CN108753945A - With Children in China obesity and/or the relevant SNP site of hypertriglyceridemia and its application - Google Patents

Abstract

The invention discloses application of the SNP site in preparing Children in China obesity and/or hypertriglyceridemia early detection product, the SNP site is rs189326455, specially chr1:233463936, c.162G>C, p.Glu54Asp.Present invention research is with fat relevant gene loci rs189326455 as fat and hypertriglyceridemia early stage biomarker, further to study the genetic molecule mechanism of fat and hypertriglyceridemia, explores fat and hypertriglyceridemia early prevention and treatment drug target and provide new direction.

Description

With Children in China is fat and/or the relevant SNP site of hypertriglyceridemia and its Using

Technical field

The present invention relates to biotechnologys and technical field of medical detection, and in particular to fat and/or high by three with Children in China The relevant SNP site of acyl glycerine mass formed by blood stasis and its application, the SNP site are rs189326455, specially chr1: 233463936, c.162G>C, p.Glu54Asp.

Background technology

Obesity is a kind of common Nutrition and Metabolism disease.With the improvement of modern society's material life condition, diet knot The change of structure, the factors such as the accelerating rhythm of life, and nervous, shortage movement, the quickening that life section gathers, and spirit is tightly , lack the factors such as movement, it is overweight, overweight people is increasing.Existing evidence is shown, fat disorderly with hypertension, diabetes, blood fat Disorderly, metabolic syndrome is closely related, and brings the high risk of angiocardiopathy.Obesity is classified as by WHO in 2002 causes mankind's disease One of the global prime risk factor of disease burden, the prevalence for preventing obesity be also before 21 century 50 years countries in the world face most One of big public health challenge.

Obesity belongs to complex disease, caused by the factors collective effects such as heredity, environment and life-form structure.It causes fat now In fat environment, neurological susceptibility of the genetic determination individual to environmental change.Pedigree analysis and twin study show that inherent cause exists Effect during obesity occurs accounts for about 40%-70%.Single nucleotide polymorphism (single nucleotide polymorphism, SNP it is) that the molecule that the human genome research center scholar Lander by Massachusetts Institute Technology in 1996 is proposed is lost Label is passed, DNA sequence polymorphism caused by a single nucleotide variation in genomic level is primarily referred to as.SNP is only related to To the variation of single base, there are the different forms such as conversion, transversion, insertion and missing.SNP is third generation genetic marker, and human body is permitted More phenotypic differences, all may be related with SNP to neurological susceptibility of drug or disease etc..Genome-wide association study (GWAS) exactly with SNP is the available strategy of the research complex disease genetic predisposition of genetic marker.Up to the present, it has been sent out using GWAS strategies The susceptibility loci of a large amount of obesities is showed.For example, FTO genes are located at No. 16 chromosome (16q12.2), containing aobvious outside 9 Son, mrna length 410.50kb, wide expression is in each stage of development of tissue, and in hypothalamus, skeletal muscle and fat High expression in equal tissues.It is that GWAS has found and fat associated first gene of general population.2007, Frayling TM Deng the initial association study for carrying out FTO and diabetes B when, after controlling BMI, contacting between FTO and diabetes B disappears It loses, to be surprised to find that FTO genes are related to obesity.Hereafter, FTO genetic mutations and BMI and obesity are associated with successively not It is repeated in agnate crowd, wherein being the best polymorphic site of repeatability positioned at the rs9939609 of the 1st introne.FTO Gene pleiomorphism is proved related with the fat or genetic predisposition of diabetes B successively.

Although pair being gradually increased with the genes of fat and fat relevant trait associations and the understanding of gene loci, clinic Above it has not been found that useful hereditary variation, which can be used as, is directed to having for fat individual especially children's progress early screening and intervention Imitate product.Nearest research is even drawn a conclusion, and the common SNP in one group of fat relevant candidate gene is in regulation and control by certain During the weight of diet induction changes, smaller effect is only played if playing a role.

In view of there is presently no early stage biomarker related to obesity and early diagnosis kits, as can finding corresponding Marker and the corresponding diagnostic kit of development will provide new direction to explore the drug target of fat early prevention and treatment, and be Its drug screening, evaluating drug effect and targeted therapy open up new approach.

Invention content

It is a primary object of the present invention to find one kind and Children in China obesity phase using high-throughput exon sequencing technologies Application of the biomarker and the offer biomarker of pass in preparing Children in China obesity early detection product.

The purpose of the present invention is what is realized by following technical proposal：

This research is designed using two benches case-control study：First stage is pilot study, selects one group of obesity youngster Virgin and one group of normal type children are that control carries out full-length genome exon sequencing.In sequencing data select exon region and The functional variants in montage region, including non-synonymous variation (nonsynonymous), nonsense make a variation (stop gain), termination is close Code variation (stop loss), base insertion/deletion (insertion/deletion), splice site (splice site) carry out Association analysis, filtering out 48 may (P related to obesity<0.05) variation.Second stage is confirmatory study, will be filtered out Variant sites carry out Genotyping in 2,045 Obese childrens and 3,338 non-prevalence of overweight children, these variations of analysis verification With being associated with for children obesity and relevant evaluation index.It is final to confirm that the sites rs189326455 are fat and high by three with Children in China The associated gene loci of acyl glycerine mass formed by blood stasis.

