CN109055542A - Predict the method and DNA methylation marker of weightless bone loss lumbar spine bmd downside risk - Google Patents
Predict the method and DNA methylation marker of weightless bone loss lumbar spine bmd downside risk Download PDFInfo
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Abstract
The present invention provides the DNA methylation marker for predicting weightless bone loss lumbar spine bmd downside risk, including 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 the excavation of haemocyte DNA methylation level and lumbar spine bmd index, it was found that horizontal by detection haemocyte DNA methylation, it can be realized the prediction to human body lumbar spine bmd decline individual difference after weightlessness, to which screening is more resistant to the individual of below-G conditions, or for carrying out scientific research to lumbar spine bmd situation of change.The DNA methylation site set for having forecast function to human body lumbar spine bmd downside risk after weightlessness is provided in the present invention, it is higher with the correlation of human body bone metabolism.
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 lumbar spine bmd downside risk.
Background technique
Bone density full name is bone mineral density (BMD, Bone mineral density), is one of bone strength
The important indicator of absolute value attribute, unit are gram/cubic centimeter.Bone density absorbs measurement Law (Dual- with dual-energy x-ray
Energy X-ray Absorptiometry) (DXA technology) measurement result it is more accurate, be International Health Organization (WHO) use
Bone density goldstandard.
In clinical use, bone density is diagnosis of osteoporosis, prediction osteoporotic fracture risk, monitoring natural history
And the best quantitative target of evaluation therapeutic effect of drug intervention.The diagnostic criteria of generally acknowledged osteoporosis is that the world defends both at home and abroad at present
Raw Organisation recommendations, defining bone density value and reducing degree to be equal to and more than 2.5 standard deviations is to be determined as osteoporosis.
In aerospace medicine research, bone density is detected as bone variation, and health risk judgement provides judgment basis.?
In 21 days by a definite date -6 degree head-down bed-rests in ground, DXA detection discovery volunteer's lumbar spine bmd, which averagely declines, is about
0.6% (SM.Smith, 2009).In space flight, space microgravity environment causes astronaut to be no longer affected by gravity.Load-bearing
The reduction of load, cause spacefarer's lumbar vertebrae bone mineral lost with the speed of 1%-2% every month (JD Sibonga, 2008；AD
Leblanc, 2012).Therefore, US National Aeronautics and Space Administration by space bone loss be all risk factors first of.In short, bone is close
The quantitative determination of degree is that bone mineral content change caused by the diagnosis and different reasons of disease both provides important diagnostic foundation.
In consideration of it, after being subjected to same weightlessness/simulated weightlessness conditions and influencing, the horizontal difference of human body lumbar spine bmd decline
It is different also to 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
Lumbar spine bmd downside risk before execution task to occupant or subject under simulated weightlessness conditions predicts, to being optimized to
Member selects, provides early stage health protection intervention for occupant and study below-G conditions lumbar spine bmd downside risk, all
With 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 lumbar spine bmd downside risk epigenetics index set.
The present invention provides the DNA methylation marker for predicting weightless bone loss lumbar spine bmd downside 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 in table (the 8th, 9 and 10 arrange)
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 lumbar spine bmd downside risk, the method not examining with disease
For the purpose of disconnected and treatment, include the following steps: the DNA methylation for determining lumbar spine bmd 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 waist
Vertebra density downside risk；
Wherein one or more genes of the lumbar spine bmd risk genes in claim 1；
The control sample is selected from the sample of below-G conditions lower lumbar spine bone density fall < 6%；
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 lumbar vertebrae in preparation
Application in bone density downside risk diagnostic products, the diagnostic products include one or more genes in detection claim 1
Methylation level detection reagent, the methylation that the reagent passes through gene described in claim 1 in detection Samples subjects
Level, to judge whether subject has downside risk in below-G conditions lower lumbar spine bone density；
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 lumbar spine bmd downside risk, the diagnostic products
Reagent including 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 further optimisation, the diagnostic products are kit, chip or microarray dataset.
