Yellow's syndrome screening system based on NOD2 gene new pathogenic mutation

Abstract

The invention discloses a system for screening Reiye's syndrome based on NOD2 gene new pathogenic mutation. The invention provides a computer device. The computer device comprises a memory, a processor and a computer program stored in the memory, the processor executes the computer program to implement the following steps: receiving NOD2 gene data (cDNA sequence) of a person to be tested; the method comprises the following steps: reacting the NOD2 gene with any one of the following mutations in SEQ ID No.2 stored in a computer: c.380Cgt; t, c, 2657Cgt; t, c.328Ggt; a, c.1295Cgt is selected from the group consisting of the T, c, 1981Ggt; c, and c, 2452Agt; c) performing comparison; and according to a comparison result, outputting information about whether the to-be-detected person is or is suspected to be a patient with the Reiderson's syndrome or about the risk of suffering from the Reiderson's syndrome. The novel pathogenic gene mutation form related to the NOD2 gene of the patient with the Reidersi syndrome is found, the novel pathogenic gene mutation form can serve as a target object to be used for developing a reagent and a computer device for diagnosing or screening the Reidersi syndrome, and the novel pathogenic gene mutation form has important significance on diagnosis and screening of the Reidersi syndrome.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medical informatics, and in particular to a screening system for Sweet's syndrome based on a new pathogenic mutation of NOD2 gene. BACKGROUND

[0002] Systemic autoinflammatory diseases (SAIDs) are a group of genetic heterogeneity diseases characterized by innate immune disorders, aseptic, periodic, and episodic systemic inflammation, which can cause serious organ damage and long-term complications such as joint deformity, hearing loss, blindness, stroke, and amyloidosis. The pathogenesis has not been fully elucidated so far. The diseases in SAIDs are included in the first and second batch of rare disease catalogs, but due to the lack of precise treatment methods and heterogeneous efficacy, it is easy to cause repeated attacks and adverse prognosis (Krainer J, Siebenhandl S, Weinhausel A. Systemic autoinflammatory diseases [J]. J Autoimmun, 2020, 109: 102421). Therefore, it is urgent to further study the pathogenesis of SAIDs and find precise drug treatment targets to achieve early diagnosis and effective control of the disease.

[0003] Abnormal activation of the nucleotide-binding oligomerization domain 2 (NOD2) signaling pathway can increase the susceptibility of various immune, inflammatory diseases and cancers. At present, the genetic SAIDs mutation registration website Infevers (https: / / infevers.umai-montpellier.fr / web / ) has reported 2695 SAIDs related gene mutations involving 52 genes, of which 187 are NOD2 gene mutations, showing the important role of NOD2 gene mutations in the pathogenesis of SAIDs. Studies have shown that about 10%-20% of SAIDs patients carry NOD2 variants, of which about 30%-80% are Yao syndrome (YAOS; OMIM#617321), which is the most common diagnosis among patients carrying NOD2 variants (Karamanakos A, Vougiouka O, Sapountzi E, Venetsanopoulou AI, Tektonidou MG, Germenis AE, Sfikakis PP, Laskari K. The expanding clinical spectrum of autoinflammatory diseases with NOD2 variants: a case series and literature review [J]. Front Immunol, 2024, 15: 1342668). YAOS was first reported by Professor Yao Qingping of Cleveland Hospital in the United States in 2011, and the prevalence is expected to be 1-10 / 100,000. Foreign studies have found that YAOS mainly affects white women, with an onset age of 20-50 years old, and the main manifestations are periodic fever, rash, joint pain / inflammation, non-specific digestive symptoms, dry mouth and eyes, lower extremity edema and serositis, and even may be complicated with chronic pain syndrome and disability (Yao Q, Shen B. A Systematic Analysis of Treatment and Outcomes of NOD2-Associated Autoinflammatory Disease [J]. Am J Med, 2017, 130: 365e313-365 e318.). However, there are only a few reports of YAOS patients worldwide, which indicates that the disease has not been fully diagnosed and is difficult to be discovered early.

[0004] The main NOD2 variants reported abroad are IVS8 +158 , IVS8 +158 / R702W, IVS8+158 / 1007fs and others (Yao Q, Shen M, McDonald C, Lacbawan F, Moran R, Shen B. NOD2-associated autoinflammatory disease: a large cohort study [J]. Rheumatology (Oxford), 2015, 54: 1904-1912.). In contrast, Chinese Han patients have a unique clinical phenotype and carry new NOD2 variants (NOD2 Q902K, R541P, Y514H) (Yang X, Wu D, Li J, Shen M, Zhang W. A Chinese case series of Yao syndrome and literature review [J]. Clin Rheumatol, 2018, 37:3449-3454), suggesting that NOD2 gene mutations are important in the diagnosis of YAOS and have ethnic heterogeneity. SUMMARY

[0005] The purpose of the present application is to provide a Yao syndrome screening system based on a new pathogenic mutation of the NOD2 gene.

[0006] In a first aspect, the present application claims a computer device.

[0007] The computer device claimed by the present application comprises a memory, a processor and a computer program stored on the memory; the processor can execute the computer program to achieve the following steps (A1)-(A3):

[0008] (A1) Data receiving: receiving NOD2 gene data of a testee, wherein the NOD2 gene data is the cDNA sequence information of the NOD2 gene;

[0009] (A2) Data comparison: comparing the NOD2 gene data of the testee with the NOD2 gene standard mutation form stored in the computer; the NOD2 gene standard mutation form is the NOD2 gene shown in SEQ ID No. 2 with any one of the following six mutations: c.380C>T, c.2657C>T, c.328G>A, c.1295C>T, c.1981G>C and c.2452A>C;

[0010] (A3) Result output: determining and outputting the detection result information according to the comparison result;

[0011] The detection result information is any one of the following:

[0012] P1, whether the subject is or is suspected to be a patient with NOD2-associated disease (including for distinguishing NOD2-associated disease patients and healthy controls);

[0013] P2, the risk level of the subject suffering from NOD2-associated disease.

[0014] For the above 6 mutations (gene level), c.380C>T means that the 380th position of the NOD2 gene (cDNA sequence) shown in SEQ ID No. 2 is mutated from C to T, and the mutated NOD2 gene is shown in SEQ ID No. 6. c.2657C>T means that the 2657th position of the NOD2 gene (cDNA sequence) shown in SEQ ID No. 2 is mutated from C to T, and the mutated NOD2 gene is shown in SEQ ID No. 14. c.328G>A means that the 328th position of the NOD2 gene (cDNA sequence) shown in SEQ ID No. 2 is mutated from G to A, and the mutated NOD2 gene is shown in SEQ ID No. 4. c.1295C>T means that the 1295th position of the NOD2 gene (cDNA sequence) shown in SEQ ID No. 2 is mutated from C to T, and the mutated NOD2 gene is shown in SEQ ID No. 8. c.1981G>C means that the 1981st position of the NOD2 gene (cDNA sequence) shown in SEQ ID No. 2 is mutated from G to C, and the mutated NOD2 gene is shown in SEQ ID No. 10. c.2452A>C means that the 2452nd position of the NOD2 gene (cDNA sequence) shown in SEQ ID No. 2 is mutated from A to C, and the mutated NOD2 gene is shown in SEQ ID No. 12. The same below.

[0015] The processor can also execute the computer program to implement the following steps (A4)-(A6):

[0016] (A4) Data receiving: receiving NOD2 protein data of the subject, wherein the NOD2 protein data is the amino acid sequence information of NOD2 protein;

[0017] (A5) Data comparison: comparing the NOD2 protein data of the subject with the NOD2 protein standard mutation form stored in the computer; the NOD2 protein standard mutation form is any one of the following 6 mutations of the NOD2 protein shown in SEQ ID No. 1: S127L, A886V, A110T, A432V, A661P and K818Q;

[0018] (A6) Result output: determining and outputting the detection result information according to the comparison result;

[0019] The detection result information is any one of the following:

[0020] P1, whether the subject is or is suspected to be a patient with Blau syndrome (including for differentiating Blau syndrome patients from healthy controls);

[0021] P2, the risk of the subject having Blau syndrome.

