CN108753945B - SNP (single nucleotide polymorphism) locus related to obesity and/or hypertriglyceridemia of Chinese children and application thereof - Google Patents

Abstract

The invention discloses application of an SNP locus in preparation of an early detection product for obesity and/or hypertriglyceridemia of Chinese children, wherein the SNP locus is rs189326455, and specifically chr1:233463936, c.162G > C and p.Glu54Asp. The invention researches the gene locus rs189326455 related to obesity as an early biomarker of obesity and hypertriglyceridemia, and provides a new direction for further researching the genetic molecular mechanism of obesity and hypertriglyceridemia and exploring the drug target for early prevention and treatment of obesity and hypertriglyceridemia.

Description

SNP (single nucleotide polymorphism) locus related to obesity and/or hypertriglyceridemia of Chinese children and application thereof

Technical Field

The invention relates to the technical field of biotechnology and medical detection, in particular to an SNP locus related to obesity and/or hypertriglyceridemia of Chinese children and application thereof, wherein the SNP locus is rs189326455, specifically chr1:233463936, c.162G > C, and p.Glu54Asp.

Background

Obesity is a common nutritional metabolic disorder. With the improvement of the material living conditions, the change of the dietary structure, the acceleration of the life rhythm, the factors of mental stress, lack of movement and the like, the acceleration of living joints, the factors of mental stress, lack of movement and the like, and the number of overweight and obese people is increasing. There is evidence that obesity is closely associated with hypertension, diabetes, dyslipidemia, metabolic syndrome, and carries a high risk of cardiovascular disease. In 2002, the WHO ranks obesity as one of the global primary risk factors responsible for the burden of human diseases, and the prevalence of obesity prevention is also one of the greatest public health challenges facing countries in the world 50 years before the 21 st century.

Obesity is a complex disease caused by the combined action of factors such as genetics, environment and life behaviors. In today's obesity-causing environment, genetics determine an individual's susceptibility to environmental changes. Pedigree analysis and twin studies have shown that genetic factors play a role in obesity development of about 40% to 70%. Single Nucleotide Polymorphism (SNP) is a molecular genetic marker proposed by Lander of the human genome research center of the American college of science and technology of Mazhou province in 1996, and mainly refers to DNA sequence polymorphism caused by single nucleotide variation on the genome level. SNPs involve only single base variations, in different forms, including transitions, transversions, insertions and deletions. SNP is a third generation genetic marker, and many phenotypic differences of human bodies, susceptibility to drugs or diseases and the like can be related to SNP. Genome-wide association studies (GWAS) are an effective strategy to study genetic susceptibility to complex diseases using SNPs as genetic markers. To date, a number of obesity-susceptible gene loci have been discovered using GWAS strategies. For example, the FTO gene is located on chromosome 16 (16q12.2), contains 9 exons, has a gene length of 410.50kb, is widely expressed in various developmental stages of human tissues, and is highly expressed in tissues such as hypothalamus, skeletal muscle, fat, and the like. It is the first gene that GWAS finds to be associated with obesity in the general population. In 2007, when Frayling TM et al first conducted a correlation study of FTO with type 2 diabetes, the association between FTO and type 2 diabetes disappeared after BMI control, thereby unexpectedly finding that the FTO gene is associated with obesity. Thereafter, the association of FTO gene variation with BMI and obesity was repeated in different ethnic groups, with rs9939609 in intron 1 being the most reproducible polymorphic site. The FTO gene polymorphism has been subsequently shown to be associated with genetic susceptibility to obesity or type 2 diabetes.

Despite the increasing knowledge of genes and gene loci associated with obesity and obesity-related traits, no clinically useful genetic variation has been found as an effective product for early screening and intervention in obese individuals, particularly children. Recent studies even conclude that common SNPs in a group of obesity-related candidate genes play only minor, if any, roles in regulating weight changes induced by certain diets.

In view of the fact that no early biomarker and early diagnosis kit related to obesity exist at present, if a corresponding marker can be found and a corresponding diagnosis kit can be developed, a new direction is provided for exploring a drug target for early prevention and treatment of obesity, and a new way is opened for drug screening, drug effect evaluation and targeted therapy of obesity.

Disclosure of Invention

The invention mainly aims to discover a biomarker related to Chinese child obesity by utilizing a high-throughput exon sequencing technology and provide application of the biomarker in preparation of a Chinese child obesity early detection product.

The purpose of the invention is realized by the following technical scheme:

the study adopted a two-stage case-control study design: the first stage was an exploratory study, with a panel of obese children and a panel of normal weight children selected as controls for whole genome exon sequencing. Functional variations of exon regions and splicing regions, including nonsynonymous variations (nonsynonymous), nonsense variations (stop gain), stop codon variations (stop loss), base insertions/deletions (insertion/deletion), and splicing sites (splice site) were selected from sequencing data for correlation analysis, and 48 possible obesity-related (P <0.05) variations were selected. The second stage is a validation study, the screened mutation sites are genotyped in 2,045 obese children and 3,338 non-overweight children, and the correlation between the mutations and the obesity and related evaluation indexes of the children is analyzed and validated. Finally, the rs189326455 locus is confirmed to be a gene locus related to obesity and hypertriglyceridemia of Chinese children.