MAP3K21 (3 kinases 21 of mitrogen-activated protein, mitogen-activatedprotein kinase kinase Kinase 21) also known as mixing lineage kinase 4 (MLK4), gene is located on No. 1 chromosome, and it is protein coding gene.Mix pedigree Kinases (MLKs) belongs to the superfamily of the kinases (MAP3Ks) of map kinase kinases.MLK family members are characterized in that its catalysis knot There are the characteristic sequences of Ser/Thr and Tyr kinases in structure domain.MLK1-4 contains N-terminal Src homologys (SH3) structural domain, followed by Kinase domain, leucine zipper region and Cdc42/Rac interaction primitives (CRIB).MLK3 is the widest MLK family of expression Family member, N-terminal has high homology in MLK1-4, and C-terminal is had nothing in common with each other, it is meant that these regions may execute different Regulatory function.At present for MLK4 researchs mainly in MLK4 gene mutations and colon cancer Prognostic significance and MLK4 as NF- κ B The upstream regulatory factor of signal transduction, to activation-inducing glioma stem cells (GSCs) mesenchyma (MEs) of NF- κ B across differentiation And radioresistance.

Based on this, present invention firstly provides application of the SNP site in preparing Children in China obesity early detection product, The SNP site is rs189326455, specially chr1:233463936, c.162G>C, p.Glu54Asp.

Preferably, the evaluation index of the obesity includes three in BMI, FMI, FFMI, FMP, WC, WHtR numerical value and serum Acyl glycerine, total cholesterol, high-density lipoprotein cholesterol, the content of low density lipoprotein cholesterol.

Preferably, in the mutation of the SNP site rs189326455 and BMI and/or serum triacylglycerol in full-length genome Level has negative incidence.

Preferably, described fat including general fat, body fat obesity and Central obesity.

Preferably, described fat for general obesity.

Preferably, the general fat evaluation index is BMI.

Preferably, the serum TG >=0.85mmol/L (< 10 years old) or 1.02mmol/L (10~18 years old) are defined as high by three Acyl glycerine mass formed by blood stasis.

Further, the application the present invention provides SNP site in preparing hypertriglyceridemia detection product, it is described SNP site is rs189326455, specially chr1:233463936, c.162G>C, p.Glu54Asp.

Further, the present invention also provides a kind of general fat early detection reagent of children, the reagent is for examining Survey the genotype of susceptible SNP site rs189326455.

Preferably, the primer such as SEQ ID of nucleotide sequence of the reagent including the sites amplification rs189326455 NO.3~5 or NO.1~2 SEQ ID.

Further, the present invention provides a kind of general fat early stage auxiliary detection kits of children comprising inspection The reagent of the genotype of SNP site rs189326455 in test sample sheet；The reagent is including the sites amplification rs189326455 Nucleotide sequence primer such as NO.1~2 SEQ ID.

Preferably, software may be used to design in the primer, such as uses Primer5, Oligo6.

Preferably, the kit can be that the reagent of SNP is detected using any technology known in the art, as long as its energy It enough detects SNP site rs189326455 in sample and whether there is allelic mutation.Its include but not limited to be set forth below it is each Embodiment.

In the first embodiment, kit of the present invention detects SNP in sample including the use of PCR sequencing PCR Reagent existing for the C allele of the sites rs189326455.Using the kit, can directly be measured in sample by PCR sequencing PCR The sequence of SNP site rs189326455 to judge whether it carries the variation of corresponding site allele, and then judges it Fat non-neurological susceptibility.The PCR sequencing PCR is technology known in the field, and required reagent can be commercially available, and this field is general Logical technical staff can be voluntarily selected as needed (referring to the companies such as ABI, Beckman sequenator explanation used in connection with).

In this second embodiment, kit of the present invention detects sample including the use of Taqman probe SNP detection methods The reagent of middle SNP site rs189326455 genotype.Taqman probes wherein used are to be set for SNP site rs189326455 The probe of meter, the probe can use the probe that Reagent Company provides；Software designed, designed, such as PREMIER can also be used The Beacon Designer 7.5 of Biosoft companies.

In the third embodiment, kit of the present invention is to be detected in sample using PCR- Single strand conformation polymorphisms The kit of SNP site rs189326455 genotype.The kit includes for expanding SNP site rs189326455 genes The primer of type, PCR reagent, check sample and detect conformation electrophoresis needed for reagent.The preferably non denatured polyacrylamide of the electrophoresis Amine gel electrophoresis.Check sample includes the homozygous negative control samples of SNP site rs189326455 genotype GG and site At least one of homozygous positive controls of CC may include additionally the check sample of the corresponding heterozygote of heterozygote. It is preferred that including above-mentioned three classes check sample simultaneously.The amplified production of sample to be tested amplified production and check sample electrophoresis simultaneously, than The testing result that corresponding allelic variation whether is carried in sample to be tested can be obtained compared with its electrophoresis result.

