KR20140148206A - PLEKHG5 as a causative gene of Charcot-Marie-Tooth disease and diagnosis method for the disease - Google Patents

Abstract

The present invention relates to pleckstrin homology domain-containing, family G member 5 (PLEKHG5) gene as a novel causative gene of Charcot-Marie-Tooth (CMT) disease and a method for diagnosing the disease using the same and, more specifically, to a compound heterozygous PLEKHG5 mutant, which has 1988th cytosine substituted by thymine and 2458th guanine substituted by cytosine from start point of ATG translation initiation codon, identified as a causative gene of CMT for a marker including PLEKHG5 mutant to be valuably used in diagnosis of CMT.

Description

PLEKHG5 as a causative gene for CMT and a diagnostic method for the disease using the same.

The present invention newly discloses a pleckstrin homology domain-containing, family G member 5 (PLEKHG5) as a causative gene of Charcot-Marie-Tooth disease (CMT) And a diagnostic method of CMT.

Charcot-Marie-Tooth disease (CMT) or hereditary kinetic neuropathy refers to a disorder in which motor neurons and sensory nerves are damaged by specific gene mutations. Hereditary motor neuropathies (HMSN), hereditary motor neuropathies (HMN), and hereditary sensory neuropathies (HSN) are hereditary peripheral neuropathies. Hereditary sensory neuropathy (HSN) It is one of them. Charcot-Marie-Tooth disease, first named after Charcoal-Marie-Tooth disease, or CMT in 1886, first reported by Charcot, Marie and Tous, and then abbreviated as CMT. Dyck in the late 20th century. Have named CMT as a hereditary kinetic neuropathy and now use CMT and HMSN together.

The incidence of CMT is high among rare diseases inherited by one person per 2,500 people. In CMT patients, the muscles of the feet and hands gradually contract and weaken, deforming the shape of the foot and hands, and causing facial paralysis and hearing impairment. The onset usually occurs around the age of 10, but rarely after the age of 30. Patients' symptoms vary from mild to near normal, depending on the type of gene mutation, to the extent that they need help with walking or are dependent on a wheelchair.

CMT is divided into CMT1, CMT2, and CMTX, which inherit autosomal dominant and recessive inheritance according to their inherited pattern. In the case of the same CMT type 1, gene mutations were named in order of 1A, 1B, 1C, etc. in the reported order. Up to now, more than 50 causative genes have been reported as the cause of CMT. They are caused by a single gene mutation of the causative gene, and the degree of symptoms is different but inherited according to Mendel's law. In addition, since genetic defects and onset are directly related, the result of CMT gene diagnosis is very suitable for utilizing a gene diagnosis system because it has a meaning of confirmation, not a simple tendency or possibility of presentation.

Previous CMT treatments have been limited mainly to rehabilitation, ancillary devices, and pain control, but the discovery of related genes has made genetic counseling and family planning possible, along with a scientific basis for clinical treatments . There is currently no real treatment or adjuvant to alter the progression of genetic kinetic neuropathy, but recent animal studies have shown promising results. Recently, gene therapy, cell replacement therapy, axonal transport, mitochondrial function, immune system, and integrin therapy have been studied.

There was a report in the transgenic mice treated with onapristone, an antagonist of progesterone receptor, that overexpresses Pmp22 mRNA and improves phenotypic phenotype of hereditary motor neuropathy without side effects. Among the drugs for CMT treatment, Ascorbic acid, an essential substance in herbal formulations, improved the phenotype of herpes reparative and hereditary kinetic neuropathy in CMT1A transgenic mice and neurotrophin-3 (NT-3) in the CMT1A patient group It was reported that the effect of improving the sensory symptoms was increased by increasing the herniated nerve fibers. Thus, CMT can be used as an adjunct to the genetic diagnosis of genetic neuropathy. However, the development of effective diagnostic methods for hereditary neurological diseases is still insufficient. For the treatment of kinematic neuropathy with genetically very heterogeneous characteristics, accurate genetic diagnosis must first be established.

Therefore, the present inventors have made efforts to identify a new causative gene mutation of CMT. As a result, the inventors of the present invention found that the mutation of the PLEKHG5 gene of the pleckstrin homology domain-containing, family G member 5 (PLEKHG5) c.1988C> T, p.Thr663Met) and 2458th guanine were replaced with cytosine (c.2458G> C, p.Gly820Arg), and the PLEKHG5 mutation was confirmed to be a causative gene of CMT. And thus the present invention has been completed.

It is an object of the present invention to provide a diagnostic marker using pleckstrin homology domain-containing, family G member 5 (PLEKHG5) which is a causative gene of Charcot-Marie-Tooth disease (CMT) .

In order to achieve the above object,

Provides the genetic cause mutation marker of the pleckstrin homology domain-containing, family G member 5 (PLEKHG5) gene for Charcot-Marie-Tooth disease (CMT) diagnosis.

The present invention also provides a heterozygous recessive inheritance marker of the PLEKHG5 gene for CMT diagnosis.

The present invention also provides the mutant marker or heterozygous recessive genetic marker.

The present invention also provides a CMT diagnostic kit comprising a primer set capable of amplifying each of the mutation sites including c.1988C > T or c.2458G > C of the PLEKHG5 gene.

In addition,

1) isolating genomic DNA from a sample derived from an individual;

2) detecting the mutation site of the PLEKHG5 gene in the genomic DNA isolated in step 1); And

3) identifying a mutation site detected in step 2), and a method for identifying a mutation site for providing information on the risk of onset of CMT.

(C.1988C> T) from the translation start point of the pleckstrin homology domain-containing, family G member 5 (PLEKHG5) gene of the present invention and 2458th guanine was replaced with cytosine (c.2458G> C) Since the complex heterozygous PLEKHG5 mutation is a causative mutation of Charcot-Marie-Tooth disease (CMT), the PLEKHG5 gene can be used for a marker for diagnosing CMT and a method for diagnosing CMT using the same.

Figure 1 is a family diagram of a family (family ID: FC 307) that includes patients with Charcot-Marie-Tooth disease (CMT) with multiple heterozygous PLEKHG5 mutations. White markers represent healthy members and black markers represent CMT patients. White and black halves represent the donor members with one heterozygous mutant allele, and the arrows indicate the CMT inducer. The genotypes of the 1988 and 2458th PLEKHG5 mutations were indicated under each test subject marker.
FIG. 2 is a view showing a nucleotide sequence chromatogram including PLEKHG5 mutations in a control group and a CMT patient. FIG. The arrow indicates the position of the variation.
FIG. 3 is an amino acid sequence analysis showing that the position of the PLEKHG5 mutation in the CMT patient is well preserved in the species.
4 shows a T1-weighted MRI image of inducer III-1. (A) shows the coronal images of the thighs, and (B) shows the coronal images of the lower legs. (C) represents an axial image of the central thigh, (D) represents an axial image of the upper thigh, and (E) represents an axial image of the lower calf. The black triangles represent the anterior tibialis, extensor halucislongus and digitorum longus of the anterior and the white triangles represent the lateral peroneus longus and the endometrium peroneus brevis). Arrows indicate the flounder (soleus) and posterior tibialis posterior (tibialis posterior).
FIG. 5 is a view showing a pathological examination of the peripheral nerve of the CMT patient. FIG. (A) shows a thin film transverse section stained with toluidine blue observed at a magnification of 400 times, and (B) shows a bar graph including a single-rod type distribution pattern of the water-distribution fibers.
6 is a diagram showing immunohistochemical analysis of CMT patients. (A) and (B) show electron micrographs showing atrophy and bundle formation of the axon, and collagen deposition in the nerve endocardium. (C) shows the Schwann cell nucleus of the CMT patient using the anti-PLEKHG5 antibody, and (D) shows the axon and Schwann cell nuclei of the control group. (A) was observed at 3000 times, (B) at 8000 times, and (C) and (D) at 400 times.
7 is a diagram showing the intracellular location of wild-type and mutant PLEKHG5 proteins.
8 is a graph showing the luciferase activity of the wild-type and mutant PLEKHG5 proteins. * Means that the Student's t-test is less than 0.05.