MAP3K21 (mitogen-activated protein 3 kinase 21, mitogen-activated protein kinase kinase 21), also known as mixed lineage kinase 4(MLK4), is located on chromosome 1 and is a protein-encoding gene. Mixed Lineage Kinases (MLKs) belong to the superfamily of kinases that are MAP kinase kinases (MAP3 Ks). Members of the MLK family are characterized by the presence of Ser/Thr and Tyr kinase signature sequences within their catalytic domains. MLK1-4 contains an N-terminal Src homology (SH3) domain followed by a kinase domain, a leucine zipper domain and a Cdc42/Rac interaction motif (CRIB). MLK3 is the most widely expressed member of the MLK family, with high homology at the N-terminus and differences at the C-terminus in MLK1-4, suggesting that these regions may perform different regulatory functions. At present, MLK4 is mainly studied on the prognosis relation between MLK4 gene mutation and colon cancer and MLK 4as an upstream regulatory factor of NF-kB signal transduction, and the activation of NF-kB induces the trans-differentiation and radiation resistance of Mesenchyme (MEs) of Glioma Stem Cells (GSCs).

Based on the above, the invention firstly provides the application of the SNP locus in the preparation of the early detection product of Chinese children obesity, wherein the SNP locus is rs189326455, and specifically chr1:233463936, c.162G > C, and p.Glu54Asp.

Preferably, the evaluation index of obesity includes BMI, FMI, FFMI, FMP, WC, WHtR values and the content of triacylglycerol, total cholesterol, high density lipoprotein cholesterol, low density lipoprotein cholesterol in serum.

Preferably, the mutation at the SNP site rs189326455 has a negative association with BMI and/or triacylglycerol in serum at the whole genome level.

Preferably, the obesity includes general obesity, body fat obesity, and abdominal type obesity.

Preferably, the obesity is general obesity.

Preferably, the evaluation index of general obesity is BMI.

Preferably, the serum TG ≥ 0.85mmol/L (< 10 years) or 1.02mmol/L (10-18 years) is defined as hypertriglyceridemia.

Further, the invention provides application of the SNP locus in preparation of a hypertriglyceridemia detection product, wherein the SNP locus is rs189326455, and specifically is chr1:233463936, c.162G > C and p.Glu54Asp.

Further, the invention also provides a reagent for detecting the early stage of the general obesity of children, wherein the reagent is used for detecting the genotype of the susceptible SNP locus rs 189326455.

Preferably, the reagent comprises primers for amplifying a nucleotide sequence including the rs189326455 locus, such as SEQ ID NO. 3-5 or SEQ ID NO. 1-2.

Furthermore, the invention provides an early auxiliary detection kit for the general obesity of children, which comprises a reagent for detecting the genotype of the SNP locus rs189326455 in a sample; the reagent comprises primers for amplifying nucleotide sequences including rs189326455 locus, such as SEQ ID NO. 1-2.

Preferably, the Primer can be designed by software, such as by using Primer5, Oligo6, and the like.

Preferably, the kit may be a reagent for detecting SNP by any technique known in the art, as long as it can detect the presence or absence of allelic mutation at SNP site rs189326455 in the sample. Including but not limited to the embodiments listed below.

In a first embodiment, the kit of the invention comprises a reagent for detecting the existence of the C allele at the site of SNP rs189326455 in a sample by using a sequencing method. By using the kit, the sequence of the SNP locus rs189326455 in a sample can be directly measured by a sequencing method, so that whether the SNP locus carries the variation of the allele of the corresponding locus or not is judged, and the non-susceptibility of obesity is further judged. The sequencing method is well known in the art, the required reagents can be purchased commercially, and the person skilled in the art can select the reagents according to the needs (see the relevant operating instructions of the sequencer of ABI, Beckman, etc.).

In a second embodiment, the kit of the invention comprises a reagent for detecting the genotype of the SNP locus rs189326455 in a sample by using a Taqman probe SNP detection method. The Taqman probe is designed aiming at the SNP locus rs189326455, and the probe provided by a reagent company can be used; it can also be designed by software, such as Beacon Designer 7.5 from PREMIER Biosoft.

In a third embodiment, the kit of the present invention is a kit for detecting the genotype of the SNP site rs189326455 in a sample by using a PCR-single strand conformation polymorphism method. The kit comprises a primer for amplifying the SNP locus rs189326455 genotype, a PCR reagent, a control sample and a reagent required by electrophoresis of detection conformation. The electrophoresis is preferably native polyacrylamide gel electrophoresis. The control sample comprises at least one of a negative control sample of SNP locus rs189326455 genotype GG homozygote and a positive control sample of locus CC homozygote, and can also comprise a control sample of heterozygote corresponding heterozygote. Preferably, the three types of control samples are included simultaneously. And (3) simultaneously carrying out electrophoresis on the amplification product of the sample to be detected and the amplification product of the control sample, and comparing the electrophoresis results to obtain the detection result of whether the sample to be detected carries the corresponding allelic variation.

