Biomarker, kit and scoring system for autism detection
By detecting the mRNA expression of AKR1C3, HMOX1, and PEX6 genes in peripheral blood lymphocytes and combining it with real-time quantitative PCR technology, the accuracy problem of existing autism diagnosis has been solved, enabling early diagnosis and intervention and improving the scientific rigor and reliability of diagnosis.
Patent Information
- Application Number
- CN202510996339.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Priority Date
- 2024-11-22
- Filing Date
- 2025-07-18
- Publication Date
- 2025-11-07
Smart Images
Figure CN120905376A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the technical field of biological medicine, in particular to a biological marker for autism detection, a kit and a scoring system. BACKGROUND
[0002] Autism spectrum disorder is a highly heterogeneous neurodevelopmental disorder, also known as autism or autism, and its main symptoms are social interpersonal communication disorders, speech communication expression disorders, stereotyped actions, limited interest activities, and intellectual disabilities. The cause of autism is complex, about 50% of which is related to genetic variation, and 40% of which may be caused by unknown environmental factors. Studies have shown that autism is a multi-stage, progressive brain development disorder that begins in the 1-2 month old embryo.
[0003] The current common method for diagnosing autism is to use the Childhood Autism Rating Scale to diagnose autism. The method of diagnosing autism using the Childhood Autism Rating Scale mainly relies on subjective experience and judgment, which can affect the accuracy of the diagnosis and the grading assessment of the severity of autism. And the diagnosis of autism is mainly based on behavioral characteristics and developmental history, and there is still a lack of consistent biological markers, so a large number of children are diagnosed after the age of 2. Because of the lack of disease-modifying drugs, the treatment of autism mainly relies on behavioral intervention. However, evidence-based medicine shows that most intervention methods have limited effect, resulting in poor prognosis and severe disability of children with autism, causing a huge spiritual and economic burden on families. Therefore, exploring the pathogenesis of autism, further searching for early diagnostic biomarkers and safe and effective intervention measures are important problems to be solved at present. SUMMARY
[0004] In order to solve the above-mentioned deficiencies existing in the prior art, the purpose of the present application is to provide a biological marker for autism detection, a kit and a scoring system to solve the existing problems.
[0005] The technical solution of the present application to solve the above technical problems is as follows: a biological marker for autism detection is provided, which comprises AKR1C3, HMOX1 and PEX6 genes.
[0006] The present application provides an autism detection kit, which comprises specific primers for detecting AKR1C3, HMOX1 and PEX6 genes.
[0007] Further, the sequences of the forward primer and the reverse primer of the specific primer of the AKR1C3 gene are shown in SEQ ID NO. 1 and SEQ ID NO. 2, respectively.
[0008] Further, the sequences of the forward primer and the reverse primer of the specific primer of the HMOX1 gene are shown in SEQ ID NO. 3 and SEQ ID NO. 4, respectively.
[0009] Further, the forward primer and reverse primer sequences of the specific primer sequence of the PEX6 gene are shown in SEQ ID NO. 5 and SEQ ID NO. 6, respectively.
[0010] The application provides a detection method of the biomarker.
[0011] Further, the amplification system of the real-time fluorescent quantitative method comprises 2xChamQ Universal SYBR qPCR MasterMix 10 muL, a forward primer 0.4 muL, a reverse primer 0.4 muL, a cDNA template 2 muL and RNase Free H2O 7.2 muL.
[0012] Further, the amplification procedure of the real-time fluorescent quantitative method comprises: Stage 1: 95 DEG C, 30s, 1 cycle; Stage 2: 95 DEG C 5s, 60 DEG C annealing 34s, 40 cycles; Stage 3: 95 DEG C 15s, 60 DEG C 1min, 95 DEG C 15s, 1 cycle.
[0013] The application further provides a scoring system for autism detection, comprising the diagnosis threshold values of the AKR1C3, HMOX1 and PEX6 genes.
