Hormone-induced femoral head necrosis high-risk prediction marker, kit and detection system

Abstract

The invention relates to a hormone-induced femoral head necrosis (SONFH) high-risk prediction marker, a kit and a detection system. The marker is ANK1 and EPB41. It is found that ANK1 and EPB41 mutations are closely associated with SONFH, early warning, prediction and screening of high-risk individuals can be conducted through sequencing, a foundation is laid for follow-up development of drugs for preventing (intervening) SONFH, and good application prospects are achieved.

Description

Technical Field

[0001] This invention belongs to the field of drug side effect early warning and prediction technology, and specifically relates to a high-risk predictive biomarker, reagent kit and detection system for hormone-induced avascular necrosis of the femoral head. Background Technology

[0002] Glucocorticoids are widely used anti-inflammatory and immunosuppressive drugs in clinical practice. However, some patients experience serious adverse reactions during their therapeutic effects. Among these, steroid-induced osteonecrosis of the femoral head (SONFH), induced by glucocorticoid therapy, is a complex and refractory bone disease and one of the most serious complications induced by hormones. Pathologically, SONFH results from microcirculatory disturbances in the blood supply area of ​​the femoral head, leading to osteonecrosis. After onset, it easily leads to limb disability and loss of working ability, making it a catastrophic bone disease. In a large patient population with various underlying diseases requiring hormone therapy, the cumulative incidence of SONFH after short-term high-dose or medium-to-long-term hormone exposure ranges from approximately 3% to 40%; the latest two domestic surveys (in patients with lupus erythematosus) estimate the cumulative incidence of SONFH to remain between 6% and 9%. Furthermore, there are significant individual differences in hormone tolerance. Currently, the mainstream academic view is that the pathogenesis of SONFH is the result of the combined effects of hormone exposure and individual genetic variations.

[0003] Previous studies on individual genetic factors have mainly mentioned the association between common genetic variations and other non-coding region variations with the risk of SONFH (these variations contribute very little to the risk burden of complex diseases). Currently, there is a lack of reported evidence of the association between rare harmful variations (gene variations located in protein-coding regions, which are usually considered pathogenic risk factors for complex diseases) and the risk of SONFH. Summary of the Invention

[0004] The technical problem to be solved by this invention is to provide a high-risk predictive biomarker, reagent kit, and detection system for hormone-induced osteonecrosis of the femoral head, and to discover... ANK1 , EPB41 Mutations are closely related to the pathogenesis of SONFH. Sequencing can be used to identify, warn, and predict high-risk individuals, and lay the foundation for the subsequent development of drugs to prevent (intervene) SONFH, showing promising application prospects.

[0005] In a first aspect, the present invention provides a high-risk predictive biomarker for hormone-induced avascular necrosis of the femoral head, wherein the biomarker is ankyrin-1 (ANK1) and erythrocyte membrane protein band 4.1 (EPB41).

[0006] Furthermore, the aforementioned ANK1 and EPB41 Rare variants are closely associated with the risk of developing hormone-induced osteonecrosis of the femoral head.

[0007] Furthermore, the rare variants include one or more of the following: ANK1 p.S1507P, ANK1 p.L1194P, ANK1 p.V991M, ANK1 p.A560T, ANK1 p.V435M, ANK1 p.D1030G, ANK1 p.R941Q, ANK1 p.D11N, EPB41p.P137L, EPB41 p.P357L, and EPB41 p.R700Q. The rare and harmful variants are defined as those with a minor allele frequency (MAF) of <0.1% in the Genome Aggregation Database (gnomAD) and the 1000 Genomes Project (1kGP), and simultaneously meeting the functional criteria for harmful prediction, including SIFT predicting it as deleteerious, PolyPhen-2 predicting it as probably_damaging or possibly_damaging, and a CADD score ≥20.

[0008] Furthermore, the primer sequences for detecting rare variants are shown in SEQ NO.1-22.

[0009]

[0010] Secondly, the present invention provides a high-risk prediction kit for hormone-induced avascular necrosis of the femoral head, including the aforementioned primer sequences.

[0011] Furthermore, the kit also includes other standard diagnostic reagents.

[0012] Thirdly, the present invention provides a risk prediction system for hormone-induced osteonecrosis of the femoral head, including a peripheral blood smear examination module, a red blood cell hypotonic hemolysis detection module, and a sequencing module of the aforementioned reagent kit.

