WO2014199944A1 - Méthode pour détecter une épilepsie résistante aux médicaments accompagnant une déficience intellectuelle sévère et un retard du développement moteur - Google Patents

Méthode pour détecter une épilepsie résistante aux médicaments accompagnant une déficience intellectuelle sévère et un retard du développement moteur Download PDF

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WO2014199944A1
WO2014199944A1 PCT/JP2014/065217 JP2014065217W WO2014199944A1 WO 2014199944 A1 WO2014199944 A1 WO 2014199944A1 JP 2014065217 W JP2014065217 W JP 2014065217W WO 2014199944 A1 WO2014199944 A1 WO 2014199944A1
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mutation
gnao1
gene
epilepsy
mutations
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直通 松本
浩智 才津
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公立大学法人横浜市立大学
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    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

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  • the present invention relates to a method for detecting refractory epilepsy accompanied by severe intellectual disability and motor developmental delay.
  • Epileptic encephalopathy is a neurological disease characterized by severe progressive cognitive dysfunction and behavioral disorder that tends to be caused or exacerbated by epileptic activity (Non-patent Document 1).
  • Otawara Syndrome (OS, MIM350308350 and 612164) is the most severe and earliest epileptic encephalopathy, mainly tonic convulsions found in the neonatal period, refractory seizures, EEG recording (EEG) suppression It is characterized by a burst pattern (Non-Patent Document 2).
  • an object of the present invention is to provide a novel means that enables a definitive diagnosis of intractable epilepsy accompanied by severe intellectual disability and motor developmental delay.
  • the inventors of the present invention screened de novo mutations in all exome analyzes of parents-derived samples for 5 cases out of 12 cases of Otawara syndrome that had been subjected to all exome analysis in the past. As a result, the de novo missense mutation in GNAO1 gene I found. Furthermore, as a result of conducting GNAO1 gene mutation screening by HRM analysis or WES in 367 cases of epileptic encephalopathy including Otawara syndrome, we found further GNAO1 gene de novo mutation. Epilepsy cases with GNAO1 gene mutations have the common characteristics of being refractory with severe intellectual disability and motor developmental delay, and involuntary movements, which are rare symptoms in normal epileptic encephalopathy It was confirmed that this was sometimes the case. Based on the above results, it was found that it was possible to detect refractory epilepsy with severe intellectual disability and motor development delay using GNAO1 gene mutation as an index, and to predict the involuntary movement merger, and completed the present invention. .
  • the present invention uses a sample separated from a living body to detect whether or not the target living body has a mutation in the GNAO1 gene, and detects refractory epilepsy accompanied by severe intellectual disability and motor developmental delay.
  • a method is provided wherein the refractory epilepsy is detected when a deleterious mutation is detected in at least one allele of the GNAO1 gene.
  • the present invention also provides a method for screening a therapeutic agent for refractory epilepsy with severe intellectual disability and motor developmental delay, comprising selecting a compound using as an index the activation of signal transduction by trimeric G protein. To do.
  • the present invention relates to refractory epilepsy with severe intellectual disability and motor development delay, comprising selecting a compound using the activation of signal transduction by a trimeric G protein as an index, and producing the selected compound.
  • a method for producing the therapeutic agent is provided.
  • the present invention is a possibility that the target living body suffers from epilepsy causing involuntary movement, including examining whether the target living body has a mutation in the GNAO1 gene using a sample separated from the living body. And a method for predicting that it is highly probable that an epilepsy causing involuntary movement is detected when a deleterious mutation is detected in at least one allele of the GNAO1 gene.
  • the present invention enables a definitive diagnosis of intractable epilepsy with severe intellectual disability and motor developmental delay.
  • Methods such as detection of heteroduplex detection and sequence analysis to detect abnormalities in the base sequence of genes and the presence / absence of chromosomal abnormalities (such as microdeletions) using DNA microarrays are well known. By analyzing this, more epilepsy cases can be diagnosed. For example, when the first symptom is confirmed, by carrying out the method of the present invention, it is possible to investigate whether or not the patient has refractory epilepsy with severe intellectual disability and motor developmental delay. Can contribute to life guidance.
