Application of SNP Loci of UCP1 as Biomarkers and Method for Detecting SNP Loci

Abstract

The present invention relates to the field of biotechnology, and particularly relates to UCP1 the application of the SNP locus as a biomarker and the SNP locus detection method. The present invention provides UCP1 the application of the SNP locus (rs45539933 and / or rs2270565) as a biomarker in the preparation of a product for predicting weight loss effect. Individuals carrying rs45539933 and rs2270565 have a 22% and 43.6% increase in weight loss compared with the wild type when undergoing a 25% energy restriction for 6 months or 12 months. The present invention also provides a SNP locus detection method and a kit, which have significant predictive significance for evaluating the weight loss effect under energy restriction conditions.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to the application of SNP sites of UCP1 as markers and a method for detecting SNP sites. Background Art

[0002] Modern populations are generally susceptible to metabolic disorders, and the "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 there is great controversy in the academic community. If its existence can be demonstrated, it will have an important impact on the understanding of the mechanism of glycolipid metabolism regulation, the transformation of new targets for the intervention of metabolic diseases, and clinical diagnosis and treatment.

[0003] The hypothesis of "thrifty genes" was first proposed by James Neel of the University of Chicago in 1962. He hypothesized that in the hunting and gathering era, without crops or livestock as a stable food supply, the "feast-famine" of food occurred alternately, and the body needed to efficiently store fat and reduce energy consumption to obtain the highest probability of survival, so that the mutations of "metabolic thrifty genes" were under positive selection of the environment and ultimately led to modern people generally carrying these mutations that "can increase energy absorption and reduce consumption", thus explaining why modern populations 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] This hypothesis paper has been cited more than 4,700 times, but so far no "thrifty gene mutations" have been recognized. Therefore, many well-known scholars (including Speakman JR, P, etc.) have questioned: Do humans have "thrifty genes" (Thrifty genes and human obesity. Are we chasing ghosts?)? So far, only in geographically isolated populations (such as the "Samoans" in the South Pacific) has a thrifty gene mutation (CREBRFp.R457Q) been found to be commonly carried, but no similar gene mutations with metabolic thrifty characteristics have been found in more extensive populations in Eurasia, Africa and the Americas. Regarding these possible "thrifty genes", can new diagnostic kits and weight loss, hypoglycemic and lipid-regulating drugs be developed? Summary of the Invention

[0005] In view of this, the technical problem to be solved by the present invention is to provide gene polymorphism sites that can effectively predict the weight loss effect. The present invention provides the application of SNP sites of UCP1 as markers and the detection method of SNP sites. 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 more necessary to combine intervention methods such as physical exercise to increase the basal metabolic rate.

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

[0007] The present invention provides the application of SNP sites of UCP1 as markers in the preparation of products for predicting the weight loss effect; the SNP sites include rs45539933 and / or rs2270565;

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

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

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

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

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

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

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

[0015] Step b: Determine the genotype of the rs45539933 locus of UCP1 by sequencing the amplification product.

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

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

[0018] Step b: Determine the genotype of the rs2270565 locus of UCP1 by sequencing the amplification product.

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

[0020]

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

[0022]

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

[0024] In the primer pair for amplifying UCP1 containing the rs45539933 locus, the forward primer 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

[0025] In the primer pair for amplifying UCP1 containing the rs2270565 locus, 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.

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

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

[0028] (I), the primers as described above; and / or

[0029] (II), the kit as described above;

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

[0031] The present invention also provides the application of using antisense oligonucleotides (ASO) to up-regulate targeted augmentation of nuclear gene output (TANGO) of the rs45539933 site of UCP1 with the genotype of AA or AG, or the rs2270565 site of UCP1 with the genotype of TT or TA, in formulating prevention and / or treatment programs for weight loss, blood sugar reduction and / or lipid regulation.