The kit further includes reagent needed for the electrophoresis of PCR reagent, check sample and detection conformation.Needed for round pcr Common agents, such as：DNTPs, MgCl₂, distilled water, Taq enzyme etc..The preferred non-denaturing polyacrylamide gel electricity of electrophoresis Swimming.

In the third embodiment, kit of the present invention is anti-using restriction fragment length polymorphism polymerase chain (PCR-RFLP) technology is answered to detect the kit of SNP site rs189326455 genotype in sample.

If the present invention provides the kit and detects that SNP site rs189326455 carries C allele in the sample Presence, then illustrate that the individual obesity-prone for providing the sample is weaker than the individual for not carrying the variation.

Advantageous effect of the present invention：

The present invention using two generation sequencing technologies in Children in China by being found that with Children in China fat relevant SNP Point rs189326455, and by its authenticity of generation sequence verification.The Mutation can directly cause coded amino acid Change, belongs to functional variants.By being associated with for site of analysis variation and children obesity and relevant evaluation index, the site is found It is not only associated with obesity, also has with fat relevant serum triglyceride level and be associated with.The present invention and fat relevant gene loci Fat and hypertriglyceridemia early stage biomarker is can be used as, it is fat and hypertriglyceridemia further to study Genetic molecule mechanism explores fat and hypertriglyceridemia early prevention and treatment drug target and provides new direction.

Specific implementation mode

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

Term is explained as follows in the present invention：

Terms used herein " primer " refer to the oligonucleotides for being present in the restrictive digestion content of purifying or being synthetically produced, When being placed in induction synthesis under conditions of the primer extension product of nucleic acid chains complementation, that is, there is nucleotide and derivant, (such as DNA gathers Synthase) and temperature appropriate and when pH, the oligonucleotides can be as the starting point of synthesis.Primer can be single-stranded or double Chain, and must have enough length, to cause the synthesis of required extension products in the presence of derivant.The definite length of primer Degree can depend on many factors, including temperature, Primer Source and the method used.For example, for diagnostic application, target is depended on The complexity of sequence, Oligonucleolide primers typically contain the nucleotide of 15-25 or more, although can also contain less nucleosides Acid.Factor involved in the determination of primer appropriate length is known to those of ordinary skill in the art.In general, according to ability The primer of standard method design and the selection present invention known to domain, refering to 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. are edited) Cold Spring HarborLaboratoryPress, NewYork, 133-155.

Constitutional index (BMI)：That is body-mass index, abbreviation constitutional index is also known as body mass index, and (English is BodyMass Index, abbreviation BMI), it is as obtained by square calculating of weight (kg) divided by height (m), is common in the world at present weigh The fat or thin degree of human body and whether health a standard.

Fat mass index (FMI)：The weight of people is made of fatty weight and lean body mass (lean body mass).By direct It measures human body component and obtains fat and non-fat content, wherein with square calculating of body fat content (kg) divided by height (m) Gained, i.e. body fat mass index (full name in English Fat Mass Index, abbreviation FMI), more accurately to evaluate human body Obese degree.

Non-fat performance figure (FFMI)：The weight of people is made of fatty weight and lean body mass (lean body mass).And it goes Fatty weight includes muscle, bone, soft tissue and moisture again.Fat is obtained by directly measuring human body component with non-fat to contain Amount, wherein the change of individual non-fat (lean body mass) is mainly the variation of Lean mass, with non-fat content (kg) divided by height (m) square calculating gained, i.e., non-fat performance figure (full name in English Fat-Free Mass Index, abbreviation FFMI), also referred to as Lean body mass volume index (full name in English Lean Mass Index, abbreviation LMI), to evaluate human body Lean mass and nutrient health State.

Body fat percentage (FMP)：Fat and non-fat content are obtained by measuring human body component, are removed with body fat content The percentage obtained with weight, i.e. body fat percentage (English is Fat Mass Percentage, abbreviation FMP), to appraiser Whether body fat and obese degree.

Waistline (WC)：Full name in English Waist Circumference are to reflect that the synthesis of amount of total fat and Fat Distribution refers to Mark, the measurement method of world health organisation recommendations are：Measured stands, and both feet separate 25 to 30 centimetres, and weight evenly distributes.It surveys Position is measured at the midpoint of horizontal position anterior superior iliac spine and the 12nd rib lower edge line.Measuring scale is close to without oppressing skin, normal Expiration final reading, measured value are accurate to 0.1 centimetre.

Waistline height ratio (WHtR)：Full name in English Waist-to-Height Ratio, i.e. number obtained by waistline divided by height Value, to reflect accumulation of the fat in abdomen indirectly.Since WHtR considers height factor, hardly by the shadow at age and gender Ring, thus in children using WHtR >=0.5 (i.e. waistline be more than height more than half) definition Central obesity it is very easy.

It is general fat：Using BMI as evaluation index, using the school-ager of International Obesity problem working group (IOTF) recommendation Teenager is overweight and fat screening BMI dividing values are diagnosed fat, normal and become thin.

