Csm d2, cacna1e and reln gene mutants and uses thereof

Abstract

The application discloses CSMD2 , CACNA1E and RELN gene mutants and application thereof, and belongs to the field of gene detection and disease diagnosis. CSMD2 The gene mutant has c.6584G>A and c.9932C>T mutations compared with a wild type sequence, CACNA1E The gene mutant has c.2767C>T mutation compared with a wild type sequence, RELN The gene mutant has c.2779A>G and c.5688T>A mutations compared with a wild type sequence. The application discovers mutation sites in CSMD2 , CACNA1E and RELN genes closely related to epilepsy, so that the mutation sites can be included in an analysis range on the basis of conventional epilepsy-related gene detection, thereby improving early identification capability for epilepsy to a certain extent, and further providing more reliable technical support for early intervention and individualized treatment of diseases.

Description

Technical Field

[0001] This invention relates to the field of gene detection and disease diagnosis technology, specifically to... CSMD2 , CACNA1E and RELN Gene mutants and their application in the diagnosis of epilepsy and related neurodevelopmental disorders. Background Technology

[0002] Epilepsy is a chronic neurological disorder characterized by recurrent epileptic-like clinical events, with abnormal synchronous discharge of neurons in the brain as its pathological basis. It can manifest in various forms, including clinical convulsions, loss of consciousness, and sensory abnormalities. The pathogenesis of epilepsy is complex, involving not only genetic factors but also structural, metabolic, immune, and infectious causes. Genetic factors play a significant role in epilepsy, especially childhood epilepsy and developmental and epileptic encephalopathy (DEE). More than 1500 related genes have been reported (including core pathogenic genes and related risk genes). Most of these genes are involved in processes such as ion channels, synaptic transmission, neural development, and signal transduction; mutations can lead to neuronal excitability imbalances and epileptic seizures. It is estimated that approximately 65 million people worldwide are affected by epilepsy. With the widespread application of high-throughput sequencing in clinical practice and research, hundreds to thousands of genes associated with epilepsy or DEE have been identified. These genes mainly involve pathways such as ion channels, synaptic transmission / plasticity, cell migration / development, and metabolism. It is evident that gene diagnosis plays an increasingly important role in clinical classification, prognosis, and personalized treatment.

[0003] Currently, although high-throughput sequencing technology has significantly advanced research on the genetic causes of epilepsy and related neurodevelopmental disorders, existing technologies still have certain limitations. Many recently reported associations between candidate genes and epilepsy or neurodevelopmental abnormalities are based on small sample or case studies, lacking large-scale, multi-center, and systemic functional validation, and have not yet formed stable, widely applicable clinical screening targets. Furthermore, existing epilepsy gene testing strategies differ in their scope and methodology. Conventional gene panels focus on known high-frequency pathogenic genes, while insufficiently covering genes related to pathogenic gene mutations or low-frequency mutations, potentially leading to missed detections of some potentially pathogenic cases. In addition, current testing procedures mostly remain at the variant identification level, lacking a standardized closed-loop system that integrates test results with family verification, functional assessment, and clinical intervention recommendations, limiting the practical application value of gene testing in early screening and precision medicine.

[0004] CSMD2(CUB and Sushi Multiple Domains 2) is located at 1p35.1 and contains 74 exons. It is a gene encoding a transmembrane protein containing multiple CUB and Sushi (CCP / complement) domains, which plays an important role in neuronal maturation, dendrite / synapse formation and homeostasis. CSMD Gene family members ( CSMD1 , CSMD2 , CSMD3 It is highly expressed in early brain development, and its spatiotemporal expression patterns in different neuronal subtypes (including inhibitory neurons) are closely related to neural network maturation. Recent clinical genetic studies have reported its detection in several cohorts of non-acquired focal epilepsy and focal cortical dysplasia (FCD). CSMD2 Rare / potentially pathogenic loci (including compound heterozygous or biallelic loci) were identified, and statistical enrichment, phenotypic association, and single-cell / spatiotemporal expression analysis were used to support this finding. CSMD2 Associated with focal epilepsy—that is CSMD2 High expression of inhibitory neurons in early development may explain its association with focal discharges / focal cortical developmental abnormalities. Although CSMD2 The specific pathogenic mechanism in epilepsy is not yet fully understood, but current evidence supports its inclusion as a candidate pathogenic gene in the epilepsy gene detection spectrum, and more and more case reports are constantly verifying its clinical significance.

