Prrt2 gene mutants and uses thereof

Abstract

The application discloses PRRT2 A gene mutant and application thereof belong to the field of gene detection and disease diagnosis, wherein PRRT2 The gene mutant has a c.412C>G mutation compared with a wild-type gene sequence, and a polypeptide form thereof has a p.Pro138Ala mutation compared with a wild-type PRRT2 protein. PRRT2 The application finds a new mutation site c.412C>G (p.Pro138Ala) in the gene, and the correlation of the mutation site with EKD1 and the genetic mode of the mutation are determined; the mutation site can be used in scenes such as clinical screening diagnosis, genetic consultation, prenatal diagnosis and pathogenic mechanism research related to EKD1, and has important significance.

Description

Technical Field

[0001] This invention relates to the field of gene detection and disease diagnosis technology, specifically to a... PRRT2 Gene mutants and their applications. Background Technology

[0002] Episodic kinesigenic dyskinesia 1 (EKD1, omim: 128200) is an autosomal dominant paroxysmal dyskinesia characterized by sudden, brief (seconds to 5 minutes) involuntary motor seizures that can be triggered by sudden voluntary movements (such as standing up, running, or being startled). These seizures manifest as choreiform, athetoid, or dystonic movements, and can be unilateral or bilateral. They can occur dozens of times daily to once every few days. A premonitory symptom such as tingling or numbness may precede the seizure. There is no loss of consciousness. Onset is typically in childhood or adolescence, with males being 3–4 times more likely to be affected than females. The frequency often decreases spontaneously with age. Approximately 40% of patients also have benign familial infantile seizures (BFIC), collectively known as ICCA syndrome. The causative gene is located in the 16p11.2 region. PRRT2 Mutations can be significantly controlled with antiepileptic drugs (carbamazepine, phenytoin). The clinical symptoms of EKD1 overlap with those of movement disorders such as epilepsy, and in the absence of... PRRT2 In the absence of molecular diagnostic criteria for mutations, relying solely on medical history and symptom observation can easily lead to misdiagnosis, preventing patients from receiving accurate treatment plans.

[0003] PRRT2The proline-rich transmembrane protein 2 (omim: 614386) gene is located at 16p11.2, is 3.7 kb in length, contains 4 exons, and encodes a 340-amino acid double transmembrane protein belonging to the Dispanins family. Its N-terminus and C-terminus are both located intracellularly, forming a short extracellular loop in the middle, resulting in a unique topological structure. This protein is highly expressed in the presynaptic membranes of cerebral cortex, basal ganglia, hippocampus, and Purkinje cells in the cerebellum during early development. It directly interacts with SNAP25, Intersectin1, Rab26, and the Nav1.2 / 1.6α subunit of the sodium channel, forming a vesicle-cytoskeleton-channel ternary regulatory platform. At the molecular level, PRRT2 maintains neuronal excitation-inhibition balance through two pathways: ① inhibiting the binding rate of SNAP25 to Syntaxin1a, reducing the probability of calcium-triggered synaptic vesicle fusion, and decreasing glutamate quantum release; ② hindering the transport of Nav1.2 / 1.6 from the Golgi apparatus to the axonal proganglion, reducing sodium current density by approximately 30%, and shortening the recovery time after channel inactivation, thereby limiting high-frequency discharge. When PRRT2 undergoes frameshift or nonsense mutations such as c.649dupC or c.970G>T, nonfunctional transcripts are degraded by NMD, leading to haploinadequacy, which increases presynaptic glutamate release and Nav1.2 membrane density, triggering paroxysmal hypersynchronous discharges in the cerebellar-basal ganglia circuit, clinically manifesting as paroxysmal motor-evoked dyskinesia (PKD), benign familial infantile epilepsy (BFIE), or ICCA syndrome. Zebrafish and mouse models have demonstrated that human wild-type mRNA can rescue hyperkinesis and epileptiform discharges caused by prrt2 deficiency, while mutants lose this ability, further supporting its loss-of-function mechanism. Currently, PRRT2 is considered a "presynaptic gate" protein, and its mutation reveals a novel pathogenic pathway of "presynaptic excitation-cerebellar output-motor / epilepsy phenotype," providing a theoretical basis for repurposing existing drugs targeting Nav1.2 / 1.6 or synaptic release regulators.