The present invention provides the method for predicting that weightless bone loss lumbar spine bmd decrease beyond 6%, and the method is not with disease
Diagnosing and treating for the purpose of, steps are as follows:
(1) measurement with probe cg13567986, cg24720336, cg26683794, cg14279528, cg01459748,
The DNA methylation of the corresponding gene loci of cg00897863, cg01015354 is horizontal；
(2) building of two disaggregated model of logistic regression is carried out, and substitutes into following formula:
Wherein, X=(- 321.69)+cg13567986 × (139.65)+cg24720336 × (121.87)+
cg26683794×(-48.06)+cg14279528×208.23+cg01459748×(-287.22)+cg00897863×(-
221.93)+cg01015354 × (381.07), cg13567986, cg24720336, cg26683794 in formula,
Cg14279528, cg01459748, cg00897863 and cg01015354 are the DNA methylation water in corresponding specific gene site
Flat, Y is lumbar spine bmd downside risk score；Such as lumbar spine bmd downside risk score Y < 0.5, then it is determined as that weightless bone is lost
It loses lumbar spine bmd and decrease beyond 6%.
The present invention passes through to haemocyte DNA methylation level and bone density index (bone mineral density BMD, Bone
Mineral density) excavation, discovery is horizontal by detection haemocyte DNA methylation, can be realized to human lumbar after weightlessness
Vertebra density declines the prediction of individual difference, thus individual of the screening more resistant to below-G conditions, or for close to lumbar vertebra
It spends situation of change and carries out scientific research.
The DNA methylation site for having forecast function to human body lumbar spine bmd downside risk after weightlessness is provided in the present invention
Set is higher with the correlation of human body bone metabolism.This is found to be system and discloses human body bone loss caused by below-G conditions influence
New clue is provided, 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 lumbar spine bmd variance analysis before and after 45 days -6 degree Head down tilt bed rests.
Fig. 2 is lumbar spine bmd change rate distribution before and after 45 days -6 degree Head down tilt bed rests.
Fig. 3 is simulated weightlessness conditions lumbar vertebra risk of missing prediction model ROC curve figure.
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 marks for the human body lumbar spine bmd after weightlessness can be predicted decreaseing beyond 6% risk
Will object 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 lumbar vertebra it is close
Spend the method for decreaseing beyond 6% risk.In a specific embodiment, by methylation marker combine cg13567986,
The methylation water of cg24720336, cg26683794, cg14279528, cg01459748, cg00897863, cg01015354
Flat, human body lumbar spine bmd declines individual difference after evaluation is weightless, and identification lumbar spine bmd decrease beyond 6% subject, into
And the individual more resistant to below-G conditions is screened, or study lumbar spine bmd for researcher.
Although the present invention is regardless of and is limited to any theory, inventor think in table 3 methylation level of methylation sites with
There are correlativities between below-G conditions lower lumbar spine bone density downside risk.
Human body lumbar spine bmd is decrease beyond in the specific example of 6% risk after a prediction is weightless in the present invention, waist
Vertebra density downside risk score Y, the horizontal cg13567986, cg24720336 of DNA methylation, cg26683794,
The relationship of cg14279528, cg01459748, cg00897863, cg01015354 meet
Wherein, X=(- 321.69)+cg13567986 × (139.65)+cg24720336 × (121.87)+
cg26683794×(-48.06)+cg14279528×208.23+cg01459748×(-287.22)+cg00897863×(-
221.93)+cg01015354 × (381.07), cg13567986, cg24720336, cg26683794 in formula,
Cg14279528, cg01459748, cg00897863, cg01015354 are that the DNA methylation in specific gene site is horizontal, and Y is
Lumbar spine bmd downside risk score.Such as Y < 0.5, then it is determined as that lumbar spine bmd decrease beyond 6%.
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；Detecting subject's lumbar spine bmd index by DXA technology, (bone mineral is close
Spend BMD, Bone mineral density).45 days bed rest experiments terminate the same day, detect subject's lumbar vertebra by DXA technology
Density index (bone mineral density BMD, Bone mineral density).
2, data acquire overall situation
It is as shown in table 1 that data acquire situation.
1 data of table acquire situation table
3, lumbar vertebra mineral density BMD detection method and quality control
BMD measurement: lumbar vertebrae (L1-4) BMD is measured using Dual-energy X-rays absorptionmetry (DXA), instrument is France MEDLINK public
Take charge of the digital dual intensity x line borne densitometers of taper two dimension flash-type, instrument model OSTEOCORESTATION.Bone density operating room
18-25 DEG C of temperature, humidity 75%-90%, instrument carries out external body mould calibration every time.
