CN113801931B - CEBPA, PPARgamma, CREBBP gene SNP detection substance related to ONFH risk and application thereof - Google Patents

CEBPA, PPARgamma, CREBBP gene SNP detection substance related to ONFH risk and application thereof Download PDF

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CN113801931B
CN113801931B CN202110918857.1A CN202110918857A CN113801931B CN 113801931 B CN113801931 B CN 113801931B CN 202110918857 A CN202110918857 A CN 202110918857A CN 113801931 B CN113801931 B CN 113801931B
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宋旸
董晓明
杜珍武
王晓楠
张桂珍
秦彦国
王金成
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Jilin University
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Abstract

The invention discloses a CEBPA, PPARgamma and CREBBP gene SNP detection substance related to ONFH risk and application thereof, in particular to a detection substance related to single nucleotide polymorphism of CEBPA, PPARgamma and CREBBP genes related to non-traumatic femoral head necrosis risk and application thereof, and particularly relates to single nucleotide polymorphism sites of rs17694108, rs3745971, rs10500264, rs2920502, rs2028759, rs3856806, rs2072381, rs9392, rs2283487, rs3751845 and rs129974. The invention firstly explains the association of the 11SNPs and the risk of non-invasive femoral head necrosis, and has great value for the molecular early warning and the molecular level prevention and treatment of the non-invasive femoral head necrosis.

Description

CEBPA, PPARgamma, CREBBP gene SNP detection substance related to ONFH risk and application thereof
Technical Field
The invention belongs to the field of biotechnology, and in particular relates to a substance for detecting single nucleotide polymorphism of a gene related to risk of non-invasive femoral head necrosis, which is further used for molecular early warning, drug treatment target and clinical molecular diagnosis and typing of the non-invasive femoral head necrosis.
Background
Femoral head necrosis (ONFH) is a complex disease caused by the co-action of genetic and environmental factors. Over the last 10 years, the incidence of ONFH has tended to increase year by year. It is estimated that the patients with the average year 20000-30000 are newly diagnosed as ONFH, the new ONFH cases are 150000 ～ 200000 in China every year, the current adult ONFH patients are 812 thousands of people, the main morbidity group is the young and strong years between 40 and 50 years, the disability rate is high, and the serious social and household economic burden is caused, so that the traditional Chinese medicine is one of the main challenges for human health. The early discovery of ONFH genetic molecular diagnosis markers and further early molecular early warning makes the prevention and treatment period of the ONFH genetic molecular diagnosis markers advance, which is a key for reducing the incidence rate of ONFH.
One of the important markers of research progress of the pathogenesis of ONFH molecules in recent years is to sequentially propose that the gene polymorphism of the plasminogen activation/inhibition series, the angiogenesis series, the lipid metabolism series, the collagen type II series, the cytokine series and the growth factor series is related to the risk of the ONFH, and the gene polymorphism form linkage disequilibrium with other genes to jointly influence the onset of the ONFH or influence the expression function of other susceptibility genes, thereby playing a synergistic role in the onset and development of the ONFH. The results of the etiology study of these genetic molecules clearly suggest that ONFH belongs to a complex disease in which multiple genes of micro-efficacy are involved in combination and induced by environmental factors.
CEBPA gene chromosome position 19q13.11, containing 1 exon. The gene encodes a transcription factor that contains a basic leucine zipper (bZIP) domain and recognizes the CCAAT motif in the target gene promoter. The encoded proteins play a role in homodimers and heterodimers containing the CCAAT/enhancer binding proteins β and γ. CEBPA gene-encoded proteins can regulate gene expression involved in cell cycle regulation and weight homeostasis. The gene is involved in the transcriptional co-activation of a variety of different transcription factors.
The chromosomal location 16p13.3 of the CREBBP gene contains 33 exons. The gene was originally isolated as a nuclear protein that binds to the cAMP response element binding protein (CREB), which plays a key role in embryo development, growth control and homeostasis by coupling chromatin remodeling with transcription factor recognition. The protein encoded by this gene has intrinsic histone acetyltransferase activity and acts as a scaffold to stabilize interactions between other proteins and the transcription complex. Chromosomal translocations involving this gene are associated with acute myeloid leukemia.
The PPARgamma gene chromosome position 3p25.2, contains 14 exons. The gene encodes a nuclear receptor peroxisome proliferator-activated receptor (PPAR) subfamily member. PPARs form heterodimers with retinoic acid X receptors (RXRs) that regulate transcription of various genes. Three PPAR subtypes, pparα, pparδ and pparγ, are known. Pparγ is the main controller of adipocyte differentiation. Furthermore, pparγ is associated with the pathology of many diseases, including ONFH, obesity, diabetes, atherosclerosis and tumors.
ONFH lesions often fill large amounts of adipose tissue in the bone marrow cavity, and thus lipid metabolism disorders have long been recognized as one of the key disease links of femoral head necrosis. In view of the full demonstration of animal experiments and clinical researches, the high-fat environment can inhibit the differentiation of osteoblasts, and after hyperlipidemia, the increase of fat cells in bone marrow presses the femoral head capillary to cause ischemic necrosis of bone cells, so that the abnormal regulation of adipogenesis and osteogenesis differentiation probably plays a key role in ONFH occurrence and development, but the molecular mechanism of fat accumulation is not clear.
In recent years, research on the differentiation microenvironment of bone marrow mesenchymal stem cells (BMSCs) has found a key role of adipogenic key transcription factor pparγ in regulating osteogenic and adipogenic differentiation. Pparγ is a class II nuclear receptor and has been defined as the primary regulator of adipocyte differentiation. In the molecular cascade of events that follow the sequence of adipocyte differentiation, upregulation of pparγ gene expression at early stages of adipocyte differentiation is widely used as a marker of adipocyte differentiation. In the event of deletion of pparγ, no other factors can restore adipogenesis, and almost all signaling pathways of pro-adipocytes are concentrated in pparγ, which is a key role in fully demonstrating the role of pparγ in the primary regulator of fat metabolism. Pparγ is well documented to antagonize bone formation, both of which mediate to a large extent the different cytokines that determine the direction of osteogenic or adipogenic differentiation of BMSCs, with increased expression of one transcription factor often being associated with down-regulation of expression of another transcription factor.
ONFH is considered to be a stem cell disorder, and causes massive fat accumulation in the diseased bone marrow cavity due to the differentiation and transdifferentiation disorder of BMSCs in osteogenesis and adipogenesis, and causes necrosis of the femoral head. The osteogenic transcription factors and adipogenic transcription factors including CEBPA, CREBBP and pparγ play a key regulatory role in the adipogenic and osteogenic differentiation process of BMSCs. The osteogenesis and adipogenic differentiation of BMSCs have common regulation pathways, and the Wnt signal pathway is a key signal pathway for controlling the conversion of adipogenic differentiation and osteogenic differentiation, and the adipogenic main transcription factor PPARgamma regulates the Wnt signal pathway towards the direction of adipogenic differentiation, and the osteogenic main transcription factor Runx2 regulates the Wnt signal pathway towards the direction of osteogenic differentiation. The Wnt signaling pathway controls the direction of bone formation and adipogenesis through a series of key events such as regulation of cell proliferation and differentiation. In bone marrow mesenchymal stem cells with osteogenic tendencies, wnt signaling pathway is activated, playing a key role in controlling the balance of osteogenic differentiation and adipogenic differentiation.