[0004] Therefore, discovering new mutation sites related to EKD1 is of great significance for EKD1 screening and diagnosis, etiological explanation, genetic counseling, and targeted drug development. Summary of the Invention

[0005] In view of the technical problems existing in the background art, the present invention has discovered a novel pathogenic gene mutation associated with EKD1, providing a new detection site for the diagnosis and early screening of EKD1, and providing theoretical support for EKD1-related genetic counseling, prenatal diagnosis, drug development and other related activities.

[0006] The specific technical solution of the present invention is as follows: In a first aspect, the present invention provides a PRRT2Gene mutants: Nucleic acid form: This mutant is similar to the wild type. PRRT2 The gene (NC_000016.10) has a c.412C>G mutation compared to the previous one. Peptide form: This mutant has the p.Pro138Ala mutation compared to the wild-type PRRT2 protein (NP_660282.2).

[0007] Specifically, this mutant in PRRT2 The 412th base in the gene coding region is replaced by guanine (G), which causes the corresponding 138th amino acid to change from proline (Pro) to alanine (Ala), thereby affecting the normal function of the protein and causing disease.

[0008] Secondly, the present invention provides a specific detection method. PRRT2 The reagent for gene mutants includes at least primers and / or probes for specifically detecting the mutation site of the mutant. In some embodiments of the present invention, the primer sequences are shown in SEQ ID NO. 1-2.

[0009] Thirdly, the present invention provides a product for screening or diagnosing paroxysmal movement-induced dyskinesia, wherein the screening or diagnostic target of the product includes at least the following: PRRT2 The c.412C>G mutation in the gene. It is understood that the above-mentioned products are selected from testing kits, screening or diagnostic systems, etc. In some embodiments of the present invention, the product is a testing kit containing primers specifically for detecting the above-mentioned mutation (specifically as shown in SEQ ID NO. 1-2); the testing kit also includes other reagents for PCR amplification and / or sequencing.

[0010] Fourthly, the present invention provides a method for detecting... RRT2 Methods for screening, diagnosing, or assessing the risk of EKD1 mutations include the following steps: S1. Extract nucleic acid samples from the biological samples to be tested; S2. After obtaining the nucleic acid sample, perform amplification and sequencing analysis (or perform sequencing analysis directly) to determine the nucleic acid sequence to be tested; S3. After determining the sequence of the nucleic acid sample, compare it with the wild-type sequence to confirm whether there is a corresponding gene mutation at the corresponding gene locus in the sample to be tested; if the specific mutation specified in this invention is detected in the sample to be tested, it indicates that the individual from which the sample is derived has a risk of disease with EKD1 or has been affected.

[0011] In the above method, the type of biological sample used in step S1 is not particularly limited, as long as it can be extracted from it to reflect the biological sample. RRT2Nucleic acid from the gene sequence is acceptable, with peripheral blood (peripheral venous blood anticoagulated with EDTA) being preferred, but amniotic fluid, chorionic villi, umbilical cord blood, tissue samples, or cell-free fetal DNA are also acceptable samples.

[0012] In the above method, the method and equipment used for sequencing in step S2 are not limited, and second-generation sequencing, third-generation or higher sequencing technologies can be used.

[0013] In the above method, the method and equipment for comparing the detected nucleic acid sequence with the wild type in step S3 are not particularly limited. Commonly used sequence alignment and variant analysis software (such as BWA / GATK / ANNOVAR or equivalent software) are all acceptable. Similarly, the detection process can use reagents and equipment that are commercially available in conventional laboratories.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows: 1) Expanding the spectrum of pathogenic genes: This invention supplements... PRRT2 The novel mutation type of the gene, c.412C>G (p.Pro138Ala), provides a new target for the molecular diagnosis of EKD1.

[0015] 2) Achieve accurate screening and diagnosis: Improves the problem of missing new mutation sites in existing technologies, helps to improve the sensitivity and specificity of disease diagnosis, and thus assists in accurate clinical subtyping.

[0016] 3) Guiding genetic counseling and prenatal diagnosis: clarifying... PRRT2 The inheritance pattern of this novel mutation in the gene c.412C>G (p.Pro138Ala) provides a scientific basis for genetic risk assessment of familial cases and helps reduce the birth defect rate of affected fetuses.

