CN113801931A - SNP detection substance related to ONFH risk genes CEBPA, PPAR gamma and CREBP and application thereof - Google Patents

SNP detection substance related to ONFH risk genes CEBPA, PPAR gamma and CREBP and application thereof Download PDF

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CN113801931A
CN113801931A CN202110918857.1A CN202110918857A CN113801931A CN 113801931 A CN113801931 A CN 113801931A CN 202110918857 A CN202110918857 A CN 202110918857A CN 113801931 A CN113801931 A CN 113801931A
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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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    • C12Q2600/156—Polymorphic or mutational markers

Abstract

The invention discloses a substance for detecting SNP of CEBPA, PPAR gamma and CREBP genes related to ONFH risk and application thereof, in particular to a substance for detecting single nucleotide polymorphism of CEBPA, PPAR gamma and CREBP genes related to non-traumatic femoral head necrosis risk and application thereof, wherein the single nucleotide polymorphism sites are rs17694108, rs3745971, rs10500264, rs2920502, rs2028759, rs3856806, rs2072381, rs9392, rs2283487, rs3751845 and rs 129974. The invention firstly expounds the correlation between the 11SNPs and the risk of the non-traumatic femoral head necrosis, and has great value on the molecular early warning and the molecular level prevention and treatment of the non-traumatic femoral head necrosis.

Description

SNP detection substance related to ONFH risk genes CEBPA, PPAR gamma and CREBP and application thereof
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a substance for detecting single nucleotide polymorphism of genes related to non-traumatic femoral head necrosis risk, which is further used for molecular early warning, drug treatment targets and clinical molecular diagnosis and typing of the non-traumatic 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 shown a trend to increase year by year. It is estimated that 20000-30000 patients are newly diagnosed as ONFH every year in the United states, the number of new ONFH cases is 150000-200000 every year in China, 812 million patients of ONFH in China are young and old between 40 and 50 years in the main morbidity population, the disability rate is high, serious social and family economic burden is caused, and the method is one of the main challenges for human health at present. The early discovery of the ONFH genetic molecular diagnosis marker and the implementation of early molecular early warning make the prevention and treatment period forward, which is the key to reduce the incidence of ONFH.
One of the important markers of the recent progress of research on the pathogenesis of ONFH molecules is that the gene polymorphisms of the plasminogen activation/inhibition series, the angiogenesis series, the lipid metabolism series, the type II collagen series, the cytokine series and the growth factor series are related to the risk of the ONFH, and the polymorphisms affect the pathogenesis of the ONFH together with linkage disequilibrium with other genes or affect the expression functions of other susceptible genes and play a synergistic role in the development of the ONFH. These genetic molecular etiology studies clearly suggest that ONFH belongs to a complex disease in which multiple micro-effective genes are jointly involved 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 promoter of the target gene. The encoded proteins play a role in homodimers and heterodimers containing CCAAT/enhancer binding proteins β and γ. The CEBPA gene encoding proteins can regulate the expression of genes involved in cell cycle regulation and body weight homeostasis. The gene is involved in the transcriptional co-activation of a number of different transcription factors.
The CREBP gene chromosome position 16p13.3, contains 33 exons. This gene was originally isolated as a nucleoprotein that binds cAMP response element binding protein (CREB), which plays a key role in embryonic development, growth control and homeostasis by coupling chromatin remodeling to transcription factor recognition. The protein encoded by this gene has intrinsic histone acetyltransferase activity and serves as a scaffold to stabilize the interaction between other proteins and the transcription complex. The chromosomal translocation involved in this gene is associated with acute myeloid leukemia.
The PPAR γ gene is at position 3p25.2, and contains 14 exons. The gene encodes a member of the peroxisome proliferator-activated receptor (PPAR) subfamily of nuclear receptors. PPARs form heterodimers with Retinoid X Receptors (RXRs), which regulate transcription of various genes. Three PPAR subtypes, PPAR α, PPAR δ and PPAR γ, are known. PPAR γ is the master controller of adipocyte differentiation. In addition, PPAR γ is involved in the pathology of many diseases, including ONFH, obesity, diabetes, atherosclerosis, and tumors.
The marrow cavity of ONFH lesion is often filled with a large amount of adipose tissues, so that the disorder of lipid metabolism has been recognized as one of the core morbidity links of femoral head necrosis. In view of animal experiments and clinical researches, it is well proven that a high lipid environment can inhibit differentiation of osteoblasts, increase of fat cells in bone marrow after hyperlipidemia compresses femoral head capillaries to cause ischemic necrosis of the osteoblasts, and it is earlier speculated that abnormal regulation and control of adipogenic and osteogenic differentiation may play a key role in generation and development of ONFH (oncofetal necrosis factor necrosis), but a molecular mechanism of fat accumulation of the ONFH is not clear.
In recent years, research on differentiation microenvironment of bone marrow mesenchymal stem cells (BMSCs) finds a key role of a lipogenic key transcription factor PPAR γ in regulating osteogenic and lipogenic differentiation. PPAR γ is a class II nuclear receptor, and has been defined as the primary regulator of adipocyte differentiation. In the molecular cascade of events that are sequential in adipocyte differentiation, the upregulation of PPAR γ gene expression in the early stages of adipocyte differentiation is widely used as a marker of adipocyte differentiation. In the event of PPAR γ deletion, no other factors can restore adipogenesis and nearly all signaling pathways of the preadipocytes will be concentrated in PPAR γ, these key roles fully demonstrate the role of PPAR γ in the master regulator of fat metabolism. PPAR γ is well documented to antagonize bone formation, both of which largely mediate the decision of the osteogenic or adipogenic differentiation direction of BMSCs by different cytokines, with increased expression of one transcription factor often being correlated with down-regulation of the expression of the other.
ONFH is considered a stem cell disease, which is characterized by accumulation of a large amount of fat in the diseased bone marrow cavity and induction of femoral head necrosis due to the dys-differentiation of osteogenic adipogenic differentiation of BMSCs. Osteogenic transcription factors and adipogenic transcription factors including CEBPA, CREBP and PPAR γ play a key regulatory role in the adipogenic and osteogenic differentiation programs of BMSCs. The osteogenesis and adipogenesis differentiation of BMSCs have a common regulation way, a Wnt signal pathway is a key signal pathway for controlling the conversion of adipogenesis and osteogenesis differentiation, an adipogenesis main transcription factor PPAR gamma regulates the Wnt signal pathway to the direction of adipogenesis differentiation, and an osteogenesis main transcription factor Runx2 regulates the Wnt signal pathway to the direction of osteogenesis differentiation. The Wnt signaling pathway controls the direction of bone formation and adipogenesis through a series of key events, such as cell proliferation and differentiation regulation. In osteogenically-prone mesenchymal stem cells, the Wnt signaling pathway is activated, playing a key role in controlling the balance of osteogenic and adipogenic differentiation.