In recent years, an important research progress of the pathogenesis of ONFH molecules is to sequentially propose that a plurality of series of gene polymorphisms are related to the risk of the occurrence of ONFH, and the gene polymorphisms and other genes form linkage disequilibrium to jointly influence the occurrence of ONFH or influence the expression function of other susceptible genes, thereby playing a synergistic role in the occurrence and development of ONFH. The results of the etiology study of these genetic molecules clearly suggest that ONFH belongs to a complex disease in which multiple genes of micro-efficacy are involved in combination and induced by environmental factors. However, the main bottleneck problem existing in the research field of the gene polymorphism of ONFH at home and abroad at present is that the data of scattered research gene loci are more, the data of systematic research on the multi-gene loci are less, and the explanation of the molecular etiology and molecular pathogenesis of the combined pathogenicity of multiple micro-effect genes is not facilitated. These bottleneck problems are serious constraints for elucidating the pathogenesis of ONFH molecules, and are also key barriers for interfacing the latest achievements of the etiology of ONFH molecules with clinical control practices.
Disclosure of Invention
In order to solve the technical problems, the innovative integration of the combined application molecular technology is firstly explained at home and abroad into the association of the lipid differentiation transcription factor PPARgamma and transcription co-factors CEBPA, CREBBP genotyping, allele frequency and gene combination with ONFH morbidity risk, a key molecular target is provided for establishing a molecular level control countermeasure of ONFH, the method is used for screening and early intervention of susceptible people of ONFH, the research result of molecular etiology is promoted to be in early butt joint with the clinical control of ONFH, and the method has important application value in non-invasive femoral head necrosis molecular early warning, molecular diagnosis and therapeutic drug target.
An object of the present invention is to provide a substance for detecting single nucleotide polymorphisms of CEBPA, pparγ and CREBBP genes associated with risk of non-invasive femoral head necrosis, use of the substance for diagnosis or auxiliary diagnosis of non-invasive femoral head necrosis, or use of the substance for preparation of a product for diagnosis or auxiliary diagnosis of risk of non-invasive femoral head necrosis; or the application of the substance in evaluating or assisting in evaluating the non-invasive femoral head necrosis or in preparing a product for evaluating or assisting in evaluating the risk of the non-invasive femoral head necrosis; the combination of the site related to non-traumatic femoral head necrosis or the site having interaction is any one or more of the following (1) to (9):
(1) The rs17694108 locus of CEBPA gene; (2) the rs3745971 locus of the CEBPA gene; (3) A combination of a PPARgamma gene rs2920502 locus and a CREBBP gene rs2072381 locus; (4) A combination of the PPARgamma gene rs2028759 locus and the CEBPA gene rs10500264 locus; (5) A combination of the PPARgamma gene rs3856806 locus and the CEBPA gene rs17694108 locus; (6) A combination of the rs9392 locus of the CREBBP gene and the rs2283487 locus of the CREBBP gene; (7) A combination of the rs3751845 locus of the CREBBP gene and the rs2283487 locus of the CREBBP gene; (8) A combination of the rs2072381 locus of the CREBBP gene and the rs10500264 locus of the CREBBP gene; (9) And (3) combining the rs129974 locus of the CREBBP gene and the rs2283487 locus of the CREBBP gene.
Further, the substance is a product for preparing a prediction or auxiliary prediction of the risk of non-invasive femoral head necrosis, or the substance is a product for preparing an evaluation or auxiliary evaluation of the risk of non-invasive femoral head necrosis, wherein the product comprises a readable carrier recorded with at least one of the following conditions A to N:
A. the risk of non-invasive femoral head necrosis of a human to be tested, carrying the rs17694108 locus as the minimum homozygous genotype GG, is reduced;
B. the minimal allele G frequency of the rs17694108 locus is lower than that of a human to be tested in a control group, so that the risk of non-traumatic femoral head necrosis is reduced;
C. the rs17694108 locus genetic dominant model (gg+agvsaa) is associated with reduced risk of traumatic femoral head necrosis;
D. the rs17694108 locus genetic recessive model (ggvsag+aa) is associated with reduced risk of traumatic femoral head necrosis;
E. the risk of non-invasive femoral head necrosis of a human to be tested carrying the minimum homozygous genotype TT of the rs3745971 locus is increased;
F. the risk of non-traumatic femoral head necrosis of a person to be tested with high minimum allele T frequency carrying the rs3745971 locus is increased;
G. the rs3745971 locus genetic dominant model (tt+tcvscc) is associated with increased risk of traumatic femoral head necrosis;
H. The risk of non-invasive femoral head necrosis of a human to be tested carrying the minimum homozygous genotype GG of the locus rs2920502 and the minimum homozygous genotype AA of the locus rs2072381 is increased;
I. the risk of non-traumatic femoral head necrosis of a human to be tested carrying the minimum homozygous genotype CC type of the rs2028759 locus and the minimum homozygous genotype AA type of the rs10500264 locus is increased;
J. the risk of non-traumatic femoral head necrosis of a human to be tested carrying the minimum homozygous genotype AA type of the rs9392 locus and the minimum homozygous genotype GG type of the rs2283487 locus is increased;
K. the risk of non-traumatic femoral head necrosis of a human to be tested carrying the minimum homozygous genotype AA type of the rs3751845 locus and the minimum homozygous genotype GG type of the rs2283487 locus is increased;
l, carrying the minimum homozygous genotype TT type of the rs129974 site and the minimum homozygous genotype GG type of the rs2283487 site, wherein the risk of the non-invasive femoral head necrosis of the human to be tested is increased;
and M, the risk of non-traumatic femoral head necrosis of a human to be tested carrying the minimum homozygous genotype TT type at the locus rs3856806 and the minimum homozygous genotype GG type at the locus rs17694108 is reduced.
And N, reducing the risk of non-traumatic femoral head necrosis of the human to be tested carrying the minimum homozygous genotype AA type of rs2072381 and the minimum homozygous genotype AA type of rs 10500264.
Further, the rs17694108 locus is 33240645 nucleotides from the 5' end of the human genome chromosome 19; the nucleotide at the rs17694108 locus is G or A. The rs3745971 locus is 33304320 nucleotides from the 5' end of a human genome chromosome 19; the nucleotide at the site of rs3745971 is C or T. The rs10500264 locus is 33259408 nucleotides from the 5' end of a human genome chromosome 19; the nucleotide at the rs10500264 locus is G or A. The rs2920502 locus is 12287696 nucleotides from the 5' end of a human genome chromosome 3; the nucleotide at the rs2920502 locus is G or C. The rs2028759 locus is 12377113 nucleotides from the 5' end of a human genome chromosome 3; the nucleotide at the site of rs2028759 is C or T. The rs3856806 locus is 12434058 nucleotides from the 5' end of a human genome chromosome 3; the nucleotide at the site of rs3856806 is C or T. The locus rs2072381 is 3731312 nucleotides from the 5' end of a human genome chromosome 16, and the nucleotide of the locus rs2072381 is G or A. The locus rs9392 is 3725168 nucleotides from the 5' end of a human genome chromosome 16, and the nucleotide of the locus rs9392 is G or A. The locus rs2283487 is 3919885 nucleotides from the 5' end of a human genome chromosome 16, and the nucleotide of the locus rs2283487 is A or G. The locus rs3751845 is 3728336 nucleotides from the 5' end of a human genome chromosome 16, and the nucleotide of the locus rs3751845 is G or A. The locus rs129974 is 3745291 nucleotides from the 5' end of a human genome chromosome 16, and the nucleotide of the locus rs129974 is C or T.
