Methylated biomarker for predicting female physiological age and application and product thereof

Abstract

The invention provides a methylated biomarker for predicting female physiological age and application and a product thereof, and belongs to the technical field of biology. The invention provides a methylated biomarker cg21294974, which is highly related to the aging process of women. The methylated biomarker provided by the invention can be used for predicting the female physiological age, has good accuracy and distinguishing ability, and provides a more accurate and sensitive detection method for predicting the female physiological age.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to methylation biomarkers for predicting the physiological age of women, and their applications and products. Background Art

[0002] Aging, as an inevitable stage of individual development, maturity, aging, and death, is also accompanied by a series of molecular changes in the body. DNA (deoxyribonucleic acid) is the basis of life that constitutes an individual human being and is also the fundamental for guiding life activities. DNA stores all the genetic information of an individual and determines the genetic composition of the individual. DNA guides gene expression and protein translation, and thus various life activities. Changes in DNA determine the fate of an individual. DNA methylation refers to the addition of a methyl group to the 5th carbon atom of cytosine (C) in DNA, forming 5-methylcytosine. DNA methylation modification belongs to the category of epigenetic regulation, which regulates whether a gene is expressed and the level of its expression. During the occurrence and development of many diseases, there is a disorder in the level of DNA methylation. Therefore, abnormal DNA methylation can often be used as an accompanying biomarker for the occurrence and development of diseases, indicating the occurrence and progression of diseases. During the aging process, there are also changes in DNA methylation. Age-related DNA methylation changes have been found in both humans and animals. For some DNA sites, their DNA methylation levels gradually increase with the increase of individual age, while for some other DNA sites, their DNA methylation levels gradually decrease with the increase of individual age. Considering the close correlation between DNA methylation levels and aging, the concept of DNA methylation clock has been proposed. By comparing the "DNA methylation clock" with the actual age, it can be judged whether aging is accelerating or slowing down. A study on humans found that physical exercise and diet can delay the DNA methylation clock. More and more evidence proves that the DNA methylation clock can indicate the aging process. Compared with the actual age, the aging age (aging process) reflected by the DNA methylation clock is closely related to the disease burden and ultimate death.

[0003] The prior art CN113373236A provides a method for obtaining the individual age of the Chinese population. This invention selects DNA methylation markers and constructs an MLR model for predicting age, thereby obtaining the individual age of the Chinese population. The method in the present invention realizes the inference of individual age using blood or blood stains, with relatively high accuracy and stability. However, this method requires calculating the methylation levels of 10 sites comprehensively, which is time-consuming and laborious. Moreover, this method is for the entire population and does not separately explore methylation markers related to women.

[0004] Searching for DNA methylation sites closely related to female aging and developing a method for predicting a female's physiological age based on these DNA methylation sites will have significant economic and social benefits. Summary of the Invention

[0005] Terms and statements of the present invention:

[0006] 1. Articles "a", "an" and "the": Unless otherwise explicitly limited to one (kind) of object, include plural objects.

[0007] 2. Numerical ranges: Unless otherwise explicitly indicated, all ranges or ratios disclosed herein will be understood to include any and all sub-ranges or sub-ratios contained therein. For example, a stated range or ratio of 1 to 30 should be considered to include between the minimum value of 1 and the maximum value of 30, and any sub-range or sub-ratio, integer, decimal, or sub-range or sub-ratio composed of integers or decimals including the endpoints.

[0008] To solve the above problems, the present invention provides methylation biomarkers for predicting a female's physiological age, their applications, and products.

[0009] On the one hand, the present invention provides the application of methylation marker cg21294974 in predicting a female's physiological age.

[0010] Specifically, the methylation site is: cg21294974.

[0011] On the other hand, the present invention provides a method for predicting a female's physiological age, the method comprising the following steps:

[0012] (1) Measuring the methylation level of site cg21294974 in a biological sample;

[0013] (2) Predicting a female's physiological age based on the methylation level measured in step (1).

[0014] Specifically, in step (1), the biological sample includes but is not limited to: blood, saliva, urine, and lymph fluid.

[0015] Preferably, the sample is: peripheral blood.

[0016] Specifically, in step (1), the method for measuring the methylation level includes: bisulfite sequencing PCR, methylation-specific PCR, pyrosequencing, Sanger sequencing, high-throughput sequencing, or third-generation sequencing or single-molecule sequencing.

[0017] On yet another hand, the present invention provides the application of the above methylation marker in the preparation of a product for predicting a female's physiological age, the product including but not limited to: reagent kits, chips, and test strips.

