0/1(1) 1/0(1) 1/1(2) rs2032582 A 0/0(1) 0/1(-1) 1/0(-1) 1/1(-2) rs1135216 C 0/0(1) 0/1(-1) 1/0(-1) 1/1(-2) rs10280623 C 0/0(1) 0/1(-1) 1/0(-1) 1/1(-2) rs10497346 C 0/0(1) 0/1(-1) 1/0(-1) 1/1(-2) rs10248420 G 0/0(1) 0/1(-1) 1/0(-1) 1/1(-2)
。
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