Further, the substance is a reagent for direct sequencing; or reagents for polymerase chain reaction in combination with restriction fragment length polymorphism analysis; or reagents for polymerase chain reaction in combination with direct sequencing; or a reagent for use in any one of the following SNP typing methods: hybridization-based methods, primer extension-based methods, conformation-based methods, or high resolution dissolution profile analysis techniques.
Further, the substance for detecting the single nucleotide gene polymorphism of the rs17694108 locus is primer pair A or complete set single-stranded DNA molecule A; the primer pair A consists of a single-stranded DNA molecule shown as SEQ ID NO.1 and a single-stranded DNA molecule shown as SEQ ID NO. 2: the complete set of single-stranded DNA molecules A consists of single-stranded DNA shown as SEQ ID NO.1, single-stranded DNA shown as SEQ ID NO.2 and single-stranded DNA shown as SEQ ID NO. 3.
Further, the substance for detecting the single nucleotide gene polymorphism of the rs3745971 locus is a primer pair B or a complete set of single-stranded DNA molecules B; the primer pair B consists of a single-stranded DNA molecule shown as SEQ ID NO.4 and a single-stranded DNA molecule shown as SEQ ID NO. 5; the complete set of single-stranded DNA molecules B consists of single-stranded DNA shown in SEQ ID NO.4, single-stranded DNA shown in SEQ ID NO.5 and single-stranded DNA shown in SEQ ID NO. 6.
Further, the substance for detecting single nucleotide gene polymorphism of rs10500264[ G/A ] locus is primer pair C or complete set single-stranded DNA molecule C; the primer pair C consists of a single-stranded DNA molecule shown as SEQ ID NO.7 and a single-stranded DNA molecule shown as SEQ ID NO. 8; the complete set of single-stranded DNA molecules consists of single-stranded DNA shown in SEQ ID NO.7, single-stranded DNA shown in SEQ ID NO.8 and single-stranded DNA shown in SEQ ID NO. 9.
Further, the substance for detecting the single nucleotide gene polymorphism of the rs2920502 locus is a primer pair D or a complete set of single-stranded DNA molecules D; the primer pair D consists of a single-stranded DNA molecule shown as SEQ ID NO.10 and a single-stranded DNA molecule shown as SEQ ID NO. 11; the complete set of single-stranded DNA molecules consists of single-stranded DNA shown in SEQ ID NO.10, single-stranded DNA shown in SEQ ID NO.11 and single-stranded DNA shown in SEQ ID NO. 12.
Further, the substance for detecting the single nucleotide gene polymorphism of the rs2028759 locus is primer pair or complete set of single-stranded DNA molecules; the primer pair consists of a single-stranded DNA molecule shown as SEQ ID NO.13 and a single-stranded DNA molecule shown as SEQ ID NO. 15; the complete set of single-stranded DNA molecules consists of single-stranded DNA shown in SEQ ID NO.13, single-stranded DNA shown in SEQ ID NO.14 and single-stranded DNA shown in SEQ ID NO. 15.
Further, the substance for detecting the single nucleotide gene polymorphism of the rs3856806 locus is a primer pair or a complete set of single-stranded DNA molecules; the primer pair consists of a single-stranded DNA molecule shown as SEQ ID NO.16 and a single-stranded DNA molecule shown as SEQ ID NO. 17; the complete set of single-stranded DNA molecules consists of single-stranded DNA shown in SEQ ID NO.16, single-stranded DNA shown in SEQ ID NO.17 and single-stranded DNA shown in SEQ ID NO. 18.
Further, the substance for detecting the single nucleotide gene polymorphism of the rs2072381 locus is a primer pair heptyl or a complete set of single-stranded DNA molecules heptyl; the primer pair heptyl consists of a single-stranded DNA molecule shown in SEQ ID NO.19 and a single-stranded DNA molecule shown in SEQ ID NO. 20; the complete set of single-stranded DNA molecules comprises single-stranded DNA shown in SEQ ID NO.19, single-stranded DNA shown in SEQ ID NO.20 and single-stranded DNA shown in SEQ ID NO. 21.
Further, the substance for detecting the single nucleotide gene polymorphism of the rs9392 locus is primer pair or complete set single-stranded DNA molecule octyl; the primer pair consists of a single-stranded DNA molecule shown as SEQ ID NO.22 and a single-stranded DNA molecule shown as SEQ ID NO. 23; the complete set of single-stranded DNA molecules consists of single-stranded DNA shown in SEQ ID NO.22, single-stranded DNA shown in SEQ ID NO.23 and single-stranded DNA shown in SEQ ID NO. 24.
Further, the substance for detecting the single nucleotide gene polymorphism of the rs2283487 locus is a primer pair or a complete set of single-stranded DNA molecules; the primer pair nonconsists of a single-stranded DNA molecule shown as SEQ ID NO.25 and a single-stranded DNA molecule shown as SEQ ID NO. 26; the complete set of single-stranded DNA molecules nonconsists of single-stranded DNA shown in SEQ ID NO.25, single-stranded DNA shown in SEQ ID NO.26 and single-stranded DNA shown in SEQ ID NO. 27.
Further, the substance for detecting the single nucleotide gene polymorphism of the rs3751845 locus is primer pair decyl or complete set single-stranded DNA molecule decyl; the primer pair decyl consists of a single-stranded DNA molecule shown as SEQ ID NO.28 and a single-stranded DNA molecule shown as SEQ ID NO. 29; the complete set of single-stranded DNA molecules consists of single-stranded DNA shown in SEQ ID NO.28, single-stranded DNA shown in SEQ ID NO.29 and single-stranded DNA shown in SEQ ID NO. 30.
Further, the substance for detecting the single nucleotide gene polymorphism of the rs129974 locus is a primer pair or a complete set of single-stranded DNA molecules; the primer pair consists of a single-stranded DNA molecule shown as SEQ ID NO.31 and a single-stranded DNA molecule shown as SEQ ID NO. 32; the complete set of single-stranded DNA molecules consists of single-stranded DNA shown in SEQ ID NO.31, single-stranded DNA shown in SEQ ID NO.32 and single-stranded DNA shown in SEQ ID NO. 33.
Further, the product for predicting or aiding in predicting or evaluating the risk of non-traumatic femoral head necrosis is a kit comprising the substance for detecting single nucleotide polymorphisms of (1) to (9) sites or combinations of sites related to the risk of non-traumatic femoral head necrosis.
The invention also provides a targeted therapeutic drug, which takes any one or more of the sites (1) to (9) related to non-traumatic femoral head necrosis or the site combination with interaction as a target gene.