[0005] CACNA1E Located on chromosome 1q25.3, containing 53 exons, it encodes the α1E subunit of the voltage-gated R-type calcium channel CaV2.3. CaV2.3 is widely expressed in the central nervous system, mediating high-voltage activated R-type currents and playing a crucial role in presynaptic calcium inflow and neurotransmitter release, as well as synaptic plasticity. Calcium channels participate in the regulation of neural excitability by modulating neurotransmitter release and synaptic plasticity; structural or functional abnormalities can promote epileptic seizures. Previous studies have shown that... CACNA1E The pathogenic variants are associated with early-onset developmental epileptic encephalopathy (OMIM: 618285). These variants often lead to functional changes in Cav2.3 calcium channels (e.g., altered protein structural stability and activity), resulting in severe clinical phenotypes such as early-onset refractory epilepsy, severe dystonia / hypotonia, significant developmental delay, often accompanied by motor disorders (choreiform involuntary movements / myoclonus), congenital joint contractures, and macrocephaly. Clinical case data show that... CACNA1E Related variants typically present with early seizures, significant developmental delays, and poor response to conventional antiepileptic drugs, suggesting... CACNA1EIt has practical significance for decision-making regarding the genetic diagnosis and treatment of epilepsy.

[0006] RELN Located on chromosome 7q22.1, it contains 65 exons and encodes the secreted large glycoprotein Reelin, which is an important factor in cell migration, stratification, and neural circuit localization during brain development. RELN Reelin participates in localization signals during the formation of the cerebral cortex and hippocampus, influencing the correct assembly of neural networks. It regulates neuronal migration, dendritic / axon localization, and synaptic plasticity through its receptors and downstream signaling pathways. Biaallelic (homozygous / compound) reelin is known. RELN Severe deficiencies can lead to abnormal development of the cerebral cortex and cerebellar dysplasia, while recent studies have also found heterozygous deficiencies. RELN Mutations can serve as a pathogenic factor in autosomal-dominant lateral temporal epilepsy (ADLTE) (OMIM: 257320): heterogeneous missense mutations identified in several families have co-segregated with familial ADLTE, and these mutations often reduce serum Reelin levels or impair protein folding and secretion, suggesting that partial functional loss is a pathogenic mechanism. Furthermore, some rapid case reports indicate... RELN Variations may also coexist with complex epileptic phenotypes (such as auditory epilepsy) and overlap with other neuropsychiatric phenotypes, suggesting... RELN It participates in multiple neural network functional circuits, and its dysfunction may lead to a wide range of neuropsychiatric manifestations. Summary of the Invention

[0007] This invention discovers new pathogenic gene mutations associated with epilepsy, providing new detection sites for the diagnosis and early screening of epilepsy-related diseases, thereby improving the effectiveness of epilepsy screening and risk assessment to a certain extent.

[0008] In a first aspect, the present invention provides the following epilepsy-related gene mutants: (1) CSMD2 Gene mutants: Nucleic acid form: its nucleotide sequence is similar to that of the wild type. CSMD2 Compared to the gene sequence (NM_001281956.2), it has c.6584G>A and c.9932C>T mutations; In polypeptide form, its amino acid sequence has the p.Gly2195Glu and p.Ser3311Phe mutations compared to the wild-type CSMD2 protein sequence (NP_001268885.1).

[0009] (2) CACNA1E Gene mutants: Nucleic acid form: its nucleotide sequence is similar to that of the wild type. CACNA1E Compared to the gene sequence (NM_001205293.3), it has a c.2767C>T mutation; In polypeptide form, its amino acid sequence has the p.His923Tyr mutation compared to the wild-type CACNA1E protein sequence (NP_001192222.1).

[0010] (3) RELN Gene mutants Nucleic acid form: its nucleotide sequence is similar to that of the wild type. RELN Compared to the gene sequence (NM_005045.4), it has c.2779A>G and c.5688T>A mutations; In polypeptide form, its amino acid sequence has the p.Met927Val and p.Asp1896Glu mutations compared to the wild-type RELN protein sequence (NP_005036.2).

[0011] Secondly, the present invention provides a method for epilepsy screening by detecting the above-mentioned gene mutants, comprising the following steps: S1. Extract nucleic acid samples from the biological samples to be tested; S2. After obtaining the nucleic acid sample, perform sequencing analysis 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; if the specific mutation specified in this invention (or a variant equivalent to or highly homologous to the mutation site described in this invention) is detected in the sample to be tested, it indicates that the individual from which the sample was obtained has a risk of developing epilepsy or has already been affected.

[0012] In the above method, the type of biological sample used in step S1 is not particularly limited, as long as the desired gene can be extracted from it (i.e., CSMD2 , CACNA1E and / or RELN Nucleic acid sequences are 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.

[0013] 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.

[0014] 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.

