KR20160110600A - Dgat2 as a causative gene responsible for an early-onset autosomal dominant axonal charcot-marie-tooth neuropathy and diagnosis method for the disease - Google Patents

Dgat2 as a causative gene responsible for an early-onset autosomal dominant axonal charcot-marie-tooth neuropathy and diagnosis method for the disease Download PDF

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KR20160110600A
KR20160110600A KR1020150032537A KR20150032537A KR20160110600A KR 20160110600 A KR20160110600 A KR 20160110600A KR 1020150032537 A KR1020150032537 A KR 1020150032537A KR 20150032537 A KR20150032537 A KR 20150032537A KR 20160110600 A KR20160110600 A KR 20160110600A
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정기화
최병옥
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공주대학교 산학협력단
사회복지법인 삼성생명공익재단
성균관대학교산학협력단
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Abstract

The present invention relates to a DGAT2 mutant gene which is a causative gene of an axon-type Charcot-Marie-Tooth disease (CMT). The present invention also provides a DGAT2 mutant protein encoded from the DGAT2 mutation gene. The present invention also provides a CMT diagnostic marker comprising a DGAT2 mutant gene or protein and a method for diagnosing CMT using the CMT diagnostic marker. The present invention enables precise diagnosis before the onset of CMT using the DGAT2 mutant gene

Description

TECHNICAL FIELD [0001] The present invention relates to DGAT2 as a causative gene for early-onset autosomal dominant axonal Charcot-Marie-Tooth neuropathy, and to a method for diagnosing the disease using the DGAT2 gene. DIAGNOSIS METHOD FOR THE DISEASE}

The present invention relates to the genetic cause DGAT2 mutant gene of Charcot-Marie-Tooth disease (CMT).

Charcot-Marie-Tooth (CMT) is a hereditary peripheral neuropathy characterized by progressive weakness and sensory loss. CMT is a group of clinically genetically heterogeneous genetic neuropathy and is the most common genetic neuromuscular disease with a prevalence of 17-40 per 100,000 individuals. CMT is basically divided into dehydrated CMT (CMT1) and axon CMT (CMT2) based on nerve conduction study and histologic findings. According to electrophysiological criteria, CMT is normal or slightly decreased (> 38 m / s) in dehydration-induced neuropathy (CMT1) or hyperosmotic rate, which is severely impaired with a motor nerve conduction velocity (MNCV) of less than 38 m / s It is classified into two major subtypes of axonal neuropathy (CMT2) with markedly reduced amplitude.

Axon CMT2 is known to have more than 30 genetic loci and about 20 causative genes (Auer-Grumbach et al. 2003; Rossor et al. 2013). In addition, recently, DYNC1H1 (MIM 600112) (Weedon et al. 2011), DHTKD1 (MIM 614984) (Xu et al. 2012), HINT1 (MIM 601314) (Zimonet al. 2012), MTATP6 (MIM 516060) (Pitceathly et al. 2012), MARS (MIM 156560) (Gonzalez et al. 2013), HARS (MIM 142810) Vester et al. 2013), HADHB (MIM 143450) (Hong et al. 2013), TFG (MIM 602498) (Tsai et al.2014), and DNAJB2 (MIM 604139) Are continuously being updated. CMT2-related genes function in a variety of processes including: microtubule-based organelles (DYNC1H1); Mitochondrial epidemiology and morphology (MFN2, and GDAP1); Structural scaffolds of nerve cells (NEFL) and nuclei (LMNA); A transcription initiation complex assembly (MED25); Endocytosis (DNM2); Regulation of cell adhesion signaling pathway, self-ubiquitylation (LRSAM1); Protein biosynthesis (GARS, AARS, MARS, and HARS); Calcium channel (TRPV4); Damage restoration - apoptosis, chaperoning (HSPB1, HSPB8, and DNAJB2); And carbohydrate and fatty acid metabolism-Krebs cycle (DHTKD1, MTATP6, and HADHB).

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 number of nerve fibers that had been exposed to several seconds. However, the above therapeutic agents are limited to CMT type 1, and CMT is caused by several hundreds of genetic mutations. In order to treat CMT, a customized treatment technique suitable for each gene defect and a model And the effects of CMT patients on drugs are significantly different, which limits the choice of medication for CMT patients and their symptoms.

Therefore, the inventors of the present invention have found that, as a causative gene for early-onset autosomal dominant axonal Charcoal-Mariotus disease of Charcot-Marie-Tooth disease (CMT), DGAT2 gene And found a new causative gene of Charcot-Marie-Tooth disease and developed a molecular diagnostic method to complete the present invention.

1. Auer-Grumbach, M., De Jonghe, P., Verhoeven, K., Timmerman, V., Wagner, K., Hartung, H.P., and Nicholson, G.A. (2003). Autosomal dominant inherited neuropathies with prominent sensory loss and mutilations: a review. Neurol. 60, 329-334. 2. Rossor, A. M., Polke, J. M., Houlden, H., and Reilly, M. M. (2013). Clinical implications of genetic advances in Charcot-Marie-Tooth disease. Nat. Rev. Neurol. 9, 562-571. 3. Newbury-Ecob, C., Greenhalgh, L., Weimon, MN, Hastings, R., Caswell, R., Xie, W., Paszkiewicz, K., Antoniadi, T., R., et al. (2011). Exome sequencing identifies a DYNC1H1 mutation in a large pedigree with dominant axonal Charcot-Marie-Tooth disease. Am. J. Hum. Genet. 89, 308-312. Wu, X. L., et al., J. Biol. ≪ / RTI > (2012). A nonsense mutation in DHTKD1 causes Charcot-Marie-Tooth disease type 2 in a large Chinese pedigree. Am. J. Hum. Genet. 91, 1088-1094. 5. Matur, Z., Guergueltcheva, V., < RTI ID = 0.0 > A. < / RTI > Tournev, I., et al. (2012), Loss-of-function mutations in HINT1 cause axonal neuropathy with neuromyotonia. Nat. Genet. 44, 1080-1083. 6. Pitceathly, RD, Murphy, SM, Cottenie, E., Chalasani, A., Sweeney, MG, Woodward, C., Mudanohwo, EE, Hargreaves, I., Heales, S., Land, J., et al . (2012). Genetic dysfunction of MT-ATP6 causes axonal Charcot-Marie-Tooth disease.Neurology. 79, 1145-1154. L., Hadjivassilious, M., Speziani, F., Yang, XL, Antonellis, A., Reilly, H., Gould, H., Houlden, H., Guo, M., Gonzalez, M., McLaughlin, MM, et al. (2013) .Exome sequencing identifies a significant variant of methionyl-tRNAsynthetase (MARS) in a family with late-onset CMT2.J. Neurol. Neurosurg. Psychiatry. 84, 1247-1249. 8. Vester, A., Velez-Ruiz, G., McLaughlin, H. M. NISC Comparative Sequencing Program, Lupski, J. R., Talbot, K., Vance, J. M., Zuchner, S., Roda, R. H., Fischbeck, K. H., et al. (2013). A loss-of-function variant in the human histidyl-tRNAsynthetase (HARS) gene is neurotoxic in vivo. 34, 191-199. Yoon, B. R., Yoo, J. H., Koo, H., Jung, S. C., Chung, K.W., and Choi, B. O. et al. (2013). A compound heterozygous mutation in HADHB gene causes an axonal Charcot-Marie-tooth disease. BMC Med. Genet. Liu, Y. T., Liu, T.T., Kao, L. S., et al., &Quot; Tsai, P. C., Huang, Y. H., Guo, Y. C., Wu, H. T., Lin, K. P., Tsai, Y. S., Liao, Y. C., A novel TFG mutation causes TFG function. Neurology 83, 903-912. 11. Gess, B., Auer-Grumbach, M., Schirmacher, A., Strom, T., Zitzelsberger, M., Rudnik-Schoneborn, S., Rohr, D., Halfter, H., Young, P. , and Senderek, J. (2014). HSJ1-related hereditary neuropathies: novel mutations and extended clinical spectrum. Neurology 83, 1726-1732.