  • an antiepileptic drug having a calcium channel inhibitory action may be effective for epilepsy patients whose pathogenesis is caused by a mutation in the GNAO1 gene
  • the present invention can also be used to select an antiepileptic drug prescribed to an epileptic patient.
  • GNAO1 gene mutation impairs trimeric G protein signal transduction, it is also possible to develop new epilepsy treatment drugs by drug discovery targeting trimeric G protein signal transduction. become.
  • a and B are EEGs in case 3 between seizures.
  • a suppression burst pattern was observed at 2 months of age (A), and transition to hypus allemia was observed at 4 months of age (B).
  • C is an EEG at the time of intermittent seizure in Case 2, and a suppression burst pattern was observed at 2 months of age.
  • D is the EEG at the time of intermittent seizures in case 4, and a scattered spike or sharp wave complex was observed at the age of 5 years.
  • T2-weighted axial images passing through the basal ganglia are shown in E, H, I, T1-weighted axial images are shown in F, and sagittal images are shown in G.
  • Case 1 showed cerebral atrophy at age 5 and 6 months (E).
  • Case 2 showed delayed myelination and hypoplasia of the corpus callosum at 10 months of age (F, G).
  • case 3 the findings at 3 months of age were normal (H).
  • Case 4 showed a decrease in white matter at age 7 (I). It is the result of expressing the wild type (WT) and five mutants of G ⁇ o1 protein in N2A cells and observing the intracellular localization.
  • the left is a GDP-binding inactive G ⁇ i ⁇ heterodimer (PDB code 1GG2)
  • the middle is a nucleotide-free G ⁇ s ⁇ complex (PDB code) complexed with an agonist-linked monomer ⁇ 2 adrenergic receptor ( ⁇ 2AR) 3SN6)
  • PDB code 3OHM transition state analog of GTP (GDP + AlF 4 ⁇ ) -linked G ⁇ q complexed with phospholipase C- ⁇ (PLC ⁇ ), an effector.
  • PDC ⁇ phospholipase C- ⁇
  • the molecular structure is shown in a space-filling model diagram drawn using PyMOL (www.pymol.org).
  • the amino acid number is the number in human G ⁇ o1 , in parentheses, the left figure is rat G ⁇ i 1 (UniProtKB / Swiss-Prot P10824), the middle figure is bovine G ⁇ s (UniProtKB / Swiss-Prot P04896), the right figure is mouse G ⁇ q ( UniProtKB / Swiss-Prot P21279).
  • the illustrations shown at the top of each model diagram show the orientation of each subunit and the molecule bound.
  • (B) It is a graph which shows the free energy change after amino acid substitution estimated from the calculation result using FoldX software.
  • “Control” is a current trace before addition of 10 ⁇ M norepinephrine
  • “norepinephrine” is a current trace 3 minutes after addition.
  • (B) is a diagram showing a current density of calcium current before norepinephrine processes in WT G.alpha o1 expressing cells and variants G.alpha o1 expressing cells. A scatter plot represents the current density in individual cells.
  • Each bar represents the mean% decrease in current density and SEM induced by the addition of 10 ⁇ M norepinephrine.
  • the disease targeted by the present invention is refractory epilepsy with severe intellectual disability and motor developmental delay that develops in childhood.
  • the inventors of the present application have identified the GNAO1 gene (MIM 139311) as a responsible gene for the intractable epilepsy. It has also been confirmed that there are cases of involuntary movement among epilepsy cases having a GNAO1 mutation (see Examples below). In epilepsy encephalopathy, involuntary movement is rare, and involuntary movement may occur. This is one of the characteristics of epilepsy cases with GNAO1 gene mutation.
  • the GNAO1 gene is a gene encoding a G ⁇ subunit, which is one of the subunits constituting the guanine nucleotide binding protein (G protein).
