CN109234378A - Predict the method and DNA methylation marker of weightless bone loss osteocalcin OC downside risk - Google Patents
Predict the method and DNA methylation marker of weightless bone loss osteocalcin OC downside risk Download PDFInfo
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Abstract
The present invention provides the DNA methylation marker for predicting weightless bone loss osteocalcin OC downside risk, including one or more genes, gene loci annotation information are shown in Table 3.The present invention also provides correspondingly prediction techniques.The excavation that the present invention passes through human body haemocyte DNA methylation level and serum bone density index (osteocalcin OC) before and after p- 6 degree of Head down tilt bed rests simulated weightlessness, it was found that horizontal by detection haemocyte DNA methylation, it can be realized the prediction to weightless bone loss human osteocalcin element decline individual difference, to which screening is more resistant to the individual of weightless bone loss, or for carrying out scientific research to osteocalcin situation of change.It is higher with the correlation of human body weightlessness bone loss bone metabolism the present invention provides the DNA methylation site for having forecast function to human body osteocalcin downside 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 osteocalcin OC downside risk.
Background technique
Osteocalcin (osteocalcin, OC) is a kind of vitamin by synthesis and secretions such as osteoblast, odontoblasts
K dependence calbindin plays an important role in adjusting bone calcium metabolism, is a biochemical marker for studying bone metabolism, right
The medicals diagnosis on disease such as osteoporosis syndrome, abnormal calcium metabolism have important value.It is detected by serum osteocalcin, it will be appreciated that skeletonization
Cell, the active state of the osteoblast especially newly formed.Since osteocalcin is generated by osteoblast, it often by with
Make the marker of bon e formation process.It has been observed that with synthesis bon e formation drug therapy osteoporosis (such as Tai Paleide)
Period, higher level of bone gla protein in serum are significant related to the increase of bone density.
In aerospace medicine research, bone amount it is rapid reduction be in long-time manned space flight human body needs face it is important
One of health risk.Osteocalcin OC is the specific index for evaluating bon e formation and bone turnover rate, is usually used in weightlessness/simulated weightlessness item
The research of the bone metabolism disturbances problem such as bone loss under part.Studies have shown that bone loss of the human body under space flight weightlessness and bone calcium
Plain horizontal abnormality is related.Discovery is being detected to the spacefarer on Mir space station, the osteocalcin OC in few members' serum contains
Amount reduce (Collet P, 1997；Smith SM, 2000).In 14 days by a definite date -6 degree head-down bed-rests in ground, aspiration
Osteocalcin OC also shows downward trend (Kim, H, 2003) in person's serum.It is found in further cytologic experiment, space
The expression and secretion of osteoblast osteocalcin OC is substantially reduced (Hughes- under microgravity and ground simulation microgravity effect environment
Fulfor M, 2006；, Bucaro MA, 2007) this also partial interpretation mechanism of serum osteocalcin OC reduction.
To sum up, under weightlessness/simulated weightlessness conditions, human serum osteocalcin level is reduced to be reduced with Human osteoblast's cell activity
And bone density decline is in close relations.In consideration of it, after being subjected to same weightlessness/simulated weightlessness conditions and influencing, human serum bone calcium
The difference of plain decline level can also reflect that human body leads to the individual difference of bone loss tolerance degree to below-G conditions.Towards manned boat
Its task, as can the osteocalcin downside risk before execution task to occupant or subject under simulated weightlessness conditions carries out in advance
It surveys, optimization member is selected, early stage health protection intervention is provided for occupant, all there is 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 osteocalcin OC downside risk epigenetics index set.
The present invention provides the DNA methylation marker of prediction below-G conditions osteocalcin OC downside risk, including one or more
A gene, gene loci annotation information are shown in Table 3.
