CN111424098A

CN111424098A - Human health longevity marker based on specific peripheral blood DNA methylation sites and application

Info

Publication number: CN111424098A
Application number: CN202010242240.8A
Authority: CN
Inventors: 孔庆鹏; 肖富辉; 王昊天
Original assignee: Kunming Institute of Zoology of CAS
Current assignee: Kunming Institute of Zoology of CAS
Priority date: 2020-03-31
Filing date: 2020-03-31
Publication date: 2020-07-17

Abstract

The invention discloses a human health longevity marker based on specific peripheral blood DNA methylation sites and application thereof, wherein the total number of the markers is 90, the markers can be used for predicting human health longevity alone or in combination of two or more than two, and the markers have the characteristics of high accuracy and high sensitivity, wherein the AUC values of 14 sites exceed 0.85, and the markers can be well used for evaluating the potential of individual health longevity.

Description

Human health longevity marker based on specific peripheral blood DNA methylation sites and application

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to a human health and longevity marker based on a specific peripheral blood DNA methylation site and application thereof.

Background

The global population ages to a great extent and cannot be reversed. The population base of China is large, and the aging situation is more severe. Elderly people often suffer from one or more of the geriatric diseases, and the medical care costs of elderly people increase dramatically with age. Statistical data show that the medical care expenditure of the old aged over 85 years old can reach 3 times of that of the old aged 65-74 years old. Therefore, the early evaluation of the potential of the old for the health and the long life is realized, the health risk early warning function can be realized, and the public health cost is saved.

In the early days, the prediction method of human longevity potential is mainly based on the detection of DNA polymorphic sites, for example, a gene detection kit related to aging or longevity is patented (application No. 200710179002.1, application date: 2007-12-07). However, genome data of many people with long life show that genetic bases such as gene mutation are difficult to completely explain the reason of long life of people with long life, because: first, fewer longevity-associated genetic variations have been identified; second, different longevity populations carry different longevity-related genetic variations. Therefore, the technology developed based on the DNA polymorphic site detection of the longevity population has many limitations in the aspect of popularization and application, and the development of a new technology which can be widely applied and can predict the potential of the health and longevity of the old is urgently needed.

The relationship between epigenetic modification (epigenetic modification) and the occurrence of aging and senile diseases is receiving wide attention. Among these, DNA methylation, the most widely-focused apparent modification, exists mainly in the form of 5mC, i.e., methylation of the 5 th carbon atom of DNA cytosine catalyzed by DNA methyltransferase. DNA methylation modification has now been used in the development of epigenetic clocks that predict, assess, the physical and physiological age of an individual. However, the technologies are developed based on DNA methylation-age correlation of the general population, and no biomarker and reference standard for evaluating human health and longevity based on DNA methylation exist at present.

Disclosure of Invention

In view of the above, the present invention provides a human health longevity marker based on specific peripheral blood DNA methylation sites and applications thereof.

In order to solve the technical problems, the invention discloses a human health longevity marker based on a specific peripheral blood DNA methylation site, wherein the specific peripheral blood DNA methylation site comprises: chr, chr, chr22:26717474, chr15:88774139, chr4:184797994, chr21:41310315, chr8:75667922, chr3:149094893, chr11:24151395, chr3:149095007, chr2:223921132, chr7:151740319, chr4:172976050, chr5:27825819, chr3:149095284, chr7:7369258, chr8:80268282, chr14:64746787, chr4:172973617, chr3:149094817, chr14:81893209, chr3:149095305, chr3:149095202, chr2:176383009, chr3:149095267, chr7: 151740384.

The invention also discloses application of the human health longevity marker based on the specific peripheral blood DNA methylation sites in preparing a human health longevity prediction product, wherein the prediction product comprises one or more reagents of the specific peripheral blood DNA methylation sites, and the reagents are used for predicting and evaluating whether the subject can realize the health longevity or not through the methylation level of the subject DNA methylation sites.

Alternatively, the methylation level of the marker chr1:154390784 is significantly increased in a healthy long-lived population.

Optionally, the markers chr, the methylation level of chr, chr is obviously reduced in healthy people with long life.

Alternatively, the reagents are those required for gene chip based detection or PCR detection of the methylation level of the one or more DNA methylation sites.