[0014] The present application has the following beneficial effects: the present application detects the mRNA expression of AKR1C3, HMOX1 and PEX6 in peripheral blood lymphocytes, finds that the expression level of AKR1C3 is significantly higher in autism patients than in the normal group, the expression level of HMOX1 is significantly lower in autism patients than in the normal group, and the expression level of PEX6 is significantly higher in autism patients than in the normal group. Therefore, the present application develops an RT-PCR kit for detecting the mRNA level changes of AKR1C3, HMOX1 and PEX6 in peripheral blood lymphocytes, and designs an autism scoring system according to the expression thereof. The kit and the scoring system of the present application have more accurate and reliable detection results, good stability and repeatability. Not only can the kit and the scoring system reflect the autism disease condition, but also can be used as an early warning for autism diagnosis, and the possibility of disease can be detected when the child has not yet shown obvious autism symptoms. The kit and the scoring system can be combined with hospitals to intervene early and hinder the further development of autism. Compared with the diagnosis of the autism rating scale based on appearance and behavior at the present stage, the present application is more scientific and can be widely applied to hospitals. BRIEF DESCRIPTION OF DRAWINGS
[0015] Figure 1 AKR1C3, HMOX1 and PEX6 are 45 iron death-related differential genes; Figure 2 AKR1C3, HMOX1 and PEX6 are 12 intersection genes related to autism; Figure 3 AKR1C3, HMOX1 and PEX6 are 6 core genes related to iron death in autism; Figure 4 AKR1C3, HMOX1 and PEX6 are co-localization analysis graphs of the genes; Figure 5 AKR1C3, HMOX1 and PEX6 are mRNA expression amounts of the genes in peripheral blood lymphocytes of autism patients; Figure 6 AKR1C3, HMOX1 and PEX6 are ROC curves of the genes. DETAILED DESCRIPTION
[0016] The following examples are only used to explain the present application, and are not used to limit the scope of the present application. If the specific conditions are not specified in the examples, the conventional conditions or the conditions recommended by the manufacturer are used. If the reagents or instruments used are not specified by the manufacturer, they are all conventional products that can be purchased on the market.
[0017] The inclusion criteria and exclusion criteria of the samples of the present application are as follows: (1) Inclusion criteria: patients diagnosed as autism by the autism rating scale; (2) Elimination criteria: ① suffering from other mental diseases (such as schizophrenia) and other neurodevelopmental disorders; ② suffering from genetic metabolic diseases; ③ suffering from severe neurological diseases and history of major physical diseases such as craniocerebral injury.
[0018] The present application is divided into two stages: the first stage is to study the differential genes in autism patients; the second stage is to verify the biomarkers, kits and scoring systems for autism detection of the present application. A total of 37 samples were collected in the second stage study; among them, 25 autism patients: 21 males and 4 females, aged 3-7 years old, all diagnosed as autism patients by autism rating scale; 12 normal samples.
[0019] Example 1: Screening of autism biomarkers The present application determines that the mRNA expression amounts of AKR1C3, HMOX1 and PEX6 in peripheral blood lymphocytes can be used as biomarkers for autism and severity grading evaluation by statistical analysis of blood samples of autism patients. The specific steps are as follows: (1) Study of differential genes in autism patients The mRNA expression data of peripheral blood lymphocytes of autism patients and normal controls from GEO data GSE25507 were used. First, batch effect removal and data normalization were performed to obtain 2673 differential genes. Then, the ferroptosis-related genes (obtained from the ferroptosis-related database FerrDb V2 website) were intersected with the above-mentioned 2673 differential genes to obtain 45 ferroptosis-related differential genes, as shown in Table 1. Figure 1 SVM-REF and LASSO machine model algorithms were used to analyze the 45 ferroptosis-related differential genes, and two algorithms were used to screen the characteristic genes related to autism, respectively. Then, the characteristic genes screened by the two algorithms were intersected to obtain 12 genes, as shown in Table 2. Figure 2 Then, the STRING database was used to perform protein interaction analysis on the 12 intersection genes of the two algorithms, and the MCC algorithm was used to generate the top 10 genes (CALM3, CAMKK2, RPTOR, RICTOR, MAPKAP1, DEPTOR, CFL1, HMOX1, DPP4 and AKR1C3) of the protein interaction network, and then the 10 protein interaction core genes were intersected with the 12 ferroptosis-related characteristic genes to obtain 6 ferroptosis-related core genes in autism, as shown in Table 3. Figure 3 Finally, co-localization analysis was performed on the 6 genes, and the results showed that 3 of them had significant relevance to autism, as shown in Table 4, which were AKR1C3, HMOX1 and PEX6. Figure 4
[0020] The mRNA expression of these three genes was analyzed, and the results are as follows: Figure 5 As shown. By Figure 5 It was found that the mRNA expression levels of AKR1C3, HMOX1, and PEX6 genes were significantly altered in peripheral blood lymphocytes of autistic patients, with AKR1C3 and PEX6 showing an increasing trend and HMOX1 showing a decreasing trend. ROC curves were plotted for the three differentially expressed genes (see...). Figure 6 ),Depend on Figure 6 The area under the curve (AUC) was 0.721, which is greater than 0.65, indicating statistical significance. This demonstrates that using the expression levels of these three genes to predict autism is effective.