[0022] For the above 6 mutations (protein level), S127L means that the 127th amino acid of the NOD2 protein shown in SEQ ID No. 1 is mutated from serine (S) to leucine (L), and the mutated NOD2 protein is shown in SEQ ID No. 5 (corresponding to the gene mutation: c.380C>T). A886V means that the 886th amino acid of the NOD2 protein shown in SEQ ID No. 1 is mutated from alanine (A) to valine (V), and the mutated NOD2 protein is shown in SEQ ID No. 13 (corresponding to the gene mutation: c.2657C>T). A110T means that the 110th amino acid of the NOD2 protein shown in SEQ ID No. 1 is mutated from alanine (A) to threonine (T), and the mutated NOD2 protein is shown in SEQ ID No. 3 (corresponding to the gene mutation: c.328G>A). A432V means that the 432nd amino acid of the NOD2 protein shown in SEQ ID No. 1 is mutated from alanine (A) to valine (V), and the mutated NOD2 protein is shown in SEQ ID No. 7 (corresponding to the gene mutation: c.1295C>T). A661P means that the 661st amino acid of the NOD2 protein shown in SEQ ID No. 1 is mutated from alanine (A) to proline (P), and the mutated NOD2 protein is shown in SEQ ID No. 9 (corresponding to the gene mutation: c.1981G>C). K818Q means that the 818th amino acid of the NOD2 protein shown in SEQ ID No. 1 is mutated from lysine (K) to glutamine (Q), and the mutated NOD2 protein is shown in SEQ ID No. 11 (corresponding to the gene mutation: c.2452A>C). The same below.

[0023] Further, in the (A3), if the NOD2 gene data of the test subject contains the standard mutation form of the NOD2 gene, the detection result information of "the test subject is or is suspected to be a patient with NOD2-associated enteropathy" or "the test subject has a relatively high risk of suffering from NOD2-associated enteropathy" is output. If the NOD2 gene data of the test subject does not contain the standard mutation form of the NOD2 gene, the detection result information of "the test subject is not or is not a candidate for a patient with NOD2-associated enteropathy" or "the test subject has a relatively low risk of suffering from NOD2-associated enteropathy" is output. Note that the NOD2-associated enteropathy herein specifically refers to the NOD2-associated enteropathy caused by the above-mentioned 6 mutations (gene level). That is, if the NOD2 gene data of the test subject does not contain the standard mutation form of the NOD2 gene, it cannot be excluded whether the test subject suffers from or is suspected to suffer from the NOD2-associated enteropathy caused by other known pathogenic mutations of the NOD2 gene and other unknown pathogenic mutations. In actual detection, a person skilled in the art can store other known pathogenic mutation forms of the NOD2 gene in the computer for comparison according to the needs.

[0024] Further, in the (A6), if the NOD2 protein data of the test subject contains the standard mutation form of the NOD2 protein, the detection result information of "the test subject is or is suspected to be a patient with NOD2-associated enteropathy" or "the test subject has a relatively high risk of suffering from NOD2-associated enteropathy" is output. If the NOD2 protein data of the test subject does not contain the standard mutation form of the NOD2 protein, the detection result information of "the test subject is not or is not a candidate for a patient with NOD2-associated enteropathy" or "the test subject has a relatively low risk of suffering from NOD2-associated enteropathy" is output. Note that the NOD2-associated enteropathy herein specifically refers to the NOD2-associated enteropathy caused by the above-mentioned 6 mutations (protein level). That is, if the NOD2 protein data of the test subject does not contain the standard mutation form of the NOD2 protein, it cannot be excluded whether the test subject suffers from or is suspected to suffer from the NOD2-associated enteropathy caused by other known pathogenic mutations of the NOD2 protein and other unknown pathogenic mutations. In actual detection, a person skilled in the art can store other known pathogenic mutation forms of the NOD2 protein in the computer for comparison according to the needs.

[0025] Furthermore, steps (A1) and (A4) further include a step of receiving clinical information data of the test subject; the clinical information data is the clinical symptom presentation of the test subject. Correspondingly, steps (A2) and (A5) further include a step of comparing the clinical information data of the test subject with typical phenotypes of autoinflammatory diseases stored in a computer. Accordingly, in step (A3), if the NOD2 gene data of the test subject contains the standard mutation form of the NOD2 gene, and the clinical information data of the test subject matches the typical phenotype of the autoinflammatory disease, then the test result information "the test subject is or is suspected of having Yao's syndrome" or "the test subject has a relatively high risk of having Yao's syndrome" is output; in step (A6), if the NOD2 protein data of the test subject contains the standard mutation form of the NOD2 protein, and the clinical information data of the test subject matches the typical phenotype of the autoinflammatory disease, then the test result information "the test subject is or is suspected of having Yao's syndrome" or "the test subject has a relatively high risk of having Yao's syndrome" is output.

[0026] The typical phenotype of the aforementioned autoinflammatory disease includes (or may be): recurrent fever with myalgia and / or periodic fever with abdominal pain, rash, serositis, lymphadenopathy, and / or arthritis without any apparent cause; elevated inflammatory markers and / or C-reactive protein during flare-ups, and normal levels between flare-ups. The same applies below.

[0027] Furthermore, to rule out the pathogenicity of MEFV gene mutations, the subjects described in this invention can be patients who exhibit the above symptoms but do not respond well to colchicine treatment. The same applies below.

[0028] Secondly, this invention claims protection for a computer program product.

[0029] The computer program product claimed in this invention includes a computer program that, when executed by a processor, performs the steps described in the first aspect above.

[0030] The computer program product may be a software product that primarily implements its solution through computer programs.

[0031] Thirdly, the present invention claims protection for a computer-readable storage medium.

[0032] The present invention claims a computer program stored on a computer-readable storage medium; said computer program, when executed by a processor, performs the steps described in the first aspect above.

[0033] The computer readable storage medium refers to a carrier storing data, which can be a magnetic tape, a magnetic disk, a floppy disk, an optical disk, a magneto-optical disk, a ROM, a PROM, a VCD, a DVD, a hard disk, a flash memory, a U disk, a CF card, an SD card, an MMC card, an SM card, a Memory Stick, or an xD card.

[0034] In a fourth aspect, the present application claims a system.

[0035] The system claimed by the present application is system 1 or system 2.

[0036] The system 1 can include the following (B1) and (B2):

[0037] (B1) a substance for detecting NOD2 gene data of a subject to be tested; the NOD2 gene data is the cDNA sequence information of the NOD2 gene;

[0038] (B2) a device; the device includes a data receiving module, a data storage module, a data comparison module, and a conclusion output module;

[0039] The data receiving module is configured to receive the NOD2 gene data of the subject to be tested;

[0040] The data storage module is configured to store a standard mutation form of the NOD2 gene; the standard mutation form of the NOD2 gene is any one of the following six mutations of the NOD2 gene shown in SEQ ID No. 2: c.380C>T, c.2657C>T, c.328G>A, c.1295C>T, c.1981G>C, and c.2452A>C;

[0041] The data comparison module is configured to receive the NOD2 gene data of the subject to be tested sent by the data receiving module, and call the standard mutation form of the NOD2 gene in the data storage module, compare the NOD2 gene data of the subject to be tested with the standard mutation form of the NOD2 gene;

[0042] The conclusion output module is configured to receive the comparison result sent by the data comparison module, and determine and output the detection result information according to the comparison result; the detection result information is any one of the following:

[0043] P1, whether the subject to be tested is or is suspected to be a NOD2-associated syndrome patient;

[0044] P2, the risk of the subject to be tested suffering from NOD2-associated syndrome.

[0045] The system 2 can include the following (B3) and (B4):

[0046] (B3) a substance for detecting NOD2 protein data of the subject to be tested; the NOD2 protein data is amino acid sequence information of NOD2 protein;

[0047] (B4) a device; the device comprises a data receiving module, a data storage module, a data comparison module and a conclusion output module;

[0048] The data receiving module is configured to receive the NOD2 protein data of the subject to be tested;

[0049] The data storage module is configured to store NOD2 protein standard mutant forms; the NOD2 protein standard mutant forms are any one of the following six mutations of NOD2 protein shown in SEQ ID No. 1: S127L, A886V, A110T, A432V, A661P and K818Q;

[0050] The data comparison module is configured to receive the NOD2 protein data of the subject to be tested sent by the data receiving module, and call the NOD2 protein standard mutant forms in the data storage module, compare the NOD2 protein data of the subject to be tested with the NOD2 protein standard mutant forms;

[0051] The conclusion output module is configured to receive the comparison result sent by the data comparison module, and determine and output detection result information according to the comparison result; the detection result information is any one of the following:

[0052] P1, whether the subject to be tested is or is suspected to be a NOD2-associated syndrome patient (including for distinguishing NOD2-associated syndrome patients and healthy controls);

[0053] P2, the risk of the subject to be tested suffering from NOD2-associated syndrome.

