CN108660205A - A kind of diabetes genetic risk assessment kit and its utilization - Google Patents
A kind of diabetes genetic risk assessment kit and its utilization Download PDFInfo
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Abstract
The present invention provides a kind of kits and its detection method for assessing diabetes genetic risk.The kit includes：For the primer of ACE2 Polymorphisms site rs1978124, rs2074192, rs4848188 and rs879922.The kit of the present invention assesses the genetic risk of individual diabetes, diabetes-related complication by detecting the polymorphic sites genotype such as ACE2 Polymorphisms site rs1978124, rs2074192, rs4848188 and rs879922, so that diabetes, diabetes-related complication inheritance susceptible crowd are screened from general population, change bad life style as early as possible, prevented with achieving the purpose that or delays to suffer from aforementioned relevant disease.
Description
Invention field
The present invention relates to a kind of diabetes genetic risks to assess kit, and for assessing individual diabetes and its correlation simultaneously
Send out the genetic risk of disease.
Background technology
Diabetes are had become by the metabolic disease under inherent cause and environmental factor collective effect characterized by hyperglycemia
For one of the public health problem of global most serious.Cut-off 2015, global maturity-onset diabetes patient populations up to 4.15 hundred million, and
It is still in rising trend, it is contemplated that the year two thousand forty, the number will reach 6.42 hundred million.Latest data is shown：Chinese residents suffer from glycosuria within 2013
Sick illness rate is 10.9%, close to U.S.'s 2011-2012 diabetes prevalences (12%-14%), prediabetes prevalence rate
Up to 35.7%, diabetes mellitus in China patient has ranked the first in the world.Diabetes mellitus in China morbidity also presentation rejuvenation trend, 2010
Early onset diabetes proportion up to 13.5%, falls ill more early complication risk more, such as：BMI highers, blood glucose is more difficult to control,
LDL levels increase in blood pressure higher, plasma lipid profile and the decline of HDL levels is apparent etc..Onset diabetes it is more early with it is higher complete because dead
It dies that rate risk is related, is mainly due to increase and the angiocardiopathy death rate in exponential increase with the age, and depend primarily on
Diabetic duration, i.e. morbidity occur that more early cardiovascular event risk is higher, and diabetes merge the quantity of angiocardiopathy with it is dead
It is significantly shorter related to age other life expectancy to die risk increase.It is event, diabetes mellitus in China prevention and control situation is very severe.
The long-term follow-up follow-up investigation of the milestones such as DCCT, UKPDS and STENO-2 research is as a result, illustrate diabetes morning
The importance that phase intervenes, is likely to become the assurance of intervention the important determinant for determining entire final result, intervention when
Machine is sometimes even more important than intervention itself.Thus, it is necessary to it determines and diabetes, especially early onset diabetes occurs for screening
High risk group, exploration how to change lifestyles to prevent or delay the generation of diabetes B.Newest patients with type Ⅰ DM heredity
Research is learned to find：Specific genetic locus and the genetic risk of entirety lead to β cell dysfunctions jointly, are to lead to Chinese
The main reason for diabetes B.From the perspective of heredity, the research of diabetes inheritance susceptible provides for the early prevention of diabetes
A kind of new strategy.
There is also national differences in China for diabetes morbidity, such as：Between Han nationality and non-Han nationality and between non-Han nationality.
In non-Chinese Han Population, either city or rural area, Xinjiang Uygur diabetes incidence higher, and awareness, treatment rate
It is very low with control rate.Unsound life style also generally existing in Uygur nationality's people with diabetes, in addition to having
Outside the common feature of diabetes mellitus in China patient general population, individual character also related with the unique eating habit of the Uygur nationality itself is special
Point, such as：Barbecue, wheaten food and milk tea etc. under high sugared high salt diet pattern high in fat.Do not considering diabetes genetic background premise
Under, healthy lifestyles and low diabetes risk and its related cardiovascular event are (such as：Hypertension, myocardial infarction, heart failure
Exhaust, cerebral apoplexy etc.) risk it is related.Although it is possible to change unsound life style, but genetic structure can not possibly be changed.It is strong
The cardiovascular benefit that health life style is brought often is offset by high genetic risk.Therefore Chinese Uyger diabetes prevention and control
Cheng Zhong, emphasize risk factor comprehensive assessment and bad life style management while, the heredity to diabetes cannot be ignored
The clinical assessment of background.