[0015] Thirdly, this invention provides the application of reagents for detecting the aforementioned gene mutants in the preparation of epilepsy screening products. Based on this epilepsy-related disease screening product, it can be used in conjunction with the following detection process: peripheral blood DNA extraction → WES (whole exome sequencing) or targeted panel sequencing → bioinformatics annotation → Sanger verification of specific mutation sites of this invention → family co-segregation analysis → functional verification (e.g., synapse formation / MEA electrophysiological detection of iPSC-derived neurons, synaptic protein interaction detection, ectopic expression and patch-clamp electrophysiological assays, cell secretion experiments, or in vitro neuronal migration experiments, etc.). Preferably, in the above applications, the reagent at least comprises primers and / or probes for detecting mutation sites in the aforementioned gene mutants. In some embodiments of the present invention, the reagent may include primers for detecting... CSMD2 Primers for gene c.6584G>A and c.9932C>T mutations, with sequences shown in SEQ ID NO.1-4; used for detection. CACNA1E Primers for the c.2767C>T mutation of the gene, with sequences shown in SEQ ID NO.5-6; used for detection. RELN Primers for gene c.2779A>G and c.5688T>A mutations, with sequences as shown in SEQ ID NO.7-10.

[0016] Fourthly, epilepsy screening products prepared according to the above applications also fall within the scope of protection of this invention, and the products include, but are not limited to, reagent kits, risk assessment systems, etc.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention, based on routine epilepsy-related gene testing, will... CSMD2 , CACNA1E , RELN The study included three genes closely related to epilepsy, providing information on mutation sites within these genes that enhance the identification of potential pathogenic variants. Compared to conventional methods relying solely on single sequencing or predictive analysis, this approach combines pedigree genetic patterns for screening and integrates conservation analysis, various harmfulness prediction software, and protein structure influence prediction results to provide a more comprehensive evaluation of candidate variants. This results in more objective and standardized variant interpretation and helps reduce the interpretability difficulties caused by some variants of unknown significance. Through these technical measures, this invention contributes to improving the efficiency and interpretation level of genetic testing for epilepsy and related neurodevelopmental abnormalities, providing supplementary molecular evidence for early clinical screening, genetic counseling, and subsequent research. 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. For those skilled in the art, other drawings can be obtained based on these drawings without any creative effort.

[0019] Figure 1 This is information about epilepsy family 1 in Example 1.

[0020] Figure 2 This is a diagram showing the Sanger sequencing verification results in Example 1.

[0021] Figure 3 The conservation of mutation sites in the CSMD2 protein in Example 1 is assessed (A), and the impact on protein structure is predicted (B).

[0022] Figure 4 This is information about epilepsy family 2 in Example 2.

[0023] Figure 5 This is a diagram showing the Sanger sequencing verification results in Example 2.

[0024] Figure 6 The conservation of mutation sites in the CACNA1E protein in Example 2 is assessed (A), and the predicted impact on protein structure is presented (B).

[0025] Figure 7 This is information about epilepsy family 3 in Example 3.

[0026] Figure 8 This is a diagram showing the Sanger sequencing verification results in Example 3.

[0027] Figure 9 The conservation of mutation sites in the RELN protein in Example 3 is assessed (A), and the impact on protein structure is predicted (B).

[0028] Figure 10 This invention aims to predict the harmfulness of mutations in the CSMD2, CACNA1E, and RELN proteins. Detailed Implementation

[0029] 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.

[0030] To address the shortcomings of existing epilepsy gene testing strategies, such as missed detections due to insufficient coverage of pathogenic gene mutations or low-frequency mutations, and the lack of integration of test results with family verification, this invention, based on clinical cases and family sequencing analysis, has discovered new pathogenic gene mutations associated with epilepsy. Based on this, a detection and analysis method has been established that covers a more comprehensive range of related genes, combines family and functional evidence, and provides standardized clinical interpretation and intervention guidance. This aims to improve the early identification of epilepsy and related neurodevelopmental disorders, providing more reliable technical support for early intervention and individualized treatment.

[0031] This invention, when comprehensively assessing the pathogenicity and functional impact of candidate variants from probands, follows the principles of the ACMG / AMP guidelines and employs a multi-level, multi-method evidence integration strategy. First, it starts with population database frequencies, querying databases such as gnomAD / ExAC / 1000G; variants that are extremely rare or unseen in these databases can be considered evidence supporting pathogenicity. Subsequently, pedigree typing and cosegregation analysis are combined; if the variant is de-degenerate or significantly cosegregates with the phenotype in the family, it is included in genetic evidence with strong weights, such as PS2 / PM6 or PP1. For bioinformatics evidence, various prediction tools (SIFT, PolyPhen-2, MutationTaster, PROVEAN, etc.) are used to predict the harmfulness of missense or splice sites. Based on the above experimental procedure, the embodiments of this invention specifically provide the following epilepsy-related gene mutants: CSMD2 Gene mutants: whose nucleotide sequences are similar to those of wild-type mutants. CSMD2 Compared with the gene sequence (NM_001281956.2), it has c.6584G>A and c.9932C>T mutations, and its amino acid sequence has p.Gly2195Glu and p.Ser3311Phe mutations compared with the wild-type CSMD2 protein sequence (NP_001268885.1).