[0018] In another aspect, the present invention provides a kit, and the kit includes reagents required for detecting the methylation level of locus cg21294974.

[0019] Specifically, the reagents include at least one of primers, Taq DNA polymerase, free nucleotides (dNTPs), MgCl2, and buffer.

[0020] In another aspect, the present invention provides a system for predicting the physiological age of a female. Among them, the system includes a detection device, a calculation device, and an output device. The detection device includes a sampler and a detector, and the detector is used to detect the methylation level of the cg21294974 methylation biomarker in the sample.

[0021] Specifically, the calculation device includes a memory and a processor, and the processor makes the following discrimination: the methylation level of the cg21294974 methylation biomarker in the sample indicates the physiological age of the subject corresponding to the sample.

[0022] More specifically, a computer program is stored in the memory, and the processor is configured to execute the computer program stored in the memory to implement the above discrimination.

[0023] Compared with the prior art, the present invention has the following advantages:

[0024] The present invention provides a methylation biomarker highly related to the aging process of females. The methylation biomarker can be used to predict the physiological age of females, and has good accuracy and discrimination ability, providing a more accurate and sensitive detection method for predicting the physiological age of females. Description of the Drawings

[0025] Figure 1 For the methylation levels of each sample in the training set in Example 1.

[0026] Figure 2 For the methylation levels of each sample in the test set in Example 2. Detailed Embodiments

[0027] The following combines specific embodiments to further elaborate on the present invention. The following embodiments are not used to limit the present invention, but only to illustrate the present invention. The experimental methods used in the following embodiments, unless otherwise specified, and the experimental methods without specific conditions indicated in the embodiments are usually in accordance with conventional conditions. The materials, reagents, etc. used in the following embodiments, unless otherwise specified, can all be obtained from commercial channels.

[0028] Example 1 Screening of Methylation Loci for Predicting the Physiological Age of Females

[0029] 1. Training set information

[0030] The training set samples are peripheral blood from 50 healthy women, including peripheral blood from 10 healthy women aged 35, peripheral blood from 10 healthy women aged 45, peripheral blood from 10 healthy women aged 55, peripheral blood from 10 healthy women aged 65, and peripheral blood from 10 healthy women aged 75.

[0031] 2. Extraction of DNA from blood samples

[0032] The human DNA in the blood samples was extracted using a kit (Qiagen, 51106), and the extraction process was carried out according to the instructions of the kit.

[0033] 3. Bisulfite conversion

[0034] Using a kit (ZYMO RESEARCH, D5002), the DNA sample obtained in step 2 was methylated, and the specific steps are as follows:

[0035] (1) Adjust the DNA to 500 ng / 45 μL in a PCR reaction tube, add 5 μL of M-Dilution Buffer, and vortex.

[0036] (2) Place the PCR reaction tube on a PCR instrument and run the program: 37 °C for 15 minutes.

[0037] (3) Centrifuge the PCR reaction tube instantaneously for 30 s. After removing the residual liquid on the upper lid, add 100 μL of CT conversion solution and vortex.

[0038] (4) Take out 75 μL and transfer it to a new PCR reaction tube. Place it on a PCR instrument and run the program: 95 °C for 30 seconds; 50 °C for 60 minutes for 16 cycles.

[0039] (5) After the reaction is completed, centrifuge instantaneously and store at 4 °C

[0040] (6) Assemble the extraction column of Zymo-Spin TM with the collection tube, and add 400 μL of M-Binding Buffer to each well of the extraction column.

[0041] (7) Aspirate the solution in the RCR reaction tube and add it to the extraction column of Zymo-Spin TM Shake it up and down to mix well, incubate at room temperature for 1 min, centrifuge at 16100×g for 1 min, and pour out the liquid in the collection tube.

[0042] (8) Add 100 μL of M-Wash Buffer, incubate at room temperature for 1 min, centrifuge at 16100×g for 1 min, and pour out the liquid in the collection tube.

[0043] (9) Add 200 μL of M-Desulphonation Buffer, incubate at room temperature for 20 min to allow sufficient infiltration, centrifuge at 16100×g for 1 min, and pour off the liquid in the collection tube.

[0044] (10) Add 200 μL of M-Wash Buffer, incubate at room temperature for 1 min, centrifuge at 16100×g for 1 min, and pour off the liquid in the collection tube.

[0045] (11) Add 200 μL of M-Wash Buffer and centrifuge at 16100×g for 1 min.

[0046] (12) Replace with a new sample collection tube, add 11 μL of M-Elution Bufer to each extraction column, centrifuge at 16100×g for 1 min, and collect the liquid in the sample collection tube.

