Application of SNP site of UCP1 as marker and SNP site detection method

Abstract

The invention relates to the technical field of biology, in particular to application of an SNP site of UCP1 as a marker and an SNP site detection method. The invention provides an application of an SNP (Single Nucleotide Polymorphism) site (rs45539933 and / or rs2270565) of UCP1 as a marker in preparation of a product for predicting a weight loss effect, and when an individual carrying rs45539933 and rs2270565 undergoes 25% energy limitation for 6 months or 12 months, compared with a wild type, the weight loss amplitude is increased by 22% and 43.6%. The invention further provides an SNP site detection method and a kit, and the method and the kit have remarkable prediction significance on weight loss effect evaluation under the prediction energy limiting condition.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to the application of a SNP site of UCP1 as a marker and a SNP site detection method. Background Art

[0002] Modern people are generally susceptible to metabolic disorders, and metabolic "thrifty genes" are often used to explain the genetic factors behind this phenomenon. However, although this hypothesis has been widely accepted and spread for a long time, it has not been fully verified and is highly controversial in the academic community. If its existence can be proven, it will have an important impact on the understanding of the regulatory mechanism of glucose and lipid metabolism, the transformation of new targets for metabolic disease intervention, and clinical diagnosis and treatment.

[0003] James Neel of the University of Chicago first proposed the "thrifty gene" hypothesis in 1962. He hypothesized that in the hunting and gathering era, there were no crops or livestock products as a stable food supply, and "food abundance and scarcity (feast-famine)" occurred alternately. The body needed to store fat efficiently and reduce energy consumption to obtain the maximum survival probability, which made the "metabolic thrifty gene" mutation under environmental positive selection, and ultimately led to the common carrying of these "mutations that can increase energy absorption and reduce consumption" by modern people, thus explaining why modern people are generally susceptible to metabolic diseases. He speculated that in today's era of excess calories, these mutations that once played a "survival benefit" in evolution have become harmful.

[0004] The paper on this hypothesis has been cited more than 4700 times, but up to now, no "thrifty gene mutation" has been recognized, so many famous scholars (including Speakman JR, P et al.) questioned whether humans have "thrifty genes" (Thrifty genes and human obesity. Are we chasing ghosts?)? At present, only geographically isolated populations (such as the "Samoans" in the South Pacific) have been found to carry a thrifty gene mutation (CREBRFp.R457Q), but no similar gene mutations with metabolic thriftiness have been found in a wider range of populations in Eurasia and Africa. Can new diagnostic kits and weight loss, blood sugar and lipid-lowering drugs be developed around these possible "thrifty genes"? Summary of the invention

[0005] In view of this, the technical problem to be solved by the present invention is to provide a gene polymorphism site that can effectively predict the weight loss effect. The present invention provides the application of the SNP site of UCP1 as a marker and a SNP site detection method. Individuals carrying rs45539933 and rs2270565 have a higher basal metabolic rate and a better weight loss effect after energy restriction. On the contrary, it is necessary to combine intervention methods such as physical exercise to increase the basal metabolic rate.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides the use of a SNP site of UCP1 as a marker in the preparation of a product for predicting weight loss effects; the SNP site includes rs45539933 and / or rs2270565;

[0008] The prediction includes: the genotype of the rs45539933 site (NM_021833.4:c.190G>A) of UCP1 is AA or AG, and the weight loss effect is good; the genotype of the rs45539933 site of UCP1 is GG, and the weight loss effect is poor; the genotype of the rs2270565 site (NM_021833.4:c.685A>T) of UCP1 is TT or TA, and the weight loss effect is good; the genotype of the rs2270565 site of UCP1 is AA, and the weight loss effect is poor.

[0009] The present invention also provides a method for predicting weight loss effect, comprising:

[0010] Step 1, detecting the genotype of rs45539933 and / or rs2270565 of UCP1 of the subject;

[0011] Step 2, predicting the weight loss effect of the subject according to the genotype of rs45539933 and / or rs2270565 of UCP1;

[0012] The prediction includes: the genotype of the rs45539933 site (NM_021833.4:c.190G>A) of UCP1 is AA or AG, and the weight loss effect is good; the genotype of the rs45539933 site of UCP1 is GG, and the weight loss effect is poor; the genotype of the rs2270565 site (NM_021833.4:c.685A>T) of UCP1 is TT or TA, and the weight loss effect is good; the genotype of the rs2270565 site of UCP1 is AA, and the weight loss effect is poor.

