CN110275026B - Molecular marker for diagnosing idiopathic inflammatory myopathy and application thereof - Google Patents
Molecular marker for diagnosing idiopathic inflammatory myopathy and application thereof Download PDFInfo
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Abstract
The invention relates to a molecular marker for diagnosing idiopathic inflammatory myopathy and application thereof. The molecular marker is an autologous heat shock transcription factor 1(HSF1) antibody in serum. The antibody is IgG; the idiopathic inflammatory myopathy is Polymyositis (PM) or Dermatomyositis (DM); the diagnosis is to differentiate idiopathic inflammatory myopathy from a healthy population or to predict disease progression of Idiopathic Inflammatory Myopathy (IIM).
Description
Technical Field
The invention belongs to the technical field of medical biology, and particularly relates to a molecular marker for diagnosing idiopathic inflammatory myopathy and application thereof.
Background
Idiopathic Inflammatory Myopathy (IIM) is a group of rare heterogeneous autoimmune diseases with proximal muscle weakness, serum myopathy abnormalities, skeletal myositis infiltration and extramuscular involvement as the main clinical manifestations, mainly including Polymyositis (PM), Dermatomyositis (DM) and Inclusion Body Myositis (IBM), [ solution of myyositis, m ]1]. Autoantibodies to IIM include Myositis Specific Antibodies (MSAs) and Myositis Associated Antibodies (MAAs), which are found in 60-80% of IIM patients and are of great value in disease diagnosis, treatment guidance and prognosis2,3]. MSAs are associated with a particular clinical syndrome and are found only in IIM; MAAs can be seen in other autoimmune diseases, such as systemic lupus erythematosusSore (SLE) and scleroderma (SSc) ("SLE")4]. In addition, it has been shown that the anti-SRP antibody and the anti-HMGCR antibody can directly participate in the injury of muscle cells, suggesting that the autoantibody to myositis may have pathogenicity [ 2 ]5,6]. In addition, high expression of multiple MSAs target antigens is found in the muscle tissue of IIM patients7,8]. Therefore, the discovery of a novel myositis autoantibody not only enables more precise typing of IIM, but also the study of autoantibody has important implications for the pathogenesis of IIM
Most of the MSAs known at present are screened by immunoprecipitation and found by a target antigen validation experiment. In addition, serological screening of recombinant cDNA expression libraries is also an effective way to find unknown antibodies and has been widely used in research to identify tumor-associated antigens. However, not only is the construction of a cDNA expression library long in time consumption and difficult in technology, but also the library depends on bacteria to express recombinant proteins, the protein expression system cannot perform post-translational modification on the proteins, and some autoantibodies capable of recognizing the post-translational modified proteins cannot be identified. The protein microarray is a new method for screening autoantibodies, thousands of eukaryotic systems are expressed and purified to obtain protein which is coated in a microarray hole, and then the serum of a patient reacts with the protein, so that the autoantibodies in the serum can be screened efficiently. The myositis autoantibodies that we recognize at present are all nonspecific proteins that are widely expressed in humans, and are not proteins that are expressed and function only in IIM, so that it is possible to discover new myositis autoantibodies by screening microarrays that contain a very large number of protein types.
In the research, a proteome chip technology (containing 9374 human full-length proteins) is used for screening unknown antibodies in IIM, heat shock resistant transcription factor 1(HSF1) antibodies are found in DM, and the existence of the antibodies is verified by combining ELISA, Immunoprecipitation (IP), Immunoblot (IB) and Dot Blot (DB) methods.
Disclosure of Invention
The invention firstly relates to a molecular marker for diagnosing Idiopathic Inflammatory Myopathy (IIM), wherein the molecular marker is an autologous heat shock transcription factor 1(HSF1) antibody in serum.
The antibody is IgG;
the idiopathic inflammatory myopathy is Polymyositis (PM) or Dermatomyositis (DM);
the diagnosis is to distinguish idiopathic inflammatory myopathy from a healthy population.
Specifically, the diagnosis is as follows: detecting the IgG antibody level of the anti-HSF 1 in the serum of the target patient, and taking the average value of the serum anti-HSF 1 antibody level of healthy people plus 3 times of standard deviation as a positive critical value, if the IgG antibody level of the anti-HSF 1 in the serum of the target patient is higher than the critical value, diagnosing the idiopathic inflammatory myopathy.
