KR20180105597A - Kit for Diagnosing Charcot-Marie-Tooth - Google Patents

Abstract

Provided are a kit for diagnosing a gene causing a Charcot-Marie-Tooth disease based on next-generation sequencing, and a diagnosis method. According to the present invention, a one-step diagnosis system is built by simplifying an existing identification algorithm of a gene causing a Charcot-Marie-Tooth disease, thereby saving cost and time while increasing the identification efficiency of the gene, and also identifying new gene mutation in the Charcot-Marie-Tooth disease.

Description

Kit for Diagnosing Charcot-Marie-Tooth}

The present invention relates to a kit for diagnosing Charcoal-Marie-tus disease.

Charcot-Marie-Tooth (CMT) disease or hereditary motor and sensory neuropathy (HMSN) is distal hereditary motor neuropathy (dHMN), hereditary sensory neuropathy. ; HSN) and Hereditary Neuropathy with a liability to pressure palsy (HNPP). Genetically, more than 70 genes of several types have been reported as the cause of CMT disease (Rossor AM, et al. Nat Rev Neurol. 2013;9:562-71, http://www.molgen.ua.ac.be /cmtmutations/ ).

The genetic diagnosis of CMT relies on direct sequencing of the main causative gene, but this has a low separation efficiency despite a difficult process. With the advent of Whole Exome Sequencing (WES) based on Next-generation Sequencing (NGS) technology, genetic studies of various diseases, especially heterogeneous diseases such as CMT, are expanding and accelerating.

Throughout this specification, a number of papers and patent documents are referenced and citations are indicated. The disclosure contents of the cited papers and patent documents are incorporated by reference in this specification as a whole, and the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly described.

The present inventors have tried to develop a new diagnostic kit that can reduce cost and time while simplifying the algorithm for identifying the causative genes of Charcot-Marie-Tooth disease, which is composed of multiple steps, and increasing identification efficiency. I did. As a result, the present invention was completed by developing a kit for diagnosing the causative gene of Sharko-Marie-tus disease, which is composed of the causative gene of Sharko-Marie-tus disease and genes that are closely related, and by identifying a new causal gene mutation.

Accordingly, an object of the present invention is to provide a kit for diagnosing the cause of the Charcoal-Marie-tus disease.

Another object of the present invention is to provide a method for diagnosing the cause of the Charcoal-Marie-tus disease.

Another object of the present invention is to provide a mutant gene for a diagnostic marker of Charcoal-Marie-tus disease.

Other objects and advantages of the present invention will become more apparent by the following detailed description, claims and drawings.

According to an aspect of the present invention, the present invention is AARS (GenBank access number NM_001605), AIEM1 (NM_004208), ARIIGER10 (NM_014629), ATL1 (NM_015915), ATL3 (NM_015459), BSCL2 (NM_032667), C12orf65 ( NM_152269), CCT5 (NM_012073), CTDP1 (NM_004715), DCTN1 (NM_004082), DHTKD1 (NM_018706), DNAJB2 (NM_006736), DNM2 (NM_004945), DNMT1 (NM_001379), EST2 (NM_DYNC1) NM_000399), FAM134B(NM_001034850), FBLN5(NM_006329), FGD4(NM_139241), FIG4(NM_014845), GAN(NM_022041), GARS(NM_002047), GDAP1_0(NM_018972), GJB1(29NB), GJB1(NM_216), NM_216 NM_000183), HARS(NM_002109), HINT1(NM_005340), HK1(NM_000188), HOXD10(NM_002148), HSPB1(NM_001540), HSPB3(NM_006308), HSPB8(NM_014365), IGRAPD1(NM_180P2), IGRAPD1(NM_KBK2P2) NM_003640), INF2(NM_022489), KARS(NM_005548), KIF1A(NM_004321), LITAF(NM_004862), LMNA(NM_170707), LRSAM1(NM_138361), MARS(NM_004990), MED25874(NM_973), MED25(NM_973) NM_000530), MTMR2(NM_016156), MYH14(NM_024729), NDRG1(NM_006096), NEFL(NM_006158), NGF(NM_002506), NTRK1(NM_002529), NRG2(NM_0139 82), PDK3(NM_005391), PLEKHG5(NM_020631), PMP22(NM_000304), PRPS1(NM_002764), PRX(NM_181882), RAB7A(NM_004637), REEP1(NM_022912), SBF1(NM_002972), SBF1(NM_002972), SBF1(NM_002972), SBF1(NM_002972) NM_015046), SCN9A(NM_002977), SH3TC2(NM_024577), SLC5A7(NM_021815), SLC12A6(NM_005135), SOX10(NM_006941), SPTLC1(NM_006415), SPTLC1(NM_006415), SPTLC2(NM_0048632), TDP1TFG(NM_0NM_006), TDP1183M_006 NM_015271), TRPV4 (NM_021625), TUBB3 (NM_006086), VAPB (NM_004738), WNK1 (NM_213655), YARS (NM_003680), COX10 (NM_001303) and TEKT3 (NM_031898). It provides a kit for diagnosing the cause of a next generation sequencing-based Charcot-Marie-Tooth disease including a primer or probe that specifically binds to it.

The present inventors have tried to develop a new diagnostic kit that can reduce cost and time while simplifying the algorithm for identifying the causative gene of Charcoal-Marie-tus disease composed of multiple steps, and increasing identification efficiency. As a result, a kit for diagnosing the causative gene of Sharko-Marie-tus disease, consisting of genes that cause Charcoal-Marie-tus disease and closely related genes, was developed, and new causative gene mutations were identified.

The Charcoal-Marie-tus disease to be diagnosed in the present invention is also referred to as hereditary motor and sensory neuropathy, and is a group of peripheral nerve diseases that are genetically and clinically very heterogeneous, and distal muscle weakness and atrophy, urinary foot ( pes caus) and sensory impairment (Reilly MM, et al., J Neurol Neurosurg Psychiatry 80: 1304-1314, 2009).

The conventional method for diagnosing genetic mutations in Charcoal-Marie-tus disease is to analyze subtypes through NCS (Nerve Conduction Study), and after double-testing of the PMP22 gene, mutations in the major causative genes (PMP22, MPZ, Cx32 and MFN2). Was investigated, and the diagnosis was confirmed through whole exome sequencing and capillary sequencing.

The present invention provides a next-generation sequencing-based kit capable of diagnosing the causative gene of Charcoal-Marie-tus disease in one step.

In the present specification, the term “next-generation sequencing (massive parallel sequencing)” refers to an analysis method for decoding the genome by dividing the genome into countless pieces and combining each nucleotide sequence.

According to one embodiment of the present invention, the next-generation sequencing analysis is exome sequencing. The exome sequencing is a technique for selectively sequencing the coding region of the genome, and is a technique for sequencing only the exon region that constitutes about 1% of the human genome and makes most of the protein (see Bahareh Rabbani et al. Journal of Humn Genetics (Reference: Bahareh Rabbani et al. Journal of Humn Genetics). 2014) 59, 5-15, https://en.wikipedia.org/wiki/Exome_sequencing).

According to another embodiment of the present invention, the exome sequencing is SBS (Sequencing-By-Synthesis) method. In the SBS method, a single-stranded DNA fragment is attached to a substrate, and then the fragments are polymerized to form a cluster. In this process, the type of base hybridized to the DNA fragment to be tested is identified through a signal to detect the sequence (see: http://www.illumina.com/technology/next-generation-sequencing/sequencing-technology. html and https://en.wikipedia.org/wiki/Illumina_dye_sequencing).