[0054] Further, in the system 1, the conclusion output module determines and outputs the detection result information according to the comparison result as follows: if the NOD2 gene data of the testee contains the standard mutation form of the NOD2 gene, the detection result information of "the testee is or is suspected to be a patient with NOD2-associated syndrome" or "the testee has a relatively high risk of suffering from NOD2-associated syndrome" is output. If the NOD2 gene data of the testee does not contain the standard mutation form of the NOD2 gene, the detection result information of "the testee is not or is not a candidate for a patient with NOD2-associated syndrome" or "the testee has a relatively low risk of suffering from NOD2-associated syndrome" is output. [Note: Here, the NOD2-associated syndrome specifically refers to the NOD2-associated syndrome caused by the above-mentioned 6 mutations (gene level). That is, if the NOD2 gene data of the testee does not contain the standard mutation form of the NOD2 gene, it cannot be excluded whether the testee suffers from or is suspected to suffer from the NOD2-associated syndrome caused by other known pathogenic mutations of the NOD2 gene and other unknown pathogenic mutations]. In actual detection, the skilled in the art can also store other known pathogenic mutation forms of the NOD2 gene into the data storage module for comparison according to the needs.

[0055] Further, in the system 2, the conclusion output module determines and outputs the detection result information according to the comparison result as follows: if the NOD2 protein data of the testee contains the standard mutation form of the NOD2 protein, the detection result information of "the testee is or is suspected to be a patient with NOD2-associated syndrome" or "the testee has a relatively high risk of suffering from NOD2-associated syndrome" is output. If the NOD2 protein data of the testee does not contain the standard mutation form of the NOD2 protein, the detection result information of "the testee is not or is not a candidate for a patient with NOD2-associated syndrome" or "the testee has a relatively low risk of suffering from NOD2-associated syndrome" is output. [Note: Here, the NOD2-associated syndrome specifically refers to the NOD2-associated syndrome caused by the above-mentioned 6 mutations (protein level). That is, if the NOD2 protein data of the testee does not contain the standard mutation form of the NOD2 protein, it cannot be excluded whether the testee suffers from or is suspected to suffer from the NOD2-associated syndrome caused by other known pathogenic mutations of the NOD2 protein and other unknown pathogenic mutations]. In actual detection, the skilled in the art can also store other known pathogenic mutation forms of the NOD2 protein into the data storage module for comparison according to the needs.

[0056] Further, in the system 1 and the system 2, the data receiving module is configured to further receive clinical information data of the subject to be tested; the clinical information data is a clinical symptom of the subject to be tested. Correspondingly, the data storage module is configured to further store a typical phenotype of the autoimmune disease; the data comparison module is configured to further receive the clinical information data of the subject to be tested sent by the data receiving module, and call the typical phenotype of the autoimmune disease in the data storage module, and compare the clinical information data of the subject to be tested with the typical phenotype of the autoimmune disease. Correspondingly, in the system 1, the conclusion output module is configured to receive the comparison result sent by the data comparison module, and determine and output the detection result information according to the comparison result as follows: if the NOD2 gene data of the subject to be tested contains the standard mutation form of the NOD2 gene, and the clinical information data of the subject to be tested is consistent with the typical phenotype of the autoimmune disease, the detection result information of "the subject to be tested is or is suspected to be a patient with Blau syndrome" or "the risk of the subject to be tested suffering from Blau syndrome is relatively high" is output. In the system 2, the conclusion output module is configured to receive the comparison result sent by the data comparison module, and determine and output the detection result information according to the comparison result as follows: if the NOD2 protein data of the subject to be tested contains the standard mutation form of the NOD2 protein, and the clinical information data of the subject to be tested is consistent with the typical phenotype of the autoimmune disease, the detection result information of "the subject to be tested is or is suspected to be a patient with Blau syndrome" or "the risk of the subject to be tested suffering from Blau syndrome is relatively high" is output.

[0057] In a fifth aspect, the present application claims a method for screening Blau syndrome by using a computer.

[0058] The method for screening Blau syndrome by using a computer claimed in the present application is method I or method II or method III or method IV:

[0059] The method I can include the following steps (C1)-(C3):

[0060] (C1) data receiving: receiving NOD2 gene data of a subject to be tested, the NOD2 gene data being cDNA sequence information of a NOD2 gene;

[0061] (C2) data comparison: comparing the NOD2 gene data of the subject to be tested with the NOD2 gene standard mutation form stored in the computer; the NOD2 gene standard mutation form is any one of the following six mutations of the NOD2 gene shown in SEQ ID No. 2: c.380C>T, c.2657C>T, c.328G>A, c.1295C>T, c.1981G>C and c.2452A>C;

[0062] (C3) result output: according to the comparison result, the detection result information is determined and output as follows: if the NOD2 gene data of the subject to be tested contains the NOD2 gene standard mutation form, the detection result information of "the subject to be tested is or is suspected to be a patient with NOD2-associated syndrome" or "the subject to be tested has a relatively high risk of NOD2-associated syndrome" is output. If the NOD2 gene data of the subject to be tested does not contain the NOD2 gene standard mutation form, the detection result information of "the subject to be tested is not or is not a candidate for NOD2-associated syndrome" or "the subject to be tested has a relatively low risk of NOD2-associated syndrome" is output

Note: here, the NOD2-associated syndrome specifically refers to the NOD2-associated syndrome caused by the above six mutations (gene level). That is, if the NOD2 gene data of the subject to be tested does not contain the NOD2 gene standard mutation form, it cannot be excluded whether the subject to be tested has or is suspected to have NOD2-associated syndrome caused by other known pathogenic mutations and other unknown pathogenic mutations of NOD2 gene

[0063] The method II can include the following steps (C4)-(C6):

[0064] (C4) data receiving: receiving the NOD2 protein data of the subject to be tested, the NOD2 protein data being NOD2 protein amino acid sequence information;

[0065] (C5) data comparison: comparing the NOD2 protein data of the subject to be tested with the NOD2 protein standard mutation form stored in the computer; the NOD2 protein standard mutation form is any one of the following six mutations of the NOD2 protein shown in SEQ ID No. 1: S127L, A886V, A110T, A432V, A661P and K818Q;

[0066] (C6) Result output: according to the comparison result, the detection result information is determined and output as follows: if the NOD2 protein data of the subject contains the NOD2 protein standard mutation form, the detection result information of "the subject is or is suspected to be a NOD2-associated disease patient" or "the subject has a relatively high risk of NOD2-associated disease" is output. If the NOD2 protein data of the subject does not contain the NOD2 protein standard mutation form, the detection result information of "the subject is not or is not a candidate for a NOD2-associated disease" or "the subject has a relatively low risk of NOD2-associated disease" is output. [Note: Here, the NOD2-associated disease specifically refers to the NOD2-associated disease caused by the above-mentioned six mutations (protein level). That is, if the NOD2 protein data of the subject does not contain the NOD2 protein standard mutation form, it cannot be ruled out whether the subject has or is suspected to have a NOD2-associated disease caused by other known pathogenic mutations of NOD2 protein and other unknown pathogenic mutations]. In actual detection, those skilled in the art can also include other known pathogenic mutations of NOD2 protein according to the needs of the subject for comparison.

[0067] The method III can include the following steps (C7)-(C9):

[0068] (C7) Data receiving: receiving the NOD2 gene data and clinical information data of the subject; the NOD2 gene data is the cDNA sequence information of the NOD2 gene; the clinical information data is the clinical symptom manifestation;

[0069] (C8) Data comparison: comparing the NOD2 gene data of the subject with the NOD2 gene standard mutation form stored in the computer; the NOD2 gene standard mutation form is that the NOD2 gene shown in SEQ ID No. 2 has any one of the following six mutations: c.380C>T, c.2657C>T, c.328G>A, c.1295C>T, c.1981G>C and c.2452A>C; and comparing the clinical information data of the subject with the typical phenotype of the autoimmune disease;

[0070] (C9) Result output: according to the comparison result, the detection result information is determined and output as follows: if the NOD2 gene data of the subject contains the NOD2 gene standard mutation form, and the clinical information data of the subject is consistent with the typical phenotype of the autoimmune disease, the detection result information of "the subject is or is suspected to be a NOD2-associated disease patient" or "the subject has a relatively high risk of NOD2-associated disease" is output.

[0071] The method IV can include the following steps (C10)-(C12):

[0072] (C10) data receiving: receiving NOD2 protein data and clinical information data of the subject to be tested; the NOD2 protein data is NOD2 protein amino acid sequence information; the clinical information data is clinical symptom manifestation;

[0073] (C11) data comparison: comparing the NOD2 protein data of the subject to be tested with NOD2 protein standard mutant forms stored in the computer; the NOD2 protein standard mutant forms are any one of the following six mutations of the NOD2 protein shown in SEQ ID No. 1: S127L, A886V, A110T, A432V, A661P and K818Q; and comparing the clinical information data of the subject to be tested with the typical phenotype of the subject's own inflammatory disease stored in the computer;

[0074] (C12) result output: according to the comparison result, the detection result information is determined and output as follows: if the NOD2 protein data of the subject to be tested contains the NOD2 protein standard mutant form, and the clinical information data of the subject to be tested is consistent with the typical phenotype of the subject's own inflammatory disease, the detection result information of "the subject to be tested is or is suspected to be a patient with Blau syndrome" or "the risk of the subject to be tested suffering from Blau syndrome is relatively high" is output.