The kit also includes PCR reagents, a control sample, and reagents required for electrophoresis to detect the conformation. Common reagents required for PCR technology, such as: dNTPs, MgCl₂Double distilled water, Taq enzyme, and the like. The electrophoresis is preferably native polyacrylamide gel electrophoresis.

In a third embodiment, the kit of the present invention is a kit for detecting the genotype of the SNP site rs189326455 in a sample by using the restriction fragment length polymorphism polymerase chain reaction (PCR-RFLP) technique.

If the kit provided by the invention detects the existence of C allele carried by SNP locus rs189326455 in a sample, the obesity susceptibility of the individual providing the sample is weaker than that of the individual not carrying the variation.

The invention has the beneficial effects that:

the invention discovers the SNP locus rs189326455 related to obesity of Chinese children by adopting a second-generation sequencing technology in the Chinese children, and verifies the authenticity of the SNP locus rs189326455 through the first-generation sequencing. The site variation can directly cause the change of the coded amino acid, and belongs to functional variation. By analyzing the association of the site variation and the obesity of children and related evaluation indexes, the site is found to be associated with not only the obesity but also the level of triacylglycerol related to the obesity. The gene locus related to the obesity can be used as an early biomarker of the obesity and the hypertriglyceridemia, and provides a new direction for further researching the genetic molecular mechanism of the obesity and the hypertriglyceridemia and exploring a drug target point for early prevention and treatment of the obesity and the hypertriglyceridemia.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

The terms in the present invention are explained as follows:

the term "primer" as used herein refers to an oligonucleotide that is present in a purified restriction digest or produced synthetically and which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product complementary to a nucleic acid strand is induced, i.e., in the presence of nucleotides and an inducing agent (e.g., a DNA polymerase) and at an appropriate temperature and pH. The primer may be single-stranded or double-stranded and must be of sufficient length to prime the synthesis of the desired extension product in the presence of the inducing agent. The exact length of the primer will depend on many factors, including temperature, source of primer, and method used. For example, for diagnostic applications, oligonucleotide primers typically contain 15-25 or more nucleotides, although fewer nucleotides may be present, depending on the complexity of the target sequence. Factors involved in determining the appropriate length of a primer are well known to those of ordinary skill in the art. In General, primers of the invention are designed and selected according to standard methods well known in the art, see Dieffenbach, C.W., Lowe, T.M.J., Dveksler, G.S (1995) General Concepts for PCR Primer design, PCR Primer, A Laboratory Manual (Dieffenbach, CW and Dveksler, G.S. eds.), Cold Spring harbor Laboratory Press, New York, 133-.

Body Mass Index (BMI): the body mass Index, also called Body Mass Index (BMI) for short, is calculated by dividing the weight (kg) by the height (m) squared, and is a standard for measuring the body fat and body weight and whether the body is healthy.

Fat Mass Index (FMI): the body weight of a person consists of fat body weight and lean body weight (lean body mass). Fat and non-Fat contents are obtained by directly measuring body components, wherein the body Fat content (kg) is divided by the height (m) to obtain a square, namely a body Fat Mass Index (FMI) for more accurately evaluating the obesity degree of a human body.

Non-fat mass index (FFMI): the body weight of a person consists of fat body weight and lean body weight (lean body mass). The fat body weight removed includes muscle, bone, soft tissue and moisture. Fat and non-Fat contents are obtained by directly measuring human body components, wherein the change of individual non-Fat (Lean body Mass) is mainly the change of muscle content, and the change is calculated by dividing the non-Fat content (kg) by the square of height (m), namely a non-Fat Mass Index (Fat-Free Mass Index, FFMI for short) and a Lean body Mass Index (Lean Mass Index, LMI for short) for evaluating the muscle content and the nutritional health state of the human body.

Percent body Fat (FMP): fat and non-Fat contents are obtained by measuring body components, and the Percentage obtained by dividing the body Fat content by the body weight, namely the body Fat Percentage (FMP) is used for evaluating whether a human body is obese and the obesity degree.

Waist Circumference (WC): the English full name Waist Circuit is a comprehensive index reflecting the total fat amount and the fat distribution, and the measurement method recommended by the world health organization is as follows: the subject stands with feet separated by 25 to 30 cm and weight is evenly distributed. The measurement position is at the midpoint of the line connecting the anterior superior iliac crest and the lower edge of the 12 th rib in the horizontal position. The dipstick is held tightly against the skin without compression, and at the end of normal expiration the measurement is taken to the nearest 0.1 cm.

Waist height ratio (WHtR): the English term is called Waist-to-Height Ratio, which is the value of Waist circumference divided by Height, and is used to indirectly reflect the accumulation of fat in abdomen. Since WHtR takes into account height factors and is hardly affected by age and sex, it is very simple to define abdominal obesity in children using WHtR ≧ 0.5 (i.e., waist circumference more than half the height of the body).