[0032] CACNA1E Gene mutants: whose nucleotide sequences are similar to those of wild-type mutants. CACNA1E The gene sequence (NM_001205293.3) has a c.2767C>T mutation, and its amino acid sequence has a p.His923Tyr mutation compared to the wild-type CACNA1E protein sequence (NP_001192222.1).

[0033] RELN Gene mutants: whose nucleotide sequences are similar to those of wild-type mutants. RELNCompared with the gene sequence (NM_005045.4), it has c.2779A>G and c.5688T>A mutations, and its amino acid sequence has p.Met927Val and p.Asp1896Glu mutations compared with the wild-type RELN protein sequence (NP_005036.2).

[0034] This invention further evaluated the impact of the aforementioned mutations on the function of the corresponding proteins. Site conservation was assessed through multi-species sequence alignment; non-conserved substitutions at conserved sites indicated greater functional importance. Combined with gene / protein domain annotation, variations located in known key functional domains (e.g., voltage-sensing regions of ion channels, receptor binding sites, or enzyme activity cores) could be included as moderately weighted evidence, such as PM1. Multi-species alignment showed that CSMD2 protein 2195Gly and 3311Ser, CACNA1E protein 923His, and RELN protein 1896Asp and 927Met are highly conserved across multiple animal populations, indicating the importance of these sites. The potential impact of amino acid substitutions on protein stability and conformational dynamics was assessed using structural / energetic tools such as DynaMut2; when multiple tools consistently indicated harmfulness and DynaMut2 showed a significant ΔΔG or conformational change, this could serve as supporting evidence, such as PP3. Protein structure prediction showed that mutations in CSMD2 protein (Gly2195Glu and Ser3311Phe), CACNA1E protein (His923Tyr), and RELN protein (Asp1896Glu and Met927Val) all caused significant changes in hydrogen bond interactions and steric hindrance, indicating that mutations can disrupt protein structural stability.

[0035] This invention also provides primers for detecting the above-mentioned gene mutants, specifically: primers for detecting... CSMD2 Primers for gene mutants, as shown in SEQ ID NO. 1-4, are used for detection. CACNA1E Primers for gene mutants, as shown in SEQ ID NO. 5-6, are used for detection. RELN The primers for gene mutants are shown in SEQ ID NO.7-10. Based on the above primers, the detection method can be as follows: extract DNA from the sample to be tested (such as peripheral blood of the subject), perform specific amplification using the above primers, and perform Sanger sequencing on the amplified fragment to obtain mutation information; on this basis, further pedigree verification and functional verification can be performed to improve the accuracy of detection.

[0036] Given the correlation between the gene mutants provided by this invention and epilepsy, these gene mutants can be used to screen for epilepsy-related diseases: At once CSMD2 Regarding the effects of gene mutants on the body, CSMD2The encoded protein is a large molecule containing CUB and Sushi domains, involved in synapse formation / homeostasis and neuronal development. If the CSMD2 protein is mutated, resulting in structural changes, abnormal folding / positioning, or decreased binding ability to its interacting proteins, it can interfere with synapse formation and the balance between inhibitory and excitatory neurons, potentially leading to phenotypes such as focal epilepsy, cortical dysplasia, or neurodevelopmental abnormalities. Accordingly, this invention clarifies... CSMD2 Gene mutations can disrupt synapse formation and microenvironment homeostasis, inducing abnormal discharges in neural circuits and manifesting as clinical symptoms such as epileptic seizures (which may be accompanied by developmental delays).

[0037] At once CACNA1E Regarding the effects of gene mutants on the body, CACNA1E The α1 subunit encodes the R-type (CaV2.3) voltage-gated calcium channel, regulating presynaptic calcium inflow and neurotransmitter release. Pathogenic variants can lead to gain-out or loss-of-function of the channel (e.g., altered activation voltage threshold, delayed inactivation, altered current density), thereby changing neural excitability and network synchrony, manifesting as early-onset refractory epilepsy, motor abnormalities, developmental delays, etc. Functional evidence (patch-clamp electrophysiology) plays a decisive role in determining the pathogenicity of the variant.

[0038] At once RELN In terms of the biological effects of gene mutants, RELN It encodes Reelin, a secreted macromolecular protein that is involved in neuronal migration, cortical stratification, and synaptic plasticity. RELN Mutations in these proteins can affect protein folding / secretion or receptor binding capacity, leading to abnormal cortical structures or defects in neural network connectivity, thereby resulting in auditory / language-induced focal seizures (such as ADLTE) or a broader cortical development-related epileptic phenotype.