Body fat is fat：Using《Chinese School-aged children and adolescents are overweight and obesity prevention with control guide》The standard of recommendation, Using FMP as evaluation index, male's FMP >=20%, women 14 years old and following FMP >=25%, women 15 years old and above FMP >=30% For obesity.

Central obesity：Using WHtR as evaluation index, Central obesity is diagnosed as with WHtR >=0.5.

Dyslipidemia：According to U.S.'s cardiopulmonary and Blood Research Institute (NHLBI) about children and youth cardiovascular health and wind Danger reduces guide, and the raising of serum triglyceride level edge is defined as hypertriglyceridemia, i.e. 10 years old or less children TG >=75mg/ Dl (0.85mmol/L), 10 years old and 10 years old or more children and youth TG >=90mg/dl (1.02mmol/L).Similarly, high cholesterol Mass formed by blood stasis is defined as TC >=170mg/dl (4.41mmol/L)；High low density lipoprotein cholesterolemia be defined as LDL-C >= 110mg/dl(2.85mmol/L)；Low high-density lipoprotein cholesterol is defined as HDL-C < 45mg/dl (1.16mmol/ L)。

Mutation Taster：Mutation Taster score values (version 2.3) indicate the variation to protein sequence Influence, including two groups of data, Score gives the calculating score after each site of submitted amino acid mutates, this point Number is 0~1, and the value the big more " harmful ", shows that the SNP causes protein structure or the possibility of function change big；Predict is Prediction result indicates with A, D, N or P, wherein A：Disease causing automatic disease cause mutations, D：disease Causing may be disease cause mutation, N：Polymorphism is polymorphic may harmless, P：Polymorphism automatic are more State is harmless.

MAF：Full name in English minor allele frequency, i.e., less gene frequency are typically referred to given Uncommon allele occurrence frequency in crowd.MAF is widely used in the genome-wide association study of complex disease.It is closing In connection research, smaller MAF will make the reduction of statistics efficiency, to cause the result of false negative.In order to rare in study population Mutation is associated with disease, usually imitates loss of energy by increasing the method for sample size to make up statistics caused by MAF reductions.

In technical scheme of the present invention：

Exon sequencing experiment is to use Roche/NimbleGen's SeqCap EZ Human Exome Kit v3.0 Exon group liquid phase captures chip and captures full-length genome exon, structure exon group library, in Illumina HiseqTM 2500 high-flux sequence platforms carry out both-end sequencing, it is desirable that sequencing depth reaches 100 times.

Genotyping experiment is that 48 variant sites that will be filtered out are compareed in 2045 fat school-agers and 3338 Genotyping is carried out in children, these variations of analysis verification are associated with children obesity.According to gene loci sequence information, design And PCR reactions are synthesized with single base amplimer, the genotyping techniques using Sequenom MassARRAY high throughputs are complete At Genotyping.

Blood lipids index of the present invention measures：It is existing in 12 hours peripheric venous blood 5ml of early morning acquisition research object empty stomach Serum (non-anticoagulant tube) is isolated in field, is used for biochemistry detection；Unified purchase reagent, is surveyed using 7020 type full automatic biochemical apparatus of Hitachi Determine blood biochemistry index.Serum total cholesterol (total cholesterol, TC) and triacylglycerol (triacylglycerol, TG) Using standard enzymatic assays, high-density lipoprotein cholesterol (high density lipoprotein cholesterol, HDL- C it) is surveyed using removing method with low density lipoprotein cholesterol (low density lipoprotein cholesterol, LDL-C) It is fixed.

The present invention is mainly to be carried out using PLINK 1.90, SPSS 22.0 and R 3.4.3 softwares using statistical analysis technique Statistical analysis, analysis method include χ²Inspection, conditional QTL analysis and multiple linear regression analysis.The quantity such as BMI, TG Character and variant sites are associated with regression coefficient (β) and standard error (SE) expression, fat and metabolic risk and variant sites Association odds ratio (OR) and 95% credibility interval (CI) indicate that association analysis uses additive inheritance model.Using control mistake Discovery rate (false discovery rate, FDR) method corrects multiple check.

The arrangement of the collection and sample data of 1 sample of embodiment

Inventor has collected a large amount of obesity in April, 2004 in December, 2014 in Beijing and In Tung-hua, Jilin middle and primary schools Case and control children's blood specimen, by the arrangement to sample data, inventor therefrom select to meet the sample of following standard into Row full-length genome exon is sequenced and the laboratory sample of single SNP Sequenom MassARRAY Genotypings：

1, the obesity that constitutional index (BMI) criteria for classification recommended through International Obesity problem working group (IOTF) is clarified a diagnosis Case；

2, Han nationality children, age 6-18 Sui；

3, control includes normal type and the children that become thin, and excludes prevalence of overweight children.

And situations such as demographic data and clinical data of system acquisition these samples.

