CN109055546A - Predict the method and DNA methylation marker of weightless bone loss cortisol Cortisol Upside Risk - Google Patents
Predict the method and DNA methylation marker of weightless bone loss cortisol Cortisol Upside Risk Download PDFInfo
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/154—Methylation markers
Abstract
The present invention provides the DNA methylation marker for predicting weightless bone loss cortisol Cortisol Upside Risk, including following one or more genes, gene loci annotation information are shown in Table 3.The present invention also provides corresponding detection methods.The present invention passes through haemocyte DNA methylation level and serum bone density index (cortisol, Cortisol excavation), it was found that horizontal by detection haemocyte DNA methylation, it can be realized the prediction for rising individual difference to human body cortisol after weightlessness, to which screening is more resistant to the individual of below-G conditions, or for carrying out scientific research to cortisol situation of change.It is higher with the correlation of human body bone metabolism the present invention provides the DNA methylation site for having forecast function to human body cortisol Upside Risk after weightlessness set.
Description
Technical field
The invention belongs to physiology technical fields, and the physiological change after being related to weightlessness condition for a period of time specifically relates to
And predict the method and DNA methylation marker of weightless bone loss cortisol Cortisol Upside Risk.
Background technique
Cortisol (cortisol) a kind of glucocorticoid as secreted by adrenal cortex zona fasciculata, in blood with knot
Two kinds of forms of state and free state are closed to exist.Cortisol joins between manipulation mood and health, immunocyte and inflammation, blood vessel and blood pressure
System, and maintenance connective tissue (such as bone, muscle and skin) etc. have effects that it is especially important, to the substance of body
The physiological function of metabolism, immune function and a variety of organs has a very important role.Existing research show cortisol levels with
There are correlations for calcium variation, prompt cortisol that may play a role in the decalcification caused by short-term weightlessness.Cortisol is on the one hand
The secretion and intestines calcium uptake, influence sexual gland and growth hormone secretion axis etc. that PTH can be adjusted by indirectly-acting influence bone metabolism；
On the other hand, many cell factors such as interleukin (IL-1), osteoprotegerin (OPG), insulin-like growth factor can be inhibited
(IGF), the formation of tumor necrosis factor (TNF), hepatocyte growth factor etc. stimulates the original marrow stromal cell of people's osteogenic
The expression of RANK-Ligand promotes bone resorption by the recruitment that above-mentioned effect adjusts osteoclast, inhibits bon e formation, in turn
It interferes bone metabolism, reduce bone amount, lead to osteoporosis.
Weightlessness bone loss is a kind of long-term, lasting progressive variation, and the result of development will lead to osteoporosis, soft group
The generation of the pathological phenomenons such as calcification, kidney stone is knitted, or even gravity occurs and adapts to obstacle again, so that the body for seriously affecting spacefarer is strong
Health and working efficiency.Existing means are difficult to prevent and delay space bone loss.In micro-gravity conditions, the rate of bone loss is super
10 times for crossing the monthly bone density mass loss rates of postmenopausal women on ground.The measuring study of bone density shows the sky at 1-6 months
Between be resident during spacefarer's bone loss amount between 0.9%-1.8%, this ratio because test position difference due to it is different, at present it is general
All over think the amount lost of sclerotin and rock salt monthly be 1%-2%.Weightlessness bone loss is bone resorption increase and bon e formation
Reduce the result of comprehensive function.Weightlessness bone loss is the various spies brought by weightless and space travel because of environment
Under the conditions of, the result that interacts between osteocyte, osteoblast and osteoclast.The reduction of function of osteoblast and osteoclastic thin
The raising of cytoactive plays a key role in the bone loss caused by weightlessness.The raising of cortisol levels means to lose in serum
The raising of weight or simulated weightlessness lower body osteoclast activity and bone resorption ability.
To sum up, under weightlessness/simulated weightlessness conditions, human serum cortisol levels are increased and human body osteoclast activity and bone
The raising of absorbability is in close relations.In consideration of it, after being subjected to same weightlessness/simulated weightlessness conditions and influencing, Human Cortex's alcohol
The difference of rising level can also reflect that human body leads to the individual difference of bone loss tolerance degree to below-G conditions.Towards manned space flight
Task, as can the cortisol Upside Risk before execution task to occupant or subject under simulated weightlessness conditions is predicted,
Optimization occupant is selected, provides early stage health protection intervention for occupant and to weightless bone loss cortisol Cortisol updraught
Danger is studied, and all has significant application value.