General obesity: and (3) screening BMI threshold values to diagnose obesity, normality and emaciation by using BMI as an evaluation index and adopting overweight and obesity of school-age children and adolescents recommended by the international obesity problem task force (IOTF).

Fat and obesity: the standard recommended by the guidelines for preventing and controlling overweight and obesity of children and teenagers of Chinese school age is adopted, FMP is taken as an evaluation index, FMP of males is more than or equal to 20 percent, FMP of females 14 years old and below is more than or equal to 25 percent, and FMP of females 15 years old and above is more than or equal to 30 percent of obesity.

Abdominal obesity: the abdominal obesity is diagnosed by using WHtR as an evaluation index and using WHtR more than or equal to 0.5.

Dyslipidemia: according to the guidelines of the American institute for cardiopulmonary and hematological (NHLBI) for cardiovascular health and risk reduction in adolescents, a marginal elevation in triacylglycerol levels is defined as hypertriglyceridemia, i.e., TG ≧ 75mg/dl (0.85mmol/L) in children under 10 years of age, and TG ≧ 90mg/dl (1.02mmol/L) in children over 10 and 10 years of age. Similarly, hypercholesterolemia is defined as TC ≥ 170mg/dl (4.41 mmol/L); high low density lipoprotein cholesterolemia is defined as LDL-C being more than or equal to 110mg/dl (2.85 mmol/L); low high density lipoprotein cholesterolemia is defined as HDL-C < 45mg/dl (1.16 mmol/L).

The Mutation Taster: a Mutation Taster Score (version 2.3) which represents the influence of the variation on a protein sequence and comprises two groups of data, wherein Score gives the calculated Score of each site of the submitted amino acid after Mutation, the Score is 0-1, and the larger the value is, the more harmful the value is, the higher the possibility that the SNP causes the structural or functional change of the protein is indicated; predict is a prediction result represented by A, D, N or P, wherein A: (ii) a disease using automatic pathogenic mutation, D: disease using may be a pathogenic mutation, N: polymorphism may be harmless, P: polymorphism automatic polymorphism is harmless.

MAF: the english term minor allele frequency, i.e. the frequency of minor alleles, generally refers to the frequency of occurrence of the unusual alleles in a given population. MAF is widely used in genome-wide association studies of complex diseases. In correlation studies, a smaller MAF will decrease the statistical performance, leading to false negative results. To study the association of rare mutations with disease in the population, the loss of statistical potency due to MAF reduction is usually compensated by increasing the sample size.

The technical scheme of the invention is as follows:

the exon sequencing experiment adopts Roche/NimbleGen's SeqCap EZ Human exon Kit v3.0 exon liquid phase capture chip to capture whole genome exons, constructs an exon library, and performs double-end sequencing on an Illumina HiseqTM 2500 high-throughput sequencing platform, wherein the sequencing depth is required to reach 100 times.

In the genotyping experiment, the screened 48 variation sites are genotyped in 2045 obese school-age children and 3338 control children, and the association of the variation and the obesity of the children is analyzed and verified. And designing and synthesizing a PCR (polymerase chain reaction) and a single-base amplification primer according to the sequence information of the gene locus, and completing genotyping by using a Sequenom MassARRAY high-flux genotype detection technology.

The blood lipid index determination of the invention comprises the following steps: collecting 5ml of peripheral venous blood of a subject fasting for 12 hours in the morning, and separating serum (non-anticoagulation tube) on site for biochemical detection; reagents are purchased in a unified mode, and a Hitachi 7020 full-automatic biochemical analyzer is used for measuring blood biochemical indexes. Serum Total Cholesterol (TC) and Triacylglycerol (TG) were measured using standard enzymatic methods, and high density lipoprotein cholesterol (HDL-C) and low density lipoprotein cholesterol (LDL-C) were measured using the clearance method.

The invention uses a statistical analysis method to mainly use PLINK 1.90, SPSS 22.0 and R3.4.3 software to carry out statistical analysis, and the analysis method comprises chi²Test, unconditional logistic regression, and multiple linear regression analysis. The correlation between quantitative traits such as BMI, TG and the like and the variation site is expressed by a regression coefficient (beta) and Standard Error (SE), the correlation between obesity and metabolic risk and the variation site is expressed by a ratio (OR) and a 95% Confidence Interval (CI), and an additive genetic model is adopted in correlation analysis. Multiple tests were corrected using the controlled False Discovery Rate (FDR) method.

EXAMPLE 1 Collection of samples and working up of sample data

The inventor collects a large number of obese cases and control children blood samples in primary and middle schools in Beijing and universalization areas from 4 months to 2014 months in 2004, and selects a sample meeting the following standards from the samples to carry out whole genome exon sequencing and single SNP Sequenom MassARRAY genotyping experiment samples by collating sample data:

1. cases of obesity that are clearly diagnosed by the Body Mass Index (BMI) classification criteria recommended by the international obesity problem task force (iot);

2. han children, age 6-18 years;

3. controls included normal weight and lean children, with overweight children excluded.