In recent years, important research progress of the pathogenesis of ONFH molecules is to continuously suggest that multiple series of gene polymorphisms are related to the pathogenesis risk of ONFH, and the polymorphisms and other genes form linkage disequilibrium to jointly influence the pathogenesis of ONFH or influence the expression function of other susceptible genes and play a synergistic role in the development of ONFH. These genetic molecular etiology studies clearly suggest that ONFH belongs to a complex disease in which multiple micro-effective genes are jointly involved and induced by environmental factors. However, the major bottleneck problem exists in the research field of ONFH gene polymorphism at home and abroad at present, the scattered research on gene sites has more data, the systematic research on multiple gene sites has less data, and the elucidation of the molecular etiology and the molecular pathogenesis of multiple micro-effect gene combined pathogenesis is not facilitated. These bottleneck problems are the serious constraints for elucidating the pathogenesis of ONFH molecules and are the key obstacles for the recent results of the etiology of ONFH molecules to interface with clinical prevention and treatment practices.
Disclosure of Invention
In order to solve the technical problems, the innovation integration of the combined application molecular technology firstly expounds the association between the adipogenic differentiation transcription factor PPAR gamma and the transcription co-factors CEBPA and CREBP genotyping, allele frequency, combination among genes and the onset risk of ONFH at home and abroad, provides a key molecular target for establishing the molecular level prevention strategy of ONFH, is used for screening and early intervention of susceptible people of ONFH, promotes the research result of molecular etiology to be in early butt joint with the clinical prevention of ONFH, and has important application values in molecular early warning of non-traumatic femoral head necrosis, molecular diagnosis and therapeutic drug targets.
The invention provides a substance for detecting single nucleotide polymorphism of CEBPA, PPAR gamma and CREBP genes related to the risk of non-traumatic femoral head necrosis, and an application of the substance in diagnosing or assisting in diagnosing the non-traumatic femoral head necrosis or preparing a product for diagnosing or assisting in diagnosing the risk of the non-traumatic femoral head necrosis; or the application of the substance in evaluating or assisting in evaluating the non-traumatic femoral head necrosis or in preparing a product for evaluating or assisting in evaluating the risk of the non-traumatic femoral head necrosis; the site related to non-traumatic femoral head necrosis or the site having interaction is combined with any one or more of the following (1) to (9):
(1) rs17694108 locus of CEBPA gene; (2) rs3745971 site of CEBPA gene; (3) the combination of the rs2920502 site of the PPAR gamma gene and the rs2072381 site of the CREBP gene; (4) the combination of the rs2028759 site of the PPAR gamma gene and the rs10500264 site of the CEBPA gene; (5) the combination of the rs3856806 site of the PPAR gamma gene and the rs17694108 site of the CEBPA gene; (6) the combination of the site rs9392 of the CREBP gene and the site rs2283487 of the CREBP gene; (7) the combination of the site rs3751845 of the CREBP gene and the site rs2283487 of the CREBP gene; (8) the combination of the site of CREBP gene rs2072381 and the site of CREBP gene rs 10500264; (9) the combination of the site of the CREBP gene rs129974 and the site of the CREBP gene rs 2283487.
Further, the substance is a product for preparing and predicting or assisting in predicting the risk of non-traumatic femoral head necrosis, or the substance is a product for preparing and evaluating or assisting in evaluating the risk of non-traumatic femoral head necrosis, and the product comprises readable carriers with at least one of the following conditions A to N:
A. the risk of the minimally homozygous genotype GG-type to-be-detected person carrying the rs17694108 locus suffering from non-traumatic femoral head necrosis is reduced;
B. the frequency of the minimum allele G carrying the rs17694108 locus is lower than that of the minimum allele G of the control group, and the risk of non-traumatic femoral head necrosis of the human to be detected is reduced;
C. the rs17694108 locus genetics dominant model (GG + AGvsAA) is related to reducing the risk of traumatic femoral head necrosis;
D. the rs17694108 locus genetic recessive model (GGvsAG + AA) is related to reducing the risk of traumatic femoral head necrosis;
E. the risk of the person to be detected carrying the minimum homozygous genotype TT type at the rs3745971 locus to suffer from non-traumatic femoral head necrosis is increased;
F. the risk of the non-traumatic femoral head necrosis of the person to be detected carrying the rs3745971 locus with high minimum allele T frequency is increased;
G. the rs3745971 site genetic dominant model (TT + TCvsCC) is related to increase the risk of traumatic femoral head necrosis;
H. simultaneously, the risk of non-traumatic femoral head necrosis of a to-be-detected person carrying the GG type with the minimum homozygous locus of rs2920502 and the AA type with the minimum homozygous locus of rs2072381 is increased;
I. simultaneously, the risk of the non-traumatic femoral head necrosis of the human to be detected carrying the CC type of the minimum homozygous genotype at the rs2028759 locus and the AA type of the minimum homozygous genotype at the rs10500264 locus is increased;
J. simultaneously, the risk of the non-traumatic femoral head necrosis of a to-be-detected person carrying the rs9392 locus minimum homozygous genotype AA and the rs2283487 locus minimum homozygous genotype GG is increased;
K. simultaneously, the risk of the non-traumatic femoral head necrosis of a to-be-detected person carrying the rs3751845 locus minimum homozygous genotype AA and the rs2283487 locus minimum homozygous genotype GG is increased;
l, increasing the risk of non-traumatic femoral head necrosis of a to-be-detected person carrying the rs129974 locus minimum homozygous genotype TT type and the rs2283487 locus minimum homozygous genotype GG type at the same time;
m. the risk of non-traumatic femoral head necrosis of a human to be detected carrying the rs3856806 locus minimum homozygous genotype TT type and the rs17694108 locus minimum homozygous genotype GG type at the same time is reduced.
N. the risk of non-traumatic femoral head necrosis of a human to be tested carrying the minimum homozygous genotype AA of rs2072381 and the minimum homozygous genotype AA of rs10500264 is reduced.