  • G protein is a heterotrimer composed of ⁇ , ⁇ , and ⁇ subunits, and forms a G ⁇ complex with G ⁇ bound to GDP in the basic state (inactive form).
  • G protein-coupled receptor with 7 transmembrane domains (G protein-coupled receptor; ⁇ GPCR, also called“ 7-transmembrane receptor ”due to its structure) binds to the ligand
  • ⁇ GPCR also called“ 7-transmembrane receptor ”due to its structure
  • G ⁇ subunits In mammals, G ⁇ subunits are classified into four groups: G ⁇ i / o , G ⁇ q / 11 , G ⁇ s and G ⁇ 12/13 .
  • G.alpha subunits GNAO1 gene encodes is G.alpha o.
  • G ⁇ o is abundant in brain tissue and accounts for approximately 0.5% of membrane proteins (Huff, RM, et al. (1985). J Biol Chem 260, 10864-10871.) And is important for brain function It is suggested that it plays a role.
  • Two transcription variants are known for the GNAO1 gene.
  • Variant 1 (GenBank accession number NM — 020988) is composed of 8 exons
  • variant 2 (GenBank accession number NM — 138736) is composed of 9 exons
  • exons 1 to 6 are the same in both variants.
  • Sequence numbers 1 and 2 in the sequence listing are the base sequence of the cDNA coding region of variant 1 (NM_020988) and the amino acid sequence encoded thereby
  • SEQ ID NOs 3 and 4 are the base sequence of the cDNA coding region of variant 2 (NM_138736). And the amino acid sequence encoded thereby.
  • SEQ ID NOs: 5 to 15 the sequences of each exon and the nearby intron are shown in Table 1.
  • GNAO1 gene it is examined whether or not the target living body has a mutation in the GNAO1 gene using a sample separated from the living body. Mutations of GNAO1 gene, change in only a small number of amino acid residues of G.alpha o subunit proteins GNAO1 gene encodes (substitutions, deletions, insertions) other, deleting at least a partial area of G.alpha o subunit protein Changes in the gene sequence that result in mutations that are lost are included, and mutations that delete all or part of the GNAO1 gene region are also included.
  • missense mutation For example, missense mutation, nonsense mutation, frameshift mutation, in-frame deletion or insertion mutation (deletion or insertion of one or more amino acids) due to base substitution, deletion, insertion, duplication, etc. in the exon or intron region .
  • Mutations that cause splicing abnormalities, or microdeletions of the chromosomal region containing the GNAO1 gene are not limited thereto.
  • the genomic sequence of GNAO1 genes in the sequence listing is a typical example of a normal GNAO1 sequences, the presence or absence of GNAO1 gene mutation Is based on the sequence of the GNAO1 gene shown in the sequence listing, and can be determined by comparison with this reference sequence.
  • GNAO1 GNAO1
  • GNAO1 mutation it is also possible to determine whether or not the GNAO1 mutation actually detected is a harmful mutation using such a known prediction tool.
  • SIFT if the score is less than 0.05, the substitution is predicted to be intolerant (affects changes in protein function).
  • PolyPhen is predicted to be pathogenic if the score exceeds 2.0.
  • PolyPhen-2 scores between 0.000 (most likely benign) to 0.999 (most likely harmful), and if the decision based on the score is possibly or probably damaging, Sex mutations are strongly suggested.
  • Align GVGD class scores are evaluated in the range of Class C0 (small possibility) to Class C65 (large possibility), and mutations with a class score of C55 or higher suggest pathogenic mutations.
  • the mutations shown in Table 2 below are pathogenic mutations of refractory epilepsy associated with severe intellectual disability and motor developmental delay, which were identified in the Examples by analysis targeting epileptic encephalopathy patients including Otawara syndrome. All of these mutations occur in evolutionarily highly conserved amino acid residues (Fig. 1), are not found in the healthy population, and are pathogenic mutations as assessed by the prediction tool described above. Is a strongly suggested mutation. However, these four types of mutations are examples of GNAO1 gene mutations that serve as indices in the present invention, and the scope of the present invention is not limited to these specific examples.