[0075] In a sixth aspect, the present application claims any of the following applications:

[0076] Application I: use of a substance for detecting whether the NOD2 gene shown in SEQ ID No. 2 of the subject to be tested has any one of the six mutations: c.380C>T, c.2657C>T, c.328G>A, c.1295C>T, c.1981G>C and c.2452A>C in the preparation of a product;

[0077] The product is a product with any of the following uses:

[0078] (D1) detecting whether the subject to be tested is or is suspected to be a patient with Blau syndrome (i.e. diagnosing or screening Blau syndrome, including distinguishing Blau syndrome patients from healthy controls);

[0079] (D2) detecting the risk of the subject to be tested suffering from Blau syndrome;

[0080] The substance can be a primer and / or a probe, etc. used for specifically detecting the above mutation sites. In an embodiment of the present application, the substance used for detecting the c.380C>T and the c.328G>A is a primer pair consisting of SEQ ID No. 15 and SEQ ID No. 16; the substance used for detecting the c.1295C>T is a primer pair consisting of SEQ ID No. 17 and SEQ ID No. 18; the substance used for detecting the c.1981G>C is a primer pair consisting of SEQ ID No. 19 and SEQ ID No. 20; the substance used for detecting the c.2452A>C is a primer pair consisting of SEQ ID No. 21 and SEQ ID No. 22; and the substance used for detecting the c.2657C>T is a primer pair consisting of SEQ ID No. 23 and SEQ ID No. 24.

[0081] Application II: use of the substance for detecting whether the NOD2 protein shown in SEQ ID No. 1 of the subject has any one of the six mutations: S127L, A886V, A110T, A432V, A661P and K818Q in the preparation of a product;

[0082] The product is a product having any one of the following uses:

[0083] (D1) detecting whether the subject is or is suspected to be a Blau syndrome patient (i.e. diagnosing or screening Blau syndrome, including distinguishing Blau syndrome patients from healthy controls);

[0084] (D2) detecting the risk of the subject suffering from Blau syndrome.

[0085] The substance can be an antibody, etc. used for specifically detecting the above mutation sites.

[0086] In the above aspects, the subject can be a person after birth or a fetus existing in a mother during pregnancy. When the former, the sample to be tested can be a blood sample; when the latter, the sample to be tested can be amniotic fluid.

[0087] In specific embodiments of the present application, the mutation in the above aspects is a heterozygous mutation, and of course can also be a homozygous mutation.

[0088] In a seventh aspect, the present application claims protection of any one of the following biological materials:

[0089] (E1) a NOD2 protein mutant, the amino acid sequence of which is shown in SEQ ID No. 5, SEQ ID No. 13, SEQ ID No. 3, SEQ ID No. 7, SEQ ID No. 9 or SEQ ID No. 11;

[0090] (E2) a NOD2 gene mutant, the amino acid sequence of which is shown in SEQ ID No. 6, SEQ ID No. 14, SEQ ID No. 4, SEQ ID No. 8, SEQ ID No. 10 or SEQ ID No. 12;

[0091] (E3) an expression cassette or a recombinant vector or a transgenic cell line containing the NOD2 gene mutant of (E2).

[0092] The present application discloses a new pathogenic gene mutation and mutant protein form related to NOD2 gene of a patient with Wiskott-Aldrich syndrome (WAS) by performing whole-exome high-throughput sequencing on a proband of WAS. The new NOD2 mutant gene and mutant protein provided by the present application can be used as a target for developing a reagent or kit for diagnosing or screening WAS. The present application has important significance for the diagnosis and screening of WAS. BRIEF DESCRIPTION OF DRAWINGS

[0093] Figure 1 A computer flowchart for screening WAS according to the present application.

[0094] Figure 2 Sanger verification results of the site in Example 1.

[0095] Figure 3 A protein conservation sequence frequency chart in Example 2.

[0096] Figure 4 Protein three-dimensional structure prediction analysis results (1) in Example 3.

[0097] Figure 5 Protein three-dimensional structure prediction analysis results (2) in Example 3.

[0098] Figure 6 Wiskott-Aldrich syndrome (YAO syndrome, YAOS) patient peripheral blood mononuclear cell transcriptome sequencing results (heat map) in Example 4. HC represents a healthy control.

[0099] Figure 7 Wiskott-Aldrich syndrome (YAO syndrome, YAOS) patient peripheral blood mononuclear cell transcriptome sequencing results (KEGG analysis and GSEA analysis) in Example 4.

[0100] Figure 8 Wiskott-Aldrich syndrome (YAO syndrome, YAOS) patient peripheral blood mononuclear cell immunoblotting results in Example 5, indicating that the WAS patient has obvious activation of the inflammatory pathway. HC represents a healthy control.

[0101] Figure 9 Results of enzyme-linked immunosorbent assay of inflammatory factors in peripheral blood mononuclear cells of a patient with YAO syndrome in Example 6. It is suggested that the patient with YAO syndrome has different degrees of release of inflammatory factors. HC represents a healthy control. * represents P < 0.05; ** represents P < 0.01; and *** represents P < 0.001. DETAILED DESCRIPTION

[0102] The application will be further described in conjunction with the specific embodiments. The examples provided below are only for the purpose of illustrating the application, and are not intended to limit the scope of the application. The examples provided below can serve as a guide for further improvement by those of ordinary skill in the art, and do not in any way constitute a limitation on the application.

[0103] The experimental methods in the following examples are all routine methods, unless otherwise specified, which are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained commercially.

[0104] The human NOD2 protein is UNIPROT (https: / / www.uniprot.org / )-Q9HC29, and the encoding gene is National Center for Biotechnology Information (https: / / www.ncbi.nlm.nih.gov / )-Gene ID: 64127, and the transcript is NM_022162.3.

[0105] The NOD2 gene (wild type) is shown as SEQ ID No. 2; and the NOD2 protein (wild type) is shown as SEQ ID No. 1.

[0106] Figure 1 A computer flowchart for screening YAO syndrome according to the application.

[0107] In step S1, NOD2 gene data of a subject to be tested is received, and the NOD2 gene data is cDNA sequence information of the NOD2 gene;

[0108] In step S2, the NOD2 gene data of the subject to be tested is compared with a standard mutation form of the NOD2 gene stored in a computer; and the standard mutation form of the NOD2 gene is any one of the following six mutations of the NOD2 gene shown as SEQ ID No. 2: c.380C>T, c.2657C>T, c.328G>A, c.1295C>T, c.1981G>C and c.2452A>C.

[0109] In step S3, according to the comparison result, the detection result information is determined and output as follows: if the NOD2 gene data of the to-be-tested person contains the standard mutation form of the NOD2 gene, the detection result information of "the to-be-tested person is or is suspected to be a patient with Blau syndrome" or "the to-be-tested person has a relatively high risk of Blau syndrome" is output.

[0110] Example 1, discovery of mutation forms of NOD2 gene

[0111] I. Proband information

[0112] Proband 1, female, 47 years old, came to the hospital for recurrent fever with myalgia for 11 months. The patient had fever for 11 months Recurrent fever without a predisposing cause Fever with myalgias The highest body temperature was 39.9℃, initially accompanied by chills and shivering. Accompanied by myalgia of limbs, sore throat. Accompanied by bilateral knee and wrist pain without swelling. Mild lower extremity edema. No skin rash, chest pain, abdominal pain, diarrhea, headache, eye inflammation, oral ulcer, periorbital edema, dry mouth, hearing loss, etc. Approximately 2-4 days per episode, 4 days to 4 weeks between episodes . Elevated inflammatory markers during episodes, normal between episodes The patient's life and work were normal. The results of infection, immunity, and tumor examination were all negative. The patient was treated with hormones, which was effective, and the symptoms recurred when the dose was reduced to 3-4 tablets / day. The patient tried colchicine for 1 week, which was ineffective. The patient had a typical phenotype of autoinflammatory diseases (underlined part), and was considered to have autoinflammatory diseases.

[0113] Proband 2, female, 24 years old, intermittent fever with abdominal pain for 6 years. Periodic fever, 3-7 days per episode, weeks to months between episodes Recurrent. Associated with abdominal pain Constipation, chest pain, myalgia, a small amount of lymphocyte infiltration around skin blood vessels, toe arthritis, Lower extremity edema . Elevated C-reactive protein during episodes, inflammation, normal between episodes The patient had a typical phenotype of autoinflammatory diseases (underlined part), and was considered to have autoinflammatory diseases.