The kit for diagnosing the cause of the next-generation sequencing-based Charcoal-Marie-tus disease of the present invention is amplified using a primer suitable for next-generation sequencing.

The primers were designed using Sureselect (Agilent Technologies, Santa Clara, CA).

* The primer used in the kit of the present invention has a sequence complementary to the nucleotide sequence of the present invention. In the present specification, the term "complementary" means having a degree of complementarity capable of selectively hybridizing to the nucleotide sequence of the present invention listed above under certain hybridization or annealing conditions. Therefore, the term “complementary” has a different meaning from the term perfectly complementary, and the primer or probe of the present invention is sufficient to selectively hybridize to the nucleotide sequence of the present invention. It can have a mismatch sequence. As used herein, the term “primer” refers to a single template that can serve as an initiation point for template-directed DNA synthesis under suitable conditions (ie, four different nucleoside triphosphates and polymerases) in a suitable buffer at a suitable temperature. -Means a stranded oligonucleotide. The suitable length of the primer varies depending on various factors such as temperature and the application of the primer, but is typically 15-30 nucleotides. Short primer molecules generally require lower temperatures to form sufficiently stable hybrid complexes with the template.

The sequence of the primer does not need to have a sequence that is completely complementary to some of the sequences of the template, and it is sufficient to have sufficient complementarity within the range capable of hybridizing with the template to perform a unique function of the primer. Therefore, the primer in the present invention does not need to have a sequence perfectly complementary to the nucleotide sequence of the present invention listed above as a template, and it is sufficient if it has sufficient complementarity within the range capable of hybridizing to this gene sequence to function as a primer. The design of such a primer can be easily carried out by a person skilled in the art by referring to the nucleotide sequence of the present invention, for example, it can be done using a primer design program (eg, SURESELECT program).

The term "amplification" as used herein refers to a reaction to amplify a nucleic acid molecule. Since the present invention analyzes (sequencing) the nucleotide sequence, the nucleotide sequence of the nucleotide sequence of the present invention is determined from a sample to be analyzed. Therefore, in principle, the present invention performs an amplification reaction using a nucleotide sequence in a sample as a template and a primer binding thereto.

The primer used in the present invention is hybridized or annealed at one site of the template to form a double-chain structure. Conditions for nucleic acid hybridization suitable for forming such a double-stranded structure are Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001) and Haymes, BD, et al., Nucleic Acid Hybridization. , A Practical Approach, IRL Press, Washington, DC (1985).

According to another aspect of the present invention, the present invention provides AARS (GenBank accession number NM_001605), AIEM1 (NM_004208), ARIIGER10 (NM_014629), ATL1 (NM_015915), ATL3 (NM_015459), BSCL2 (NM_032667), C12orf65 ( NM_152269), CCT5 (NM_012073), CTDP1 (NM_004715), DCTN1 (NM_004082), DHTKD1 (NM_018706), DNAJB2 (NM_006736), DNM2 (NM_004945), DNMT1 (NM_001379), EST2 (NM_DYNC1) NM_000399), FAM134B(NM_001034850), FBLN5(NM_006329), FGD4(NM_139241), FIG4(NM_014845), GAN(NM_022041), GARS(NM_002047), GDAP1_0(NM_018972), GJB1(29NB), GJB1(NM_216), NM_216 NM_000183), HARS(NM_002109), HINT1(NM_005340), HK1(NM_000188), HOXD10(NM_002148), HSPB1(NM_001540), HSPB3(NM_006308), HSPB8(NM_014365), IGRAPD1(NM_180P2), IGRAPD1(NM_KBK2P2) NM_003640), INF2(NM_022489), KARS(NM_005548), KIF1A(NM_004321), LITAF(NM_004862), LMNA(NM_170707), LRSAM1(NM_138361), MARS(NM_004990), MED25874(NM_973), MED25(NM_973) NM_000530), MTMR2(NM_016156), MYH14(NM_024729), NDRG1(NM_006096), NEFL(NM_006158), NGF(NM_002506), NTRK1(NM_002529), NRG2(NM_013 982), PDK3(NM_005391), PLEKHG5(NM_020631), PMP22(NM_000304), PRPS1(NM_002764), PRX(NM_181882), RAB7A(NM_004637), REEP1(NM_022912), SBF1(NM_002972), SBF1(NM_002972), SBF1(NM_002972), SBF1(NM_002972) NM_015046), SCN9A(NM_002977), SH3TC2(NM_024577), SLC5A7(NM_021815), SLC12A6(NM_005135), SOX10(NM_006941), SPTLC1(NM_006415), SPTLC1(NM_006415), SPTLC2(NM_0048632), TDP1TFG(NM_0NM_006), TDP1183M_006 NM_015271), TRPV4 (NM_021625), TUBB3 (NM_006086), VAPB (NM_004738), WNK1 (NM_213655), YARS (NM_003680), COX10 (NM_001303), and TEKT3 (NM_031898). It provides a next-generation sequencing-based method for diagnosing the causative gene of Charcoal-Marie-tus disease, comprising the step of amplifying using a primer or probe that specifically binds to the sequence.

According to an embodiment of the present invention, the next-generation sequencing is exome sequencing. According to another embodiment of the present invention, the exome sequencing is SBS method.

The primers were designed using Sureselect (Agilent Technologies, Santa Clara, CA).

The present invention is a method for diagnosing the causative gene using the same primers or probes as the kit for diagnosing the causative gene of the Next Generation Sequencing-based Charcot-Marie-Tooth disease. Description of the common content between the two is omitted in order to avoid excessive complexity of the present specification.

The present invention provides a novel diagnostic marker of the Charco-Marie-tus disease identified while confirming the clinical effectiveness using the next-generation sequencing-based kit for diagnosing the cause of the Charcoal-Marie-tus disease.

The selection and application of significant diagnostic markers determines the reliability of the diagnostic results. Significant diagnostic markers refer to markers with high reliability so that results obtained through diagnosis are accurate and thus have high validity and consistent results even when repeated measurements are made. The mutant gene as a diagnostic marker according to the present invention is a highly reliable marker that is detected only in a patient group exhibiting Charcoal-Marie-tus disease and has little probability of being detected in a normal control group. Therefore, the diagnosis result based on the result obtained by detecting the presence or absence of a mutant gene as a significant diagnostic marker of the present invention can be reasonably reliable.

According to another aspect of the present invention, the present invention provides a mutant gene in which nucleotide 47 of the wild-type PMP22 gene is substituted with cytosine as a diagnostic marker for Charcoal-Marie-tus disease.

According to one embodiment of the present invention, the wild-type PMP22 gene is encoded by the nucleotide sequence of SEQ ID NO: 1.

The 47th nucleotide of the wild-type PMP22 gene is substituted with cytosine, so that the 16th amino acid of the wild-type PMP22 protein becomes a mutant protein in which arginine is substituted.

According to another aspect of the present invention, the present invention provides a mutant gene in which nucleotides 929 and 3272 of the wild-type SH3TC2 gene are substituted with adenine and thymine, respectively, as a diagnostic marker for Charcoal-Marie-tus disease. .

According to one embodiment of the present invention, the wild-type SH3TC2 gene is encoded by the nucleotide sequence of SEQ ID NO: 2.

The 929th and 3272th nucleotides of the wild-type SH3TC2 gene are replaced with adenine and thymine, respectively, so that the 310th and 1091th amino acids of the wild-type SH3TC2 protein are replaced with glutamic acid and valine, respectively.

According to another aspect of the present invention, the present invention provides a mutant gene in which the 154th nucleotide of the wild-type MPZ gene is substituted with guanine as a diagnostic marker for Charcoal-Marie-tus disease.