2 peripheral blood DNA of embodiment extracts

In the blood sample of above-mentioned qualified Obese children and normal type children, using centrifugal column method, (DNA is a small amount of Extracts kit, QIAGEN companies, Germany) extraction human genome DNA.The specific steps are：

1,20 μ L Proteinase Ks are added to 1.5mL centrifuge tubes；

2,200 μ L leucocyte mixed liquors are added, if fruit volume is less than 200 μ L, with phosphate buffered saline solution (phosphate Buffer saline, PBS) it supplies；

3,200 μ L buffer AL (lysate), vortex oscillation 15 seconds, rapid centrifugation is added；

4,56 DEG C of water-baths 10 minutes；

5, rapid centrifugation, avoiding pipe from covering, there are solution；

6,200 μ L absolute ethyl alcohols, vortex oscillation 15 seconds, rapid centrifugation is added；

7, all solution are transferred in QIAamp centrifugal columns, 10000rpm is centrifuged 2 minutes, collecting pipe is abandoned, by centrifugal column New collecting pipe (kit offer) is provided；

8,500 μ L bufferAW1 (cleaning solution 1) are added on centrifugal column, 10000rpm is centrifuged 2 minutes, abandons collecting pipe, Centrifugal column is transferred to new collecting pipe (kit offer)；

9,500 μ L bufferAW2 (cleaning solution 2) are added on centrifugal column, 10000rpm is centrifuged 5 minutes, abandons collecting pipe, Centrifugal column is transferred to new collecting pipe (kit does not provide, and can be replaced with 1.5mL centrifuge tubes), 12000rpm centrifuges 3 points Clock；

10, centrifugal column is transferred to new 1.5mL Eppendorf pipes, 200 μ L bufferAE are added on centrifugal column (DNA lysates) is placed at room temperature for 5 minutes, and 13000rpm is centrifuged 3 minutes；

11, centrifugal column, lid upper tube cap are discarded.- 20 DEG C save backup.

The full exon group detection of SNP in 3 peripheral blood DNA of embodiment

The blood sample DNA of 96 Obese childrens in embodiment 2 and 96 normal type children is carried out outside full-length genome Aobvious son sequencing, compares two groups of children's exon sequencing datas and obtains candidate gene site.The specific steps are：

1, DNA quality inspections：Using ultraviolet/visible spectrophotometer (DU800, Beckman, the U.S.) or (NanoDrop 2000, Sai Mo fly generation you, the U.S.), read A230nm, A260nm, A280nm absorbance value, be calculated by formula DNA concentration (g/L)= A260nm × 50, and pass through OD ratios (A260/A280, A260/A230) identification of dna purity.The low prompt of A260/A280 ratios has Protein residues, but if having used phenol in operating process, then it is more likely that phenol remains, A260/A280 ratios are higher to be carried Showing may be since ribonucleic acid (ribonucleic acid, RNA) be clean without removal.The low prompt remaining of A260/A230 ratios Salt or small molecular weight impurity pollution, it is excessively high centainly to have unknown impurity.

On the preliminary quantitative basis of DNA, further agarose gel electrophoresis is used to detect (gum concentration：0.8%, voltage： 120V, time：20min) accurate quantification DNA.Occur illustrating that DNA there may be drop there are miscellaneous band except weak band or master tape after electrophoresis It solves phenomenon or there are impurity, if occurring a strong band after electrophoresis, illustrate that the DNA mass of extraction is higher.Exon sequencing is wanted Seek DNA concentration >=50ng/ μ L, total amount not less than 5 μ g, OD260/280 between 1.8~2.0, OD260/230 2.0 or so, Sample is polluted without RNA, no degradation or slight degradation.Sequenom SNP Genotypings require DNA concentration to be more than 25ng/ μ L, DNA volumes are more than 10 μ L, and between purity requirement OD 260/280 is 1.7~2.0, agarose electrophoresis must have bright more than 10kb Single band, no RNA and protein contamination.

2, exon trapping：Using Roche/NimbleGen's SeqCap EZ Human Exome Kit v3.0 liquid phases Exon trapping technology carries out hybrid capture, 85% or more average capture efficiency.Its substantially flow be first by genomic DNA with Machine is broken into the segment of 150-220bp or so, and being then separately connected top connection at segment both ends prepares Hybrid Library.Library is through pure It carries out hybridizing enrichment with Biotinylated DNA Library by the linear amplification of LM-PCR after change, using LM-PCR's Linear amplification, be available on the machine after library detection qualification sequencing (Hiseq2500 sequenators).

3, exon group library construction：To the DNA sample of quality inspection qualification, using the DNA true-seq of Illumina standards Library development flow builds exon group library.Library construction process description is as follows：

(1) 5 μ g genomic DNAs are taken, random mechanization is done with Bioruptor in appropriate system and is interrupted, segment master tape is made Near 200bp, gel extraction 150-250bp segments；

(2) end reparation is done to DNA fragmentation and 3 ' ends adds A tails；

(3) connection sequence measuring joints (adapter), connection product carry out PCR (polymerase after purification Chain reaction, PCR) amplification, amplified production purifying is pre- library；

(4) probe other than a certain amount of pre- library in aobvious subgroup capture agent box is taken to do hybrid capture.According to NimbleGen The flow of capture chip is captured.Hybrid product does PCR amplification after elution is recycled, and product recycling is whole library, agar Sugared gel electrophoresis is run sample and is confirmed；

(5) final quality inspection is carried out to library using the method for quantitative PCR, to judge library insert size and The ultimate density in the upper preceding library of machine, qualified library arrange upper machine sequencing.