[0114] Proband 3, male, 45 years old, periodic fever, abdominal pain for 1.5 years, Periodic fever, 3-10 days per episode, 2 months between episodes, associated with abdominal pain Abdominal distension, constipation, small intestinal erosion, small intestinal inflammation, joint pain, inflammation, Elevated C-reactive protein during episodes The patient had a typical phenotype of autoinflammatory diseases (underlined part), and was considered to have autoinflammatory diseases.

[0115] Proband 4, male, 27 years old, Recurrent fever with abdominal pain for 9 years accompanied by upper abdominal discomfort, chills, chills, back pain, accompanied by abdominal distension, nausea, vomiting and constipation, improved for 4-10 days, diarrhea for a few days after the attack stopped, relapsed after 1-2 months, Elevated white blood cell and C-reactive protein during episodes, normal between episodes Periodic fever with abdominal pain for 3 years The patient had a typical phenotype of autoinflammatory diseases (underlined part), and was considered to have autoinflammatory diseases.

[0116] Proband 5, male, 50 years old, Elevated C-reactive protein . Periodic fever, abdominal pain, abdominal distension, diarrhea, nausea, vomiting, weight loss, inflammation, Periodic fever for 5 years . Father often fever, suspicious hearing loss. This patient has a typical phenotype of autoinflammatory disease (underlined part), consider autoinflammatory disease.

[0117] Proband 6, male, 71 years old, 1 week per episode, weeks or months between episodes . Periodic fever, Figure 2 Figure 3 , fatigue, headache, inflammation, once suffered from pulmonary interstitial disease and ankylosing spondylitis. This patient has a typical phenotype of autoinflammatory disease (underlined part), consider autoinflammatory disease.

[0118] II. Whole exome sequencing of the proband (sanger verification)

[0119] For the subject's complaint, the genes with clear pathogenic relationship collected in OMIM database were analyzed. For a part of patients, the genomic DNA from the blood of the subjects was used as the detection material. First, the DNA was broken to prepare a library, and then the DNA of the target gene exon and the adjacent splicing region was captured and enriched by Roche KAPA HyperExome chip, and finally the variant detection was performed by using MGISEQ-2000 sequencing platform. The quality control index of the sequencing data was that the average sequencing depth of the target region was ≥180X, and the proportion of the sites with an average depth of >20X in the target region was >95%. Another part of the patients was completed on the Illumina sequencing platform. The average sequencing depth of the obtained data on the known gene exons and the upstream and downstream 5bp sequences of the human genome was ≥90X, and about 98% of the sequencing depth of the target sequence was >20X. The base recognition was performed on all sequencing fragments. The sequencing fragments were aligned with the UCSC hg19 human reference genome by using BWA, and the duplicates were removed. GATK was used for base quality correction, SNV, INDEL and genotype detection. ExomeDepth was used for copy number variation detection at the exon level. The gene naming was in accordance with the naming standard of the Human Genome Organization Gene Nomenclature Committee (HGNC); the variation naming was in accordance with the naming standard of the Human Genome Variation Society (HGVS). Based on the clinical information of the subjects, the population database, the disease database and the biological information prediction tool, the variation annotation and screening were performed. The pathogenicity classification of the variation was based on the American College of Medical Genetics and Genomics (ACMG) and the American Society of Molecular Pathologists (AMP) sequence variation interpretation guidelines, and reference was made to the detailed interpretation of the guidelines by ClinGen sequence variation interpretation working group and the British Clinical Genomics Science Society (ACGS). Proband 1 and 3 found MEFV and NOD2 genes, but proband 1 and 3 had poor treatment effect after receiving colchicine, so the pathogenicity of MEFV was excluded. No gene mutation other than NOD2 gene that could explain the clinical symptoms of proband 2, 4, 5 and 6 was found in the whole exome sequencing of proband 2, 4, 5 and 6.

[0120] The NOD2 gene of proband 1 had the following mutation: NM_022162.3: c.2452A>C (p.Lys818Gln), which was located at position 50712363 of chromosome 16 (Human GRCh38 / hg38 genome database). That is, the proband had a single nucleotide mutation of A→C in the 4th exon of the NOD2 gene, which caused the corresponding amino acid residue in the encoded protein to be mutated from lysine to glutamine. The nucleotide sequence of the mutated NOD2 gene is shown as SEQ ID No. 12, and the corresponding encoded protein is shown as SEQ ID No. 11.

[0121] The NOD2 gene of the proband 2 has the following mutation: NM_022162.3: c.380C>T (p.Ser127Leu), which is located at the position of 50699794 of chromosome 16 (Human GRCh38 / hg38 genome database). That is, the proband has a single nucleotide mutation of C→T in the 2nd exon of the NOD2 gene, thereby causing the corresponding amino acid residue in the encoded protein to be mutated from serine to leucine. The nucleotide sequence of the NOD2 gene after mutation is shown as SEQ ID No. 6, and the corresponding encoded protein is shown as SEQ ID No. 5.

[0122] The NOD2 gene of the proband 3 has the following mutation: NM_022162.3: c.2657C>T (p.Ala886Val), which is located at the position of 50719951 of chromosome 16 (Human GRCh38 / hg38 genome database). That is, the proband has a single nucleotide mutation of C→T in the 7th exon of the NOD2 gene, thereby causing the corresponding amino acid residue in the encoded protein to be mutated from alanine to valine. The nucleotide sequence of the NOD2 gene after mutation is shown as SEQ ID No. 14, and the corresponding encoded protein is shown as SEQ ID No. 13.

[0123] The NOD2 gene of the proband 4 has the following mutation: NM_022162.3 c.1295C>T (p.Ala432Val), which is located at the position of 50711206 of chromosome 16 (Human GRCh38 / hg38 genome database). That is, the proband has a single nucleotide mutation of C→T in the 4th exon of the NOD2 gene, thereby causing the corresponding amino acid residue in the encoded protein to be mutated from alanine to valine. The nucleotide sequence of the NOD2 gene after mutation is shown as SEQ ID No. 8, and the corresponding encoded protein is shown as SEQ ID No. 7. The sanger verification result of the site is shown as Figure 4

[0124] The NOD2 gene of the proband 5 has the following mutation: NM_022162.3: c.1981G>C (p.Ala661Pro), which is located at the position of 50711892 of chromosome 16 (Human GRCh38 / hg38 genome database). That is, the proband has a single nucleotide mutation of G→C in the 4th exon of the NOD2 gene, thereby causing the corresponding amino acid residue in the encoded protein to be mutated from alanine to proline. The nucleotide sequence of the NOD2 gene after mutation is shown as SEQ ID No. 10, and the corresponding encoded protein is shown as SEQ ID No. 9.

[0125] ​The NOD2 gene of the proband 6 has the following mutation: NM_022162.3 c.328G>A (p.Ala110Thr), which is located at position 50699742 of chromosome 16 (Human GRCh38 / hg38 genome database). That is, the proband has a G→A single nucleotide mutation in the 2nd exon of the NOD2 gene, causing the corresponding amino acid residue in the encoded protein to mutate from alanine to threonine. The nucleotide sequence of the mutated NOD2 gene is shown in SEQ ID No. 4, and the corresponding encoded protein is shown in SEQ ID No. 3.

[0126] Both of the above mutations are heterozygous mutations, that is, the proband has a normal NOD2 gene transcript (cDNA as shown in SEQ ID No. 2, WT protein as shown in SEQ ID No. 1) in one chromosome and a mutant NOD2 gene transcript in the other chromosome.

[0127] Wein syndrome belongs to multifactorial autoinflammatory diseases, and NOD2 gene mutation increases the susceptibility of the disease, which can be used as a diagnostic marker of the disease. Based on the above, the clinical phenotype of the proband meets the clinical diagnostic criteria of Wein syndrome, and no other gene mutations that can explain the patient's clinical manifestations are found in addition to the NOD2 gene heterozygous mutation. The NOD2 gene mutation can also fully explain the clinical phenotype of the proband. Therefore, by combining steps one and two, other autoinflammatory diseases can be excluded, and the proband is finally diagnosed as Wein syndrome.

[0128] Example 2, Protein Conservation Sequence Frequency Mapping Using Online Tools

[0129] The NOD2 protein transcript NM_022162.3 homologous amino acid sequence was downloaded from the NCBI protein database, and multiple sequence alignment analysis was performed, and protein conservation frequency analysis was performed. The analysis steps are as follows:

[0130] 1. Search for NOD2 in the protein database of the NCBI website, and search for the corresponding transcript NM_022162.3.

[0131] 2. Select homologous sequences: click blast- blastp, screen for amino acid sequences similar in length to human amino acids, select 44 sequences including humans, click Protein alignment, and perform online alignment analysis.