[0039] Specifically, CSMD2, CACNA1E and RELN Gene mutants can be used in clinical genetic diagnosis, early screening for complex epilepsy, prenatal / prenatal risk assessment, and family genetic counseling, thus providing a reference for disease diagnosis, prenatal intervention, and drug selection. It is understood that when a subject is found to have a mutation site disclosed in this invention, they can be considered... CSMD2 / CACNA1E / RELN Individuals with epilepsy or at high risk.

[0040] 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.

[0041] The patient samples involved in the following examples were all from Tongji Hospital affiliated to Tongji Medical College of Huazhong University of Science and Technology. All patients and their families signed informed consent forms. Peripheral venous blood was collected from patients in EDTA-K2 negative pressure blood collection tubes, and the information was accurately recorded and stored at 4°C. Systematic sampling of families was carried out and pedigree charts were drawn. The clinical phenotype of the proband (seizure type, age of onset, developmental assessment, brain imaging, EEG, etc.) was recorded to facilitate phenotype-gene matching analysis.

[0042] Example 1 This example provides information related to epilepsy. CSMD2 The gene mutation sites are as follows: (1) Sample collection and family history data.

[0043] Collected such as Figure 1 The epilepsy pedigree shown is pedigree 1. □ represents a normal male, ○ represents a normal female, and ● represents a female with epilepsy. Proband II-1: Age of first onset: 10 months; Seizure type: loss of consciousness, fixed gaze, cyanosis, trismus, frothing at the mouth, and limb tremors; followed by one unexplained seizure, without fever or cough, accompanied by 3 episodes of vomiting and slightly loose, grayish-white stools; serum lactate 2.6 mmol / L; Seizure type: loss of consciousness, fixed gaze, cyanosis, trismus, frothing at the mouth, and rigid tremors of the limbs, lasting approximately 2 minutes before subsiding; ALT: 24 U / L; AST: 49 U / L; MRI: abnormal, with decreased CBF in the left cerebral hemisphere compared to the contralateral side. The parents are phenotypically normal, indicating a recessive inheritance pattern.

[0044] (2) DNA extraction and whole exome sequencing.

[0045] DNA was extracted and its concentration determined, then stored at -20°C. First, whole-exome sequencing was achieved through targeted probe hybridization, followed by massively parallel sequencing using a next-generation sequencing platform. Finally, computational biology methods were used to process the raw sequence data. In this example, the Illumina sequencing platform was specifically used for whole-exome sequencing to identify pathogenic genes.

[0046] Exon region sequence enrichment: Genomic DNA was randomly fragmented using physical methods to obtain shorter DNA fragments. The resulting DNA molecules were then blunted using an end repair system. Adapters containing specific sequences were then ligated to both sides of the blunt-ended DNA double strands. The fragments were then hybridized with an Agilent Sure Select Human All Exon V4 library for enrichment. Unhybridized DNA fragments were washed away, and the fragments were then amplified and purified by ligation-mediated PCR.

[0047] High-throughput sequencing: The captured library fragments were sequenced using a HiSeq 2000 sequencer.

[0048] Bioinformatics analysis: Raw data quality control was performed to remove low-quality data. The filtered data was then compared with a reference genome to identify variations such as single nucleotide polymorphisms (SNPs) and insertions / deletions (InDels). Detected mutations were further analyzed and annotated using NCBI CCDS, RefSeq, Ensembl, and Encode databases. Bioinformatics prediction employed SIFT, PolyPhen-2, MutationTaster, and PROVEAN to assess pathogenicity, and DynaMut2 to predict the impact on protein stability / kinetics. Finally, all evidence was preliminarily scored according to ACMG guidelines.

[0049] (3) Whole exome sequencing results of the proband.

[0050] Sample analysis of proband II-1 revealed that, CSMD2 The gene (transcript: NM_001281956.2) contains compound heterozygous variants: c.6584G>A (p.Gly2195Glu) and c.9932C>T (p.Ser3311Phe). This variant has not been reported in normal population gene databases, and the clinical symptoms of this case are consistent with epilepsy.

[0051] (4) Sanger sequencing verification.

[0052] The identified mutations were validated using Sanger sequencing. PCR primers were designed using SnapGene software and NCBI Primer-BLAST according to primer design principles (as shown in Table 1). Peripheral blood genomic DNA was collected from family members and then validated using first-generation sequencing.

[0053] Table 1

[0054] Sanger sequencing results as follows Figure 2 As shown, the proband carries CSMD2 The gene contains compound heterozygous mutations c.6584G>A (p.Gly2195Glu) and c.9932C>T (p.Ser3311Phe). The proband's mother carries the c.6584G>A (p.Gly2195Glu) mutation and has a normal phenotype, while the proband's father carries the c.9932C>T (p.Ser3311Phe) mutation and also has a normal phenotype, consistent with an autosomal recessive inheritance pattern.