  • GNAO1 gene mutation can be detected by analyzing the nucleotide sequence using a nucleic acid sample such as genomic DNA or RNA.
  • a nucleic acid sample such as genomic DNA or RNA.
  • genomic DNA can be easily prepared from peripheral blood, oral mucosa swabs, and the like by conventional methods.
  • various prenatal genetic testing methods are known, and it is possible to examine whether a fetus has a GNAO1 gene mutation.
  • a maternal blood sample containing fetal cells corresponds to “a sample separated from a living body”, and a fetus corresponds to a “target living body”.
  • the amino acid sequence of a protein can be affected not only by the exon region but also by mutations in the intron region.
  • the exon and its vicinity usually include several tens to several hundred bases, for example, an intron region of about 30 to 50 bases. It is common to inspect.
  • sequence analysis may be performed on exons and introns in the vicinity thereof.
  • detecting mutations by analyzing the genome sequence refer to SEQ ID NOS: 5 to 14 in the sequence listing of the present application and the genome sequence of the GNAO1 gene available from publicly known databases, design primers appropriately, and use a genomic DNA sample. Therefore, sequencing may be performed by a conventional method.
  • the mutation is homo or hetero can be confirmed from the waveform data of the sequence. If there is a heterozygous mutation, two types of signals will overlap at the same site.
  • GNAO1 gene mutations of interest in the present invention are mainly hetero mutations, it is effective to screen for GNAO1 gene mutations by detecting heteroduplexes.
  • heterozygous mutation exists, recombining the genomic DNA sample after heat denaturation results in heteroduplex in which normal DNA and mutated DNA are hybridized.
  • Heteroduplexes (1) show different mobility in non-denaturing polyacrylamide gels, (2) mismatched bases are susceptible to cleavage by chemicals and enzymes, (3) different denaturation temperatures during denaturation It has the characteristic that shows.
  • Methods for detecting heteroduplexes using these characteristics are known in this field, and have been put into practical use as mutation testing methods. Specifically, for example, a method for detecting heteroduplexes using denaturing high performance liquid chromatography (dHPLC) and a High-Resolution-Melt method are known.
  • the High-Resolution-Melt method is a method for melting double-stranded DNA using fluorescent dyes (SYTO (registered trademark) 9, LC Green (registered trademark), EvaGreen (registered trademark), etc.) that bind to double-stranded DNA at high density.
  • This is a method of detecting heteroduplexes by treating the process of (thermal denaturation) as a change in fluorescence intensity. That is, when a double-stranded DNA is stained with a fluorescent dye that binds to the double-stranded DNA at a high density, when the double-stranded DNA is melted (thermally denatured), the fluorescent dye is drawn from the site where the double strand is dissociated.
  • the primer to be used can be appropriately designed based on the sequence of the exon + neighboring intron region of the GNAO1 gene described in the Sequence Listing of the present application. In the following examples, examples of primers and reaction conditions that can be used for screening for GNAO1 gene mutation by High Resolution Melt method are shown.
  • all the base sequences of the exon of the GNAO1 gene and the nearby intron region may be determined, and the presence or absence of mutation may be examined.
  • the inspection can be carried out more efficiently.
  • the detection method of the present invention can also be applied to predicting whether or not there is a possibility of having epilepsy with involuntary movement.
  • the GNAO1 gene in the target organism By examining the GNAO1 gene in the target organism and detecting a deleterious mutation in at least one allele, it can be predicted that the target organism is likely to suffer from epilepsy that causes involuntary movements. For example, even if there is no involuntary movement at the time when the first symptom is confirmed, if there is a harmful mutation in the GNAO1 gene, it can be predicted that the involuntary movement may be combined later.
  • an antiepileptic drug having an action of suppressing a calcium channel may be effective for an epilepsy case having a GNAO1 mutation.
  • Known antiepileptic drugs with calcium channel inhibitory action include pregabalin and gabapentin that act as selective calcium channel blockers, and topiramate with the action of regulating high voltage activated calcium channels in dentate granule cells .