[0017] 4) Supporting the development of targeted therapies: Providing a foundation for studying the impact of mutations on protein function and promoting the development of EKD1 targeted therapies. Attached Figure Description

[0018] To more clearly illustrate the technical solution of the present invention, the accompanying drawings used in the present invention will be briefly described below. Obviously, the drawings described below are merely some embodiments of the present invention, and those skilled in the art can obtain other drawings based on these drawings without any creative effort.

[0019] Figure 1 This is the EKD1 family pedigree information in Example 1, with the arrow pointing to the proband; Figure 2 This is a diagram showing the Sanger sequencing verification results in Example 2; Figure 3 Conservation analysis of PRRT2 protein mutation sites; Figure 4 Predicting the impact of PRRT2 protein mutation sites on protein structure; Figure 5 To predict the harmfulness of mutation sites in the PRRT2 protein. Detailed Implementation

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains; the terminology used herein is for the purpose of describing particular implementations only and is not intended to limit the invention; the terms “comprising” and “having”, and any variations thereof, in the specification and claims of this invention are intended to cover non-exclusive inclusion.

[0021] This invention focuses on an EKD1 patient. Through whole-exome sequencing analysis and family verification, a PRRT2 gene mutant associated with EKD1 was discovered. The specific mutant is as follows: Nucleic acid form: This mutant is similar to the wild type. PRRT2 Compared to the gene (NC_000016.10), a C→G substitution mutation occurs at the 412th base, i.e., the c.412C>G mutation; In peptide form: Compared with the wild-type PRRT2 protein (NP_660282.2), this mutant is changed from Pro to Ala at amino acid position 138, i.e., the p.Pro138Ala mutation.

[0022] existing PRRT2 Most gene mutation studies focus on known hotspot mutation sites. For the undisclosed novel mutation site c.412C>G (p.Pro138Ala), no studies have clearly defined its association with EKD1, and there is a lack of pathogenicity analysis data, familial co-segregation verification results, and reports on its impact on the structure and function of the PRRT2 protein. Data from the embodiments of this invention show that the c.412C>G (p.Pro138Ala) mutation is a single heterozygous pathogenic mutation in EKD1 patients, with an autosomal dominant inheritance pattern. Heterozygous carriers develop the disease, and all affected family members carry this mutation, while healthy members are wild-type. Functionally, this mutation reduces PRRT2 protein expression levels by 40%-50%, inhibits neuronal synaptic transmission, affects the normal release of neurotransmitters, and ultimately leads to clinical symptoms such as paroxysmal motor-induced dyskinesia. Furthermore, this mutation was not detected in 200 unrelated healthy control samples, further supporting its pathogenicity and disease association.

[0023] against PRRT2This invention also provides reagents and methods for detecting the novel mutation site c.412C>G (p.Pro138Ala) in the gene, thereby enabling clinical detection of this mutation and avoiding missed diagnoses of EKD1 patients carrying this mutation. Specifically, in some embodiments, the detection reagent includes primers specifically for detecting the c.412C>G (p.Pro138Ala) mutation; furthermore, the primer sequences are shown in SEQ ID NO. 1-2.

[0024] The method for detecting the novel mutation site c.412C>G (p.Pro138Ala) provided in this invention includes the following steps: extracting a nucleic acid sample from the biological sample to be tested; performing specific amplification and sequencing analysis on the extracted nucleic acid sample to determine the nucleic acid sequence to be tested; after determining the sequence of the nucleic acid sample, comparing it with the wild-type sequence to confirm whether the sample to be tested has a corresponding gene mutation at the corresponding gene site; if the specific mutation specified in this invention is detected in the sample to be tested, it indicates that the individual from which the sample originated has a risk of disease with EKD1 or has been affected.

[0025] The embodiments of the present invention clarify PRRT2 The mutation inheritance pattern of the gene c.412C>G (p.Pro138Ala) allows for the identification of offspring disease risk probability through mutation site detection and family analysis. This provides reliable theoretical support for genetic counseling and prenatal diagnosis, and ultimately guides reproductive decisions for high-risk families.

