CN111518894B - Reagent for detecting rs9273471 site polymorphism and its application - Google Patents

Abstract

The invention belongs to the fields of molecular biology and medicine, and relates to a reagent for detecting rs9273471 locus polymorphism and application thereof. Prediction of risk of latent autoimmune diabetes in adults (LADA) or prediction of discrimination between LADA and type 2 diabetes (T2 DM) is performed by detection of the genotype of the locus. The invention also discloses a corresponding detection kit, which contains a primer for amplifying the rs9273471 locus and a primer for detecting the locus genotype. The method for detecting the genotype of the rs9273471 locus is simple, easy, quick and efficient, has low cost and provides a simple and novel approach for diagnosis and treatment of LADA.

Description

Reagent for detecting rs9273471 site polymorphism and its application

Technical field:

The invention relates to the fields of molecular biology and medicine, and relates to a detection reagent for the single nucleotide polymorphism site rs9273471 of human HLA gene and its application in the preparation of LADA-related risk assessment reagents.

Background technique:

Latent autoimmune diabetes in adults (LADA) is an adult-onset autoimmune diabetes with diabetes-related autoantibodies that does not require insulin treatment for a period of time after diagnosis. It is the most common type of adult-onset autoimmune diabetes. It is also probably the most common form of autoimmune diabetes. Immunologically, glutamic acid decarboxylase autoantibodies are the most common autoantibodies in adults with diabetes.

As a special subtype of type 1 diabetes (T1DM), LADA is also a T cell-mediated autoimmune disease, which is related to the abnormal expression of HLA class II antigen on the surface of pancreatic beta cells. The histocompatibility complex is located on the short arm of the sixth pair of chromosomes. There are nearly 60 loci in the entire complex. According to the different coding molecular characteristics, the genes of the entire complex can be divided into three categories: class I, class II Class, Class III. The class II gene region is located at the proximal end of the centromere, and is the most complex region, mainly composed of three subregions, DR, DQ, and DP, and each subregion has several sites. The locus is a locus in the DQ subregion of a class II gene. CD4 ⁺ T cell surface receptors combine with the complex formed by HLA class II molecules and antigenic peptides to stimulate helper T cells to secrete cytokines, etc., thus participating in the pathogenesis of LADA. The infiltration of islets by T cells and antigen-presenting cells is a lengthy process, leading to the onset of LADA that can occur months to years after the onset of the immune response.

Diabetes is a multifactorial disease caused by complex interactions between environmental and genetic risk factors. The genetic determinants of type 1 diabetes (T1DM) and type 2 diabetes (T2DM) have been extensively studied, whereas studies of latent autoimmune diabetes in adults (LADA) have been sparse. In order to finally realize the treatment of LADA, there is an urgent need in this field to find LADA susceptibility genes, and to develop methods, kits, and related therapeutic drugs for detecting LADA susceptibility genes. In addition, LADA patients are often misdiagnosed as type 2 diabetes due to similar clinical symptoms. In order to distinguish LADA from other types of diabetes and to treat patients with LADA more precisely, the detection and treatment of LADA require specific strategies.

Invention content:

The primary purpose of the present invention is to provide an application of a reagent for detecting rs9273471 site polymorphism in preparing preparations for predicting LADA disease risk. This polymorphic site was found to be associated with the risk of LADA for the first time, and the genotype of this site was used to determine the risk of LADA, with high prediction accuracy and high reliability.

Further, the polymorphism at the rs9273471 site is: AA, AG or GG.

The present invention found that the number of normal people in the population carrying the rs9273471GG genotype is much higher than the number of LADA patients, suggesting that patients with the GG genotype have a low risk of suffering from LADA. The other two genotypes, AA and AG, are based on the GG genotype.