4, sequencing of extron group：It is high-throughput using Illumina HiseqTM 2500 to the exon group library built Sequenator carries out double ends (pair-end) sequencings, and sequencing pattern is 150PE, sequencing reagent V3, sequencing depth for 100 ×. Illumina Hiseq2500 sequencing systems are a kind of high throughput sequencing technologies, and sequencing principle is closed using the side of reversible cessation method (sequencing by synthesis, SBS) technology is sequenced at side.

5, data analysis and processing：Quality control is carried out to full sequencing of extron group data：1. sample recall rate>95%, Reject the sample that genotype miss rate is more than 5%；2. site recall rate>95%, reject the position that genotype miss rate is more than 5% Point；③MAF>0.01；4. control group should meet Hardy-Weinberg equilibrium (P>1.0E-6).It is carried out using the data of Quality Control qualification Association analysis finds the genotype distribution frequency SNP that there were significant differences, wherein SNP site in fat case group and control group The results are shown in Table 1 for the association analysis of MAP3K21rs189326455 exons.

1 exon of table is sequenced the frequency of mutation in the sites MAP3K21rs189326455 in sample and is associated with fat

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/mutation allele；F_case：Equipotential is mutated in case group Gene frequency；F_control：Mutation allele frequency in control group.

The Sequenom MassARRAY Genotypings of 4 single SNP of embodiment

Above-mentioned qualified 2045 fat school-agers and 3338 controls (2840 normal type children and 498 children that become thin) in, above-mentioned full exon sequencing is found with fat 48 significantly correlated SNP in Sequenom It is detected on MassARRAY Genotyping platforms, the specific steps are：

1, Sequenom MassARRAY Genotypings are carried out.To the discovery of full sequencing of extron group and obesity related 48 A SNP design specificity amplification primers and specific extension primer；The system of amplified reaction includes：0.625μL PCRbuffer (10*), 0.1 μ L dNTP mix (25mM), 0.325 μ L MgCl₂(25mM), 0.2L HotStar Taq (5U/ μ L), Mei Duizheng To with the 1 μ L (0.5uM) of mixture of reversed amplimer and 1.75 μ L distilled waters, the DNA sample that 1 μ L are added carries out PCR amplification Reaction.The system of extension includes：2 μ L of EXTENDMix liquid (wherein each 0.94 μ L, iPLEX enzymes of extension primer mixture 0.041 μ L extend 0.2 μ L of mixture).It is added at SAP (shrimp alkaline phosphatase, shrimp alkaline phosphotase) 9 μ L of PCR product after reason carry out single base extension.The instrument used is ABI9700 type PCR instruments.The product of purifying 4000rpm is centrifuged 4 minutes, and 384 are transferred to using MassARRAY Nanodispenser RS1000 point sample instruments after precipitated resin On hole SpectroCHIP (Sequenom) chip, MALDI-TOF mass spectral analyses are carried out.Wherein, MAP3K21 genes The SNP design specificity amplification primers of rs189326455 and specific extension primer are as shown in table 2.

Primer sequence in 2 sites MAP3K21rs189326455 mass spectrography of table

2, genotype interpretation：It is carried out using TYPER4.0 softwares (sequenom).

3, data processing and analysis：Following quality control is carried out to genotype data：1. sample recall rate>90%, it picks Except genotype miss rate is more than 10% sample；2. site recall rate>90%, reject the position that genotype miss rate is more than 10% Point；3. Detection accuracy>99%；④MAF>0.01；5. control group should meet Hardy-Weinberg equilibrium (P>0.001).

After data Quality Control, the difference of three kinds of genotype distribution frequencies in case group and control group of more each SNP It is different, and be associated with fat and correlated traits using genetic model (additive model) analysis SNP is added.Using multifactor Logistic analysis of regression model site is associated with children's difference obesity phenotype, strength of association with odds ratio (odds ratio, OR it) is indicated with 95% credibility interval (confidence interval, CI).Using multiple linear regression model site of analysis with Fat correlated quantitative traits constitutional index (BMI), fat mass index (FMI), non-fat performance figure (FFMI), body fat percentage Than the association of (FMP), waistline (WC) and waistline height ratio (WHtR), often increased with partial regression coefficient (β) and standard error (SE) expression The unit value for adding BMI, FMI, FFMI, FMP, WC and WHtR caused by an allele to change.Similarly, SNP site and blood fat The association analysis of index quantitative character uses multiple linear regression model, and SNP site and dyslipidemia qualitative character are associated with point Analysis uses multivariate logistic regression analysis model.Age and gender are adjusted as covariant.When case or control number are small When 5, examined using Fisher exact probabilities.

