CN112094905A - Primer group and kit for detecting SNP of diabetic nephropathy related susceptibility gene ALDOB - Google Patents
Primer group and kit for detecting SNP of diabetic nephropathy related susceptibility gene ALDOB Download PDFInfo
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Abstract
The invention discloses a primer group and a kit for detecting SNP of diabetic nephropathy related susceptibility gene ALDOB. The primer composition is selected from one or more PCR primers in any one of sites rs118168553, rs146360505, rs1588170400, rs760339672, rs182003715, rs 37397621, rs759204107 and rs141267935 of ALDOB genes. The kit comprises the primer composition. The mutation sites discovered by the invention can be used as detection targets to be applied to the preparation of an SNP screening kit of susceptibility genes related to diabetic nephropathy. The detection primer can be used as an important component of the kit.
Description
Technical Field
The invention relates to the technical field of genetic engineering, in particular to a primer group and a kit for detecting SNP of diabetic nephropathy related susceptibility gene ALDOB.
Background
The aldolase B (ALDOB) gene is located at 9q22.3 for a total of 9 exons. Aldolase B consists of 364 amino acid residues. Under normal conditions, this enzyme catalyzes the cleavage of fructose-1-phosphate to dihydroxyacetone phosphate and D-glyceraldehyde. When ALDOB gene is mutated, ALDOB structure and activity are changed, 1-phosphofructose is accumulated in organs such as liver, small intestine, kidney and the like, and further activity of enzymes such as phosphorylase, fructose 1, 6-bisphosphatase, liver aldolase, fructokinase and the like in cells is inhibited, so that metabolism of organs such as liver, small intestine, kidney and the like is disturbed, and organ damage is caused. By 1995, Tolan concluded that 21 mutations in the aldolase B gene have been identified in northern europe, elsewhere and in minority, 15 of which were single base substitutions, a total of 9 amino acid substitutions in the aldolase B protein by other amino acids, 4 codons with nonsense mutations, 2 splicing defects, 2 large deletions and 2 deletions of 4 bases per 1 base insertion, with the most common mutations occurring in exons 5 and 9. In 2005, Santen et al screened 72 family 80 patients for fructose intolerance gene mutations, which resulted in: the most common enzyme protein mutations A150P account for 65%, A175O accounts for 11%, N33K accounts for 8%, and then 360-363 delCAAA, R60X, Y204X and 865delC, and 8 new mutations are found, wherein the deletion mutation: 1044 to 1049delTTCTGG, C345 to 372del28, C841 to 842 delAC; insertion mutation: insACACT; splicing mutation: 113-1G is greater than A, C799+2T is greater than K; nonsense mutation: 612T > G (Y204X); missense mutation: 532T > G (C178R), 851T > C (L284P). Based on these data, the prevalence of HFI with few ALDOB gene mutations in Central Europe was considered to be 1: 26000 (95% confidence interval of 1: 12600-1: 78000). Esposito et al reported 6 novel mutations in the ALDOB gene: 3 missense, 1 nonsense and 2 splice mutations [ g6846T > CLPI74T, g10236G > T (PV222F), g10258T > C (PL229p), g8584C > T, g8180G > C, g10196A > C ]. However, the mutation of ALDOB in Asian population, especially Chinese population, has only been reported in small sample size.
Diabetic Nephropathy (DN) is a major microvascular complication of diabetes and is one of the most common causes of end stage renal disease and an independent risk factor for cardiovascular events. Diabetic patients had 30 times as many concurrent DNs as patients without concurrent DNs. Since diabetics cannot take too much glucose and because fructose has a very low glycemic index and is metabolized differently than glucose and cannot undergo insulin regulation, fructose is often used clinically for the prevention and control of diabetes, and is called "ideal sweetener for diabetics". However, currently, in Asia, particularly China, when fructose is administered to diabetic patients, the ALDOB mutant gene is not detected, and thus the conditions of sensitivity or intolerance to fructose existing in the individuals are ignored.
Disclosure of Invention
The invention aims to provide application of a group of SNPs of predisposing genes ALDOB related to diabetic nephropathy.
Another objective of the invention is to provide a group of SNP detection primer combinations of the susceptibility gene ALDOB related to diabetic nephropathy.
It is still another object of the present invention to provide a kit for detecting SNPs in susceptibility genes associated with diabetic nephropathy.
The purpose of the invention can be realized by the following technical scheme:
the SNP of a group of ALDOB genes is used as a detection target point in the preparation of an ALDOB gene mutation screening kit for diabetics, and the SNP of the ALDOB genes is selected from one or more of the following SNP sites: rs118168553, rs146360505, rs1588170400, rs760339672, rs182003715, rs 373975, rs759204107 and rs 141267935.
