Application of KCNJ10 as target in diagnosis of paroxysmal motor-induced dyskinesia

Abstract

The invention discloses an application of KCNJ10 gene mutation as a detection target in preparation of a kit for diagnosing narcoleptic motor-induced dyskinesia. The KCNJ10 gene mutation in the invention causes the destruction of the Kir4.1 channel function and further causes the narcolepsy motor-induced dyskinesia, so that the KCNJ10 gene mutation can be used as a target for diagnosing the narcolepsy motor-induced dyskinesia.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to the application of KCNJ10 as a target in diagnosing paroxysmal kinesigenic dyskinesia. Background Art

[0002] Paroxysmal Kinesigenic Dyskinesia (PKD), also known as Paroxysmal Kinesigenic Choreoathetosis (PKC), was first reported and named by Japanese scholars in 1967. PKD is characterized by the ability of sudden movements to induce brief involuntary movements in a static state, with manifestations including dystonia, chorea, ballismus, athetosis, etc. It is estimated that the prevalence of PKD is about 1:150,000, making it the most common type of paroxysmal movement disorder. The disease often begins in childhood, with symptoms being most severe in adolescence. Patients may experience sudden twisting spasms or choreic movements when changing postures in any occasion or place, and are difficult to control.

[0003] These symptoms occur frequently, sometimes up to hundreds of times a day, seriously endangering adolescents' social interaction, study and work. Because PKD is relatively rare and has the characteristics of repeated attacks and sudden cessation, it is very easy to be misdiagnosed as epilepsy in clinical practice; coupled with the mixture of various factors such as mental and psychological factors, some patients are also misdiagnosed as mental illnesses. However, PKD is completely different from epilepsy in terms of seizure triggers, seizure forms and EEG characteristics, and the response to different anti-epileptic drugs is also uneven. Only by more in-depth research on the molecular mechanism and pathogenic mechanism of PKD can more and more PKD patients receive timely and effective treatment.

[0004] According to the etiology, PKD can be divided into primary and secondary (caused by stroke, multiple sclerosis or brain injury). Primary PKD is mostly caused by genetic factors, and its inheritance is usually autosomal dominant with incomplete penetrance. In 2011, the PRRT2 gene was identified as the first pathogenic gene causing PKD, accounting for about one-third of the total number of PKD patients. A recent cohort study of 493 Chinese PKD patients showed that PRRT2 mutations accounted for 69.0% of familial cases and 22.6% of sporadic cases. Several other genes associated with paroxysmal movement disorders (such as PNKD and SLC2A1), as well as epilepsy-related genes (such as SCN8A, DEPDC5, KCNA1, ADCY5, CHRNA4 and KCNMA1) have also been partially reported to be associated with PKD. However, all these genes together explain less than 4% of PRRT2-negative cases. Therefore, in approximately 60% of cases, the genetic basis of PKD remains unknown. Summary of the invention

[0005] The present invention aims to provide the use of KCNJ10 as a target in diagnosing paroxysmal kinesigenic dyskinesia.

[0006] The technical solution of the present invention is as follows:

[0007] The invention relates to the use of a KCNJ10 gene mutation as a detection target in the preparation of a kit for diagnosing paroxysmal kinesigenic dyskinesia, wherein the KCNJ10 gene mutation leads to the destruction of Kir4.1 channel function.

[0008] In a preferred embodiment of the present invention, the KCNJ10 gene mutation includes at least one of c.504G>C[p.K168N], c.596G>A[p.R199Q], c.649C>G[p.L217V], c.323G>C[p.C108S], c.263T>C[p.L88P], c.601G>A[p.A201T], c.643G>A[p.G215R] and c.511C>T[p.R171W].

[0009] Further preferably, the KCNJ10 gene mutation is at least one of c.504G>C[p.K168N], c.596G>A[p.R199Q], c.649C>G[p.L217V], c.323G>C[p.C108S], c.263T>C[p.L88P], c.601G>A[p.A201T], c.643G>A[p.G215R] and c.511C>T[p.R171W].

