A surface hsa-miR-4516 marker, kit and application for diagnosing fragile X syndrome
By screening hsa-miR-4516 as a miRNA marker and preparing a diagnostic kit, the problem of low diagnostic efficiency of fragile X syndrome in the existing technology was solved, and a rapid and accurate diagnosis effect was achieved.
Patent Information
- Application Number
- CN202411740806.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-29
- Publication Date
- 2025-09-19
- Estimated Expiration
- 2044-11-29
AI Technical Summary
The existing technology lacks effective miRNA markers for diagnosing fragile X syndrome (FXS), resulting in low diagnostic efficiency.
hsa-miR-4516 was screened as a significantly overexpressed miRNA marker, and by designing specific primer pairs and universal primer pairs, combined with basic PCR reagents, a diagnostic kit was prepared for the rapid and accurate diagnosis of fragile X syndrome.
Through the application of the hsa-miR-4516 marker and kit, rapid and accurate diagnosis of fragile X syndrome was achieved, diagnostic efficiency was improved, and clinicians were helped to quickly and accurately understand the patient's condition and improve treatment outcomes.
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Figure CN119433016B_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of molecular biological diagnosis, and specifically relates to a miRNA marker combination, a kit and an application thereof that can be used to diagnose fragile X syndrome (FXS). Background Art
[0002] Fragile X syndrome (FXS) is a hereditary mental retardation syndrome second only to idiopathic schizophrenia, affecting 0.05% of all children. It is inherited through an X-linked mechanism and accounts for 40% of X-linked mental retardation. Its penetrance varies by sex, with a prevalence of 80% in males and 30% in females. Clinically, the incidence of Fragile X syndrome also differs between males and females, with a prevalence of 1 in 4,000 males and 1 in 6,000 females. The neurodevelopmental impairments caused by FXS are associated with a variety of behavioral features, including delayed neuropsychomotor development (ADNPM), anxiety, aggression, self-injury, attention deficit disorder, social withdrawal, and physical comorbidities. The molecular genetic basis of Fragile X syndrome in over 95% of patients is caused by dynamic mutations in the (CGG)n structural expansion of the FMR1 gene. Less than 5% of cases are caused by missense and deletion mutations in the FMR1 gene that affect the normal structure of FMRP.
[0003] miRNAs (microRNAs) are a class of short, non-coding RNAs that participate in post-transcriptional gene regulation by promoting messenger RNA (mRNA) silencing and influencing protein translation. Evidence from multiple studies indicates that miRNAs are involved in the pathogenesis of various diseases, and disease-specific miRNA profiles have been isolated from body fluids (circulating miRNAs), including miRNAs selectively expressed in blood and urine. MiRNAs play a role in neural development and regulate important biological processes in central nervous system function. Dysregulated expression of miRNAs has been found to play an important role in the pathogenesis of neurological diseases (see: Im HI, Kenny PJ. MicroRNAs in neuronal function and dysfunction. Trends Neurosci. 2012; 35(5): 325-334.). Due to the stability and accessibility of circulating miRNAs, their use as diagnostic and prognostic biomarkers for patient stratification has gained attention and has improved the efficacy of targeted therapies for neurological diseases (such as fragile X syndrome, FXS).
[0004] Studies have also shown that miRNAs can serve as a new class of potential biomarkers for the diagnosis and prognostication of common central nervous system (CNS) diseases, including neurodevelopmental disorders, and provide new treatments (see: Jin XF, Wu N, Wang L, Li J. Circulating microRNAs: A novel class of potential biomarkers for diagnosing and prognosing central nervous system diseases Cell. Mol. Neurobiol. 2013; 33(5): 601–613.). However, to date, there have been few reports of miRNA biomarkers that can effectively diagnose FXS. Summary of the Invention
[0005] The technical problem to be solved by the present invention is to provide a serum miRNA marker for diagnosing fragile X syndrome (FXS).
[0006] To solve the above technical problems, the present invention adopts the following technical solutions: A serum miRNA marker for the diagnosis of fragile X syndrome (FXS)
[0007] The present invention collected serum samples from patients with a clinical diagnosis of Fragile X syndrome and healthy controls. The case samples were collected from patients undergoing physical examinations during a lecture. Through testing of these two serum samples, a miRNA (hsa-miR-4516) was screened and identified as significantly overexpressed in Fragile X syndrome. hsa-miR-4516 was further validated for its effectiveness in diagnosing FXS using validation samples collected from additional patients. Statistical calculations and receiver operating characteristic (ROC) curves revealed an AUC value of 0.942 for distinguishing FXS patients from healthy controls, demonstrating its effectiveness as a biomarker for diagnosing FXS.