[0013] Furthermore, the peripheral blood smear examination module is used to determine whether there is an increase in overt or potential abnormal red blood cell morphologies such as spherical or oval shapes; the red blood cell hypotonic hemolysis detection module is used to determine whether there is an increase in red blood cell membrane fragility; and the sequencing module is used to confirm whether there are latent red blood cell membrane defects.

[0014] Fourthly, this invention provides a method for early warning and prediction of patients at high risk of developing hormone-induced avascular necrosis of the femoral head after hormone therapy, comprising the following steps: (1) For new patients who need long-term hormone therapy, blood tests should be conducted before hormone therapy is planned. In addition to routine red blood cell (including blood smear) and reticulocyte tests, the red blood cell hypotonic hemolysis detection module should be used to preliminarily screen for increased red blood cell membrane fragility. If increased fragility is found, the patient's medical history can be further questioned to rule out whether there is a history of diabetes.

[0015] (2) For individuals with increased fragility, the presence of latent red blood cell membrane defects can be confirmed through sequencing modules (exon sequencing, Westblot protein expression examination). ANK1 , EPB41 There may even be other cytoskeleton gene defects and mutations.

[0016] Beneficial effects This invention discovers ANK1 , EPB41 Rare mutations are closely associated with SONFH and can be screened by sequencing before planned high-dose or medium- to long-term hormone therapy to determine whether an individual is at high risk of hormone sensitivity, providing timely risk warnings. This lays the foundation for assisting clinicians in making decisions regarding hormone medication regimens and for the subsequent development (intervention) of drugs (such as enhancing / stabilizing erythrocyte membrane properties and maintaining microcirculatory homeostasis), as well as preventing or reducing SONFH events caused by hormone use. Overall, hormones are used extensively in clinical practice. Given the current lack of effective early warning, prediction, and intervention methods for identifying and screening hormone-induced SONFH events, this invention fills a gap in this field and has significant social and economic value in preventing or reducing the occurrence of SONFH, thus showing promising application prospects. Attached Figure Description

[0017] Figure 1 A indicates that individuals carrying the ANK1 / EPB41 mutation have significantly decreased ANK1 protein expression levels. Figure 1 B indicates that individuals carrying the ANK1 / EPB41 mutation have a significantly higher proportion of abnormal red blood cell morphology than the control group; Figure 1 C indicates that individuals carrying the ANK1 / EPB41 mutation have a significantly increased degree of erythrocyte membrane rupture (fragility) under hypotonic conditions. Detailed Implementation

[0018] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0019] Example 1 1. A close association was found between ANK1 and EPB41 mutations and hormone-induced osteonecrosis of the femoral head (SONFH): Peripheral blood samples were collected from two groups of patients who developed SONFH (case group) and did not develop SONFH (control group) after hormone therapy, based on a common primary disease background (systemic lupus erythematosus, SLE). The samples were partly from the blood sample bank donated by patients with femoral head necrosis in the Department of Orthopedics of Shanghai Sixth People's Hospital, and partly from the blood samples of the observation subjects in the SLE hormone exposure and SONFH cohort study conducted by Shanghai Sixth People's Hospital and Shanghai Renji Hospital. All the above blood sample collection processes were conducted with informed consent from the patients and were approved by the ethics committees of the above hospitals. Whole-exome sequencing analysis revealed that rare and harmful variants (according to the criteria listed above) in the genes encoding cytoskeleton proteins ANK1 and EPB41 (clearly related to the erythrocyte membrane, both linked to spectroscopy proteins that act as cytoskeleton links) were significantly more prevalent in the SONFH group than in the control group [SONFH group data first, control group data second, and so on; 10.8% (9 / 83 cases) vs 0% (0 / 91 cases), P=0.001]. Furthermore, there was no significant difference in the frequency of these rare variants between the control group and the healthy Han Chinese population. In addition, these rare variants were replicated at a certain proportion (4.1%, 10 / 246 cases) in the exome sequencing of another independent non-SLE SONFH cohort sample, suggesting that these variants are prevalent in SONFH and are not related to any specific disease. Analysis of hormone exposure dose and duration in SLE-SONFH case-control groups revealed that the SONFH group had significantly lower median hormone dose (9.9g vs 25.2g) and exposure duration (26 months vs 123 months) than the control group (P<0.001), suggesting that the SONFH group was more sensitive to hormone exposure. Individuals carrying these two rare gene variants had a significantly increased risk of developing SONFH (adjusted for Firth's logistic regression odds ratio: aOR). ANK1 =11.3, aOR EPB41 =32.3, aOR ANK11 / EPB41=36.3, P<0.05). Furthermore, family history confirmed that these two gene variations can be stably inherited by the next generation after occurring in either parent; and that these gene variations caused mild damage to erythrocyte membrane function (leading to an increased proportion of abnormal erythrocyte morphology, increased erythrocyte membrane fragility, and decreased erythrocyte deformability), while also affecting the reduced expression levels of encoded proteins (decreased expression of ANK1 protein in the erythrocyte membrane). Figure 1 ).