According to the statistical analysis of about 500 cases of LADA cases and normal controls, the present invention finds that when the genotype of the rs9273471 locus is AA, the odds ratio OR of the genotype of GG is 4.14, and the p value is less than 0.05, which has a significant difference . That is, the risk of LADA in the population carrying the rs9273471AA genotype was significantly increased, which was 4.14 times that of the GG genotype population; when the genotype of the rs9273471 locus was AG, the odds ratio OR was 1.31 relative to the genotype GG, but The p value is greater than 0.05, and there is no significant difference. Therefore, when the genotype of rs9273471 is AG, it is not enough to judge the risk of LADA.

Further, the conclusion obtained in the present invention is: when the genotype of the rs9273471 locus of the detected object is AA, the risk of LADA is higher than that of the genotype GG.

Furthermore, the above reagents for detecting polymorphisms at the rs9273471 site can predict the following situations:

(1) It is used to predict the risk of LADA in ordinary people without clinical symptoms of diabetes.

(2) It is used to predict the risk of LADA in people who have clinical symptoms of diabetes but have not yet been diagnosed with diabetes.

(3) It is used to predict the risk of LADA in patients who have been diagnosed with diabetes but have not yet been diagnosed with a specific type of diabetes.

The second object of the present invention is to provide an application of a reagent for detecting rs9273471 site polymorphism in preparing a predictive preparation for distinguishing LADA and T2DM. This polymorphic locus is found for the first time and can be used to predict the difference between LADA and T2DM, and the genotype of this locus is used to determine the probability of suffering from LADA instead of T2DM, with high prediction accuracy and high reliability.

Further, the polymorphism at the rs9273471 site is: AA, AG or GG.

Since the number of T2DM patients in the population carrying the rs9273471GG genotype is much higher than the number of LADA patients, it is suggested that the probability of GG genotype suffering from LADA rather than T2DM is low. The other two genotypes AA and AG are based on the GG genotype.

The present invention carries out statistical analysis with about 500 cases of LADA cases and T2DM cases and finds that:

When the genotype of rs9273471 locus is AA, the odds ratio OR is 3.45 relative to the genotype of GG.

When the genotype of rs9273471 locus was AG, the odds ratio OR was 1.44 relative to the genotype of GG.

That is, people carrying the rs9273471AA genotype have a significantly higher probability of developing LADA instead of T2DM, and the probability of suffering from LADA instead of T2DM is 3.45 times that of the GG genotype; 1.44 times that of the crowd.

Furthermore, when distinguishing between LADA and T2DM, the probability of rs9273471 locus genotype suffering from LADA instead of T2DM is AA>AG>GG genotype, and the p values are all less than 0.05, which has a significant difference.

The above reagents for detecting rs9273471 polymorphism can predict the following situations:

(1) It is used to predict the probability of LADA instead of T2DM in people who have clinical symptoms of diabetes but have not yet been diagnosed with diabetes.

(2) It is used to predict the probability of LADA instead of T2DM in patients who have been diagnosed with diabetes but have not yet been diagnosed with a specific type of diabetes.

(3) It is used to predict the probability of LADA instead of T2DM in patients who are diagnosed with diabetes but have not yet been diagnosed with LADA or T2DM.

In the case of (3), LADA patients are often misdiagnosed as type 2 diabetes due to similar clinical symptoms. Therefore, this distinction in application is of great medical significance.

The third object of the present invention is to provide a kit for predicting the risk of LADA or for predicting and distinguishing between LADA and T2DM. The kit is designed based on the newly discovered SNP site of the present invention for predicting the risk of LADA or predicting the difference between LADA and T2DM, and has high prediction accuracy and high reliability.

The kit is specifically a reagent for detecting the polymorphism of the rs9273471 site.

Further, the kit:

Contains primers for specifically amplifying the rs9273471 site and sequencing primers.

Further, the preferred sequencing primer sequence is: TGGGAATTCTGGGCAGG, see SEQ ID NO.1; but the kit of the present invention is not limited to this sequencing sequence.

Further, the preferred amplification primer sequence is: F: TGCATCAAGCTGAAGTTCTGTG, see SEQ ID NO.2; R: GGTGGGGATGAAAGGAGATG, see SEQ ID NO.3; but the kit of the present invention is not limited to the amplification primer sequence.