It is an object of the present invention to provide a DGAT2 mutant gene which is a causative gene of Charcot-Marie-Tooth disease (CMT).

Another object of the present invention is to provide a DGAT2 mutant protein which is encoded from the DGAT2 mutation gene.

It is yet another object of the present invention to provide a CMT diagnostic marker comprising a DGAT2 mutant gene or protein.

It is still another object of the present invention to provide a method for diagnosing CMT using the CMT diagnostic marker.

In order to accomplish the above object, the present invention provides a method for producing a DGAT2 gene, which is characterized in that mutation is induced in the DGAT2 gene represented by the nucleotide sequence of SEQ ID NO: 1 from the adenine (A) base of the ATG translation initiation codon to the 667th base (DGAT2) mutation gene of Charcot-Marie-Tooth disease (CMT).

The term " DGAT2 gene " of the present invention is a diacylglycerol O-acyltransferase 2 gene. In the present invention, DGAT2 is located on chromosome 11q13.5 and has an acyl-CoA diacylphosphatase catalyzing the final step of the triglyceride (TG) biosynthetic pathway. Encodes glycerol acyltransferase (DGAT) enzyme (EC 2.3.1.20) (Cases et al. 2001). Despite similar enzyme activities, DTAT2 has no sequence homology with DGAT1 (MIM 604,900). The DGAT2 enzyme is a membrane transport protein located in the ER (ER) and is associated with lipid droplets and mitochondria-associated membranes. DGAT2 plays a key role in creating and maintaining a pool of triglycerides. Triglyceride is a major storage molecule of fatty acids for energy utilization and plays an important role in many physiological processes.

The mutation of the present invention may be that the thymine is replaced with cytosine at the 667th base from the adenine (A) base of the ATG translation initiation codon in the DGAT2 gene represented by the nucleotide sequence of SEQ ID NO: 1. According to the Human Genome Variation Society (HGVS: http://www.hgvs.org/mutnomen/recs.html) guidelines, the DGAT2 mutation can be represented by c.667T> C. Specifically, the DGAT2 mutation is characterized in that c.667T > C in the DGAT2 gene represented by the nucleotide sequence of SEQ ID NO: 1. Wherein the DGAT2 mutation is a new missense mutation having c.667T > C identified in the DGAT2 gene.

The term "diagnosis" of the present invention means identifying the presence or characteristic of a pathological condition. For the purpose of the present invention, the diagnosis is to confirm the onset of CMT.

The mutant gene of the present invention may be an autosomal dominant axon type CMT characterized by early onset of CMT and slow disease progression but not limited thereto and may be a major genetic cause gene of peripheral neuropathy. To date, many genes have been identified as genetic causes of CMT, but this is the first time that a mutation of DGAT2 has been reported that causes early onset, slow progressive autosomal dominant axon CMT.

The present invention also provides a DGAT2 mutant protein (p.Y223H) which is encoded from the DGAT2 mutant gene. The mutant protein may be a DGAT2 mutant protein (p.Y223H) wherein the 223rd amino acid of the amino acid sequence represented by SEQ ID NO: 2 contains a sequence substituted by histidine in tyrosine.

The present invention also provides a CMT diagnostic marker comprising said DGAT2 mutant gene or said protein.

The term "diagnosis marker" of the present invention is a substance capable of diagnosing the onset of the hereditary neuropathy showing the complex symptoms. For the purpose of the present invention, A DGAT2 mutant gene according to the present invention and a DGAT2 mutant protein encoded from the mutant gene.

The present invention also provides a CMT diagnostic composition comprising the agent capable of detecting the expression of the DGAT2 mutant gene or a protein encoded from the gene. The preparation may include, but is not limited to, a primer set for specifically amplifying the DGAT2 mutation gene represented by SEQ ID NO: 3 and SEQ ID NO: 4. The agent may also include, but is not limited to, an antibody specific for a protein encoded by the DGAT2 mutant gene.

Based on the base sequence of the DGAT2 gene, a primer or a probe that specifically amplifies a specific region of the gene can be devised. Since a mutation site used as a diagnostic marker of hereditary neurological diseases showing symptoms of CMT in the nucleotide sequence of DGAT2 gene of SEQ ID NO: 1 of the present invention has been identified by the present invention, a person skilled in the art will be able to identify the DGAT2 mutant gene A primer or a probe capable of specifically amplifying a specific region can be easily designed.

The term "primer " of the present invention refers to a single-stranded oligonucleotide capable of forming a base pair with a complementary template with a nucleic acid sequence having short free 3'-terminal hydroxyl groups and acting as a starting point for template strand replication . The primer can serve as a starting point for template-directed DNA synthesis under appropriate conditions in suitable buffer (e.g., four different nucleoside triphosphates and polymerase such as DNA, RNA polymerase or reverse transcriptase) and appropriate temperature . The appropriate length of the primer may vary depending on the intended use, but is usually 15 to 30 nucleotides. Short primer molecules generally require lower temperatures to form stable hybrids with the template. The primer sequence need not be completely complementary to the template, but should be sufficiently complementary to hybridize with the template. In the present invention, it is possible to diagnose the onset of CMT genetic neuropathy by PCR amplification using forward and reverse primers for the MYH14 mutant gene and amplification of the PCR product.

In the diagnostic composition according to the present invention, the agent for detecting the presence or absence of the DGAT2 mutation gene may be a primer designed to detect the substitution of cytosine for the 667th base from the initiation codon in the DGAT2 gene of SEQ ID NO: But are not limited thereto.

The term "probe" in the present invention means a nucleic acid fragment such as RNA or DNA corresponding to a few to several hundred bases, which can form a specific binding with mRNA. The probe may be prepared in the form of an oligonucleotide probe, a single-stranded DNA probe, a double-stranded DNA probe, an RNA probe, or the like. In the present invention, hybridization can be performed using a probe complementary to the DGAT2 mutant gene, and the possibility of CMT development can be diagnosed through hybridization. The selection and hybridization conditions of suitable probes can be suitably modified on the basis of those known in the art.

The primers or probes according to the present invention can be chemically synthesized using methods well known in the art, including the phosphoramidite solid support method. Such nucleic acid sequences may also be modified using many means known in the art. Non-limiting examples of such modifications include, but are not limited to, methylation, capping, substitution with one or more of the natural nucleotide analogs, and modifications between nucleotides such as uncharged linkers (e.g., methylphosphonate, phosphotriester, Amidates, carbamates, etc.) or charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.).