[0010] A kit for diagnosing paroxysmal kinesigenic dyskinesia comprises a reagent capable of detecting whether KCNJ10 has a gene mutation, wherein the KCNJ10 gene mutation leads to the destruction of Kir4.1 channel function.

[0011] In a preferred embodiment of the present invention, the KCNJ10 gene mutation includes at least one of c.504G>C[p.K168N], c.596G>A[p.R199Q], c.649C>G[p.L217V], c.323G>C[p.C108S], c.263T>C[p.L88P], c.601G>A[p.A201T], c.643G>A[p.G215R] and c.511C>T[p.R171W].

[0012] Further preferably, the KCNJ10 gene mutation is at least one of c.504G>C[p.K168N], c.596G>A[p.R199Q], c.649C>G[p.L217V], c.323G>C[p.C108S], c.263T>C[p.L88P], c.601G>A[p.A201T], c.643G>A[p.G215R] and c.511C>T[p.R171W].

[0013] The beneficial effect of the present invention is that the KCNJ10 gene mutation in the present invention leads to the destruction of Kir4.1 channel function, thereby causing paroxysmal kinesigenic dyskinesia. Therefore, the KCNJ10 gene mutation can be used as a target for diagnosing paroxysmal kinesigenic dyskinesia. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 KCNJ10 mutations in 8 unrelated PKD families in Example 1 of the present invention are shown. (A) Black, blue, and orange symbols represent subjects with PKD, benign infantile seizures (BIS), or EAST / SeSAME syndrome, respectively; hollow symbols represent unaffected subjects. The patient number of each case and the KCNJ10 genotype of each subject are shown; WT, wild type; ND, not detected; (B) Typical seizure manifestations exhibited by three cases: facial spasms, abnormal finger postures, and limb twisting; PKD103-1 (left), PKD67-1 (middle), and EAST-2 (right). (C) KCNJ10 mutation localization map; (D) Conservation analysis of changed amino acids in different species; (E) Strip representation of the human WT Kir4.1 channel model based on the rat Kir4.1 channel structure (PDB: 8i5m) 31, showing its homotetrameric structure. The position of the variant is emphasized with a black border in the overall framework and highlighted in red in the enlarged view. For the p.C108S mutation, the disulfide bond between C108 and C140 is indicated by a yellow line. For the p.I129N mutation, a top-down view is shown to illustrate the ion pathway (left panel). For the p.K168N and p.R171W variants, the pip2 binding pocket on the Kir4.1 channel is detailed. Hydrogen bonds are indicated by yellow dashed lines and distances are indicated by black dashed lines (right panel).

[0015] Figure 2It shows that the reduction of potassium ion macrocurrent was detected in HEK293T cells overexpressing mutant Kir4.1 protein in Example 1 of the present invention. (A) Representative trace of potassium ion macrocurrent recorded in the range of -150 to 60mV on HEK293T cells overexpressing wild-type and mutant Kir4.1 proteins. (BC) Current-voltage relationship diagram from -150 to 60mV (with 10mV as step), n represents the number of cells recorded. Compared with the wild type, all mutants (except I209T) were significantly reduced. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; ns, not significant. DETAILED DESCRIPTION

[0016] The technical solution of the present invention is further illustrated and described below through specific implementation modes in combination with the accompanying drawings.

[0017] Example 1

[0018] 1 Materials and methods

[0019] 1.1 Research subjects

[0020] A total of 106 PKD cases with a clinical diagnosis of paroxysmal kinesigenic dyskinesia and negative PRRT2 gene test results from the Department of Neurology of the First Affiliated Hospital of Fujian Medical University from January 2010 to September 2021 were collected. This example has been approved by the ethics committee of the relevant hospital. All participants obtained the consent of themselves or their guardians and signed the relevant informed consent forms.

[0021] Inclusion criteria: 1. All patients met the diagnostic criteria for primary PKD established by Bruno et al. in 2004: induced by sudden movements; short duration of seizures (<1 min); clear consciousness during seizures; age of onset 1 to 20 years old, if there is a family history, the age of onset can be appropriately relaxed; phenytoin sodium or carbamazepine can effectively control the seizures; neurological examinations and neuroelectrophysiological examinations are normal, and other secondary factors such as epilepsy, multiple sclerosis, central nervous system infections, metabolic diseases (such as hypothyroidism or hyperthyroidism), and cardiogenic movement disorders are excluded.