Hereinafter, the present invention will be described in detail.

The present invention provides a genetic source mutant marker of the pleckstrin homology domain-containing, family G member 5 (PLEKHG5) gene for Charcot-Marie-Tooth disease (CMT) diagnosis.

The PLEKHG5 gene (GeneBank registration number: NM_020631.3) is preferably a nucleotide sequence of SEQ ID NO: 1, but is not limited thereto.

Preferably, the genetic cause mutation marker is c.1988C> T or c.2458G> C in the PLEKHG5 gene, but is not limited thereto.

In a specific embodiment of the present invention, the present inventors selected CMT patients having no known major CMT gene mutation among CMT patients and analyzed the nucleotide sequences of exons from the genomic DNA of CMT patients and their families. As a result, Of the PLEKHG5 gene have a complex heterozygous mutation in which the c.1988C > T in the father gene and the c.2458G > C mutation in the mother gene cause the protein mutation of p.Thr663Met and p.Gly820Arg, respectively (See Figs. 1 to 3, Table 1 and Table 2).

In addition, the present inventors confirmed the clinical symptoms of the CMT patients, and the CMT patients of the present invention are patients with lower motor neuron disease (LMND) having a mutation of PLEKHG5 (Maystadt I et al., Neurology 2006, 67 : 120-124). The nerve conduction velocity was measured and electrophysiologic symptoms were observed. As a result, the CMT patients showed gradual weakening and atrophy of the leg muscles, and sensory and motor nerve conduction It was confirmed that the speed was reduced or not measured (see Table 3 and Table 4).

In addition, the present inventors have taken MRI images of the brain, hips, thighs, and legs of the CMT patients, and have found various muscular atrophy and fat replacement in the leg muscles (see FIG. 4).

We also performed biopsy of the distal sural nerve for histopathologic analysis of the CMT patients to determine the length of the myelinated fibers (MFs), the length of the axonal Diameters and thickness of myelin were measured. For immunohistochemical analysis, the number of MFs, the size of MFs, the diameter of the axons were measured using an electron microscope. (Fig. 5 and Fig. 6). As a result, it was confirmed that the degenerated fibers were clustered, the bands of bands were formed, and atrophy and concentration of positive cells in Schwann cells were observed.

In addition, the present inventors produced a gene vector for expressing the mutant protein due to the mutation of PLEKHG5 gene of the CMT patient. As a result of expressing the mutant protein in HEK293 cell, the mutant protein is present in the cytoplasm and the pathway of NK- (See Figs. 7 and 8).

Therefore, it was confirmed that the heterozygous mutation marker of PLEKHG5 comprising c.1988C> T or c.2458G> C of the present invention can be used as a marker for diagnosing CMT.

The present invention also provides a heterozygous recessive inheritance marker of the PLEKHG5 gene for CMT diagnosis.

The heterozygous recessive inheritance marker is preferably, but not limited to, the 1988 base from the starting point of the ATG translation initiation codon in the PLEKHG5 gene is thymine and the 2458th base is cytosine.

The PLEKHG5 gene of the present invention was found to be the causative gene of CMT from the starting point of translation, and the complex heterozygous PLEKHG5 mutation in which 2458th guanine was replaced with cytosine thymine and 2458th guanine was replaced with cytosine. Thus, the heterozygous recessive inheritance marker of PLEKHG5 was diagnosed as CMT It can be useful as a marker.

The present invention also provides a CMT diagnostic kit comprising the heterozygous mutation marker or the heterozygous recessive genetic marker.

In addition, the present invention provides a CMT diagnostic kit comprising a primer set capable of amplifying mutant sites each of which is characterized by c.1988C> T or c.2458G> C of the PLEKHG5 gene.

In addition,

1) isolating genomic DNA from a sample derived from an individual;

2) detecting the mutation site of the PLEKHG5 gene in the genomic DNA isolated in step 1); And

3) identifying a mutation site detected in step 2), and a method for identifying a mutation site for providing information on the risk of onset of CMT.

The sample of step 1) is preferably blood, but is not limited thereto.

Preferably, the mutation in step 2) is c.1988C> T or c.2458G> C, but is not limited thereto.

In step 2), detection may be performed by sequencing analysis, microarray hybridization, allele specific PCR, dynamic allele-specific hybridization (DASH), PCR extension analysis, or TaqMan But it is not limited thereto.

Among the above methods, sequencing analysis can be performed using a conventional method for nucleotide sequencing, and can be performed using an automated gene analyzer (Sanger, F. et al ., Proc. Natl . Acad . Sci . USA . , 74 (12), 5463-5467, 1977; Maxam, AM and Gilbert, W., Proc . Natl . Acad . Sci . USA , 74 (2), 560-564, 1997).

Allele-specific PCR analysis refers to a PCR method in which a DNA fragment in which the mutation is located is amplified with a primer set including a primer designed with a base at the 3 'end at which the mutation is located. The principle of the above method is that, for example, when a specific base is substituted by G to A, an opposite primer capable of amplifying a primer containing the G as a 3 'terminal base and a DNA fragment of an appropriate size is designed, When the base at the mutation position is G, the amplification reaction is normally performed and a band at a desired position is observed. When the base is substituted with A, the primer can be complementarily bound to the template DNA, '(Newton, CR et al. , ≪ RTI ID = 0.0 > al ., Nucleic Acids Res . , 17 (1), 2503-2516, 1989).

TaqMan probe PCR analysis (Livak, KJ, Genet . Anal ., 14, 143-149, 1999) was performed by 1) designing and constructing a primer and a TaqMan probe to amplify a desired DNA fragment; 2) labeling probes of different alleles with FAM and VIC dyes (Applied Biosystems, USA); 3) performing PCR using the above primers and probes using the DNA as a template; 4) After completion of the PCR reaction, analysis and confirmation of the TaqMan assay plate with a sequencer; And 5) determining the genotype of the polynucleotides of step 1 from the analysis results.

Dynamic allele hybridization (DASH) analysis can be performed by a method designed by Prince et al. (Prince, JA et al ., Genome Res . 11 (1), 152-162, 2001).