And the system collects the conditions of demographic data, clinical data and the like of the samples.

Example 2 peripheral blood DNA extraction

Human genomic DNA was extracted from blood samples of the above-mentioned eligible obese children and normal weight children by the centrifugal column method (DNA miniprep kit, QIAGEN, Germany). The method comprises the following specific steps:

1. adding 20 mu L of proteinase K into a 1.5mL centrifuge tube;

2. adding 200 μ L of leukocyte mixture, and if the volume is less than 200 μ L, supplementing with Phosphate Buffer Saline (PBS);

3. adding 200 mu L buffer AL (lysate), carrying out vortex oscillation for 15 seconds, and carrying out rapid centrifugation;

4. water bath at 56 deg.c for 10 min;

5. performing rapid centrifugation to avoid solution remaining on the tube cover;

6. adding 200 mu L of absolute ethyl alcohol, carrying out vortex oscillation for 15 seconds, and quickly centrifuging;

7. all solutions were transferred to a QIAamp spin column, centrifuged at 10000rpm for 2 minutes, the collection tube was discarded, and the spin column was transferred to a new collection tube (provided with the kit);

8. adding 500. mu.L of buffereAW 1 (washing solution 1) to the centrifugal column, centrifuging at 10000rpm for 2 minutes, discarding the collection tube, and transferring the centrifugal column to a new collection tube (provided by the kit);

9. adding 500 μ L of buffereaw 2 (washing solution 2) to the centrifugal column, centrifuging at 10000rpm for 5 minutes, discarding the collection tube, transferring the centrifugal column to a new collection tube (the kit is not provided, and can be replaced by a 1.5mL centrifugal tube), and centrifuging at 12000rpm for 3 minutes;

10. transferring the column to a new 1.5mL Eppendorf tube, adding 200. mu.L of bufferaE (DNA lysate) to the column, standing at room temperature for 5 minutes, and centrifuging at 13000rpm for 3 minutes;

11. discard the column and cover the tube. Storing at-20 deg.C for use.

Example 3 Whole exome detection of SNPs in peripheral blood DNA

The DNA of the blood samples of 96 obese children and 96 normal weight children in example 2 were subjected to whole genome exon sequencing, and two groups of children exon sequencing data were compared to obtain candidate gene loci. The method comprises the following specific steps:

1. and (3) DNA quality inspection: using an ultraviolet/visible spectrophotometer (DU800, beckman, usa) or (NanoDrop 2000, seimer feishel, usa), absorbance values of a230nm, a260nm, a280nm were read, the DNA concentration (g/L) ═ a260nm × 50 was calculated according to the formula, and the DNA purity was identified by the OD ratio (a260/a280, a260/a 230). A low A260/A280 ratio indicates protein residues, but if phenol is used in the process, phenol residues are more likely, and a high A260/A280 ratio indicates that RNA is not removed completely. A low A260/A230 ratio indicates contamination with residual salts or small molecule impurities, and an excessively high ratio must have unknown impurities.

On the basis of preliminary DNA quantification, agarose gel electrophoresis detection (gel concentration: 0.8%, voltage: 120V, time: 20min) is further adopted to accurately quantify DNA. If a strong band appears after electrophoresis, the extracted DNA has higher quality. The exon sequencing requires that the concentration of DNA is more than or equal to 50 ng/mu L, the total amount is not less than 5 mu g, the OD260/280 is between 1.8 and 2.0, the OD260/230 is about 2.0, and a sample has no RNA pollution, no degradation or slight degradation. Sequenom SNP genotyping requires that the DNA concentration is more than 25 ng/muL, the DNA volume is more than 10 muL, the purity requirement OD260/280 is 1.7-2.0, and agarose electrophoresis needs a bright single band of more than 10kb without RNA and protein pollution.

2. Exon capture: hybrid capture is carried out by adopting Roche/NimbleGen's SeqCap EZ Human exterior Kit v3.0 liquid-phase exon capture technology, and the average capture efficiency is more than 85%. The general process is to break the genome DNA into about 150-220bp fragments randomly, and then to connect the two ends of the fragments with adapters to prepare the hybrid library. The Library is purified, subjected to LM-PCR linear amplification, hybridized and enriched with Biotinylated DNA Library, subjected to LM-PCR linear amplification, and subjected to on-machine sequencing (Hiseq2500 sequencer) after qualified Library detection.

3. Exome library construction: and (3) constructing an exome library by adopting a DNA true-seq library construction process of an Illumina standard on DNA samples qualified by quality inspection. The library construction procedure is briefly described as follows:

(1) taking 5 mu g of genome DNA, randomly and mechanically breaking the genome DNA by a Bioruptor in a proper system to ensure that the main band of the fragment is close to 200bp, cutting the gel and recovering a 150-bp fragment;

(2) carrying out end repair and adding A tail to the 3' end of the DNA fragment;

(3) connecting a sequencing adaptor (adapter), purifying a connecting product, performing Polymerase Chain Reaction (PCR) amplification, and purifying an amplification product into a pre-library;

(4) taking a certain amount of pre-library to perform hybridization capture by using a probe in the exome capture kit. The capture was performed according to the procedure of the NimbleGen capture chip. Carrying out PCR amplification on the hybrid product after elution and recovery, wherein the product recovery is a final library, and confirming a small sample by agarose gel electrophoresis;

(5) and performing final quality inspection on the library by using a quantitative PCR method to judge the size of the library insert and the final concentration of the pre-computer library, and arranging the on-computer sequencing on the qualified library.