[0022] The clinical data collection and neurological examination of PKD patients were performed by more than two neurologists. In addition, 1,000 local healthy subjects were selected as negative controls in this example.

[0023] 1.2 Collection, storage, and processing of peripheral blood samples

[0024] About 10 ml of peripheral venous blood was collected from the patient in an EDTA-K2 anticoagulant tube. The sample was aliquoted and incorporated into the sample library within 6 hours after collection. After peripheral blood was collected, it was centrifuged at 4°C 4000rpm for 5 minutes, and the upper plasma was aliquoted into 0.5 ml portions and frozen at -80°C. 1 ml of the lower blood sample was taken for DNA extraction. After 1 ml of red blood cells were removed with red blood cell lysis buffer, the white blood cells were treated with Trizol and frozen at -80°C for subsequent RNA extraction. The remaining blood sample was aliquoted into 1.5 ml portions and frozen at -80°C.

[0025] In this example, Tiangen's TGuid S16 fully automatic nucleic acid extraction and purification instrument was used in conjunction with OSR-M102 whole genome extraction reagent for DNA extraction, and the extraction steps were strictly carried out in accordance with the standard instructions. The extracted genomic DNA was preliminarily inspected by agarose gel electrophoresis, and the DNA concentration was determined by NanoDrop 2000 spectrophotometer. The OD260 / OD260 of the standard DNA was between 1.8 and 2.0 to determine whether there was RNA, protein, and organic solvent contamination; Qubit 4.0 was used for accurate quantification to ensure that the DNA content used for subsequent library construction was above 1 μg.

[0026] 1.3 Whole exome sequencing

[0027] Patients with negative PRRT2 first-generation sequencing screening underwent whole-exome gene sequencing. The brief operation process included DNA sample quality inspection, library construction, capture, machine sequencing, quality control, and information analysis. The summary is as follows:

[0028] (1) DNA sample quality inspection: The DNA sample standard for library construction is that the sample concentration is greater than 60ng / μL, the volume is greater than 20μL, and the OD260 / OD280 value is 1.8-2.0. The following three methods can be used for detection: Qubit accurately quantifies DNA concentration; UV spectrophotometer detects OD260 / OD280; agarose gel electrophoresis detects whether the DNA state is degraded and whether there is protein or RNA contamination.

[0029] (2) Library construction: Qualified DNA samples were randomly broken into small fragments of 180-280 bp using an ultrasonic disruptor. The ends of the DNA fragments were made blunt-ended by end repair, a phosphate group was added to the 5' end, and a ploy A tail was added to the 3' end. Adapter sequences were added to both ends of the DNA fragments by TA ligation. After one round of PCR amplification of the DNA fragments containing sequencing adapters, a sequenceable fragment library was obtained.

[0030] (3) Capture: First, the Agilent liquid phase chip capture system Sure Select Human AllExon V6 is used to efficiently enrich the DNA of specific target regions of humans, covering a target size of 58Mb, including exomes and flanking regions, part of the UTR and miRNA regions. The purified and enriched DNA fragments are then mixed with biotinylated RNA Sure Select probes and incubated for 24 hours to allow the DNA fragments to be specifically captured. Then, streptavidin-labeled magnetic beads are added, and the RNA probe-target DNA hybrids are adsorbed. The magnetic beads are eluted, and the RNA Sure Select probes are degraded to capture the target DNA.

[0031] (4) Sequencing: The DNA library was subjected to high-throughput sequencing using the Illumina HiSeq 2500 (Illumina, San Diego, United States) sequencing platform, with an average sequencing depth of 100×.

[0032] (5) Quality Control: The average base accuracy is greater than 99% at Q20, the base accuracy is greater than 85% at Q30, and the average clean data accounts for more than 90% of the raw data. About 90% of the exon regions have a coverage of more than 10×, ensuring the highest sequencing uniformity.

[0033] (6) Information analysis: The raw data obtained by WES sequencing is filtered, aligned, identical reads are removed, variants are identified, variants are filtered, variants are annotated, etc., for subsequent analysis.