The invention firstly explains the association of the 11SNPs and the ONFH morbidity risk, and has great value for ONFH molecular early warning and molecular level control. The invention provides application of CEBPA gene single nucleotide polymorphic locus rs17694108, rs3745971, rs10500264 marker, PPAR gamma gene single nucleotide polymorphic locus rs2920502, rs2028759, rs3856806 marker, CREBBP gene single nucleotide polymorphic locus rs2072381, rs9392, rs2283487, rs3751845, rs129974 marker combination in detection of non-traumatic femoral head necrosis onset risk, and further application in ONFH molecular early warning, clinical molecular diagnosis and parting, and drug treatment target.
The invention provides an application of a combination of rs17694108, rs3745971, rs10500264, rs2920502, rs2028759, rs3856806, rs2072381, rs9392, rs2283487, rs3751845, rs129974 locus and interaction thereof in ONFH morbidity risk and molecular early warning, clinical molecular diagnosis and parting, or as an ONFH drug treatment target and a product thereof.
According to the invention, through the research of an ONFH clinical case control system of Chinese Han population, the minimum homozygous genotype GG type of CEBPA gene mononucleotide polymorphic site rs17694108 and the minimum allele G frequency are found to be obviously related to the reduction of the risk of ONFH, and the dominant model (GG+AGvsaA) and the recessive model (GGvsaG+AA) of CEBPArs17694108 site are also obviously related to the reduction of the risk of ONFH; the interaction of rs2072381 and rs10500264 and the interaction of rs3856806 and rs17694108 are also obviously related to the reduction of the incidence risk of ONFH, and can be used as a molecular protection marker for reducing the incidence risk of ONFH. Whereas, the minimum allele T frequency of the single nucleotide polymorphism locus rs3745971 of the CEBPA gene and the genetic dominant model (TT+TCvsCC) are all obviously related to increasing the risk of onFH; the interaction of rs2920502 and rs2072381, the interaction of rs2028759 and rs10500264, the interaction of rs9392 and rs2283487, the interaction of rs3751845 and rs2283487 and the interaction of rs129974 and rs2283487 are found to be obviously related to increasing the incidence risk of ONFH, and the molecular marker can be used as an ONFH molecular early warning, molecular diagnosis and clinical drug treatment target application molecular marker.
Drawings
FIG. 1 is a flow chart of MassarraySNP typing experiments.
Detailed Description
The experimental methods used in the following examples are conventional methods unless otherwise specified.
The experimental materials and reagents used in the examples described below were commercially available unless otherwise specified.
Application of example 1, CEBPA, PPARg, CREBBP transcription factor gene combination in non-traumatic femoral head necrosis molecular pre-warning, molecular diagnosis and therapeutic drug targets.
1. Establishing an ONFH case contrast research system; based on informed consent, the ONFH case control study system was 600 cases total, with 300 healthy control groups and 300 ONFH patient groups. ONFH cases were from hospitalized clinical patients in orthopaedics of the second, third, and third clinical college of medicine, respectively, at the ages of about 54.49±11.8 years, from 6 months 2012 to 10 months. The clinical diagnosis and the stage of ONFH are carried out according to the Ficat diagnosis and the stage standard by eliminating the obvious trauma history of the hip joint, the congenital diseases of the hip joint, the infectious diseases of the hip joint and the tumors of the hip joint. The healthy control group was from healthy physical examination persons at physical examination center of second clinical medical college of Jilin university during 11 months 2013 to 3 months 2014, of which men were 131 and women were 169, and aged 56.5.+ -. 8.61 years. The fasting blood sugar, serum triglyceride and total cholesterol of the method are all at the reference value level of normal people, and the abdominal organs are subjected to ultrasonic examination and chest X-ray film taking without abnormality, so that the history of serious diseases such as cardiovascular and cerebrovascular diseases is eliminated. The above case-control study systems are all Chinese Han people, and individuals have no blood relationship.
2. Extracting peripheral blood leukocyte genome DNA;
2ml of elbow vein blood is collected in a fasting state, and the method is operated according to the specification of a whole blood genome DNA extraction kit, and comprises the following main steps in sequence: anticoagulation 2ml, centrifugation at 2000rpm for 10min, taking plasma and preserving at-80 ℃; adding 1ml of erythrocyte lysate A into the residual cells, and gently reversing and uniformly mixing; 10000g of the mixture is centrifuged for 2min at room temperature, and the supernatant is discarded; re-suspending the precipitate in 500 μl of erythrocyte lysate A, gently blowing and mixing, centrifuging at 10000g at room temperature for min, and removing supernatant; adding 0.6mL of solution A, gently blowing and mixing uniformly, and reacting for 5min at room temperature; adding 0.2mL of chloroform and 0.3mL of solution B, immediately reversing and uniformly mixing; centrifuging at 13000g for 3min at room temperature, transferring the supernatant to a new 1.5mL plastic centrifuge tube; adding 0.7ml of isopropanol, and mixing for 5-8 times in a reverse way to obtain flocculent DNA precipitate; adding 1mL of 75% ethanol, centrifuging for min at 13000g, volatilizing ethanol in the air after removing supernatant, and adding 0.2mL of Tris-EDTA to react and dissolve DNA; taking 1 μl of extracted DNA, and detecting the content and purity of the DNA by using Nanodrop 2000; packaging DNA, and storing at-80deg.C.
3. Optimizing and screening target genes and SNPs thereof;
the SNPs of CEBPA, PPARg, CREBBP genes are queried by using a Hapmap database and related documents, the distribution of the SNPs sites in different countries, nations and regions is compared through a plurality of bioinformatics databases, particularly the distribution data of Asian populations, the distribution of the gene polymorphism accords with Hardway balance, the gene coding region, the promoter region and the introns are respectively selected, 11SNPs are selected as target sites for research, and the selected target gene SNPs sites are shown in Table 1.
TABLE 1
The sequence information of the CREBPA, PPARg, CREBBP gene 11SNPs is shown in Table 2.
TABLE 2
4. MassarraySNP typing experiment flow is shown in figure 1.
Primer design, synthesis and dilution
1. Acquisition of the Gene sequence: (1) registering as a user in a Myagena website; (2) inputting the name of the SNP locus in NCBI webpage (http:// www.ncbi.nlm.nih.gov/projects/SNP /), and displaying according to the dbSNPbachtreport format; (3) the sequence of the SNP locus is sent to a mailbox registered by a Myagena website; (4) selecting a genogyping in the toolbars of the TOOLS of myogena website; (5) clicking RSformat, and selecting a file sent to a mailbox by the NCBI website in a Browse button; (6) after the formatting of the sequence by the website is completed, selecting ProxSNP in a Sentto column; (7) starting ProxSNP, clicking BeginStart; (8) after the above steps are completed, preXTEND is selected in the Sentto column; (9) starting ProxSNP, clicking BeginStart; in the generated results, OUTPUT is selected and the file content is copied into the new text file txt.