Further, the diagnosis is: and detecting the level of the anti-HSF 1 IgG antibody in the serum of the target patient, and taking the average value of the serum anti-HSF 1 antibody level of a healthy human group plus 3 times standard deviation as a positive critical value, wherein if the level of the anti-HSF 1 IgG antibody in the serum of the target patient is higher than the critical value and the autoantibodies of other known IIMs in the serum of the target patient are increased, the idiopathic inflammatory myopathy is diagnosed, and the other autoantibodies are preferably anti-TIF 1 gamma antibodies.
The invention also relates to a molecular marker for predicting the disease process of Idiopathic Inflammatory Myopathy (IIM), wherein the molecular marker is an autologous heat shock transcription factor 1(HSF1) antibody in serum.
The antibody is IgG;
the idiopathic inflammatory myopathy is Polymyositis (PM) or Dermatomyositis (DM);
the disease process for predicting the Idiopathic Inflammatory Myopathy (IIM) is as follows:
(1) predicting risk of IIM secondary tumor associated myositis (CAM);
(2) predicting the prognosis of treatment for non-CAM IIM patients;
the prediction predicts the risk of IIM secondary tumor associated myositis (CAM) as the level of IgG antibodies against HSF1 in the serum of the target patient, the positive cut-off value being the mean value plus 3-fold standard deviation of the serum anti-HSF 1 antibody level of the healthy population, e.g. the IgG antibody level against HSF1 in the serum of the target patient is higher than the cut-off value, and the high risk of CAM is the case when the autoantibodies against other known IIMs in the target patient are elevated, said IIM autoantibodies including anti-transcriptional mediator 1 γ (TIF1 γ) antibody, anti-nuclear matrix protein 2(NXP2) antibody, anti-small ubiquitin-like modification activating enzyme (SAE) antibody and anti-histaminyl tRNA synthetase (Jo-1) antibody.
The prognosis of the non-CAM IIM patient is determined by detecting the anti-HSF 1 IgG antibody level in the target patient serum, and if the anti-HSF 1 IgG antibody level in the target patient serum is decreased after treatment, the prognosis is good.
The present invention also relates to a diagnostic kit for diagnosing or prognosing the course of an Idiopathic Inflammatory Myopathy (IIM) disease, said kit comprising: detecting an effective amount of heat shock transcription factor 1(HSF1) and necessary related reagents, wherein the related reagents comprise but are not limited to protein preservation solution, diluent, developing solution, contrast reagent and the like, and the kit is a detection kit based on the antigen-antibody interaction principle.
Preferably, the kit is an ELISA kit or an immunoblotting kit.
The invention also relates to application of the molecular marker for diagnosing the Idiopathic Inflammatory Myopathy (IIM) in the preparation of the kit, wherein the molecular marker is an autologous heat shock transcription factor 1(HSF1) antibody in serum.
The antibody is IgG;
the idiopathic inflammatory myopathy is Polymyositis (PM) or Dermatomyositis (DM);
the diagnosis is to distinguish idiopathic inflammatory myopathy from a healthy population.
Specifically, the diagnosis is as follows: detecting the IgG antibody level of the anti-HSF 1 in the serum of the target patient, taking 3 times of the average serum anti-HSF 1 antibody level of healthy people as a positive critical value, and if the IgG antibody level of the anti-HSF 1 in the serum of the target patient is higher than the critical value, diagnosing the idiopathic inflammatory myopathy.
Further, the diagnosis is: detecting the IgG antibody level of the anti-HSF 1 in the serum of the target patient, and taking 3 times of the average serum anti-HSF 1 antibody level of healthy people as a positive critical value, if the IgG antibody level of the anti-HSF 1 in the serum of the target patient is higher than the critical value, and the autoantibodies of other known IIMs in the target patient are increased, then diagnosing the idiopathic inflammatory myopathy, wherein the other autoantibodies are preferably anti-TIF 1 gamma antibodies.
The invention also relates to application of the molecular marker for predicting the disease process of the Idiopathic Inflammatory Myopathy (IIM) in preparation of a kit, wherein the molecular marker is an autologous heat shock transcription factor 1(HSF1) antibody in serum.