The inheritance susceptible candidate gene for being currently known diabetes has very much, but gene studies related with RAAS is less.
Angiotensin converting enzyme2 (angiotensin converting enzyme 2, ACE2) adjusts enzyme, people as RAAS keys
The class ACE2 assignments of genes gene mapping are on the sites Xp22 of X chromosome, and full length gene about 39.98kb, including 20 intrones, 18 aobvious outside
Son.The albumen of ACE2 codings is I type film combination glycoprotein, is made of 805 amino acid, including c-terminus transmembrane anchor area, ammonia
Tri- structure divisions of metalloproteinases zinc-binding domain HEXXH outside cardinal extremity signal peptide area, after birth.ACE2 gene orders have height
Polymorphism, and cohort study's result shows that ACE2 gene pleiomorphisms are associated with diabetes and its related complication, can be used as
The inheritance susceptible candidate gene of diabetes.Although having confirmed that ACE2 gene pleiomorphisms and diabetes are closely related, it exists
The genetic heterogeneity of height.The EARLY RECOGNITION of ACE2 genetic heterogeneities in Uygur nationality crowd is contributed in rear era gene
Implementing diabetes early stage, accurately individuation prevents and benefits for a long time.
Invention content
For foregoing problems, the present invention provides a kind of for detecting the genetic risk of diabetes and its related complication
Kit.
The kit includes：
Detect the Specific PCR primers pair and extension primer of ACE2 Polymorphisms site rs1978124；
Detect the Specific PCR primers pair and extension primer of ACE2 Polymorphisms site rs2074192；
Detect the Specific PCR primers pair and extension primer of ACE2 Polymorphisms site rs4848188；
Detect ACE2 Polymorphisms site rs879922 Specific PCR primers pair and extension primer；
PCR reaction components (including Taq archaeal dna polymerases, NTP mixtures, MgCl2Solution, 10 × PCRBuffer solution,
Deionized water etc.)；
PCR product purification componentry (including shrimp alkaline phosphotase (SAP), SAP Buffer, deionized water etc.)
PCR single base extensions component (including iPLEX enzymes, iPLEX primer mixtures, iPLEX terminate liquids, iPLEX
Buffer, deionized water etc.)
Specific primer described in this kit to be for ACE2 Polymorphisms site rs1978124,
Rs2074192, rs4848188 and rs879922, energy specific amplification go out comprising this 4 mononucleotide polymorphic site DNA fragmentations
Primer pair.It is that those skilled in the art can be unlabored to design this kind of primer pair.Comprising with SEQ ID in kit
The primer pair of sequence shown in NQ.1 and 2,3 and 4,5 and 6,7 and 8.Specific primer synthesizes available conventional technique, this
The primer of invention is not limited to this 4 pairs of primers, and all round pcrs that can be used for detect drawing for this 4 ACE2 mononucleotide polymorphic sites
Object is within the scope of the present invention.
Extension primer described in this kit be for ACE2 Polymorphisms site rs1978124,
Rs2074192, rs4848188 and rs879922 draw for carrying out genotyping to this 4 mononucleotide polymorphic sites
Object.It is that those skilled in the art can be unlabored to design this kind of primer pair.In kit comprising with SEQ ID NQ.9,10,
The extension primer of sequence shown in 11 and 12.Specific extension primer synthesizes available conventional technique, primer of the invention
This 4 extension primers are not limited to, all DNA sequencing technologies that can be used for detect this 4 ACE2 mononucleotide polymorphic site genes point
The extension primer of type is within the scope of the present invention.
Further, the component of this kit and content include：1. PCR reaction components：10ng/μL DNA 1.0μL、5U/
0.2 μ L of μ L Taq archaeal dna polymerases, 0.5 μm of 1.0 μ L of ol/L primer mixtures, 0.1 μ L of 25mmol/L dNTP mixtures,
25mmol/L MgCl20.4 μ L, 10 × PCR Buffer, 0.5 μ L, 1.8 μ L of deionized water, each reaction system total volume are
5.0μL；2. PCR product purification componentry：Step 1. 5.0 μ L of final product, 0.3 μ L of 1.7U/ μ L SAP, 0.17 μ of SAP Buffer
L, 1.53 μ L of deionized water, each reaction system total volume are 7.0 μ L；3. PCR single base extension components：2. step is produced eventually
7.0 μ L of object, 0.041 μ L of iPLEX enzymes, 0.94 μ L of iPLEX primer mixtures, 0.2 μ L of iPLEX terminate liquids, iPLEX Buffer
0.2 μ L, 0.619 μ L of deionized water, each reaction system total volume are 9.0 μ L.This kit is detected for a person-portion and is used, reagent
Box storage temperature is -20 DEG C.