[0132] 3. Open the aligned sequence in MEGA11 (https: / / www.megasoftware.net / dload_win_beta), click alignment to perform ClustalW multiple sequence alignment, download it and open it in WebLogo 3 (http: / / weblogo.threeplusone.com), adjust the corresponding parameters to draw a protein conserved sequence frequency map.

[0133] See protein conserved sequence frequency map Figure 5 The proband gene mutations A110, K818, and A886 were found to be relatively conserved in species 43 / 44, while S127, A432, and A661 were conserved sequences in species 44 / 44, suggesting that site alterations may be pathogenic.

[0134] Example 3: Prediction of Protein Three-Dimensional Structure

[0135] The online software swiss-model (https: / / swissmodel.expasy.org / ) was used to perform wild-model predictions on the amino acid sequences corresponding to the reference sequence (shown in SEQ ID No. 1), and mutation model predictions were performed on the mutated amino acid sequences (shown in SEQ ID Nos. 3, 5, 7, 9, 11, and 13). The prediction steps are as follows:

[0136] 1. Search for NOD2 in the protein database on the NCBI website, select NOD2 nucleotide binding oligomerization domain containing 2 [Homo sapiens (human)] - NCBI Reference Sequences (RefSeq) - NP_071445.1 - download the FASTA sequence, and modify it according to the mutation site.

[0137] 2. Start modelling - Enter the FASTA sequence of the protein to be predicted, and click Build Model.

[0138] 3. Model Selection: Initial screening is performed based on similarity and model score (GMQE). After screening, click on structureassessment to view the Ramachandran Plots for evaluation. The Ramachandran Plots should show that most residues are located in the Ramachandran Favoured region (region area > 90%).

[0139] 4. Download the qualified model and open it with the open source version of the molecular three-dimensional structure display software Pymol (https: / / www.lfd.uci.edu / ~gohlke / pythonlibs / #pymol-open-source).

[0140] The analysis results are shown in Figure 6 and Figure 7 The predicted structure of the wild-type protein is shown in green, and the predicted structure of the mutant protein is shown in blue. The results show that the polar interaction force of the mutated amino acid site and other amino acid sites changes to varying degrees, which can explain that these mutation sites may change the function of the protein, thereby causing the occurrence of diseases.

[0141] Example 4, transcriptome sequencing analysis of peripheral blood mononuclear cells of patients with Tay-Sachs syndrome

[0142] Peripheral blood mononuclear cells (PBMCs) of patients with Tay-Sachs syndrome and healthy controls (HC) were routinely isolated, and cell suspensions were prepared with 1640 culture medium containing 10% fetal bovine serum, and inoculated into 24-well plates at 1.35 x 10 6 cells / well. After 24 h of culture, the cells were collected, and total RNA was extracted using Trizol reagent according to the manufacturer's procedure. The quantity and purity of total RNA were analyzed using Bioanalyzer 2100 and RNA 6000 Nano LabChip Kit, and high-quality RNA samples with RIN values > 7.0 were used to construct sequencing libraries. After total RNA extraction, mRNA was purified from total RNA (5 μg) using Dynabeads Oligo(dT), with a total of two rounds of purification. After purification, the mRNA fragments were fragmented into short fragments at high temperature using divalent cations. Then, using Superscript TM II reverse transcriptase, E. coli DNA polymerase I, RNase H and dUTP Solution to synthesize U-labeled second-strand DNA. Then, A bases were added to the blunt ends of each strand, preparing to link them to the adapters. Each adapter contains a T base overhang for linking the adapter to the fragment DNA with an A tail. Double adapters are linked to the fragments, and size selection is performed using AMPureXP magnetic beads. The U-labeled second-strand DNA is treated with a heat-labile UDG enzyme, and the linked product is amplified by PCR, with the following conditions: 95°C initial denaturation for 3 min; 98°C denaturation for 15 seconds, 60°C annealing for 15 seconds, 72°C extension for 30 seconds for 8 cycles; and finally, amplification at 72°C for 5 min. The average insert length of the cDNA library is 300 ± 50 bp. Finally, according to the recommended protocol of the supplier, the cDNA library was sequenced on the Illumina Novaseq TM6000 (LC-Bio Technology CO, Ltd, Hangzhou, China) for 2x150bp paired-end sequencing (PE150). The results were subjected to Gene Ontology (GO) functional annotation and enrichment analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis and Gene Set Enrichment Analysis (GSEA) analysis.

[0143] Figure 7 The heat map shows that the differentially expressed genes of the NOD2 mutation-carrying Weismann patient PBMCs are significantly different from those of the HC. Figure 8 The KEGG analysis (left) shows that the differentially expressed genes can be enriched in metabolic and interleukin-17 related signaling pathways. Figure 9 The GSEA analysis (right) shows that the differentially expressed genes are enriched in innate immunity and adaptive immunity related pathways, suggesting that there may be immune disorders.

[0144] Example 5, Western blotting experiment to detect the mutation site of the inflammation pathway of peripheral blood mononuclear cells of Weismann syndrome patients in vitro

[0145] The peripheral blood mononuclear cells (PBMCs) of Weismann syndrome patients and healthy controls (HC) were routinely isolated, and cell suspensions were prepared with 1640 culture medium containing 10% fetal bovine serum, and inoculated into 24-well plates at 1.8x10 6 cells / well.

[0146] The stimulation groups are:

[0147] control (ctrl) group: only medium was added;

[0148] lipopolysaccharide (LPS) group: LPS (final concentration 100 ng / mL) was added;

[0149] NOD2 ligand muramyl dipeptide (MDP) group: MDP (final concentration 10 μg / mL) was added;

[0150] LPS+MDP (L+M) group: LPS (final concentration 100 ng / mL) and MDP (final concentration 10 μg / mL) were added.

[0151] After 24 hours of incubation at 37°C in a 5% CO2 incubator, the cells were collected, and the number of cells in the culture wells was gently blown several times with a pipette, and then transferred to 1.5 mL centrifuge tubes, centrifuged, and 100 μL of a pre-prepared RIPA containing a protease inhibitor cocktail and a protein phosphatase inhibitor was added (the dilution ratio of the protease inhibitor cocktail was 1:50, and the dilution ratio of the protein phosphatase inhibitor was 1:100), and then placed on ice for lysis for 15 minutes. After centrifugation (4°C, 14000g, 10 minutes), the supernatant was collected, and 1 / 4 volume of 5x protein loading buffer was added. After vortex mixing, the sample was placed in a constant temperature metal bath for heating (100°C, 6-10 minutes), and after cooling, it was aliquoted. The expression levels of inflammatory pathway molecules in the PBMC lysate were detected by Western blot (WB). The gel was prepared, the electrophoresis buffer and the electrophoresis buffer were prepared, the protein was loaded according to the BCA results, 160V, 45 minutes constant voltage electrophoresis, and when the target band was completely separated, the electrophoresis was terminated; 300mA constant current electrophoresis for 2 hours, 5% defatted milk was used for room temperature blocking for 50 minutes, and the primary antibody (p-p38, p38, p-ERK, ERK and β-actin) was incubated overnight, and then washed with TBST for 10 minutes on a shaker for 3 times. The corresponding secondary antibody of the primary antibody was incubated at room temperature for 50 minutes, and then ECL developing solution was added, and the Tian Neng chemiluminescence instrument (5800) was used for development. After development, the regeneration stripping solution was used to repeat the above steps.

[0152] The results are shown in ​ Fig. 6. The PBMCs of the NOD2 mutation-carrying Wechsler patient (red box) showed significantly increased p38 and phosphorylated extracellular regulated protein kinase (ERK) protein expression (deeper blotting) compared to the HC before and after stimulation, indicating inflammatory activation of the p38-mitogen-activated protein kinase (MAPK) pathway and the ERK pathway, suggesting the pathogenic significance of the mutation site.

[0153] Example 6: Enzyme-linked immunosorbent assay (ELISA) detection of inflammatory factors in the plasma and PBMC culture supernatant of Wechsler syndrome patients for in vitro functional verification of the mutation site

[0154] The plasma of Wechsler syndrome patients and healthy controls (HC) was routinely separated and stored at -80°C; the peripheral blood mononuclear cells (PBMCs) of Wechsler syndrome patients and HC were routinely separated, and a cell suspension was prepared with 1640 culture medium containing 10% fetal bovine serum, and then inoculated into 24-well plates at a concentration of 1.8 x 10 6 cells / well.

[0155] The stimulation groups were as follows: the control (ctrl) group: only the culture medium was added;

[0156] MDP group: MDP was added (final concentration 10 μg / mL).