[0013] In some specific embodiments of the present invention, step 1 comprises:

[0014] Step a, extracting DNA from the oral mucosal swab of the subject, and performing PCR amplification using a forward primer having a nucleotide sequence as shown in SEQ ID NO.1 and a reverse primer having a nucleotide sequence as shown in SEQ ID NO.2 to obtain an amplified product;

[0015] Step b: The amplified product is sequenced to determine the genotype of the rs45539933 site of UCP1.

[0016] In some specific embodiments of the present invention, step 1 comprises:

[0017] Step a, extracting DNA from the oral mucosal swab of the subject, and performing PCR amplification using a forward primer having a nucleotide sequence as shown in SEQ ID NO.3 and a reverse primer having a nucleotide sequence as shown in SEQ ID NO.4 to obtain an amplified product;

[0018] Step b: The amplified product is sequenced to determine the genotype of the rs2270565 site of UCP1.

[0019] In some specific embodiments of the present invention, the reaction procedure of the PCR amplification in step 1 comprises:

[0020]

[0021] In some specific embodiments of the present invention, based on a 20 μL reaction system, the PCR amplification reaction system comprises:

[0022]

[0023] The present invention also provides primers used in the method, including a primer pair for amplifying UCP1 containing the rs45539933 site and / or a primer pair for amplifying UCP1 containing the rs2270565 site;

[0024] The forward primer of the primer pair for amplifying UCP1 containing the rs45539933 site has a nucleotide sequence as shown in SEQ ID NO.1, and the reverse primer has a nucleotide sequence as shown in SEQ ID NO.2; and / or

[0025] In the primer pair for amplifying UCP1 containing the rs2270565 site, the forward primer has a nucleotide sequence as shown in SEQ ID NO.3, and the reverse primer has a nucleotide sequence as shown in SEQ ID NO.4.

[0026] The invention also provides a kit, comprising the primers.

[0027] The present invention also provides a method for detecting the SNP site of UCP1, comprising detecting by any of the following items:

[0028] (I), the primer; and / or

[0029] (II), the kit;

[0030] The SNP sites include rs45539933 and / or rs2270565.

[0031] The present invention also provides the use of antisense oligonucleotides (ASO) to upregulate the targeted augmentation of nuclear gene output (TANGO) of the genotype of the rs45539933 site of UCP1 being AA or AG, or the genotype of the rs2270565 site of UCP1 being TT or TA, in formulating a plan for preventing and / or treating weight loss, hypoglycemic and / or lipid regulation.

[0032] The present invention also provides a method for using CRISPR-Cas9 gene editing technology to edit the gene GG at the rs45539933 site (NM_021833.4: c.190G>A) of UCP1 in primary adipocytes to AA; or to edit the gene AA at the rs2270565 site (NM_021833.4: c.685A>T) to TT, thereby restoring the thermogenic effect, and using the method in formulating a plan for preventing and / or treating weight loss, lowering blood sugar and / or regulating lipids.

[0033] The present invention includes but is not limited to providing the following beneficial effects:

[0034] 1. The present invention found that two single nucleotide polymorphisms (SNP, referring to the genomic variation of a single base position in DNA) rs45539933 and rs2270565 of the thermogenic gene UCP1 are enriched in lean people;

[0035] 2. The present invention found that rs45539933 and rs2270565 coexist in the population, that is, individuals with rs45539933 also coexist with rs2270565;

[0036] 3. Individuals carrying rs45539933 and rs2270565 experienced 22% and 43.6% increased weight loss compared with controls when undergoing 25% energy restriction for 6 or 12 months. Therefore, this locus can fully predict the weight loss effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0038] Figure 1 indicates rs45539933 wild type;

[0039] Figure 2 It indicates rs45539933 heterozygous type;

[0040] Figure 3 indicates rs2270565 wild type;

[0041] Figure 4 It indicates rs2270565 heterozygous type;

[0042] Figure 5 It shows that rs45539933 and rs2270565 are common in thin people; A shows the frequency of rs45539933 and rs2270565 carriers in obese people; B shows the influence of rs45539933 and rs2270565 carriers on the body mass index of the population;