[0032] The present invention also provides the application of using the 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 into AA; or editing the gene AA at the rs2270565 site (NM_021833.4:c.685A>T) into TT to restore the thermogenic effect, in formulating prevention and / or treatment programs for weight loss, blood sugar reduction and / or lipid regulation.

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

[0034] 1. The present invention discovers that two single nucleotide polymorphisms (SNP, which refers to genomic variations at single base positions in DNA) of the thermogenic gene UCP1, rs45539933 and rs2270565, are enriched in lean populations;

[0035] 2. The present invention discovers that rs45539933 and rs2270565 coexist in the population, that is, individuals with rs45539933 also have rs2270565 at the same time;

[0036] 3. Individuals carrying rs45539933 and rs2270565 have a 22% and 43.6% increase in weight loss compared with the control when undergoing 25% energy restriction for 6 months or 12 months. Therefore, this locus can fully predict the weight loss effect. Description of the Drawings

[0037] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art.

[0038] Figure 1 Indicates rs45539933 wild type;

[0039] Figure 2 Indicates rs45539933 heterozygous;

[0040] Figure 3 Indicates rs2270565 wild type;

[0041] Figure 4 Indicates rs2270565 heterozygous;

[0042] Figure 5 Indicates that rs45539933 and rs2270565 are prevalent in lean populations; among them, A indicates the frequency of rs45539933 and rs2270565 carriers in obese populations; B indicates the impact of rs45539933 and rs2270565 carriers on the body mass index of the population;

[0043] Figure 6 Indicates that individuals carrying rs45539933 and rs2270565 are more sensitive to the energy restriction weight loss program; among them, A indicates a 6-month 25% energy restriction diet test program; B indicates the change in body mass index of rs45539933 and rs2270565 carriers within 6 months of a 25% energy restriction diet; C indicates the change in body weight of rs45539933 and rs2270565 carriers within 6 months of a 25% energy restriction diet; D indicates a 12-month 25% energy restriction diet test program; E indicates the change in body mass index of rs45539933 and rs2270565 carriers within 12 months of a 25% energy restriction diet; F indicates the change in body weight of rs45539933 and rs2270565 carriers within 12 months of a 25% energy restriction diet;

[0044] Figure 7 Indicates that the present invention combines a homologous point mutation reverse knock-in mouse model to construct a mouse that mimics the wild-type sequence of human UCP1; it is found that 8-week-old male mice mimicking human rs45539933 and rs2270565 have lower body weight and fat weight compared to mice mimicking the wild-type sequence of human UCP1; further demonstrating the weight loss prediction effect of rs45539933 and rs2270565; among them, * indicates P value < 0.05, ** indicates P value < 0.01, *** indicates P value < 0.001. Detailed implementation manners

[0045] The present invention discloses the application of SNP sites of UCP1 as markers and a method for detecting SNP sites. Those skilled in the art can draw on the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all regarded as included in the present invention. The methods and applications of the present invention have been described through preferred embodiments. It is obvious that relevant personnel can make changes or appropriate alterations and combinations to the methods and applications 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] Term Explanation:

[0047] Uncoupling protein 1 (UCP1): Also known as thermogenin, it is a mitochondrial inner membrane thermogenic protein that dissipates the proton gradient across the mitochondrial inner membrane and releases this gradient as heat (the thermogenesis process). This protein is located in human adipose tissue.

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

[0049] Uncoupling protein 1 (UCP1) is a mitochondrial thermogenic protein. In the early stage of the present invention, relying on the large genetic resource of "Chinese Adolescent Extreme Obesity Cohort (GOCY)", the cohort, genetics, and evolution were fully integrated, and it was first discovered that:

[0050] 1. The present invention found that two single nucleotide polymorphisms (SNPs, which refer to genomic variations at single base positions in DNA) of the thermogenic gene UCP1, rs45539933 and rs2270565, are enriched in lean populations;

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

[0052] 3. Individuals carrying rs45539933 and rs2270565 showed a 22% and 43.6% increase in weight loss compared to the control when undergoing 25% energy restriction for 6 months or 12 months. Therefore, this locus can fully predict the weight loss effect.