Further, the rs17694108 locus is 33240645 th nucleotide from the 5' end of chromosome 19 of the human genome; the nucleotide at the rs17694108 site is G or A. The rs3745971 site is 33304320 th nucleotide from the 5' end of the chromosome 19 of the human genome; the nucleotide at the rs3745971 site is C or T. The rs10500264 locus is 33259408 th nucleotide from the 5' end of the 19 th chromosome of the human genome; and the nucleotide at the rs10500264 site is G or A. The rs2920502 locus is 12287696 th nucleotide from the 5' end of the chromosome 3 of the human genome; and the nucleotide at the rs2920502 site is G or C. The rs2028759 locus is 12377113 th nucleotide from the 5' end of the chromosome 3 of the human genome; and the nucleotide at the rs2028759 site is C or T. The rs3856806 locus is 12434058 th nucleotide from the 5' end of the chromosome 3 of the human genome; and the nucleotide at the rs3856806 site is C or T. The rs2072381 site is 3731312 th nucleotide from the 5' end of the human genome No.16 chromosome, and the nucleotide of the rs2072381 site is G or A. The rs9392 site is 3725168 th nucleotide from the 5' end of the human genome chromosome 16, and the rs9392 site nucleotide is G or A. The rs2283487 site is the 3919885 th nucleotide from the 5' end of the chromosome 16 in the human genome, and the nucleotide at the rs2283487 site is A or G. The rs3751845 site is 3728336 th nucleotide from the 5' end of the human genome No.16 chromosome, and the nucleotide of the rs3751845 site is G or A. The rs129974 site is 3745291 th nucleotide from the 5' end of chromosome 16 of the human genome, and the nucleotide of the rs129974 site is C or T.
Further, the substance is a reagent for direct sequencing; or reagents for polymerase chain reaction coupled with restriction fragment length polymorphism analysis; or reagents for polymerase chain reaction coupled 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 melting curve analysis techniques.
Further, the substance for detecting the single nucleotide gene polymorphism at 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 set of single-stranded DNA molecules A consists of single-stranded DNA shown by SEQ ID NO.1, single-stranded DNA shown by SEQ ID NO.2 and single-stranded DNA shown by SEQ ID NO. 3.
Further, the substance for detecting the single nucleotide gene polymorphism at the rs3745971 site 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 by SEQ ID NO.4, single-stranded DNA shown by SEQ ID NO.5 and single-stranded DNA shown by SEQ ID NO. 6.
Further, the substance for detecting the single nucleotide gene polymorphism of the rs10500264[ G/A ] 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 C consists of single-stranded DNA shown by SEQ ID NO.7, single-stranded DNA shown by SEQ ID NO.8 and single-stranded DNA shown by SEQ ID NO. 9.
Further, the substance for detecting the single nucleotide gene polymorphism of the rs2920502 site 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 by SEQ ID NO.10, single-stranded DNA shown by SEQ ID NO.11 and single-stranded DNA shown by SEQ ID NO. 12.
Further, the substance for detecting the single nucleotide gene polymorphism at the rs2028759 site is primer pair E or complete set of single-stranded DNA molecule E; the primer pair E 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 by SEQ ID NO.13, single-stranded DNA shown by SEQ ID NO.14 and single-stranded DNA shown by SEQ ID NO. 15.
Further, the substance for detecting the single nucleotide gene polymorphism of the rs3856806 site 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 set of single-stranded DNA molecules consists of single-stranded DNA shown by SEQ ID NO.16, single-stranded DNA shown by SEQ ID NO.17 and single-stranded DNA shown by SEQ ID NO. 18.
Further, the substance for detecting the single nucleotide polymorphism of the rs2072381 site is a primer pair G or a complete set of single-stranded DNA molecules G; the primer pair G 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 molecule G consists of single-stranded DNA shown by SEQ ID NO.19, single-stranded DNA shown by SEQ ID NO.20 and single-stranded DNA shown by SEQ ID NO. 21.
Further, the substance for detecting the single nucleotide gene polymorphism of the rs9392 locus is primer pair octyl or a complete set of single-stranded DNA molecule octyl; the primer pair octyl 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 by SEQ ID NO.22, single-stranded DNA shown by SEQ ID NO.23 and single-stranded DNA shown by SEQ ID NO. 24.
Further, the substance for detecting the single nucleotide gene polymorphism of the rs2283487 site is a primer pair nonyl or a complete set of single-stranded DNA molecules nonyl; the primer pair nonane 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 consists of single-stranded DNA shown by SEQ ID NO.25, single-stranded DNA shown by SEQ ID NO.26 and single-stranded DNA shown by SEQ ID NO. 27.
Further, the substance for detecting the single nucleotide gene polymorphism at the rs3751845 locus is a primer pair deca or a complete set of single-stranded DNA molecules deca; the primer pair 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 by SEQ ID NO.28, single-stranded DNA shown by SEQ ID NO.29 and single-stranded DNA shown by SEQ ID NO. 30.
Further, the substance for detecting the single nucleotide gene polymorphism at the rs129974 site is a primer pair or a 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 by SEQ ID NO.31, single-stranded DNA shown by SEQ ID NO.32 and single-stranded DNA shown by SEQ ID NO. 33.
Further, the product for predicting or assisting in predicting the risk of non-traumatic femoral head necrosis or the product for evaluating or assisting in evaluating the risk of non-traumatic femoral head necrosis is a kit, and the kit comprises the substance for detecting single nucleotide polymorphisms of (1) to (9) sites or a combination of the sites related to the risk of non-traumatic femoral head necrosis.
The invention also provides a targeted therapeutic drug, which is a drug taking any one or more of the (1) to (9) sites or the site combination with interaction related to the non-traumatic femoral head necrosis as a target gene.
The invention firstly expounds the correlation between the 11SNPs and the onset risk of ONFH, and has great value on ONFH molecular early warning and molecular level prevention and treatment. The invention provides application of CEBPA gene mononucleotide polymorphism sites rs17694108, rs3745971 and rs10500264 markers, PPAR gamma gene mononucleotide polymorphism sites rs2920502, rs2028759 and rs3856806 markers, CREBP gene mononucleotide polymorphism sites rs2072381, rs9392, rs2283487, rs3751845 and rs129974 marker combinations in detecting the risk of non-traumatic femoral head necrosis, and further used for ONFH molecule early warning, clinical molecule diagnosis and typing and drug therapy targets.
The invention provides a product for detecting rs17694108, rs3745971, rs10500264, rs2920502, rs2028759, rs3856806, rs2072381, rs9392, rs2283487, rs3751845 and rs129974 sites and interaction combination thereof in ONFH (oncogenic acute respiratory syndrome) morbidity risk and molecular early warning, clinical molecular diagnosis and typing, or an application and a product thereof as an ONFH drug treatment target.