4. Exome sequencing: and (3) carrying out double-end (pair-end) sequencing on the constructed exome library by adopting an Illumina HiseqTM 2500 high-throughput sequencer, wherein the sequencing mode is 150PE, the sequencing reagent is V3, and the sequencing depth is 100 x. The Illumina Hiseq2500 sequencing system is a high-throughput sequencing technology, and the sequencing principle adopts a Sequencing By Synthesis (SBS) technology of a reversible termination method.

5. Data analysis and processing: quality control of whole exome sequencing data: firstly, the detection rate of the sample is more than 95%, and the sample with the genotype deletion rate of more than 5% is rejected; secondly, the detection rate of the loci is more than 95 percent, and loci with the deletion rate of the removed genotypes exceeding 5 percent are removed; ③ MAF > 0.01; the control group should satisfy Hardy-Weinberg equilibrium (P > 1.0E-6). And performing association analysis by using data qualified in quality control, and finding SNP (Single nucleotide polymorphism) with remarkably different genotype distribution frequencies in an obesity case group and a control group, wherein the association analysis result of the SNP locus MAP3K21rs189326455 exon is shown in Table 1.

TABLE 1 mutation frequency of MAP3K21rs189326455 site in exon sequencing samples and correlation with obesity

Chr

Gene

SNP

BP(hg19)

Allele(Ref/Alt)

F_case

F_control

P

OR

1

MAP3K21

rs189326455

233463936

G/C

0.0105

0.0474

0.045

0.203

Note: allele (Ref/Alt): reference allele/mutant allele; f _ case: mutation allele frequency in the case group; f _ control: mutant allele frequency in the control group.

Example 4 Sequenom MassARRAY genotyping of Single SNPs

In the 2045 eligible obese school-aged children and 3338 control (2840 normal-weight children and 498 lean children), 48 SNPs that were found to be significantly associated with obesity by whole exon sequencing were detected on the Sequenom MassARRAY genotyping platform, with the specific steps:

1. sequenom MassARRAY genotyping was performed. Design specificity of 48 SNPs related to obesity found by sequencing of full exomeAn amplification primer and a specific extension primer; the system of the amplification reaction comprises: 0.625. mu.L of PCRbuffer (10), 0.1. mu.L of dNTPmix (25mM), 0.325. mu.L of MgCl₂(25mM), 0.2L HotStar Taq (5U/. mu.L), 1. mu.L (0.5uM) of a mixture of each pair of forward and reverse amplification primers and 1.75. mu.L of double distilled water, 1. mu.L of a DNA sample was added to conduct PCR amplification reaction. The system for the extension reaction comprises: 2. mu.L of EXTENDMix solution (0.94. mu.L of each extension reaction primer mixture, 0.041. mu.L of iPLEX enzyme, 0.2. mu.L of extension mixture). Single base extension reaction was performed by adding 9. mu.L of PCR product after SAP (shrimp alkaline phosphatase) treatment. The apparatus used was an ABI9700 type PCR apparatus. The purified product was centrifuged at 4000rpm for 4 minutes, the resin was precipitated and transferred to 384-well SpectroCHIP (Sequenom) chips using a MassARRAY Nanodispenser RS1000 spotting machine for MALDI-TOF mass spectrometry. The SNP design specific amplification primers and specific extension primers of the MAP3K21 gene rs189326455 are shown in Table 2.

TABLE 2 primer sequences in MAP3K21rs189326455 site Mass Spectrometry

2. Genotype interpretation: the method is carried out by using TYPER4.0 software (sequenom).

3. Data processing and analysis: the following quality controls were performed on the genotyping data: firstly, the detection rate of the sample is over 90 percent, and the sample with the genotype deletion rate of over 10 percent is removed; the detection rate of the loci is more than 90 percent, and loci with the deletion rate of the removed genotypes exceeding 10 percent are removed; the detection accuracy rate is more than 99 percent; fourthly, MAF is more than 0.01; the control group should satisfy Hardy-Weinberg equilibrium (P > 0.001).