[0034] 1.4 Raw data processing of whole exome sequencing

[0035] (1) Data quality control and preprocessing: FastQC software and MμLtiQC software were used to control the quality of preprocessed data. Based on the distribution characteristics of low-quality scores in sequencing data concentrated at the ends, Fastp software was used to dynamically remove adapter sequence fragments and low-quality fragments from the 3' end of the raw data.

[0036] (2) Sequence alignment: The effective sequencing data were aligned to the reference genome (GRCh37 / hg19) using BWA to obtain the initial alignment results in BAM format. Then, SAMtools was used to sort the alignment results; and Picard software was used to mark duplicate reads.

[0037] (3) Identification of SNPs and INDELs: Based on the initial alignment result BAM file, SAMtoolsmpileup and bcftools were used to identify SNP and INDEL sites.

[0038] (4) Variant annotation and statistics: ANNOVAR was used to annotate variant sites at the gene-based, region-based, and filter-based levels. This included annotation information from the dbSNP database, the 1000 Genomes Project, and other existing databases. The annotation content included seven parts: priority information, gene and region annotation, database (frequency) annotation, conservative (harmful) prediction, variant site information, gene function and pathway annotation, and tissue-specific expression of genes.

[0039] (5) Definition of rare malignant sites:

[0040] MetaSVM and CADD algorithms were used to predict the impact of missense variants. Malignant missense mutations (misD) were defined as those predicted to be harmful by MetaSVM or with a CADD score of ≥ 20. In this example, only misD and loss-of-function (LoF) variants (start-losses, stop-gains, stop-losses, canonical splice-sites, and frameshift indels) were considered potentially harmful.

[0041] Rare and high-quality heterozygous variants were filtered using the following criteria:

[0042] 1) MAF of all samples in EXAC and gnomAD databases ≤ 2×10 -5 ;

[0043] 2) Calibration of variant quality scores through GATK;

[0044] 3) genotype quality score ≥20;

[0045] 4) Minimum site coverage depth ≥10;

[0046] 5) Alternative allele ratio ≥ 20%. All variants were manually verified using IGV (Integrative Genomics Viewer) to exclude false positives. Sanger sequencing was performed for candidate variants of interest.

[0047] 1.5 Case-control analysis of rare malignant mutations at the genetic level

[0048] The two-tailed Fisher's exact test was used to compare whether the number of rare deleterious variants observed in the PKD group for both genes was significantly different from the number carried by East Asians in gnomAD, regardless of inheritance mode. The control group in gnomAD was screened for variants using the same criteria. The total allele number was taken as the median number of alleles reported at all positions in the coding region across the gene.

[0049] 1.6 Conservation analysis of different species

[0050] Clustal Omega is a multiple sequence alignment tool developed by the European Bioinformatics Institute (EMBL-EBI), which can perform multiple sequence alignments on DNA or proteins. Its URL is https: / / www.ebi.ac.uk / Tools / msa / clustalo / . First, download the KCNJ10 protein sequence of humans and other vertebrates from the official website of the National Center for Biotechnology Information (NCBI). Its URL is https: / / www.ncbi.nlm.nih.gov / gene / 4758 / ortholog / ?scope=7742. Then import the sequence into the multiple sequence alignment page of Clustal Omega for conservation analysis.

[0051] 1.7 Protein structure analysis

[0052] Rat Kir4.1 channel in complex with PIP2 (PDB: 8i5m) was downloaded from the Protein Data Bank (https: / / www.rcsb.org / ). The structures of human WT and mutant Kir4.1 channels were modeled using alphafold2 and Swiss-Model (https: / / swissmodel.expasy.org).21), and the sequence identity of human Kir4.1 channels with the template was 98%. The effects of variants on the interaction between PIP2 and the channel were predicted using PyMOL (https: / / pymol.org / ), and hydrogen bonds and disulfide bonds were analyzed and visualized using UCSF Chimera X (https: / / www.cgl.ucsf.edu / chimerax).