The LADA susceptibility gene detected by the present invention is the genotype of the rs9273471 site of the DQ subregion of the HLA class II gene. The nucleotide sequence of rs9273471 can be found at http://genome.ucsc.edu/.

The supporting detection method of the present invention comprises the following steps:

(a) extracting the genomic DNA in the sample;

(b) PCR amplification to obtain a product comprising the rs9273471 site;

(c) Sequencing the product, and analyzing the genotype of the site.

The detection object of the present invention is Asian race, especially Chinese.

Techniques such as extraction of genomic DNA, amplification, and sequencing involved in the above methods can all adopt conventional operating methods in the art.

After years of research, the present invention proves for the first time that the single nucleotide polymorphism at the rs9273471 site of the DQ subregion of the HLA class II gene is related to the risk of LADA. The change of rs9273471 genotype will increase the risk of LADA, and the results of association studies show that there is a significant difference in the distribution of rs9273471 (GG→AA) between cases and controls (P<0.05); and this locus is used for The determined standard genotype has a higher prediction accuracy. In addition, the present invention also found that LADA patients were often misdiagnosed as type 2 diabetes due to similar clinical symptoms, and this polymorphic site was found for the first time to be used to predict and distinguish LADA and T2DM, and the criteria for determining this site were also It is a genotype, with high prediction accuracy and high reliability; therefore, it is of great significance to be able to distinguish LADA from type 2 diabetes simply and efficiently, and to provide more precise treatment for LADA patients.

The invention can be used for early diagnosis of individual LADA susceptibility, comprising the steps of: (1) extracting genomic DNA of a sample. (2) The genomic DNA of the sample is amplified by PCR with specific amplification primers for rs9273471 to obtain amplification products. (3) Design sequencing primers to sequence the rs9273471 site of the amplified product. Detect the genotype of the individual's HLA gene rs9273471 site, and the individual with the AA genotype of rs9273471 has a significantly higher susceptibility to LADA than the general population; in order to determine whether the individual's risk of LADA is higher than the general population, in addition , can also be used to predict and distinguish between two types of diabetes, LADA and T2DM. The invention provides a simpler and easier method for the diagnosis and treatment of LADA.

The present invention also develops a corresponding detection kit based on the above findings, which includes specific primers for amplifying the site and sequencing primers. Using the invention to detect the genotype of the rs9273471 site, the method is simple, fast, efficient and low in cost, and provides a simple new way for the diagnosis and treatment of LADA.

Description of drawings:

Fig. 1 is the genomic DNA concentration and quality detection result of the sample in embodiment 1.

Fig. 2 shows that the genomic DNA lengths of the samples in Example 1 are all greater than 10 kb detected by agarose gel electrophoresis.

Fig. 3 is the electrophoresis result of amplification of the rs9273471 site of some samples in Example 2.

Figure 4 is a screenshot of the amplification and sequencing results of the rs9273471 site in Example 2; three genotype illustrations; the upper figure is the rs9273471AA genotype; the middle figure is the rs9273471AG genotype; the lower figure is the rs9273471GG genotype.

Figure 5 is a screenshot of the amplification and sequencing results of the rs9273471 site in Example 3; the upper figure is the rs9273471AA genotype; the middle figure is the rs9273471AG genotype; the lower figure is the rs9273471GG genotype.

Detailed ways:

The present invention will be further described below in conjunction with specific implementation cases. These embodiments are only used to describe the present invention and do not limit the scope of the present invention. Experimental methods without specific conditions are in accordance with conventional conditions or in accordance with the conditions suggested by the manufacturer.

Embodiment 1: PCR amplification

1. Experimental materials

PCR instrument: Eppendorf, Germany; polymerase chain reaction solution: Gold Mix, Beijing Qingke Biotechnology Co., Ltd.