After data quality control, the difference of the distribution frequency of the three genotypes of each SNP in a case group and a control group is compared, and the association of the SNP with obesity and related traits is analyzed by adopting an additive genetic model. The association of loci with different obesity phenotypes in children was analyzed using a multifactorial logistic regression model, with the strength of association expressed as Odds Ratio (OR) and 95% Confidence Interval (CI). The association of sites with obesity-related quantitative trait Body Mass Index (BMI), Fat Mass Index (FMI), non-fat mass index (FFMI), percent body Fat (FMP), Waist Circumference (WC), and waist circumference to height ratio (WHtR) was analyzed using a multiple linear regression model, with the unit values of BMI, FMI, FFMI, FMP, WC, and WHtR changes per addition of one allele being expressed in terms of partial regression coefficient (β) and Standard Error (SE). Similarly, the correlation analysis of the SNP loci and the quantitative traits of the blood lipid indexes adopts a multiple linear regression model, and the correlation analysis of the SNP loci and the quality traits of the blood lipid abnormality adopts a multi-factor logistic regression model. Age and gender were adjusted as covariates. When the number of cases or controls is less than 5, Fisher's exact probability test is used.

4. Results

4.1 association of rs189326455 with the obesity phenotype: nonsynonymous coding variation rs189326455(chr1:233463936, c.162G) located in chromosome 1 MAP3K21 gene after adjustment of age and sex>C, p.glu54asp) was associated with general obesity to a genome-wide significance level, OR 0.26 (95% CI: 0.17-0.42, P-9.605 × 10^-9) After multiple tests are corrected by a control False Discovery Rate (FDR) method, the correlation still has statistical significance, and the correlation directions of the boys and the girls are consistent. However, the association of this site with body fat obesity was not statistically significant after correction of multiple tests, and was not seen in association with abdominal obesity. The results are shown in Table 3.

TABLE 3 Association of MAP3K21rs189326455 with different obesity phenotypes in children

Note: adjusting age and gender, wherein font is thickened to indicate that P is less than 0.05, and P is still less than 0.05.OR after multiple tests is corrected by adopting a method for controlling error discovery rate established by Benjamini and Hochberg, and a ratio is obtained; CI, trusted interval.

4.2 association of rs189326455 with obesity assessment index: after adjusting age and sex, MAP3K21 gene rs189326455(chr1:233463936, c.162G>C, p.glu54asp) with BMI (β ═ 2.22, P ═ 8.916 × 10^-8) In whole genome waterAverage has negative correlation, consistent between men and women; after multiple tests corrected by the FDR method, the associations remained statistically significant in the general population as well as in boys, but disappeared in girls. rs189326455 has a negative association trend with FMI and FMP, but the association disappears after multiple tests and corrections. rs189326455 has no relation with FFMI, WC and WHtR. The results are shown in Table 4.

TABLE 4 correlation between MAP3K21rs189326455 and different obesity assessment indices

Note: adjusting age and gender, wherein font bold indicates P <0.05, P <0.05. beta, partial regression coefficient is remained after multiple tests are corrected by adopting a method for controlling error discovery rate established by Benjamini and Hochberg; SE, standard error of partial regression coefficients; BMI, body mass index; FMI, fat mass index; FFMI, non-fat mass index; FMP, percent body fat; WC, waist circumference; WHtR, waist height ratio.

4.3 correlation of rs189326455 with obesity-related lipid indicators: after adjusting age and sex, MAP3K21 gene rs189326455(chr1:233463936, c.162G>C, p.glu54asp) and the natural logarithm of the level of triacylglycerol (β -0.25, P-5.990 × 10^-11) Negative associations were found at the genome-wide level, consistent between men and women; after multiple tests corrected by the FDR method, the association was still statistically significant in the general population as well as in males and females. rs189326455 is in negative association with TC, and after multiple test correction, the association in boys disappears but does not reach the significant level of the whole genome. After multiple test correction, rs189326455 has no correlation with HDL-C, LDL-C. The results are shown in Table 5.

TABLE 5 correlation between MAP3K21rs189326455 and blood lipid indices

Note: adjusting age and gender, wherein font bold indicates P <0.05, P <0.05. beta, partial regression coefficient is remained after multiple tests are corrected by adopting a method for controlling error discovery rate established by Benjamini and Hochberg; SE, standard error of partial regression coefficients; # TG, triacylglycerol, were subjected to statistical analysis after conversion to normal distribution by natural logarithm; TC, total cholesterol; HDL-C, high density lipoprotein cholesterol; LDL-C, low density lipoprotein cholesterol.

4.4 association of rs189326455 with dyslipidemia: after adjusting age and sex, MAP3K21 gene rs189326455(chr1:233463936, c.162G>C, p.glu54asp) has a significant negative association with hypertriglyceridemia (OR ═ 0.47, 95% CI: 0.33-0.67, P2.75X 10^-5) After multiple tests are corrected by a control False Discovery Rate (FDR) method, the correlation still has statistical significance, and the correlation directions of the boys and the girls are consistent. However, the association of this site with increased TC and decreased HDL-C was not statistically significant after correction of the multiplex assay, and was not found to be associated with increased LDL-C. The results are shown in Table 6.

TABLE 6 Association between MAP3K21rs189326455 and childhood dyslipidemia

Note: adjusting age and gender, wherein font is thickened to indicate that P is less than 0.05, and P is still less than 0.05.OR after multiple tests is corrected by adopting a method for controlling error discovery rate established by Benjamini and Hochberg, and a ratio is obtained; CI, trusted interval. TG, triacylglycerols; TC, total cholesterol; HDL-C, high density lipoprotein cholesterol; LDL-C, low density lipoprotein cholesterol.