According to the invention, through the research of an ONFH clinical case control system of Chinese Han nationality population, the GG type and the G frequency of the minimum homozygous genotype of the mononucleotide polymorphic site rs17694108 of the CEBPA gene are found to be obviously related to the reduction of the risk of suffering from ONFH, and the dominant model (GG + AGvsAA) and the recessive model (GGvsAG + AA) of the CEBPARs17694108 site are also obviously related to the reduction of the risk of suffering from ONFH; the interaction between rs2072381 and rs10500264 and the interaction between rs3856806 and rs17694108 are also obviously related to the reduction of the ONFH onset risk, and can be used as molecular protection markers for reducing the ONFH risk. The frequency of the minimum allele T of the mononucleotide polymorphic site rs3745971 of the CEBPA gene and a genetic dominant model (TT + TCvsCC) are obviously related to the increase of the ONFH risk; the interaction between rs2920502 and rs2072381, the interaction between rs2028759 and rs10500264, the interaction between rs9392 and rs2283487, the interaction between rs3751845 and rs2283487 and the interaction between rs129974 and rs2283487 are all found to be remarkably related to the increase of the onset risk of ONFH, and the molecular markers can be used as the application molecular markers of ONFH molecular early warning, molecular diagnosis and clinical drug therapy targets.
Drawings
FIG. 1 is a flow chart of MassarraySNP typing experiment.
Detailed Description
The experimental procedures used in the following examples are conventional unless otherwise specified.
The test materials and reagents used in the following examples were commercially available unless otherwise specified.
Example 1, application of transcription factor gene combination of CEBPA, PPARg and CREBP in molecular early warning, molecular diagnosis and therapeutic drug target of non-traumatic femoral head necrosis.
Firstly, establishing an ONFH case contrast research system; based on informed consent, the ONFH case-control study system consisted of 600 cases, 300 healthy controls and 300 ONFH patients. The ONFH cases were from hospitalized clinical patients in orthopedics department of the second and third clinical medical college of the university of jilin during months 6 to 2018, respectively, of which 204 were male, 96 were female, and the ages were 54.49 ± 11.8 years. Excluding obvious history of trauma of hip joint, congenital disease of hip joint, infectious disease of hip joint and tumor of hip joint, and carrying out clinical diagnosis and staging of ONFH according to Ficat diagnosis and staging standard. The healthy control group was from healthy examiners at the second clinical medical college health center of the Jilin university during the period from 11 months 2013 to 3 months 2014, with 131 men, 169 women, and the age of 56.5 ± 8.61 years. The fasting blood sugar, the serum triglyceride and the total cholesterol are all at the reference value level of normal people, the ultrasonic examination of abdominal viscera and the chest X-ray radiography are not abnormal, and the history of major diseases such as cardiovascular and cerebrovascular diseases and the like is eliminated. The case-contrast research systems are all Chinese Han nationalities, and individuals have no blood relationship.
Secondly, extracting peripheral blood leukocyte genome DNA;
2ml of cubital vein blood is collected in an empty stomach state, and the operation is carried out according to the specification of the whole blood genome DNA extraction kit, and the main steps are as follows: anticoagulation blood 2ml, centrifuging at 2000rpm for 10min, collecting blood plasma, and storing at-80 deg.C; adding 1ml of erythrocyte lysate A into the residual cells, and slightly reversing and uniformly mixing; centrifuging at room temperature of 10000g for 2min, and removing the supernatant; resuspending the precipitate in 500 μ l erythrocyte lysate A, gently blowing, mixing, centrifuging at 10000g at room temperature for min, and removing supernatant; adding 0.6mL of the solution A, gently blowing, beating and uniformly mixing, and acting at room temperature for 5 min; adding 0.2mL of chloroform and 0.3mL of solution B, immediately reversing and uniformly mixing; centrifuging at 13000g for 3min at room temperature, and transferring the supernatant to a new 1.5mL plastic centrifuge tube; adding 0.7ml of isopropanol, reversing and uniformly mixing for 5-8 times to generate flocculent DNA precipitate; adding 1mL of 75% ethanol, centrifuging for min at 13000g, removing the supernatant, volatilizing the ethanol in the air, and adding 0.2mL of Tris-EDTA to react and dissolve the DNA; taking 1 mu l of extracted DNA, and detecting the content and purity of the DNA by using Nanodrop 2000; the DNA was aliquoted and stored at-80 ℃.
Thirdly, optimizing and screening target genes and SNPs thereof;
the SNPs of CEBPA, PPARg and CREBP genes are inquired 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 biological information bases, particularly the distribution data of Asian people, the distribution of gene polymorphism accords with Hardway balance, 11SNPs are respectively selected as target sites for research in a gene coding region, a promoter region and an intron, and the selected target gene SNPs sites are shown in a table 1.
TABLE 1
Figure BDA0003206688540000081
Figure BDA0003206688540000091
The sequence information of the CREBA, PPARg and CREBP gene 11SNPs is shown in Table 2.
TABLE 2
Figure BDA0003206688540000092
Figure BDA0003206688540000101
Fourthly, MassarraySNP typing experimental process, which is shown in figure 1 specifically.
Primer design, synthesis and dilution
1. Acquisition of gene sequence: registering a user in a Myagena website; inputting the name of the SNP site in an NCBI webpage (http:// www.ncbi.nlm.nih.gov/projects/SNP /), and displaying according to the format of dbSNPbatchreport; thirdly, sending the sequence of the SNP locus to a mailbox registered by a Myagena website; fourthly, selecting Genotyping in the TOOLS tool bar of the Myagena website; clicking RSformat, selecting a file sent to a mailbox by an NCBI website in a Browse button; sixthly, after the formatting of the sequence is finished by the website, selecting ProxSNP in a Sentto column; seventhly, starting ProxSNP, and clicking BeginStart; eighthly, selecting PreXTEND in a Sentto column after the steps are finished; ninthly, beginning ProxSNP, clicking BeginStart; the r selects OUTPUT in the resulting result and copies the file contents in the new text file txt.
2. PCR reaction and single base extension primer design are carried out by adopting AssayDesigner3.1 software, and the primers are synthesized by biological companies: selecting a Browse button in a SNPGroup column of the software to find the txt file generated by the step I; selecting SBEMASSExtend in the column of AssyDesign, selecting iPLex in the column of SBEsetoprox, and selecting different reaction weights in the M mu multiplexLevel according to actual conditions; thirdly, SNPpath, Extendedprimersesign and MASSM [ mu ] plexing all select default parameters; fourthly, clicking Run after setting parameters; fifthly, finding the generated primer sequence file at the corresponding position of the txt file directory, wherein the designed primer sequence is shown in a table 3.