[0020] These results suggest that rare harmful variants in ANK1 and EPB41 are likely pathogenic genetic factors leading to SONFH, but require secondary hormonal effects to cause microcirculatory disturbances to result in SONFH.

[0021] 2. ANK1 and EPB41 mutations impair erythrocyte function: ① Proteins were extracted from the patient's red blood cell membranes, and Westblot analysis showed ( Figure 1 A): ANK1 mutations lead to decreased expression of ankylosing protein (ANK1); EPB41 mutations show normal expression of protein 4.1 (EPB41), but decreased ANK1 expression. This is because both ANK1 and EPB41 are important key proteins on the erythrocyte membrane, exhibiting complex interdependence on the erythrocyte membrane, existing as multiple complexes linked by spectroscopy.

[0022] ② The proportion of abnormal red blood cell morphology increases in mutant patients. Normal red blood cells are biconcave disc-shaped, while mutants usually cause spherical or oval changes. Of course, this is related to the severity of the damage. The damage we found was relatively mild, and no abnormalities were observed under routine microscopy. However, flow cytometry revealed a significant increase in the proportion of abnormal red blood cells in the mutant group. Figure 1 B).

[0023] ③ In a hypotonic environment, the mutated erythrocyte membrane is more prone to rupture and hemolysis, suggesting increased osmotic fragility. Figure 1 C).

[0024] 3. In the non-SLE hormone therapy-induced SONFH cohort, the proportion of patients carrying ANK1 or EPB41 mutations was again found to be 4.1%, which, although slightly lower than the SLE-SONFH group, was still significantly higher than the mutation proportion in the normal population (mutation proportions in the general Chinese population: ANK1 mutation approximately 7 per 10,000, EPB41 mutation approximately 6 per 1,000) (Table 1). This suggests that this mutation is present in a high proportion in the hormone-induced femoral head necrosis group and is a pathogenic risk factor for hormone-induced necrosis.

[0025] Table 1. Frequency distribution of rare variants of ANK1 and EPB41 in major global ethnic groups (races)*

Claims

1. A biomarker for predicting high risk of hormone-induced avascular necrosis of the femoral head, characterized in that, The markers are ankyrin-1ANK1 and erythrocyte membrane protein band 4.1EPB41.

2. The predictive biomarker for high risk of hormone-induced avascular necrosis of the femoral head according to claim 1, characterized in that, The ANK1 and EPB41 Rare variants are closely associated with the risk of developing hormone-induced osteonecrosis of the femoral head.

3. The high-risk predictive biomarker for hormone-induced avascular necrosis of the femoral head according to claim 2, characterized in that, The rare variants include one or more of the following: ANK1 p.S1507P, ANK1 p.L1194P, ANK1 p.V991M, ANK1 p.A560T, ANK1 p.V435M, ANK1 p.D1030G, ANK1 p.R941Q, ANK1 p.D11N, EPB41 p.P137L, EPB41 p.P357L, and EPB41 p.R700Q.

4. The predictive biomarker for high risk of hormone-induced avascular necrosis of the femoral head according to claim 2, characterized in that, The primer sequences for detecting rare variants are shown in SEQ NO.1-22.

5. A high-risk prediction kit for hormone-induced avascular necrosis of the femoral head, characterized in that: Includes the primer sequence as described in claim 4.

6. The high-risk prediction kit for hormone-induced avascular necrosis of the femoral head according to claim 5, characterized in that, The kit also includes other standard diagnostic reagents.

7. A risk prediction system for hormone-induced avascular necrosis of the femoral head, characterized in that: It includes a peripheral blood smear examination module, a red blood cell hypotonic hemolysis detection module, and a sequencing module containing the kit as described in claim 5.

8. The risk prediction system for hormone-induced avascular necrosis of the femoral head according to claim 7, characterized in that: The peripheral blood smear examination module is used to determine whether there is an increase in overt or potential abnormal red blood cell morphology, the red blood cell hypotonic hemolysis detection module is used to determine whether there is an increase in red blood cell membrane fragility, and the sequencing module is used to confirm whether there is a latent red blood cell membrane defect.

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