4, result

4.1 rs189326455 are associated with obesity phenotype：After adjusting age and gender, it is located at No. 1 chromosome The non-synonymous coding variation rs189326455 (chr1 of MAP3K21 genes:233463936, c.162G>C, p.Glu54Asp) with General fat association reaches full-length genome significance, and OR is 0.26 (95%CI：0.17-0.42, P=9.605 × 10^-9), being associated with after control false discovery rate (FDR) method correction multiple check still has statistical significance, and man, young girl's association Direction is consistent.But the site does not have statistical significance after being associated in correction multiple check with body fat obesity, with Central obesity Have no association.It the results are shown in Table 3.

3 MAP3K21rs189326455 of table is associated with children's difference obesity phenotype

Note：Age and gender are adjusted, font-weight indicates P<0.05, the * control established using Benjamini and Hochberg Still P after false discovery rate method correction multiple check processed<0.05.OR, odds ratio；CI, credibility interval.

4.2 rs189326455 are associated with assessment of adiposity index：After adjusting age and gender, MAP3K21 genes rs189326455(chr1:233463936, c.162G>C, p.Glu54Asp) and BMI (β=- 2.22, P=8.916 × 10^-8) Have negative incidence, men and women consistent in full-length genome level；After FDR methods correct multiple check, in total crowd and boy Middle association is still statistically significant, but is associated with and disappears in young girl.Though rs189326455 and FMI, FMP have negative incidence trend, It is associated with after multiple testing adjustment and disappears.Rs189326455 has no with FFMI, WC, WHtR and is associated with.It the results are shown in Table 4.

Association between 4 MAP3K21rs189326455 of table and different assessment of adiposity indexs

Note：Age and gender are adjusted, font-weight indicates P<0.05, the * control established using Benjamini and Hochberg Still P after false discovery rate method correction multiple check processed<0.05. β, partial regression coefficient；SE, the standard error of partial regression coefficient； BMI, constitutional index；FMI, fat mass index；FFMI, non-fat performance figure；FMP, body fat percentage；WC, waistline； WHtR, waistline height ratio.

4.3 rs189326455 are associated with fat relevant blood lipids index：After adjusting age and gender, MAP3K21 bases Because of rs189326455 (chr1:233463936, c.162G>C, p.Glu54Asp) with the natural logrithm value (β of serum triglyceride level =-0.25, P=5.990 × 10^-11) in full-length genome level there is negative incidence, men and women is consistent；It is corrected through FDR methods multiple After inspection, it is associated in total crowd and man, young girl still statistically significant.Rs189326455 and TC is in negative incidence, through more It is associated with and disappears after examining correction again, in boy, but do not reach the full-length genome level of signifiance.After multiple testing adjustment, Rs189326455 has no with HDL-C, LDL-C and is associated with.It the results are shown in Table 5.

Being associated between 5 MAP3K21rs189326455 of table and blood lipids index

Note：Age and gender are adjusted, font-weight indicates P<0.05, the * control established using Benjamini and Hochberg Still P after false discovery rate method correction multiple check processed<0.05. β, partial regression coefficient；SE, the standard error of partial regression coefficient；# TG, triacylglycerol enter statistical analysis after natural logrithm is converted to normal distribution；TC, total cholesterol；HDL-C, high density fat Protein cholesterol；LDL-C, low density lipoprotein cholesterol.

4.4 rs189326455 are associated with dyslipidemia：After adjusting age and gender, MAP3K21 genes rs189326455(chr1:233463936, c.162G>C, p.Glu54Asp) with hypertriglyceridemia have notable negative customers (OR=0.47,95%CI：0.33-0.67, P=2.75 × 10^-5), correct multiple inspection through controlling false discovery rate (FDR) method Be associated with after testing still have statistical significance, and man, young girl's relating heading it is consistent.But the site is increased with TC, HDL-C is reduced Be associated in correction multiple check after do not have statistical significance, have no and be associated with LDL-C raisings.It the results are shown in Table 6.

Being associated between 6 MAP3K21rs189326455 of table and children's dyslipidemia

Note：Age and gender are adjusted, font-weight indicates P<0.05, the * control established using Benjamini and Hochberg Still P after false discovery rate method correction multiple check processed<0.05.OR, odds ratio；CI, credibility interval.TG, triacylglycerol； TC, total cholesterol；HDL-C, high-density lipoprotein cholesterol；LDL-C, low density lipoprotein cholesterol.

The protein function of the application Mutation Taster programs prediction of embodiment 5 SNP

The present invention to the SNP site MAP3K21rs189326455 of positive association, using Mutation Taster programs into Row protein function prediction, Mutation Taster results show that rs189326455 is pathogenicity variation, and specifying information is referring to table 7。

The function prediction result of 7 MAP3K21rs189326455 of table

6 generation sequence verification of embodiment

In the effective sample of above-mentioned completion rs189326455 Genotypings, 100 (including fat groups 40 are randomly selected Example, regular recombinate 60) carry out Sanger sequencings.Genome is carried out to the peripheral blood sample of patient as described in Example 2 The preparation of DNA.PCR reactions are carried out after concentration is adjusted to 25ng/ μ L.