  • Such an antiepileptic drug can be actively prescribed for epilepsy patients having a GNAO1 gene mutation as a pathogenic mutation.
  • the present invention can also be used to select antiepileptic drugs prescribed for epileptic patients.
  • the GNAO1 gene mutation impairs trimeric G protein signaling
  • compounds that activate trimeric G protein signaling are refractory with severe intellectual disability and motor developmental delay. It may be useful as an epilepsy treatment or symptom relief agent. That is, it is possible to screen for novel antiepileptic drugs using the activation of trimeric G protein signaling as an index.
  • the treatment of epilepsy includes alleviation of symptoms of epilepsy.
  • the compound obtained by screening can be produced by a known method in the chemical field.
  • genomic DNA sample was extracted from peripheral blood leukocytes by a conventional method. In order to detect the mosaic mutation in Case 2, genomic DNA was extracted from saliva using Oragene DNA kit (DNA Genotek) and from nails using ISOHAIR kit (Nippon Gene).
  • Genomic DNA was amplified using illustra GenomiPhi V2 DNA Amplification Kit (GE Healthcare). Exons 1 to 2 that cover the coding region of two transcription variants of GNAO1 gene (transcription variant 1, GenBank accession number NM_020988.2, encoding G ⁇ o1 ; transcription variant 2, GenBank accession number NM_138736.2, encoding G ⁇ o2 ) 8 (SEQ ID NOs: 5-14) were screened by HRM analysis. These two transcription variants differ in the last two exons. HRM analysis was performed using a Light Cycler 480 (Roche Diagnostics). Sequences were determined for samples that showed abnormal melting curves. PCR primers and reaction conditions are shown in Table 3. All new mutations were verified using the original genomic DNA and searched against a mutation database of 408 control exomes in the laboratory.
  • Deep sequencing of mosaic mutation using IonTorrent A PCR product (chain length 178-bp) across the c.521A> G mutation was amplified using the blood, saliva and nail DNA samples from case 2 and the blood DNA samples of their parents as templates.
  • Adapter ligation, nick repair and amplification reactions were performed using the Ion XpressPlus Fragment Library Kit (Life Technologies) according to the protocol (Part Number 4471989 Rev. B). Indexing was performed using Xpress (trade name) Barcode Adapters 1-16 Kit (Life Technologies).
  • the Emulsion PCR and enrichment process was performed using Ion OneTouch (trade name) 200 Template Kit v2 (Life Technologies) according to the protocol (Part Number 4478371 Rev. A).
  • Ion Torrent Personal Genome Machine recommends using the Ion 314 sequencing chip and Ion PGM (trade name) 200 Sequencing Kit (Life Technologies) (Part Number 4474246). Rev. B) and used. Torrent Suite 2.2 was used for all analyses. Mosaic rates were measured using Integrative Genomics Viewer (Robinson, JT, et al. (2011). Nat Biotechnol 29, 24-26. And Thorvaldsdottir, H., et al. (2013). Brief Bioinform 14, 178-192.). We investigated using.
  • GNAO1 cDNA clone full length human GNAO1 cDNA clone (encoding the transcriptional variants 1, G.alpha o1) were purchased from Kazusa DNA Research Institute. Human GNAO1 cDNA was inserted into the pEF6 / V5-His-C vector to introduce a C-terminal V5 epitope (Life Technologies).
  • N2A cells were grown as previously described (Saitsu, H., et al. (2008). Nat Genet 40, 782-788.). Using X-tremeGENE 9 DNA Transfection Reagent (Roche Diagnostics), N2A cells on a cover glass were transfected with 200 ng of plasmid DNA. After 24 hours, the cells were fixed in 4% paraformaldehyde / PBS for 15 minutes and permeabilized in 0.1% Triton X-100 / PBS for 5 minutes. Cells were then blocked with 10% normal goat serum for 30 minutes.