Optionally, the predictive product is a kit, chip or assay platform.

The invention also discloses a kit for predicting health and longevity, which comprises one or more of the specific peripheral blood DNA methylation sites.

Compared with the prior art, the invention can obtain the following technical effects:

1) according to the method, based on DNA methylation data of peripheral blood leukocytes of the elderly with long life, key DNA methylation sites relevant to the health and long life of human beings are identified, and a reference standard for the health and long life of the elderly is formulated, so that the method can be well used for evaluating the potential of the health and long life of individuals.

2) The specific DNA methylation locus biomarker for the longevity population has high accuracy and sensitivity, wherein the AUC values of 14 loci exceed 0.85.

Of course, it is not necessary for any one product in which the invention is practiced to achieve all of the above-described technical effects simultaneously.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention and not to limit the invention. In the drawings:

FIG. 1 is a graph showing that the methylation level of the specific site of the longevity population of the invention is independent of age;

FIG. 2 shows that the methylation level of the specific loci of the longevity population of the invention is significantly different between longevity and control population.

Detailed Description

The following embodiments are described in detail with reference to the accompanying drawings, so that how to implement the technical features of the present invention to solve the technical problems and achieve the technical effects can be fully understood and implemented.

Among them, the world health organization defines healthy aging (or longevity) as: in the elderly stage, a series of functional abilities (including self-satisfying self-needs; learning and decision making; acting, establishing and maintaining interpersonal relationships; and contributing to society) can be maintained, which can lead to the process of valuable life for oneself (https:// www.who.int/serving/health-serving/en /). To achieve healthy aging, it is essential that senile diseases (aged disease [ M ]. scientific press, 2011, 1/1) and serious diseases (defined in "standard for defining disease for insurance of serious diseases" started from 4/3/2007) be avoided during aging, and that the elderly stage is passed with little morbidity and extremely low hospitalization rate.

Example 1 human health longevity markers based on specific peripheral blood DNA methylation sites

Based on the comparison of DNA methylation group data of peripheral blood leukocytes of 53 healthy and long-lived elderly people (age range of 100-111 years, average age: 102.4 years, life of over 95 years, and no serious senile disease (defined in "definition of disease insurance usage standard for serious disease started from 4/3 th of 2007)) and 36 control people (age range of 45-71 years, average age: 59.3 years) with matched living environments (consistent living places and similar dietary habits), 90 long-lived population specific DNA methylation sites can be obtained, the markers can be used alone or in combination of two or more of the markers to predict the human healthy and long-lived people, the methylation level and change of each DNA methylation marker site in the healthy and long-lived people can be shown in Table 1, and the corresponding area AUC under the prediction line can be shown as the area under the table 1, so that the 90 methylation sites can be used to better distinguish normal people from healthy people And (4) clustering.

Wherein 53 women healthy and long-lived elders (average age: 102.4 years) are long-lived people, and 36 women with similar living environments (average age: 59.3 years) are common control people.

Of the 90 longevity population-specific peripheral blood DNA methylation sites, 89 methylation levels are significantly reduced in longevity populations, and 1 methylation level is significantly increased in longevity populations (i.e., chr1: 154390784).

TABLE 1 methylation difference between Long-lived and general control populations for each methylation marker site and corresponding area under prediction line AUC

Example 2 prediction of health longevity using specific peripheral blood DNA methylation sites as markers

Step 1, carrying out whole blood genome DNA extraction after anticoagulation peripheral blood of a subject is adopted according to a standard and reasonable flow, and carrying out DNA sequencing after whole genome bisulfite treatment on the obtained DNA.

Step 2, data processing: and (3) obtaining an original fastq file after sequencing the whole genome bisulfite, filtering and cleaning the fastq file by using a Trimmomatic software with default parameters, comparing the filtered fastq file to a human reference genome hg19 by using a Bismark, and dividing the number of methylated cytosines of each site and the total coverage number of the site by the Bismark software to obtain the methylation level of the site (the range is 0-100%, namely the unmethylated site is 0%; and the completely methylated site is 100%).