These bases are identified by gene symbol (the 10th column) and at least one chromosomal foci (the 7th, 8,9 column) in table
The preferred methylated nucleotide position of cause.These genes are by probe id (column 1) further identification, in these genes, especially
It is 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 osteocalcin OC downside risk, and the method is not with the diagnosis of disease
For the purpose for the treatment of, include the following steps: the DNA methylation water for determining osteocalcin OC risk genes site in Samples subjects
It is flat, and the methylation level of the gene loci is compared with control sample, to predict subject in below-G conditions bone calcium
Plain OC downside risk；
Wherein one or more genes of the osteocalcin OC risk genes in claim 1；
The control sample is selected from the sample that osteocalcin OC fall under below-G conditions < 20% or do not declined；
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 bone calcium in preparation
Application in plain OC downside risk diagnostic products, the diagnostic products include one or more genes in detection claim 1
DNA methylation assay reagent, the reagent are come by the methylation level of gene described in claim 1 in detection Samples subjects
Judge whether subject osteocalcin OC under below-G conditions has downside risk；
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 another preferred embodiment, 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 osteocalcin OC downside risk, the diagnostic products packet
Include 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 method that below-G conditions osteocalcin decrease beyond 20%, and the method is not with the diagnosis of disease
For the purpose for the treatment of, 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 are under osteocalcin
Risk score drops；Such as Y > 0.5, then it is determined as that osteocalcin decrease beyond 20%.
The present invention passes through the excavation to haemocyte DNA methylation level and serum bone density index (osteocalcin, OC), this hair
Clear proof is bright, and discovery is horizontal by detection haemocyte DNA methylation, can be realized and declines individual difference to human body osteocalcin after weightlessness
Prediction, thus screening more resistant to below-G conditions individual, or for osteocalcin situation of change carry out scientific research.
The DNA methylation site collection for having forecast function to human body osteocalcin downside risk after weightlessness is provided in the present invention
It closes, 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 mentions
New clue has been supplied, scientific research personnel is worth further to study.
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 that 45 days -6 degree Head down tilt bed rests test the different corresponding sites cg13989999 of osteocalcin change levels grouping
The horizontal box figure of DNA methylation.
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 osteocalcin after weightlessness can be predicted decreaseing beyond 20% risk
List (gene location annotation information and estimated performance are shown in Table 3), and provide a kind of prediction it is weightless after the decline of human body osteocalcin it is super
The method for crossing 20% risk.In a specific embodiment, by methylate marker cg13989999 methylation level,
Human body osteocalcin declines individual difference after evaluation is weightless, and identification osteocalcin decrease beyond 20% subject, and then screens more
It is resistant to the individual of below-G conditions, or carries out the further research for osteocalcin for researcher.
Although the present invention, which is regardless of, is limited to any theory, inventor thinks DNA methylation marker levels and mistake in table 3
There are correlativities between osteocalcin downside risk under the conditions of weight.
In a specific embodiment in the present invention, osteocalcin downside risk score Y, DNA methylation level
The relationship of cg13989999 meets
Wherein, X=1768.701+cg13989999 × (- 2486.653), the cg13989999 in formula are specific gene position
The DNA methylation of point is horizontal.Such as Y > 0.5, then it is determined as that osteocalcin decrease beyond 20%.
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 subjects 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 1 day and bed the before bed test are detected by elisa technique
45 days serum osteocalcin indexs.
2, data acquire overall situation
It is as shown in table 1 that data acquire situation.
1 data of table acquire situation table
3, osteocalcin OC detection method and quality control
Subject's bone is detected using the Human Gla-Osteocalcin Sensitive EIA kit of Takara company
Calcium element OC.Each 100 microlitres of sample and reagent are added in coated 96 orifice plate of antibody, is incubated at room temperature 1 hour.Subsequent eluent
The antibody-solutions of 100 microlitres of POD label are added in cleaning 3 times, are incubated at room temperature 1 hour.TMBZ is added after eluent cleans 4 times
Solution reacts at room temperature 10-15 minutes.Then 100 microlitres of terminate liquids are added, detect 450nm wavelength absorption using spectrophotometer
Value.Osteocalcin OC content is calculated eventually by the standard curve that standard items are drawn.
The variation tendency of subject's osteocalcin OC index is as shown in table 2 before and after 45 days -6 degree Head down tilt bed rests.To every by
Examination person analyzes its osteocalcin OC with respect to amplitude of variation.Wherein, 9 subject's osteocalcin falls are obvious, osteocalcin after test
20% or more level decline, 6 subject's osteocalcin level declines are no more than 5% or are promoted, based under osteocalcin level
Have the characteristics that relevance and experimental data between drop and osteoporosis, subject is divided into osteocalcin and is remarkably decreased group (decline
Amplitude > 20% amounts to 9 subjects) and be not remarkably decreased group (fall < 20% does not decline, and total 6 tested
Person), the DNA methylation level of statistical analysis discovery moiety site, there are significant difference (Fig. 1), can carry out subsequent spy between two groups
Sign assessment and modeling work.