The SNP of a group of ALDOB genes is used as a detection target point in the preparation of an SNP detection kit of a susceptibility gene related to diabetic nephropathy, and the SNP of the ALDOB genes is selected from one or more of the following SNP sites: rs118168553, rs146360505, rs1588170400, rs760339672, rs182003715, rs 373975, rs759204107 and rs 141267935.
The application of the reagent for detecting the SNP of the ALDOB gene in preparing the ALDOB gene mutation screening kit for the diabetic patient is disclosed.
The application of the reagent for detecting the SNP of the ALDOB gene in preparing the SNP detection kit of the susceptibility gene related to diabetic nephropathy.
Preferably, the reagent for detecting SNP of ALDOB gene is selected from the group consisting of a reagent for detecting SNP of ALDOB gene according to claim 1 by Taqman method, DHPLC technique and mass spectrometry, single strand conformation polymorphism analysis, allele-specific oligonucleotide hybridization, DNA chip, micro-sequencing, high temperature ligase method, SNaPshot method and DNA sequencing method.
Preferably, the reagent for detecting the SNP of the ALDOB gene is a PCR primer for detecting any one or more of sites rs118168553, rs146360505, rs1588170400, rs760339672, rs182003715, rs 373975, rs759204107 and rs141267935 of the ALDOB gene.
As a further preferred aspect of the present invention, the PCR primers are as follows:
a kit for detecting SNP of susceptibility genes related to diabetic nephropathy comprises PCR primers of any one or more of sites of ALDOB genes rs118168553, rs146360505, rs1588170400, rs760339672, rs182003715, rs 373975, rs759204107 and rs 141267935.
In a preferred embodiment of the present invention, the primer sequences are as follows:
the kit also comprises MgCl2、PCR Enzym、dNTP Mix
A primer composition for detecting SNP of diabetic nephropathy related susceptibility gene ALDOB is selected from PCR primer of any one or more of ALDOB gene rs118168553, rs146360505, rs1588170400, rs760339672, rs182003715, rs 373974, rs759204107 and rs141267935 locus; preferred are the primers shown below:
has the advantages that:
according to the invention, through whole genome sequencing of diabetic patients (who do not have diabetic nephropathy within 10 years) and diabetic nephropathy patients in Chinese population, it is found that certain ALDOB mutations are abnormally high in diabetic nephropathy patients compared with diabetic patients who do not have diabetic nephropathy. These SNPs, which are differentially present in diabetic patients who do not develop diabetic nephropathy and diabetic nephropathy patients, are the first reports of the present invention. Further in vivo and in vitro experimental studies show that the ALDOB mutation can cause fructose metabolism abnormality to cause kidney injury, and the detection of the mutation at the site can predict the risk of diabetic nephropathy. Therefore, it is considered that the detection of ALDOB gene mutation is particularly required for diabetic patients. If a diabetic carries some mutation of the ALDOB gene, the diabetic needs to avoid the intake of fructose especially in daily life and clinical treatment to avoid diabetic nephropathy due to abnormal fructose metabolism.
Drawings
FIG. 1 immunofluorescence results on cryo-sections of kidney tissue: compared with normal kidney tissues, the ALDOB protein is obviously down-regulated in the glomerulus of stage IIa, IIb, III and IV diabetic nephropathy.
Detailed Description
Example 1
1. Blood samples of 199 diabetic nephropathy patients and 200 diabetic nephropathy-free patients within 10 years are collected, DNA is extracted for whole genome sequencing, and sequencing results show that in 199 diabetic patients, several mutation sites of ALDOB are remarkably higher than those of diabetic nephropathy-free patients. Analysis shows that the sites can cause the reduction of ALDOB expression content/activity, so that fructose cannot be metabolized normally, and further kidney damage is caused.
TABLE 1
Example 2
2.1 extracting 1ml of blood from the subject, extracting the blood DNA with a blood DNA extraction kit (for example, 0.1-20 ml (DP319) of blood genome DNA extraction System available from Tiangen corporation);
2.2A 0.5. mu.M (per primer) PCR primer mix was prepared containing S (forward primer) and R (reverse primer) for each SNP site in the multiplex reaction (Table 2).
TABLE 2
2.3DNA samples (volume 2. mu.l; concentration 10 ng/. mu.l) have been preloaded in 96-well plates. To each empty portion was added a PCR reaction solution (the reaction solution composition is shown in Table 3).
Table 3: PCR reaction system
2.4 after completion, the plate was sealed with a membrane, vortexed for 2min and centrifuged for 2min at 3000 rpm.
2.5 Place 96-well plate on PCR instrument for thermal cycling as follows:
95℃，5min
40 cycles (95 ℃, 30 s; 60 ℃, 30 s; 72 ℃, 30s)
72℃，5min
4 ℃ heat preservation
2.6 performing NGS sequencing on the PCR product to obtain sequence information, and judging the mutation carried by the ALDOB gene of the subject.