Further, the application method of kit of the present invention includes step：(1) extraction subject DNA；(2) PCR expansions are carried out
Increasing reaction, (reaction condition is：94 DEG C of 15min → (94 DEG C of 20s → 56 DEG C 30s → 72 DEG C 60s, 45 cycles) → 72 DEG C of 3min → 4
DEG C keep)；(3) PCR after reaction, PCR product is handled with shrimp alkaline phosphotase, with remove in reaction system dissociate
(reaction condition is dNTPs：37 DEG C of 40min → 85 DEG C 5min → 4 DEG C are kept)；(4) (reaction condition is single base extension：
94 DEG C of 30s → (94 DEG C of 5s → 52 DEG C 5s → 80 DEG C 5s, 40 cycles) → 72 DEG C of 3min → 4 DEG C are kept)；(5) purifying resin；(6)
Chip point sample and Mass Spectrometer Method determine the genotype of this 4 ACE2 mononucleotide polymorphic sites.
Further, kit involved in the present invention is occurred by detection with diabetes and its related complication closely related
ACE2 Polymorphisms site, diabetes and its related complication genetic risk rank occurs to assess subject,
It instructs subject's early stage to intervene from life style level from gene level, targetedly prevents diabetes and its correlation simultaneously
Disease is sent out, to reduce the incidence of diabetes.
Description of the drawings
Fig. 1 is using 4.0 softwares of TYPER to ACE2 Polymorphisms site rs1978124 parting schematic diagrames
Fig. 2 is using 4.0 softwares of TYPER to ACE2 Polymorphisms site rs2074192 parting schematic diagrames
Fig. 3 is using 4.0 softwares of TYPER to ACE2 Polymorphisms site rs4848188 parting schematic diagrames
Fig. 4 is using 4.0 softwares of TYPER to ACE2 Polymorphisms site rs879922 parting schematic diagrames
Specific embodiment
1 diabetes genetic risk of embodiment assesses the use of kit
Step 1: being included in subject
It is included in 136 normal control populations's (Normal group) and 128 diabetic populations (diabetes group) altogether, two groups are removed
Sex composition, age, diastolic pressure, blood lipid level (LDL, CHOL, Lp (a), ApoA1/Apo ratios), renal function it is horizontal (Cr,
BUN, UA), liver function (ALT, AST, AIb), in terms of HsCRP and RAAS active (ACE, renin, ALD (clinostatism)) difference without system
Meter is learned outside meaning (all ＞ 0.05), in systolic pressure, BMI, blood lipid level (TRIG, HDL), blood glucose (FBS, HbA1C), blood electrolysis
Matter (Serum Na+ concentration, serum potassium concentration), the RAAS activity differences such as (AngI, AngII) and 24 hours microalbuminurias are equal
Statistically significant (all P ＜ 0.05), is shown in Table 1.
1 Normal group of table and glycosuria group group subject's clinic baseline characteristic
Step 2: acquisition subject's DNA sample
Each subject blood sample 2ml is acquired, with phenol/chloroform genomic DNA.