[0047] (13) Measure the concentration of the purified sample using nano and record it.

[0048] 4. DNA Methylation Chip Detection

[0049] The commercial chip used is the Illumina Infinium MethylationEPIC BeadChip (935K DNA methylation chip). Among them, the process of using the DNA methylation chip for detection includes amplification - fragmentation - precipitation - resuspension - hybridization - extension - washing - scanning, and finally the raw data of the chip and the chip scan image are obtained.

[0050] (1) Amplification

[0051] The reagents used are shown in Table 1.

[0052] Table 1

[0053] Name Purchase Source MA1 Illumina MA2 Illumina MSM Illumina

[0054] Experimental operation:

[0055] 1) Paste the MSAI barcode label on the new MIDI plate for marking.

[0056] 2) Add 20 μL of MA1 per well to the wells of the MSA4 plate.

[0057] 3) Transfer 4 μL of the sample per well to the wells of the MSA4 plate, and record the sample numbers corresponding to the wells on the MAS4 plate.

[0058] 4) Add 4 μL of 0.1 M NaOH per well to the wells of the MSA4 plate.

[0059] 5) Mix at 1600 rpm on an oscillator for 1 min; centrifuge at 280 x g for 1 min.

[0060] 6) Incubate at room temperature and let stand for 10 min.

[0061] 7) Gently remove the lid and dispense 68 μL of RPM into each well of the MSA1 plate containing the sample.

[0062] 8) Dispense 75 μL of MSM into each well of the MSA1 plate containing the sample.

[0063] 9) Mix at 1600 rpm on an oscillator for 1 min; centrifuge at 280 × g for 1 min

[0064] 10) Incubate in a hybridization oven at 37 °C for 22 h.

[0065] (2) Breakage - precipitation - resuspension

[0066] The reagents used are shown in Table 2.

[0067] Table 2

[0068] tem Supplied By FMS Illumina PM1 Illumina RA1 Illumina

[0069] 1) Add 50 μL per well of FMS to the wells of the MSA4 plate, mix by shaking at 600 rpm for 1 min, centrifuge at 50 x g for 1 min, and incubate in a 37 °C heat block for 1 hour.

[0070] 2) Add 100 μL per well of PM1 to the wells of the MSA4 plate, mix at 1600 rpm on an oscillator for 1 min, incubate in a 37 °C heat block for 5 min, and centrifuge at 280 × g for 1 min in a centrifuge.

[0071] 3) Add 300 μL per well of 100% isopropyl alcohol to the wells of the MSA4 plate, invert up and down at least 10 times to mix thoroughly, incubate in a 4 °C refrigerator for 30 min, centrifuge at 3000 × g for 20 min at 4 °C, then immediately remove the MSA4 plate from the centrifuge, immediately invert to remove the supernatant, place it upside down horizontally on absorbent paper and tap gently until there is no water mark, about 1 min, without moving the blue beads, keep the MSA4 in the upside - down state for 1 hour, and air - dry

[0072] 4) Add 46 μL / well of RA1 to the wells of the MSA4 plate. After covering the MSA4 with a metal foil, apply it under a heat sealer for 5 seconds. Then, change the up-and-down order of the MSA4 and apply it for another 5 seconds. Subsequently, slightly lift the metal foil to verify the sealing effect.

[0073] 5) Place the sealed MSA4 in a hybridization oven at 48 °C and incubate for 1 hour. Mix it on an oscillator at 1800 rmp for 1 minute, and centrifuge it at 280 × g for 30 seconds in a centrifuge.

[0074] (3) Hybridization

[0075] The reagents used are shown in Table 3.

[0076] Table 3

[0077] Name Purchase Source PB2 Illumina BeadChips Illumina Hyb Chambers Illumina Hyb Chamber gaskets Illumina Hyb Chamber inserts Illumina XC4 Illumina

[0078] 1) Open the hybridization cassette, place the hybridization cassette liner into the hybridization cassette. Add 400 μL of PB2 to the upper and lower grooves of the hybridization cassette (one chip corresponds to the upper and lower grooves). Immediately close the hybridization cassette to prevent volatilization. Fasten it in the diagonal direction and let it stand at room temperature for use. After resuspension, seal the MSA4 and place it in a heat block at 95 °C for denaturation for 20 minutes.

[0079] 2) Take out the chip and let it stand at room temperature for use. Let the denatured MSA4 cool at room temperature for 30 minutes, and then centrifuge it at 280 xg for 30 seconds in a centrifuge.