The PCR extension analysis first amplifies a DNA fragment containing a base in which the mutation is located to a pair of primers, inactivates all the nucleotides added to the reaction by dephosphorylation, and adds thereto an extension primer specific for the mutation, a dNTP mixture, And then performing primer extension reaction by adding an oxynucleotide, a reaction buffer, and a DNA polymerase. At this time, the extension primer has a base immediately adjacent to the 5 'direction of the base in which the mutation is located at the 3' end, and the nucleic acid having the same base as the didyoxynucleotide is excluded in the dNTP mixture, and the didyoxynucleotide shows mutation Base type. For example, in the case of the substitution of G to A, when the dATP, dCTP and TTP mixture and ddGTP are added to the reaction, the primer is extended by the DNA polymerase in the substituted base, The primer extension reaction is terminated by ddGTP at the position where the G base first appears. If the substitution has not occurred, the extension reaction is terminated at the position, so that it is possible to discriminate the type of the base showing the mutation by comparing the lengths of the extended primers.

At this time, as a detection method, when the extension primer or the dioxynucleotide is fluorescently labeled, the mutation is detected by detecting fluorescence using a gene analyzer (for example, Model 3700 manufactured by ABI) used for general nucleotide sequence determination (Chen, J., Genome Res ., 10 (4), 549-557, 2000). When unlabeled extension primers and dodecyloxynucleotides are used, the molecular weight is measured using MALDI-TOF (matrix assisted laser desorption ionization-time of flight) The mutation can be detected (Ross, PL , Anal . Chem , 69 (20), 4197-4202, 1997).

The PLEKHG5 gene of the present invention was found to be a causative gene of CMT from the translation initiation point of the 1988 catechin thymine and the 2458th guanine-substituted PLEKHG5 mutant in which the guanine was substituted with cytosine, thereby diagnosing CMT using the heterozygous mutation of PLEKHG5 It can be useful.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are illustrative of the present invention, and the contents of the present invention are not limited by the following examples.

< Example  1> CMT  Identification of neuropathic genes

&Lt; 1-1 > CMT  Gene preliminary screening

To identify the genes responsible for Charcot-Marie-Tooth disease (CMT), we screened patients with CMT neuropathy and extracted DNA from the blood to preliminarily screen the patient's CMT-related genes.

Specifically, 160 families and 248 patients with CMT neuropathy who did not have a blood relationship from the physician and neurologist of the primary care institution were included in the study. The present invention includes 300 healthy control subjects and all participants were informed prior to the protocol approved by the Institutional Audit Committee of Ewha Womans University Mokdong Hospital (ECT 11-58-37). We identified negative responders to the overlapping of major myelin protein (PMP22) and major CMT mutations, which are known to be causative agents of CMT neuropathy.

Then, 10 ml of the blood of the selected patients was collected in a tube containing EDTA and the DNA was purified using a QIAamp blood DNA purification kit (Qiagen, Hilden, Germany). Using the extracted DNA, overlapping of the 17p12 region identified as a major genetic cause of dehydrative CMT was confirmed by hexaplex microsatellite PCR method using the primer shown in Table 1 (Choi BO et. al., Mol Cells 2007, 23: 39-48.). The sequences of MPZ, PMP22, GJB1 and MFN2, known as CMT-related genes, were analyzed together for the purpose of sequencing. PCR conditions and primer sequences used for the respective exon analyzes were as shown in [Table 2] 5] (Tables 2 to 5).

As a result, as shown in FIG. 1, a family (family ID: FC 307) including a CMT patient of a 19-year-old woman was selected as an object of the present invention (FIG.

Analysis of 17p12 using Hexaflex Simultaneous Amplification PCR and sequencing of primers used gene
location primer
Size (bp) label
(labeling) Name of the primer The base sequence (5 '-> 3')
D17S921
D17S921_F
(SEQ ID NO: 2) GTGTTGTATTAGGCAGAGTTCTCC
139-151

FAM
D17S921_R
(SEQ ID NO: 3) GGCAGTAGATGGTGACTTTATGGC
D17S9B
D17S9B_F
(SEQ ID NO: 4) TCTCAGTCCTGATTTCTTGATTTTG
99-135

HEX
D17S9B_R
(SEQ ID NO: 5) CCAGAGCTAACACCACATTCA
D17S9A
D17S9A_F
(SEQ ID NO: 6) CAACCATCAGTGATTTGATGGTTTAC
164-204

HEX
D17S9A_R
(SEQ ID NO: 7) GAGTTGTCACTAGAACCCTGTTC
D17S918
D17S918_F
(SEQ ID NO: 8) TCCTGTAATCTGTCCCCAAACGTC
235-250

HEX
D17S918_R
(SEQ ID NO: 9) TTCCTCACACAACCTATTGATAGTC
D17S4A
D17S4A_F
(SEQ ID NO: 10) CTGTGGAGGAAAGAAAACACTGCC
160-188

FAM
D17S4A_R
(SEQ ID NO: 11) GCACTAAAGTAGCTTGTAACTCTG
D17S2230
D17S2230_F
(SEQ ID NO: 12) AGGAAACTGATGTCTAAAACTATCC
204-314

FAM
D17S2230_R
(SEQ ID NO: 13) GTGAATCCAGGAGGCAGAGCTTGC

PCR conditions for the exon analysis of MPZ and the sequence of the primers used Gene location
primer Size (bp)
Tm temperature
(° C) Name of the primer The base sequence (5 '-> 3')
Exon 1
MPZ-N1F
(SEQ ID NO: 14) tccatatctatccctcagagaagt
573

60
MPZ-N1R
(SEQ ID NO: 15) ctgaatataatgtcaccttcctgc
Exon 2
MPZ-2F
(SEQ ID NO: 16) ctgatctcacttcctctgtatcc
254

57
MPZ-2R
(SEQ ID NO: 17) ctttgaagcactttctgttatcc
Exon 3
MPZ-3F
(SEQ ID NO: 18) acagctgtgttctcattagggtc
312

57
MPZ-3R
(SEQ ID NO: 19) ctcccaaactgcttcccatacc
Exon 4, 5
MPZ-4F
(SEQ ID NO: 20) ctaggaaccacagatacagggc
460

57
MPZ-4R
(SEQ ID NO: 21) gggttctccttcccatcttgtc
Exon 6 MPZ-6F
(SEQ ID NO: 22) aacagtcaagccccagtcgctc
250
57 MPZ-6R
(SEQ ID NO: 23) gctcatcctttcgtagctccatc

PCR conditions for the exon analysis of PMP22 and the sequence of the primers used Gene location
primer Size (bp)
Tm temperature
(° C) Name of the primer The base sequence (5 '-> 3')
Promoter
PMP-P1F
(SEQ ID NO: 24) CACAGATGCATGGGATAGGGGTCC
721