[0053] Unless otherwise specified, the raw materials and reagents used in the application of the SNP sites of UCP1 as markers and the method for detecting SNP sites provided by the present invention can 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 Measurement

[0056] 1. Experimental Materials

[0057] Table 1 Experimental Instruments

[0058] Equipment Name Equipment Supplier 3730xl First-generation Sequencer Applied Biosystems Nano-100 Micro-spectrophotometer Hangzhou Allsheng Instruments Co., Ltd. PCR Instrument 310A Hangzhou Longji Scientific Instruments Co., Ltd. L-550 Desktop Centrifuge Changsha Xiangyi Centrifuge Instruments Co., Ltd. MIX-100 Shaker Hangzhou Allsheng Instruments Co., Ltd. Micropipettor Eppendorf FR-110 Ultraviolet Analyzer Shanghai Furi Technology Co., Ltd. FR-250 Electrophoresis Instrument Shanghai Furi Technology Co., Ltd. 1-10μL 12-channel Pipettor Eppendorf Laminar Flow Hood Shanghai Yipu Laboratory Equipment Co., Ltd.

[0059] Table 2 Experimental Reagents

[0060] Reagent Name Reagent Provider Primer Shanghai Mapbiotech Co., Ltd. 2×Taq Master Mix (Dye Plus) Novoprotein Scientific Inc. Ethanol Sinopharm Chemical Reagent Co., Ltd. Isopropanol Sinopharm Chemical Reagent Co., Ltd. BigDye Terminator Applied Biosystems Hi-Di Formamide Applied Biosystems Agarose Sangon Biotech (Shanghai) Co., Ltd. Boric Acid Sangon Biotech (Shanghai) Co., Ltd. Ethidium Bromide Sangon Biotech (Shanghai) Co., Ltd. Glacial Acetic Acid Sinopharm Chemical Reagent Co., Ltd. Ethylenediaminetetraacetic Acid Sinopharm Chemical Reagent Co., Ltd. DNA ladder marker (DL 2000) Sangon Biotech (Shanghai) Co., Ltd. Blood / Tissue / Cell Genomic DNA Extraction Kit Tiangen Biotech (Beijing) Co., Ltd.

[0061] Table 3 Amplification Primer Sequences

[0062]

[0063] Table 4 Single - strand Sequencing Primer Sequences

[0064]

[0065] 2. Experimental Procedures

[0066] (1) Obtain the oral mucosa of the subject: One hour before sampling, refrain from smoking, drinking, and fasting to reduce the influence of food or exogenous factors on the sampling process. Ten seconds before sampling, all participants rinse their mouths with clean water. Use the first oral mucosa swab to scrape and sample the right cheek / right upper and lower gums of the participant; use the second oral mucosa swab to scrape and sample the corresponding position on the left side of the participant; use the third oral mucosa swab to scrape and sample the corresponding position on the right side of the participant again. After sampling, dry the swab in a clean and well - ventilated place and then put it into a sealed and dry plastic bag. The sample is quickly sent back to the laboratory and stored at 4°C.

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

[0068] (3) PCR amplify the DNA of the oral mucosa swab extracted in (2) to obtain the PCR product.

[0069] Reaction System:

[0070] Composition System Premix Taq 10μL Forward Primer 1μL (concentration: 10μmol / L) Reverse Primer 1μL (concentration: 10μmol / L) DNA 1μL (concentration: 20 - 200ng / μL) <![CDATA[ddH2O]]> 7μL Total 20μL

[0071] Reaction Program: 94°C for 5 min; 28 cycles: 94°C for 30 s, 58°C for 30 s, 72°C for 1 min; 72°C for 10 min.

[0072] (4) Gel purification of the PCR product

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

[0074] (5) Sanger sequencing experiment.

[0075] Sequencing reaction system: The total volume was 5 μL, including 1 μL of sequencing primer (3.2 μM), 1-3 μL of purified PCR products, and 1 μL of BigDyeMix kit.