The antibody is IgG;
the idiopathic inflammatory myopathy is Polymyositis (PM) or Dermatomyositis (DM);
the disease process for predicting the Idiopathic Inflammatory Myopathy (IIM) is as follows:
(1) predicting risk of IIM secondary tumor associated myositis (CAM);
(2) predicting the prognosis of treatment for non-CAM IIM patients;
the prediction predicts the risk of IIM secondary tumor associated myositis (CAM) by detecting the level of anti-HSF 1 IgG antibodies in the serum of the target patient at a positive cut-off value of 3-fold the mean serum anti-HSF 1 antibody level of healthy population, e.g., the level of anti-HSF 1 IgG antibodies in the serum of the target patient is higher than the cut-off value, and the risk of CAM is high if the level of autoantibodies to other known IIMs in the target patient is elevated, including anti-transcriptional mediator 1 γ (TIF1 γ) antibody, anti-nuclear matrix protein 2(NXP2) antibody, anti-small ubiquitin-like modification activating enzyme (SAE) antibody, and anti-histaminyl tRNA synthetase (Jo-1) antibody.
The prognosis of the non-CAM IIM patient is determined by detecting the anti-HSF 1 IgG antibody level in the target patient serum, and if the anti-HSF 1 IgG antibody level in the target patient serum is decreased after treatment, the prognosis is good.
The invention has the beneficial effects that:
this study screened for a novel type of IIM autoantibody, anti-HSF 1 antibody, using human proteome chip technology and further examined the distribution of this antibody in large samples of IIM and other autoimmune disease patients. It was found that,anti-HSF 1 antibodies in phase with CAM Closing deviceHowever, the presence of this antibody was not detected in simple tumors.Serum anti-HSF 1 antibody levels and disease in non-CAM patients Disease activity is moderately positively correlated, and anti-HSF 1 antibody can be turned negative as the patient treatment is relieved。
In humans, HSF1 is constitutively expressed in many cells, and HSF1 is mostly present in the cytoplasm and nucleus as an inactive monomer in a non-stressed state. When cells are subjected to harmful stimuli, such as heat stress, ischemia, infection, etc., HSF1 accumulates into the nucleus and undergoes phosphorylation and oligomerization, eventually forming active trimers. The active HSF1 trimer can regulate the expression of heat shock protein gene by binding Heat Shock Element (HSE) in the promoter of the target gene15,20]. Unlike normal tissue cells, the high expression of HSF1 can be found in various tumor tissues such as ovarian cancer, breast cancer, endometrial cancer and liver cancer21-24]. Furthermore, it was found that HSF1 is mostly in the active form of trimer in cancer cells and is involved in malignant transformation of cancer cells25]. Wilson et al detected IgA against phosphorylated HSF1 in the serum of patients with early stage high grade ovarian cancer (HGSOC), but no other Ig class [ 2 ] was found against this antigen26]。
Although IgG was not detected against non-phosphorylated HSF1 in ovarian cancer-only patients, the antibody was detected in 3 cases of CAM-combined ovarian cancer patients. It is noted that we also merge other classes in 8 cases of CAMIgG was detected against non-phosphorylated HSF1 in patients with type tumors, including 2 esophageal, 2 thyroid, 1 gastric, 1 breast, 1 cervical and 1 nasopharyngeal carcinomas, but the presence of this antibody was also not detected in matched tumor-only patients. Thus, the relative immune responses of HSF1 in CAM and simple tumors were different,IgG against non-phosphorylated HSF1 can be used as a vaccine Serological markers for CAM。
A plurality of epidemiological surveys show that IIM patients often have concurrent tumors, and the incidence rate of the tumors is about 20 to 25 percent27]. Certain MSAs in IIM, such as anti-TIF 1 gamma, anti-NXP 2, anti-HMGCR and anti-SAE antibodies, are associated with the development of CAM28-31]. Interestingly, these tumor-associated MSAs do not predict the risk of developing simple tumors32,33]. In addition, studies have reported that patients with MSAs-negative IIM have an increased risk of developing tumors, suggesting that other unknown autoantigens [ 2 ] may be present in the CAM34]. Thus, new CAM-associated autoantibodies remain to be explored further. The study found that 16.9% of the anti-HSF 1 positive IIM patients were CAM and MSAs were detectable in the sera of this group of patients, including anti-TIF 1 γ, anti-NXP 2, anti-SAE and anti-Jo-1 antibodies. After correction of age distribution and sex ratio for anti-HSF 1 positive and anti-HSF 1 negative IIM patients by multifactorial analysis,discovery The anti-HSF 1 antibody was still associated with tumorigenesis, suggesting that the antibody is a risk factor for IIM tumorigenesis。
Recent clinical studies have found that MSAs/MAAs in the serum of IIM patients disappear as the disease is alleviated, suggesting that serum myositis antibody levels can be used to monitor patient disease activity3]. Antibodies against SRP, HMGCR, MDA5, Jo-1 and NXP2 in MSAs have been shown to be associated with myositis disease activity35-39]. This study found that by studying both the lateral and longitudinal against HSF 1-positive IIM patientsSerum anti-HSF 1 antibody levels and myositis disease activity in non-CAM IIM patients Is in moderate positive correlation。
Drawings
FIG. 1, immunoprecipitation results of DM patients
FIG. 2, distribution characteristics of anti-HSF 1 antibody in IIM and other autoimmune diseases
(2A) Distribution of anti-HSF 1 antibodies in IIM (612 cases), Healthy Controls (HCs) (65 cases).