57
PMP-P1R
(SEQ ID NO: 25) ATGGAGATGAGGTACAGTGTGGGC
Exon 1A
PMP-1AF
(SEQ ID NO: 26) ATCTCTGCAGAATTCACTGGGAG
528

57
PMP-1AR
(SEQ ID NO: 27 GCATAGGCACACATCACCCAGAG
Exon 1B
PMP-1BF
(SEQ ID NO: 28 TCGTAGGTGCAGACAGTAATAGC
729

57
PMP-1BR
(SEQ ID NO: 29) ATGCCCACATTTCTCCTCCATCTC
Exon 2
PMP-2F
(SEQ ID NO: 30) CGCAGGCAGAAACTCCGCTGAGC
234

58
PMP-2R
(SEQ ID NO: 31) CGTCTGAGACTTCCAACTGTCTGC
Exon 3
PMP-3F
(SEQ ID NO: 32) AACGTTGGCTCTTACCATGCAGG
390

66
PMP-3R
(SEQ ID NO: 33) TGGACTCATGGCTCCCTGTCAC
Exon 4
PMP-4F
(SEQ ID NO: 34) ATGTATGTAATGTGTACATATTGGCAC
481

64
PMP-4R
(SEQ ID NO: 35) TTGGATGCACCCCGCTTCCACA
Exon 5
PMP-5F
(SEQ ID NO: 36) TCTGCCATGGACTCTCCGTCAC
314

57
PMP-5R
(SEQ ID NO: 37) GTTTGGGATTTTGGGCTAGCTC

PCR conditions for the exon analysis of GJB1 and the sequence of the primers used Gene location
primer size
(bp) Tm temperature
(° C) Name of the primer The base sequence (5 '-> 3')
Promoters 1 and
Exon 1a P1F
(SEQ ID NO: 38) tgcagcttgcccgcactgtggatc
477

62
P1R
(SEQ ID NO: 39) ccggcccactgtgccacatcagc
Promoters 2 and
Exon 1b P2F
(SEQ ID NO: 40) tcccctcttcacatccacct
440

62
P2R
(SEQ ID NO: 41) gctccttaactccagacctg
Exon 2a
Cx-E2aF
(SEQ ID NO: 42) gctactggctcttggaagagttga
461

62
Cx-E2aR
(SEQ ID NO: 43) tccccatggccctcaagccgtagc
Exon 2b
Cx-E2bF
(SEQ ID NO: 44) tgtggtccctgcagctcatcctag
419

62
Cx-E2bR
(SEQ ID NO: 45) ccggatgatgaggtacaccacctc
Exon 2c
Cx-E2cF
(SEQ ID NO: 46) tcttcaccgtcttcatgctagctg
385

64
Cx-E2cR
(SEQ ID NO: 47) tccccagcaggcagaggcctgtgc

The PCR conditions for the exon analysis of MFN2 and the sequence of the primers used location primer size
(bp) Tm temperature
(° C) name Base sequence Exon 3
3F (SEQ ID NO: 48) AGCATTCCGCCATCTCCCTAGCAG 326
56
3R (SEQ ID NO: 49) CACCCGCAAATCCCACTAGCTAA Exon 4
4F (SEQ ID NO: 50) TCCAGACTTGGGACTGTGGAAC 284
56
4R (SEQ ID NO: 51) TGGAACGTTCTGTGACCTTGCAC Exon 5
5F (SEQ ID NO: 52) CCAGGCTGGTATCTGCGTTGTGA 306
56
5R (SEQ ID NO: 53) GTGTCACAACGGAGGACTTGCTC Exon 6
6F (SEQ ID NO: 54) TGTGATGCAGCGGCACAGGAAATC 248
56
6R (SEQ ID NO: 55) TTCCAGTTTGGACCTC Exons 7 and 8 7F (SEQ ID NO: 56) GGCACCATAGGGCTCCTGCTCTGCCTGATGA 425
56
8R (SEQ ID NO: 57) TCCCTCGGGGTTGCATTC Exon 9
9F (SEQ ID NO: 58) GCACTGCCCAGCCTCTTATGACCTATTC 323
56
9R (SEQ ID NO: 59) TGACAGACTCCTCAGCACGAGAC Exons 10 and 11 10F (SEQ ID NO: 60) CTGCTGCCAAGTTGTTTCTGGAC 409
60
11R (SEQ ID NO: 61) TCCACCTATCTGCAGTCTTGGAC Exon 12
12F (SEQ ID NO: 62) CCTCTGCTTAGTCAGACAGGAAC 233
60
12R (SEQ ID NO: 63) GGACTCTACTCGGAGTCCAAATC Exons 13 and 14 13F (SEQ ID NO: 64) TGCTGCAGGAGTGAACTTTGGTC 517
60
14R (SEQ ID NO: 65) CAGGGCCACAGCTGCCCAGTTC Exon 15
15F (SEQ ID NO: 66) GTGCAGGGCTGAGCTGATAAG 342
60
15R (SEQ ID NO: 67) ATGGTTCCCGTGTCTGGAGGCAG Exxon 16
16F (SEQ ID NO: 68) ACTAGGGCAACACTGAGGGTTCAC 349
60
16R (SEQ ID NO: 69) TATGCAGTGGACTGTGGAGTGTG Exxon 17
17F (SEQ ID NO: 70) GGCCACAGAGAGGCTGCACTCCA 378
94
17R (SEQ ID NO: 71) CCTGAAGGCCCAGGGTCTACGA Exon 18
18F (SEQ ID NO: 72) TAAGCCACCAGTGGCATCTTGC 296
60
18R (SEQ ID NO: 73) TTTGTGTCCACACCCAAGACGC Exxon 19
19F (SEQ ID NO: 74) TCAAGCGTCCTTAGGATGGTGC 296
60
19R (SEQ ID NO: 75) ATGGTACGAGACTGGGTGCTTC

Since exon 1 and exon 2 are un-translated regions (UTR), no analysis was performed.

<1-2> CMT  Identification of root cause mutations

We analyzed the nucleotide sequence of the exome of the patient in order to confirm the root cause mutation of the CMT of the patients screened through the preliminary screening.

Specifically, the nucleotide sequence of the exon was analyzed from the DNA isolated from the blood sample of the CMT terminal proband of the FC 307 family selected in Example <1-1> (Lee SS et. al., JAMA Neurol, 2013, 70: 607-615). Functionally significant mutations such as mismatch, nonsense, exonic indel, and splice location shifts are first screened into WES data, and either new or dbSNP137 (http://www.ncbi.nlm.nih.gov) and Variants with a minor allele frequency (MAF) of less than 0.1 were enrolled in the 1000 Genome Project database (http://www.1000genomes.org/). Finally, homozygous or compound heterozygous mutations were finally selected. Protein sequences were analyzed using the MEGA5 ver 5.05 program (Tamura K et al., Mol Biol Evol 2011, 28: 2731-2739), and in silico analysis was performed using the PolyPhen 2 program to determine the effect of the resulting mutations on protein function.

As a result, as shown in Figs. 1 to 3, there exist c.1988C > T in the father gene and c.2458G > C mutation in the mother gene in the PLEKHG5 gene of the Korean CMT patient family, It was confirmed that a protein mutation of p.Gly820Arg occurs (FIGS. 1 and 2). The genetic variation of the father and the mother was inherited from grandmother and grandmother and the gene mutation was found to be new to dbSNP137 and the 1000 genome project database. The p.Thr663Met mutation was not confirmed in 300 Korean control groups, and the p.Gly820Arg mutation was confirmed in three control groups, and it was generally confirmed that both mutations were well conserved in species (Fig. 3). In the silico analysis, the p.Thr663Met mutation was 0.993 and the p.Gly820Arg mutation was 1.000, so that both mutations affected the function of the protein.