TABLE 3
Figure BDA0003206688540000111
3. Primer dilution:
preparing a PCRMastermix primer, diluting a single tube of PCRMaser to the concentration of 100 mu M, adding deionized water and mixing all the single tubes of PCRMaser to ensure that the concentration of the final reaction PCRMastermix is 0.5 mu M; ② EXTENDMix primer configuration: the single-tube extension primers were diluted to a final concentration of 500. mu.M, and the primers were added and mixed so that the concentrations of the primers were 8. mu.M, 10. mu.M, and 15. mu.M, respectively. And calculating the molecular weight, the mass number and the mole number of the primer according to the use instruction of the DNA synthesis product, and further calculating the amount of the deionized water to be added according to the required concentration. Secondly, the mixed single-tube extension primers are respectively 1 time (less than 6300Da) and 1.2 times (more than 7200Da) of the single-tube extension primers and 1.5 times of the single-tube extension primers according to the molecular weights for standby.
(II) AgenaMassArray system genotyping step
The parting principle is as follows: amplifying a target fragment containing an SNP site to be detected through PCR reaction, removing residual deoxyribonucleoside triphosphate (dNTP) and primers in a PCR system by SAP enzyme, wherein the sequence of PPAR gamma, CEBPA and CREBPPCR primers and a sequencing primer is shown in a table 3, adding a single-base extension primer, wherein the 3' terminal base of the single-base extension primer is close to the SNP site and is completely complementary with the base on the target fragment, and replacing the dNTP with four ddNTPs, so that the probe only extends one base at the SNP site, and the connected ddNTPs correspond to alleles of the SNP site. The base at this point was determined by detecting the difference in molecular weight between the extended product and the unextended primer using matrix assisted laser Desorption ionization time of flight Mass Spectrometry (MALDI-TOFMS).
PCR amplification reaction:
firstly, taking 1.5ml of EP tube to prepare PCRmastermix, oscillating and centrifuging at low speed, and obtaining reaction components shown in the following table 4; secondly, an 8-channel pipettor is selected, 4 mu l of PCRmastermix is added into each sample adding hole of the 384-hole plate, finally 1 mu l of template DNA (20 ng/mu l) is added and mixed evenly, the 384-hole sealing plate film is carefully covered, each hole is firmly pressed, and the phenomena of evaporation and the like during the PCR process are prevented. Centrifuging at 1000rpm for 1 minute; setting the following PCR amplification reaction program: 5min at 94 ℃; 94 ℃ 20sec, 56 ℃ 30sec, 72 ℃ 1min, 45 cycles; 3min at 72 ℃; infinity at 4 ℃. The PCR reaction plate was placed on the PCR instrument and the program was started.
TABLE 4
Figure BDA0003206688540000121
2. Product alkaline phosphatase treatment:
firstly, after the PCR reaction is finished, treating the PCR product with SAP (SAP, shrimp alkaline phosphatase) to remove free dNTPs in the system; secondly, preparing alkaline phosphatase treatment reaction liquid in a new 1.5ml EP tube, wherein the reaction components of SAPMix are shown in the following table 5; ③ adding SAPmix to a 384-well PCR reaction plate, wherein the total reaction volume is 7. mu.l for each alkaline phosphatase-treated reaction well, wherein the PCR product is 5. mu.l, and the amount of SAPmix is 2. mu.l; fourthly, after the liquid transfer is finished, carefully covering a 384-hole sealing plate membrane, firmly pressing each hole to prevent evaporation and other phenomena during a PCR procedure, and carrying out the following reaction procedure after centrifugation; setting an SAP reaction program: 20min at 37 ℃; 5min at 85 ℃; infinity at 4 ℃. And a 384 well reaction plate was 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: firstly, after the alkaline phosphatase treatment is finished, carrying out single base extension reaction, wherein the total volume of a reaction system is 9 mu l; preparing single base extension reaction liquid in a new 1.5ml EP tube, wherein the EXTENDMix reaction components are shown in the following table 6; ③ add EXTENDMix to 384-well reaction plates. For each reaction well, one base extension reaction system EXTENDMix2 μ l; SAP + PCRinteraction 7 μ l, Totalvolume9 μ; fourthly, after the liquid transfer is finished, carefully covering a 384-hole sealing plate membrane, firmly pressing each hole to prevent evaporation and the like during a PCR procedure, and carrying out a reaction procedure after centrifugation, wherein the reaction procedure is carried out at 94 ℃ for 30 sec; [94 ℃ 5sec, (52 ℃ 5sec,80 ℃ 5sec)5cycles ]40 cycles; infinity at 4 ℃.
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. Resin purification: evenly filling resin in an 384/6MGDimple plate, and standing for 10 minutes to dry; ② add 16. mu.l of water to each well of 384 sample plates; ③ gently flip the 384 sample plate over and snap onto the sample plate, and then tap to drop the resin into each well of the sample plate; and fourthly, placing the 384 sample plate in a turnover centrifuge to rotate and mix evenly for 30 minutes at room temperature.
5. Chip spotting: the MassARRAYANODispenseserRS 1000 spotting instrument was started and the resin purified extension product was transferred to 384-well SpectroCHIPbioarray.
6. Mass spectrum detection and data output: analyzing the spotted SpectroCHIP chip by using a MALDI-TOF mass spectrometer, acquiring original data and a genotyping chart by using TYPER4.0 software according to a detection result, checking the integrity and correctness of a data file, storing the result into a corresponding storage medium and submitting the result to a biological information room for analysis.
(III) carrying out statistical analysis;
genotype frequency and allele frequency calculations, Hardy-Weinberg equilibrium and MAF were tested and genotype and allele distributions were compared between case and control groups by the Person chi-square test.
Single SNP association analysis, through Pearson chi-square test or Fisher's exact test, analysis of ONFH groups and disease groups at each site of genotype and allelic differences, search for disease associated sites.
The correlation between the pairwise interaction between multiple SNPs and the onset risk of ONFH (oncotic disease) is analyzed and completed through the gene interaction (epistatic effect) of the PLINK software.
Fifth, analysis of experimental results
The correlation between the genotyping of 11SNPs of the ONFH genes and the genotyping of the control groups CEBPA, PPAR gamma and CREBP genes and the onset risk of ONFH, and the distribution of the genotypes of the 11SNPs in the ONFH groups and the control groups is shown in a table 7.