Subject prepares: unlined garment unlined trousers, no metal accessories, and both legs are bent and are placed on and check on foot pad, lies on the back and lies low.Laser
Anchor point is moved to 3 finger positions under navel and starts to scan, and whole process is not moved, and avoids acutely breathing.After to be detected, choosing
It selects suitable " ROI " and calculates lumbar vertebrae BMD.Instrument operator trueness error (LSC) is 2.5%, and operator and analysis personnel are equal
Receive training class's training that international clinical bone density measurement association (ISCD) is held, and obtains respective certificate.
Variation tendency such as 2 institute of table of subject's lumbar vertebra mineral density BMD index before and after 45 days -6 degree Head down tilt bed rests
Show.It is analyzed by paired t-test, bed front and back subject's lumbar spine bmd is remarkably decreased (p=1e-5) (Fig. 1).Each subject
Lumbar spine bmd relative drop amplitude distribution is as shown in Figure 2.By subject be divided into lumbar spine bmd decline high risk group (H group,
Fall > 6% amounts to 8 subjects) and low-risk group (L group, fall < 6% amount to 7 subjects).
Fig. 1 is lumbar spine bmd variance analysis before and after 45 days -6 degree Head down tilt bed rests.
Fig. 2 is lumbar spine bmd change rate distribution before and after 45 days -6 degree Head down tilt bed rests.
The variation tendency of 2 lumbar vertebra mineral density BMD 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, between lumbar spine bmd decline high risk group (H group) and low-risk group (L group) into
Row t check analysis.It is threshold value with p-value < 0.05, the probe of methylation level significant difference between screening group.Thus it filters out
2301 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 2301 probes.Using staying
One verification method evaluates the performance of each site model, output model AUC, Odd-Ratio value, model accuracy Accuracy, model
The parameter informations such as precision confidence interval and p value.It is threshold value with p-value < 0.05, filtering out 419 has higher forecasting performance
Methylation sites.Wherein 303 site annotations are on known (table 3).
Table 3 there is the methylation characteristic site of predictive ability to gather below-G conditions lower lumbar spine bone loss risk
Function enrichment analysis is done for gene where above-mentioned 303 probes filtered out using Logic Regression Models.In table 4
Analysis is the results show that p- 6 degree of Head down tilt bed rests simulated weightlessness conditions lower lumbar spine bone loss risk has the methylation of predictive ability
Gene most significant enrichment is in Dorso-ventral axis formation (dorsoventral axis is formed) signal path (p- where site
Value=4.4e-4).
The function of gene is enriched with result where 4 below-G conditions lower lumbar spine bone loss risk profile methylation sites of table
(4) aspect of model screening 3 --- the logistic regression modeling based on LASSO homing method
LASSO recurrence be while be fitted generalized linear model progress Variable Selection (variable selection) and
Complexity adjusts (regularization).This analysis is intended to using the punishment discriminant function in LASSO regression analysis to all
The regression coefficient of 303 methylation probe variables carries out restrict, some meaningless or minimum meaning variation coefficient
It is compressed to 0, finishing screen selects the characteristic set that input variable number is less and classification performance is optimal.Using 5 times of intersections in analysis
The ratio of authentication policy, training set and inspection set is selected as 4: 1, i.e., randomly selects 12 sample conducts from 15 samples every time
Training set, 3 samples are as test set, assessment classification estimated performance.The analysis uses glmnet kit (v2.0- in R language
16).Parameter type.measure=" class " is set in analysis, the error rate for limiting category of model is minimum.Analyze result table
It is bright, when λ value takes 0.186115, it can get the best performance feature being made of 7 methylation probes and combine.