In addition, as shown in the following Tables 6 and 7, the entire exon sequence of the CMT patient represented by III-1 in the diagram of FIG. 1 was analyzed to confirm the defect of the CMT-related gene (Table 6 ), PLEKHG5, and more than 50 variants of CMT-related genes, but all of the mutations were polymorphic, and did not have the feature of recessive inheritance and thus could not be the root cause of CMT (Table 7) .

Overall exon sequencing of patient (III-1) Item FC307  ( III -One) The total number of base sequences (Gbp) 6.5 % Mapped reaction (/ total reaction) 94.0 % Range of target area (10X or more) 93.4 The center reaction depth of the target area
(Mean Read Depth of Target Region) 88.5X Total confirmed SNPs (SNs) 57,356 Total verified insertion-deletion (Total observed indices) 9,592 Filtering    Encrypted SNPs 19,700    Encryption Insert - Fruit 526    Functionally significant variants 9,136    Functionally significant mutants in the CMT gene 30

CMT: Charcoal-Marie-Tooth disease;

SNP: single nucleotide polymorphism

^a gene bank of source sequence (gene bank) registration number

^b cDNA number was calculated by taking A of the ATG start codon as +1.

^c gene bank registration number / allele frequency of 1000 genomic database (-; not reported)

< Example  2> CMT  Clinical symptoms and electrophysiological analysis of patient

<2-1> CMT  Identification of patient's clinical symptoms

To confirm the clinical symptoms of CMT patients, motor and sensory impairments, deep tendon reflexes, and muscle atrophy of CMT patients and their families were identified.

Specifically, the CMT foot terminal III-1, the parents II-3 and II-7 and the grandparents I-1, I-2, I-3 and I- 4). The strength of flexor and extensor muscles was measured by the medical research council (MRC). Physical disabilities were measured using the CMT neuropathy score (CMTNS) (Shy ME et al., Neurology 2005, 64: 1209-1214). Sensory disorders were measured for severity of pain, temperature, vibration, and location. In addition, age at onset was confirmed by examining the onset of symptoms such as weakening of the distal muscle, deformity of the foot, or change of sensation. Compared with the symptoms of CMT, patients with lower motor neuron disease (LMND) with mutations in PLEKHG5 (Maystadt I et al., Neurology 2006, 67 : 120-124) .

As a result, the clinical symptoms of the CMT and LMND patients of the present invention were compared as shown in Table 8 (Table 8). The CMT inducer was a 19-year-old female, the first child born to a healthy non-blood-born Korean parent, whose parents had no abnormalities in neurological and electrophysiological tests. The inducer was born during normal birth at birth, and showed no abnormality during the early stages of development, and could walk one year after birth. At the age of 8, the muscles began to fall frequently, and at the age of 16, short leg braces were used. At the age of 19, neurological examination revealed weakening of the muscles below the upper part and atrophy of the bilateral distal muscles. We observed atrophic changes in both pescavus, steppage gait, and intrinsic foot and calf muscles, but no scoliosis was seen. Sensitivity to pinprick, tactile sense, position and vibration using a needle as a whole, and sensitivity to vibration and position sense was lower than vibration and tactile sense. No knee or Achilles tendon reflexes were seen, but pyramidal or cerebellar signs were not seen. CMTNS was 15, and creatine kinase concentration of serum was 246 μmol / L, which was significantly higher than that of normal control (185 μmole / L) (Table 8).

Comparison of clinical symptoms of patients with PLEKHG5 mutations Item Measures Standard value Inspection age (years) 14 15 16 19 Side (Side) right right right right - Median nerve TL (ms) 6.1 5.4 5.5 7.0 <3.9 CMAP (mV) 8.2 9.5 9.6 6.4 > 6.0 MNCV (m / s) 25.3 24.7 25.6 29.3 > 50.5 Ulnar nerve TL (ms) 4.9 4.9 4.9 5.7 <3.0 CMAP (mV) 9.7 10.2 10.0 10.0 > 8.0 MNCV (m / s) 25.0 22.4 20.4 25.0 > 51.1 Peroneal nerve TL (ms) A A A A <5.3 CMAP (mV) A A A A > 1.6 MNCV (m / s) A A A A > 41.2 Tibial nerve TL (ms) 7.2 6.0 7.4 A <5.4 CMAP (mV) 0.6 0.3 0.2 A > 6.0 MNCV (m / s) 19.2 15.8 13.4 A > 41.1 Median sensory nerve SNAP (mV) 6.5 4.8 7.8 8.0 > 8.8 SNCV (m / s) 27.0 25.9 27.3 25.6 > 39.3 Ulnar sensory nerve SNAP (mV) 7.4 6.9 5.0 4.5 > 7.9 SNCV (m / s) 23.4 21.9 22.3 23.7 > 37.5 Calf nerves SNAP (mV) A A A A > 6.0 SNCV (m / s) A A A A > 32.1

A dark letter means an abnormal value; A, absent potentials; TL, terminal latency; CMAP, compound muscle action potential; MNCV, motor nerve conduction velocity; SNAP, sensory nerve action potential; SNCV, sensory nerve conduction velocity.

< Example  3> CMT  Fat replacement of patient's leg muscles fatty replacement ) Confirmation of

MRI images of the brain, hips, thighs, and legs of CMT patients were taken to confirm the finding that the muscle of the CMT patient was replaced with fat.

Specifically, axial and coronal planes were imaged using a 1.5-T system (Siemens, Erlangen, Germany). The field of view (FOV) of the axial surface was 24 to 32 cm, the slice thickness was 6 mm, and the slice gap was 0.5 to 1.0 mm. The field of view of the coronal plane was 38 to 40 cm, the section thickness was 4 to 5 mm, and the intervals of the sections were 0.5 to 1.0 mm. T1-weighted spin-echo (SE) (TR / TE 570-650 / 14-20, 512 matrix), T2-weighted SE (TR / TE 2800-4000 / 96-99, 512 matrix) , And fat-suppressed T2-weighted SE (TR / TE 3090-4900 / 85-99, 512 matrix).

As a result, as shown in FIG. 4, hyperintense signal abnormalities were observed on the MRI images of the thigh and the lower leg of the CMT patient. On T1-weighted images, it was confirmed that axonal degeneration was dependent on the length, as compared to the thigh, in that it showed displacements of the upper and lower parts of various muscles in the leg muscles (Figs. 4A and 4B ). In the thigh, the vastus lateralis was replaced with fat and semimembranous muscles were confirmed, but muscle was more abundant than fat. Some fat streaks were observed in Sartorius muscles, gracillis muscles, biceps femoris muscles and rectus femoris muscles (Fig. 4C). In addition, the anterior and lateral compartment muscles in the leg MRI showed a preferential and dominant relationship, but normal findings were observed in the superficial and deep posterior compartment muscles (FIGS. 4D and 4D) 4E).

< Example  4> CMT  Histopathological analysis of patients

<4-1> CMT  Peripheral nerve biopsy of patient

To identify the histopathologic features of CMT patients, biopsy was performed on distal sural nerve of 19 year old CMT patients.