[0080] 3) Take out the chip from the box, carefully remove its packaging without touching the sample-adding position of the chip. Place it in the Hyb chamber inserts corresponding to the barcode position. Add 26 μL of the sample to the sample-adding area of the chip in the order of the chip samples. Note that during the slow inflow process, no bubbles should be generated. At the same time, record the corresponding numbers of the sample and the chip. Place the Hyb chamber inserts with the chip in the hybridization cassette corresponding to the barcode position, close the hybridization cassette, and incubate it in the hybridization cassette at 48 °C for 18 hours.

[0081] 4) Add 330 μL of 100% ethanol to XC4, mix it vigorously for 15 seconds, and let it stand overnight at room temperature for use.

[0082] (4) Washing and Scanning

[0083] The reagents used are shown in Table 4.

[0084] Table 4

[0085] Name Purchase Source RA1 Illumina XC1 Illumina XC2 Illumina TEM Illumina XC3 Illumina STM Illumina ATM Illumina PB1 Illumina PB1 Illumina Multi-Sample BeadChip Alignment Fixture Illumina Te-Flow Flow-Through Chambers Illumina Wash Dish Illumina Wash Rack Illumina

[0086] 1) Take out the hybridization cassette from the hybridization oven without opening it, and let the hybridization cassette cool to room temperature for 25 minutes.

[0087] 2) 200 mL of PB1 per Wash Dish (two in total). Add 150 mL of PB1 to the Bead Chip Alignment Fixture. Prepare clear plastic spacers and glass back plates, and install the Wash Rack.

[0088] 3) Take out RA1, XC1, XC2, TEM, ST, and ATM, thaw them, and centrifuge at 3000 x g for 1 min for later use.

[0089] 4) Prepare 95% formamide / 1 mM EDTA: 1 mL per chip.

[0090] 5) Turn on Te-Flow, expel air bubbles, and adjust the temperature to be stable at 44 °C. Place the Wash Rack in the first Wash Dish and ensure it is completely immersed in PB1.

[0091] 6) Take out the Hyb Chamber inserts with the chip in the hybridization box. Remove the chip from the inserts, lift off the IntelliHyb seal, and immediately place the chip in the Wash Rack. Lift the Wash Rack with the chip up and down for 1 min, noting to be slow and gentle, and leave the liquid surface each time. Place the Wash Rack with the chip in the second Wash Dish and lift it up and down for 1 min as in the previous step.

[0092] 7) Assemble the Flow-Through Chambers: Place the black frame in the Bead Chip Alignment Fixture, place the chip in the black frame (note that the chip is completely immersed in PB1), then correctly place the clear plastic spacer, then place the Alignment bar above the Fixture, and then correctly place the clean glass black plate (the bevel corresponds to the chip barcode and there are no air bubbles between the plate and the chip), and then correctly place the clamps at both ends, 5 mm away from the upper and lower edges.

[0093] 8) Take out the Flow-Through Chambers, cut off the excess spacer, and place the Flow-Through Chambers horizontally until the next step.

[0094] 9) The temperature of Te-flow is stabilized at 44 °C. Place the Flow-Through Chambers correctly on the Chambers Rack with the chip barcode facing up. Then, continue to add the following substances to the inclined plane of the glass black plate in the following order: 150 μL of RA1, incubate for 30 seconds, repeat five times; 450 μL of XC1, incubate for 10 min; 450 μL of XC2, incubate for 10 min; 200 μL of TEM, incubate for 15 min; 450 μL of 95% formamide / 1 mM EDTA, incubate for 1 min, repeat once, and then incubate for 5 min.

[0095] 10) Adjust the temperature of Te-flow to 32 °C (consistent with the STM required temperature). Add 450 μL of XC3, incubate for 1 min, repeat once, and wait for the temperature of Te-flow to stabilize at 32 °C. Then, turn on the iScan and continue to add the following substances to the inclined plane of the Glass back plate in the following order: 250 μL of STM, incubate for 10 min; 450 μL of XC3, incubate for 1 min, repeat once, and then incubate for 5 min; 250 μL of ATM, incubate for 10 min;. 450 μL of XC3, incubate for 1 min, repeat once, and then incubate for 5 min; 250 μL of STM, incubate for 10 min; 450 μL of XC3, incubate for 1 min, repeat once, and then incubate for 5 min; 250 μL of ATM, incubate for 10 min; 450 μL of XC3, incubate for 1 min, repeat once, and then incubate for 5 min; 250 μL of STM, incubate for 10 min; 450 μL of XC3, incubate for 1 min, repeat once, and then incubate for 5 min.