[0043] Figure 6 The results showed that individuals carrying rs45539933 and rs2270565 were more sensitive to the energy restriction weight loss program; A showed a 6-month 25% energy restriction diet test program; B showed the changes in body mass index of rs45539933 and rs2270565 carriers on a 25% energy restriction diet for 6 months; C showed the changes in body weight of rs45539933 and rs2270565 carriers on a 25% energy restriction diet for 6 months; D showed a 12-month 25% energy restriction diet test program; E showed the changes in body mass index of rs45539933 and rs2270565 carriers on a 25% energy restriction diet for 12 months; F showed the changes in body weight of rs45539933 and rs2270565 carriers on a 25% energy restriction diet for 12 months;

[0044] Figure 7 The present invention combines the homologous point mutation reverse knock-in mouse model to construct mice that simulate the human UCP1 wild-type sequence; it is found that 8-week-old male mice that simulate human rs45539933 and rs2270565 have lower body weight and fat weight than mice that simulate the human UCP1 wild-type sequence; the weight loss prediction effect of rs45539933 and rs2270565 is further demonstrated; wherein, * indicates P value <0.05, ** indicates P value <0.01, and *** indicates P value <0.001. DETAILED DESCRIPTION

[0045] The present invention discloses the application of the SNP site of UCP1 as a marker and a method for detecting the SNP site. A person skilled in the art can refer to the content of this article and appropriately improve the process parameters for implementation. It should be particularly noted that all similar substitutions and modifications are obvious to a person skilled in the art and are considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the method and application described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0046] Terminology explanation:

[0047] Uncoupling protein 1 (UCP1): also called thermogenic protein, is a thermogenic protein in the inner membrane of mitochondria that dissipates the proton gradient of the inner membrane of mitochondria and releases this gradient as heat (thermogenesis). This protein is located in human adipose tissue.

[0048] Thrifty genes: These genes help people store extra energy easily and would have an evolutionary advantage during famines. Obese and overweight people have "thrifty genes".

[0049] Uncoupling protein 1 (UCP1) is a mitochondrial heat-producing protein. In the early stage of this invention, relying on the large genetic resource of "Chinese Youth Extreme Obesity Cohort (GOCY)", the cohort, genetics and evolution were fully integrated, and the following were discovered for the first time:

[0050] 1. The present invention found that two single nucleotide polymorphisms (SNP, referring to the genomic variation of a single base position in DNA) rs45539933 and rs2270565 of the thermogenic gene UCP1 are enriched in lean people;

[0051] 2. The present invention found that rs45539933 and rs2270565 coexist in the population, that is, individuals with rs45539933 also coexist with rs2270565;

[0052] 3. Individuals carrying rs45539933 and rs2270565 experienced 22% and 43.6% increased weight loss compared with controls when undergoing 25% energy restriction for 6 or 12 months. Therefore, this locus can fully predict the weight loss effect.

[0053] Unless otherwise specified, the use of the SNP site of UCP1 as a marker and the raw materials and reagents used in the SNP site detection method provided by the present invention can all be purchased from the market.

[0054] The present invention will be further described below in conjunction with embodiments:

[0055] Example 1 DNA sequencing and body mass index determination

[0056] 1. Experimental Materials

[0057] Table 1 Experimental instruments

[0058] Equipment Name Equipment Supplier 3730xl first generation sequencer Applied Biosystems Nano-100 Microvolume Spectrophotometer Hangzhou Aosheng Instrument Co., Ltd. PCR instrument 310A Hangzhou Langji Scientific Instrument Co., Ltd. L-550 Benchtop Centrifuge Changsha Xiangyi Centrifuge Instrument Co., Ltd. MIX-100 Oscillator Hangzhou Aosheng Instrument Co., Ltd. Micropipette Eppendorf FR-110 UV analysis device Shanghai FuRi Technology Co., Ltd. FR-250 electrophoresis apparatus Shanghai FuRi Technology Co., Ltd. 1-10μL 12-channel pipette Eppendorf Clean bench Shanghai Yipujian Laboratory Equipment Co., Ltd.

[0059] Table 2 Experimental reagents

[0060] Reagent name Reagent Provider Primers Shanghai Maipu Biotechnology Co., Ltd. 2×Taq Master Mix (Dye Plus) Novozyme Biotechnology Co., Ltd. Ethanol Sinopharm Chemical Reagent Co., Ltd. Isopropyl alcohol Sinopharm Chemical Reagent Co., Ltd. BigDye Terminator Applied Biosystems Hi-Di Formamide Applied Biosystems Agarose Shanghai Bioengineering Co., Ltd. Boric acid Shanghai Bioengineering Co., Ltd. Ethidium bromide Shanghai Bioengineering Co., Ltd. Glacial acetic acid Sinopharm Chemical Reagent Co., Ltd. EDTA Sinopharm Chemical Reagent Co., Ltd. DNA ladder marker (DL 2000) Shanghai Bioengineering Co., Ltd. Blood / Tissue / Cell Genomic Extraction Kit Tiangen Biochemical Technology (Beijing) Co., Ltd.