(2B) Coli-derived recombinant HSF1(Abcam) was further verified by ELISA using HEK 293-derived recombinant HSF1(OriGene) coated with anti-HSF 1-positive IIM (62 cases), randomly selected anti-HSF 1-negative IIM (81 cases) and HCs (31 cases).
(2C) ELISA blocking experiments tested 3 anti-HSF 1 Positive (PS) and 2 anti-HSF 1 Negative (NS) patients for changes in anti-HSF 1 antibody levels in serum.
(2D) Immunoblot results of sera from anti-HSF 1-positive IIM patients (49) and HCs (9).
(2E) Dot blot results of sera from anti-HSF 1 positive IIM patients (65) and HCs (7).
The dotted line of levels in 2A and 2C is the positive cutoff for anti-HSF 1 antibody, and the horizontal line of levels in B is the mean, i.e., the mean serum anti-HSF 1 antibody level of HCs plus 3-fold standard deviation. HSF1, heat shock transcription factor 1; IIM, idiopathic inflammatory myopathy; HCs, healthy control; ELISA, enzyme-linked immunosorbent assay; PS, anti-HSF 1 positive serum samples; NS, anti-HSF 1 negative serum sample.
FIG. 3 distribution of anti-HSF 1 antibodies in CAM and tumor-only patients
anti-HSF 1 antibody was found in 21.6% (11/51) of CAM patients, 0% (0/27) of tumor-free patients (x)25.1, P0.024). The dashed horizontal line in the figure is the positive cutoff for anti-HSF 1 antibody (0.271). HSF1, heat shock transcription factor 1; CAM, tumor-associated myositis.
FIG. 4, serum anti-HSF 1 antibody levels in anti-HSF 1 positive non-CAM patients correlated with disease activity (cross-sectional and longitudinal analysis)
(4A) anti-HSF 1 positive non-CAM patients (51 cases) serum anti-HSF 1 antibody levels positively correlated with patient PGA VAS (r 0.325, P0.02), typically VAS (r 0.312, P0.026) and muscle VAS (r 0.399, P0.004).
(4B) anti-HSF 1 positive non-CAM patients (10 cases) serum anti-HSF 1 antibody levels varied with disease treatment.
The dashed horizontal line in the figure is the positive cutoff for anti-HSF 1 antibodies, i.e., the mean serum anti-HSF 1 antibody levels of HCs plus 3-fold standard deviation. P < 0.05; HSF1, heat shock transcription factor 1; IIM, idiopathic inflammatory myopathy; PGA, physician's overall assessment of patient disease activity; VAS, visual analog score.
FIG. 5, serum anti-HSF 1 antibody levels in CAM patients were not correlated with disease activity
(5A) Correlation analysis of anti-HSF 1 antibody levels with PGA VAS;
(5B) correlation analysis of anti-HSF 1 antibody levels with general case VAS;
(5C) correlation analysis of anti-HSF 1 antibody levels with cutaneous VAS;
(5D) correlation analysis of anti-HSF 1 antibody levels with skeletal joint VAS;
(5E) correlation analysis of anti-HSF 1 antibody levels with gut VAS;
(5F) correlation analysis of anti-HSF 1 antibody levels with lung VAS;
(5G) correlation analysis of anti-HSF 1 antibody levels with cardiovascular VAS;
(5H) correlation analysis of anti-HSF 1 antibody levels with muscle VAS.