Summary of the invention
In order to solve the problems in the existing technology, Head down tilt bed rest human experimentation model, sieve are spent in use -6 of the present invention
Select and identify can predict weightlessness after human body serum cortisol Upside Risk epigenetics index set.
The present invention provides the DNA methylation marker for predicting weightless bone loss cortisol Cortisol Upside Risk, including
Following one or more genes, gene loci annotation information are shown in Table 3.
These are identified by gene symbol (the 11st column) and at least one chromosomal foci (the 8th, 9,10 column) in table
The preferred methylated nucleotide position of gene.These genes are further identified by probe id (column 1), special in these genes
It is not the identification site CpG in their controlling element (in chromosomal foci).
Hereditary bibliography herein is typically referenced to genome human hg38.In table, probe id refers in array
The sequence indicated on platform, each for inquiring the specific site CpG, chromosome (chr), start site (start) and
End locus (end) can uniquely identify first position nt of each probe.
The present invention provides the method for predicting weightless bone loss cortisol Cortisol Upside Risk, and the method is not with disease
Diagnosing and treating for the purpose of, include the following steps: determine Samples subjects in cortisol risk genes site DNA methylation
Level, and the methylation level of the gene loci is compared with control sample, to predict subject in below-G conditions skin
Matter alcohol Cortisol Upside Risk；
Wherein one or more genes of the cortisol risk genes in claim 1；
The control sample is selected from the sample of below-G conditions hypopallium alcohol Cortisol decline；
The method of the determining DNA methylation level can be determining by any method known in the art, the method
Methylating including full-length genome, screening is analyzed, the methylation profiles of chip platform are analyzed, methylation status of PTEN promoter analysis, flight
The analysis of Mass Spectrometer Method (Sequenome), sulphite treated gene order-checking, joint bisulfites restriction enzyme
Analysis, methylation-specific endonuclease digestion and quantitative polyase chain reaction Conjoint Analysis, methylation sensitive high-resolution are molten
Solution curve analysis or pyrosequencing determination method.
The DNA methylation assay reagent that the present invention provides gene described in claim 1 predicts weightless bone loss cortex in preparation
Application in alcohol Cortisol Upside Risk diagnostic products, the diagnostic products include one or more in detection claim 1
The DNA methylation assay reagent of gene, the methylation that the reagent passes through gene described in claim 1 in detection Samples subjects
Level, to judge whether subject has Upside Risk in below-G conditions hypopallium alcohol；
Preferably, the reagent is to methylate to screen the methylation figure of analysis, chip platform based on full-length genome
Spectrum analysis, methylation status of PTEN promoter analysis, flight mass spectrum detection (sequenome), sulphite treated gene order-checking
Analysis, joint bisulfites restriction endonuclease analysis, methylation-specific endonuclease digestion and quantitative polyase chain reaction
Conjoint Analysis, methylation sensitive high-resolution melting curve analysis or pyrosequencing test and analyze required reagent.
As another preferred embodiment, the methylation level of the gene is in the upstream of the open reading frame of marker gene
What region, especially promoter region determined；Or one of following:
(a) nucleic acid defined in the chromosomal foci as shown in claim 1；
(b) comprising the site CpG of nucleic acid a；
(c) nucleic acid (a) being no more than with above-mentioned nucleic acid a distance in 1000 nucleotide.
As another preferred embodiment, the reagent including specific amplification include methylation sites including claim 1
Described in gene primer；
As further optimisation, the diagnostic products are kit, chip or microarray dataset.
The present invention provides one group of nucleic acid primer and/or hybridization probe, to one or more gene described in claim 1
Potential methylation region be specific；
Preferably, the primer and/or hybridization probe are specific for amplification marker gene open reading frame
Methylate upstream region, especially promoter region；Or methylation is one of following:
(a) nucleic acid defined in the chromosomal foci as shown in claim 1；
(b) comprising the site CpG of nucleic acid a；
(c) nucleic acid (a) being no more than with above-mentioned nucleic acid a distance in 1000 nucleotide.
Primer and/or probe are used to target a potential first in the DNA molecular of the one or more genes selected in table 3
Base region.
The present invention provides a kind of kit, and the kit includes one group of nucleic acid primer as claimed in claim 6 or hybridization
Probe further includes methylation-specific restriction enzyme and/or takes off the reagent of amine for bisulfites nucleotide.