[0061] Table 3 Amplification primer sequences

[0062]

[0063] Table 4 Unidirectional sequencing primer sequences

[0064]

[0065] 2. Experimental steps

[0066] (1) Obtaining oral mucosa of the subjects: 1 hour before sampling, smoking, drinking and eating are prohibited to reduce the impact of food or exogenous factors on the sampling process. 10 seconds before sampling, all participants rinse their mouths with water, and use the first oral mucosal swab to scrape the right cheek / right upper and lower gums of the participants; the second oral mucosal swab is used to scrape the corresponding position on the left side of the participant; the third oral mucosal swab is used to scrape the corresponding position on the right side of the participant again. After sampling, the swab is dried in a clean and ventilated place and placed in a sealed dry plastic bag. The sample is quickly returned to the laboratory and stored at 4°C.

[0067] (2) Extract DNA from oral mucosal swabs (Jiangsu Yimi Biotechnology Co., Ltd., model YMKT-2).

[0068] (3) PCR amplification of the oral mucosal swab DNA extracted in (2) to obtain PCR products.

[0069] Reaction system:

[0070] Element system Premix Taq 10μL Forward Primer 1μL (concentration 10μmol / L) Reverse primer 1μL (concentration is 10μmol / L) DNA 1μL (concentration 20~200ng / μL) <![CDATA[ddH2O]]> 7μL total 20μL

[0071] Reaction program: 94℃5min; 28 cycles: 94℃30s, 58℃30s, 72℃1min; 72℃10min.

[0072] (4) PCR product purification

[0073] The PCR products were subjected to 2% agarose gel electrophoresis, and target bands of equal size were cut out and purified and recovered using the Tiangen Recovery Kit (DP214-03) to obtain purified PCR products.

[0074] (5) Sanger sequencing experiment.

[0075] Sequencing reaction system: total system 5 μL, sequencing primer 1 μL (3.2 μM), purified PCR product 1-3 μL, BigDyeMix kit 1 μL.

[0076] Reaction program: 94℃1min; 28 cycles: 94℃20s, 50℃10s, 60℃4min; 4℃30min.

[0077] NH4AC.EDTA purification: After the sequencing reaction is completed, the 96-well plate is centrifuged at 3700 rpm for 0.5 min; 1 μL NH4AC.EDTA solution is added to each well and centrifuged at 3700 rpm for 30 s; the plate is placed on a mixer and shaken for 30 s and centrifuged at 3700 rpm for 30 s.

[0078] 100% alcohol precipitation: add 18 μL of 100% ethanol solution, precipitate at -20℃ for 30 min, centrifuge at 4500 rpm for 30 min, and invert centrifuge at 400 rpm for 2 min.

[0079] 75% alcohol wash: add 50 μL of 75% alcohol, centrifuge at 4500 rpm for 10 min, and invert and centrifuge at 400 rpm for 2 min.

[0080] Denaturation: Add 6 μL Hi-Di, centrifuge at 3700 rpm for 1 min, and store at -20°C until loading on 3730xl sequencer.

[0081] (6) Sequencing and data analysis:

[0082] Sequencing was performed using a 3730xl sequencer. The sequencing results were analyzed using DNA Sequencing Analysis software, interpreted using Sequencing Analysis 5.2.0 software, and aligned using Sequencher 5.1 software package.

[0083] (7) Measure the subject’s weight and waist circumference and record them.

[0084] (8) Test results:

[0085] The test results are as follows Figures 1 to 5As shown, with the help of the genetic resources of "Chinese adolescent extreme obesity cohort and golden control (1994 cases vs 2161 cases)", the present invention first discovered that the frequency of homozygous carriers of rs45539933 and rs2270565 in obese people is half that of normal people (A). Through DNA sequencing and body mass index detection, it was found that homozygous and heterozygous carriers of rs45539933 and rs2270565 had a lower body mass index than the wild type, with an average weight reduction of 11.39 kg and a waist circumference reduction of 9.31 cm (B).