In the present invention, "measurement of protein expression" is a process for confirming the presence and expression level of a DGAT2 mutant protein expressed from a DGAT2 mutant gene in an individual sample. Preferably, the antibody specifically binds to the protein of the gene Can be used to confirm the amount of protein. Analysis methods include Western blotting, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, and rocket Immunoprecipitation Assay, Complement Fixation Assay, Fluorescence Actuated Cell Sorter (FACS), and Protein Chip are examples of the immunoassay method. .

The present invention also provides a CMT diagnostic kit comprising a CMT diagnostic composition comprising the agent capable of detecting the expression of the DGAT2 mutant gene or a protein encoded from the gene.

The kit of the present invention can diagnose the expression of a DGAT2 mutant gene or the expression of a DGAT2 mutant protein encoded from the gene in a subject to be tested for the possibility of inducing CMT.

The kit of the present invention includes a primer or a probe capable of detecting the expression of the DGAT2 mutant gene and an antibody selectively recognizing the DGAT2 mutant protein encoded from the gene and further comprising one or more other components suitable for analysis Compositions, solutions, or devices.

As a specific example, the kit for measuring the expression of the DGAT2 mutant gene in the present invention may be a kit containing the essential elements required for conducting RT-PCR. RT-PCR kits can also be used in addition to the respective primer pairs specific for the DGAT2 mutation gene, as well as enzymes such as test tubes or other appropriate containers, reaction buffers, deoxynucleotides (dNTPs), Taq-polymerase and reverse transcriptase, DNase, RNase inhibitors, DEPC Water (DEPC-water), sterile water, and the like.

In addition, the kit of the present invention may be in the form of a microarray comprising the DGAT2 mutation gene according to the present invention. The microarray may comprise DNA or RNA polynucleotide probes. The microarray comprises a conventional microarray configuration except that it contains a probe specific for the base sequence of the DGAT2 mutant gene according to the present invention. The microarray of the present invention can detect the presence of the DGAT2 mutation gene according to the present invention and provide useful information for diagnosing the possibility of CMT.

The present invention also relates to a method for isolating genomic DNA comprising: isolating genomic DNA from a sample derived from an individual; And identifying a mutation at position 667 from the ATG translation initiation site of the DGAT2 gene represented by SEQ ID NO: 1 in the separated genomic DNA; The method comprising the steps of: The present invention also relates to a method of isolating genomic DNA from a sample derived from an individual; And detecting the expression of the DGAT2 mutant gene or the protein encoded by the mutant gene in the separated genomic DNA. The present invention also provides a method for providing information necessary for the early diagnosis of CMT. The mutation may be one in which the thymine is replaced with a cytosine at the 667th base (c.667T> C) from the adenine (A) base of the ATG translation initiation codon in the DGAT2 gene represented by the nucleotide sequence of SEQ ID NO: The sample is preferably blood, but is not limited thereto.

The present invention also provides a method for detecting a CMT diagnostic marker comprising detecting the expression of the DGAT2 mutant gene or a protein encoded by the mutant gene to provide information necessary for the diagnosis of CMT or early diagnosis before onset thereof do.

The present invention also relates to a method of preparing a sample, And contacting the antibody specifically binding to the mutant protein of the present invention with the sample to confirm expression of the protein; The method comprising the steps of:

The detection can be performed by sequencing, hybridization by microarray, allele specific PCR, dynamic allele-specific hybridization (DASH), PCR extension analysis or TaqMan probe PCR analysis But it is not limited thereto.

Among these methods, the nucleotide sequence analysis can be performed using a conventional method for determining the nucleotide sequence, and can be performed using an automated gene analyzer (Sanger, F. et al., Proc. Natl. Acad. , 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., Nucleic Acids Res., 17 (1), 2503-2516, 1989).

TaqMan probe PCR analysis (Livak, K. J., Genet. Anal., 14, 143-149, 1999) was performed by 1) designing and constructing primers and TaqMan probes to amplify the desired DNA fragments; 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, J. A. 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), MALDI-TOF (matrix assisted laser desorption ionization-time of flight) is used when unlabeled extension primers and didoxynucleotides are used. (Ross, PL, Anal. Chem, 69 (20), 4197-4202, 1997).

In an embodiment of the present invention, a Korean chromosome dominant axon CMT family characterized by early onset, sensory ataxia, tremor and slow disease progression was studied. In CMT patients, large myelinated fibers were significantly reduced in the sural nerve, and lower limb MRI showed length-dependent axonal degeneration. In an embodiment of the present invention, diacylglycerol O-acyltransferase 2 (DGAT2) encoding acyl-CoA: diacylglycerol acyltransferase (DGAT) enzyme (EC 2.3.1.20) catalyzed through exome sequencing, A novel heterozygous mutation (p.Y223H) was identified in the gene. In an embodiment of the present invention, patients with DGAT2 mutations have consistently decreased serum triglyceride levels and overexpression of the DGAT2 mutation has been shown to significantly inhibit the proliferation of mouse motor neurons. Also, in embodiments of the invention, the mutant form of human DGAT2 has been shown to inhibit axon branching in the zebrafish peripheral nervous system. Thus, in the present invention, it was found that DGAT2 mutation is a new root cause of autosomal dominant axon CMT2 neuropathy.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.

The DGAT2 mutant gene according to the present invention is a new causative gene for axonal CMT peripheral neuropathy, which is inherited as an autosomal dominant gene caused by a single genetic defect with strong inheritance. Therefore, the DGAT2 mutant gene test of the present invention enables accurate early diagnosis before CMT. In addition, it is possible to understand the mechanism of CMT onset and disease progression according to the accurate genetic diagnosis, and further, it is possible to make customized treatment according to the etiology.