  • V5-tagged G ⁇ o1 was detected using mouse anti-V5 antibody (1: 200 dilution; Life Technologies) and Alexa-488-conjugated goat anti-mouse IgG (1: 1000 dilution; Life Technologies). Cover glass was enclosed with Vectashield (Vector Laboratories) containing 4,6-diamidino-2-phenylindole (DAPI) and visualized with an inverted FV1000-D confocal microscope (Olympus).
  • the expression vector was introduced by electroporation using a Lonza Nucleofector apparatus and Cell Line Nucleofector Kit V (Lonza) according to the manufacturer's protocol (Program X-023). 2 ⁇ g of plasmid DNA was used per transfection.
  • Transfected cells were plated on a poly L lysine-coated plastic cover slip (Cell Desk LF, MS-0113L; Sumitomo Bakelite) at a cell density of about 5 ⁇ 10 4 cells / cm 2 and 10% fetal bovine serum (FBS ) Cultured in Dulbecco's modified Eagle medium (DMEM).
  • FBS fetal bovine serum
  • the cells were differentiated for 3-7 days using DMEM supplemented with 10 ⁇ M prostaglandin E1 (PGE1), 50 ⁇ M IBMX and 1% FBS, and calcium current was recorded. During the culture period, half of the medium was changed every other day.
  • PGE1 prostaglandin E1
  • IBMX 50 ⁇ M IBMX
  • FBS 1% FBS
  • the calcium current was recorded by a perforated patch clamp whole cell voltage recording method using amphotericin B.
  • Cells on the coverslip were placed in a bath solution containing 140 mM NaCl, 5 mM CaCl 2 , 4 mM KCl, 1 mM MgCl 2 , 10 mM HEPES, 10 mM TEACl, 8 mM glucose and 0.0002 mM tetrodotoxin under an Olympus BX51W upright microscope (Olympus). (Adjusted to pH 7.3 with NaOH).
  • a solution containing 100 mM CsCl, 10 mM EGTA and 40 mM HEPES (adjusted to pH 7.3 with CsOH) was used as the patch pipette solution.
  • Amphotericin B was added to the pipette solution at 2 ⁇ l / ml just before the experiment.
  • As the pipette a pipette assembled with a borosilicate glass capillary and exhibiting a resistance value of 4 to 8 M ⁇ in a state filled with an amphotericin B-containing pipette solution was used. Recording started when the series resistance dropped to ⁇ 150 M ⁇ after the giga seal was formed and a capacitive surge of cells appeared clearly.
  • a voltage-dependent calcium current was generated by applying a 50 ms depolarizing pulse from a holding potential of -65 mV to +10 mV and recorded using Multiclamp 700B (MolecularDevices) under the control of pCLAMP10 software (MolecularDevices). . Data was filtered at 2 kHz, sampled at 10 kHz, and series resistance was corrected by 50%. Inhibition of current mediated G.alpha o was generated by giving 10 ⁇ M norepinephrine bath solution via the. After 3-5 minutes, current inhibition was evaluated by measuring the change in current density just before the end of the depolarization pulse. Recording was performed at room temperature.
  • a de novo missense mutation (c.836T> A [p.Ile279Asn]) in the GNAO1 gene located at 16q12.2 was identified in case 1.
  • Known early-onset epileptic encephalopathy (EOEE) genes (SLC25A22 [MIM 609302], PNPO [MIM 610090], PNKP [MIM 613402], PLCB1 [MIM 613722] and ST3GAL3 [MIM 615006]) (Edvardson, S., et al. al. (2013). Epilepsia 54, e24-27 .; Molinari, F., et al. (2005).
  • Phenotypes associated with GNAO1 mutations The neurological characteristics of 4 cases with GNAO1 mutations are shown in Table 5.
  • cases 1, 2 and 3 tonic seizures with suppression burst were observed at the onset (4 to 29 days of age), and Otawara syndrome (OS) was diagnosed.
  • Cases 2 and 3 transitioned to West syndrome, which is common as an epileptic syndrome in infancy, as is evident from the fact that it showed a hypusvoluic pattern at the age of 3-4 months (FIGS. 2A-2C).