HSF1, heat shock transcription factor 1; CAM, tumor-associated myositis; IIM, idiopathic inflammatory myopathy; PGA, physician's assessment of patient overall disease activity; VAS, visual analog score.
FIG. 6 variation of serum antibody levels against HSF1 positive non-CAM patients with disease treatment
(6A) The change in serum anti-HSF 1 antibody levels and muscle VAS with disease treatment in non-CAM patients (10 cases);
(6B) levels of serum anti-HSF 1 antibody and lung VAS in non-CAM patients (10 cases) were varied with disease treatment.
The dashed horizontal line in the figure is the positive cutoff for anti-HSF 1 antibody (0.271). HSF1, heat shock transcription factor 1; IIM, idiopathic inflammatory myopathy; VAS, visual analog score.
Detailed Description
Materials and methods
1. Study object
This study analyzed the distribution characteristics and clinical significance of anti-HSF 1 antibodies in IIM by cross-sectional and longitudinal studies.
(1) Cross sectional study
A total of 612 IIM patients, including 499 DM and 113 PM patients, were enrolled to visit the central friendly hospital from 12 months 2009 to 12 months 2017. And (3) inclusion standard: DM and PM diagnosis meet the Bohan & Peter diagnostic criteria in 1975. Exclusion criteria: patient clinical data is missing; a subtype of IIM that is less common in chinese populations, such as Inclusion Body Myositis (IBM).
The diagnostic criteria for tumor associated myositis (CAM) is that the tumor is diagnosed within 3 years of the onset of myositis9]. Clinical data including age, sex, medical history, general condition, physical examination, laboratory examination, medication, etc. were collected from the patients.
The diagnostic criteria for Interstitial Lung Disease (ILD) is high resolution ct (hrct) suggesting ILD signs and/or restricted ventilation dysfunction (total lung volume, lung capacity, carbon monoxide transport factor values less than 80% of predictive values) ]10]。
The diagnostic criteria for weight loss is that the patient's weight suddenly loses more than 5% of the original weight within 6 months11]。
The 6 extramuscular system activities were evaluated according to the Myositis disease activity assessment visual analogue scales (myoacs), including general case, skin, skeletal joints, gastrointestinal tract, lung and cardiac system activities.
The patient is assessed for global disease activity (PGA), muscle involvement, and extramuscular global activity using a 10cm Visual Analog Scale (VAS)12]。
(2) Longitudinal study
A total of 10 anti-HSF 1 positive IIM patients were enrolled, followed by a visit of 16m (6-34 m), and serum collected from each patient admission (1-20 m). The disease treatment status of the patients was evaluated according to 3 core indices (myooct, PGA and muscle score). The standard for judging whether the patient's disease is improved or not is such that at least 2 of the 3 core indicators are improved by not less than 20%, and the remaining 1 is deteriorated by not less than 25%13]。
(3) Control group
65 Healthy Controls (HCs) and 27 tumor-free patients were included.
The diagnosis of tumors in both CAM and tumor-only patients was confirmed pathologically (see Table 1 for details). Both sex ratios and age distribution of HCs were matched to IIM patients. All serum samples were collected prior to hospitalization of the patient and stored at-80 ℃.
TABLE 1 tumor types and demographic characteristics of CAM and tumor-only patients
aCAM (tumor associated myositis): the patient's tumor diagnosis is within 3 years of the onset of IIM.
The study was approved by the ethical committee of the central friendly hospital (No.: 2013-6), and all patients enrolled in the study signed informed consent.