Figure 1 shows the phylogenetic and clinical characteristics of the axon CMT family. (A) axon CMT family diagram. An empty figure represents an unaffected entity (normal person), and a filled figure represents an affected entity (patient). The leader is indicated by an arrow. The asterisk indicates an individual with DNA used in exome sequencing. The genotype of the DGAT2 mutation was indicated under each tested member. (B) T1-weighted MRI results of both pes cavus and muscle wasting pictures (CD) of the distal calf of the axon CMT foot terminal (II-2). (C) Axis scan of the thigh. The spinal muscles were relatively sparing compared to the lower leg muscles. (D) Axial scan of the lower leg. Superior atrophy and fatty replacements were observed in the muscles of the soleus (marked with arrowheads) and calves (marked with arrows).
Fig. 2 shows the histopathological analysis results of the distal calf nerve biopsy (foot terminal). (A) Toluidine blue Dyeed semi-thin cross section. The lack of large elongated fibers and the remaining middle and small-sized elongated fibers are seen with scattered thin elongated fibers. The regenerated axon clusters were scarcely observed (X400 magnification). (B) Histogram results showing unimodal distribution patterns of water fibers. (C) Electron microscope results. Abnormal watery fibers with small axon diameter, thick myelin and focal myelin are frequently present (round magnification: x10000). (D) A thin watery fiber was observed infrequently with a few seconds collapse (circle magnification: x20000).
Figure 3 shows the identification in vitro characteristics of the p.Y223H mutation of DGAT2. (A) A wild type (WT) and Y223H mutant (Mut) sequencing chromatogram. (B) Conservation analysis of amino acid sequences in vertebrate species (H. sapiens: NP_115953.2, P. abelii: XP_002822303.1, M. musculus: NP_080660.1, C. lupus familiaris: XP_542303.3, B. Taurus: NP_991362.2, G. gallus: XP_419374.3, X. laevis: NP_001083204.1 and D. rerio: NP_001025367.1). (C) Expression of DGAT2 in the distal calf nerve by transcript analysis. Expression levels of DGAT1 and MFN2 as well as DGAT2 were expressed in fractions per kilobyte (kb) per million mapped reads (FPKM). (D) Expression of the mutant DGAT2 protein. The duplicated wild type (WT) and mutant (Y223H) DGAT2 proteins were overexpressed in NSC34 cells after being transduced into each vector or control (Ctr) vector and detected by Western blot. (E) Effect of DGAT2 protein on cell proliferation after overexpression. Expression of mutant DGAT2 suppressed cell proliferation more than wild type or empty vector. (F) TG levels of fibroblasts from foot (II-2) and control (Ctr1-3: healthy male of 30, 31, 37 year old) The TG level (μg) of the cell lysate was adjusted to the amount of protein (mg).
Figure 4 shows axonal defects of the human DGAT2 mutation (Y223H) in zebrafish. (A, D) Larvae of zebrafish injected with human DGAT (WT) or DGAT2 (Y223H) were morphologically normal during development. The box representation of each image represents the axon phenotype analysis area (scale bar: 200 mm). (B) Zebrafish larvae expressing human DGAT2 (Y223H) showed abnormal formation of nerve bundles with normal muscle fibers of the neuromuscular junction (NMJ) at 3 days (dpf) after fertilization. * Neuronal fascicle (scale bar: upper panel, 50 mm, lower panel, 20 mm). (C) The graph shows quantitative data of nerve bundles analyzed in 3 dpf zebrafish (N = 20 in each genotype). (E) Zebrafish larvae expressing human DGAT2 (Y223H) ) Showed a decrease in axons branching from the bundle (yellow line) with NMJ normal muscle fibers. (F) Branching axon (purple line) 15dpf Zebra fish traces were traced using Image J plugin Neuron J in both abdominal and dorsal regions. The graph shows quantified data of branches per fascicle (BPF) in the abdominal region of the trunk (N = 20 in each genotype).
Figure 5 shows the cytoplasmic localization of wild-type and mutant DGAT2 proteins. HEK293 cells were transfected with wild-type or mutant DGAT2 expression vectors, after which the cells were stained with DAPI (nucleus) and C-Myc antibody. Both wild-type and mutant DGAT2 proteins were expressed in the cytoplasm and no difference in the expression level was observed in the cells.
FIG. 6 shows the ATG start codon, the TGA stop codon, and the c.667T> C position in the DGAT2 gene (GenBank Accession No. NM_032564.4) shown in SEQ ID NO: 1 of the present invention.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are merely illustrative of the present invention and that the scope of the present invention is not construed as being limited by these embodiments.

Materials and methods

patient

Two patients (family ID: FC490) and seven family members of the Korean axon CMT family were studied (FIG. 1A). We also studied 500 healthy controls who were recruited from neurology clinics who were found to have no family history of neurological disease after accurate clinical and electrophysiological testing. I recruited from neurology. Written consent was obtained from all participants under the protocol approved by the Bioethics Committee of Kongju National University (Industry-Academic Cooperation Foundation) and Samsung Medical Center.

Clinical evaluation

Clinical information included motor and sensory disturbances, deep keystroke and amyotrophic assessments. The strength of the flexor and extensor muscles was assessed manually using a standard medical research committee (MRC) scale. To determine the physical impairment, the CMT Neuropathy Score (CMTNS) was used. Sensory disorders assessed the severity and level of pain, temperature, vibration and location, and compared pain and vibration sensations.

Electrophysiological study

The motor conduction velocities (MCVs) of the median nerve and ulnar nerve were recorded on the abductor pollicis brevis and the abductor digiti quinti, respectively, while the complex muscle activity potentials (CMAPs) It was decided by stimulation. The calf and tibial nerves were determined by stimulating the knees and ankles in the same manner, recording CMAPs for the short extensor digitorum brevis and the adductor hallucis, respectively. Sensory conduction velocities (SCVs) and amplitudes of sensory nerve action potentials (SNAPs) were obtained for a finger-wrist segment derived from the central and ulnar nerves by orthodromic scoring, (sural nerves).

MRI of the buttocks, thighs, and legs

MRI was obtained from the buttocks, thighs and legs using a 1.5-T system (Siemens Vision, Siemens, Germany). Imaging was performed using axial protocol (field of view 24-32 cm, slice thickness 10 mm slice interval 0.5-1.0 mm) and coronal plane (FOV 38-40, slice thickness 4-5 mm, slice spacing T1-weighted spin echo (SE) (TR / TE 570-650 / 14-20, 512 matrixes), T2-weighted SE (TR / TE 2800-4000 / 96-99, 512 matrixes) and fat-suppressed T2-weighted SE (TR / TE 3090-4900 / 85-99, 512 matrixes).

Histopathology

Peripheral calf nerve was biopsied in a 38-year-old patient (II-2). The density of watery fibers (MFS), axon diameter and myelin thickness was measured on a semi-thin cross section using a computer assisted image analyzer (AnalySIS; Soft Imaging System, Germany). Ultrathin cut samples , 60 to 65 nm) were compared with uranyl acetate and lead citrate for electron microscopy (H-7650, Hitachi, Japan).

17 p12  DNA preparation and testing for replication

DNA was purified from the blood using QIAamp blood DNA purification kit (Qiagen, Hilden, Germany). Patient samples were pre-screened for 17p12 redundancy, a major genetic cause of dehydrative CMT using hexaplex microsatellite PCR (Choi et al., 2007).

Exome ( Exome ) And Transcript ( transcriptome Analysis and Variation of Filtering

(2 patients: II-2, III-1; 2 normal subjects) using the SeqCap EZ Human Exome Library v3.0 (SeqCap EZ Human Exome Library v3.0, Roche / NimbleGen, Madison, WI) : I-1, -2) and sequenced by HiSeq 2000 Genome Analyzer (HiSeq 2000 Genome Analyzer, Illumina, San Diego, Calif.). The UCSC assembly hg19 (UCSC assembly hg19) was used as a reference standard sequence. Choose functional variants (mist, nonsense, exonic indel and splice site mutation) in the WES data and then use the dbSNP142 (http://www.ncbi.nlm.nih.gov), the 1000 Genomes project database http://www.1000genomes.org/) and the Exome Variant Server (http://evs.gs.washington.edu/EVS/) or the rare variation (MAF≤0.01). Because the parents of the proband were normal, only the dominant inherited mutations were selected from the parent to the originator (patient) of the de novo mutation. For transcript analysis, polyA RNA was extracted from peripheral calf neurobiology obtained from two normal controls, and the cDNA library was prepared using a TruSeq RNA library kit (Illumina).

Candidate mutations were confirmed by Sanger's sequencing method using an ABI3130XL genetic analyzer (Life Technologies, Foster City, Calif.). The genomic evolution rate profiling (GERP) score was determined by the GERP ++ program (http://mendel.stanford.edu/SidowLab/downloads/gerp/index.html). Conservation of protein sequences was performed using MEGA5, ver 5.05 (http://www.megasoftware.net/).