[0055] (5) Effects of mutations on the function of CSMD2 protein.

[0056] The conservation of mutation sites was determined by multi-species sequence alignment. The results showed that the 2195Gly and 3311Ser of the CSMD2 protein are highly conserved in evolution across multiple animal species. Figure 3 A) indicates that this site is functionally important. Further evaluation of the potential impact of amino acid substitutions on protein stability and conformational dynamics showed that mutations in Gly2195Glu and Ser3311Phe in the CSMD2 protein both caused significant changes in hydrogen bond interactions and steric hindrance. Figure 3 B) indicates that mutations can disrupt protein structural stability.

[0057] Example 2 This example provides information related to epilepsy. CACNA1E The gene mutation sites are as follows: (1) Sample collection and family history data.

[0058] Collected such as Figure 4 The epilepsy pedigree shown is 2, where □ represents a normal male and ● represents an affected female. The proband, II-1, first experienced seizures at seven months of age and was admitted to the hospital due to "three intermittent seizures." On May 14, 2023, the child experienced three seizures. An EEG (3 hours) showed an abnormal infant EEG, detecting one partial seizure. On the afternoon of June 7, 2023, the child experienced seizures without any obvious cause, without fever, exhibiting altered consciousness, fixed gaze, drooling, and cyanosis of the lips, lasting approximately one minute before subsiding. The child then experienced two more seizures without fever, exhibiting the same symptoms, each lasting approximately one minute before subsiding. The family stated that both seizures occurred after the child received a "vaccine." The patient's mother, I-2, had a history of suspected seizures when she was 2 years old, indicating a dominant inheritance pattern.

[0059] (2) DNA extraction and whole-exome sequencing. This step is the same as in Example 1.

[0060] (3) Whole exome sequencing results of the proband.

[0061] Sample analysis of proband II-1 revealed that, CACNA1E The gene (transcript: NM_001205293.3) contains a heterozygous variant: c.2767C>T (p.His923Tyr); this variant has not been reported in normal population gene databases and the clinical symptoms of this case are consistent with epilepsy symptoms.

[0062] (4) Sanger sequencing verification.

[0063] The identified mutations were validated using Sanger sequencing. PCR primers were designed using SnapGene software and NCBI Primer-BLAST according to primer design principles (as shown in Table 2). Peripheral blood genomic DNA was collected from family members and then validated using first-generation sequencing.

[0064] Table 2

[0065] Sanger sequencing results as follows Figure 5 As shown, the proband carries CACNA1E The heterozygous mutation c.2767C>T (p.His923Tyr) was also carried by the proband's mother. CACNA1E The patient has a heterozygous mutation c.2767C>T (p.His923Tyr) and a history of seizures in childhood, which is consistent with an autosomal recessive-dominant inheritance pattern.

[0066] (5) Effects of mutations on the function of CACNA1E protein.

[0067] The conservation of mutation sites was determined by multi-species sequence alignment. The results showed that the 923His mutation in the CACNA1E protein is highly conserved in evolution across multiple animals. Figure 6 A) indicates that this site is functionally important. Further evaluation of the potential impact of amino acid substitutions on protein stability and conformational dynamics showed that the His923Tyr mutation in the CACNA1E protein causes significant changes in hydrogen bond interactions and steric hindrance. Figure 6 B) indicates that mutations can disrupt protein structural stability.

[0068] Example 3 This example provides information related to epilepsy. RELN The gene mutation sites are as follows: (1) Sample collection and family history data.

[0069] Collected such as Figure 7 The epilepsy pedigree shown is 3. □ represents a normal male, ○ represents a normal female, and ■ represents an affected male. The proband, II-1, a six-month-old infant, experienced 8 seizures within 4 days without any obvious cause, each lasting 1-2 minutes. EEG: Abnormal infant EEG (burst-inhibition) with a 4-5 Hz 0-wave rhythm in the bilateral occipital regions during awake, quiet, and feeding conditions. Cranial MRI: The right temporal horn is slightly larger than the contralateral one. Both parents are phenotypically normal, indicating a recessive inheritance pattern.

[0070] (2) DNA extraction and whole-exome sequencing. This step is the same as in Example 1.

[0071] (3) Whole exome sequencing results of the proband.

[0072] Sample analysis of proband II-1 revealed that, RELNThe gene (transcript: NM_005045.4) contains compound heterozygous variants: c.5688T>A (p.Asp1896Glu) and c.2779A>G (p.Met927Val); this variant has not been reported in normal population gene databases and the clinical symptoms of this case are consistent with epilepsy symptoms.