2. Primer design PCR reaction and single base extension primer are carried out by adopting AssayDesigner3.1 software, and the primer is synthesized by biological company: (1) selecting a Browse button in a software SNPGroup column to find the generated txt file; (2) selecting SBEMassExtend in AassyDesign column, selecting iPLex in SBEstopmix column, and selecting different reaction weight numbers in M mu-multiplexing level according to actual conditions; (3) SNPcapture, extennprimerdiesign, MASSM μmultiplexing all select default parameters; (4) after parameter setting, clicking Run; (5) the generated primer sequence file is found at the corresponding position of the txt file catalog, and the designed primer sequence is shown in table 3.
TABLE 3 Table 3
3. Primer dilution:
(1) PCRmasttermix primer configuration, diluting a single tube PCRmastter to a concentration of 100 mu M, adding deionized water to mix all single tubes PCRmastter so that the final reaction PCRmasttermix concentration is 0.5 mu M; (2) extndmix primer configuration: the single tube extension primers were diluted to a final concentration of 500. Mu.M, and the primers were mixed so that each primer had a concentration of 8. Mu.M, 10. Mu.M, 15. Mu.M. The molecular weight, mass number and mole number of the primer were calculated according to the instructions of the DNA synthesis product, and the amount of deionized water to be added was calculated according to the desired concentration. (2) And (3) respectively taking 1 times (less than 6300 Da) of the mixed single-tube extension primer (6300 Da to 7200 Da) 1.2 times (more than 7200 Da) of the mixed single-tube extension primer according to the molecular weight, and mixing the single-tube extension primer with 1.5 times of the mixed single-tube extension primer for later use.
(II) AgenaMassArray System genotyping step
Parting principle: amplifying a target fragment containing SNP loci to be detected by PCR reaction, removing residual deoxyribonucleoside triphosphates (dNTPs) and primers in a PCR system by SAP enzyme, wherein PPARgamma, CEBPA, CREBBPPCR primers and a sequencing primer sequence table are shown in table 3, adding a single-base extension primer, wherein the base at the 3' end of the single-base extension primer is close to the SNP loci and is completely complementary with the base on the target fragment, and adopting four ddNTPs to replace the dNTPs, so that the probe extends only one base at the SNP loci, and the connected ddNTPs correspond to alleles of the SNP loci. The difference in molecular weight between the extension product and the unextended primer was detected using matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOFMS) to determine the base at this point.
PCR amplification reaction:
(1) PCRmasttermix was prepared in a 1.5ml EP tube and centrifuged at low speed with shaking, the reaction components being shown in Table 4 below; (2) 8 pipettes are selected, 4 mu.l of PCRmasttermix is added into each sample adding hole of a 384-well plate, and finally 1 mu.l of template DNA (20 ng/. Mu.l) is added for uniform mixing, 384 Kong Fengban films are carefully covered, each hole is firmly pressed, and the phenomena of evaporation and the like are prevented during the PCR procedure. Centrifuging at 1000rpm for 1minute; (3) the following PCR amplification reaction procedure was set up: 94 ℃ for 5min;94℃20sec,56℃30sec,72℃1min,45cycles;72 ℃ for 3min;4 ℃ is infinity. The PCR reaction plate was placed on a PCR instrument and the procedure was started.
TABLE 4 Table 4
2. Product alkaline phosphatase treatment:
(1) after the PCR reaction is finished, the PCR product is treated with SAP (shrimp alkaline phosphatase) to remove free dNTPs in the system; (2) preparing an alkaline phosphatase treatment reaction solution in a new 1.5ml EP tube, the reaction components of SAPMix being shown in Table 5 below; (3) SAPmix was added to 384-well PCR plates, with a total reaction volume of 7. Mu.l for each alkaline phosphatase treated reaction well, where PCR products were 5. Mu.l, SAPmix 2. Mu.l; (4) after pipetting, 384 Kong Fengban films are carefully covered and each hole is firmly pressed to prevent evaporation and other phenomena during the PCR process, and the following reaction process is carried out after centrifugation; (5) setting an SAP reaction program: 20min at 37 ℃;85 ℃ for 5min;4 ℃ is infinity. And 384 well reaction plates were placed on the PCR instrument and the procedure was started.
TABLE 5
SAPmixofReagent Concentration Volume(1rxn)
Water(HPLCgrade) NA 1.53μl
SAPBuffer 10x 0.17μl
SAPEnzyme 1U/μl 0.30μl
Totalvolume - 2.00μl
3. Single base extension reaction: (1) after the alkaline phosphatase treatment is finished, single-base extension reaction is carried out, and the total volume of a reaction system is 9 mu l; (2) preparing a single base extension reaction solution in a new 1.5ml EP tube, wherein the EXTENDMix reaction components are shown in the following table 6; (3) EXTENDMix was added to 384 well reaction plates. For each reaction well, 2. Mu.l of the single base extension reaction system EXTENDMix; SAP+PCRreaction 7. Mu.l, totalVolume 9. Mu.l; (4) after pipetting, 384 Kong Fengban films were carefully covered and each well was pressed to prevent evaporation and the like during the PCR procedure, and after centrifugation, the following reaction procedure was performed at 94℃for 30sec; [94℃for 5sec, (52℃for 5sec,80℃for 5 sec) 5cycles ]40cycles;4 ℃ is infinity.
TABLE 6
EXTENDMixofReagent Conc.in9μl Volume(1rxm)
Water(HPLCgrade) NA 0.619μl
iPLEXBufferPlus 0.222x 0.200μl
iPLEXTerminationmix 1x 0.200μl
PrimerMix(7μM:14μM) 0.625uM:1.25uM 0.940μl
iPLEXEnzyme 1x 0.041μl
4. And (3) purifying resin: (1) uniformly filling resin in a 384/6MGDimple plate and standing for 10 minutes to air-dry; (2) add 16. Mu.l of water to each well of 384 sample plates; (3) the 384 sample plates were gently flipped over and snapped onto the simple plates, and then tapped to drop the resin into each well of the sample plates; (4) the 384 sample plates were placed in a flip centrifuge and mixed for 30 minutes at room temperature.
5. Chip sample application: the MassarynanodipenserRS 1000 spotter was started and the resin purified extension product was transferred to 384-well spectrochipbioarray.
6. Mass spectrum detection and data output: the spotted SpectroCHIP chip is analyzed by using a MALDI-TOF mass spectrometer, the detection result is used for acquiring original data and a genotyping chart by using TYPER4.0 software, the integrity and the correctness of a data file are checked, and the result is stored in a corresponding storage medium and submitted to a biological information room for analysis.
(III) statistical analysis;
genotype frequency and allele frequency calculations, hardy-Weinberg equilibrium and MAF were examined, and genotype and allele distribution between the comparative case group and control group were compared by the Person chi-square test.
Single SNP association analysis, by Pearson chi-square or Fisher exact test, the differences in genotype and allele at each site of ONFH and disease groups were analyzed to find sites associated with disease.
Correlation of pairwise interactions between SNPs and ONFH risk was accomplished by PLINK software gene interactions (superordinate effects), analysis.
5. Analysis of experimental results
Correlation of ONFH group and control group CEBPA, PPARgamma, CREBBP gene 11SNPs genotyping and ONFH incidence risk, and distribution of specific 11SNPs genotypes in ONFH group and control group is shown in Table 7.