DGAT2  Vector production and transduction transfection )

The total mRNA was purified from HEK293 cells using an RNeasy mini kit (Qiagen), and the cDNAs were amplified using Superscript reverse transcriptase (Invitrogen, Carlsbad, Calif.) And 5'-ccatgaagaccctcatagcc-3 '(DGAT2- -gctcagttcacctccaggac-3 '(DGAT2-R, SEQ ID NO: 6) primer. After PCR amplification, the product was cloned into pCR2.1-TOPO vector (Invitrogen) and transferred to expression vector pCMV-myc (Clontech, Mountain View CA) using EcoRI restriction enzyme. After preparation of wild-type DGAT2, site-directed mutagenesis was performed using QuikChange Positioning Mutation Kit (Stratagene, La Jolla, Calif.) And 5'-cgggacaccatagaccatttgctttc-3 '(DGAT2-MutF, SEQ ID NO: 3) and 5'-gaaagcaaatggtctatggtgtcccg -3 ' (DGAT2-MutR, SEQ ID NO: 4) to obtain Y223H-DGAT2. The nucleotide sequence of all the prepared vectors was confirmed by Sanger 's sequencing method. NSC34 cells (2 x 10 5 ) were transfected with the control vector pCMV-myc, and DGAT2 DNA was cloned using Lipofectamine 2000 reagent (Invitrogen).

Western Blot  And immunocytochemistry ( immunocytochemistry )

Protein expression of NSC34 cells (mouse motor neuron cell line) was determined by standard Western blotting. Anti-actin Ab, anti-mouse secondary Ab, and anti-rabbit secondary Ab (Sigma, St. Louis, Mo.) were used for protein detection. Expression and localization of DGAT2 protein was analyzed by immunocytochemistry using anti-myc antibody after transfection into HEK293 cells.

Proliferation and Triglyceride ( triglyceride ) Level measurement

To measure cell proliferation, NSC34 cells were cultured in 24-well plates (2 x 10 5 cells) for 24 hours and then transfected with control and DGAT2 vector. Cell proliferation was determined directly by microscope at 24 hour intervals. The total amount of triglycerides (TG) in the cells was measured using a triglyceride quantification kit (Abcam) according to the manufacturer's protocol. Normal control and patient fibroblasts were used to determine the TG level of the cells.

Zebrafish of housing housing and handling

Zebrafish (AB strain) grows according to the standard protocol (Westerfield 2007) and distinguishes developmental stages. The embryos were obtained by natural spawning and culturing in 1X E3 solution (60X: 300 mM NaCl, 10.2 mM KCl, 19.8 mM CaCl 2 and 19.8 mM MgSO 4 (adjusted to pH 7.2) plus 28.5? C 0.0001% methylene blue) . To obtain a clear image, the embryos were anesthetized with 0.04% Tricaine (Sigma, St. Louis, MO) in E3 solution.

Zebra fish  Into the embryo mRNA  Microinjection

Both wild type and mutant (Y223H) of human DGAT2 were subcloned into pCS2 + vector. MRNA of the DNA construct was synthesized in vitro using mMessage mMachine SP6 kit (Ambion, Austin, Tex.) According to the manufacturer's instructions. 100 pg / nl of each of the mRNAs were injected into one or two cell-stage embryos by microinjection.

Zebra fish  Immunohistochemistry of larvae Immunohistochemistry )

The zebrafish larvae collected at 3 and 15 days post-fertilization (DPF) were fixed with 4% paraformaldehyde (PFA) for 2 hours at room temperature (RT), permeated to ice-cold acetone for 7 minutes . Immobilized larvae were blocked with blocking solution (1% DMSO, 1% BSA, 0.5% TritonX-100 and 5% normal goat serum in 0.1 M PBS) for 1 h at RT and then incubated with Alexa 647-conjugated alpha-BTX (1: 100, Molecular Probes, Eugene, OR). The larvae were washed three times for 15 minutes per wash with PBDT buffer (1% DMSO, 1% BSA, 0.5% Triton X-100) and incubated with early mouse monoclonal anti-SV2 antibody (1: 200, Developmental Studies Hybridoma Bank, Boston, MA) overnight at 4 ° C. The larvae were washed six times with PBST (0.5% Triton X-100 in 0.1 M PBS) for 15 minutes per wash and incubated with Alexa Fluor 488 goat anti-mouse antibody (1: 250, Life technologies, Carlsbad, CA) Lt; / RTI > Larvae were mounted on slides in 70% glycerol in 0.1 M PBS and fluorescence images were taken with a fluorescence confocal microscope (Carl Zeiss LSM700; Zeiss, Jena, Germany).

Zebra fish  The nerves of the trunks Of the neuronal axons  analysis

Confocal images were performed and analyzed using Zeiss ZEN imaging software (Zeiss). Trunk neuronal fascicles and branched axons derived from fascicles were analyzed in each of 3 and 15 dpf zebrafish larvae. The axon branch was analyzed using an Image J plugin Neuron J (NIH, Bethesda, Md.). For quantification, branches per bundle of zebrafish were separated and counted in the fins and abdomen.

< Example  1> Clinical symptom check

Patients 1. A 38-year-old man (Fig. 1B, II-2) was born healthy from non-patient parents, but initially weak at distal lower limbs at age 8 and collapsed due to ataxia The phenomenon appeared frequently. I experienced tremor both hands when I was 12 years old. The difficulty of walking progressed gradually, and at the age of 37, he started using ankle foot orthosis. Neurological examination at 38 years of age showed that the most common leg weakness and atrophy occurred without involvement of the proximal muscle. The distal leg showed more severe muscle atrophy than the distal upper end. Bilateral pes cavus, broad-based gait, and intrinsic foot muscles and atrophy of the calf muscles were observed, but scoliosis was not observed. Needle pinprick, touch, position and sense of vibration have been reduced. Vibration and position sensation were more disturbed than strict pain and touch sensation. Sensory ataxia and positive Romberg sign were observed. Deep tendon reflexes were absent and no pathological reflections were found. He had a mild category of 10 CMTNS.

Serum TG levels were slightly reduced (63.3 ± 1.7 mg / dL) in the trials compared to the age-matched controls (mean value of 120 men aged 38 years in the laboratory: 125 ± 65 mg / dL). However, serum levels of total cholesterol and LDL cholesterol were normal with a reference value of 130-240 mg / dL (172 mg / dL) and LDL cholesterol (reference value: <120 mg / dL) of 106 mg / dl. Serum creatine kinase (CK) levels were elevated (348 IU / L, reference <185 IU / L). Levels of blood glucose, HbA1C, lactate, and pyruvate were normal. Neurological and electrophysiological examinations showed no symptoms of his parents (I-1, I-2), no disturbance of movement, sensory deficits, and abnormal electrophysiologic findings.

Patient 2. The son of the originator (Fig. 1A, III-1) was also normal and could walk at age one. At the age of 5, he had weakened dorsiflexion of the big toe, often falling. The results of the neurological examination at the age of 6 showed significant weakening in the leg girdle. He underwent ankle dorsiflexion on both sides of the limb, but his upper limb was normal. Sensory dyskinesia and a positive Romberg sign appeared like his father. Deep tendon reflexes were reduced and pathological reflexes were not found.