[0096] 11) Remove the Flow-Through Chambers from the Chamber Rack and place them horizontally. Add 310 μL of PB1 to the first Wash Dish, place the staining rack in it, remove the Flow-Through Chambers, take out the chip, and place it in the staining rack (note that the chip barcode faces away from the experimenter and the locking arm of the staining rack faces the experimenter). Lift the staining rack with the chip up and down 10 times, note to be slow and gentle, and leave the liquid surface each time, then soak for 5 min. After shaking XC4 vigorously, add 310 ml of XC4 to the second wash Dish, take out the staining rack from the first wash dish and put it into the second wash dish, lift the staining rack with the chip up and down 10 times, note to be very slow and gentle, and leave the liquid surface each time, then soak for 5 min.

[0097] 12) After taking out the staining rack with the chip, immediately place the chip face-up horizontally on the tuberack and dry the chip in a 508 mmHg (0.68 bar) vacuum pump for 50 - 55 min. Wipe the back of the chip, i.e., the XC4 dried on both sides, with Kimwipes to keep the back of the chip flat.

[0098] 13) Place the chip in its original packaging and place it in a vacuum pump at room temperature. Perform an iScan within 72 hours: Start the software iScan to scan the chip. Pre-download the chip decoding file (dmap). Pre-download the chip manifest file (bpm).

[0099] The experimental results are shown in Figure 1 , Figure 1 which lists the methylation levels of the methylation probe sites in the training set (peripheral blood of 10 healthy 35-year-old women, peripheral blood of 10 healthy 45-year-old women, peripheral blood of 10 healthy 55-year-old women, peripheral blood of 10 healthy 65-year-old women, peripheral blood of 10 healthy 75-year-old women). Each point represents a mixture of 10 samples.

[0100] According to the experimental results of the test set, the determination criteria recorded in Table 5 can be obtained.

[0101] Table 5

[0102] Methylation Site <35 35-45 45-55 55-65 65-75 >75 cg21294974 >0.62 0.54-0.62 0.52-0.54 0.44-0.52 0.39-0.44 <0.39

[0103] Example 2 Verification of Methylation Sites for Predicting Women's Physiological Age

[0104] The verification set samples include: peripheral blood of 10 healthy 40-year-old women, peripheral blood of 10 healthy 50-year-old women, peripheral blood of 10 healthy 60-year-old women, peripheral blood of 10 healthy 70-year-old women, peripheral blood of 10 healthy 80-year-old women. The methylation level detection method is the same as that recorded in Example 1. The experimental results are shown in Figure 2 .

[0105] The experimental results show that the female physiological age prediction model provided by the present invention has good accuracy.

[0106] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than a limitation on the protection scope of the present invention. The sites of the present invention are intended to reflect the differences between different age groups. However, for specific difference determination values, there may be certain differences among different populations. But in the case of clear sites, it is easy for those skilled in the art to confirm the determination criteria for personnel in different regions or with specific inclusion criteria through conventional technical means. Any simple modification or equivalent replacement of the technical solution of the present invention by those of ordinary skill in the art does not depart from the essence and scope of the technical solution of the present invention.

Claims

1. Use of methylation marker cg21294974 in predicting the physiological age of females.

2. A method for predicting a woman's physiological age, characterized in that, It includes the following steps: measuring the methylation level of locus cg21294974 in a biological sample, and predicting the physiological age of a female based on the methylation level.

3. The method according to claim 2, characterized in that, The biological sample includes: blood and lymph fluid.

4. The method according to claim 3, characterized in that The biological sample is: peripheral blood.

5. The method according to claim 2, wherein The measuring method of the methylation level includes: bisulfite sequencing PCR, methylation-specific PCR, pyrosequencing, Sanger sequencing, high-throughput sequencing, or third-generation sequencing or single-molecule sequencing.

6. Use of the methylation marker according to claim 1 in the preparation of a product for predicting the physiological age of a female, said product comprising: Kit, chip or test strip.

7. A kit for predicting the physiological age of women, characterized in that, The kit includes reagents required for detecting the methylation level of locus cg21294974.

8. The kit according to claim 7, characterized in that, The reagent includes at least one of: primers, Taq DNA polymerase, free nucleotides (dNTPs), MgCl2 and buffer.

9. A system for predicting a female's physiological age, wherein, The system includes a detection device, a computing device and an output device. The detection device includes a sampler and a detector, and the detector is used for detecting the methylation level of the cg21294974 methylation biomarker in the sample.

10. The system according to claim 9, wherein The computing device includes a memory and a processor, and the processor makes the following discrimination: the methylation level of the cg21294974 methylation biomarker in the sample indicates the physiological age of the subject corresponding to the sample.

Images