TABLE 7
Sequencing results show that the frequency of the minimum homozygous genotype GG (Minor genotype) of the ONFH group CEBPA gene rs17694108 (G/A) locus is obviously lower than that of a control group, and P= 0.0002345 is shown in table 7; that is, the CEBPA gene rs17694108 (G/A) locus GG type carrier significantly reduces the risk of ONFH onset, lower than carriers of GG or GA genotypes.
Correlation of ONFH group and control group CEBPA, PPARgamma, CREBBP gene 11SNPs allele frequency and ONFH incidence risk, and distribution frequency of SNP allele in case group and control group samples is shown in Table 8.
TABLE 8
Sequencing results show that the frequency of the minimum allele G of the ONFH group CEBPA gene rs17694108 (G/A)) locus is obviously lower than that of a control group, and P= 6.516E-00; OR (95% CI) 0.311 (0.189-0.511), that is, the frequency of the G allele at the Ars17694108 (G/A) locus of the CEBP gene significantly reduces the risk of ONFH onset, and the probability of the human being tested for the allele G having ONFH is less than the human being tested for the allele A.
Sequencing results also found that the frequency of the minimum allele T of the CEBPA gene rs3745971 (T/C) site of the ONFH group was significantly higher than that of the control group, p=0.012; OR (95% CI) 1.636 (1.113-2.406), see Table 8, that is, the frequency of the allele at position T of rs3745971 (T/C) of the CEBPA gene significantly increases the risk of ONFH onset, and the probability of the allele being T of the human being tested for ONFH is high as Yu Dengwei gene being C of the human being tested for ONFH.
And (III) correlation between different genetic models of CEBPA, PPARgamma and CREBBP gene 11SNPs and ONFH morbidity risk.
The analysis results of the different genetic models of the CEBPA gene rs17694108 (G/a)) locus further revealed that the dominant model of the CEBPArs17694108 locus (gg+agvsaa) of the ONFH group was significantly lower than that of the control group, p= 6.099e-005, that is, the minimum homozygous genotype GG frequency of the CEBPA gene rs17694108 (G/a)) locus significantly reduced the ONFH risk of onset, as shown in table 9.
The recessive model (ggvsag+aa) of the CEBPA gene rs17694108 (G/a) locus of the ONFH group was also significantly lower than that of the control group, see table 8, p= 0.005164, that is, the minimal homozygous genotype GG frequency of the CEBPA gene rs17694108 (G/a) locus significantly reduced the risk of ONFH onset, see table 9.
The dominant model of the ONFH group CEBPA gene rs3745971 (G/A) locus (TT+TCvsCC) was significantly higher than that of the control group, P=0.0019, that is, the minimum homozygous genotype TT frequency of CEBPA gene rs3745971 (G/A) locus significantly increased the risk of ONFH onset, as shown in Table 9.
TABLE 9
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* Chi-square statistical analysis
(IV) CEBPA, PPARgamma, CREBBP gene 11SNPs interactions and ONFH risk correlation.
And (3) carrying out pairwise interaction analysis on 11SNPs (rs 17694108, rs3745971, rs10500264, rs2920502, rs2028759, rs3856806, rs2072381, rs9392, rs2283487, rs3751845 and rs 129974) by using a PLINK software gene interaction analysis method. The rs17694108 locus is 33240645 nucleotides from the 5' end of a human genome chromosome 19; the nucleotide of the rs17694108 locus is G or A; the rs3745971 locus is 33304320 nucleotides from the 5' end of a human genome chromosome 19; the nucleotide of the rs3745971 locus is C or T; the rs10500264 locus is 33259408 nucleotides from the 5' end of a human genome chromosome 19; the nucleotide of the rs10500264 locus is G or A; the rs2920502 locus is 12287696 nucleotides from the 5' end of a human genome chromosome 3; the nucleotide of the rs2920502 locus is G or C; the rs2028759 locus is 12377113 nucleotides from the 5' end of a human genome chromosome 3; the nucleotide of the rs2028759 locus is C or T; the rs3856806 locus is 12434058 nucleotides from the 5' end of a human genome chromosome 3; the nucleotide of the rs3856806 locus is C or T; the rs2072381 locus is 3731312 th nucleotide from the 5' end of a human genome chromosome 16, and the nucleotide of the rs2072381 locus is G or A; the rs9392 locus is 3725168 th nucleotide from the 5' end of a human genome chromosome 16, and the nucleotide of the rs9392 locus is G or A; the rs2283487[ A/G ] locus is 3919885 th nucleotide from the 5' end of a human genome chromosome 16, and the nucleotide of the rs2283487 locus is A or G; the rs3751845[ G/A ] locus is 3728336 th nucleotide from the 5' end of a human genome chromosome 16, and the nucleotide of the rs3751845 locus is G or A; the locus rs129974 is 3745291 nucleotides from the 5' end of a human genome chromosome 16, and the nucleotide of the locus rs129974 is C or T. Correlation analysis of different SNP interactions with femoral head necrosis is shown in Table 10.
Table 10
* PLINK chi-square test; ▲ interaction; # PLINK gene interactions (upper effects, epistasis) analysis.
The results of PLINK software analysis of gene interactions indicate that:
interaction between the PPARgamma gene rs2920502[ G/C ] site and the CREBBP gene rs2072381[ G/A ] site significantly increases the risk of developing ONFH, OR:1.812, p=0.0222, see sequence number 1 in table 10, that is, individuals carrying both minimum homozygous genotype GG of pparγ gene rs2920502[ G/C ] locus and minimum homozygous genotype AA of CREBBP gene rs2072381[ G/a ] locus significantly increase the risk of ONFH onset, and can be used as molecular markers for ONFH molecular early warning, molecular diagnosis, clinical drug treatment targets.
Interaction between the pparγ gene rs2028759[ C/T ] site and the CEBPA gene rs10500264[ G/a ] site significantly increases the risk of ONFH onset, OR:1.973, p= 0.0487, see sequence number 2 in table 10, that is, individuals carrying both the minimum homozygous genotype CC of the ppary gene rs2028759[ C/T ] locus and the minimum homozygous genotype AA of the CEBPA gene rs10500264[ G/a ] locus significantly increase the risk of ONFH onset, and can be used as molecular markers for ONFH molecular early warning, molecular diagnosis, and clinical drug treatment targets.
Interaction between the rs9392[ G/A ] locus of the CREBBP gene and the rs2283487[ A/G ] locus of the CREBBP gene significantly increases the risk of ONFH onset, OR:1.472, P= 0.02315, see the sequence number 4 in Table 10, that is, the individuals carrying both the minimum homozygous genotype AA type of the rs9392[ G/A ] locus of the CREBBP gene and the minimum homozygous genotype GG type of the rs2283487[ A/G ] locus of the CREBBP gene obviously increase the incidence risk of ONFH, and can be used as targets of ONFH molecular early warning, molecular diagnosis and clinical drug treatment to apply molecular markers.