[0073] (4) Sanger sequencing verification.

[0074] The identified mutations were validated using Sanger sequencing. PCR primers were designed using SnapGene software and NCBI Primer-BLAST according to primer design principles (as shown in Table 3). Peripheral blood genomic DNA was collected from family members and then validated using first-generation sequencing.

[0075] Table 3

[0076] Sanger sequencing results as follows Figure 8 As shown, the proband carries RELN The gene contains compound heterozygous mutations c.5688T>A (p.Asp1896Glu) and c.2779A>G (p.Met927Val). The proband's mother carries the c.2779A>G (p.Met927Val) mutation and has a normal phenotype, while the proband's father carries the c.5688T>A (p.Asp1896Glu) mutation and also has a normal phenotype, consistent with an autosomal recessive inheritance pattern.

[0077] (5) Effects of mutations on the function of RELN proteins.

[0078] The conservation of mutation sites was determined by multi-species sequence alignment. The results showed that the 1896Asp and 927Met of the RELN protein are highly conserved in evolution across multiple animals. Figure 9 A) indicates that this site is functionally important. Further evaluation of the potential impact of amino acid substitutions on protein stability and conformational dynamics showed that the Asp1896Glu and Met927Val mutations in the RELN protein both caused significant changes in hydrogen bond interactions and steric hindrance. Figure 9 B) indicates that mutations can disrupt protein structural stability.

[0079] Example 4 The mutation in Example 2 CACNA1E Taking the application of c.2767C>T (p.His923Tyr) in the diagnostic analysis of developmental epileptic encephalopathy as an example, this invention illustrates the application method of gene mutants in the screening and diagnosis of epilepsy-related diseases.

[0080] Based on the disclosure of this invention CACNA1E Gene mutants and their corresponding detection reagents can be used for molecular detection of collected samples to determine whether specific mutants exist in the sample. CACNA1E Mutation sites are used to determine whether the individuals from which the sample was obtained are likely to have the same disease. CACNA1E The related developmental epileptic encephalopathy may be a high-risk carrier / onset population, thus providing important reference for clinical diagnosis, treatment decisions and genetic counseling.

[0081] In clinical applications, the detection and analysis methods described in this invention include the following steps: 1) Extracting nucleic acid samples from biological samples; 2) After obtaining the nucleic acid sample, perform sequencing analysis to determine the nucleic acid sequence to be tested; 3) After determining the sequence of the nucleic acid sample, compare it with the wild-type sequence; if the specific mutation specified in this invention (or a variant equivalent to or highly homologous to the mutation site described in this invention) is detected in the sample to be tested, it indicates that the individual from which the sample was obtained has a risk of developing epilepsy or has already been affected.

[0082] The detection and interpretation of this mutation can be achieved through at least the following two analytical approaches: (1) Direct molecular detection method: Sanger sequencing or NGS (including WES / WGS or targeted panel) can be performed directly on the sample to determine the molecular markers. CACNA1E The presence of c.2767C>T (p.His923Tyr) or other exemplary pathogenic variants within the scope of this invention is investigated in the coding region. If the individual being tested is an isolated case and this variant is detected: if the variant is confirmed by Sanger testing to be real and de novo (neither parent carries the variant), then this variant can serve as strong evidence of pathogenicity; if the individual being tested is a familial case and this variant is isolated from the disease within the family, then it can also serve as evidence of pathogenicity or highly probable pathogenicity. This direct molecular detection method is rapid and convenient, with low dependence on disease familial lineage, and is suitable for first-line clinical screening.

[0083] (2) Risk assessment method based on family / genetic model: When an individual is a member of a bloodline and there are multiple testable relatives, the risk of disease or reproduction is determined by systematic family pedigree collection and molecular testing combined with a genetic logic model. Because CACNA1E The related lesions are mostly autosomal dominant, and many pathogenic variants are de novo. Therefore, it may be necessary to construct the following analytical scenarios to clarify the risk: (a) If the proband (patient) tests positive for c.2767C>T (p.His923Tyr), and neither parent carries the variant (verified by Sanger), it is presumed to be a de novo variant. This variant is highly suggestive of pathogenicity. The risk to the proband's siblings is low, but the risk of inheritance in the proband's offspring is assessed according to the dominant mutation propagation pattern (if the proband is of reproductive age and is heterozygous, the probability of inheritance in each pregnancy of their offspring is approximately 50%). This type of case is suitable as a basis for clinical diagnosis and for use in patient genetic counseling and prenatal monitoring.

[0084] (b) If the variant segregates among the proband and one or more relatives with similar phenotypes (i.e., familial carrier and consistent with the disease phenotype), it can be identified as familial dominant inheritance. The pathogenicity and penetrance of the variant need to be assessed, and risk prediction and prenatal counseling should be conducted in the family.