[0053] 1.8 Plasmids and mutagenesis

[0054] Human KCNJ10 cDNA was obtained by gene synthesis with a C-terminal HA tag and inserted into the pCDH-CMV-MCS-EF1-copGFP vector (System Biosciences). The mutations C.263T>C, C.323G>C, C.386T>A, C.504G>C, C.511G>T, C.557T>C, C.596G>A, C.601G>A, C.626T>C, C.643G>A, and C.649C>G were introduced into the expression construct using the Fast Mutagenesis Kit (Vazyme).

[0055] 1.9 HEK293T cell transfection and preparation

[0056] HEK293T cells were cultured in a carbon dioxide cell culture incubator at 37°C, 5% CO2. The culture medium consisted of DMEM containing 10% FBS. The cells were passaged every 2-3 days and the medium was changed every other day.

[0057] The day before transfection, plate the cells onto a 3.5cm dish, and transfect them when the cells grow to 90% confluence after 24 hours. PEI is used as the transfection reagent. During transfection, 125μl Opti-MEM is used to mix 3μg of plasmid and 9μl of PEI (1μg / μL), respectively. Then, the two tubes of liquid are mixed and allowed to stand at room temperature for 20 minutes. The mixture is then evenly added to the culture dish, and the cell culture medium is replaced after 6-8 hours. Electrophysiological recordings and protein immunoblotting can be performed after 16 hours. Before recording, the cells need to be digested with 0.125% trypsin and then re-plated on a round glass slide (the round glass slide is placed in a 24-well plate, coated with 100ug / ml poly-lysine overnight in advance, and washed 3 times with double distilled water, and then dried in a clean bench for later use). 1x 10 5 After 30 minutes, when the cells are firmly attached to the round glass slide, the glass slide can be moved to the electrophysiological recording table for electrophysiological recording.

[0058] 1.10 Electrophysiological recordings from HEK293T cells

[0059] In this embodiment, borosilicate fiberglass tubes produced by Sutter Instrument are used to make glass microelectrodes (containing filament, 10 cm long, 1.5 mm outer diameter, and 0.86 mm inner diameter), and the P-1000 horizontal electrode puller produced by the company is used for step-by-step pulling. After the electrode liquid is poured into the electrode, the water resistance of the electrode is about 3-5 MΩ. The amplifier used in this embodiment is Axopatch 200B, and the digital-to-analog converter is Axon Digidata 1440A. The data acquisition software is Clampex, the noise is controlled within 20pA, the sampling frequency is 10kHz, and the data analysis uses Clampfit 10.7 and Origin2018.

[0060] Before recording, cells were digested with 0.125% trypsin and then re-plated on a round glass slide. The round glass slide was placed in a 24-well plate and 1x 10 5 cells. After 30 minutes, the cells are firmly attached to the round glass slide and the glass slide can be moved to the electrophysiological recording station for electrophysiological recording. Place the glass slide in a culture dish containing extracellular solution on the electrophysiological recording station. Extracellular solution: 135mM NaCl, 5mM KCl, 2mM CaCl2, 1mM MgCl2, 5mM Glucose, 10mM HEPES, pH 7.4 (NaOH). Electrode solution: 135mM KCl, 2mM MgCl2, 2mM NaATP, 10mM HEPES, pH 7.3 (KOH). Find the target cell to be recorded under a low-power microscope. In this part of the experiment, cells with GFP fluorescence positive are selected and the area is moved to the center of the field of view. Fill the glass electrode with electrode solution and flick the microelectrode to remove the bubbles at its tip. Then install the glass electrode on the microelectrode holder, apply positive pressure and then enter the solution, check whether the electrode resistance is appropriate, and compensate for the offset potential. Adjust the position of the glass electrode so that its tip moves to the center of the field of view. Adjust the micromanipulator to bring the tip of the microelectrode close to the target cell, and make the tip of the microelectrode touch and gently press the cell until the resistance Rt increases by 0.2MΩ. Release the positive pressure in the microelectrode, and the amplitude of the current square wave will be significantly reduced. Switch the stage from Bath to Patch mode, and adjust the amplifier Holding voltage to -80mV, and then gently apply negative pressure to make the sealing resistance reach GΩ. At this time, the square wave current shrinks to the baseline. After compensating for the fast capacitance, switch the stage to Cell mode, and then apply negative pressure to the microelectrode to break the cell membrane. At this time, the appearance of the whole-cell capacitance current with a larger time constant and the increase of the square wave current can be seen. Check the membrane resistance and access resistance values, and record after the cell membrane potential is stable.