[0157] After 24 hours of culture at 37℃ in a 5% CO2 incubator, the cells were collected, centrifuged, and the cell precipitate was avoided. The culture supernatant was aspirated into another 1.5 mL centrifuge tube, and the culture supernatant was immediately frozen at -80℃ for testing. The ELISA kit was equilibrated to room temperature in advance, the ELISA kit standard (used to draw a standard curve to realize concentration quantification) was prepared, the sample was diluted to a certain concentration and added to the enzyme-labeled plate, the biotinylated antibody working solution was added according to the instructions and incubated for a certain time, the plate washer was used to wash the plate, and the liquid was discarded. Streptavidin-HRP working solution was added and incubated for 20 minutes, the plate was washed again, and the liquid was discarded. TMB color developing liquid was added, and after the color changed to dark blue, the reaction was terminated by adding a stop solution. Within 10 minutes, the absorbance at 450 nm (detection wavelength) and 620 nm (calibration wavelength) was read by an enzyme-labeled instrument. The absorbance results were fitted with a four-parameter equation standard curve, and the corresponding detection concentration was calculated by substituting the sample OD value; the detection concentration was multiplied by the dilution factor to obtain the actual concentration of the sample.

[0158] The results are shown in Table 1. ​ As shown in Table 1, the plasma and PBMC supernatant of the Blau patient carrying the NOD2 mutation showed a significant increase in inflammatory factors compared with HC before and after stimulation (p<0.05), suggesting that the mutation site has pathogenic significance.

[0159] As can be seen from the results of Examples 1 to 6, the in vitro functional experiment of the Blau syndrome patient carrying the mutation site suggests that the mutation site has pathogenic significance and can be used as a disease marker for the development of diagnostic and therapeutic kits.

[0160] In practical applications, it is detected whether the NOD2 gene of the testee carries any one of the following six mutations: c.380C>T, c.2657C>T, c.328G>A, c.1295C>T, c.1981G>C and c.2452A>C; if so, the testee is or is suspected to be a Blau syndrome patient or the testee has a relatively high risk of Blau syndrome.

[0161] The specific detection primers for the above six mutation sites are as follows:

[0162] The primers for detecting c.328G>A are as follows:

[0163] Forward primer: 5'-CTGGACACCGTCTGGAATAAG-3' (SEQ ID No. 15);

[0164] Forward primer: 5'-CTGGACACCGTCTGGAATAAG-3' (SEQ ID No. 15);

[0165] Primers for detecting c.1295C>T:

[0166] Forward primer: 5'-CTTCCAGTTACTCCTTGACCAC-3' (SEQ ID No. 17);

[0167] Reverse primer: 5'-TCATACTGGCTGACGAAACC-3' (SEQ ID No. 16).

[0168] Primers for detecting c.1295C>T:

[0169] Forward primer: 5'-CTTCCAGTTACTCCTTGACCAC-3' (SEQ ID No. 17);

[0170] Reverse primer: 5'-GACACCATCCATGAGAAGACAG-3' (SEQ ID No. 18).

[0171] Primers for detecting c.1981G>C:

[0172] Forward primer: 5'-TGATGTGCCACCAGCTTT-3' (SEQ ID No. 19);

[0173] Reverse primer: 5'-GGATGGAGTGGAAGTGCTTG-3' (SEQ ID No. 20).

[0174] Primers for detecting c.2452A>C:

[0175] Forward primer: 5'-ATGTTGGGCACCTCAAGTT-3' (SEQ ID No. 21);

[0176] Reverse primer: 5'-GATGCCTCGGTCTGAGATATTG-3' (SEQ ID No. 22).

[0177] Primers for detecting c.2657C>T:

[0178] Forward primer: 5'-GCGAGCAATTGCAGAAGTTAG-3' (SEQ ID No. 23);

[0179] Reverse primer: 5'-CCACACTGCCAATGTTGTTC-3' (SEQ ID No. 24).

[0180] The application has been described in detail. Those skilled in the art will understand that they can make modifications and alterations to this application without departing from the spirit and scope of the application. Although this application presents specific examples, it is to be understood that further modifications can be made. In general, the application is intended to cover any adaptations or variations of the present application including modifications based on the disclosure in the patent application in its broadest form.

Claims

1. A computer device comprising a memory, a processor, and a computer program stored in the memory, characterized in that: The processor executes the computer program to perform the following steps (A1)-(A3): (A1) Data reception: Receive the NOD2 gene data of the subject; the NOD2 gene data is the cDNA sequence information of the NOD2 gene; (A2) Data Comparison: The NOD2 gene data of the subject is compared with the standard mutation form of the NOD2 gene stored in the computer; the standard mutation form of the NOD2 gene is any one of the following 6 mutations in the NOD2 gene shown in SEQ ID No.2: c.380C>T, c.2657C>T, c.328G>A, c.1295C>T, c.1981G>C and c.2452A>C; (A3) Result Output: Determine and output the detection result information based on the comparison results; The detection result information is any one of the following: P1. Whether the subject of the test is or is suspected of being a patient with Yao's syndrome; P2. The risk of the subject having Johns Hopkins syndrome; or The processor executes the computer program to perform the following steps (A4)-(A6): (A4) Data reception: Receive NOD2 protein data from the subject, wherein the NOD2 protein data is the amino acid sequence information of the NOD2 protein; (A5) Data Comparison: The NOD2 protein data of the subject is compared with the standard mutation form of NOD2 protein stored in the computer; the standard mutation form of NOD2 protein is any one of the following 6 mutations in the NOD2 protein shown in SEQ ID No.1: S127L, A886V, A110T, A432V, A661P and K818Q; (A6) Result Output: Determine and output the detection result information based on the comparison results; The detection result information is any one of the following: P1. Whether the subject of the test is or is suspected of being a patient with Yao's syndrome; P2, the risk level of the subject having Yale syndrome.

2. The computer device according to claim 1, characterized in that: In (A1) and (A4), the method further includes the step of receiving clinical information data of the test subject; the clinical information data is the clinical symptom manifestation of the test subject. In (A2) and (A5), the method further includes a step of comparing the clinical information data of the subject with a typical phenotype of an autoinflammatory disease stored in a computer.

3. The computer device according to claim 1 or 2, characterized in that: In claim 1 (A3), if the NOD2 gene data of the test subject contains the standard mutation form of the NOD2 gene, the test result information "the test subject is or is suspected to be a patient with Yao's syndrome" or "the test subject has a relatively high risk of having Yao's syndrome" is output. Alternatively, in claim 1 (A6), if the NOD2 protein data of the test subject contains the standard mutation form of the NOD2 protein, then the test result information "the test subject is or is suspected to be a patient with Yao's syndrome" or "the test subject has a relatively high risk of having Yao's syndrome" is output; or In claim 2 (A3), if the NOD2 gene data of the test subject contains the standard mutation form of the NOD2 gene, and the clinical information data of the test subject is consistent with the typical phenotype of the autoinflammatory disease, then the test result information of "the test subject is or is suspected to be a patient with Yao's syndrome" or "the test subject has a relatively high risk of having Yao's syndrome" is output. Alternatively, in claim 2 (A6), if the NOD2 protein data of the test subject contains the standard mutation form of the NOD2 protein, and the clinical information data of the test subject matches the typical phenotype of the autoinflammatory disease, then the test result information "the test subject is or is suspected to be a patient with Yao's syndrome" or "the test subject has a relatively high risk of having Yao's syndrome" is output.

4. A computer program product, comprising a computer program, characterized in that: When the computer program is executed by the processor, it performs the steps described in any one of claims 1-3.

5. A computer-readable storage medium having a computer program stored thereon, characterized in that: When the computer program is executed by the processor, it performs the steps described in any one of claims 1-3.