< Example  2> Confirm electrophysiological characteristics

The results of nerve conduction studies (NCSs) are summarized in Table 1. The electrophysiological results of proband and his son were consistent with axon neuropathy. The initiator showed prolonged motor latencies in both the median and ulnar nerves and CMAPs of the peroneal and tibial nerves were not elicited. SNAPs were absent in bilateral sural nerves, and decreased in both medial and ulnar nerves. The originator's son also showed sensory-dominant axon neuropathy; SNAPs and SNCVs were reduced in all upper and lower test nerves. Needle EMG revealed a neurogenic pattern of muscle regression. Visual evoked potentials and brainstem auditory evoked potentials were normal. Table 1 shows the electrophysiological characteristics of the patients in the FC490 axon CMT family.

Figure pat00001

Asterix ( * ) indicates abnormal abnormalities. A: absent potential, 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. Length of lower limb - dependent Muscle atrophy  Confirm

The rider revealed muscle atrophy in the leg muscles. The T1-weighted MRI results proved to be severe atrophy in the calf muscles rather than in the thighs, i.e., predominantly distal. At the thigh level, semitendinosus, adductor muscles, vastus medialis, and intermedius muscles muscles spared relatively (Figure 1C). In the lower legs, the soleus (arrow), gastrocnemius and peronei (arrowhead) muscles were found to be severely impaired. However, the tibialis anterior and posterior muscles showed weak fat infiltration (Fig. 1D).

Example  4. Distal  Distal sural  nerve) Biopsy Axon  Neuropathy Axonal  neuropathy) confirmation

Light microscopic examination of the length and cross section of the nerve fibers revealed slight nerve bundle size reduction, mild perineurium thickening, and neuronal cell proliferation. Giant MFs markedly decreased the number of endoneurial fibrosis, which was evidenced by a special stain (Luxol fast blue and modified Masson's trichrome). Myelin digestion chambers were occasionally observed. Semi-thin transverse sections showed focal subperineurial edema, medium sized and small MFs, scattered thin MFs, and rare but prominent regenerating axonal clusters 2A). The remaining MFs decreased slightly to 8,383 / mm 2 (normal mean distal calf nerve at the age of 33 years: 8,600 / mm 2 ). The range and mean diameters of MFs were 0.8 to 9.1 μm and 2.7 μm, respectively (range and mean diameters of normal distal calf nerves in a 33 year-old male were 1.8-14.0 μm and 5.4 μm, respectively). The histograms showed MF diameters of 3 탆 and less than 6 탆 consisting of 70.9% and 96.6%, respectively, and a unimodal distribution pattern (Fig. 2B). MFs with diameters larger than 8 μm were 0.3%. In this case, the MF% area was 5.9% (27.1% of normal calf nerves in a 33 year-old male). The range and mean of the g-ratio (g-ratio, axon diameter / MF diameter) were 0.36-0.84 and 0.66 ± 0.08, respectively (21-50 years g- ratio average: 0.66). The g-ratio greater than 0.7 (an abnormally thin aquatic plant) was 34.96% of the MFs and the g-ratio of less than 0.4 (abnormally thick aquatic plant) was 0.29%. Electron microscopy revealed scattered myelinated and unmyelinated axons with swelling or vacuolization of the axoplasm and membrane structures. The presence of MFs with small axon diameter, thick myelin and cluster of Schwann cell cytoplasmic processes containing axon-free collagen pockets was compatible with axonopathy (Fig. 2C). There was hardly any degradation of the myelin and axon collapse and MFs (FIG. 2D). However, no demyelinated axons or onion bulb formation was observed.

Thus, the clinical symptoms of the axon CMT family of the invention are characterized by early onset, sensory dysfunction, tremor, and slow disease progression. Even early onset, Patient 1 was able to walk without orthopedic orthoses at age 37, suggesting a slow disease progression. During the 30-year disease period, MRI results of patient 1 revealed mild muscle atrophy and fat infiltration in both calf muscles; However, at the hip and thigh level, the lower body muscles were almost intact. This function was consistent with the slow disease progression symptoms of axon CMT and also implied length-dependent axonal degeneration. PRX- and FGD4-associated dehydration-induced neuropathy CMT is characterized by slow clinical progression, early onset and sensory dyskinesia. Some clinical aspects of DGAT2-related CMT have been shown to be similar to those of PRX and FGD4 mutant CMT, although demyelinating and axon neuropathy represent different neurological types.

Example  5. DGAT2 Identification of heterozygous mutations in

The average total sequence sequencing yield of the four WES samples was approximately 8.83 Gbp / sample with a 91.25% coverage rate of the target exon region (≥ 10X) (Table 2). First, dbSNP142 and the 1000 genome project were used to remove all reported SNPs from exomes. Among the novel or rare functional mutations, mutations were selected only in patients within the family. (I-1, -2), while the parental foot of the family and his son were the patients (II-2, III-1) Comparing these SNPs with the mutations found in normal parents, c.667T> C (p.Y223H) of DGAT2 gene (MIM 606983) was the only selected. Capillary DNA sequencing showed that the DGAT2 mutation was very well co-disassociated only in patients with extended family members (Figure 3A). The Y223H mutation was located in a well-conserved region of the N-acetyltransferase superfamily (NAT-SF) domain (Fig. 3B). The GERP at the base mutation site was measured at a high score of 5.99.

Transcriptome analysis of healthy controls (n = 2) showed relatively high levels of DGAT2 expression in the distal sural nerve compared to MFN2, which is most frequently mutated in DGAT1 and axotomized CMT patients Figure 3C) (Hong et al., 2014). All other functional mutations observed in peripheral neuropathy-associated genes were considered to be genetic polymorphisms that did not cause them, either because they were not found in the control or co-localized with the patients in the experimental family (Table 3).

As described above, a novel p.Y223H mutation of DGAT2 was confirmed from the autosomal dominant Korean axon CMT family by pedigree analysis and exon sequence sequencing. No new mutations could be identified in the normal control and many known human genome mutation databases. The mutations were well conserved in the N-acetyltransferase superfamily (NAT-SF) domain and were able to imply pathogenic predictions through multiple in silico analyzes. Thus, we have determined that the identified DGAT2 mutation is a novel fundamental cause of axon CMT.

Summary of exon sequencing data for the FC490 family Filtering steps / samples Affected members Unaffected members II-2 III-1 I-1 I-2 Total yields (Gbp) 7.19 6.29 10.39 11.43 Mappable reads of total reads (%) 93.60% 75.00% 99.40% 99.40% Coverage of target region (= 1X) 97.40% 94.60% 95.40% 95.20% Coverage of target region (= 10X) 94.10% 89.30% 90.80% 90.80% Mean read depth of target region 75.2X 45.8X 46.7X 49.3X Total number of SNPs 58,649 84,893 86,404 88,153 Number of coding SNPs 19,693 20,018 20,308 20,382 Total number of indels 9,888 8,214 8,145 8,324 Number of coding indels 556 406 406 414

Indels: insertion / deletions, SNP: single nucleotide polymorphism.