The present invention provides a kind of diagnostic products for predicting weightless bone loss cortisol Cortisol Upside Risk, the diagnosis
Product includes the reagent for being able to detect the level of gene methylation described in claim 1；
Preferably, the reagent of gene methylation level described in the detection claim 1 includes being used for full base
Because group methylation screening analysis a reagent, for based on chip methylation profiles analysis reagent, be used for methylation-specific
The reagent of PCR analysis is surveyed for the reagent of flight mass spectrum detection (sequenome), for sulphite treated genome
The reagent of sequence analysis, is used for methylation-specific restriction endonuclease at the reagent for combining bisulfites restriction endonuclease analysis
The reagent of digestion and quantitative polyase chain reaction Conjoint Analysis, for the examination of methylation sensitive high-resolution melting curve analysis
Agent or the reagent tested and analyzed for pyrosequencing.
As another preferred embodiment, the reagent including specific amplification include methylation sites including claim 1
Described in genetic fragment primer；
As another preferred embodiment, the diagnostic products are kit, chip or microarray dataset.
The present invention provides the prediction weightlessness bone loss cortisol Cortisol method that is increased beyond 7%, the method not with
For the purpose of the diagnosing and treating of disease, steps are as follows:
(1) DNA methylation for measuring DNA methylation site is horizontal；
(2) building of two disaggregated model of logistic regression is carried out, and substitutes into following formula:
Wherein, X=intercept coefficient+DNA methylation site DNA methylation level × independent variable coefficient, Y is cortisol
Cortisol Upside Risk score；Such as Y < 0.5, then it is determined as that cortisol Cortisol is increased beyond 7%.
The present invention passes through the digging to haemocyte DNA methylation level and serum bone density index (cortisol, Cortisol)
Pick, discovery is horizontal by detection haemocyte DNA methylation, can be realized and rises the pre- of individual difference to human body cortisol after weightlessness
It surveys, thus individual of the screening more resistant to below-G conditions, or for carrying out scientific research to cortisol situation of change.
The present invention provides the DNA methylation site for having forecast function to human body cortisol Upside Risk after weightlessness set,
It is higher with the correlation of human body bone metabolism.This is found to be human body bone loss caused by system announcement below-G conditions influence and provides
New clue is worth further investigation.
Detailed description of the invention
Attached drawing is used to provide further understanding of the present invention, and constitutes part of specification, with reality of the invention
It applies example to be used to explain the present invention together, not be construed as limiting the invention.In the accompanying drawings:
Fig. 1 is the cortisol change level grouping box figure of corresponding DNA methylation level before and after 45 days -6 degree Head down tilt bed rests
(by taking cg08569786 as an example).
Specific embodiment
Embodiment below facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method is unless otherwise specified conventional method.Test material as used in the following examples is unless otherwise specified city
It sells.
The present invention provides the DNA methylation markers for the human body cortisol after weightlessness can be predicted being increased beyond 7% risk
List (gene location annotation information and estimated performance are shown in Table 3), and provide a kind of prediction it is weightless after human body cortisol rise it is super
The method for crossing 7% risk.In a specific embodiment, by methylate marker cg08569786 methylation level,
Human body cortisol rises individual difference after evaluation is weightless, and identification cortisol is increased beyond 7% subject, and then screens more resistance to
Cortisol Upside Risk is studied by the individual of below-G conditions, or for researcher.
Although the present invention, which is regardless of, is limited to any theory, inventor thinks methylation marker levels and simulation in table 3
There are correlativities between below-G conditions hypopallium alcohol Upside Risk.
In a specific embodiment in the present invention, cortisol Upside Risk score Y, DNA methylation level
The relationship of cg08569786 meets
Wherein, X=1312+cg08569786 × (- 2048), the cg08569786 in formula are the DNA in specific gene site
Methylation level.Such as Y < 0.5, then it is determined as that cortisol is increased beyond 7%.
Embodiment 1
1, -6 degree Head down tilt bed rest simulated weightlessness model
(1) volunteer
Testing volunteer is 15 healthy non-smoking males, special secondary school and the above schooling of special secondary school.Age was at 22 years old to 34
Between year (M=26.62, SD=4.21), height (M=170.31, SD=4.02) between 160cm to 174cm, weight 53~
72Kg (M=62.81, SD=5.90).It without special medical history, eyesight or corrects defects of vision normal, is dextro manuality, non athlete.Institute
Some test volunteers fill in informed consent form before receiving experiment.