According to one embodiment of the present invention, the wild-type MPZ gene is encoded by the nucleotide sequence of SEQ ID NO: 3.

The 154th nucleotide of the wild-type MPZ gene is replaced with guanine, so that the 52nd amino acid of the wild-type MPZ protein becomes a mutant protein in which valine is substituted.

According to another aspect of the present invention, the present invention provides a mutant gene in which nucleotide 262 of the wild-type MPZ gene is substituted with cytosine as a diagnostic marker for Charcoal-Marie-tus disease.

According to one embodiment of the present invention, the wild-type MPZ gene is encoded by the nucleotide sequence of SEQ ID NO: 3.

The 262nd nucleotide of the wild-type MPZ gene is substituted with cytosine, and the 88th amino acid of the wild-type MPZ protein becomes a mutant protein in which histidine is substituted.

According to another aspect of the present invention, the present invention provides a mutant gene in which nucleotide 286 of the wild-type GJB1 gene is substituted with cytosine as a diagnostic marker for Charcoal-Marie-tus disease.

* According to one embodiment of the present invention, the wild-type GJB1 gene is encoded by the nucleotide sequence of SEQ ID NO: 4.

The 286th nucleotide of the wild-type GJB1 gene is replaced with cytosine, and the 96th amino acid of the wild-type GJB1 protein becomes a mutant protein in which proline is substituted.

According to another aspect of the present invention, the present invention provides a mutant gene in which nucleotide 435 of the wild-type SPTLC2 gene is substituted with thymine as a diagnostic marker for Charcoal-Marie-tus disease.

According to one embodiment of the present invention, the wild-type SPTLC2 gene is encoded by the nucleotide sequence of SEQ ID NO:5.

Nucleotide 435 of the wild-type SPTLC2 gene is substituted with thymine to become a mutant protein in which the 145th amino acid of the wild-type SPTLC2 protein is substituted with serine.

According to another aspect of the present invention, the present invention provides a mutant gene in which nucleotide 1019 of the wild-type DCTN1 gene is substituted with guanine as a diagnostic marker for Charcoal-Marie-tus disease.

According to one embodiment of the present invention, the wild-type DCTN1 gene is encoded by the nucleotide sequence of SEQ ID NO:6.

The 1019th nucleotide of the wild-type DCTN1 gene is replaced with guanine, so that the 340th amino acid of the wild-type DCTN1 protein becomes a mutant protein in which glycine is substituted.

According to another aspect of the present invention, there is provided a mutant protein encoded by the mutant gene as a diagnostic marker for Charcoal-Marie-tus disease.

The mutant protein is a mutant protein by amino acid substitution according to the nucleotide substitution of the mutant gene, and descriptions of the contents in common between the two are omitted in order to avoid undue complexity in the present specification.

According to another aspect of the present invention, there is provided a kit for diagnosing a cause of Charcoal-Marie-tus disease gene comprising an agent capable of detecting the mutant gene or the mutant protein.

According to another aspect of the present invention, there is provided a method for diagnosing a cause gene of Charcoal-Marie-tus disease, comprising an agent capable of detecting the mutant gene or the mutant protein.

The features and advantages of the present invention are summarized as follows:

(a) The present invention provides a kit and a diagnostic method for diagnosing the causative gene of Next Generation Sequencing-based Charcot-Marie-Tooth disease.

(b) The diagnostic kit and method of the present invention simplifies the existing algorithm for identifying the causative gene of the complex Charcoal-Marie-tus disease to construct a one-step diagnostic system.

(c) The diagnostic kit and method of the present invention can reduce cost and time while increasing the efficiency of identification of the causative gene.

(d) In addition, the present invention has identified a novel causative gene mutation of Charcoal-Marie-tus disease.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for describing the present invention in more detail, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Example

Experimental materials and test methods

object

A total of 243 Charcot-Marie-Tooth (CMT) disease patients or normal household members of the patient were enrolled in this experiment. Clinical evaluation was conducted by two independent neurologists. All participants provided informed consent according to the procedure approved by the Institutional Bioethics Committee of Sungkyunkwan University and Samsung Medical Center.

Design of the gene panel

Target genes were selected through extensive literature searches and databases. Primer design was performed with Sureselect (Agilent Technologies, Santa Clara, CA). A total of 1669 sites were read with the 6298 probe. With 99.5% coverage, the total size is 433 Mb.

Sequencing and identification of causal variations

Peripheral blood DNA was purified using a QIAamp blood DNA purification kit (Qiagen, Hilden, Germany). Patient samples were pre-screened for 17p12, a major genetic cause of dehydration CMT, using hexaplex microsatellite PCR (6). Sequencing was performed with a HiSeq2500 Gemon Analyzer (Illumina, San Diego, CA, USA), and UCSC assembly hg19 was used as a reference sequence. Functionally significant variants (misense, nonsense, exon indel and splicing site variants) were selected and dbSNP142 ( http://www.ncbi.nlm.nih.gov ), 1000 Genome Project Database ( http: //www.1000genomes.org/ ) and exome variant servers ( http://evs.gs.washington.edu/EVS/ ) were additionally selected and excluded as new or rare (MAF≤0.01) variants. Sanger sequencing was used to identify candidate variants.

result

Preparation and calibration of the CMT gene panel

To prepare a CMT gene panel, a total of 81 genes (Table 1) were selected, and primers were designed according to Sureselect (Agilent) optimized for HiSeq2500 Genome Analyzer (Illumina). To calibrate the newly developed gene panel, 114 patients with pre-determined causal mutations were tested in our hospital. As expected, all mutations could be detected from the sample. Thus, the effectiveness of the newly developed CMT gene panel was sufficiently confirmed.