6. A system, characterized in that: The system is either System 1 or System 2; The system 1 includes the following (B1) and (B2): (B1) A substance used to detect the NOD2 gene data of the subject; the NOD2 gene data is the cDNA sequence information of the NOD2 gene; (B2) Device; the device includes a data receiving module, a data storage module, a data comparison module, and a conclusion output module; The data receiving module is configured to receive the NOD2 gene data of the subject. The data storage module is configured to store standard mutation forms of the NOD2 gene; the standard mutation forms of the NOD2 gene are any one of the following six mutations in the NOD2 gene shown in SEQ ID No. 2: c.380C>T, c.2657C>T, c.328G>A, c.1295C>T, c.1981G>C, and c.2452A>C; The data comparison module is configured to receive the NOD2 gene data of the subject sent by the data receiving module, and to call the standard mutation form of the NOD2 gene in the data storage module to compare the NOD2 gene data of the subject with the standard mutation form of the NOD2 gene. The conclusion output module is configured to receive the comparison results sent by the data comparison module, and determine and output detection result information based on the comparison results; the detection result information is any one of the following: P1. Whether the subject of the test is or is suspected of being a patient with Yao's syndrome; P2. The risk of the subject having Johns Hopkins syndrome; or The system 2 includes the following (B3) and (B4): (B3) A substance used to detect NOD2 protein data in the subject; the NOD2 protein data is the amino acid sequence information of the NOD2 protein; (B4) Device; the device includes a data receiving module, a data storage module, a data comparison module, and a conclusion output module; The data receiving module is configured to receive the NOD2 protein data of the subject. The data storage module is configured to store standard mutant forms of the NOD2 protein; the standard mutant forms of the NOD2 protein are any one of the following six mutations in the NOD2 protein shown in SEQ ID No.1: S127L, A886V, A110T, A432V, A661P, and K818Q; The data comparison module is configured to receive the NOD2 protein data of the subject sent by the data receiving module, and to call the standard mutation form of the NOD2 protein in the data storage module to compare the NOD2 protein data of the subject with the standard mutation form of the NOD2 protein. The conclusion output module is configured to receive the comparison results sent by the data comparison module, and determine and output detection result information based on the comparison results; the detection result information is any one of the following: P1. Whether the subject of the test is or is suspected of being a patient with Yao's syndrome; P2, the risk level of the subject having Yale syndrome.

7. The system according to claim 6, characterized in that: In System 1, the conclusion output module determines and outputs the detection result information according to the comparison results as follows: if the NOD2 gene data of the test subject contains the standard mutation form of the NOD2 gene, then the detection result information "the test subject is or is suspected to be a patient with Yao's syndrome" or "the test subject has a relatively high risk of having Yao's syndrome" is output. In System 2, the conclusion output module determines and outputs the detection result information according to the comparison results as follows: if the NOD2 protein data of the test subject contains the standard mutation form of the NOD2 protein, then the detection result information "the test subject is or is suspected to be a patient with Yao's syndrome" or "the test subject has a relatively high risk of having Yao's syndrome" is output. Furthermore, in both System 1 and System 2, the data receiving module is configured to also receive the clinical information data of the test subject; the clinical information data is the clinical symptom manifestation of the test subject. Each data storage module is configured to also store typical phenotypes of autoinflammatory diseases; each data comparison module is configured to also receive the clinical information data of the test subject sent by the data receiving module, and call the typical phenotypes of autoinflammatory diseases in the data storage module to compare the clinical information data of the test subject with the typical phenotypes of autoinflammatory diseases; in System 1, the conclusion output module is configured to receive the comparison results sent by the data comparison module, and determine and output the detection result information according to the comparison results as follows: if the NOD2 gene data of the test subject contains the standard mutation form of the NOD2 gene, and the clinical information data of the test subject... If the test result matches the typical phenotype of the autoinflammatory disease, the system outputs the test result information as "the test subject is or is suspected of having Yao's syndrome" or "the test subject has a relatively high risk of having Yao's syndrome". In the system 2, the conclusion output module is configured to receive the comparison results sent by the data comparison module, and determine and output the test result information according to the comparison results as follows: if the test subject's NOD2 protein data contains the standard mutation form of the NOD2 protein, and the test subject's clinical information data matches the typical phenotype of the autoinflammatory disease, the system outputs the test result information as "the test subject is or is suspected of having Yao's syndrome" or "the test subject has a relatively high risk of having Yao's syndrome".

8. A method for screening for Yao syndrome using a computer, namely, Method I, Method II, Method III, or Method IV: Method I includes the following steps (C1)-(C3): (C1) Data reception: Receive the NOD2 gene data of the subject, wherein the NOD2 gene data is the cDNA sequence information of the NOD2 gene; (C2) Data Comparison: The NOD2 gene data of the subject was compared with the standard mutation forms of the NOD2 gene stored in the computer; the standard mutation forms of the NOD2 gene are any one of the following 6 mutations in the NOD2 gene shown in SEQ ID No. 2: c.380C>T, c.2657C>T, c.328G>A, c.1295C>T, c.1981G>C and c.2452A>C; (C3) Result Output: Based on the comparison results, the detection result information is determined and output as follows: If the NOD2 gene data of the subject contains the standard mutation form of the NOD2 gene, the detection result information "the subject is or is suspected to be a patient with Yao's syndrome" or "the subject has a relatively high risk of having Yao's syndrome" is output. Method II includes the following steps (C4)-(C6): (C4) Data reception: Receive NOD2 protein data from the subject, wherein the NOD2 protein data is the NOD2 protein amino acid sequence information; (C5) Data Comparison: The NOD2 protein data of the subject is compared with the standard mutation form of NOD2 protein stored in the computer; the standard mutation form of NOD2 protein is any one of the following 6 mutations in the NOD2 protein shown in SEQ ID No.1: S127L, A886V, A110T, A432V, A661P and K818Q; (C6) Result Output: Based on the comparison results, the detection result information is determined and output as follows: If the NOD2 protein data of the subject contains the standard mutation form of the NOD2 protein, the detection result information "the subject is or is suspected to be a patient with Yao's syndrome" or "the subject has a relatively high risk of having Yao's syndrome" is output. Method III includes the following steps (C7)-(C9): (C7) Data Reception: Receive the NOD2 gene data and clinical information data of the subject; the NOD2 gene data is the cDNA sequence information of the NOD2 gene; the clinical information data is the clinical symptoms; (C8) Data Comparison: The NOD2 gene data of the test subject is compared with the standard mutation forms of the NOD2 gene stored in the computer; the standard mutation forms of the NOD2 gene are any one of the following six mutations in the NOD2 gene shown in SEQ ID No. 2: c.380C>T, c.2657C>T, c.328G>A, c.1295C>T, c.1981G>C, and c.2452A>C; and the clinical information data of the test subject is compared with the typical phenotype of autoinflammatory diseases stored in the computer. (C9) Result Output: Based on the comparison results, the test result information is determined and output as follows: If the NOD2 gene data of the test subject contains the standard mutation form of the NOD2 gene, and the clinical information data of the test subject is consistent with the typical phenotype of the autoinflammatory disease, then the test result information "the test subject is or is suspected to be a patient with Yao's syndrome" or "the test subject has a relatively high risk of having Yao's syndrome" is output. Method IV includes the following steps (C10)-(C12): (C10) Data Reception: Receive NOD2 protein data and clinical information data from the subject; the NOD2 protein data is the NOD2 protein amino acid sequence information; the clinical information data is the clinical symptom presentation. (C11) Data Comparison: The NOD2 protein data of the test subject is compared with the standard mutation forms of NOD2 protein stored in the computer; the standard mutation forms of NOD2 protein are any one of the following six mutations in the NOD2 protein shown in SEQ ID No.1: S127L, A886V, A110T, A432V, A661P, and K818Q; and the clinical information data of the test subject is compared with the typical phenotype of autoinflammatory diseases stored in the computer. (C12) Result Output: Based on the comparison results, the detection result information is determined and output as follows: If the NOD2 protein data of the test subject contains the standard mutation form of the NOD2 protein, and the clinical information data of the test subject is consistent with the typical phenotype of the autoinflammatory disease, then the detection result information "the test subject is or is suspected to be a patient with Yao's syndrome" or "the test subject has a relatively high risk of having Yao's syndrome" is output.

9. Application, either Application I or Application II; Application I is the application of a substance used to detect whether the NOD2 gene shown in SEQ ID No. 2 of the test subject has any one of six mutations in the preparation of the product; the six mutations are: c.380C>T, c.2657C>T, c.328G>A, c.1295C>T, c.1981G>C and c.2452A>C; The product is one that has any of the following uses: (D1) To determine whether the subject is or is suspected of having Yao's syndrome; (D2) To determine the risk of the subject having Johns Hopkins syndrome; Application II is the application of a substance used to detect whether the NOD2 protein shown in SEQ ID No. 1 of the test subject has any one of six mutations in the preparation of the product; the six mutations are: S127L, A886V, A110T, A432V, A661P and K818Q; The product is one that has any of the following uses: (D1) To determine whether the subject is or is suspected of having Yao's syndrome; (D2) To determine the risk of the subject having Yale syndrome.

10. Any of the following biological materials: (E1) NOD2 protein mutant, the amino acid sequence of which is shown in SEQ ID No. 5, SEQ ID No. 13, SEQ ID No. 3, SEQ ID No. 7, SEQ ID No. 9 or SEQ ID No. 11; (E2)NOD2 gene mutant, the amino acid sequence of which is shown in SEQ ID No. 6, SEQ ID No. 14, SEQ ID No. 4, SEQ ID No. 8, SEQ ID No. 10 or SEQ ID No. 12; (E3) An expression cassette, recombinant vector, or transgenic cell line containing the NOD2 gene mutant described in (E2).

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