PCR primer sequence is as shown in table 8 below：

8 rs189326455 generation sequencing primer sequences of table

25 μ L of PCR reaction systems, including：12.5 μ L PCR Mix (TaKaRa), each 0.5 μ L of upstream and downstream primer, 0.5 μ L bases Because of a group DNA (25ng/ μ L), 11 μ LddH₂O。

PCR reaction conditions are as follows：95 DEG C of pre-degeneration 3 minutes；94 DEG C of 30s, 58 DEG C of 30s, 72 DEG C it is 1 minute each；Totally 32 are followed Ring；Last 72 DEG C extend 10 minutes.

PCR reaction products are carried out by QIAquick PCR purification kits (Qiagen) after purification by ABI PRISM 3730 Automatic sequencer (Applied Biosystems) is sequenced.

Sanger sequencings transfer to Sinogenomax Co., Ltd. according to known to those skilled in the art Sanger sequencing technologies complete.Compared with aforementioned Sequenom MassARRAY methods of genotyping, two kinds of detection methods Concordance rate be 89%, prompt the site be necessary being variation, further demonstrate the sites rs189326455 with obesity phase It closes.

Therefore, inventors demonstrated that rs189326455 can forecast China children be general fat well suffers from The horizontal close associations of TG in risk, with BMI and serum.

Embodiment 7 is used for the making of children obesity early stage auxiliary diagnosis SNP kits

A, kit forms：

Primer：The primer pair of nucleotide sequence including the sites specific amplified rs189326455 such as SEQ ID NO:1 He SEQ ID NO:Shown in 2；

Other compositions：PCR reaction buffers, MgCl₂(25mmol/L), dNTP, Taq enzyme, ddH₂O。

B, application method

1) PCR reactions are carried out after sample to be tested is added in the constituent of kit；

2) sequencing, obtained sequence and MAP3K21 normal gene sequences is compared, really after PCR reaction products are purified Rs189326455 is determined with the presence or absence of mutation.

If the present invention provides the kit and detects that SNP site rs189326455 carries C allele in the sample Presence, then the individual children obesity neurological susceptibility for illustrating to provide the sample weakens.

The value of this kit is only to need peripheral blood without other tissue samples, by most simplifying and special Primer pair detects SNP, then composes auxiliary judgment obesity-prone by SNP, not only stablizes, easy to detect and accurate, greatly improves The sensibility and specificity that obesity diagnoses, therefore this kit is put into and is put into practice, it can help to instruct diagnosis and more effective Bodyization is treated.

The kit of the present invention can be used for the purposes in early prediction hypertriglyceridemia kit.

Although above the present invention is described in detail with a general description of the specific embodiments, On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause This, these modifications or improvements, belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.

Sequence table

<110>Shoudu Inst. of Pediatrics

<120>With Children in China obesity and/or the relevant SNP site of hypertriglyceridemia and its application

<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 (10)

1. Application of the sites 1.SNP in preparing Children in China obesity early detection product, the SNP site are rs189326455, Specially chr1:233463936, c.162G>C, p.Glu54Asp.
2. 2. as described in claim 1 application, which is characterized in that the evaluation index of the obesity include BMI, FMI, FFMI, FMP, Triacylglycerol, total cholesterol, high-density lipoprotein cholesterol, low density lipoprotein cholesterol in WC, WHtR numerical value and serum Content.
3. 3. application as described in claim 1, which is characterized in that the mutation of the SNP site rs189326455 and BMI and/or blood Triacylglycerol has negative incidence in full-length genome level in clear.
4. 4. application as described in claim 1, which is characterized in that the obesity includes that generality is fat, body fat is fat and abdomen type is fertile It is fat.
5. 5. application as claimed in claim 4, which is characterized in that the obesity is general fat；Described general fat comments Valence index is BMI.
6. Application of the sites 6.SNP in preparing hypertriglyceridemia detection product, the SNP site are rs189326455, tool Body is chr1:233463936, c.162G>C, p.Glu54Asp.
7. 7. a kind of general fat early detection reagent of children, which is characterized in that the reagent is for detecting SNP site The genotype of rs189326455.
8. 8. reagent as claimed in claim 7, which is characterized in that the reagent is including the sites amplification rs189326455 The primer of nucleotide sequence such as SEQIDNO.3~5 or SEQIDNO.1~2.
9. 9. a kind of general fat early stage auxiliary detection kit of children, which is characterized in that it includes SNP site in detection sample The reagent of the genotype of rs189326455；Nucleotide sequence of the reagent including the sites amplification rs189326455 draws Object such as SEQIDNO.1~2.
10. 10. kit as claimed in claim 9, which is characterized in that the kit is including the use of PCR sequencing PCR, Taqman probes Method, restriction fragment length polymorphism polymerase chain reaction (PCR-RFLP) technology or PCR- Single strand conformation polymorphisms detect sample The genotype of SNP site rs189326455 in this.