[0085] (c) If the variant is detected in the proband, but the same variant is also detected in the clinically asymptomatic parents, low penetrance or incomplete dominance, mild phenotype of the parents or phenotypic variability of the variant should be considered. False positives, sequencing errors or chimerism should not be ignored. In this case, it is recommended to conduct more extensive family testing, retrospective clinical phenotype analysis and necessary functional validation to determine the clinical classification of the variant.

[0086] (d) If no known pathogenic loci are detected in the proband, the WES / WGS data are thoroughly screened based on clinical phenotype, family pattern and gene function to identify possible new pathogenic loci, and Sanger validation and functional experiments are performed on candidate variants to determine their pathogenicity; a dynamic reanalysis and follow-up mechanism is established for new highly suspected loci.

[0087] In clinical promotion and application, the limitations of the detection technology should be considered, explaining that a negative test does not completely rule out all pathogenic variants (e.g., deep structural variants, complex structural repetitive regions, or very low proportions of chimeras may not be detected by routine WES). If necessary, supplementary testing can be performed using methods such as increased sequencing depth, targeted capture for enhanced coverage, MLPA, or specialized structural variant detection methods. For variants determined to be pathogenic or potentially pathogenic, it is recommended to provide corresponding clinical recommendations in the report: family follow-up, prenatal / prenatal testing options, referral recommendations for targeted drugs or clinical trials, and, if necessary, functional validation to further clarify the functional impact of the variant.

[0088] Example 5 This example evaluates using multiple prediction tools. CSMD2 The gene mutation sites c.6584G>A (p.Gly2195Glu) and c.9932C>T (p.Ser3311Phe) CACNA1EThe pathogenic potential of the gene c.2767C>T (p.His923Tyr) mutation site, and the RELN gene c.5688T>A (p.Asp1896Glu) and c.2779A>G (p.Met927Val) mutation sites.

[0089] Specific results are as follows Figure 10 As shown: different prediction tools operate on different principles, and multiple tools predict the three gene mutations disclosed in this invention as harmful mutations, indicating that these mutations will affect the function of the corresponding proteins, thereby causing epileptic symptoms.

[0090] 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 CSMD2 , CACNA1E and / or RELN The application of reagents containing gene mutants in the preparation of epilepsy screening products is characterized by, The CSMD2 The mutant gene sequence exhibits c.6584G>A and c.9932C>T mutations compared to the wild-type gene sequence, and its polypeptide form exhibits p.Gly2195Glu and p.Ser3311Phe mutations compared to the wild-type CSMD2 protein; CACNA1E The mutant gene sequence has a c.2767C>T mutation compared to the wild-type gene sequence, and its polypeptide form has a p.His923Tyr mutation compared to the wild-type CACNA1E protein; RELN The mutant gene sequence has c.2779A>G and c.5688T>A mutations compared to the wild-type gene sequence, and its polypeptide form has p.Met927Val and p.Asp1896Glu mutations compared to the wild-type RELN 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 CSMD2 The reagents for gene mutants include primers with sequences as shown in SEQ ID NO.1-4.

4. The application according to claim 2, characterized in that, Detect the CACNA1E The reagents for gene mutants include primers with sequences as shown in SEQ ID NO.5-6.

5. The application according to claim 2, characterized in that, Detect the RELN The reagents for gene mutants include primers with sequences as shown in SEQ ID NO.7-10.

6. An epilepsy screening product, characterized in that, It contains at least the reagents for detecting any of the following mutants: CSMD2 The gene mutant has c.6584G>A and / or c.9932C>T mutations compared to the wild-type gene sequence, and its polypeptide form has p.Gly2195Glu and / or p.Ser3311Phe mutations compared to the wild-type CSMD2 protein; CACNA1E The gene mutant has a c.2767C>T mutation compared to the wild-type gene sequence, and its polypeptide form has a p.His923Tyr mutation compared to the wild-type CACNA1E protein; RELN The gene mutant has c.2779A>G and / or c.5688T>A mutations compared to the wild-type gene sequence, and its polypeptide form has p.Met927Val and / or p.Asp1896Glu mutations compared to the wild-type RELN protein.

7. The epilepsy screening product according to claim 7, characterized in that, It also contains reagents for detecting other epilepsy-causing genes or mutation sites.

8. The epilepsy screening product according to claim 7, characterized in that, Contains the following reagents: as shown in SEQ ID NO.1-4 for the detection of... CSMD2 Primers for detecting gene mutants, as shown in SEQ ID NO. 5-6. CACNA1E Primers for detecting gene mutants, as shown in SEQ ID NO.7-10. RELN Primers for gene mutants.

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