Step 3: PCR reacts
Use the PCR reaction components in kit.Wherein, it is directed to ACE2 Polymorphisms site containing following
The specific primer pair of rs1978124, rs2074192, rs4848188 and rs879922：
(1) it is directed to the specific primer pair of ACE2 gene mononucleotides site rs1978124：
Sense primer：ACGTTGGATGAAGCTGCTGATGTAGAAGTG(SEQ ID NQ.1)
Downstream primer：ACGTTGGATGGAGAGAACTTTGGAAACCTG(SEQ ID NQ.2)
(2) it is directed to the specific primer pair of ACE2 gene mononucleotides site rs2074192：
Sense primer：ACGTTGGATGCCCTTAAACACAGCAGTCAC(SEQ ID NQ.3)
Downstream primer：ACGTTGGATGTTAGGTTCATCAACAGCTCC(SEQ ID NQ.4)
(3) it is directed to the specific primer pair of ACE2 gene mononucleotides site rs4848188：
Sense primer：ACGTTGGATGCTCTGTGTTCCCTTCTGTTG(SEQ ID NQ.5)
Downstream primer：ACGTTGGATGGATATTCTACTCAGAAACG(SEQ ID NQ.6)
(4) it is directed to the specific primer pair of ACE2 gene mononucleotides site rs879922：
Sense primer：ACGTTGGATGGGCAGTTTATTGTACATTGTG(SEQ ID NQ.7)
Downstream primer：ACGTTGGATGGCTCCAGCAAATTCAAGGAC(SEQ ID NQ.9)
4 independent PCR reactions, each reaction system are carried out respectively for aforementioned 4 ACE2 mononucleotide polymorphic sites
Total volume is 5.0 μ L.Including：1.0 μ L of 10ng/ μ L DNA, 0.2 μ L of 5U/ μ L Taq archaeal dna polymerases, 0.5 μm of ol/L primer are mixed
Close object 1.0 μ L, 0.1 μ L of 25mmol/L dNTP mixtures, 0.4 μ L of 25mmol/L MgCl2,10 × PCR Buffer, 0.5 μ L,
1.8 μ L of deionized water.Reaction condition is：94 DEG C of 15min → (94 DEG C of 20s → 56 DEG C 30s → 72 DEG C 60s, 45 cycles) → 72 DEG C
3min → 4 DEG C are kept.
Step 4: PCR product purifies.
PCR after reaction, PCR product is purified using the PCR purification componentries in this kit.Each PCR
Product purification reaction system total volume is 7.0 μ L, including：5.0 μ L of final product, 1.7U/ μ L shrimp alkaline phosphotases in step 3
(SAP) 0.3 μ L, 0.17 μ L of SAP Buffer, 1.53 μ L of deionized water.Reaction condition is：37℃40min→85℃5min→4
DEG C keep.
Step 5: DNA sequencing reaction
Use the PCR single base extension components in this kit.Wherein, more for ACE2 mononucleotides containing following
The specific extension primer of state site rs1978124, rs2074192, rs4848188 and rs879922：
(1) it is directed to the specific extension primer of ACE2 gene mononucleotides site rs1978124：
CCATATCTCTATCTGATGGAC(SEQ ID NQ.9)；
(2) it is directed to the specific extension primer of ACE2 gene mononucleotides site rs2074192：
CAAGGGTGGAAATGTATAAATGGTTGG(SEQ ID NQ.10)；
(3) it is directed to the specific extension primer of ACE2 gene mononucleotides site rs4848188：
GGGGAACGTAGAATTTTAGTTGAATG(SEQ ID NQ.11)；
(4) it is directed to the specific extension primer of ACE2 gene mononucleotides site rs879922：CTCAAGGACTGGGGTTA
(SEQ ID NQ.12)。
Each PCR single base extension system total volumes are 9.0 μ L.Including：7.0 μ L of final product, iPLEX in step 4
0.041 μ L of enzyme, specific 0.94 μ L of extension primer mixture, 0.2 μ L of iPLEX terminate liquids, 0.2 μ L of iPLEX Buffer, go from
0.619 μ L of sub- water.Reaction condition is：94 DEG C of 30s → (94 DEG C of 5s → 52 DEG C 5s → 80 DEG C 5s, 40 cycles) → 72 DEG C of 3min → 4
DEG C keep.
Step 6: purifying resin.
The uniform potting resin and placing makes it dry in 10 minutes in 384/6 MG Dimple plates, uses Liquid afterwards
Handler robotic arms continue gently to turn 384 sample planes and are buckled in the 16 μ L water of each Kong Zhongjia of 384 sample planes
On Dimple plates, then tapping makes resin fall into each hole of sample plane, and 384 sample planes are then placed room temperature in centrifuge
Rotate mixing 60 minutes.
Step 7: chip point sample and Mass Spectrometer Method Genotyping.