Functionally important mutations observed in the FC490 family of CMT-related genes (patient 1) Gene Ref sequence Variants dbSNP142 1000G b ESP c Comments Nucleotide - Amino acid EA AA KIF1B NM_015074.3 c.3649C> T p.P1217S rs121908163 <0.001 - - Non-segregation c.4384A> C p.T1462P rs78662124 - - - Polymorphism NGF NM_002506.2 c.104C> T p.A35V rs6330 0.306 0.452 0.207 Polymorphism SCN9A NM_002977 c.3448 T> C p.W1150R rs6746030 0.891 0.871 0.891 Polymorphism CCT5 NM_012073.3 c.1243G> A p.D415N rs11557654 - 0.000 <0.001 Non-segregation NRG2 NM_004883.2 c.1072G> A p.V358I rs188700714 0.002 - - Polymorphism DST


NM_015548.4


c.13108C> G p.L4370V rs80260070 0.047 0.002 0.002 Polymorphism c.8439G> A p.M2813I rs4715630 0.808 0.742 0.908 Polymorphism c.8176A> G p.T2726A rs4715631 0.688 0.702 0.642 Polymorphism c.5831C> T p.A1944V - - - - Non-segregation FIG4
NM_014845.5
c.1090A> T p.M364L rs2295837 0.099 0.036 0.022 Polymorphism c.1961T> C p.V654A rs9885672 0.374 0.152 0.668 Polymorphism ARHGEF10
NM_014629.2
c.1110G > C p.L370F rs9657362 0.135 0.144 0.037 Polymorphism c.3960G> C p.R1320S rs117084443 0.007 - - Polymorphism NEFL NM_006158.4 c.1413delC p.P471fs rs11300136 - - - Polymorphism IKBKAP



NM_003640.3



c.3473C> T p.P1158L rs1538660 0.223 0.165 0.303 Polymorphism c.3214T> A p.C1072S rs3204145 0.223 0.165 0.304 Polymorphism c.2490A> G p.I830M rs2230794 0.080 0.047 0.016 Polymorphism c.2446A> C p.I816L rs2230793 0.286 0.183 0.441 Polymorphism c.2294G> A p.G765E rs2230792 0.278 0.182 0.414 Polymorphism LRSAM1 NM_138361.5 c.952A> G p.N318D rs1539567 0.735 0.773 0.682 Polymorphism SETX



NM_015046.5



c.7834A> G p.S2612G rs3739927 0.155 0.026 0.165 Polymorphism c.7759A> G p.I2587V rs1056899 0.508 0.297 0.732 Polymorphism c.5563A> G p.T1855A rs2296871 0.414 0.155 0.603 Polymorphism c.3455T> G p.F1152C rs3739922 0.099 0.034 0.048 Polymorphism c.1979C> G p.A660G rs882709 0.209 0.058 0.223 Polymorphism DHTKD1

NM_018706.6

c.58T> C p.F20L rs1279138 0.981 0.999 0.905 Polymorphism c.814T> G p.Y272D rs3740015 0.467 0.589 0.268 Polymorphism c.1821C> G p.I607M rs2062988 0.721 0.814 0.601 Polymorphism HK1 NM_033500.2 c.20A> G p.H7R rs906220 0.909 0.922 0.848 Polymorphism BSCL2 NM_001122955.3 c.133G> A p.G45S rs3763853 - - - Polymorphism IGHMBP2

NM_002180.2

c.602T> C p.L201S rs560096 0.703 0.844 0.650 Polymorphism c.2011A> G p.T671A rs622082 0.223 0.308 0.127 Polymorphism c.2636C> A p.T879K rs17612126 0.235 0.278 0.078 Polymorphism WNK1

NM_213655.4

c.3922A> C p.T1308P rs956868 0.848 0.851 0.855 Polymorphism c.5273G> C p.C1758S rs7955371 0.990 1,000 0.959 Polymorphism c.6180G> T p.M2060I rs12828016 0.387 0.391 0.510 Polymorphism DYNC1H1 NM_001376.4 c.7979T> G p.V2660G rs201272954 - - - Polymorphism LITAF NM_004862.3 c.274A> G p.I92V rs4280262 0.129 0.213 0.072 Polymorphism SEPT9 NM_006640.4 c.1672A> G p.M464V rs2627223 0.913 0.940 0.798 Polymorphism CTDP1 NM_004715.4 c.1019C> T p.T340M rs2279103 0.112 0.179 0.039 Polymorphism DNM2 NM_004945.3 c.T1946G p.V649G rs201780856 - - - Polymorphism PRX
NM_181882.2
c.3394G> A p.G1132R rs268674 0.956 0.936 0.989 Polymorphism c.T2612C p.V871A rs201389706 - - - Polymorphism ATP7A
NM_000052.5
c.G2299C p.V767L rs2227291 0.274 0.214 0.329 Polymorphism c.4048G> A p.E1350K rs4826245 1,000 - - Polymorphism

a Variant allele frequencies in 1000 Genome database (Nov, 2014).

b Variant allele frequencies in NHLBI Exome Sequencing Project (EA: European American, AA: African American) (Nov, 2014).

Example  6. DGAT2  Identification of cell proliferation inhibition by mutation

Transfection of DGAT2 wild type and Y223H mutants into NSC34 cells resulted in good expression of both in NSC34 without significant difference (FIG. 3D). As a result of measuring cell proliferation after overexpression of NSC34, Y223H mutation significantly inhibited cell proliferation as compared with wild type DGAT2 (FIG. 3E).

To determine whether the Y223H mutation affected enzyme activity, the TG concentration of fibroblasts derived from patient (II-2) was compared to the matched control group, sex and age (30-37 years) (Fig. 3F) in fibroblasts compared to control fibroblasts (Fig. 3F). This result is consistent with a decrease in the serum TG level of the initiator, suggesting that the Y223H mutation degrades the enzyme activity. Immunocytochemical results showed that neither wild-type nor mutant DGAT2 were present in the HEK293 cytoplasm, and there was no characteristic aggregation or abnormal distribution (FIG. 5).

As a result, the serum triglyceride (TG) levels in serum and fibroblasts of the patients were significantly lower than those in the control group matched with age and gender. These data suggest that mutations in DGAT2 are likely to induce loss of enzyme activity. On the other hand, the Y223H overexpression mutation, which greatly inhibits the cell proliferation of NSC34 cells, implies that the mutant protein causes an anti-proliferative effect on the motor nerve. In addition, a decrease in TG levels can affect some neurological functions. Thus, a mutation in DGAT2 can cause haploinsufficiency or dominant negative effects.

Example  7. Zebra fish  In the peripheral nervous system Axon  A human being obstructing a branch DGAT2  Mutation confirmation

Human DGAT2 (Y223H) was overexpressed in zebrafish and compared with the phenotype of human wild-type DGAT2 (WT) expressing larvae in order to confirm pathological effects of the mutation of DGAT2 (Y223H).

The appearance of the mutant Y223H-overexpressed larvae was normal on all 3 days (dpf) and 15 days after fertilization (Fig. 4A and D). Immunohistochemical analyzes were performed with zebrafish anti-SV2 antibody and anti-alpha-bivalirotoxin (BTX) antidote to label the neuromuscular junction (NMJ). In DGAT2 normal protein (WT) and mutant - overexpressing zebrafish larval 3dpf, bundles of muscle fibers and neurons were observed. As a result, mutant Y223H overexpressed larvae showed no change in muscle fibers, while loss of trunk fascicles was observed at 3dpf (FIG. 4B-C).

In the zebrafish mutant Y223H-induced tubular bundle, 15 days larvae were observed to determine its effect in later development. As a result, it was confirmed that zebrafish larvae expressing the human DGAT2 mutation (Y223H) showed a significant decrease in axon branches derived from bundles of neuromuscular junction (NMJ) without defect of muscle fibers (DGAT2 (DGAT2 WT-introduced larvae: 22.65 + 0.14, DGAT2 Y223H-introduced larval: 16.65 + 0.17) (Fig. 4E-F).