Specifically, the length of myelinated fibers (MFs), axonal lengths from semi-thin transverse sections using a computer-based image analyzer (AnalySIS, Munster, Germany) And the thickness of myelin were measured.

As a result, as shown in Fig. 5, the MFs of medium or large size were lost in the thin film transverse section, only small MFs remained (Fig. 5A), and the number of MFs was 9,800 / mm ² compared with 297 / mm ² (Fig. 5B). The range of MF diameters was 1.2 to 4.6 mu m and the average was 2.4 mu m, confirming that the MF of the control group was reduced in the range of 2 to 12.5 mu m and the average of 4.5 mu m. It was confirmed that the distribution pattern of the MF diameter was composed of unimodal 75% of 3 탆 MFs or less. In addition, the area of MF% was 0.2%, which was lower than that of the normal distal appendicular nerve in the 20-year-old female control group. The range and average of the axon diameter / MF diameter were 0.38 to 0.73 and 0.56 ± 0.09, and the average g-ratio of 21 to 50 years was 0.66. A g-ratio of 0.7 or more, representing an abnormally thin myelin sheath, constitutes 10% of the MFs, and a g-ratio of 0.4 or more representing an abnormally thick aqueduct is 1.7%.

<4-2> CMT  Immunohistochemical analysis of patients

For the immunohistochemical analysis of CMT patients, we confirmed the medullary nerve fibers and Schwann cells of patients and controls by electron microscopy.

Ultrathin cut samples (60 to 65 nm) were analyzed with an electron microscope (H-7650, Hitachi, Japan) and compared with uranyl acetate and lead citrate Respectively. Immunohistochemistry was performed using anti-PLEKHG5 antibody (N-15: sc-130100, 1:50 dilution, Santa Cruz Biotech. Santa Cruz, Calif. Respectively.

As a result, as shown in Fig. 6, the remaining small MFs showed focal folding of myelin, a very rare evidence of degradation, through the formation of degenerated fibers forming clusters (Figs. 6A and 6B) 6B). The unmyelinated axons were confirmed to be bundle formation and atrophy, a reinnervated bands bungner formed by re-curing anhydrous fibers. In addition, it was confirmed that fibroblast differentiation and collagen deposition of endoneurial were performed many times. In addition, a positive reaction was observed in the nucleus of axons and Schwann cells of the control group (FIG. 6C), but a concentrated positive reaction was observed in the nucleus of schwann cells of CMT patients (FIG. 6D).

< Example  5> CMT  Cause of PLEKHG5  Due to mutation Mutant  Identification of Protein Expression

<5-1> PLEKHG5  Due to mutation Mutant  Intracellular localization of proteins

In order to confirm the intracellular location of the mutant protein expressed by the mutation of the CMT cause in the PLEKHG5 gene, the PLEKHG5 gene was cloned so as to have the same mutation as the mutation of the CMT patient (III-1) confirmed in Example 1 above To express the protein.

Specifically, the pCMV-myc vector containing the PLEKHG5 gene (BC015231) linked to the c-myc epitope was purchased from Capital Biosciences (Rockville, Md.) And analyzed using a QuickChange location-directed mutation kit (Stratagene, La Jolla, CA) PCR was carried out using the primers shown in Table 9 below to induce p.Thr663Met and p.Gly820Arg mutations in the PLEKHG5 gene. Then, the plasmid was transformed into E. coli, and the corresponding mutation sequence was confirmed using the primer shown in Table 10, and 2 ㎍ of the vector containing the mutated sequence and Lipofectamine 2000 (Invitrogen , Carlsbad, Calif.), And HEK293 cells were cultured for 24 hours in a medium containing 6 ㎕ of DNA.

Then, the HEK293 cells were fixed with methanol to express the protein and analyzed by immunocytochemistry. An anti-myc antibody (Abcam, Cambridge, UK) was added and allowed to stand for 1 hour, HEK293 cells were stained with a fluorescently labeled secondary antibody, and the nuclei of the cells were stained with DAPI and observed with a fluorescence microscope.

As a result, as shown in FIG. 7, it was confirmed that the mutant protein was present in the cytoplasm and no characteristic aggregate was found in all three kinds of proteins (FIG. 7).

The nucleotide sequence of the primer used to induce the position-directed mutation name The base sequence (5 '-> 3')  PLEKHG5_T663M-F (Sequence Listing: 76)  GGGGCCTACATGTTCCAGGCCAGTG  PLEKHG5_T663M-R (Sequence Listing: 77)  GCCTGGAACATGTAGGCCCCTACAGC  PLEKHG5_G820R-F (Sequence Listing: 78)  CTCTGCCTACCGCACCCTCTCCCC  PLEKHG5_G820R-R (Sequence Listing: 79)  AGAGGGTGCGGTAGGCAGAGTCCATG

The nucleotide sequence of the primer used to identify the location-directed mutation name The base sequence (5 '-> 3')  PLEKHG5-seq_961 (Sequence Listing: 80)  GGCCAGTCGCTTCAGCGGCT  PLEKHG5-seq_1921 (Sequence Listing: 81)  GCACCTGGACTTGACAGCGC  PLEKHG5-seq_2401 (SEQ ID NO: 82)  CAGT0GCACTTCAGCTGCCA

<5-2> PLEKHG5  Due to mutation Mutant  Protein NF - kB  Route Activation ability  Confirm

In order to confirm the NF-kB pathway activating ability of the mutant protein expressed by the mutation of the CMT cause in the PLEKHG5 gene, the PLEKHG5 gene had the same mutation as the mutation of the CMT patient (III-1) confirmed in Example 1 above And the protein was expressed.

Specifically, the PLEKHG5 mutant gene used in Example <5-1> was cloned into a pNF-κB-Luc-reporter vector provided from Sung Kyun Kwan University and then transformed into HEK293 cells in the same manner as in Example <5-1> Transfected to express the mutant protein. Then, the cells were lysed with a Luciferase assay kit (Promega, Madison, Wis.), Reacted with a substrate, and the degree of luminescence was measured with a luminometer to confirm the expression amount of the mutant protein.

As a result, as shown in FIG. 8, it was confirmed that the activity of luciferase was decreased in cells expressing the mutant protein compared to wild-type PLEKHG5 (FIG. 8).