Fig. 1 is that 45 days -6 degree Head down tilt bed rests test different osteocalcin change levels grouping corresponding DNA methylation level casees
Formula figure (by taking cg13989999 as an example).
The variation tendency of 2 osteocalcin OC 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: m > 50i (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, osteocalcin OC risk of missing prediction methylation sites screening under Head down tilt bed rest simulated weightlessness conditions
(1) aspect of model screens
To 412345 methylation probes, t is carried out between osteocalcin decline high risk group (H group) and low-risk group (L group)
Check analysis.It is threshold value with p-value < 0.001, the probe of methylation level significant difference between screening group.Thus it filters out
604 probes are used for subsequent analysis.
(2) 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 604 probes.Using staying one
Verification method evaluates the performance of each site model, 95% confidence interval of output model AUC, AUC, Odds-Ratio value, model essence
Spend the parameter informations such as Accuracy.It is threshold value with AUC >=0.75, filters out 476 methylation positions with higher forecasting performance
Point.Wherein 333 site annotations are on known (table 3).
Table 3 there is the methylation characteristic site of predictive ability to gather osteocalcin OC downside risk under below-G conditions
Function enrichment analysis is done for gene where above-mentioned 333 probes filtered out using Logic Regression Models.In table 4
Analysis is the results show that osteocalcin downside risk has the methylation of predictive ability under p- 6 degree of Head down tilt bed rest simulated weightlessness conditions
Gene most significant enrichment is in signal paths such as Ras signaling pathway (p-value=9.96e-4) where site.
The function of gene is enriched with result where osteocalcin downside risk prediction methylation sites under 4 below-G conditions of table
(3) weightless front and back osteocalcin OC downside risk prediction model example
It is input with methylation sites cg13989999 in table 3.Probe cg13989999 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:
> chr20:31861674-31861725 (cg13989999)
It is " 1 " that osteocalcin, which is remarkably decreased group (fall > 20% amounts to 9 subjects) labeled bracketing symbol, is not shown
Writing group of decreased (fall < 20% does not decline, amounts to 6 subjects) labeled bracketing symbol is " 0 ".Use R language platform
(v3.4.4) and kit caret (v6.0.80) carries out the building of two disaggregated model of logistic regression, simulates to 45 days Head down tilt bed rests
Osteocalcin decline high risk group and low-risk group crowd predict under below-G conditions.Prediction model coefficient such as table 5.
5 prediction model coefficient of table
| ? | Coefficient |
| Cg13989999 (independent variable) | -2486.653 |
| Intercept | 1768.701 |
Calculation formula used is as follows:
Wherein, X=1768.701+cg13989999 × (- 2486.653), the cg13989999 in formula are specific gene position
The DNA methylation of point is horizontal.Such as Y > 0.5, then it is determined as that osteocalcin decrease beyond 20%.
By staying a verifying to analyze, prediction model AUC=1.The result shows that the prediction model can concentrate bone close data
It spends high risk/low-risk crowd and carries out Accurate Prediction.
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. predict the DNA methylation marker of weightless bone loss osteocalcin OC downside risk, including following one or more genes,
Its site annotation information is as follows:
2. the method for predicting weightless bone loss osteocalcin OC downside risk, the method is not using the diagnosing and treating of disease as mesh
, it is characterised in that: include the following steps: the DNA methylation water for determining osteocalcin OC risk genes site in Samples subjects
It is flat, and the methylation level of the gene loci is compared with control sample, to predict subject in below-G conditions bone calcium
Plain OC downside risk；
Wherein one or more genes of the osteocalcin OC risk genes in claim 1；
The control sample is selected from the sample that osteocalcin OC fall under below-G conditions < 20% or do not declined；
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 predicts weightless bone loss osteocalcin OC downside risk in preparation
Application in diagnostic products, it is characterised in that: the diagnostic products include the first of one or more genes in detection claim 1
Base detection reagent, the reagent passes through the methylation level of gene described in claim 1 in detection Samples subjects, to sentence
Whether disconnected subject osteocalcin OC under below-G conditions has downside risk；
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 osteocalcin OC downside risk, it is characterised in that: the diagnostic products packet
Include 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. predicting the method that weightless bone loss osteocalcin decrease beyond 20%, the method is not with the diagnosing and treating of disease
Purpose, 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 are osteocalcin canyon wind
Dangerous score；Such as Y > 0.5, then it is determined as that osteocalcin decrease beyond 20%.
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