[0008] The sequence of hsa-miR-4516 is: GGGAGAAGGGUCGGGGC (SEQ ID NO: 1).
[0009] In a first aspect, the present invention provides a serum miRNA marker for detecting fragile X syndrome: hsa-miR-4516.
[0010] In a second aspect of the present invention, use of the miRNA markers described above in preparing a diagnostic kit for FXS is provided.
[0011] In a third aspect of the present invention, there is provided use of a reagent for detecting the expression level of the miRNA described above in the preparation of a product for diagnosing fragile X syndrome, wherein the miRNA is hsa-miR-4516.
[0012] Preferably, the miRNA expression level is the expression level in a serum sample.
[0013] Preferably, the product is a detection kit.
[0014] Preferably, the reagent further comprises a specific primer pair for detecting the expression level of hsa-miR-4516. The sequences of the primers for detecting hsa-miR-4516 of the present invention can be obtained by conventional design methods in the art.
[0015] Specifically, the sequences of the specific primer pair for detecting hsa-miR-4516 are: 5'-GGACGGTAGCAAGCAAAGAGTGTGGCCCCGACCCT-3' and 5'-GGGATTCTGGAAGATGATGATGACGGGAGAAGGGT-3'. Furthermore, the present invention also provides a universal primer pair, whose sequences are: 5'-GGACGGTAGCAAGCAAAGAGTGTG-3' and 5'-GGGATTCTGGAAGATGATGATGAC-3'.
[0016] In a fourth aspect, the present invention also provides a miRNA detection kit for diagnosing FXS, comprising primers for detecting hsa-miR-4516; further, the kit also includes other basic PCR reagents. The primers for detecting hsa-miR-4516 include a specific primer pair for detecting the expression level of hsa-miR-4516. Specifically, the sequences of the specific primer pair are: 5'-GGACGGTAGCAAGCAAAGAGTGTGGCCCCGACCCT-3' and 5'-GGGATTCTGGAAGATGATGATGACGGGAGAAGGGT-3'. Furthermore, the present invention also provides a universal primer pair, whose sequences are: 5'-GGACGGTAGCAAGCAAAGAGTGTG-3' and 5'-GGGATTCTGGAAGATGATGATGAC-3'.
[0017] Preferably, the basic reagents include at least one of a positive quality control, a negative quality control, a poly-A tailing enzyme, a reverse transcriptase, dNTPs, a reverse transcription buffer, RNase-free water, a qPCR buffer, magnesium chloride, a DNA polymerase, and a SYBR Green fluorescent dye.
[0018] In a fifth aspect, the present invention further provides use of a reagent for detecting hsa-miR-4516 in preparing a kit for diagnosing fragile X syndrome.
[0019] The technical effects of the present invention are as follows: Through the development and application of serum miRNA markers and diagnostic kits, the miRNA markers provided by the present invention can quickly and accurately diagnose fragile X syndrome, thereby improving diagnostic efficiency. This makes the diagnosis of fragile X syndrome more convenient and easy, helping clinicians to quickly and accurately understand the patient's condition and improve clinical treatment efficacy. BRIEF DESCRIPTION OF THE DRAWINGS
[0020] Figure 1 Differences in expression of related miRNAs in patients with fragile X syndrome (Case represents FXS patient samples, Control represents healthy samples; * indicates the degree of significant difference; ns indicates no significant difference).
[0021] Figure 2 Differential expression of hsa-miR-4516 in the Fragile X syndrome validation set (Case represents FXS patient samples, Control represents healthy samples; * indicates the degree of significant difference; ns indicates no significant difference).
[0022] Figure 3 ROC curve of hsa-miR-4516 in diagnosing patients with fragile X syndrome. DETAILED DESCRIPTION
[0023] The following non-limiting examples may enable those skilled in the art to more fully understand the present invention, but are not intended to limit the present invention in any way. Any person skilled in the art who, within the technical scope disclosed herein, makes equivalent substitutions or modifications based on the technical solutions and inventive concepts of the present invention falls within the scope of protection of the present invention.
[0024] Example 1 Collection of samples
[0025] Fifteen unrelated individuals were collected: eight male FXS patients and seven male controls. Serum samples were removed from a -80°C freezer, placed in an ice bath at 0-4°C until completely thawed, and centrifuged at 15,000 g for 10 minutes at 4°C. 200 μL of the supernatant was collected and placed in a new RNase-free EP tube. Blood samples from both cases and healthy controls were used for molecular diagnosis of FXS using the Fragile X PCR assay to detect the CGG repeat sequence in the FMR1 gene. Controls had normal FMR1 alleles.