Start MassARRAY Nanodispenser RS1000 point sample instruments, the extension products after purifying resin are moved to
On 384 hole Spectro CHIP (Sequenom) chips, MALDI-TOF MS is used in combination to analyze, testing result uses TYPER 4.0
Software (sequenom) parting simultaneously exports result.
Be familiar with DNA sequencing technology those skilled in the art can cross by recognize DNA sequencing collection of illustrative plates determine that subject is examined
The genotype in the aforementioned 4 ACE2 Polymorphisms site surveyed.
2 diabetes inheritance susceptible risk of embodiment is assessed
Step 1：Acquire subject DNA.Subject's blood sample sample 2mL is acquired by professional.
Step 2: Genotyping detects.Using kit provided by the invention, by correlation step in embodiment 1, to tested
ACE2 Polymorphisms site rs1978124, rs2074192, rs4848188 entrained by person's genome and
Rs879922 carries out DNA sequencing analysis, determines the genotype in this 4 ACE2 Polymorphisms sites.
Step 3: to ACE2 Polymorphisms site rs1978124, rs2074192, rs4848188 and
The genotype of rs879922 carries out relative risk (OR) assignment, as shown in table 2.
Diabetes relative risk occurs for 2 difference ACE2 Polymorphism loci gene types of table
Step 3: to ACE2 Polymorphisms site rs1978124, rs2074192, rs4848188 and
The genotype of rs879922 carries out relative risk (OR) assignment, as shown in table 2.
Step 4: according to detected ACE2 Polymorphisms site rs1978124, rs2074192,
The genotype of rs4848188 and rs879922 judges that the degree (or rank) of diabetes risk occurs for subject by following standard：
OR≤1：Low-risk；1 ＜ OR ＜ 2：Risk；≥2：High risk.
Step 5: by identify subject's ACE2 Polymorphisms site rs1978124, rs2074192,
The genotype of rs4848188 and rs879922 provides the inheritance susceptible risk analysis report that diabetes occur for individual.In report in detail
Describe the inheritance susceptible risk that diabetes and diabetes-related complication (as described in claim 8-10) occur for bright subject in detail
Degree (or rank), and be described in detail from medical professional to subject or understand and give corresponding medical speciality meaning
See.
Although the present invention discloses the practical range for being as above not limited to the present invention with preferably embodiment.It is any
Those skilled in the art, it is without departing from the scope of the present invention, when a little improvement can be made, i.e., every according to institute of the present invention
The same improvement done, should be the scope of the present invention and is covered.
Claims (10)
1. a kind of diabetes genetic risk assesses kit, which includes：For 2 gene of angiotensin converting enzyme
(ACE2) primer of mononucleotide polymorphic site rs1978124, rs2074192, rs4848188 and rs879922.
2. the primer in the kit of claim 1 for ACE2 gene mononucleotides site rs1978124 includes：
Rs1978124 sense primers：
ACGTTGGATGAAGCTGCTGATGTAGAAGTG(5’-3’)
Rs1978124 downstream primers：
ACGTTGGATGGAGAGAACTTTGGAAACCTG(5’-3’)
Rs1978124 extension primers：
CCATATCTCTATCTGATGGAC(5’-3’)。
3. being directed to the specific primer packet of ACE2 gene mononucleotides site rs2074192 in the kit of claim 1
It includes：
Rs2074192 sense primers：
ACGTTGGATGCCCTTAAACACAGCAGTCAC(5’-3’)
Rs2074192 downstream primers：
ACGTTGGATGTTAGGTTCATCAACAGCTCC(5’-3’)
Rs2074192 extension primers：
CAAGGGTGGAAATGTATAAATGGTTGG(5’-3’)。
4. being directed to the specific primer packet of ACE2 gene mononucleotides site rs4848188 in the kit of claim 1
It includes：
Rs4848188 sense primers：
ACGTTGGATGCTCTGTGTTCCCTTCTGTTG(5’-3’)
Rs4848188 downstream primers：
ACGTTGGATGGATATTCTACTCAGAAACG(5’-3’)
Rs4848188 extension primers：GGGGAACGTAGAATTTTAGTTGAATG (5’-3’).