[0021] Example 2: An autism detection kit and its detection method An autism detection kit includes peripheral blood lymphocyte extract, mRNA separation solution, dilution solution, washing solution, reverse transcription reaction solution, specific primers, standard positive template, and SYBR Green real-time quantitative PCR reaction solution; the detection method includes the following steps: (1) Extraction of total RNA from human peripheral blood lymphocytes using RNA extraction reagent (Trizol): Blood was collected using disposable anticoagulated vacuum aspiration tubes, with a blood volume of 1-2 mL, and stored at room temperature. Fresh anticoagulated whole blood was taken and diluted with an equal volume of diluent. 5 mL of lymphocyte separation medium was added to a sterile plastic centrifuge tube, and then the diluted blood sample was slowly added to the lymphocyte separation medium, ensuring that the blood sample was on the upper layer of the separation medium to prevent blood cells from spreading to the lower layer. Centrifuged at 2000 rpm for 20 min at room temperature. After centrifugation, a thin, relatively dense white membrane, i.e., a mononuclear cell (including lymphocytes and monocytes) layer, was found between the plasma layer and the separation medium layer. The white membrane layer was then carefully aspirated into another centrifuge tube using a pipette. The aspirated cells were mixed by inverting with 5 mL of washing buffer and centrifuged again at 1500 rpm for 10 min. The supernatant was discarded. Repeat washing 1-2 times, discard the supernatant, add 1 mL Trizol and incubate on ice for 30 min to fully extract RNA. After incubating on ice for 30 min, add 200 μL of chloroform, vortex vigorously for 15 s, incubate at room temperature for 5 min, centrifuge at 12000 rpm at 4℃ for 10 min, transfer the supernatant to a new Eppendorf tube, add an equal volume of isopropanol, mix gently, incubate at room temperature for 10 min, centrifuge at 12000 rpm at 4℃ for 10 min, discard the supernatant. Add 1 mL of 75% absolute ethanol (pre-cooled), mix by inversion, centrifuge at 12000 rpm at 4℃ for 10 min, discard the supernatant, then wash twice with ethanol, invert and air dry. Preheat RNase-free H2O to 55℃, add 30 μL to each tube to dissolve RNA, and measure the concentration.
[0022] (2) Using the extracted total RNA of human peripheral blood lymphocytes, cDNA of each mRNA sample is obtained by reverse transcription: reverse transcription reaction solution configuration: 4xgDNA Wiper Mix 4 μL, RNA sample 1 ug, RNase Free H2O 7.2 μL, 5xHiScript Ⅲ qRT SuperMix 4 μL.
[0023] (3) SYBR Green reaction solution configuration: 2xChamQ Universal SYBR qPCR MasterMix 10 μL, 0.4 μL of forward primer and reverse primer (10 μM) respectively, 2 μL of cDNA template, RNase Free H2O 7.2 μL, a total of 20 μL.
[0024] Table 1 Specific primer sequence
[0025] (4) The reaction steps of the fluorescence real-time quantitative PCR system are as follows: Stage 1: DNA polymerase activation and pre-denaturation, 95℃, 30s; Stage 2: Cycle reaction, 95℃ 5s, 60℃ annealing 34s, 40 cycles, fluorescence acquisition is performed in this stage; Stage 3: Melting curve, 95℃ 15s, 60℃ 1min, 95℃ 15s, 1 cycle.
[0026] (5) Data analysis: the data is analyzed by using the relative quantification method (2-△△Ct method).