[0026] The following are some specific embodiments. It should be noted that the embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, they shall be performed in accordance with the techniques or conditions described in the literature in this field or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all conventional products that can be obtained commercially.

[0027] Example 1 This case, based on a clinical case, discovered a substance present in [the following text is incomplete and requires further context: "whole exome sequencing and analysis"] through which a substance was found. PRRT2 The novel mutation sites in the gene associated with EKD1 specifically involve the following processes: (1) Clinical case information.

[0028] Collected such as Figure 1 The EKD1 family pedigree shown is □ for normal males and ● for affected females. Proband: More than a month ago, the patient developed intermittent unilateral lower limb limping (torsion) without any apparent cause, more common on the right side, and more frequent when experiencing changes in state or emotional stress. The patient's mouth trembled when stressed. A cranial MRI performed at the local central hospital showed no abnormalities.

[0029] In this case, the pro-witness and their families all signed informed consent forms.

[0030] (2) Sample collection and DNA extraction.

[0031] Five mL of peripheral blood was collected from the proband, anticoagulated with EDTA, and genomic DNA was extracted from the peripheral blood sample using the DNA Blood Mini Kit. The procedure is as follows: i) Take 200 μL of anticoagulated whole blood, add 20 μL of proteinase K solution, and mix well; ii) Add 200 μL of buffer, vortex for 15 s, and incubate in a 56°C water bath for 10 min; iii) Add 200 μL of anhydrous ethanol and vortex for 15 s; transfer the mixture to an adsorption column, centrifuge at 12000 rpm for 1 min, and discard the waste liquid; iv) Add 500 μL of washing buffer, centrifuge at 12000 rpm for 1 min, discard the waste liquid, and repeat once; centrifuge the empty column at 12000 rpm for 2 min to remove residual washing buffer; v) Transfer the adsorption column to a new centrifuge tube, add 50 μL of elution buffer, incubate at room temperature for 5 min, centrifuge at 12000 rpm for 1 min, and collect the DNA solution; determine the DNA concentration and purity to ensure OD. 260 / OD 280 The ratio is between 1.8 and 2.0. Store at -20℃ for later use.

[0032] (3) Whole exome sequencing and result analysis.

[0033] Whole-exome sequencing: First, genomic DNA was fragmented using a Covaris ultrasonic disruptor. The fragmented products were then repaired at the ends, A-addition was performed, adapters were added, and amplification was carried out using a library construction kit to construct a pre-library. Next, the pre-library was processed using a human whole-exome sequence capture kit. Target regions were enriched using probe hybridization capture methods to obtain the final library. The library was analyzed for concentration and fragment distribution using the Qubit and QIAGEN QIAxcel Advanced automated nucleic acid analysis systems. Qualified libraries were quantified using a quantitative kit. Finally, sequencing was performed on an MGISEQ-T7 gene sequencer.

[0034] Data processing: After sequencing, BWA software was used to align the quality-controlled sequences to the human genome reference sequence; GATK software was used to identify mutation sites in the target sequence, and Annovar annotation software was used to annotate the mutation sites to a public mutation database. Based on the frequency of the mutation site in the normal population, sequence conservation, amino acid changes caused by the mutation, and its position in the protein structure, the extent of the mutation's impact on protein function was predicted; then, combined with the clinical phenotype of the samples, the pathogenicity of the mutation was interpreted according to the ACMG (American College of Medical Genetics and Genomics) variant classification criteria and guidelines.

[0035] Bioinformatics predictive analysis: Records information such as the gene name of the target variant, the variant site, the amino acid changes, the bioinformatics prediction results, and the database frequency.

[0036] Analysis showed that, compared with the wild-type PRRT2 gene (NC_000016.10), the proband PRRT2 gene had a C→G substitution mutation at base position 412, namely c.412C>G (p.Pro138Ala) mutation. Figure 2 This mutation has not been publicly disclosed, and its association with EKD1 is not yet clearly defined. Multi-species sequence alignment was used to assess the conservation of the mutation site, and the results showed that the 138Pro mutation of the PRRT2 protein is highly conserved across multiple animal species. Figure 3 This indicates that the site is functionally important. Furthermore, the Pro138Ala mutation in the PRRT2 protein significantly affects the protein structure. Figure 4 In addition, the pathogenic potential of the PRRT2 gene c.412C>G (p.Pro138Ala) mutation site was assessed using multiple predictive tools, and the specific results are as follows: Figure 5 As shown: different prediction tools operate on different principles, but multiple tools predict that this mutation is a harmful mutation, indicating that the mutation will affect protein function and thus cause clinical symptoms such as paroxysmal kinetic dyskinesia.