The methylation probe combinations of the best performance feature are as follows: cg13567986, cg24720336, cg26683794,
cg14279528,cg01459748,cg00897863,cg01015354.Each probe homologue sequence and corresponding positions confidence
Breath is following (italic overstriking font shows that sequence includes probe location upstream and downstream 25bp nucleic acid sequence), is human with reference to genome
Hg38:
> chr1:37859188-37859239 (cg13567986)
> chr1:151281590-151281641 (cg24720336)
> chr13:97068069-97068120 (cg26683794)
> chr9:37261056-37261107 (cg14279528)
> chr11:114258453-114258504 (cg01459748)
> chr17:32342432-32342483 (cg00897863)
> chr17:22552837-22552888 (cg01015354)
(5) the multidigit point prediction model construction of logic-based regression model and performance evaluation
With the probe combinations cg13567986, cg24720336 that methylates, cg26683794, cg14279528,
Cg01459748, cg00897863, cg01015354 are input pointer, and lumbar spine bmd is declined high risk group (H group, decline
Amplitude > 6%, amount to 8 subjects) labeled bracketing symbol be " 0 ", low-risk group (L group, fall < 6%, amount to 7 by
Examination person) labeled bracketing symbol be " 1 ".Logistic regression is carried out using R language platform (v3.4.4) and kit caret (v6.0.80)
Two disaggregated models building, to 45 days Head down tilt bed rest simulated weightlessness conditions lower lumbar spine bone loss high risk groups (H group) and low-risk
Group (L group) crowd predicts.Prediction model coefficient such as table 5.
5 prediction model coefficient of table
| Variable | Coefficient |
| Cg13567986 (independent variable) | 139.65 |
| Cg24720336 (independent variable) | 121.87 |
| Cg26683794 (independent variable) | -48.06 |
| Cg14279528 (independent variable) | 208.23 |
| Cg01459748 (independent variable) | -287.22 |
| Cg00897863 (independent variable) | -221.93 |
| Cg01015354 (independent variable) | 381.07 |
| Chang Bianliang | -321.69 |
Calculation formula used is as follows:
Wherein, X=(- 321.69)+cg13567986 × (139.65)+cg24720336 × (121.87)+
cg26683794×(-48.06)+cg14279528×208.23+cg01459748×(-287.22)+cg00897863×(-
221.93)+cg01015354×(381.07).Cg13567986, cg24720336, cg26683794 in formula,
Cg14279528, cg01459748, cg00897863, cg01015354 are that the DNA methylation in specific gene site is horizontal, and Y is
Lumbar spine bmd downside risk score.Such as lumbar spine bmd downside risk score Y < 0.5, then it is determined as that lumbar spine bmd declines
More than 6%.
By staying a verifying to analyze, prediction model AUC==1 (p-value=8.035e-05), other model performances
See Table 6 for details for parameter output.The result shows that the prediction model can concentrate bone density high risk/low-risk crowd to carry out quasi- data
Really 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 lumbar spine bmd downside risk, including following one or more bases
Cause, site annotation information are as follows:
2. the method for predicting weightless bone loss lumbar spine bmd 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 for determining lumbar spine bmd 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 waist
Vertebra density downside risk；
Wherein one or more genes of the lumbar spine bmd risk genes in claim 1；
The control sample is selected from the sample of below-G conditions lower lumbar spine bone density fall < 6%；
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 lumbar spine bmd canyon wind in preparation
Application in dangerous diagnostic products, it is characterised in that: the diagnostic products include one or more genes in detection claim 1
Methylation level detection reagent, 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 downside risk in below-G conditions lower lumbar spine bone density；
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 lumbar spine bmd downside risk, it is characterised in that: the diagnostic products
Reagent including 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 lumbar spine bmd decrease beyond 6%, the method is not with the diagnosing and treating of disease
For the purpose of, it is characterised in that: steps are as follows:
(1) measurement with probe cg13567986, cg24720336, cg26683794, cg14279528, cg01459748,
The DNA methylation of the corresponding gene loci of cg00897863, cg01015354 is horizontal；
(2) building of two disaggregated model of logistic regression is carried out, and substitutes into following formula:
Wherein, X=(- 321.69)+cg13567986 × (139.65)+cg24720336 × (121.87)+cg26683794 ×
(-48.06)+cg14279528×208.23+cg01459748×(-287.22)+cg00897863×(-221.93)+
Cg01015354 × (381.07), cg13567986, cg24720336, cg26683794, cg14279528 in formula,
Cg01459748, cg00897863 and cg01015354 are that the DNA methylation in corresponding specific gene site is horizontal, and Y is lumbar vertebrae
Bone density downside risk score；Such as lumbar spine bmd downside risk score Y < 0.5, then it is determined as that weightless bone loss lumbar vertebra is close
Degree decrease beyond 6%.
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