Interaction between the rs3751845[ G/A ] locus of the CREBBP gene and the rs2283487[ A/G ] locus of the CREBBP gene significantly increases the risk of ONFH onset, OR:2.125, p= 0.006302, see sequence number 5 in table 10, that is, individuals carrying both the minimum homozygous genotype AA of the rs3751845[ G/a ] locus of the CREBBP gene and the minimum homozygous genotype GG of the rs2283487[ a/G ] locus of the CREBBP gene significantly increase the risk of ONFH onset, and can be used as molecular markers for ONFH molecular early warning, molecular diagnosis, and clinical drug treatment targets.
The interaction between the CREBBP gene rs129974[ C/T ] locus and the CREBBP gene rs2283487[ A/G ] locus obviously increases the incidence risk of ONFH, OR is 1.831, P= 0.01471, the serial number 7 in Table 10 is shown, that is, an individual carrying the minimum homozygous genotype TT of the CREBBP gene rs129974[ C/T ] locus gene and the minimum homozygous genotype GG of the CREBBP gene rs2283487[ A/G ] locus gene obviously increases the incidence risk of ONFH, and the compound can be used as an ONFH molecular early warning, molecular diagnosis and clinical drug treatment target application molecular marker.
The interaction of the CREBBP gene rs2072381[ G/A ] locus and the CEBPA gene rs10500264[ G/A ] locus is obviously related to reducing the incidence risk of ONFH, OR is 0.3681, P= 0.04758, the serial number 6 in Table 10 is shown, that is, an individual carrying the minimum homozygous genotype AA type of the CREBBP gene rs2072381[ G/A ] locus and the minimum homozygous genotype AA type of the CREBBP gene rs10500264[ G/A ] locus obviously increases the incidence risk of ONFH, and the molecular protection marker can be used for reducing the incidence risk of ONFH.
The interaction between the PPARgamma gene rs3856806[ C/T ] locus and the CEBPA gene rs17694108[ G/A ] locus obviously increases and reduces the incidence risk of ONFH, OR:0.4148, P= 0.04181, see the sequence number 3 in Table 10, that is, individuals carrying the minimum homozygous genotype TT of the PPARgamma gene rs3856806[ C/T ] locus and the minimum homozygous genotype GG of the CEBPA gene rs17694108[ G/A ] locus obviously reduce the incidence risk of ONFH, and the individuals can be used as molecular protection markers for reducing the incidence risk of ONFH.
The invention discovers that the interaction of 14SNPs is obviously related to the ONFH morbidity risk for the first time, the risk correlation is partially or completely different from the action of single SNP on the ONFH morbidity risk, the result of gene interaction more clearly discovers the risk action or protection effect of a plurality of SNPs on the ONFH, further verifies that the ONFH is a complex disease caused by the combination of a plurality of micro-effective genes, explains the combined effect of the molecular genetic loci, and has important value for the establishment of early molecular early warning and molecular prevention and control countermeasures of the ONFH. The gene interaction, also known as the superior effect, refers to the action exhibited by two pairs of non-alleles affecting the same trait, one of which (dominant or recessive) inhibits (or masks) the action of the other pair of dominant genes.
The skilled artisan will be able to conduct an analysis of the nucleotides present on the SNP markers disclosed herein in an individual's nucleic acid by any method or technique for detecting SNP sites. For example, one can detect SNP site markers by performing Tagman, mass spectrometry, DNA microarray, micro-sequencing, hybridization, restriction analysis, oligonucleotide ligation detection, allele-specific PCR-HRM, or a combination thereof using the methods of the invention, although this list is merely exemplary and in no way limiting of the invention.
Any suitable method may be used by those skilled in the art to effect such detection.
The main laboratory instruments and equipment involved in the examples of the present invention are shown in table 11 below.
TABLE 11
The major reagents or software involved in the examples of the present invention are as shown in table 12 below.
The above-described embodiments of the present invention do not limit the scope of the present invention. Any of various other corresponding changes and modifications made according to the technical idea of the present invention should be included in the scope of the claims of the present invention.
Sequence listing
<110> Jilin university
<120> CEBPA, PPAR ɣ, CREBBP gene SNP detection substance related to ONFH risk and application thereof
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Claims (4)

1. The use of a substance for detecting single nucleotide polymorphisms of (1) a site, and (2) a site, and (3) a site combination, and (4) a site combination, and (5) a site combination, and (6) a site combination, and (7) a site combination, and (8) a site combination, and (9) a site combination, in the preparation of a product for diagnosing or aiding in diagnosing a human subject with risk of non-invasive femoral head necrosis, or in the preparation of a product for evaluating or aiding in evaluating a human subject with risk of non-invasive femoral head necrosis;
(1) The rs17694108 locus of CEBPA gene;
(2) The rs3745971 locus of CEBPA gene;
(3) The combination of the rs2920502 locus of the PPAR ɣ gene and the rs2072381 locus of the CREBBP gene;
(4) Combination of PPAR ɣ gene rs2028759 locus and CEBPA gene rs10500264 locus;
(5) Combination of PPAR ɣ gene rs3856806 locus and CEBPA gene rs17694108 locus;
(6) A combination of the rs9392 locus of the CREBBP gene and the rs2283487 locus of the CREBBP gene;
(7) A combination of the rs3751845 locus of the CREBBP gene and the rs2283487 locus of the CREBBP gene;
(8) A combination of the rs2072381 locus of the CREBBP gene and the rs10500264 locus of the CEBPA gene;
(9) And (3) combining the rs129974 locus of the CREBBP gene and the rs2283487 locus of the CREBBP gene.
2. The use according to claim 1, wherein: detecting the single nucleotide polymorphism of the loci in (1), and (2), and (3), and (4), and (5), and (6), and (7), and (8), and (9), and the use of a readable carrier describing the conditions in A to N as follows, in the preparation of a product for diagnosing or aiding in diagnosing the risk of non-traumatic femoral head necrosis in a human subject, or in the preparation of a product for evaluating or aiding in evaluating the risk of non-traumatic femoral head necrosis in a human subject;
The product comprises a readable carrier recorded with the following conditions A to N:
A. the risk of non-invasive femoral head necrosis of a human to be tested, carrying the rs17694108 locus as the minimum homozygous genotype GG, is reduced;
B. the minimal allele G frequency of the rs17694108 locus is lower than that of a human to be tested in a control group, so that the risk of non-traumatic femoral head necrosis is reduced;
C. the rs17694108 locus genetic dominant model (gg+agvsaa) is associated with reduced risk of traumatic femoral head necrosis;
D. the rs17694108 locus genetic recessive model (ggvsag+aa) is associated with reduced risk of traumatic femoral head necrosis;
F. the risk of non-traumatic femoral head necrosis of a person to be tested with high minimum allele T frequency carrying the rs3745971 locus is increased;
G. the rs3745971 locus genetic dominant model (tt+tcvscc) is associated with increased risk of traumatic femoral head necrosis;
H. the risk of non-invasive femoral head necrosis of a human to be tested carrying the minimum homozygous genotype GG of the locus rs2920502 and the minimum homozygous genotype AA of the locus rs2072381 is increased;
I. the risk of non-traumatic femoral head necrosis of a human to be tested carrying the minimum homozygous genotype CC type of the rs2028759 locus and the minimum homozygous genotype AA type of the rs10500264 locus is increased;
J. The risk of non-traumatic femoral head necrosis of a human to be tested carrying the minimum homozygous genotype AA type of the rs9392 locus and the minimum homozygous genotype GG type of the rs2283487 locus is increased;
K. the risk of non-traumatic femoral head necrosis of a human to be tested carrying the minimum homozygous genotype AA type of the rs3751845 locus and the minimum homozygous genotype GG type of the rs2283487 locus is increased;
l, carrying the minimum homozygous genotype TT type of the rs129974 site and the minimum homozygous genotype GG type of the rs2283487 site, wherein the risk of the non-invasive femoral head necrosis of the human to be tested is increased;
m, the risk of non-invasive femoral head necrosis of a human to be tested carrying the minimum homozygous genotype TT type at the locus rs3856806 and the minimum homozygous genotype GG type at the locus rs17694108 is reduced;
and N, reducing the risk of non-traumatic femoral head necrosis of the human to be tested carrying the minimum homozygous genotype AA type of rs2072381 and the minimum homozygous genotype AA type of rs 10500264.