[0076] Reaction procedure: 94°C for 1 min; 28 cycles: 94°C for 20 s, 50°C for 10 s, 60°C for 4 min; 4°C for 30 min.

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

[0078] 100% alcohol precipitation: 18 μL of 100% ethanol solution was added, precipitated at -20°C for 30 min, centrifuged at 4500 rpm for 30 min, and inverted centrifuged at 400 rpm for 2 min.

[0079] 75% alcohol washing: 50 μL of 75% alcohol was added, centrifuged at 4500 rpm for 10 min, and inverted centrifuged at 400 rpm for 2 min.

[0080] Denaturation: 6 μL of Hi-Di was added, centrifuged at 3700 rpm for 1 min, and stored at -20°C for loading onto the 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 compared and analyzed using the Sequencher 5.1 software package.

[0083] (7) Measure the body weight and waist circumference data of the subjects and record them.

[0084] (8) Test results:

[0085] The test results are as Figures 1 to 5As shown in the figure, by leveraging the genetic resources of the "Extremely Obese Cohort of Chinese Adolescents and the Golden Control (1994 cases vs 2161 cases)", the present invention first discovered that the frequency of homozygous carriers of rs45539933 and rs2270565 in the obese population is half that of the normal population (A). Through DNA sequencing and body mass index detection, it was found that homozygous and heterozygous carriers of rs45539933 and rs2270565 have a lower body mass index than the wild type, with an average body weight 11.39 kg lighter and a waist circumference reduced by 9.31 cm (B).

[0086] Weight loss response of the population in Example 2 to energy-restricted diet

[0087] As Figure 6 shown in the figure, the present invention utilized two energy-restricted cohorts of the population to observe the weight loss responses of homozygous and heterozygous carriers of rs45539933 and rs2270565 and wild-type individuals to a 6-month (A-C) and a 12-month 25% energy-restricted diet (D-F). Compared with the wild type, the weight loss amplitude increased by 22% and 43.6%. Therefore, this locus can fully predict the weight loss effect, which is also the only discovery so far that can predict the weight loss effect.

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

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

[0090] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A method for predicting the weight loss effect of subjects under energy-restricted diet, wherein the prediction method is not used for disease diagnosis and / or treatment purposes, characterized in that Comprising: Step 1, Detect the genotypes of the SNP loci of the subject UCP1 ; The SNP locus consists of rs45539933 and rs2270565; Step 2. Predict the weight loss effect of the subject according to the genotypes of UCP1 rs45539933 and rs2270565; The prediction includes: UCP1 The genotype of the rs45539933 locus of UCP1 is AA or AG, and the weight loss effect is good; UCP1 The genotype of the rs45539933 locus of UCP1 is GG, and the weight loss effect is poor; UCP1 The genotype of the rs2270565 locus of UCP1 is TT or TA, and the weight loss effect is good; UCP1 The genotype of the rs2270565 locus of UCP1 is AA, and the weight loss effect is poor; Step 1 comprises: Step a, extracting the DNA of the oral mucosal swab of the subject, and performing PCR amplification using a forward primer with the nucleotide sequence shown in SEQ ID NO.1 and a reverse primer with the nucleotide sequence shown in SEQ ID NO.2 to obtain an amplification product; Step b, the genotype of the rs45539933 locus determined by sequencing the amplification product; and UCP1 ; and Step 1 comprises: Step a, extracting the DNA of the oral mucosal swab of the subject, and performing PCR amplification using a forward primer with the nucleotide sequence shown in SEQ ID NO.3 and a reverse primer with the nucleotide sequence shown in SEQ ID NO.4 to obtain an amplification product; Step b: The genotype of the rs2270565 locus of the amplified product is determined by sequencing. UCP1 ​ 2. The prediction method according to claim 1, wherein The reaction procedure of the PCR amplification in Step 1 comprises: 。

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