2. Primer design and synthesis

Using the partial sequence of the HLA class II gene including the rs9273471 site as a template, Primer 3 software was used to analyze and design primers, which were synthesized by Beijing Qingke Biotechnology Co., Ltd.

The sequencing primer sequence is: TGGGAATTCTGGGCAGG

The amplification primer sequence is: F: TGCATCAAGCTGAAGTTCTGTG

R: GGTGGGGATGAAAGGAGATG.

3. Sample testing

1. Experimental detection LADA, T2DM patients and normal control population, each case collected 5ml blood samples, extracted genomic DNA, and the extraction results were detected by Thermo Fisher Nanodrop2000.

2. Carry out PCR amplification according to the following system (Table 1, Table 2):

Table 1 PCR reaction system

Add reagent Amount added Gold medal PCR mix 27ul f 1ul R 1ul dna 1ul

Table 2 PCR reaction program

4. Genotype test results

The test results of genomic DNA extraction:

The concentration and quality of genomic DNA of all samples meet the detection requirements, 260/280>1.8, concentration>10ng/ul, see Figure 1, and the length of genomic DNA detected by agarose gel electrophoresis is greater than 10kb, as shown in Figure 2.

Direct sequencing is recognized as the most accurate genotyping method, and it is also the "gold standard" for HLA typing recommended by the World Health Organization's HLA Nomenclature Committee. PCR products can be directly used for sequencing, but some SNP polymorphic sites are missing in some individuals, resulting in the inability to obtain amplification products, and also resulting in double peaks in the sequencing results, making it difficult to distinguish. One approach is to sequence the PCR products after cloning. However, the overall process of sequencing PCR products after cloning is very troublesome, and the cost of sequencing is high, so it is difficult to be widely used. Another method is amplification and sequencing with specific primers, which is relatively simple and inexpensive. The present invention adopts the latter method, has carried out several attempts, used multiple pairs of primers for amplification and sequencing, and designed specific primers for the common region to solve this problem.

The primers used are as follows:

First group:

Amplification primer rs9273471-1F: CATCAAGCTGAAGTTCTGTGTC, see SEQ ID NO.4;

rs9273471-1R: GGGTGGGGATGAAAGGAGAT, see SEQ ID NO.5;

Sequencing primer rs9273471-seq1: CATCAAGCTGAAGTTCTGTGTC, see SEQ ID NO.6;

Second Group:

Amplification primer rs9273471-2F: TCTGCATCAAGCTGAAGTTCT, see SEQ ID NO.7;

rs9273471-2R: TGCTTCTCTTGAGCAGTCTGA, see SEQ ID NO.8;

Sequencing primer rs9273471-seq2: TCTGCATCAAGCTGAAGTTCT; see SEQ ID NO.9;

The third group:

Amplification primer rs9273471-3F: TCTGCATCAAGCTGAAGTTCTG, see SEQ ID NO.10;

rs9273471-3R: TGGGGATGAAAGGAGATGAC, see SEQ ID NO.11;

Sequencing primer rs9273471-seq3: TCTGCATCAAGCTGAAGTTCTG; see SEQ ID NO.12;

Fourth group:

Amplification primer rs9273471-4F: TGCATCAAGCTGAAGTTCTGTG

rs9273471-4R: GGTGGGGATGAAAGGAGATG

Sequencing primer rs9273471-seq4: TGGGAATTCTGGGCAGG.

Finally, only the fourth group of primers can be well amplified and sequenced, and the other groups of primers have double peaks, which are difficult to distinguish.

Example 2

The rs9273471 site of the HLA class II gene was detected by sequencing. A total of 488 LADA patients and 501 normal controls were selected for sequencing to determine the genotype of rs9273471.

A total of 488 LADA patients, 286 males, 202 females, aged 42.5 (33-53) years, 501 normal controls, 258 males, 243 females, 49 (38-58) years old, age data were all Expressed as median (interquartile range).