2. The statistical method comprises the following steps:
those with continuous variables that fit a normal distribution are described by means of the mean ± standard deviation, and those with non-normal distributions are described by the median and the interquartile range (IQR). Categorical variables are expressed as percentages and absolute frequencies. Comparison between the measurement data groups applied either the independent sample t-test or the Mann-Whitney U-test. Counting data is applied to x2Test or Fisher exact test. Logistic regression analysis assessed the risk of tumorigenesis in anti-HSF 1 positive IIM patients. Spearman correlation analysis and Generalized Estimation Equation (GEE) analyzes the correlation of serum anti-HSF 1 antibody levels with disease activity in IIM patients. Using SPSS 23.0(SPSS institute, USA) and GraphPad Prism 5.0(GraphPad Software, USA) for data management and statistical analysis.
Example 1 screening for highly specific antibodies in IIM and determining the Presence of HSF1 antibody
Applications of
Human Protein Microarray v5.1(Life Technologies, USA) measures antibody distribution in serum of 10 DM patients and 10 HCs and performs statistical analysis. The specific method comprises the following steps:
sealing, preparing a sealing solution (50mM HEPES,200mM NaCl, 0.01% Triton X-100, 25% glycerol,20mM reduced glutamthione, 1.0mM DTT,1X Synthetic Block), placing the chip in a chip incubation box, and sealing for 1h at 4 ℃;
② incubating the serum sample, adding prepared sample diluent (1: 500; 10 DM patients, 10 HCs) into a chip incubation box, and incubating for 1.5h at 4 ℃;
③ incubation of secondary antibody, Alexa
647-conjugated coat anti-human IgG fluorescent secondary antibody is added into the chip incubation box, and incubated for 1.5h at 4 ℃ in the dark;
drying, namely, centrifugally drying the chip by using a chip centrifugal machine (200g,2 min);
scanning and data collection, the chip is scanned by a Tecan fluorescent microarray PowerScanner, and data is collected by using GenePix Pro 6.0 software (Molecular Devices, Sunnyvale, Calif.);
data analysis, using Life Technologies' prodirery
The data was analyzed by the Prospector software to screen for unknown antibodies in the sera of DM patients.
Unknown antibodies in IIM were screened by testing antibody profiles in serum from 10 DM patients (2 positive for MDA5 and 8 negative for myositis) and 10 HCs. High signals of MDA5 antigen (anti-melanoma differentiation-associated gene-5) were detected in 2 anti-MDA 5 positive DM patient sera, confirming the feasibility of the protein chip for large-scale screening of autoantibodies. High signals for HSF1 antigen were detected in 2/8 DM patient sera, and presence of anti-HSF 1 antibodies in patient sera was also found by IP (immunoprecipitation) (fig. 1). Therefore, we selected anti-HSF 1 antibody for further expansion studies.
Example 2 ELISA detection of anti-HSF 1 antibodies
Commercial recombinant HSF1 protein (Abcam, Cambridge, UK) was diluted and coated in wells (200 ng/well) of a polystyrene plate (Thermo Scientific, Roskilde, Denmark) and ELISA detected anti-HSF 1 antibody levels in serum. In addition, the serum anti-HSF 1 antibody level was measured by ELISA using another commercially available recombinant HSF1 protein (OriGene, Maryland, USA). Gradient dilution ELISA (1:2) assay serum was optimized for dilution ratio, ELISA blocking assay ELISA was used to determine the specificity of anti-HSF 1 antibody. All serum samples were tested 2 times. Before testing serum for anti-HSF 1 antibodies by ELISA, diluted anti-HSF 1 positive IIM patients (3) and HCs (2) sera were incubated with recombinant HSF1 protein (Abcam, Cambridge, UK) overnight at 4 ℃ before testing anti-HSF 1 antibody levels.
The results show that: coli-derived commercial HSF1(Abcam) was used as the coating protein, and the optimal dilution factor was 1:200 as determined by gradient dilution of patient serum. Antibody positivity was calculated as the mean serum anti-HSF 1 antibody levels of HCs plus 3-fold standard deviation as the positivity threshold (0.271). anti-HSF 1 antibodies were found at 10.6% (65/612) IIM, and 3.1% (2/65) HCs (fig. 2A). The results of HSF1 antigen detection in 10 DM patients in the human proteome chip study were consistent with the results of ELISA detection, confirming the feasibility of the two experimental methods. Meanwhile, another commercial HSF1 from HEK293 is used as a coating protein, and ELISA (enzyme-linked immunosorbent assay) is used for detecting anti-HSF 1 antibodies in serum of IIM patients (shown in figure 2B), and the results of ELISA detection of two HSF1 proteins are better in consistency and correlation.