[0061] The ion channel current-voltage relationship curve is an important parameter for reflecting the kinetic properties of ion channels, which can reflect the activation process, threshold potential, reversal potential and rectification characteristics of the channel. In order to obtain the IV curve, a continuously changing step pulse current is required. During electrophysiological recording, the voltage is first kept at the cell resting membrane potential, i.e. -80mV, and then the cell is stimulated with a voltage of -150mV to +60mV in steps of 10mV for 800ms, and finally maintained at the resting membrane potential to record the whole-cell macrocurrent.

[0062] 1.11 Statistical analysis

[0063] Statistical analysis was performed using GraphPad Prism software. The D'Agostino and Shapiro-Wilk normality tests were used to assess whether the data conformed to a Gaussian distribution. If the data passed the Gaussian distribution test, a parametric unpaired two-tailed t-test was used for two groups of data, and a one-way analysis of variance (ANOVA) was performed, followed by Tukey's multiple comparison test for three or more groups. Otherwise, the nonparametric unpaired Mann-Whitney test was used to compare the two groups. Data are presented as mean ± SEM. The following conventions were used: *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; ns, not significant.

[0064] 2 Results

[0065] 2.1 Identification of KCNJ10 mutations in PKD patients

[0066] This example first analyzed three families (PKD-13, PKD-118, and PKD-148) with multiple family members diagnosed with PKD. Based on the assumed autosomal dominant inheritance pattern in these families, this example prioritized rare harmful heterozygous variants, which are shared among all affected individuals but not present in their unaffected family members. This strategy identified 34 candidate genes in PKD-13, 31 candidate genes in PKD-118, and 23 candidate genes in PKD-148 (see Table 1), of which KCNJ10 (NM_002241) is the only gene with intersection in these three families, with three missense variants (c.504G>C[p.K168N], c.596G>A[p.R199Q], c.649C>G[p.L217V]).

[0067] Table 1. Candidate genes identified in PKD families using whole exome sequencing

[0068]

[0069]

[0070]

[0071] Subsequently, this example confirmed the loci of five other probands with rare malignant mutations of KCNJ10, PKD-46 (c.323G>C [p.C108S]), PKD-67 (c.263T>C [p.L88P]), PKD-81 (c.601G>A [p.A201T]), PKD-103 (c.643G>A [p.G215R]) and PKD-123 (c.511C>T [p.R171W]) by Sanger sequencing, and performed family co-segregation analysis in family members with DNA samples. In total, this example identified 11 individuals with PKD phenotype in 8 families ( Figure 1 A). None of the 11 affected individuals carried pathogenic or likely pathogenic variants in other genes previously implicated as PKD pathogens, including rare variants (MAF < 0.001) in TMEM151A, PNKD, SLC2A1, SCN8A, KCNMA1, ADCY5, KCNA1, DEPDC5, or CHRNA4.

[0072] To assess the frequency of rare deleterious heterozygous KCNJ10 variants in the general population, this example first screened whole exome sequencing data of 1000 internal controls. No predicted deleterious mutations were detected in these individuals. Then, this example compared the number of rare deleterious heterozygous KCNJ10 variants screened in all PKD probands with the East Asian control group in gnomAD v2.1.1. In PKD cases, rare deleterious KCNJ10 variants were significantly enriched compared with the matched gnomAD East Asian dataset (8 / 336 vs. 17 / 18394, 2-tailed Fisher P = 9.89 × 10 -9 , see Table 2).

[0073] Table 2. Rare damaging heterozygous KCNJ10 variants found in gnomAD v2.1.1 controls

[0074]

[0075]

[0076]

[0077] Abbreviations: gnomAD=the Genome Aggregation Database; CADD=The CombinedAnnotation Dependent Depletion.