gene Phenotype Hereditary form Loci GenBank access number Coverage AARS CMT2N AD 16q22 NM_001605.2 100% AIFM1 CMTX4 XR Xq26.1 NM_004208.3 100% ARHGEF10 CMT1 AD 8p23 NM_014629.2 100% ATL1 HSN1D AD 14q22.1 NM_015915.4 100% ATL3 HSAN AD 11q13.1 NM_015459.3 100% BSCL2 dHMN5 AD 11q13 NM_032667.6 100% C12orf65 CMT6 AD 12q24.31 NM_152269.4 100% CCT5 HSN AR 5p15.2 NM_012073.3 100% CTDP1 Nuropathy, cataract AR 18q23 NM_004715.4 100% DCTN1 dHMN7B AD 2p13 NM_004082.4 100% DHTKD1* CMT2Q AD 10p14 NM_018706.6 100% DNAJB2 CMT2T AR 2q32 NM_006736.5 100% DNM2 DI-CMTB, CMT2M AD 19p13.2 NM_004945.3 100% DNMT1 HSN1E AD 19p13.2 NM_001379.2 98% DST HSAN6 AR 6p12.1 NM_001723.5 100% DYNC1H1 CMT2O AD 14q32 NM_001376.4 100% EGR2 CMT1D, DSS AD 10q21.1 NM_000399.3 100% FAM134B HSN2B AR 5p15.1 NM_001034850.2 100% FBLN5 CMT1 AD 14q32.1 NM_006329.3 92% FGD4 CMT4H AR 12p11.21 NM_139241.2 98% FIG4 CMT4J AR 6q21 NM_014845.5 100% GAN GAN AR 16q24.1 NM_022041.3 100% GARS CMT2D, dHMN5A AD 7p15 NM_002047.2 100% GDAP1 CMT2K, CMT4A, RI-CMTA AR, AD 8q21.11 NM_018972.2 100% GJB1 CMTX1 XD Xq13.1 NM_000166.5 100% GNB4 CMT-DIF AD 3q26.33 NM_021629.3 100% HADHB CMT2 AR 2p23 NM_000183.2 100% HARS Axonal peripheral neuropathy AD 5q31.3 NM_002109.4 99% HINT1 AR-CMT2 AR 5q31.2 NM_005340.6 94% HK1* HMSN-Russe/CMT4G) AR 10q22 NM_000188.2 97% HOXD10 CMT with congenital vertical talus AD 2q31.1 NM_002148.3 100% HSPB1 CMT2F, dHMN2B AD 7q11.23 NM_001540.3 100% HSPB3 dHMN2C AD 5q11.2 NM_006308.2 100% HSPB8 CMT2L, dHMN2A AD 12q24.23 NM_014365.2 100% IFRD1 HMSN with ataxia (SMNA) AD 7q31.1 NM_001550.3 100% IGHMBP2 dHMN6 AR 11q13.3 NM_002180.2 100% IKBKAP HSAN3 AR 9q31 NM_003640.3 100% INF2 CMT-DIE AD 14q32.33 NM_022489.3 99% KARS RI-CMT2C AD 16q23.1 NM_005548.2 100% KIF1A HSN2C AR 2q37.3 NM_004321.6 100% LITAF CMT1C AD 16p13.13 NM_004862.3 88% LMNA CMT2B1 AD>AR 1q22 NM_170707.3 100% LRSAM1 CMT2P AR 9q33.3 NM_138361.5 100% MARS CMT2 AD 12q13.3 NM_004990.3 100% MED25 CMT2B2 AR 19q13.3 NM_030973.3 98% MFN2 CMT2A, CMT6 AD 1p36.22 NM_014874.3 100% MPZ CMT1B, DI-CMT CMT2, DSS AD 1q23.3 NM_000530.6 100% MTMR2 CMT4B1 AR 11q22 NM_016156.5 98% MYH14 PN with myopathy AD 19q13.33 NM_024729.3 100% NDRG1 CMT4D AR 8q24.3 NM_006096.3 100% NEFL CMT1F, CMT2E AD 8q21 NM_006158.4 100% NGF HSAN5 AR 1p13.1 NM_002506.2 100% NTRK1 HSN IV AR 1q21-q22 NM_002529.3 100% NRG2 CMT4 AR 5q23-q33 NM_013982.2 100% PDK3 CMTX6 XD Xp22.11 NM_005391.4 100% PLEKHG5 RI-CMT AR, AD 1p36.31 NM_020631.4 99% PMP22 CMT1A, HNPP AD 17p12 NM_000304.3 100% PRPS1 CMTX5 XR Xq22.3 NM_002764.3 100% PRX CMT4F, DSS AR 19q13.2 NM_181882.2 100% RAB7A CMT2B AD 3q21.3 NM_004637.5 100% REEP1 dHMN5 AD 2p11.2 NM_022912.2 94% SBF1 CMT4B3 AR 22q13.33 NM_002972.2 98% SBF2 CMT4B2 AR 11p15.4 NM_030962.3 99% SETX dHMN AD 9q34.13 NM_015046.5 100% SCN9A* HSAN2D AR 2q24 NM_002977.3 100% SH3TC2* CMT4C AR 5q32 NM_024577.3 97% SLC5A7 dHMN7A AD 2q12 NM_021815.2 100% SLC12A6 PN with Andermann syndrome AR 15q13 NM_005135.2 100% SOX10 CMT1 AD 22q13.1 NM_006941.3 100% SPTLC1 HSAN1A AD 9q22.2 NM_006415.3 100% SPTLC2 HSAN1C AD 14q24.3 NM_004863.3 100% TDP1 CMT2 AR 14q32.11 NM_018319.3 100% TFG HMSN-P AD 3q12.2 NM_006070.5 100% TRIM2 CMT2 AR 4q31.3 NM_015271.3 100% TRPV4 CMT2C AD 12q24.1 NM_021625.4 100% TUBB3 CMT2 AD 16q24.3 NM_006086.3 100% VAPB SMA-late onset, ALS8 AD 20q13.33 NM_004738.4 100% WNK1* HSAN II AR 12p13.3 NM_213655.4 100% YARS DI-CMTC AD 1p35.1 NM_003680.2 100% COX10*, ^# 17p12 NM_001303.3 100% TEKT3 ^# 17p12 NM_031898.2 100%

Genetic diagnosis using a gene panel

Genetic diagnostic tests were performed on patients who were not genetically identified. A total of 117 samples from 64 CMT households were analyzed (Table 2).

shape Number of households Genetic confirmation unidentified CMT1 13 One 12 CMT2 29 4 25 CMT4 One One 0 CMTX 9 9 0 dHMN 4 One 3 HSAN 2 One One etc 5 0 5 total 64 17 47

After filtering and capillary sequencing, 14 causal mutations could be isolated from PMP22, SH3TC2, MARS, MFN2, GJB1, SPTLC2 and DCTN1 (Table 3).

shape Genetic classification gene Variant family heredity Lee Mark and References Nucleotide amino acid CMT1 CMT1E PMP22 c.47T>C L16R FC541 dominant new CMT4 CMT4C SH3TC2 c.929G>A + c.3272G>T G310E + G1091V FC703 zeal new CMT2 CMT2 MARS c.2398C>A P800T FC495 dominant 12 CMT2A2 MFN2 c.839G>A R280H FC527 dominant 13 CMT2I MPZ c.154T>G F52V FC156 dominant new c.262T>C Y88H FC141 dominant new CMTX CMTX1 GJB1 c.20A>G Y7C FC718 dominant 14 c.43C>T R15W FC751 dominant 15 c.283G>A V95M FC565, FC687, FC698 dominant 16 c.286G>C A96P FC725 dominant new c.490C>T R164W FC714 dominant 14 c.491G>A R164Q FC254, FC722 dominant 14 HSAN HSAN1C SPTLC2 c.435G>T R145S FC459 dominant new dHMN dHMN7B DCTN1 c.1019A>G E340G FC180 dominant new

Seven of the mutations in Table 3 are novel mutations, and the other seven are known mutations. All causal mutations were dominantly inherited, except for complex heterozygous mutations in SH3TC2. Interestingly, the GJB1 mutation was markedly separated (6 variants in 9 families).

PMP22 duplicate test

Next, the efficiency of the gene panel to detect the copy number variation of 1.4 Mb overlap at the CMT1A, 17p12 site was calculated. 10 samples of CMT1A patients and 5 samples of normal subjects tested by the hexaflex method were applied. A method of comparing the read depth of the PMP22 gene and TEKT3 gene in the 1.4 Mb overlap region with the average read depth of the other parts was applied. Compared with the normal subjects, the PMP22 gene was 1.496±0.098 and the TEKT gene 1.472±0.119 was significantly increased in CMT1A patients. In addition, considering the fact that CMT1A patients have 1.5 times the gene mass in the 17p12 region due to gene duplication, the average value is 1.484±0.105, so when this gene panel was applied, almost similar values could be obtained. These results mean that the genetic panel of the present invention is suitable for detecting 1.4 Mb duplication in CMT1A patients.