ELISA blocking experiments found that anti-HSF 1 positive serum samples had significantly reduced anti-HSF 1 antibody levels after blocking by e.coli-derived HSF1, confirming the specific presence of this antibody in the serum (fig. 2C). In addition, we found better agreement with the ELISA results for IIM positive against HSF1 (fig. 2D, E).
Example 3 clinical features of anti-HSF 1 Positive IIM patients
Patients with HSF 1-positive IIM had a higher proportion of CAM (16.9% vs 7.3%, P ═ 0.008) and were frequently associated with weight loss (P ═ 0.046) and skin itching (P ═ 0.039). Meanwhile, anti-HSF 1 antibodies were associated with anti-TIF 1 γ antibody (P ═ 0.046), hypercholesterolaemia (P <0.001), elevation of C-reactive protein (P ═ 0.039), and elevation of blood sedimentation (ESR, P ═ 0.006). We did not find that anti-HSF 1 antibodies were associated with other typical IIM clinical features, such as skin involvement, pulmonary interstitial lesions, and elevated myozymes (table 2). Furthermore, the ANA karyotype of sera from patients with HSF 1-positive IIM was predominantly characterized as either a speckled or a cytoplasmic granular pattern.
TABLE 2 clinical characteristics of anti-HSF 1 antibody-positive IIM patients
HSF1, heat shock transcription factor 1; IIM, idiopathic inflammatory myopathy; PM, polymyositis; DM, dermatomyositis; ANA, antinuclear antibodies; CK, creatine kinase; LDH, lactate dehydrogenase;aweight loss: the accidental weight loss is more than or equal to 5 percent;bCAM (tumor associated myositis): the patient's tumor is diagnosed within 3 years before and after the onset of IIM; myositis antibodies including anti-Mi-2, anti-TIF 1 γ, anti-MDA 5, anti-NXP 2, anti-Jo-1, anti-PL-7, anti-EJ, anti-OJ, anti-SRP, anti-HMGCR, anti-SAE, anti-Ku, anti-Scl-100, anti-Scl-75 antibodies.cThe data can be seen in 611 patients,dthe data can be found in 594 patients,ethe data can be seen in 601 patients,fthe data can be seen in 551 patients with,gthe data can be seen in 600 patients with,hthe data can be found in 541 patients,ithe data can be seen in 588 patients,jdata ofAs can be seen in the 590 cases of patients,kdata are available in 382 patients.
Example 4 anti-HSF 1 antibody and IIM tumorigenesis
In this study, 8.3% (51/612) IIM patients developed tumors, including urogenital (23/51, 45.1%), digestive (9/51, 17.6%), respiratory (7/51, 13.7%), hematologic (3/51, 5.9%) and endocrine (9/51, 17.6%). Of these, 21.6% (11/51) of the CAM patients were positive for HSF1, yet the presence of the antibody was not detected in their corresponding tumor-only patients (FIG. 3). 11 anti-HSF 1 positive CAM patients were MSAs positive, including anti-TIF 1 γ 7 (63.6%), anti-NXP 21 (9.1%), anti-SAE 1 (9.1%) and anti-Jo-12 (18.2%). Univariate analysis found that increased age (OR 3.6[ 95% CI 2.0-6.4], P <0.001), anti-HSF 1 positive (OR 2.6[ 95% CI 1.3-5.3], P ═ 0.008), anti-HSF 1 OR anti-TIF 1 γ positive (OR 10.4[ 95% CI 5.4-20.0], P <0.001) was associated with tumorigenesis. Multivariate analysis after correcting for the effects of age distribution, patients with increased risk of developing tumors were still found to be positive for anti-HSF 1(OR 2.4[ 95% CI 1.1-5.0], P ═ 0.022), anti-HSF 1, OR anti-TIF 1 γ (OR 9.5[ 95% CI 4.9-18.4], P <0.001) (table 3).
TABLE 3 risk of tumorigenesis in anti-HSF 1-positive IIM patients
OR, ratio; 95% CI, 95% confidence interval; IIM, idiopathic inflammatory myopathy; HSF1, heat shock transcription factor 1; TIF1 γ, transcription mediator 1 γ; ROC curve analysis determined the optimal age cutoff for IIM-pooled tumor patients to be 56 years old.