<110> EWHA UNIVERSITY - INDUSTRY COLLABORATION FOUNDATION          KONGJU NATIONAL UNIVERSITY INDUSTRY-UNIVERSITY COOPERATION FOUNDATION <120> PLEKHG5 as a causative gene of Charcot-Marie-Tooth disease and          diagnosis method for the disease <130> 13P-05-019 <160> 82 <170> Kopatentin 2.0 <210> 1 <211> 2793 <212> DNA <213> Homo sapiens <400> 1 atgcattatg atgggcatgt ccgcttcgac cttcccccac aaggctctgt gctggcccgg 60 aacgtgtcca cccggtcatg cccgccgcgc accagccccg cagtggactt ggaggaggag 120 gaggaggaga gctctgtgga tggcaaaggg gaccggaaga gcacaggcct gaaactctcc 180 aagaagaaag caaggaggag acacacggat gacccaagca aggaatgctt cactctgaaa 240 tttgacctga atgtggacat tgagacagag atcgtcccag ccatgaagaa gaagtcactg 300 ggggaggtgc tgctgcctgt atttgaaagg aagggcattg cgctgggcaa agtggacatc 360 tacctggacc agtccaacac acccctgtcc ctcaccttcg aggcctacag gttcggggga 420 cactaccttc gtgtcaaagc cccagccaag cctggagatg agggcaaggt ggagcagggc 480 atgaaggact ccaagtccct gagtttgccg attctgcggc cagctgggac cgggcccccc 540 gccctggagc gtgtggacgc ccagagccgc cgggagagcc tggacatctt ggcccctggc 600 cgccgccgca agaacatgtc ggagttcctg ggggaggcga gcatccccgg gcaggagccc 660 cccacgccct ccagctgctc tctgcccagc ggcagcagtg gcagcaccaa cactggcgac 720 agctggaaga accgggcggc cagtcgcttc agcggctttt tcagctccgg ccccagcacc 780 agcgcctttg gccgggaggt agacaagatg gagcagctgg agggcaagct gcacacctac 840 agcctcttcg ggctgcccag gctgccccgg gggctgcgct tcgaccatga ctcctgggag 900 gaggagtacg atgaagacga ggatgaggac aatgcctgcc tgaggctgga ggacagctgg 960 cgggagctca ttgatgggca tgagaagctg acccggcggc agtgccacca gcaggaggcg 1020 gtgtgggagc tgctgcacac ggaggcctcc tacatcagga aactgcgggt gatcatcaac 1080 ctgttcctgt gctgcctcct gaacctgcaa gagtcagggc tgctgtgtga ggtggaggcg 1140 gagcgcctgt tcagcaacat cccggagatc gcgcagctgc accgcaggct gtgggctagc 1200 gtgatggcgc cggtgctgga gaaggcgcgg cgcacgcgag cgctgctaca gcccggggac 1260 ttcctcaaag gcttcaagat gttcggctcg ctcttcaagc cctacatccg ctactgcatg 1320 gaggaggagg gctgcatgga gtacatgcgc ggcctgctgc gcgacaacga cctcttccgg 1380 gcctacatca cgtgggcgga gaagcaccca cagtgccaga ggctgaagct gagcgacatg 1440 ctggccaaac cccaccagcg gctcaccaag tacccgctgc tgctcaagtc ggtgctgagg 1500 aagaccgagg agccgcgcgc caaggaggcc gtcgtcgcca tgatcggctc cgtggagcgc 1560 ttcatccacc acgtgaacgc gtgcatgcgg cagcggcagg agcggcagcg gctggcggcc 1620 gtggtgagcc gcatcgacgc ctacgaggtg gtggaaagca gcagcgacga agtggacaag 1680 ctcctgaagg aatttctgca cctggacttg acagcgccca tccctggcgc ctccccggag 1740 gagacgcggc agctgctgct ggaggggagc ctgaggatga aggaggggaa ggacagcaag 1800 atggatgtgt actgcttcct cttcacggat ctgctgttgg tgaccaaagc agtgaagaag 1860 gcagagagga ccagggtcat caggccaccc ctgctcgtgg acaagattgt gtgccgggag 1920 ctacgggacc ctgggtcctt cctccttatc tacctgaatg agtttcacag tgctgtaggg 1980 gcctacacgt tccaggccag tggccaggcc ttgtgccgtg gctgggtgga caccatttac 2040 aatgcccaga accagctgca acagctgcgt gcacaggagc ccccaggcag tcagcagccc 2100 ctgcagagcc tggaagagga ggaggatgag caggaggagg aagaggagga ggaggaggag 2160 gaggaggaag gcgaggacag tggcacttca gctgccagct cccctaccat catgcggaaa 2220 agcagcggca gccccgactc tcagcactgt gcctcagatg gctccacgga gaccctggcc 2280 atggttgtgg tagagcctgg ggacacgctg tcctcccccg agttcgacag cggtcctttc 2340 agctcccagt ctgatgagac ctctctcagc accactgcct catctgccac gcccaccagt 2400 gagctgctgc ccctgggtcc ggtggacggc cgctcctgct ccatggactc tgcctacggc 2460 accctctccc caacctcctt acaagacttt gtggccccag gcccaatggc agagctagtg 2520 cctcgggccc cagagtcccc acgagttcct tcccctccac cctcgccccg tctccgccgc 2580 cgcacccctg tccagctgtt gagctgcccg ccccacctgc tcaagtctaa gtccgaggcc 2640 agcctcctcc agctgctggc aggggctggc acccatggga caccctctgc ccccagccgc 2700 agcctgtcag agctctgcct ggctgttcca gccccagcac aggaagctga ccctggccca 2760 gctctaccga atcaggacca ccctgctgct taa 2793 <210> 2 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> D17S921_F <400> 2 gtgttgtatt aggcagagtt ctcc 24 <210> 3 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> D17S921_R <400> 3 ggcagtagat ggtgacttta tggc 24 <210> 4 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> D17S9B_F <400> 4 tctcagtcct gatttcttga ttttg 25 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> D17S9B_R <400> 5 ccagagctaa caccacattc a 21 <210> 6 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> D17S9A_F <400> 6 caaccatcag tgatttgatg gtttac 26 <210> 7 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> D17S9A_R <400> 7 gagttgtcac tagaaccctg ttc 23 <210> 8 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> D17S918_F <400> 8 tcctgtaatc tgtccccaaa cgtc 24 <210> 9 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> D17S918_R <400> 9 ttcctcacac aacctattga tagtc 25 <210> 10 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> D17S4A_F <400> 10 ctgtggagga aagaaaacac tgcc 24 <210> 11 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> D17S4A_R <400> 11 gcactaaagt agcttgtaac tctg 24 <210> 12 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> D17S2230_F <400> 12 aggaaactga tgtctaaaac tatcc 25 <210> 13 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> D17S2230_R <400> 13 gtaatccag gaggcagagc ttgc 24 <210> 14 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> MPZ-N1F <400> 14 tccatatcta tccctcagag aagt 24 <210> 15 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> MPZ-N1R <400> 15 ctgaatataa tgtcaccttc ctgc 24 <210> 16 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> MPZ-2F <400> 16 ctgatctcac ttcctctgta tcc 23 <210> 17 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> MPZ-2R <400> 17 ctttgaagca ctttctgtta tcc 23 <210> 18 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> MPZ-3F <400> 18 acagctgtgt tctcattagg gtc 23 <210> 19 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> MPZ-3R <400> 19 ctcccaaact gcttcccata cc 22 <210> 20 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> MPZ-4F <400> 20 ctaggaacca cagatacagg gc 22 <210> 21 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> MPZ-4R <400> 21 gggttctcct tcccatcttg tc 22 <210> 22 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> MPZ-6F <400> 22 aacagtcaag ccccagtcgc tc 22 <210> 23 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> MPZ-6R <400> 23 gctcatcctt tcgtagctcc atc 23 <210> 24 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> PMP-P1F <400> 24 cacagatgca tgggataggg gtcc 24 <210> 25 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> PMP-P1R <400> 25 atggagatga ggtacagtgt gggc 24 <210> 26 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> PMP-1AF <400> 26 atctctgcag aattcactgg gag 23 <210> 27 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> PMP-1AR <400> 27 gcataggcac acatcaccca gag 23 <210> 28 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> PMP-1BF <400> 28 tcgtaggtgc agacagtaat agc 23 <210> 29 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> PMP-1BR <400> 29 atgcccacat ttctcctcca tctc 24 <210> 30 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> PMP-2F <400> 30 cgcaggcaga aactccgctg agc 23 <210> 31 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> PMP-2R <400> 31 cgtctgagac ttccaactgt ctgc 24 <210> 32 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> PMP-3F <400> 32 aacgttggct cttaccatgc agg 23 <210> 33 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> PMP-3R <400> 33 tggactcatg gctccctgtc ac 22 <210> 34 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> PMP-4F <400> 34 atgtatgtaa tgtgtacata ttggcac 27 <210> 35 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> PMP-4R <400> 35 ttggatgcac cccgcttcca ca 22 <210> 36 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> PMP-5F <400> 36 tctgccatgg actctccgtc ac 22 <210> 37 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> PMP-5R <400> 37 gtttgggatt ttgggctagc tc 22 <210> 38 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> P1F <400> 38 tgcagcttgc ccgcactgtg gatc 24 <210> 39 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> P1R <400> 39 ccggcccact gtgccacatc agc 23 <210> 40 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> P2F <400> 40 tcccctcttc acatccacct 20 <210> 41 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> P2R <400> 41 gctccttaac tccagacctg 20 <210> 42 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Cx-E2aF <400> 42 gctactggct cttggaagag ttga 24 <210> 43 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Cx-E2aR <400> 43 tccccatggc cctcaagccg tagc 24 <210> 44 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Cx-E2bF <400> 44 tgtggtccct gcagctcatc ctag 24 <210> 45 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Cx-E2bR <400> 45 ccggatgatg aggtacacca cctc 24 <210> 46 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Cx-E2cF <400> 46 tcttcaccgt cttcatgcta gctg 24 <210> 47 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Cx-E2cR <400> 47 tccccagcag gcagaggcct gtgc 24 <210> 48 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> 3F <400> 48 agcattccgc catctcccta gcag 24 <210> 49 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> 3R <400> 49 cacccgcaaa tcccactagc taa 23 <210> 50 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> 4F <400> 50 tccagacttg ggactgtgga ac 22 <210> 51 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> 4R <400> 51 tggaacgttc tgtgaccttg cac 23 <210> 52 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> 5F <400> 52 ccaggctggt atctgcgttg tga 23 <210> 53 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> 5R <400> 53 gtgtcacaac ggaggacttg ctc 23 <210> 54 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> 6F <400> 54 tgtgatgcag cggcakhga aatc 24 <210> 55 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> 6R <400> 55 ttccagtttg gacctc 16 <210> 56 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> 7F <400> 56 ggcaccatag ggctcctgct ctgcctgatg a 31 <210> 57 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> 8R <400> 57 tccctcgggg ttgcattc 18 <210> 58 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> 9F <400> 58 gcactgccca gcctcttatg acctattc 28 <210> 59 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> 9R <400> 59 tgacagactc ctcagcacga gac 23 <210> 60 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> 10F <400> 60 ctgctgccaa gttgtttctg gac 23 <210> 61 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> 11R <400> 61 tccacctatc tgcagtcttg gac 23 <210> 62 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> 12F <400> 62 cctctgctta gtcagacagg aac 23 <210> 63 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> 12R <400> 63 ggactctact cggagtccaa atc 23 <210> 64 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> 13F <400> 64 tgctgcagga gtgaactttg gtc 23 <210> 65 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> 14R <400> 65 cagggccaca gctgcccagt tc 22 <210> 66 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> 15F <400> 66 gtgcagggct gagctgataa g 21 <210> 67 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> 15R <400> 67 atggttcccg tgtctggagg cag 23 <210> 68 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> 16F <400> 68 actagggcaa cactgagggt tcac 24 <210> 69 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> 16R <400> 69 tatgcagtgg actgtggagt gtg 23 <210> 70 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> 17F <400> 70 ggccacagag aggctgcact cca 23 <210> 71 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> 17R <400> 71 cctgaaggcc cagggtctac ga 22 <210> 72 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> 18F <400> 72 taagccacca gtggcatctt gc 22 <210> 73 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> 18R <400> 73 tttgtgtcca cacccaagac gc 22 <210> 74 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> 19F <400> 74 tcaagcgtcc ttaggatggt gc 22 <210> 75 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> 19R <400> 75 atggtacgag actgggtgct tc 22 <210> 76 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> PLEKHG5_T663M-F <400> 76 ggggcctaca tgttccaggc cagtg 25 <210> 77 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> PLEKHG5_T663M-R <400> 77 gcctggaaca tgtaggcccc tacagc 26 <210> 78 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> PLEKHG5_G820R-F <400> 78 ctctgcctac cgcaccctct cccc 24 <210> 79 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> PLEKHG5_G820R-R <400> 79 agagggtgcg gtaggcagag tccatg 26 <210> 80 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PLEKHG5-seq_961 <400> 80 ggccagtcgc ttcagcggct 20 <210> 81 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PLEKHG5-seq_1921 <400> 81 gcacctggac ttgacagcgc 20 <210> 82 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> PLEKHG5-seq_2401 <400> 82 cagtgcactt cagctgcca 19