2.7 results of detection
The whole genome detection results are shown in table 4:
TABLE 4
The NGS test results are shown in table 5:
TABLE 5
The experimental results show that the mutation results detected by NGS are 100% identical to the mutation conditions detected by whole genome sequencing.
Example 3
We performed immunofluorescent marker staining of kidney tissue sections of diabetic nephropathy patients due to ALDOB mutation, and the results (FIG. 1) showed that ALDOB protein expression was significantly reduced in tissues of diabetic nephropathy patients due to mutation of ALDOB gene.
Sequence listing
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Claims (10)
1. The SNP of a group of ALDOB genes is used as a detection target point in the preparation of an ALDOB gene mutation screening kit for diabetics, and the SNP of the ALDOB genes is selected from one or more of the following SNP sites: rs118168553, rs146360505, rs1588170400, rs760339672, rs182003715, rs 373975, rs759204107 and rs 141267935.
2. The SNP of a group of ALDOB genes is used as a detection target point in the preparation of an SNP detection kit of a susceptibility gene related to diabetic nephropathy, and the SNP of the ALDOB genes is selected from one or more of the following SNP sites: rs118168553, rs146360505, rs1588170400, rs760339672, rs182003715, rs 373975, rs759204107 and rs 141267935.
3. The application of the reagent for detecting the SNP of the ALDOB gene in claim 1 in preparing the ALDOB gene mutation screening kit for diabetics.
4. Use of a reagent for detecting SNP of ALDOB gene as set forth in claim 1 in the preparation of a SNP detection kit for susceptibility genes associated with diabetic nephropathy.
5. The use according to claim 4, wherein the reagent for detecting SNP of ALDOB gene as defined in claim 1 is selected from the group consisting of Taqman method, DHPLC technique and mass spectrometry, single strand conformation polymorphism analysis, allele-specific oligonucleotide hybridization, DNA chip, micro-sequencing, high temperature ligase method, SNaPshot method and DNA sequencing method for detecting SNP of ALDOB gene as defined in claim 1.
6. The use according to claim 4, characterized in that the reagent for detecting SNP of ALDOB gene in claim 1 is PCR primer for detecting any one or more of sites rs118168553, rs146360505, rs1588170400, rs760339672, rs182003715, rs 37397621, rs759204107 and rs141267935 of ALDOB gene.
7. The use according to claim 6, wherein the PCR primers are as follows:
8. a kit for detecting SNP of susceptibility genes related to diabetic nephropathy is characterized by comprising PCR primers of any one or more of sites of ALDOB genes rs118168553, rs146360505, rs1588170400, rs760339672, rs182003715, rs 373974, rs759204107 and rs 141267935.
9. The kit according to claim 8, wherein the primer sequences are as follows:
10. a primer composition for detecting SNP of diabetic nephropathy related susceptibility gene ALDOB is characterized in that the primer composition is selected from one or more PCR primers in any one of sites of ALDOB gene rs118168553, rs146360505, rs1588170400, rs760339672, rs182003715, rs 37397621, rs759204107 and rs 141267935; preferred are the primers shown below:
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004084797A2 (en) * | 2003-02-28 | 2004-10-07 | Hubit Genomix Inc | Type 2 diabetes mellitus-associated gene and utilization of the same |
| KR100930027B1 (en) * | 2009-07-06 | 2009-12-07 | 경북대학교 산학협력단 | Composition and kit for the diagnosis of iga nephropathy and tgbm nephropathy |
| US20150204874A1 (en) * | 2012-07-26 | 2015-07-23 | Joslin Diabetes Center, Inc. | Predicting and treating diabetic complications |
| CN109790643A (en) * | 2016-03-09 | 2019-05-21 | 分子听诊器公司 | For detecting the method and system of organization factors |
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- 2020-10-15 CN CN202011101905.XA patent/CN112094905A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004084797A2 (en) * | 2003-02-28 | 2004-10-07 | Hubit Genomix Inc | Type 2 diabetes mellitus-associated gene and utilization of the same |
| KR100930027B1 (en) * | 2009-07-06 | 2009-12-07 | 경북대학교 산학협력단 | Composition and kit for the diagnosis of iga nephropathy and tgbm nephropathy |
| US20150204874A1 (en) * | 2012-07-26 | 2015-07-23 | Joslin Diabetes Center, Inc. | Predicting and treating diabetic complications |
| CN109790643A (en) * | 2016-03-09 | 2019-05-21 | 分子听诊器公司 | For detecting the method and system of organization factors |
Non-Patent Citations (1)
| Title |
|---|
| 陈姗等: "糖尿病肾病疾病进展相关基因的基因组学研究", 《中华肾脏病杂志》, vol. 22, no. 9, pages 528 - 534 * |
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