TABLE 7
Figure BDA0003206688540000141
Figure BDA0003206688540000151
Sequencing results show that the frequency of GG (minor genotype) with the minimum homozygous genotype at rs17694108(G/A) locus of CEBPA gene in the ONFH group is obviously lower than that of GG (minor genotype) in a control group, and P-0.0002345 is shown in a table 7; that is, carriers of the CEBPA gene rs17694108(G/A) site GG type significantly reduce the risk of ONFH onset, lower than carriers of GG or GA genotypes.
And (II) the relativity between the allele frequencies of the 11SNPs of the CEBPA, PPAR gamma and CREBP genes of the ONFH group and the control group and the onset risk of ONFH, wherein the distribution frequency of the SNP allele types in the samples of the case group and the control group is shown in a table 8.
TABLE 8
Figure BDA0003206688540000152
Figure BDA0003206688540000161
Sequencing results show that the frequency of the minimum allele G at the locus of the ONFH group CEBPA gene rs17694108(G/A)) is obviously lower than that of a control group, and P is 6.516E-00; OR (95% CI)0.311(0.189-0.511), that is, the G allele frequency at the position of CEBP gene Ars17694108(G/a) significantly reduces the risk of onset of ONFH, and the probability that a human to be tested with allele G suffers ONFH is smaller than that of a human to be tested with allele a.
Sequencing results also show that the T frequency of the minimum allele at the rs3745971(T/C) locus of the ONFH group CEBPA gene is obviously higher than that of a control group, and P is 0.012; OR (95% CI)1.636(1.113-2.406), see table 8, that is, T allele frequency at the rs3745971(T/C) site of the CEBPA gene significantly increases the risk of onset of ONFH, and the probability that a person to be tested having an allele T suffers from ONFH is higher than that of a person to be tested having an allele C.
(III) CEBPA, PPAR gamma, CREBP gene 11SNPs different genetic models and ONFH onset risk correlation.
The analysis results of different genetic models of CEBPA gene rs17694108(G/A)) sites further find that the dominant model (GG + AGvsAA) of the CEBPARs17694108 sites of the ONFH group is obviously lower than that of a control group, and P is 6.099e-005, namely, the GG frequency of the minimum homozygous genotype of the rs17694108(G/A)) sites of the CEBPA gene obviously reduces the onset risk of ONFH, which is shown in Table 9.
The ONFH group has a CEBPA gene rs17694108(G/A)) locus recessive model (GGvsAG + AA) which is also obviously lower than that of a control group, and the frequency of the GG is shown in table 8, and the P is 0.005164, namely, the minimum homozygous genotype of the CEBPA gene rs17694108(G/A) locus GG obviously reduces the onset risk of the ONFH, and is shown in table 9.
The dominant model (TT + TCvsCC) of the rs3745971(G/A) site of the CEBPA gene in the ONFH group is obviously higher than that in the control group, and P is 0.0019, namely, the TT type frequency of the minimum homozygous genotype at the rs3745971(G/A) site of the CEBPA gene obviously increases the ONFH risk, and the table 9 shows.
TABLE 9
Figure BDA0003206688540000162
Figure BDA0003206688540000171
Chi square statistical analysis
(IV) CEBPA, PPAR γ, CREBP gene 11SNPs interactions and ONFH risk correlation.
And (3) analyzing the pairwise interaction of the 11SNPs (rs17694108, rs3745971, rs10500264, rs2920502, rs2028759, rs3856806, rs2072381, rs9392, rs2283487, rs3751845 and rs129974) by using a PLINK software gene interaction analysis method. The rs17694108 locus is 33240645 th nucleotide from the 5' end of the chromosome 19 of the human genome; the nucleotide at the rs17694108 site is G or A; the rs3745971 site is 33304320 th nucleotide from the 5' end of the chromosome 19 of the human genome; the nucleotide at the rs3745971 site is C or T; the rs10500264 locus is 33259408 th nucleotide from the 5' end of the 19 th chromosome of the human genome; the nucleotide at the rs10500264 locus is G or A; the rs2920502 locus is 12287696 th nucleotide from the 5' end of the chromosome 3 of the human genome; the nucleotide at the rs2920502 site is G or C; the rs2028759 locus is 12377113 th nucleotide from the 5' end of the chromosome 3 of the human genome; the nucleotide at the rs2028759 site is C or T; the rs3856806 locus is 12434058 th nucleotide from the 5' end of the chromosome 3 of the human genome; the nucleotide at the rs3856806 site is C or T; the rs2072381 site is 3731312 th nucleotide from the 5' end of the human genome No.16 chromosome, and the nucleotide of the rs2072381 site is G or A; the rs9392 site is 3725168 th nucleotide from the 5' end of the human genome No.16 chromosome, and the nucleotide of the rs9392 site is G or A; the rs2283487[ A/G ] locus is 3919885 th nucleotide from the 5' end of the chromosome 16 of the human genome, and the nucleotide at the rs2283487 locus is A or G; the rs3751845[ G/A ] locus is 3728336 th nucleotide from the 5' end of the chromosome 16 of the human genome, and the nucleotide of the rs3751845 locus is G or A; the rs129974 site is 3745291 th nucleotide from the 5' end of chromosome 16 of the human genome, and the nucleotide of the rs129974 site is C or T. The correlation analysis of different SNP interactions with femoral head necrosis is shown in table 10.
Watch 10
Figure BDA0003206688540000181
Figure BDA0003206688540000191
PLINK chi-square test;▲interaction;#PLINK gene interaction (epistasis) analysis.
The results of the PLINK software analysis of gene interactions show that:
the interaction between the rs2920502[ G/C ] site of PPAR gamma gene and the rs2072381[ G/A ] site of CREBP gene obviously increases the risk of ONFH, OR: 1.812, P is 0.0222, see serial number 1 in table 10, that is to say, carry PPAR γ gene rs2920502[ G/C ] locus minimum homozygous genotype GG type and CREBP gene rs2072381[ G/A ] locus minimum homozygous genotype AA type individual and increase ONFH onset risk notably at the same time, can be used as ONFH molecular early warning, molecular diagnosis, clinical drug treatment target application molecular marker.
The interaction between the rs2028759[ C/T ] site of the PPAR γ gene and the rs10500264[ G/A ] site of the CEBPA gene significantly increases the risk of developing ONFH, OR: 1.973, P is 0.0487, see serial No.2 in table 10, that is to say, the individuals carrying the minimal homozygous genotype CC of PPAR γ gene rs2028759[ C/T ] site and the minimal homozygous genotype AA of CEBPA gene rs10500264[ G/a ] site significantly increase the risk of ONFH, and can be used as molecular markers for ONFH molecular early warning, molecular diagnosis and clinical drug therapy target application.