[0078] Biallelic KCNJ10 pathogenic variants are associated with EAST / SeSAME syndrome (omim#612780), manifested by seizures, ataxia, sensorineural deafness, mental retardation, and electrolyte imbalance. Of note, this example retrospectively studied another Chinese EAST / SeSAME family. The proband, EAST-1, was a 6-year-old boy with compound heterozygous mutations in KCNJ10. Generalized seizures began at 3 months of age, auditory brainstem response testing showed bilateral hearing loss, and physical examination showed severe ataxia and mental retardation. His father, who carried the p.I129N mutation, was a typical PKD patient who had experienced dystonia attacks triggered by sudden intentional movements. His mother, who carried the p.V186A mutation, was asymptomatic. Some asymptomatic parents (the father of PKD-103 and the mother of PKD-123) were also found to be mutation carriers, suggesting that, like other PKD genes (e.g., PRRT2, TMEM151A), KCNJ10 may cause a PKD phenotype with variable penetrance.

[0079] The KCNJ10 gene encodes the inward rectifier potassium channel Kir4.1, which is expressed in the central nervous system, inner ear, and kidney

[20] In the brain, Kir4.1 is specifically expressed in glial cells and has been shown to play multiple functions, including extracellular potassium homeostasis, maintenance of astrocyte resting membrane potential, cell volume regulation, and promotion of glutamate uptake.

[24] Kir4.1 consists of an intracellular N-terminus and C-terminus, two transmembrane α-helices, and an extracellular loop containing the characteristic potassium ion selective sequence "TIGYG". The activity of Kir4.1 is regulated by cell membrane phosphatidylinositol 4,5-bisphosphate (PIP2) and protons. The variants detected in PKD patients in this example are located in the extracellular, intracellular and transmembrane regions of the protein ( Figure 1 C). All variants in PKD patients affect highly conserved amino acid residues ( Figure 1 D). Model comparison shows that in the WT channel, cysteine ​​108 forms a disulfide bond with cysteine ​​140, whereas in the p.C108S mutant channel, cysteine ​​108 is disrupted ( Figure 1E). This disulfide bond is conserved in the Kiel channel to ensure proper folding and function. The variant p.I129N is located in a critical K+ selectivity filter motif. The mutation from the hydrophobic residue isoleucine to the hydrophilic residue asparagine affects potassium permeability. Lysine 168 and arginine 171 are involved in the formation of a positively charged pocket to which PIP2 binds. These mutations (p.K168N and p.R171W) alter the electrical properties of the residues and disrupt hydrogen bonding with PIP2 ( Figure 1 E).

[0080] 2.2 Clinical characteristics of PKD patients with KCNJ10 mutations

[0081] A detailed summary of the clinical presentation of each patient is shown in Table 3. The mean age of onset was 13.75 ± 3.44 years (range, 8–19 years). Sudden movement from a stationary position was a common trigger for all 12 patients examined. Dystonia was the most common form of seizure, manifested by facial spasms, abnormal finger postures, and limb distortions ( Figure 1 B). The duration of the attacks was usually short, lasting no longer than 10 seconds in most patients (10 / 12). The frequency of the attacks was extremely erratic, ranging from more than 50 attacks per day to once every 2-3 days. Seven patients experienced spontaneous remission after the age of 20 years. A total of nine patients underwent electroencephalogram (EEG) and brain magnetic resonance imaging (MRI): no abnormalities were detected. Six patients were treated with carbamazepine or oxcarbazepine, with good responses: four patients had complete remission (seizure freedom, with or without aura), and two patients had partial remission (seizure frequency decreased but still present). Of note, PKD 13-1 had been treated with daily levodopa 100 mg and sodium valproate 25 mg; however, the patient did not respond to these medications.

[0082] Table 3 Clinical characteristics of PKD patients with KCNJ10 mutations

[0083]

[0084]

[0085]

[0086] Abbreviations: SM = sudden movement, S = stress, SC = change in speed, D = dystonia, NA = not available, L-dopa = levodopa, BSZ = benserazide, CBZ = carbamazepine, OXC = oxcarbazepine.