Example 5 serum anti-HSF 1 antibody levels in IIM patients
Cross-sectional studies found that PGA VAS was significantly higher in anti-HSF 1 positive non-CAM patients than in anti-HSF 1 negative patients (P ═ 0.014). Spearman correlation analysis found that serum antibody levels in anti-HSF 1 positive non-CAM patients were positively correlated with PGA VAS (r 0.325, P0.02), general VAS (r 0.312, P0.026) and muscle VAS (r 0.399, P0.004) (fig. 4A), whereas serum antibody levels in anti-HSF 1 positive CAM patients were not found to correlate with disease activity (fig. 5, P > 0.05). Longitudinal studies analyzed the changes in serum anti-HSF 1 antibody levels in 10 anti-HSF 1 positive non-CAM patients during disease treatment. During the follow-up period, 9/10 (90%) patients responded well and achieved clinical remission, and had decreased anti-HSF 1 antibody levels in their sera, with 7 patients turning negative for anti-HSF 1 antibody at the last visit; 1/10 (10%) of the patients had relapsed and increased anti-HSF 1 antibody levels in their sera (FIG. 4B, FIG. 6). Statistical analysis of the GEE model revealed that serum anti-HSF 1 antibody levels were positively correlated with PGA VAS, muscle VAS, and lung VAS.
Finally, it should be noted that the above embodiments are only used to help those skilled in the art understand the essence of the present invention, and do not limit the protection scope.
Claims (5)
1. Use of a molecular marker for the preparation of a diagnostic kit for the diagnosis of idiopathic inflammatory myopathy,
the molecular marker is an autologous heat shock transcription factor 1 antibody in serum;
the idiopathic inflammatory myopathy is polymyositis or dermatomyositis;
the diagnosis is as follows: and detecting the level of the anti-HSF 1 IgG antibody in the serum of the target patient, and if the level of the anti-HSF 1 IgG antibody in the serum of the target patient is higher than a positive critical value, diagnosing the idiopathic inflammatory myopathy by taking the average serum anti-HSF 1 antibody level of healthy people as reference.
2. The use of claim 1, wherein the positive cutoff value is the mean serum anti-HSF 1 antibody level plus 3-fold standard deviation in healthy humans.
3. Use according to claim 1 or 2,
the diagnosis is as follows: detecting the IgG antibody level of anti-HSF 1 in the serum of the target patient, taking the serum anti-HSF 1 antibody level of healthy people as reference, if the IgG antibody level of anti-HSF 1 in the serum of the target patient is higher than a positive critical value,
and autoantibodies to other known IIMs are elevated in the subject patient, then the diagnosis is idiopathic inflammatory myopathy.
4. Use according to claim 3,
the other autoantibodies to known IIMs are anti-TIF 1 γ antibodies.
5. Use of a molecular marker for the preparation of a diagnostic kit for predicting the course of an idiopathic inflammatory myopathy disease,
the molecular marker is an autologous heat shock transcription factor 1 antibody in serum,
the prediction of the idiopathic inflammatory myopathy disease process is as follows:
(1) predicting risk of IIM secondary tumor-associated myositis;
(2) predicting the prognosis of treatment for non-CAM IIM patients;
the antibody is IgG;
the idiopathic inflammatory myopathy is polymyositis or dermatomyositis;
the prediction predicts the risk of secondary tumor-associated myositis of IIM as follows: detecting the level of anti-HSF 1 IgG antibody in the serum of the target patient, and taking the average value of the serum anti-HSF 1 antibody level of healthy people plus 3 times standard deviation as a positive critical value, if the level of anti-HSF 1 IgG antibody in the serum of the target patient is higher than the critical value and the autoantibodies of other known IIM in the target patient are increased, the CAM is high risk;
the autoantibodies of the IIM comprise an anti-transcription mediator 1 gamma antibody, an anti-nucleoplasmin 2 antibody, an anti-small ubiquitin-like modification activating enzyme antibody and an anti-histaminyl tRNA synthetase antibody;
the prognosis of the non-CAM IIM patient is determined by detecting the anti-HSF 1 IgG antibody level in the target patient serum, and if the anti-HSF 1 IgG antibody level in the target patient serum is decreased after treatment, the prognosis is good.
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