sample Read depth Mean PMP22 TEKT3 Normal person (n=5) 1.000 ± 0.060 1.000 ± 0.082 1.000 ± 0.069 FC425-1 1.081 1.046 1.064 ± 0.025 FC453-1 0.989 0.962 0.976 ± 0.019 FC565-1 1.064 1.079 1.072 ± 0.011 FC703-1 0.872 0.935 0.904 ± 0.045 FC707-1 0.993 0.977 0.985 ± 0.011 CMT1A patient (n=15) 1.496 ± 0.098 1.472 ± 0.119 1.484 ± 0.105 FC045-12 1.435 1.440 1.438 ± 0.003 FC144-1 1.468 1.439 1.453 ± 0.020 FC168-1 1.615 1.630 1.623 ± 0.010 FC175-1 1.468 1.459 1.463 ± 0.006 FC179-1 1.518 1.568 1.543 ± 0.035 FC214-3 1.678 1.705 1.692 ± 0.018 FC215-2 1.395 1.381 1.388 ± 0.009 FC226-1 1.548 1.528 1.538 ± 0.014 FC287-1 1.578 1.418 1.498 ± 0.113 FC339-1 1.623 1.649 1.636 ± 0.018 FC498-1 1.328 1.303 1.315 ± 0.017 FC511-1 1.414 1.348 1.381 ± 0.047 FC512-1 1.414 1.422 1.418 ± 0.005 FC561-1 1.504 1.436 1.470 ± 0.048 FC589-1 1.449 1.356 1.402 ± 0.065

Review

In the present invention, a novel diagnostic kit using a one-stem detection system has been proposed. Conventionally, a detection system arranged in a row was used for genetic diagnosis of CMT, which took considerable time and cost. The detection of chromosome 17p region duplication and the type of diagnosis based on the motor neuron conduction rate depended on the capillary sequencing of the major genes with high frequency. Even in the complex process of each step, the success rate was less than 60%.

Compared with the previous method, the novel diagnostic system developed in the present invention has the advantage of higher detection efficiency and reduced time and cost. It was confirmed by applying to causal mutations in 75 non-CMT1A patients and 38 patients. Considering that the CMT1A family in the Korean population is 35.1%, applying the gene panel of the present invention will increase the separation rate to 68%. The present inventor applied Whole Exome Sequencing (WES) to non-CMT1A patients, and the detection rate was 68.6%. Therefore, when the target gene panel for CMT of the present invention is applied, time and cost can be reduced while exhibiting a similar separation rate compared to the conventional method and WES.

The one-stem detection system of the present invention also includes detection of copy number variation (CNV) for the chromosome 17p site. Conventional short nucleotide read (short nucleotide read)-based NGS has a disadvantage in the detection of copy number variation, but the present invention overcomes this by using 8 markers. In a previous study, we applied 6 markers to detect PMP22 duplicates with 99.9% efficiency (6). In the present invention, CNV was measured by comparing the read-depth of two markers for the overlapping site and 6 markers in different chromosomes. Since the mean difference between the markers had a significant difference, CNV was detected for the overlapping region satisfactory for diagnosis. The present inventors have not tested the effectiveness of HNPP (Hereditary Neuropathy with a liability to Pressure Palsy), but since it is a deletion of the same site, it may be applied to disease diagnosis.

As described above, specific parts of the present invention have been described in detail, and it is obvious that these specific techniques are only preferred embodiments, and the scope of the present invention is not limited thereto for those of ordinary skill in the art. Therefore, it will be said that the practical scope of the present invention is defined by the appended claims and their equivalents.