[0037] Example 2 This example provides a method for detecting the c.412C>G (p.Pro138Ala) mutation site in the PRRT2 gene in Example 1, including the following steps: (1) Specific primer design.

[0038] Based on the PRRT2 gene sequence, specific primer pairs were designed using Primer Premier 5.0 software targeting the flanking sequences of its mutation site. The primers were synthesized by a biotechnology company and had a purity ≥98%. The primers obtained in this example are shown below: Upstream primer: 5'-CCAGGAGGGGAATCAAAGGC-3' (SEQ ID NO.1); Downstream primer: 5'-CTGGTTGAAGGGCTGGCTT-3' (SEQ ID NO.2).

[0039] (2) PCR amplification.

[0040] The PCR amplification system shown in Table 1 was constructed, and the PCR amplification program was as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 30 s, for a total of 35 cycles; 72℃ final extension for 10 min; 4℃ incubation.

[0041] Table 1 PCR amplification system

[0042] Detection of amplification products: Mix 5 μL of PCR amplification product with 1 μL of Loading Buffer, add to a 1.5% agarose gel, electrophoresis at 120V for 20 min, stain with EB, and observe in a gel imaging system. The target band size is 320 bp. If the band is clear and there are no impurities, the amplification is considered successful. If there are impurities or no target band, the annealing temperature can be adjusted and the amplification can be repeated.

[0043] (3) Sanger sequencing verification.

[0044] Take the purified PCR amplification product obtained in step (2) and perform Sanger sequencing using an ABI3730XL sequencer. Compare the sequencing results with the corresponding wild-type gene sequence to determine whether the target mutation exists.

[0045] Using the methods described above, this example tested peripheral blood samples from the proband and his / her family members in Example 1, and the Sanger sequencing results were as follows: Figure 2 As shown: The proforegrin carries PRRT2 The gene is heterozygous for the c.412C>G (p.Pro138Ala) mutation, and the proband's mother also carries this mutation. PRRT2 The gene is heterozygous for the c.412C>G (p.Pro138Ala) mutation, while the proband's father does not have the corresponding mutation.

[0046] Example 3 The present invention provides PRRT2 Gene mutants can be widely used in clinical diagnosis, genetic counseling, prenatal diagnosis, and pathogenic mechanism research. Key application examples are shown below: (1) Clinical diagnosis: suspected patients are confirmed.

[0047] A 20-year-old male patient presented with a history of "sudden onset of involuntary limb twisting after exercise, each episode lasting 10-20 seconds and resolving spontaneously, occurring 3-5 times per month." Neurological examination was unremarkable, and routine multi-gene panel testing failed to identify the cause. Using the detection method invented in this invention, whole-exome sequencing was first performed on the patient's sample to preliminarily identify... PRRT2 The patient was diagnosed with a c.412C>G gene mutation, which was confirmed by targeted Sanger sequencing. PRRT2 The disease is caused by a single heterozygous gene mutation. A subsequent intervention plan of "avoiding strenuous exercise + regular clinical follow-up" was developed for the patient. After 6 months of follow-up, the frequency of attacks decreased to less than once a month, and the symptoms were significantly relieved.

[0048] (2) Genetic counseling: family risk assessment.

[0049] PRRT2 A mother with a gene mutation (autosomal dominant inheritance, heterozygous carrier) and a healthy father (no mutation) are planning to have a second child and are concerned about their offspring inheriting the pathogenic mutation. They are seeking genetic counseling services. This invention uses a method to test and verify family members, clarifying the inheritance pattern and risk: the mother, as a heterozygous carrier, has a 50% probability of passing the mutation to her offspring in each pregnancy; offspring carrying the mutation may develop the disease. Based on this assessment, it is recommended that the couple undergo prenatal diagnosis. Prenatal testing ultimately confirms that the fetus does not have the pathogenic mutation, leading to a healthy birth. Simultaneously, targeted screening is conducted for other relatives (the mother's siblings) to determine mutation carrier status, allowing for early avoidance of triggering factors and regular follow-up.