Inclusion criteria for LADA patients: ①diagnosed with diabetes; ②positive for one or more islet autoantibodies: the radioligand method was used for antibody detection. Threshold: GADA≥0.05, IA-2A≥0.0078, ZnT8A≥0.011; ③Not dependent on insulin therapy within 6 months after diagnosis of diabetes; ④Age ≥18 years old. Exclusion criteria: ①gestational diabetes, other special types of diabetes; ②severe infection, trauma, surgery and other stressful situations; ③combined with other autoimmune diseases; ④malignant tumors; ⑤serious cardiovascular and cerebrovascular diseases; ⑥pregnant or lactating women; ⑦Hepatic and renal insufficiency, etc.; ⑧Other conditions that are not suitable for inclusion in the case group.

Inclusion criteria for 501 normal controls: ① Unrelated Han people in Hunan; ② Oral glucose tolerance test (OGTT) showed fasting blood glucose <5.6mmol/L, and blood glucose <7.8mmol/L after 2 hours. Exclusion criteria: ①Heart, brain, liver, kidney and other chronic diseases and endocrine diseases; ②Combined with other types of autoimmune diseases; ③Have a family history of diabetes; ④Combined with malignant tumors.

1. Experimental method

The amplification primer sequence is: F: TGCATCAAGCTGAAGTTCTGTG

R: GGTGGGGATGAAAGGAGATG.

The amplified product was detected by agarose gel electrophoresis, and the amplified product was sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The sequencing primer was: TGGGAATTCTGGGCAGG. Analysis was performed with Lasergene software.

2. Amplification results of rs9273471 site of HLA class II gene: see attached figure 3.

3. The detection results of the genotype at the rs9273471 site of the HLA class II gene: see Figure 4.

4. Association analysis of HLA class II gene rs9273471 genotype and LADA susceptibility

The distribution of HLA class II gene rs9273471 in LADA patients and normal controls was compared using Chi-square test, and SPSS software was used for statistical analysis. Since the number of normal people in the population carrying the rs9273471GG genotype is much higher than the number of LADA patients, it is suggested that the GG genotype patients The risk of LADA was low, and the other two genotypes AA and AG were compared with GG genotype. The test results and SPSS software analysis results are shown in Table 3 below. The risk of LADA in people carrying the rs9273471AA genotype is significantly increased, which is 4.14 times that of the GG genotype group. The p value is less than 0.05, and there is a significant difference; The risk of LADA in the AG genotype group was 1.31 times that of the GG genotype group, with a p value greater than 0.05, and the difference was not significant.

Table 3 Comparison of rs9273471 genotype frequency between LADA patients and normal controls

Note: OR: odds ratio; 95% CI: 95% confidence interval, * represents significant difference.

Example 3

The rs9273471 site of HLA class II genes in LADA and T2DM patients was detected by sequencing. 488 LADA patients and 501 T2DM patients were selected for sequencing to determine the genotype of rs9273471.

A total of 488 patients with LADA, 286 males, 202 females, aged 42.5 (33-53) years, 501 T2DM patients, 256 males, 245 females, 52 (44-60) years old, age data Expressed as median (interquartile range).

Inclusion criteria for LADA patients: ①diagnosed with diabetes; ②positive for one or more islet autoantibodies: the radioligand method was used for antibody detection. Threshold: GADA≥0.05, IA-2A≥0.0078, ZnT8A≥0.011; ③Not dependent on insulin therapy within 6 months after diagnosis of diabetes; ④Age ≥18 years old. Exclusion criteria: ①gestational diabetes, other special types of diabetes; ②severe infection, trauma, surgery and other stressful situations; ③combined with other autoimmune diseases; ④malignant tumors; ⑤serious cardiovascular and cerebrovascular diseases; ⑥pregnant or lactating women; ⑦Hepatic and renal insufficiency, etc.; ⑧Other conditions that are not suitable for inclusion in the case group.