<110> SAMSUNG LIFE PUBLIC WELFARE FOUNDATION <120> Kit for Diagnosing Charcot-Marie-Tooth <130> PN150255D <150> KR 10-2015-0167973 <151> 2015-11-27 <160> 6 <170> KopatentIn 2.0 <210> 1 <211> 483 <212> DNA <213> PMP22 <400> 1 atgctcctcc tgttgctgag tatcatcgtc ctccacgtcg cggtgctggt gctgctgttc 60 gtctccacga tcgtcagcca atggatcgtg ggcaatggac acgcaactga tctctggcag 120 aactgtagca cctcttcctc aggaaatgtc caccactgtt tctcatcatc accaaacgaa 180 tggctgcagt ctgtccaggc caccatgatc ctgtcgatca tcttcagcat tctgtctctg 240 ttcctgttct tctgccaact cttcaccctc accaaggggg gcaggtttta catcactgga 300 atcttccaaa ttcttgctgg tctgtgcgtg atgagtgctg cggccatcta cacggtgagg 360 cacccggagt ggcatctcaa ctcggattac tcctacggtt tcgcctacat cctggcctgg 420 gtggccttcc ccctggccct tctcagcggt gtcatctatg tgatcttgcg gaaacgcgaa 480 tga 483 <210> 2 <211> 3867 <212> DNA <213> SH3TC2 <400> 2 atgggtggct gcttctgcat ccccagggag cggagtctga cccggggccc aggtaaagaa 60 actccttcca aggatccaac tgtatcgagt gagtgtatag cctcatctga atacaaggaa 120 aaatgttttc tgccacagaa cattaatcca gacctgacac tctccttctg tgtaaagagc 180 cgctccagga ggtgtgtaaa tggaccccta caggaagctg ctcggaggcg gctctgggca 240 ctggagaatg aggaccagga ggtgcgcatg ctgtttaagg acctctcagc aaggttggtc 300 agtatccagt ctcagagggc ccagtttctc atcaccttca agaccatgga ggaaatctgg 360 aagttctcca cctaccttaa tttaggctac gtatccatgt gtctagaaca tctcctcttt 420 gaccacaagt actggctcaa ctgcatattg gtggaggata cagagatcca agtgtctgta 480 gatgataaac acctggaaac aatatacctg ggactcctga tacaggaagg ccacttcttc 540 tgcagagccc tgtgctccgt gactccacca gccgagaagg aaggggaatg cttgacactt 600 tgcaagaatg agttaatctc agtgaagatg gcagaagctg gctccgagtt ggaaggcgtg 660 tctttggtga caggtcagcg gggcctggta ctggtgtcag ccttggagcc tctgcctctc 720 cctttccacc agtggttcct aaagaattat ccaggaagct gtggcctttc caggaagagg 780 gattggacag gctcctatca gattggcaga ggacgctgta aggccttgac gggttatgag 840 ccaggagaaa aggatgaact gaatttctac cagggagaaa gcattgagat catcggcttt 900 gtcatacctg ggcttcagtg gttcattgga aagtcgacaa gttcaggaca agtgggcttt 960 gtccccacca ggaacataga tcctgattct tattccccaa tgagcaggaa ctctgccttt 1020 ctcagtgatg aggagagatg ctccctgttg gccctgggaa gtgataagca gactgagtgt 1080 tccagcttcc tccacactct tgctcgcact gacatcacat ctgtctaccg gctcagtggg 1140 tttgaatcca tccagaatcc tccaaatgat ctgagtgcat cccagcctga aggtttcaag 1200 gaggtcaggc ctggcagagc ctgggaggag catcaggccg tggggtccag acagtccagc 1260 agctctgagg actccagcct ggaggaggag ctcctctcgg ccacctcaga cagctatcgc 1320 ctgccggagc ctgatgacct tgatgacccg gaactgctca tggacctaag cactggtcag 1380 gaggaggagg ctgagaactt cgcccccata ttggcttttc tggatcatga gggttatgct 1440 gaccacttta agagtctcta tgacttctcc ttctctttcc tcacttcttc cttttatagc 1500 ttctctgagg aggatgagtt tgtggcctac ctggaggcat caagaaagtg ggccaagaag 1560 agccacatga cctgggccca tgcccgtctc tgcttcctcc tgggccggct gagcatcagg 1620 aaggtcaaac tctctcaggc cagggtgtac ttcgaggagg ccatccacat tctcaatgga 1680 gcatttgagg acctatcctt ggtggccact ctgtacatca atttggctgc catctacctg 1740 aaacagaggc tgagacataa aggctccgcc ctgttggaaa aggcaggtgc cctgctggcc 1800 tgcctgcctg accgtgagtc tagtgccaag catgaactcg acgtggtggc ctacgtgctg 1860 cgccagggga ttgtggtggg cagcagcccg ctggaggcca gggcctgctt tctggccatc 1920 cgcttgctcc tgagcctagg ccggcacgag gaggtcctgc cctttgccga gcgcctgcag 1980 ctcctctctg gacaccctcc tgcctctgag gctgtggcca gtgttttgag ttttctgtat 2040 gacaagaaat atcttccaca ccttgcagtg gcctctgtcc agcaacatgg tatccagagt 2100 gcccaaggga tgtctcttcc tatttggcag gtccaccttg tcctccagaa cacaaccaag 2160 ctccttggct ttccttcccc aggctggggt gaagtttctg ccttggcctg cccaatgctc 2220 agacaggccc tggctgcctg tgaggaacta gcagaccgga gcacccagag ggccctgtgt 2280 ctcatccttt ccaaagtgta cctcgagcac aggtctcctg acggtgccat ccactacctg 2340 agccaggcct tggtgctagg gcagctgctg ggtgagcagg aatcctttga gtcttctctc 2400 tgcctggcat gggcctatct cttagccagc caggccaaga aggctttgga tgtgcttgag 2460 ccactgctat gctccctgaa ggagacagag agtctcactc aaaggggagt catctataac 2520 ctcctgggac ttgcactcca aggtgaaggc cgggtgaaca gggcagccaa gagctatctt 2580 cgggccttga acagagccca ggaggtggga gatgtgcata accaggcagt ggctatggcc 2640 aatcttggcc acctgagcct taagtcctgg gctcagcatc cagccagaaa ctatctcctg 2700 caggctgtac gactctattg tgaacttcag gccagtaagg agacagacat ggaattagta 2760 caggtgtttc tctggttggc ccaagttctg gtgtctggac accagctgac ccatggcctt 2820 ctttgttatg aaatggcatt gctgtttggc ttaaggcatc gacatctaaa gagtcagctt 2880 caggccacca aatccctctg ccatttctac agctctgtgt ccccaaaccc tgaggcatgc 2940 atcacctacc atgagcactg gctggccctg gctcagcaac tcagggaccg ggagatggaa 3000 gggaggctgc tggagtccct ggggcagctt tatcggaacc taaataccgc caggtccctc 3060 aggaggtcac tcacatgcat caaggagagc ctgcgtatct tcattgacct gggggagaca 3120 gacaaggctg ctgaggcctg gcttggggcg gggcgactcc actacctcat gcaggaagac 3180 gagctggtgg agctgtgcct gcaggcagcc atccagacag ccctgaagtc agaggagcct 3240 ttgctggctc tcaaacttta tgaagaagca ggtgatgtgt tcttcaatgg gacccgccac 3300 aggcatcatg cagtggagta ctaccgagct ggagctgttc ctttagcaag gaggttgaag 3360 gcggtgagaa ctgagctccg gattttcaat aagctgacag agctgcagat tagcctcgaa 3420 ggctatgaga aggctttgga atttgccacc ctggccgcca ggctcagcac agtcacagga 3480 gatcagaggc aagagctggt ggcctttcac cgcctggcta cagtgtacta ctccctgcac 3540 atgtatgaga tggctgagga ctgctacctg aagaccctgt ccctctgtcc accatggctg 3600 cagagtccca aggaggccct gtactatgcc aaggtgtatt atcgcctggg cagactcacc 3660 ttctgccagc tgaaggatgc ccatgatgcc actgagtact tccttctggc cctggcagca 3720 gcggtcctgc tgggtgatga ggagcttcag gacaccatta ggagcaggct ggacaacatc 3780 tgccagagcc ccctgtggca cagcaggccc tccgggtgct cctcagagag ggcgcggtgg 3840 ctgagtggtg gtggcctggc cctctga 3867 <210> 3 <211> 747 <212> DNA <213> MPZ <400> 3 atggctcctg gggctccctc atccagcccc agccctatcc tggctgtgct gctcttctct 60 tctttggtgc tgtccccggc ccaggccatc gtggtttaca ccgacaggga ggtccatggt 120 gctgtgggct cccgggtgac cctgcactgc tccttctggt ccagtgagtg ggtctcagat 180 gacatctcct tcacctggcg ctaccagccc gaagggggca gagatgccat ttcgatcttc 240 cactatgcca agggacaacc ctacattgac gaggtgggga ccttcaaaga gcgcatccag 300 tgggtagggg accctcgctg gaaggatggc tccattgtca tacacaacct agactacagt 360 gacaatggca cgttcacttg tgacgtcaaa aaccctccag acatagtggg caagacctct 420 caggtcacgc tgtatgtctt tgaaaaagtg ccaactaggt acggggtcgt tctgggagct 480 gtgatcgggg gtgtcctcgg ggtggtgctg ttgctgctgc tgcttttcta cgtggttcgg 540 tactgctggc tacgcaggca ggcggccctg cagaggaggc tcagtgctat ggagaagggg 600 aaattgcaca agccaggaaa ggacgcgtcg aagcgcgggc ggcagacgcc agtgctgtat 660 gcaatgctgg accacagcag aagcaccaaa gctgtcagtg agaagaaggc caaggggctg 720 ggggagtctc gcaaggataa gaaatag 747 <210> 4 <211> 852 <212> DNA <213> GJB1 <400> 4 atgaactgga caggtttgta caccttgctc agtggcgtga accggcattc tactgccatt 60 ggccgagtat ggctctcggt catcttcatc ttcagaatca tggtgctggt ggtggctgca 120 gagagtgtgt ggggtgatga gaaatcttcc ttcatctgca acacactcca gcctggctgc 180 aacagcgttt gctatgacca attcttcccc atctcccatg tgcggctgtg gtccctgcag 240 ctcatcctag tttccacccc agctctcctc gtggccatgc acgtggctca ccagcaacac 300 atagagaaga aaatgctacg gcttgagggc catggggacc ccctacacct ggaggaggtg 360 aagaggcaca aggtccacat ctcagggaca ctgtggtgga cctatgtcat cagcgtggtg 420 ttccggctgt tgtttgaggc cgtcttcatg tatgtctttt atctgctcta ccctggctat 480 gccatggtgc ggctggtcaa gtgcgacgtc tacccctgcc ccaacacagt