[0087] 2.3 Electrophysiological detection of HEK293T cells overexpressing wild-type and mutant Kir4.1 proteins

[0088] The KCNJ10 gene encodes the inward rectifier potassium channel Kir4.1. Potassium channels are ion channels that selectively allow potassium ions to pass through. Rectification is a term in physics that refers to the tendency of electric current to flow in one direction but not in the opposite direction. Inward rectification means that cations flow easily from the extracellular space into the intracellular space, but not easily from the intracellular space out of the extracellular space. The main mechanism may be that intracellular magnesium ions or polyamine ions enter the channel pores, block the ion channels, and thus prevent ion penetration. Bi-allelic loss of function of KCNJ10 has been reported to cause a multi-system disease called EAST / SeSAME syndrome, which is characterized by epileptic seizures, sensorineural hearing loss, ataxia, mental retardation, and electrolyte imbalance (OMIM 612780). Do the eight missense mutations that cause the phenotype of PKD patients also cause loss of function of KCNJ10? In order to determine whether the rare KCNJ10 variants identified in this example in PKD patients would disrupt Kir4.1 channel function, this example overexpressed wild-type and mutant Kir4.1 in HEK293T cells and evaluated channel function by whole-cell patch clamp recording of potassium ion macrocurrents. It can be seen that compared with wild-type controls, all 9 patient-derived variants showed a significant reduction in potassium ion currents, while one I209T variant from an asymptomatic mother did not show a reduction ( Figure 2 A). Based on the degree of current reduction, this example further divides these 9 variants into 3 groups: complete Kir4.1 dysfunction (the current is negligible, similar to that of mock-transfected cells, including L88P, I129N, K168N, V186A, R199Q, A201T, G215R, and L217V), partial Kir4.1 dysfunction (the detectable current is reduced by about 90%, including C108S and R171W), and normal Kir4.1 function (the current is indistinguishable from that of wild-type controls, including I209T) ( Figure 2 AC). Therefore, all 10 patient-derived variants resulted in disruption of Kir4.1 channel function, indicating that the functional effect of the mutations was loss-of-function. Since no functional effect was found for the I209T variant, the site of alteration was considered benign.

[0089] The above description is only a preferred embodiment of the present invention, and therefore cannot be used to limit the scope of the present invention. That is, equivalent changes and modifications made according to the patent scope of the present invention and the contents of the specification should still fall within the scope of the present invention.

Claims

1. Use of KCNJ10 gene mutation as a detection target in the preparation of a kit for diagnosing paroxysmal kinesigenic dyskinesia, characterized in that: The KCNJ10 gene mutation leads to disruption of Kir4.1 channel function.

2. The use according to claim 1, characterized in that: The KCNJ10 gene mutation includes c.504G>C [p.K168N], c.596G>A[p.R199Q], c.649C>G[p.L217V], c.323G>C[p.C108S], c.263T>C[p.L88P], c.601G>A[p.A201T], c.643G>A[p.G215R] and c.511C>T[p.R171W].

3. The use according to claim 2, characterized in that: The KCNJ10 gene mutation is at least one of c.504G>C[p.K168N], c.596G>A[p.R199Q], c.649C>G[p.L217V], c.323G>C[p.C108S], c.263T>C[p.L88P], c.601G>A[p.A201T], c.643G>A[p.G215R] and c.511C>T[p.R171W].

4. A kit for diagnosing paroxysmal kinesigenic dyskinesia, characterized in that: The invention includes a reagent capable of detecting whether a KCNJ10 gene mutation occurs, wherein the KCNJ10 gene mutation results in a disruption of the Kir4.1 channel function.

5. The kit according to claim 1, wherein: The KCNJ10 gene mutation includes c.504G>C [p.K168N], c.596G>A[p.R199Q], c.649C>G[p.L217V], c.323G>C[p.C108S], c.263T>C[p.L88P], c.601G>A[p.A201T], c.643G>A[p.G215R] and c.511C>T[p.R171W].

6. The kit according to claim 5, characterized in that: The KCNJ10 gene mutation is at least one of c.504G>C[p.K168N], c.596G>A[p.R199Q], c.649C>G[p.L217V], c.323G>C[p.C108S], c.263T>C[p.L88P], c.601G>A[p.A201T], c.643G>A[p.G215R] and c.511C>T[p.R171W].

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