[0026] Example 2 Extraction of small RNA
[0027] Using the kit method (PureLinkTM Small RNA was extracted using a miRNA isolation kit (Invitrogen) according to the manufacturer's instructions. The isolated small RNA samples were eluted in 40 μl of RNase-free water. The miRNA extracts were stored at −80°C until processing. RNA concentration was measured using a Nanodrop spectrophotometer, and equal amounts of miRNA (10 ng) were used in RT-PCR quantification reactions.
[0028] Example 3 Detection of differentially expressed miRNAs in FXS patients
[0029] The miRNAs (hsa-miR-575, hsa-miR-1248, hsa-miR-4516, hsa-miR-3609, hsa-miR-133b) that have been reported in the literature to be highly expressed in neurodevelopmental disorders were detected. Specific primers and universal primers were designed for RT-PCR detection to detect the expression levels of the above miRNAs in the two groups of samples in Example 1. Specifically, the specific primers designed were used to perform real-time quantitative PCR (RT-qPCR) on the test samples, using 0.8-1.02 μg of RNA. The expression level of miRNA was analyzed by real-time quantitative PCR in a 96-well plate using a real-time PCR system.
[0030] Table 1 shows the reaction system for RT-PCR detection of target miRNA; Table 2 lists the RT-PCR detection primers for detecting the corresponding miRNA. The reaction procedure for RT-PCR detection is: 45℃, 4min; 37℃, 5min, 1 cycle; 94℃, 30s; 37℃, 45s; 60℃ 30s, 1 cycle; 94℃, 30s; 72℃, 30s, 35 cycles, 35 cycles. Three replicate wells were set up for each sample, and the expression level of the miRNA to be tested was calculated. The results showed that the expression level of hsa-miR-4516 in FXS patients was significantly increased ( Figure 1 ), which can be considered as a potential diagnostic marker for the diagnosis of fragile X syndrome.
[0031] Table 1 Reaction system for RT-PCR detection of target miRNA
[0032] Total RNA 0.8-1.02 μg 10X PCR Buffer* 2.5 μL 5X M-MLV Buffer** 5μL 20X SYBR Green I 1.25 μL Reverse Transcriptase M-MLV 20U RNase inhibitor 4U primer P1 (10 μM) 0.5μL primer P2 (10 μM) 0.5μL primer RP1 (2 μM) 0.5μL primer RP2 (2 μM) 0.5μL <![CDATA[ddH2O]]> to 25 μL
[0033] *10X PCR Buffer (100mM Tris-HCl, pH 8.3, 500mM KCl, 15mM MgCl2)
[0034] **5X M-MLV Buffer (250mM Tris-HCl, pH 8.3, 375mM KCl, 15mM MgCl2, 50mM Dithiothreitol)
[0035] Table 2: RT-PCR detection primers
[0036]
[0037] Example 4: Further verification of the diagnostic effect of hsa-miR-4516 on FXS
[0038] Using clinical samples collected from other batches, a total of 85 unrelated individuals: 50 male FXS patients and 35 male control individuals. Referring to the detection methods of Examples 1-3, the expression levels of hsa-miR-4516 in FXS patient and control samples were detected ( Figure 2 ), draw the ROC curve ( Figure 3 ), with an AUC value of 0.942; the experimental results show that hsa-miR-4516 can effectively diagnose fragile X syndrome; it makes the diagnosis of fragile X syndrome more convenient, helping clinicians to quickly and accurately understand the patient's condition and improve clinical treatment effects.
[0039] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.
Claims
1. Use of a reagent for detecting miRNA expression levels in the preparation of a product for diagnosing fragile X syndrome, characterized in that: The miRNA is hsa-miR-4516.
2. The application according to claim 1, characterized in that The reagent includes a specific primer pair for detecting the expression level of hsa-miR-4516.
3. The use according to claim 2, wherein the sequences of the specific primer pair are: 5'-GGACGGTAGCAAGCAAAGAGTGTGGCCCCGACCCT-3' and 5'-GGGATTCTGGAAGATGATGATGACGGGAGAAGGGT-3'.
4. The application according to claim 3, characterized in that The miRNA expression level is the expression level in serum samples.
5. The application according to claim 1, characterized in that: The product is a detection kit.