(2) experiment condition
15 volunteers downward -6 degree state of holding head, continuous life 45 days on -6 degree bed for lying on.It is held in experimentation
Continuation of insurance holds body axial -6 and spends the downward state in head.
(3) experimental arrangement
Bed rest experiment starts first 1 day, acquires 15 subject's blood cell samples by EDTA anticoagulant tube, freezes in -80 DEG C
Refrigerator remains subsequent DNA methylation chip detection；Subject is detected before bed test by Micro-particle enzyme immunoassay
1 day and the 45th day serum cortisol index of lying in bed.
2, data acquire overall situation
It is as shown in table 1 that data acquire situation.
1 data of table acquire situation table
3, cortisol Cortisol detection method and quality control
Using the microparticle enzyme immunoassay (MEIA) of Abbott of the U.S., i.e., with anti-Cortisol M-Ab
(M-Ab) M-Ab-Ag compound is formed with the BNP in sample, when compound is transferred on fiber cup, particulate just can not
It is integrated on the glass fibre on fiber cup surface, and in conjunction with the alkali phosphatase enzyme mark anti-Cortisol of addition, washes inversely
Unbonded dissociant is removed, substrate tetramethyl umbelliferone phosphate is added, alkaline phosphatase is sloughed the phosphate of substrate and sent out
Fluorescence out detects to obtain cortisol quantitative result by MEIA light path element.
The variation tendency of subject's cortisol Cortisol index is as shown in table 2 before and after 45 days -6 degree Head down tilt bed rests.It is right
Every subject analyzes its cortisol Cortisol with respect to amplitude of variation.Wherein, 7 subject's cortisol ascensional ranges are obvious,
Cortisol levels rise 7% or more after test；7 subject's cortisol levels declines；1 subject does not change.Based on cortex
Alcohol level, which rises, has the characteristics that relevance and experimental data between osteoporosis, and subject is divided on cortisol and increases wind
Dangerous group (ascensional range > 7% amounts to 7 subjects) and rise low-risk group (not rising, amount to 7 subjects), statistical
There are significant difference (Fig. 1) between two groups for the DNA methylation level of analysis discovery moiety site, can carry out subsequent characteristics assessment and build
Die worker makees.
Fig. 1 is the cortisol change level grouping box figure of corresponding DNA methylation level before and after 45 days -6 degree Head down tilt bed rests
(by taking cg08569786 as an example).
The variation tendency of 2 cortisol Cortisol of table
4, DNA methylation express spectra detects
It is detected using Illumina Human 450k DNA methylation chip.
In view of biochip technology relative maturity, and there is the commercial technologies service platform of operation maturation in the country, using outer
The mode of association's test completes Illumina Human 450k DNA methylation chip testing.Main technologies include:
(1) genome DNA is extracted.It is extracted using commercial kit QIAamp DNA Mini Kit kit total
DNA。
(2) DNA quality inspection.Using NanoDrop and Agrose Gel quality detecting method, pass through A260/280 signal, A260/230
The quality inspections parameters such as signal, DNA total amount (μ g) extract the quality control of DNA.
(3) bisulfite conversion is handled using Zymo EZ DNA Methylation Kit kit.
(4) WGA mode expand, fragmentation, using Illumina chip scanner, with GenomeStudio software control.
(5) chip hybridization, elution, extension, imaging, using GenomeStudio software.
(6) data are analyzed.
Above-mentioned experimental implementation is carried out by the standard practice instructions of related kit and company.
5, DNA methylation chip of expression spectrum data prediction
Using the Illumina Human 450k DNA methylation Chip data output file * .idat of standard
In file, data analysis is carried out using R language platform (v3.4.4) and Rstudio tool (v1.1.442).Pre-process institute's recruitment
Tool includes: minfi (v1.24.0), ChAMP (v2.9.10).By filtering following probe: (1) detecting p value and be greater than 0.01；
(2) be more than 5% sample middle probe bead-count number less than 3；(3) probe of non-CG beginning；(4) site SNPs starts
Probe；(5) probe in multiple gene locations is annotated；(6) probe of the annotation on X and Y chromosome, finally obtains 412345
Probe, 15 subjects DNA methylation express modal data, be used for subsequent analysis.
6, Head down tilt bed rest simulated weightlessness conditions lower lumbar spine bone loss risk profile methylation sites screen
(1) aspect of model screening 1 --- standard deviation filtering
Standard deviation 15 subjects is calculated between each probe, probe is ranked up by standard deviation, is filtered out
The probe that benchmark methylation level differs greatly in subject population.Screening threshold value is set as sd > 0.02, thus filters out
117872 probes are used for subsequent analysis.