  • Case 4 showed developmental delay and a posterior bowing reversal posture at 7 months of age, and a complex partial seizure with epilepsy firing at 5 years of age (FIG. 2D).
  • the p.Thr191_Phe197del mutant (case 3) was expressed in the cell matrix compartment.
  • the p.Asp174Gly mutant (case 2) and p.Ile279Asn mutant (case 1) were localized on the cell surface, but a weak signal was also observed on the cell substrate, and the cell substrate signal was the p.Asp174Gly mutant. It was stronger. Similar localization pattern in the case of using a G.alpha o1 was tagged AcGFP1 the C-terminus was observed (data not shown). These localization patterns suggest that the function of the p.Thr191_Phe197del mutant is most severely impaired.
  • the region corresponding to amino acids 191 to 197 in human G ⁇ o1 is in the ⁇ -strand and in the connecting loop region involved in the interaction with the G protein-coupled receptor (GPCR) in the G ⁇ - ⁇ 2AR complex. Located ( Figure 4A). Therefore, it is considered that the deletion of this region damages the secondary structure of the molecule and not only impairs the interaction with GPCR but also significantly destabilizes the folding of G ⁇ subunit.
  • GPCR G protein-coupled receptor
  • Residues corresponding to Asp174 and Ile279 of the human G ⁇ o1 subunit are both buried within the protein (FIG. 4A) and are involved in hydrogen bonding and hydrophobic interactions, respectively.
  • the FoldX calculation showed that the free energy change increased by 2 kcal / mol or more by the p.Asp174Gly mutation and the p.Ile279Asn mutation (FIG. 4B). From these facts, it is considered that the folding of the G ⁇ subunit is destabilized by the p.Asp174Gly mutation and the p.Ile279Asn mutation.
  • the residue corresponding to human G ⁇ o1 at position 203 Gly is located within the highly conserved switch II region, a region involved in downstream effector activation by GTP binding (FIG. 4A).
  • the conformation of the switch region differs depending on the complex state of the G protein.
  • the glycine residue is surrounded in close proximity to the switch I region and GTP ( Figure 4A).
  • the calculation by FoldX showed that the free energy change was significantly increased by the p.Gly203Arg mutation.
  • G ⁇ o1 mutants N-type calcium channels have been reported to be inhibited, at least in part, through a G ⁇ o- mediated signaling system (Wettschureck, N., and Offermanns, S (2005). Physiol Rev 85, 1159-1204.).
  • NG108-15 cells calcium current inhibition of norepinephrine induced in cells is mediated by G.alpha o (FIG. 5A) (Waldo, GL, et al. (2010). Science 330, 974-980.) Using, The functional properties of G ⁇ o1 mutant were analyzed.

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Abstract

La présente invention concerne la détection de l'épilepsie résistante aux médicaments accompagnant une déficience intellectuelle sévère et un retard du développement moteur en utilisant un échantillon prélevé sur un organisme vivant pour voir si une mutation du gène GNAO1 est présente dans l'organisme vivant. L'épilepsie résistante aux médicaments est détectée dans les cas où une mutation nocive est détectée dans un allèle au moins du gène GNAO1. Dans les cas d'épilepsie où les mutations GNAO1 sont présentes, on observe des cas associés à un mouvement involontaire, un symptôme rare dans l'encéphalopathie épileptique ; il est donc possible que le mouvement involontaire soit aussi prédit en utilisant la mutation du gène GNAO1 comme un indice.
PCT/JP2014/065217 2013-06-12 2014-06-09 Méthode pour détecter une épilepsie résistante aux médicaments accompagnant une déficience intellectuelle sévère et un retard du développement moteur WO2014199944A1 (fr)

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JP7352904B2 (ja) 2019-10-08 2023-09-29 国立大学法人 東京大学 分析装置、分析方法及びプログラム
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WO2023102518A1 (fr) * 2021-12-03 2023-06-08 The Board Of Regents Of The University Of Texas System Vecteurs de thérapie génique gna01 et leurs utilisations

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