ggactgcttc 540 gtgtcccgcc ccaccgagaa aaccgtcttc accgtcttca tgctagctgc ctctggcatc 600 tgcatcatcc tcaatgtggc cgaggtggtg tacctcatca tccgggcctg tgcccgccga 660 gcccagcgcc gctccaatcc accttcccgc aagggctcgg gcttcggcca ccgcctctca 720 cctgaataca agcagaatga gatcaacaag ctgctgagtg agcaggatgg ctccctgaaa 780 gacatactgc gccgcagccc tggcaccggg gctgggctgg ctgaaaagag cgaccgctgc 840 tcggcctgct ga 852 <210> 5 <211> 1689 <212> DNA <213> SPTLC2 <400> 5 atgcggccgg agcccggagg ctgctgctgc cgccgcacgg tgcgggcgaa tggctgcgtg 60 gcgaacgggg aagtacggaa cgggtacgtg aggagcagcg ctgcagccgc agccgcagcc 120 gccgccggcc agatccatca tgttacacaa aatggaggac tatataaaag accgtttaat 180 gaagcttttg aagaaacacc aatgctggtt gctgtgctca cgtatgtggg gtatggcgta 240 ctcaccctct ttggatatct tcgagatttc ttgaggtatt ggagaattga aaagtgtcac 300 catgcaacag aaagagaaga acaaaaggac tttgtgtcat tgtatcaaga ttttgaaaac 360 ttttatacaa ggaatctgta catgaggata agagacaact ggaatcggcc aatctgtagt 420 gtgcctggag ccagggtgga catcatggag agacagtctc atgattataa ctggtccttc 480 aagtatacag ggaatataat aaagggtgtt ataaacatgg gttcctacaa ctatcttgga 540 tttgcacgga atactggatc atgtcaagaa gcagccgcca aagtccttga ggagtatgga 600 gctggagtgt gcagtactcg gcaggaaatt ggaaacctgg acaagcatga agaactagag 660 gagcttgtag caaggttctt aggagtagaa gctgctatgg cgtatggcat gggatttgca 720 acgaattcaa tgaacattcc tgctcttgtt ggcaaaggtt gcctgattct gagtgatgaa 780 ctgaatcatg catcactggt tctgggagcc agactgtcag gagcaaccat tagaatcttc 840 aaacacaaca atatgcaaag cctagagaag ctattgaaag atgccattgt ttatggtcag 900 cctcggacac gaaggccctg gaagaaaatt ctcatccttg tggaaggaat atatagcatg 960 gagggatcta ttgttcgtct tcctgaagtg attgccctca agaagaaata caaggcatac 1020 ttgtatctgg atgaggctca cagcattggc gccctgggcc ccacaggccg gggtgtggtg 1080 gagtactttg gcctggatcc cgaggatgtg gatgttatga tgggaacgtt cacaaagagt 1140 tttggtgctt ctggaggata tattggaggc aagaaggagc tgatagacta cctgcgaaca 1200 cattctcata gtgcagtgta tgccacgtca ttgtcacctc ctgtagtgga gcagatcatc 1260 acctccatga agtgcatcat ggggcaggat ggcaccagcc ttggtaaaga gtgtgtacaa 1320 cagttagctg aaaacaccag gtatttcagg agacgcctga aagagatggg cttcatcatc 1380 tatggaaatg aagactctcc agtagtgcct ttgatgctct acatgcctgc caaaattggc 1440 gcctttggac gggagatgct gaagcggaac atcggtgtcg ttgtggttgg atttcctgcc 1500 accccaatta ttgagtccag agccaggttt tgcctgtcag cagctcatac caaagaaata 1560 cttgatactg ctttaaagga gatagatgaa gttggggacc tattgcagct gaagtattcc 1620 cgtcatcggt tggtacctct actggacagg ccctttgacg agacgacgta tgaagaaaca 1680 gaagactga 1689 <210> 6 <211> 3837 <212> DNA <213> DCTN1 <400> 6 atggcacaga gcaagaggca cgtgtacagc cggacgccca gcggcagcag gatgagtgcg 60 gaggcaagcg cccggcctct gcgggtgggc tcccgtgtag aggtgattgg aaaaggccac 120 cgaggcactg tggcctatgt tggagccaca ctgtttgcca ctggcaaatg ggtaggcgtg 180 attctggatg aagcaaaggg caaaaatgat ggaactgttc aaggcaggaa gtacttcact 240 tgtgatgaag ggcatggcat ctttgtgcgc cagtcccaga tccaggtatt tgaagatgga 300 gcagatacta cttccccaga gacacctgat tcttctgctt caaaagtcct caaaagagag 360 ggaactgata caactgcaaa gactagcaaa ctgcggggac tgaagcctaa gaaggcaccg 420 acagcccgaa agaccacaac tcggcgaccc aagcccacgc gcccagccag tactggggtg 480 gctggggcca gtagctccct gggcccctct ggctcagcgt cagcaggtga gctgagcagc 540 agtgagccca gcaccccggc tcagactccg ctggcagcac ccatcatccc cacgccggtc 600 ctcacctctc ctggagcagt ccccccgctt ccttccccat ccaaggagga ggagggacta 660 agggctcagg tgcgggacct ggaggagaaa ctagagaccc tgagactgaa acgggcagaa 720 gacaaagcaa agctaaaaga gctggagaaa cacaaaatcc agctggagca ggtgcaggaa 780 tggaagagca aaatgcagga gcagcaggcc gacctgcagc ggcgcctcaa ggaggcgaga 840 aaggaagcca aggaggcgct ggaggcaaag gaacgctata tggaggagat ggctgatact 900 gctgatgcca ttgagatggc cactttggac aaggagatgg ctgaagagcg ggctgagtcc 960 ctgcagcagg aggtggaggc actgaaggag cgggtggacg agctcactac tgacttagag 1020 atcctcaagg ctgagattga agagaagggc tcagatggcg ctgcatccag ttatcagctc 1080 aagcagcttg aggagcagaa tgcccgcctg aaggatgccc tggtgaggat gcgggatctt 1140 tcttcctcag agaagcagga gcatgtgaag ctccagaagc tcatggaaaa gaagaaccaa 1200 gagctggaag ttgtgaggca acagcgggag cgtctgcagg aggagctaag ccaggcagag 1260 agcaccattg atgagctcaa ggagcaggtg gatgctgctc tgggtgctga ggagatggtg 1320 gagatgctga cagatcggaa cctgaatctg gaagagaaag tgcgcgagtt gagggagact 1380 gtgggagact tggaagcgat gaatgagatg aacgatgagc tgcaggagaa tgcacgtgag 1440 acagaactgg agctgcggga gcagctggac atggcaggcg cgcgggttcg tgaggcccag 1500 aagcgtgtgg aggcagccca ggagacggtt gcagactacc agcagaccat caagaagtac 1560 cgccagctga ccgcccatct acaggatgtg aatcgggaac tgacaaacca gcaggaagca 1620 tctgtggaga ggcaacagca gccacctcca gagacctttg acttcaaaat caagtttgct 1680 gagactaagg cccatgccaa ggcaattgag atggaattga ggcagatgga ggtggcccag 1740 gccaatcgac acatgtccct gctgacagcc ttcatgcctg acagcttcct tcggccaggt 1800 ggggaccatg actgcgttct ggtgctgttg ctcatgcctc gtctcatttg caaggcagag 1860 ctgatccgga agcaggccca ggagaagttt gaactaagtg agaactgttc agagcggcct 1920 gggctgcgag gagctgctgg ggagcaactc agctttgctg ctggactggt gtactcgctg 1980 agcctgctgc aggccacgct acaccgctat gagcatgccc tctctcagtg cagtgtggat 2040 gtgtataaga aagtgggcag cctgtaccct gagatgagtg cccatgagcg ctccttggat 2100 ttcctcattg aactgctgca caaggatcag ctggatgaga ctgtcaatgt ggagcctctc 2160 accaaggcca tcaagtacta tcagcatctg tacagcatcc accttgccga acagcctgag 2220 gactgtacta tgcagctggc tgaccacatt aagttcacgc agagtgctct ggactgcatg 2280 agtgtggagg taggacggct gcgtgccttc ttgcagggtg ggcaggaggc tacagatatt 2340 gccctcctgc tccgggatct ggaaacttca tgcagtgaca tccgccagtt ctgcaagaag 2400 atccgaaggc gaatgccagg gacagatgct cctgggatcc cagctgcact ggcctttgga 2460 ccacaggtat ctgacacgct cctagactgc aggaaacact tgacgtgggt cgtggctgtg 2520 ctgcaggagg tggcagctgc tgctgcccag ctcattgccc cactggcaga gaatgagggg 2580 ctacttgtgg ctgctctgga ggaactggct ttcaaagcaa gcgagcagat ctatgggacc 2640 ccctccagca gcccctatga gtgtctgcgc cagtcatgca acatcctcat cagtaccatg 2700 aacaagctgg ccacagccat gcaggagggg gagtatgatg cagagcggcc ccccagcaag 2760 cctccaccgg ttgaactgcg ggctgctgcc cttcgtgcag agatcacaga tgctgaaggc 2820 ctgggtttga agctcgaaga tcgagagaca gttattaagg agttgaagaa gtcactcaag 2880 attaagggag aggagctaag tgaggccaat gtgcggctga gcctcctgga gaagaagttg 2940 gacagtgctg ccaaggatgc agatgagcgc atcgagaaag tccagactcg gctggaggag 3000 acccaggcac tgctgcgaaa gaaggagaaa gagtttgagg agacaatgga tgcactccag 3060 gctgacatcg accagctgga ggcagagaag gcagaactaa agcagcgtct gaacagccag 3120 tccaaacgca cgattgaggg actccggggc cctcctcctt caggcattgc tactctggtc 3180 tctggcattg ctggtgaaga acagcagcga ggagccatcc ctgggcaggc tccagggtct 3240 gtgccaggcc cagggctggt gaaggactca ccactgctgc ttcagcagat ctctgccatg 3300 aggctgcaca tctcccagct ccagcatgag aacagcatcc tcaagggagc ccagatgaag 3360 gcatccttgg catccctgcc ccctctgcat gttgcaaagc tatcccatga gggccctggc 3420 agtgagttac cagctggagc gctgtatcgt aagaccagcc agctgctgga gacattgaat 3480 caattgagca cacacacgca cgtagtagac atcactcgca ccagccctgc tgccaagagc 3540 ccgtcggccc aacttatgga gcaagtggct cagcttaagt ccctgagtga caccgtcgag 3600 aagctcaagg atgaggtcct caaggagaca gtatctcagc gccctggagc cacagtaccc 3660 actgactttg ccaccttccc ttcatcagcc ttcctcaggg ccaaggagga gcagcaggat 3720 gacacagtct acatgggcaa agtgaccttc tcatgtgcgg ctggttttgg acagcgacac 3780 cggctggtgc tgacccagga gcagctgcac cagcttcaca gtcgcctcat ctcctaa 3837