Claims (11)

Genetic causes of mutations in the G-member 5 (PLEKHG5) gene, a pleckstrin homology domain-containing, family of Charcot-Marie-Tooth disease (CMT)
2. The genetic mutation marker of claim 1, wherein the PLEKHG5 gene comprises the nucleotide sequence of SEQ ID NO: 1.
2. The genetic mutation marker of claim 1, wherein the mutation marker is c.1988C > T or c.2458G > C in the PLEKHG5 gene.
Heterozygous recessive inheritance marker of the PLEKHG5 gene for CMT diagnosis.
5. The heterozygous recessive inheritance marker according to claim 4, wherein the heterozygous recessive inheritance marker is the 1988 th base from the starting point of the ATG translation initiation codon in the PLEKHG5 gene, and the 2458th base is cytosine.
A CMT diagnostic kit comprising the marker of claim 1 or claim 4.
A CMT diagnostic kit comprising a primer set capable of amplifying each of the mutation sites characterized by c.1988C > T or c.2458G > C of the PLEKHG5 gene.
1) isolating genomic DNA from a sample derived from an individual;
2) detecting the mutation site of the PLEKHG5 gene in the genomic DNA isolated in step 1); And
3) Identifying the mutation site detected in step 2). A method for identifying a mutation site to provide information on the diagnosis or onset risk prediction of CMT.
[9] The method according to claim 8, wherein the sample in step 1) is blood. [9] A method for identifying a mutation site for providing information on the diagnosis or risk prediction of CMT.
9. The method according to claim 8, wherein the mutation in step 2) is c.1988C > T or c.2458G > C.
9. The method of claim 8, wherein the detection in step 2) includes sequencing analysis, microarray hybridization, allele specific PCR, dynamic allele-specific hybridization (DASH) , PCR extension analysis, or TaqMan probe PCR analysis. The method for identifying a mutation site to provide information on the diagnosis or risk prediction of CMT.

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