(2) aspect of model screening 2 --- differential expression screening
To 117872 methylation probes, t is carried out between elevated risk group (H group) and low-risk group (L group) on cortisol
Check analysis.It is threshold value with p-value < 0.01, the probe of methylation level significant difference between screening group.Thus it filters out
3045 probes are used for subsequent analysis.
(3) building of unit point prediction model and performance evaluation of logic-based regression model
Using Logic Regression Models, disaggregated model building is carried out respectively with each site in above-mentioned 3045 probes.Using staying
One verification method evaluates the performance of each site model, output model AUC, Odds-Ratio value, model accuracy Accuracy, model
The parameter informations such as precision confidence interval and p value.With AUC >=0.85, and p-value < 0.01 is threshold value, and filtering out 480 has
The methylation sites of higher forecasting performance.Wherein 351 site annotations are on known (table 3).
Table 3 there is the methylation characteristic site of predictive ability to gather below-G conditions hypopallium alcohol Cortisol Upside Risk
Function enrichment analysis is done for gene where above-mentioned 351 probes filtered out using Logic Regression Models.In table 4
Analysis is the results show that have gene where the methylation sites of predictive ability most significant below-G conditions hypopallium alcohol Upside Risk
It is enriched in Pancreatic secretion signal path (p-value=6.57E-03) etc..
The signal path function of gene where 4 below-G conditions hypopallium alcohol Cortisol Upside Risk of table predicts methylation sites
Result can be enriched with
(3) cortisol Cortisol Upside Risk prediction model example before and after simulated weightlessness
It is input with methylation sites cg08569786 in table 3.Probe cg08569786 homologue sequence and corresponding
Location information is following (italic overstriking font shows that sequence includes probe location upstream and downstream 25bp nucleic acid sequence), with reference to genome
For human hg38:
> chr2:23527140-23527191 (cg08569786)
It is " 0 " by elevated risk group on cortisol (H group, ascensional range > 7% amount to 7 subjects) labeled bracketing symbol,
Low-risk group (L group, decline amount to 7 subjects) labeled bracketing symbol is " 1 ".Use R language platform (v3.4.4) and kit
Caret (v6.0.80) carries out the building of two disaggregated model of logistic regression, to elevated risk group and low wind on below-G conditions hypopallium alcohol
Dangerous group of crowd predicts.Prediction model coefficient such as table 5.
5 prediction model coefficient of table
| Variable | Coefficient |
| Cg08569786 (independent variable) | -2048 |
| Intercept | 1312 |
Calculation formula used is as follows:
Wherein, X=1312+cg08569786 × (- 2048), the cg08569786 in formula are the DNA in specific gene site
Methylation level.Such as Y < 0.5, then it is determined as that cortisol is increased beyond 7%.
By staying a verifying to analyze, prediction model AUC=1 (p-value=6.104e-05), other model performances ginseng
See Table 6 for details for number output.The result shows that the prediction model can concentrate elevated risk/low-risk crowd on cortisol to carry out to data
Accurate Prediction.
6 prediction model performance parameter of table
Finally, it should be noted that the foregoing is only a preferred embodiment of the present invention, it is not intended to restrict the invention,
Although the present invention is described in detail referring to the foregoing embodiments, for those skilled in the art, still may be used
To modify the technical solutions described in the foregoing embodiments or equivalent replacement of some of the technical features.
All within the spirits and principles of the present invention, any modification, equivalent replacement, improvement and so on should be included in of the invention
Within protection scope.
Claims (10)
1. predicting the DNA methylation marker of weightless bone loss cortisol Cortisol Upside Risk, including following one or more
A gene, site annotation information are as follows:
。
2. the method for predicting weightless bone loss cortisol Cortisol Upside Risk, the method is not with the diagnosing and treating of disease
For the purpose of, it is characterised in that: include the following steps: the DNA methylation for determining cortisol risk genes site in Samples subjects
Level, and the methylation level of the gene loci is compared with control sample, to predict subject in below-G conditions skin
Matter alcohol Cortisol Upside Risk；
Wherein one or more genes of the cortisol risk genes in claim 1；
The control sample is selected from the sample of below-G conditions hypopallium alcohol Cortisol decline；
Preferably, the method for the determining DNA methylation level includes full-length genome methylation screening analysis, chip platform
Methylation profiles analysis, methylation status of PTEN promoter analysis, flight mass spectrum detection (Sequenome), sulphite treated base
Because of a group sequencing analysis, joint bisulfites restriction endonuclease analysis, methylation-specific endonuclease digestion and quantitative polymerization
Enzyme chain reaction Conjoint Analysis, methylation sensitive high-resolution melting curve analysis or pyrosequencing determination method.