Example 5 prediction of protein function of SNP Using the Mutation Taster program

According to the invention, a Mutation Taster program is applied to the positively associated SNP site MAP3K21rs189326455 to predict protein functions, and the Mutation Taster result shows that rs189326455 is pathogenic Mutation, and the specific information is shown in Table 7.

TABLE 7 functional prediction results of MAP3K21rs189326455

Example 6 Generation sequencing validation

Of the above-mentioned effective samples for rs189326455 genotyping, 100 samples (including 40 samples in the obese group and 60 samples in the normal body recombination) were randomly selected for Sanger sequencing. The preparation of genomic DNA was carried out on a patient's peripheral blood sample as in example 2. The concentration was adjusted to 25 ng/. mu.L and then PCR reaction was carried out.

The PCR primer sequences are shown in table 8 below:

TABLE 8 rs189326455 Generation sequencing primer sequences

PCR reaction 25 μ L, containing: 12.5. mu.L of PCR Mix (TaKaRa), 0.5. mu.L of each of the upstream and downstream primers, 0.5. mu.L of genomic DNA (25 ng/. mu.L), 11. mu.L of ddH₂O。

The PCR reaction conditions were as follows: pre-denaturation at 95 ℃ for 3 min; 30s at 94 ℃, 30s at 58 ℃ and 1 minute at 72 ℃ respectively; a total of 32 cycles; final extension at 72 ℃ for 10 min.

PCR reaction products were purified by QIAquick PCR purification kit (Qiagen) and then sequenced by ABI PRISM 3730 automatic sequencer (Applied Biosystems).

Sanger sequencing was performed by the genome research center of Beijing Nonsula, Inc. according to Sanger sequencing techniques well known to those skilled in the art. Compared with the Sequenom MassARRAY genotyping method, the consistency rate of the two detection methods is 89%, which indicates that the locus is a true variation, and further verifies that the rs189326455 locus is related to obesity.

Therefore, the inventor proves that rs189326455 can well predict the risk of the common obesity of Chinese children and is closely related to BMI and TG level in serum.

EXAMPLE 7 preparation of SNP kit for early auxiliary diagnosis of childhood obesity

A. The kit comprises the following components:

primer: the primer pair for specifically amplifying the nucleotide sequence including the rs189326455 locus is shown as SEQ ID NO.1 and SEQ ID NO. 2;

other components: buffer for PCR reaction, MgCl₂(25mmol/L), dNTP, Taq enzyme, ddH₂O。

B. Application method

1) Adding a sample to be detected into the components of the kit and then carrying out PCR reaction;

2) and (3) purifying and sequencing a PCR reaction product, and comparing the obtained sequence with a MAP3K21 normal gene sequence to determine whether rs189326455 has mutation.

The kit provided by the invention indicates that the individual child providing the sample has reduced susceptibility to obesity if the existence of the C allele carried by the SNP locus rs189326455 is detected in the sample.

The kit has the value that only peripheral blood is needed, other tissue samples are not needed, SNP is detected through the simplest and most specific primer pair, and then obesity susceptibility is judged in an auxiliary mode through the SNP spectrum, so that the kit is stable, convenient and accurate to detect, and the sensitivity and specificity of obesity diagnosis are greatly improved.

The kit provided by the invention can also be used for the application in the kit for early prediction of hypertriglyceridemia.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Sequence listing

<110> institute of first-capital science

<120> SNP site related to obesity and/or hypertriglyceridemia of children in China and application thereof

<130> 18039

<160> 5

<170> SIPOSequenceListing 1.0

<210> 1

<211> 20

<212> DNA

<213> synthesis

<400> 1

agctagcccg tgatctctgc 20

<210> 2

<211> 20

<212> DNA

<213> synthesis

<400> 2

gatgagctcc ttcagctcca 20

<210> 3

<211> 29

<212> DNA

<213> synthesis

<400> 3

acgttggatg gcgctctatg actacgagg 29

<210> 4

<211> 29

<212> DNA

<213> synthesis

<400> 4

acgttggatg tcctgcgaca gcacctcca 29

<210> 5

<211> 22

<212> DNA

<213> synthesis

<400> 5

gatacgcagg ctcagctcgt cg 22

Claims (3)

1. The application of the reagent for detecting the SNP locus in preparing the early detection product of the general obesity of children in China is characterized in that the SNP locus is rs189326455, specifically chr1:233463936, c.162G > C and p.Glu54Asp, and the evaluation index of the general obesity is the BMI numerical value.

2. The use of claim 1, wherein the mutation at SNP site rs189326455 has a negative association with BMI and/or triacylglycerols in serum at the whole genome level.

3. The application of the reagent for detecting the SNP locus in preparing the hypertriglyceridemia detection product is characterized in that the SNP locus is rs189326455, specifically chr1:233463936, c.162G > C, and p.Glu54Asp.