3. The use according to claim 1, wherein:
the substance for detecting the single nucleotide gene polymorphism of the rs17694108 locus is a primer pair A or a complete set of single-stranded DNA molecules A; the primer pair A consists of a single-stranded DNA molecule shown as SEQ ID NO.1 and a single-stranded DNA molecule shown as SEQ ID NO. 2: the complete set of single-stranded DNA molecules A consists of single-stranded DNA shown as SEQ ID NO.1, single-stranded DNA shown as SEQ ID NO.2 and single-stranded DNA shown as SEQ ID NO. 3; the substance for detecting the single nucleotide gene polymorphism of the rs3745971 locus is a primer pair B or a complete set of single-stranded DNA molecules B; the primer pair B consists of a single-stranded DNA molecule shown as SEQ ID NO.4 and a single-stranded DNA molecule shown as SEQ ID NO. 5; the complete set of single-stranded DNA molecules B consists of single-stranded DNA shown as SEQ ID NO.4, single-stranded DNA shown as SEQ ID NO.5 and single-stranded DNA shown as SEQ ID NO. 6; the substance for detecting the single nucleotide gene polymorphism of the rs10500264 locus is a primer pair C or a complete set of single-stranded DNA molecules C; the primer pair C consists of a single-stranded DNA molecule shown as SEQ ID NO.7 and a single-stranded DNA molecule shown as SEQ ID NO. 8; the complete set of single-stranded DNA molecules consists of single-stranded DNA shown as SEQ ID NO.7, single-stranded DNA shown as SEQ ID NO.8 and single-stranded DNA shown as SEQ ID NO. 9; the substance for detecting the single nucleotide gene polymorphism of the locus rs2920502 is a primer pair D or a complete set of single-stranded DNA molecule D; the primer pair D consists of a single-stranded DNA molecule shown as SEQ ID NO.10 and a single-stranded DNA molecule shown as SEQ ID NO. 11; the whole set of single-stranded DNA molecules consists of single-stranded DNA shown as SEQ ID NO.10, single-stranded DNA shown as SEQ ID NO.11 and single-stranded DNA shown as SEQ ID NO. 12; the substance for detecting the single nucleotide gene polymorphism of the locus rs2028759 is primer pair or complete set of single-stranded DNA molecules; the primer pair consists of a single-stranded DNA molecule shown as SEQ ID NO.13 and a single-stranded DNA molecule shown as SEQ ID NO. 15; the whole set of single-stranded DNA molecules consists of single-stranded DNA shown as SEQ ID NO.13, single-stranded DNA shown as SEQ ID NO.14 and single-stranded DNA shown as SEQ ID NO. 15; the substance for detecting the single nucleotide gene polymorphism of the rs3856806 locus is a primer pair or a complete set of single-stranded DNA molecules; the primer pair consists of a single-stranded DNA molecule shown as SEQ ID NO.16 and a single-stranded DNA molecule shown as SEQ ID NO. 17; the complete set of single-stranded DNA molecules consists of single-stranded DNA shown as SEQ ID NO.16, single-stranded DNA shown as SEQ ID NO.17 and single-stranded DNA shown as SEQ ID NO. 18; the substance for detecting the single nucleotide gene polymorphism of the rs2072381 locus is a primer pair heptyl or a complete set of single-stranded DNA molecules heptyl; the primer pair heptyl consists of a single-stranded DNA molecule shown as SEQ ID NO.19 and a single-stranded DNA molecule shown as SEQ ID NO. 20; the complete set of single-stranded DNA molecules comprises single-stranded DNA shown as SEQ ID NO.19, single-stranded DNA shown as SEQ ID NO.20 and single-stranded DNA shown as SEQ ID NO. 21; the substance for detecting the single nucleotide gene polymorphism at the locus rs9392 is primer pair octyl or complete set single-stranded DNA molecule octyl; the primer pair consists of a single-stranded DNA molecule shown as SEQ ID NO.22 and a single-stranded DNA molecule shown as SEQ ID NO. 23; the complete set of single-stranded DNA molecules consists of single-stranded DNA shown as SEQ ID NO.22, single-stranded DNA shown as SEQ ID NO.23 and single-stranded DNA shown as SEQ ID NO. 24; the substance for detecting the mononucleotide gene polymorphism of the rs2283487 locus is a primer pair or a complete set of single-stranded DNA molecules; the primer pair nono consists of a single-stranded DNA molecule shown as SEQ ID NO.25 and a single-stranded DNA molecule shown as SEQ ID NO. 26; the complete set of single-stranded DNA molecules nonuses consist of single-stranded DNA shown as SEQ ID NO.25, single-stranded DNA shown as SEQ ID NO.26 and single-stranded DNA shown as SEQ ID NO. 27; the substance for detecting the single nucleotide gene polymorphism of the locus rs3751845 is primer pair decyl or complete set single-stranded DNA molecule decyl; the primer pair decyl consists of a single-stranded DNA molecule shown as SEQ ID NO.28 and a single-stranded DNA molecule shown as SEQ ID NO. 29; the complete set of single-stranded DNA molecules consists of single-stranded DNA shown as SEQ ID NO.28, single-stranded DNA shown as SEQ ID NO.29 and single-stranded DNA shown as SEQ ID NO. 30; the substance for detecting the single nucleotide gene polymorphism of the locus rs129974 is a primer pair or a complete set of single-stranded DNA molecules; the primer pair consists of a single-stranded DNA molecule shown as SEQ ID NO.31 and a single-stranded DNA molecule shown as SEQ ID NO. 32; the complete set of single-stranded DNA molecules consists of single-stranded DNA shown as SEQ ID NO.31, single-stranded DNA shown as SEQ ID NO.32 and single-stranded DNA shown as SEQ ID NO. 33.
4. The use according to claim 1, wherein: the diagnostic or auxiliary diagnostic product for the risk of non-invasive femoral head necrosis of a human to be tested or the product for evaluating or auxiliary evaluating the risk of non-invasive femoral head necrosis is a kit comprising a substance for detecting single nucleotide polymorphisms of (1) to (9) sites or combinations of sites related to the risk of non-invasive femoral head necrosis as claimed in claim 1.
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