Inclusion criteria for T2DM patients: ①Diabetes mellitus diagnosed; ②Insulin-independent therapy since the onset, islet autoantibody negative. Exclusion criteria: ①gestational diabetes, other special types of diabetes; ②severe infection, trauma, surgery and other stressful situations; ③combined with other autoimmune diseases; ④malignant tumors; ⑤serious cardiovascular and cerebrovascular diseases; ⑥pregnant or lactating women; ⑦Hepatic and renal insufficiency, etc.; ⑧Other conditions that are not suitable for inclusion in the case group.

1. Experimental method

The amplification primer sequence is: F: TGCATCAAGCTGAAGTTCTGTG

R: GGTGGGGATGAAAGGAGATG.

The sequencing primer is: TGGGAATTCTGGGCAGG. The amplified products were sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. Analysis was performed with Lasergene software.

2. The schematic diagram of the detection results of the genotype at the rs9273471 locus of the HLA class II gene is shown in Figure 5.

3. Association analysis of HLA class II gene rs9273471 genotype and LADA susceptibility

Chi-square test was used to compare the distribution of HLA class II gene rs9273471 in LADA patients and T2DM patients, and SPSS software was used for statistical analysis. Since the number of T2DM patients in the population carrying the rs9273471GG genotype is much higher than the number of LADA patients, it is suggested that the GG genotype patients The probability of LADA instead of T2DM is low, and the other two genotypes AA and AG are all based on GG genotype. The test results and SPSS software analysis results are shown in the table below. When distinguishing between LADA and T2DM, the probability of suffering from LADA instead of T2DM in people carrying the rs9273471AA genotype is significantly increased, and the probability of suffering from LADA instead of T2DM is 3.45 times that of the GG genotype , the p value is less than 0.05, there is a significant difference; the probability of people carrying the rs9273471AG genotype suffering from LADA instead of T2DM is 1.44 times that of the GG genotype group, the p value is less than 0.05, there is a significant difference.

Table 4 Comparison of rs9273471 genotype frequency between LADA patients and T2DM patients

Note: OR: odds ratio; 95% CI: 95% confidence interval, * represents significant difference.

Example 4:

200 normal controls were detected, including 99 males and 101 females, aged 44 (34-55) years old, and the age data were expressed by median (interquartile range).

200 confirmed LADA patients and T2DM patients were detected respectively, and the diagnostic criteria were the same as in Example 3. Among them, there were 200 LADA patients, 117 males, 83 females, aged 49 (38.5-58) years old, 200 T2DM patients, 97 males, 103 females, 52 (42-58) years old, age data All are expressed as median (interquartile range).

The genotypes of rs9273471 were detected according to the methods in Examples 2 and 3, and the frequencies of the genotypes in LADA, T2DM patients and normal controls were basically consistent with those in Examples 2 and 3, further proving that the experimental data is true and credible. The frequency is as follows:

Table 5 Comparison of rs9273471 genotype frequency of 200 cases of LADA patients and normal controls

Note: OR: odds ratio; 95% CI: 95% confidence interval, * represents significant difference.

Table 6 Comparison of rs9273471 genotype frequency of 200 LADA patients and T2DM patients

Note: OR: odds ratio; 95% CI: 95% confidence interval, * represents significant difference.

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Claims (6)

1. The application of the reagent for detecting the polymorphism of rs9273471 site in the preparation of preparations for predicting the risk of LADA. 2. The application according to claim 1, wherein the polymorphism of the rs9273471 site is: AA, AG or GG. 3. The application according to claim 1 or 2, wherein when the genotype of the rs9273471 locus of the detected object is AA, the risk of LADA is higher than that of the genotype GG. 4. The application of the reagent for detecting the polymorphism of the rs9273471 site in the preparation of the prediction preparation for distinguishing LADA and T2DM. 5. The application according to claim 4, characterized in that the polymorphism of the rs9273471 site is: AA, AG or GG. 6. The application according to claim 4 or 5, wherein when distinguishing LADA and T2DM, the probability of rs9273471 locus genotype suffering from LADA instead of T2DM is in the order of AA>AG>GG genotype.

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