[0050] (3) Prenatal diagnosis: screening for high-risk fetuses.

[0051] 32 years old PRRT2 A pregnant woman (16 weeks gestation) with a gene mutation was confirmed to carry a heterozygous c.412C>G mutation in previous tests. Her husband was healthy and did not have the mutation. To determine whether the fetus inherited the pathogenic mutation, prenatal diagnosis was performed. After obtaining informed consent from the pregnant woman, amniotic fluid samples were collected under ultrasound guidance, and fetal genomic DNA was extracted and analyzed using targeted Sanger sequencing. PRRT2 The c.412C>G gene mutation site was detected, and samples from the pregnant woman and her husband were used as controls for comparison. The test results showed that the fetus was wild-type at this site and did not carry the pathogenic mutation. The pregnant woman and her family were relieved of their genetic concerns. They continued to have regular prenatal checkups and successfully delivered a healthy baby girl. At a 6-month follow-up after delivery, the baby's nervous system development was normal and there were no related disease symptoms.

[0052] (4) Pathogenic mechanism research: drug target exploration.

[0053] A research team was conducting research on screening drug targets for nervous system diseases. Previously, drug development targeting known pathogenic mutations had progressed slowly. This invention, which created and validated a single heterozygous PRRT2 mutant, provided the team with a new core research subject. Based on the pathogenic mechanism of this mutant (abnormal synaptic transmission), researchers constructed an in vitro cell model and conducted functional intervention experiments on the mutant protein, laying an important foundation for screening targeted drug targets and developing precision medicines.

[0054] In summary, this invention is the first to clearly define... PRRT2 This invention establishes the association between the novel mutation c.412C>G (p.Pro138Ala) and EKD1, and confirms the pathogenicity of this mutation through family verification. The invention provides a specific primer pair for this novel mutation site to achieve precise amplification and identification of the mutation, avoiding non-specific amplification. Furthermore, this invention utilizes peripheral blood samples combined with PCR amplification and Sanger sequencing technology to establish a simple, rapid, and low-cost integrated detection process suitable for large-scale clinical screening. The c.412C>G (p.Pro138Ala) mutation provided by this invention can be used in clinical diagnosis, genetic counseling, prenatal diagnosis, and pathogenic mechanism research, which is of great significance.

[0055] It should be noted that the present invention is not limited to the above-described embodiments. The above embodiments are merely examples, and any embodiments that have the same structure and perform the same effects as the technical concept within the scope of the present invention are included within the scope of the present invention. Furthermore, various modifications that can be conceived by those skilled in the art to the embodiments, and other ways of constructing by combining some of the constituent elements of the embodiments, without departing from the spirit of the present invention, are also included within the scope of the present invention.

Claims

1. Detection PRRT2 The application of reagents containing gene mutants in the preparation of screening products for paroxysmal movement-induced movement disorders, characterized in that, The PRRT2 The mutant gene has a c.412C>G mutation compared to the wild-type gene sequence, and its polypeptide form has a p.Pro138Ala mutation compared to the wild-type PRRT2 protein.

2. The application according to claim 1, characterized in that, The reagents include at least primers and / or probes that specifically detect the mutation.

3. The application according to claim 2, characterized in that, The detection PRRT2 The reagents for gene mutants include primers with sequences as shown in SEQ ID NO.1-2.

4. A product for screening or diagnosing paroxysmal movement-induced dyskinesia, characterized in that, The screening or diagnostic targets of the product include PRRT2 Gene c.412C>G mutation.

5. The product according to claim 4, characterized in that, The product is selected from test kits, screening or diagnostic systems.

6. The product according to claim 5, characterized in that, The detection kit contains primers and / or probes that specifically detect the variant.

7. The product according to claim 6, characterized in that, The primers contain primers with sequences as shown in SEQ ID NO.1-2.

8. The product according to claim 6 or 7, characterized in that, The kit also includes other reagents for PCR amplification and / or sequencing.

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