[0027] Example 3: Result verification of the autism detection kit of the application The application receives a total of 37 clinical samples; among them, 25 cases of autistic children, 21 cases of boys and 4 cases of girls; the age is 3-7 years old; all are diagnosed as autistic patients by autism rating scale; 12 cases of normal samples. The 37 clinical samples are diagnosed by the kit and the autism rating scale of the application, and the results are shown in Tables 2-4. As shown in Tables 2-4, the HMOX1 expression level detected by the kit of the application is significantly higher in autistic patients than in the normal group, 2 patients do not meet the diagnosis results of the autism rating scale, and the detection efficiency is 92%. The AKR1C3 expression level in autistic patients is significantly lower than that in the normal group, 2 patients do not meet the diagnosis results of the autism rating scale, and the detection efficiency is 92%. The PEX6 expression level in autistic patients is significantly higher than that in the normal group, 3 patients do not meet the diagnosis results of the autism rating scale, and the detection efficiency is 88%.
[0028] Table 2 Verification results of AKR1C3
[0029] Table 3 verification results of HMOX1
[0030] Table 4 verification results of PEX6
[0031] Further statistics of the above results show that the diagnostic efficiency of patients 1-25 using the kit of the present application is shown in Table 5. We found that there were differences in the severity of autism among the patients, and the autism of patients 6, 9, 10, 16, 19 and 23 was milder than that of the other 19 patients, which indicated that the three genes AKR1C3, HMOX1 and PEX6 in the autism patients can not be simultaneously abnormally expressed.
[0032] Table 5 diagnostic efficiency of the kit
[0033] Based on the detection results and statistical results of the above-mentioned kit, an autism scoring system is designed, as shown in Table 6. That is, if one of the three genes AKR1C3, HMOX1 and PEX6 is dysregulated (i.e. the expression of AKR1C3 or PEX6 is higher than the threshold value, and the expression of HMOX1 is lower than the threshold value), it can be judged as mild (symptoms are not obvious) or suspected autism, and further judgment is needed in combination with clinical features. If two or more genes are dysregulated, it can be diagnosed as autism and the symptoms are obvious.
[0034] Table 6 autism scoring system
[0035] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.
Claims
1. A biomarker for autism detection, characterized in that, AKR1C3, TU 6 and PEX6 genes.
2. An autism detection kit, characterized by, The specific primers for detecting the AKR1C3, HMOX1 and PEX6 genes in claim 1.
3. The autism detection kit according to claim 2, characterized by, The forward primer and reverse primer sequences of the specific primers of the AKR1C3 gene are shown in SEQ ID NO. 1 and SEQ ID NO. 2, respectively.
4. The autism detection kit according to claim 2, characterized by, The forward primer and reverse primer sequences of the specific primers of the HMOX1 gene are shown in SEQ ID NO. 3 and SEQ ID NO. 4, respectively.
5. The autism detection kit according to claim 2, characterized by, The forward primer and reverse primer sequences of the specific primers of the PEX6 gene are shown in SEQ ID NO. 5 and SEQ ID NO. 6, respectively.
6. A method of detecting the biomarker of claim 1, characterized by, The autism detection kit of any one of claims 2-5 is used for detection by real-time fluorescent quantitative method.
7. The detection method according to claim 5, characterized in that, The amplification system of the real-time fluorescent quantitative method comprises: 2xChamQ Universal SYBR qPCR MasterMix 10 μL, forward primer 0.4 μL, reverse primer 0.4 μL, cDNA template 2 μL, RNase Free H2O 7.2 μL.
8. The detection method of claim 5, wherein, The amplification procedure of the real-time fluorescent quantitative method comprises: Stage 1: 95℃, 30s, 1 cycle; Stage 2: 95℃ 5s, 60℃ annealing 34s, 40 cycles; Stage 3: 95℃ 15s, 60℃ 1min, 95℃ 15s, 1 cycle.
9. A scoring system for autism detection, characterized by, The diagnostic thresholds of the AKR1C3, HMOX1 and PEX6 genes in claim 1; wherein the diagnostic threshold of the AKR1C3 gene is 0.965; the diagnostic threshold of the HMOX1 gene is 1.312; the diagnostic threshold of the PEX6 gene is 0.963; when the expression level of the AKR1C3 or the PEX6 gene is higher than the threshold or the expression level of the HMOX1 gene is lower than the threshold, it can be judged as suspected autism; when the expression of two or more genes among the AKR1C3, HMOX1 and PEX6 genes is dysregulated, it can be diagnosed as autism.