The invention utilizes the Whole Genome Bisulfite Sequencing (WGBS) technology to obtain the single base difference rate methylation group data of healthy and long-life people and common control people, and screens the specific DNA methylation sites of the long-life people: i.e., the methylation levels of these sites were age independent (Spearman correlation coefficient <0.1& P value > 0.05; fig. 1), i.e., the differences between the longevity and control populations of the sites employed in the present invention were not due to aging, thereby indicating that these sites are longevity-related but not aging-related. And the significant difference exists between the healthy and long-life population and the common control population (the methylation level change difference is greater than 20% & P value is less than 0.05; figure 2). And detecting the methylation level of the sites of the tested individual, and if the methylation level of the site of the tested individual is obviously different from the distinguishing threshold value and the methylation change direction is consistent with that of the elderly of centenaries, indicating that the tested individual can realize good health and long life. Otherwise, the health and longevity can not be realized, and whether the health and longevity can be realized is predicted and evaluated.

Example 3 taking the peripheral blood DNA methylation site chr14:81893209 as an example, the prediction of health longevity is carried out:

step 1-2 corresponds to step 1-2 of example 2.

And 3, obtaining that the methylation level of chr14:81893209 of the subject is 80%, and looking up a table 1, the distinguishing threshold value is 51.83%, and the methylation level of the elderly with long life is lower than that of the control population, and if the measured value of 80% is higher than the distinguishing threshold value of 51.83%, the individual represented by the sample cannot realize good health and long life.

Example 4 the peripheral blood DNA methylation sites chr14:81893209 and chr1:154390784 are taken as examples to illustrate the combined sites for prediction of health longevity:

step 1-2 corresponds to step 1-2 of example 2.

Step 3, obtaining the methylation levels of chr14:81893209 and chr1:154390784 of the subject, wherein the methylation levels are respectively measured to be 40% and 95%, and the distinguishing threshold values are 51.83% and 90.69% respectively by referring to the table 1. Compared with the control population, the methylation level of the elderly with good health and long life who are in good health and long life at chr14:8189320 is lower, the methylation level of the elderly with good health and long life who are in good health and long life at chr1:1543907840 is higher, 40% of the methylation level of the elderly with good health and long life who are in good health and long life at chr14:8189320 is lower than a distinguishing threshold value 51.83%, and the methylation level of the elderly with good health and long life at chr1:1543907840 is higher than a distinguishing threshold value 90%, the individuals represented by the sample can achieve.

While the foregoing description shows and describes several preferred embodiments of the invention, it is to be understood, as noted above, that the invention is not limited to the forms disclosed herein, but is not to be construed as excluding other embodiments and is capable of use in various other combinations, modifications, and environments and is capable of changes within the scope of the inventive concept as expressed herein, commensurate with the above teachings, or the skill or knowledge of the relevant art. And that modifications and variations may be effected by those skilled in the art without departing from the spirit and scope of the invention as defined by the appended claims.

Claims

1. A human health longevity marker based on specific peripheral blood DNA methylation sites, wherein the specific peripheral blood DNA methylation sites are: chr, chr, chr22:26717474, chr15:88774139, chr4:184797994, chr21:41310315, chr8:75667922, chr3:149094893, chr11:24151395, chr3:149095007, chr2:223921132, chr7:151740319, chr4:172976050, chr5:27825819, chr3:149095284, chr7:7369258, chr8:80268282, chr14:64746787, chr4:172973617, chr3:149094817, chr14:81893209, chr3:149095305, chr3:149095202, chr2:176383009, chr3:149095267, chr7: 151740384.

2. Use of a specific peripheral blood DNA methylation site based human health longevity marker according to claim 1 in the manufacture of a human health longevity prediction product comprising one or more agents specific for peripheral blood DNA methylation sites that are predictive of assessing whether a subject is able to achieve health longevity by the methylation level of the subject's DNA methylation sites.

3. The use according to claim 2, wherein the level of methylation of the marker chr1:154390784 is significantly increased in a healthy long-lived population.

4. The application of claim 2, wherein the markers are, the designations chr, the chr, the methylation levels of chr, and chr are remarkably reduced in healthy and long-life people.

5. The use of claim 2, wherein the reagents are reagents required for gene chip based detection or PCR detection of the methylation level of said one or more DNA methylation sites.

6. The use according to any one of claims 2 to 5, wherein the predictive product is a kit, chip or assay platform.

7. A kit for predicting health and longevity, which comprises one or more of the specific peripheral blood DNA methylation sites of claim 1.