Claims (6)

A mutation gene in which the 286th nucleotide of the wild-type GJB1 gene is replaced with a cytosine as a diagnostic marker for Charcot-Marie-Tooth disease.
Mutation protein as a Charcoal-Marie-Tooth disease veneer marker encoded by a mutated gene in which the 286th nucleotide of the wild-type GJB1 gene is replaced by a cytosine as a diagnostic marker for Charcot-Marie-Tooth disease.
A diagnostic marker for Charcot-Marie-Tooth disease, which contains an agent capable of detecting a gene consisting of a nucleotide sequence in which the 286th nucleotide of the wild type GJB1 gene has been replaced with a cytosine, Diagnostic kit for Tus disease.
4. The method of claim 3, wherein the kit further comprises at least one of AARS (NM_001605), AIEM1 (NM_04208), ARIIGER10 (NM_014629), ATL1 (NM_015915), ATL3 (NM_015459), BSCL2 (NM_032667), C12orf65 (NM_004715), DTN1 (NM_001376), EGR2 (NM_000399), FAM134B (NM_001034850), FBLN5 (NM_001034850), DCTN1 (NM_004082), DHTKD1 (NM_018706), DNAJB2 (NM_006736), DNM2 (NM_006329), FGD4 (NM_139241), FIG4 (NM_014845), GAN (NM_022041), GARS (NM_002047), GDAP1 (NM_018972), GNB4 (NM_021629), HADHB (NM_000188), HSPB1 (NM_001540), HSPB3 (NM_006308), HSPB8 (NM_014365), IGRD1 (NM_001550), IGHMBP2 (NM_002180), IKBKAP (NM_003640), INF2 (NM_022489), KARS (NM_004861), LITAF (NM_004862), LMNA (NM_170707), LRSAM1 (NM_138361), MARS (NM_004990), MED25 NM_030973, MFN2 NM_014874, MTMR2 NM_016156, MYH14 NM_024729, NDRG1 (NM_006158), NGF (NM_002506), NTRK1 (NM_002529), NRG2 (NM_013982), PDK3 (NM_005391), PLEKHG5 REF1 (NM_022912), SBF1 (NM_002972), SBF2 (NM_030962), SETX (NM_015046), SCN9A (NM_002977), SLC5A7 (NM_002977), PMP22 (NM_000304), PRPS1 (NM_002764), PRX (NM_181882), RAB7A NM_021815), SLC12A6 (NM_005135), SOX10 (NM_006941), SPTLC1 (NM_006415), SPTLC2 (NM_004863), TDP1 (NM_018319), TFG (NM_006070), TRIM2 (NM_015271), TRPV4 (NM_021625), TUBB3 Wherein the method further comprises an agent capable of detecting a gene selected from the group consisting of NM_004738, WNK1 (NM_213655), YARS (NM_003680), COX10 (NM_001303) and TEKT3 (NM_031898) Kits.
As a diagnostic marker for Charcot-Marie-Tooth disease, nucleic acid molecules are amplified using a preparation capable of detecting a gene consisting of a nucleotide sequence in which the 286th nucleotide of the wild-type GJB1 gene has been replaced with a cytosine Wherein the method comprises the steps of:
The kit according to claim 5, wherein the kit comprises at least one of AARS (NM_001605), AIEM1 (NM_04208), ARIIGER10 (NM_014629), ATL1 NM_015915, ATL3 NM_015459, BSCL2 NM_032667, C12orf65 NM_152269, CCT5 (NM_004715), DTN1 (NM_001376), EGR2 (NM_000399), FAM134B (NM_001034850), FBLN5 (NM_001034850), DCTN1 (NM_004082), DHTKD1 (NM_018706), DNAJB2 (NM_006736), DNM2 (NM_006329), FGD4 (NM_139241), FIG4 (NM_014845), GAN (NM_022041), GARS (NM_002047), GDAP1 (NM_018972), GNB4 (NM_021629), HADHB (NM_000188), HSPB1 (NM_001540), HSPB3 (NM_006308), HSPB8 (NM_014365), IGRD1 (NM_001550), IGHMBP2 (NM_002180), IKBKAP (NM_003640), INF2 (NM_022489), KARS (NM_004861), LITAF (NM_004862), LMNA (NM_170707), LRSAM1 (NM_138361), MARS (NM_004990), MED25 NM_030973, MFN2 NM_014874, MTMR2 NM_016156, MYH14 NM_024729, NDRG1 (NM_006158), NGF (NM_002506), NTRK1 (NM_002529), NRG2 (NM_013982), PDK3 (NM_005391), PLEKHG5 REF1 (NM_022912), SBF1 (NM_002972), SBF2 (NM_030962), SETX (NM_015046), SCN9A (NM_002977), SLC5A7 (NM_002977), PMP22 (NM_000304), PRPS1 (NM_002764), PRX (NM_181882), RAB7A NM_021815), SLC12A6 (NM_005135), SOX10 (NM_006941), SPTLC1 (NM_006415), SPTLC2 (NM_004863), TDP1 (NM_018319), TFG (NM_006070), TRIM2 (NM_015271), TRPV4 (NM_021625), TUBB3 And amplifying the nucleic acid molecule using a preparation capable of detecting a gene selected from the group consisting of NM_004738, WNK1 (NM_213655), YARS (NM_003680), COX10 (NM_001303) and TEKT3 (NM_031898) , A method of providing information for diagnosis of Charcot-Marie-Tooth disease.