[0086] Example 2 Weight loss response of a population to an energy restricted diet

[0087] like Figure 6 As shown, the present invention utilizes two kinds of crowd energy restriction cohorts, observes rs45539933 and rs2270565 homozygous and heterozygous carriers and wild-type individuals for a period of 6 months (AC) and 12 months 25% energy restriction diet (DF) weight loss response. Compared with wild-type, weight loss increases by 22% and 43.6%, therefore, this site can fully predict weight loss effect, which is also the only discovery that can predict weight loss effect so far.

[0088] Example 3 In vivo verification of the weight loss prediction effect of rs45539933 and rs2270565

[0089] In order to further demonstrate the weight loss prediction effect of rs45539933 and rs2270565, the present invention combined the homologous point mutation reverse knock-in mouse model to construct mice that simulated the human UCP1 wild-type sequence. It was found that 8-week-old male mice that simulated human rs45539933 and rs2270565 had lower body weight and fat weight ( Figure 7 ).

[0090] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. UCP1 Application of SNP sites as markers in the preparation of products for predicting weight loss effects; The SNP sites include rs45539933 and / or rs2270565; The forecasts include: UCP1 The genotype of rs45539933 locus is AA or AG, which has a good weight loss effect; UCP1 The genotype of rs45539933 locus was GG, and the weight loss effect was poor; UCP1 The genotype of rs2270565 locus is TT or TA, which has a good weight loss effect; UCP1 The genotype of the rs2270565 locus is AA, and the weight loss effect is poor.

2. A method for predicting weight loss effect, characterized in that: include: Step 1: Test the subjects UCP1 genotype of rs45539933 and / or rs2270565; Step 2: According to UCP1 The genotypes of rs45539933 and / or rs2270565 predicted the weight loss effect of the subjects; The forecasts include: UCP1 The genotype of rs45539933 locus is AA or AG, which has a good weight loss effect; UCP1 The genotype of rs45539933 locus was GG, and the weight loss effect was poor; UCP1 The genotype of rs2270565 locus is TT or TA, which has a good weight loss effect; UCP1 The genotype of the rs2270565 locus is AA, and the weight loss effect is poor.

3. The method according to claim 2, characterized in that Step 1 includes: Step a, extracting DNA from the oral mucosal swab of the subject, and performing PCR amplification using a forward primer having a nucleotide sequence as shown in SEQ ID NO.1 and a reverse primer having a nucleotide sequence as shown in SEQ ID NO.2 to obtain an amplified product; Step b: the amplified product is sequenced and determined UCP1 The genotype of the rs45539933 locus.

4. The method according to claim 2, characterized in that Step 1 includes: Step a, extracting DNA from the oral mucosal swab of the subject, and performing PCR amplification using a forward primer having a nucleotide sequence as shown in SEQ ID NO.3 and a reverse primer having a nucleotide sequence as shown in SEQ ID NO.4 to obtain an amplified product; Step b: the amplified product is sequenced and determined UCP1 The genotype of the rs2270565 locus.

5. The method according to claim 3, characterized in that The reaction procedure of PCR amplification in step 1 includes: 。 6. The primer used in the method according to any one of claims 3 to 5, characterized in that Including amplification of the rs45539933 locus UCP1 Primer pairs and / or amplification of the rs2270565 site UCP1 Primer pairs; The amplified UCP1 The forward primer in the primer pair has the nucleotide sequence shown in SEQ ID NO.1, and the reverse primer has the nucleotide sequence shown in SEQ ID NO.2; and / or The amplified UCP1 In the primer pair, the forward primer has the nucleotide sequence shown in SEQ ID NO.3, and the reverse primer has the nucleotide sequence shown in SEQ ID NO.

4.

7. A kit, characterized in that Comprising the primer as claimed in claim 6.

8. UCP1 A method for detecting a SNP site, characterized in that: This includes testing using any of the following: (I), the primer according to claim 6; and / or (II), the kit as claimed in claim 7; The SNP sites include rs45539933 and / or rs2270565.

9. Upregulation of targeted enhancement using antisense oligonucleotides UCP1 The genotype of rs45539933 is AA or AG, or UCP1 The genotype of the rs2270565 locus is TT or TA in the nuclear gene output, and is used in formulating prevention and / or treatment programs for weight loss, blood sugar lowering and / or lipid regulation.

10. Using CRISPR-Cas9 gene editing technology, UCP1 The gene GG at the rs45539933 site is edited to AA; or the gene AA at the rs2270565 site is edited to TT, so as to restore the thermogenic effect, and is used in formulating prevention and / or treatment plans for weight loss, blood sugar reduction and / or lipid regulation.

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