5. being directed to the specific primer packet of ACE2 gene mononucleotides site rs879922 in the kit of claim 1
It includes：
Rs879922 sense primers：
ACGTTGGATGGGCAGTTTATTGTACATTGTG(5’-3’)
Rs879922 downstream primers：
ACGTTGGATGGCTCCAGCAAATTCAAGGAC(5’-3’)
Rs879922 extension primers：
CTCAAGGACTGGGGTTA(5’-3’)。
6. the appraisal procedure of the testing result of one of the claim 1-5 kits comprising following steps：
(1) DNA of subject is extracted；
(2) pcr amplification reaction is carried out using following primer：
1. rs1978124 polymorphic site specific forward primers：
ACGTTGGATGAAGCTGCTGATGTAGAAGTG(5’-3’)；
Rs1978124 polymorphic site specific downstream primers：
ACGTTGGATGGAGAGAACTTTGGAAACCTG(5’-3’)；
2. rs2074192 polymorphic site specific forward primers：
ACGTTGGATGCCCTTAAACACAGCAGTCAC(5’-3’)；
Rs2074192 polymorphic site specific downstream primers：
ACGTTGGATGTTAGGTTCATCAACAGCTCC(5’-3’)；
3. rs4848188 polymorphic site specific forward primers：
ACGTTGGATGCTCTGTGTTCCCTTCTGTTG(5’-3’)；
Rs4848188 polymorphic site specific downstream primers：
ACGTTGGATGGATATTCTACTCAGAAACG(5’-3’)；
4. rs879922 polymorphic site specific forward primers：
ACGTTGGATGGGCAGTTTATTGTACATTGTG(5’-3’)；Rs879922 polymorphic site specific downstream primers：
ACGTTGGATGGCTCCAGCAAATTCAAGGAC(5’-3’)。
(3) PCR after reaction, PCR product is handled with shrimp alkaline phosphotase, with remove in reaction system dissociate
dNTPs；
(4) single base extension is carried out using following extension primer：
1. rs1978124 polymorphic site specificity extension primers：
CCATATCTCTATCTGATGGAC(5’-3’)；
2. rs2074192 polymorphic site specificity extension primers：
CAAGGGTGGAAATGTATAAATGGTTGG(5’-3’)；
3. rs4848188 polymorphic site specificity extension primers：
GGGGAACGTAGAATTTTAGTTGAATG(5’-3’)；
4. rs879922 polymorphic site specificity extension primers：
CTCAAGGACTGGGGTTA(5’-3’)。
(5) purifying resin (desalting)；
(6) chip point sample and Mass Spectrometer Method determine ACE2 Polymorphisms site entrained by subject invidual
The genotype of rs1978124, rs2074192, rs4848188 and rs879922；
(7) not by ACE2 Polymorphisms site rs1978124, rs2074192, rs4848188 and rs879922
Homogenic type carries out relative risk assignment；
(8) according to analyzed ACE2 Polymorphisms site rs1978124, rs2074192 in step (7),
The value-at-risk of rs4848188 and rs879922 genotype judges that the hereditary wind of diabetes and its related complication occurs for subject
Danger.
7. the subject in claim 1-6, the preferably Uygur nationality.
8. diabetes-related complication occurs for the subject in claim 1-6：Diabetes and blood pressure increase or hypertension,
Diabetes and dyslipidemia, diabetes and macrovascular complications and diabetes and microvascular complication.
9. it includes that myocardial infarction or other acute coronaries are comprehensive that diabetes and macrovascular complications, which occur, for the subject in claim 8
Simulator sickness, coronary artery or other reconstructive vascular operations, cerebral arterial thrombosis, transient ischemic attack, Peripheral atherosclerosis
The property atherosclerotic cardiovascular diseases such as disease and other atherosclerosis.
10. it includes diabetic retinopathy, glycosuria that diabetes and microvascular complication, which occur, for the subject in claim 8
Characteristic of disease neuropathy, diabetic nephropathy change and diabetes related cardiac microangiopathies etc..
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| RU2787273C1 (en) * | 2022-03-04 | 2023-01-09 | Федеральное государственное бюджетное образовательное учреждение высшего образования "Курский государственный медицинский университет" Министерства здравоохранения Российской Федерации | Method for predicting the risk of developing diabetic distal polyneuropathy in women with type 2 diabetes mellitus based on rs7784465 genotyping of the rac1 gene polymorphism rs7784465 |
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