The interaction between the rs9392[ G/A ] site of the CREBP gene and the rs2283487[ A/G ] site of the CREBP gene obviously increases the onset risk of ONFH, OR: 1.472, P is 0.02315, see serial number 4 in table 10, that is to say, the individual who carries CREBP gene rs9392[ G/A ] site minimum homozygous genotype AA type and CREBP gene rs2283487[ A/G ] site minimum homozygous genotype GG type simultaneously increases ONFH onset risk remarkably, can be used as ONFH molecular early warning, molecular diagnosis, clinical drug treatment target application molecular marker.
The interaction between the rs3751845[ G/A ] site of the CREBP gene and the rs2283487[ A/G ] site of the CREBP gene obviously increases the onset risk of ONFH, OR: 2.125, P is 0.006302, see serial number 5 in table 10, that is to say, the individual who carries minimum homozygous genotype AA type at CREBP gene rs3751845[ G/A ] site and minimum homozygous genotype GG type at CREBP gene rs2283487[ A/G ] site simultaneously increases the outbreak risk of ONFH significantly, and can be used as an application molecular marker of ONFH molecular early warning, molecular diagnosis and clinical drug therapy target.
The interactive action between the rs129974[ C/T ] locus of the CREBP gene and the rs2283487[ A/G ] locus of the CREBP gene obviously increases the onset risk of ONFH, OR:1.831, P ═ 0.01471 is shown as the serial number 7 in the table 10, namely, an individual carrying the minimum homozygous genotype TT type of the gene at the CREBP gene rs129974[ C/T ] locus and the minimum homozygous genotype GG type of the gene at the CREBP gene rs2283487[ A/G ] locus simultaneously obviously increases the onset risk of ONFH, and the gene can be used as an application molecular marker of ONFH molecular early warning, molecular diagnosis and clinical drug treatment targets.
The interaction between the rs2072381[ G/A ] locus of the CREBP gene and the rs10500264[ G/A ] locus of the CEBPA gene is obviously related to the reduction of the onset risk of ONFH, OR is 0.3681, and P is 0.04758 which is shown as the serial number 6 in the table 10, namely, the individual carrying the minimum homozygous genotype AA type of the rs2072381[ G/A ] locus and the minimum homozygous genotype AA type of the rs10500264[ G/A ] locus of the CREBP gene obviously increases the onset risk of ONFH at the same time, and can be used as a molecular protection marker for reducing the onset risk of ONFH.
The interaction between the rs3856806[ C/T ] locus of the PPAR gamma gene and the rs17694108[ G/A ] locus of the CEBPA gene obviously increases and reduces the onset risk of ONFH, OR:0.4148, P ═ 0.04181 is shown as the serial number 3 in the table 10, namely, an individual carrying the TT type of the minimum homozygous genotype of the rs3856806[ C/T ] locus of the PPAR gamma gene and the GG type of the minimum homozygous genotype of the rs17694108[ G/A ] locus of the CEBPA gene simultaneously obviously reduces the onset risk of ONFH, and the individual can be used as a molecular protection marker for reducing the risk of ONFH.
The invention discovers for the first time that the interaction of 14SNPs is obviously related to the ONFH morbidity risk, the risk correlation is partially or completely different from the action of single SNP on the ONFH morbidity risk, the result of the gene interaction discovers the risk action or the protective effect of a plurality of SNPs on ONFH more clearly, further verifies that ONFH is a complex disease caused by the combination of a plurality of micro-effect genes, explains the combined effect of the molecular genetic loci, and has important value on the establishment of molecular early warning and molecular prevention and control strategies of ONFH. The gene interaction, also known as epistatic effect, refers to two pairs of non-allelic genes affecting the same trait, where one pair of genes (dominant or recessive) suppresses (or masks) the effect exhibited by the other pair of dominant genes.
One skilled in the art will be able to perform the analysis of the nucleotides present on the SNP markers disclosed herein in the nucleic acid of an individual by any method or technique for detecting SNP sites. For example, one can use the methods of the present invention to detect SNP site markers by performing Tagman, mass spectrometry, DNA microarray, microsequencing, hybridization, restriction analysis, oligonucleotide ligation detection, allele-specific PCR-HRM, or a combination thereof, although this list is exemplary only and is in no way intended to limit the present invention.
Any suitable method may be used by those skilled in the art to effect such detection.
The main experimental equipment and equipment involved in the examples of the present invention are shown in Table 11 below.
TABLE 11
Figure BDA0003206688540000201
Figure BDA0003206688540000211
The main reagents or software involved in the examples of the present invention are as follows in table 12.
Figure BDA0003206688540000212
Figure BDA0003206688540000221
The above-described embodiments of the present invention should not be construed as limiting the scope of the present invention. Any other corresponding changes and modifications made according to the technical idea of the present invention should be included in the protection scope of the claims of the present invention.
Sequence listing
<110> Jilin university
<120> CEBPA, PPAR ɣ and CREBP gene SNP detection substance related to ONFH risk and application thereof
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Claims (5)

1. A substance for detecting single nucleotide polymorphisms of CEBPA, PPAR γ and CREBP genes related to the risk of non-traumatic femoral head necrosis, which is characterized in that: the substance is applied to diagnosis or auxiliary diagnosis of non-traumatic femoral head necrosis or applied to preparation of products for diagnosis or auxiliary diagnosis of risk of non-traumatic femoral head necrosis; or the application of the substance in evaluating or assisting in evaluating the non-traumatic femoral head necrosis or in preparing a product for evaluating or assisting in evaluating the risk of the non-traumatic femoral head necrosis; the site or combination of sites related to non-traumatic femoral head necrosis is any one or more of the following (1) to (9): (1) rs17694108 locus of CEBPA gene; (2) rs3745971 site of CEBPA gene; (3) the combination of the rs2920502 site of the PPAR gamma gene and the rs2072381 site of the CREBP gene; (4) the combination of the rs2028759 site of the PPAR gamma gene and the rs10500264 site of the CEBPA gene; (5) the combination of the rs3856806 site of the PPAR gamma gene and the rs17694108 site of the CEBPA gene; (6) the combination of the site rs9392 of the CREBP gene and the site rs2283487 of the CREBP gene; (7) the combination of the site rs3751845 of the CREBP gene and the site rs2283487 of the CREBP gene; (8) the combination of the site of CREBP gene rs2072381 and the site of CREBP gene rs 10500264; (9) the combination of the site of the CREBP gene rs129974 and the site of the CREBP gene rs 2283487.