3. the DNA methylation assay reagent of gene described in claim 1 is predicted on weightless bone loss cortisol Cortisol in preparation
Rise the application in diagnosis of risk product, it is characterised in that: the diagnostic products include one or more bases in detection claim 1
The DNA methylation assay reagent of cause, the methylation water that the reagent passes through gene described in claim 1 in detection Samples subjects
It is flat, to judge whether subject has Upside Risk in below-G conditions hypopallium alcohol；
Preferably, the reagent is methylated based on full-length genome screening analysis, the methylation profiles analysis of chip platform, first
The analysis of base specific PCR, flight mass spectrum detection (sequenome), the analysis of sulphite treated gene order-checking, joint
Bisulfites restriction endonuclease analysis, methylation-specific endonuclease digestion and quantitative polyase chain reaction Conjoint Analysis,
Methylation sensitive high-resolution melting curve analysis or pyrosequencing test and analyze required reagent.
4. application according to claim 3, it is characterised in that: the methylation level of the gene is opening in marker gene
Put what the upstream region of reading frame, especially promoter region determined；Or one of following:
(a) nucleic acid defined in the chromosomal foci as shown in claim 1；
(b) comprising the site CpG of nucleic acid a；
(c) nucleic acid (a) being no more than with above-mentioned nucleic acid a distance in 1000 nucleotide.
5. application according to claim 3 or 4, it is characterised in that: the reagent includes that specific amplification includes methylation
The primer of gene described in claim 1 including site；
Preferably, the diagnostic products are kit, chip or microarray dataset.
6. one group of nucleic acid primer and/or hybridization probe, the potential methylation to one or more gene described in claim 1
Region is specific；
Preferably, the primer and/or hybridization probe are specific in the methylation of amplification marker gene open reading frame
Swim region, especially promoter region；Or methylation is one of following:
(a) nucleic acid defined in the chromosomal foci as shown in claim 1；
(b) comprising the site CpG of nucleic acid a；
(c) nucleic acid (a) being no more than with above-mentioned nucleic acid a distance in 1000 nucleotide.
7. a kind of kit, it is characterised in that: the kit includes that one group of nucleic acid primer as claimed in claim 6 or hybridization are visited
Needle further includes methylation-specific restriction enzyme and/or takes off the reagent of amine for bisulfites nucleotide.
8. a kind of diagnostic products for predicting weightless bone loss cortisol Cortisol Upside Risk, it is characterised in that: the diagnosis
Product includes the reagent for being able to detect the level of gene methylation described in claim 1；
Preferably, the reagent of gene methylation level described in the detection claim 1 includes being used for full-length genome methyl
Change screening analysis reagent, for based on chip methylation profiles analysis reagent, for methylation status of PTEN promoter analysis
Reagent, for the reagent of flight mass spectrum detection (sequenome), for the examination of sulphite treated gene order-checking analysis
Agent, the reagent for combining bisulfites restriction endonuclease analysis are used for methylation-specific endonuclease digestion and quantify
The reagent of polymerase chain reaction Conjoint Analysis, for methylation sensitive high-resolution melting curve analysis reagent or for coke
The reagent that phosphoric acid PCR sequencing PCR tests and analyzes.
9. diagnostic products according to claim 8, it is characterised in that: the reagent includes that specific amplification includes methylation
The primer of genetic fragment described in claim 1 including site；
Preferably, the diagnostic products are kit, chip or microarray dataset.
10. the prediction weightlessness bone loss cortisol Cortisol method that is increased beyond 7%, the method not with the diagnosis of disease with
For the purpose for the treatment of, it is characterised in that: steps are as follows:
(1) DNA methylation for measuring DNA methylation site is horizontal；
(2) building of two disaggregated model of logistic regression is carried out, and substitutes into following formula:
Wherein, X=intercept coefficient+DNA methylation site DNA methylation level × independent variable coefficient, Y is cortisol
Cortisol Upside Risk score；Such as Y < 0.5, then it is determined as that cortisol Cortisol is increased beyond 7%.
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