2. Use according to claim 1, characterized in that: the substance is a product for preparing diagnosis or auxiliary diagnosis of the risk of non-traumatic femoral head necrosis, or the substance is a product for preparing evaluation or auxiliary evaluation of the risk of non-traumatic femoral head necrosis, and the product comprises a readable carrier on which at least one condition of the following A to M is recorded:
A. the risk of the minimally homozygous genotype GG-type to-be-detected person carrying the rs17694108 locus suffering from non-traumatic femoral head necrosis is reduced;
B. the frequency of the minimum allele G carrying the rs17694108 locus is lower than that of the minimum allele G of the control group, and the risk of non-traumatic femoral head necrosis of the human to be detected is reduced;
C. the rs17694108 locus genetics dominant model (GG + AGvsAA) is related to reducing the risk of traumatic femoral head necrosis;
D. the rs17694108 locus genetic recessive model (GGvsAG + AA) is related to reducing the risk of traumatic femoral head necrosis;
E. the risk of the person to be detected carrying the minimum homozygous genotype TT type at the rs3745971 locus to suffer from non-traumatic femoral head necrosis is increased;
F. the risk of the non-traumatic femoral head necrosis of the person to be detected carrying the rs3745971 locus with high minimum allele T frequency is increased;
G. the rs3745971 site genetic dominant model (TT + TCvsCC) is related to increase the risk of traumatic femoral head necrosis;
H. simultaneously, the risk of non-traumatic femoral head necrosis of a to-be-detected person carrying the GG type with the minimum homozygous locus of rs2920502 and the AA type with the minimum homozygous locus of rs2072381 is increased;
I. simultaneously, the risk of the non-traumatic femoral head necrosis of the human to be detected carrying the CC type of the minimum homozygous genotype at the rs2028759 locus and the AA type of the minimum homozygous genotype at the rs10500264 locus is increased;
J. simultaneously, the risk of the non-traumatic femoral head necrosis of a to-be-detected person carrying the rs9392 locus minimum homozygous genotype AA and the rs2283487 locus minimum homozygous genotype GG is increased;
K. simultaneously, the risk of the non-traumatic femoral head necrosis of a to-be-detected person carrying the rs3751845 locus minimum homozygous genotype AA and the rs2283487 locus minimum homozygous genotype GG is increased;
l, increasing the risk of non-traumatic femoral head necrosis of a to-be-detected person carrying the rs129974 locus minimum homozygous genotype TT type and the rs2283487 locus minimum homozygous genotype GG type at the same time;
m. the risk of non-traumatic femoral head necrosis of a human to be detected carrying the rs3856806 locus minimum homozygous genotype TT type and the rs17694108 locus minimum homozygous genotype GG type at the same time is reduced.
N. the risk of non-traumatic femoral head necrosis of a human to be tested carrying the minimum homozygous genotype AA of rs2072381 and the minimum homozygous genotype AA of rs10500264 is reduced.
3. Use according to claim 1, characterized in that:
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 set of single-stranded DNA molecules A consists of single-stranded DNA shown by SEQ ID NO.1, single-stranded DNA shown by SEQ ID NO.2 and single-stranded DNA shown by SEQ ID NO. 3; the substance for detecting the single nucleotide gene polymorphism of the rs3745971 site 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 by SEQ ID NO.4, single-stranded DNA shown by SEQ ID NO.5 and single-stranded DNA shown by 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 C consists of single-stranded DNA shown by SEQ ID NO.7, single-stranded DNA shown by SEQ ID NO.8 and single-stranded DNA shown by SEQ ID NO. 9; the substance for detecting the single nucleotide gene polymorphism of the rs2920502 site 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 by SEQ ID NO.10, single-stranded DNA shown by SEQ ID NO.11 and single-stranded DNA shown by SEQ ID NO. 12; the substance for detecting the single nucleotide gene polymorphism of the rs2028759 site is primer pair E or complete set of single-stranded DNA molecule E; the primer pair E 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 by SEQ ID NO.13, single-stranded DNA shown by SEQ ID NO.14 and single-stranded DNA shown by 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 whole set of single-stranded DNA molecules consists of single-stranded DNA shown by SEQ ID NO.16, single-stranded DNA shown by SEQ ID NO.17 and single-stranded DNA shown by SEQ ID NO. 18; the substance for detecting the single nucleotide gene polymorphism of the rs2072381 locus is a primer pair G or a complete set of single-stranded DNA molecules G; the primer pair G 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 molecule G consists of single-stranded DNA shown by SEQ ID NO.19, single-stranded DNA shown by SEQ ID NO.20 and single-stranded DNA shown by SEQ ID NO. 21; the substance for detecting the single nucleotide gene polymorphism of the rs9392 locus is primer pair octyl or a complete set of single-stranded DNA molecule octyl; the primer pair octyl 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 by SEQ ID NO.22, single-stranded DNA shown by SEQ ID NO.23 and single-stranded DNA shown by SEQ ID NO. 24; the substance for detecting the single nucleotide gene polymorphism of the rs2283487 site is a primer pair nonyl or a complete set of single-stranded DNA molecules nonyl; the primer pair nonane 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 whole set of single-stranded DNA molecules consists of single-stranded DNA shown by SEQ ID NO.25, single-stranded DNA shown by SEQ ID NO.26 and single-stranded DNA shown by SEQ ID NO. 27; the substance for detecting the single nucleotide gene polymorphism of the rs3751845 locus is a primer pair deca or a complete set of single-stranded DNA molecules deca; the primer pair 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 by SEQ ID NO.28, single-stranded DNA shown by SEQ ID NO.29 and single-stranded DNA shown by SEQ ID NO. 30; the substance for detecting the single nucleotide gene polymorphism of the rs129974 site 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 by SEQ ID NO.31, single-stranded DNA shown by SEQ ID NO.32 and single-stranded DNA shown by SEQ ID NO. 33.
4. A kit, characterized in that: an agent comprising the single nucleotide polymorphism of at least one site or a combination of sites (1) to (9) according to claim 1, for detecting a risk of non-traumatic femoral head necrosis.
5. A targeted therapeutic agent, characterized by: the drug is a drug targeting any one or more of the (1) to (9) sites or the combination of the sites related to the non-traumatic femoral head necrosis according to claim 1.
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