As described above, the zebrafish overexpressing the human DGAT2 mutation (Y223H) caused not only axon fasciculation of neurons in the neuromuscular junction (NMJ) but also axonal branching. More importantly, axonal defects observed in the early developmental stages of zebrafish were compatible with early onset CMT in patients studied.

Taken together, these data suggest that DGAT2 is not responsible for the formation of muscle fibers in neuromuscular junctions (NMJs), but rather for axon branching in motor neurons. These results also indicate that human mutations in DGAT2 (Y223H) have an adverse effect on inducing CMT, especially CMT2.

<110> Kongju National University Industry-University Cooperation Foundation          Samsung Medical Center <120> DGAT2 ASA CAUSATIVE GENE RESPONSIBLE FOR AN EARLY-ONSET          AUTOSOMAL DOMINANT AXONAL CHARCOT-MARIE-TOOTH NEUROPATHY AND          DIAGNOSIS METHOD FOR THE DISEASE <160> 6 <170> Kopatentin 2.0 <210> 1 <211> 2465 <212> DNA <213> Homo sapiens <400> 1 tgccccgttg tgaggtgata aagtgttgcg ctccgggacg ccagcgccgc ggctgccgcc 60 tctgctgggg tctaggctgt ttctctcgcg ccaccactgg ccgccggccg cagctccagg 120 tgtcctagcc gcccagcctc gacgccgtcc cgggacccct gtgctctgcg cgaagccctg 180 gccccggggg ccggggcatgg ggccaggggc gcggggtgaa gcggcttccc gcggggccgt 240 gactgggcgg gcttcagcca tgaagaccct catagccgcc tactccgggg tcctgcgcgg 300 cgagcgtcag gccgaggctg accggagcca gcgctctcac ggaggacctg cgctgtcgcg 360 cgaggggtct gggagatggg gcactggatc cagcatcctc tccgccctcc aggacctctt 420 ctctgtcacc tggctcaata ggtccaaggt ggaaaagcag ctacaggtca tctcagtgct 480 ccagtgggtc ctgtccttcc ttgtactggg agtggcctgc agtgccatcc tcatgtacat 540 attctgcact gattgctggc tcatcgctgt gctctacttc acttggctgg tgtttgactg 600 gaacacaccc aagaaaggtg gcaggaggtc acagtgggtc cgaaactggg ctgtgtggcg 660 ctactttcga gactactttc ccatccagct ggtgaagaca cacaacctgc tgaccaccag 720 gaactatatc tttggatacc acccccatgg tatcatgggc ctgggtgcct tctgcaactt 780 cagcacagag gccacagaag tgagcaagaa gttcccaggc atacggcctt acctggctac 840 actggcaggc aacttccgaa tgcctgtgtt gagggagtac ctgatgtctg gaggtatctg 900 ccctgtcagc cgggacacca tagactattt gctttcaaag aatgggagtg gcaatgctat 960 catcatcgtg gtcgggggtg cggctgagtc tctgagctcc atgcctggca agaatgcagt 1020 caccctgcgg aaccgcaagg gctttgtgaa actggccctg cgtcatggag ctgacctggt 1080 tcccatctac tcctttggag agaatgaagt gtacaagcag gtgatcttcg aggagggctc 1140 ctggggccga tgggtccaga agaagttcca gaaatacatt ggtttcgccc catgcatctt 1200 ccatggtcga ggcctcttct cctccgacac ctgggggctg gtgccctact ccaagcccat 1260 cccactgtt gtgggagagc ccatcaccat ccccaagctg gagcacccaa cccagcaaga 1320 catcgacctg taccacacca tgtacatgga ggccctggtg aagctcttcg acaagcacaa 1380 gaccaagttc ggcctcccgg agactgaggt cctggaggtg aactgagcca gccttcgggg 1440 ccaattccct ggaggaacca gctgcaaatc acttttttgc tctgtaaatt tggaagtgtc 1500 atgggtgtct gtgggttatt taaaagaaat tataacaatt ttgctaaacc attacaatgt 1560 taggtctttt ttaagaagga aaaagtcagt atttcaagtt ctttcacttc cagcttgccc 1620 tgttctaggt ggtggctaaa tctgggccta atctgggtgg ctcagctaac ctctcttctt 1680 cccttcctga agtgacaaag gaaactcagt cttcttgggg aagaaggatt gccattagtg 1740 acttggacca gttagatgat tcactttttg cccctaggga tgagaggcga aagccacttc 1800 tcatacaagc ccctttattg ccactacccc acgctcgtct agtcctgaaa ctgcaggacc 1860 agtttctctg ccaaggggag gagttggaga gcacagttgc cccgttgtgt gagggcagta 1920 gtaggcatct ggaatgctcc agtttgatct cccttctgcc acccctacct cacccctagt 1980 cactcatatc ggagcctgga ctggcctcca ggatgaggat gggggtggca atgacaccct 2040 gcaggggaaa ggactgcccc ccatgcacca ttgcagggag gatgccgcca ccatgagcta 2100 ggtggagtaa ctggtttttc ttgggtggct gatgacatgg atgcagcaca gactcagcct 2160 tggcctggag cacatgctta ctggtggcct cagtttacct tccccagatc ctagattctg 2220 gatgtgagga agagatccct cttcagaagg ggcctggcct tctgagcagc agattagttc 2280 caaagcaggt ggcccccgaa cccaagcctc acttttctgt gccttcctga gggggttggg 2340 ccggggagga aacccaaccc tctcctgtgt gttctgttat ctcttgatga gatcattgca 2400 ccatgtcaga cttttgtata tgccttgaaa ataaatgaaa gtgagaatcc tctaaaaaaa 2460 aaaaa 2465 <210> 2 <211> 388 <212> PRT <213> Homo sapiens <400> 2 Met Lys Thr Leu Ile Ala Ala Tyr Ser Gly Val Leu Arg Gly Glu Arg   1 5 10 15 Gln Ala Glu Ala Asp Arg Ser Gln Arg Ser Ser Gly Gly Pro Ala Leu              20 25 30 Ser Arg Glu Gly Ser Gly Arg Trp Gly Thr Gly Ser Ser Ile Leu Ser          35 40 45 Ala Leu Gln Asp Leu Phe Ser Val Thr Trp Leu Asn Arg Ser Ser Val Val      50 55 60 Glu Lys Gln Leu Gln Val Ile Ser Val Leu Gln Trp Val Leu Ser Phe  65 70 75 80 Leu Val Leu Gly Val Ala Cys Ser Ala Ile Leu Met Tyr Ile Phe Cys                  85 90 95 Thr Asp Cys Trp Leu Ile Ala Val Leu Tyr Phe Thr Trp Leu Val Phe             100 105 110 Asp Trp Asn Thr Pro Lys Lys Gly Gly Arg Arg Ser Gln Trp Val Arg         115 120 125 Asn Trp Ala Val Trp Arg Tyr Phe Arg Asp Tyr Phe Pro Ile Gln Leu     130 135 140 Val Lys Thr His Asn Leu Leu Thr Thr Arg Asn Tyr Ile Phe Gly Tyr 145 150 155 160 His Pro His Gly Ile Met Gly Leu Gly Ala Phe Cys Asn Phe Ser Thr                 165 170 175 Glu Ala Thr Glu Val Ser Lys Lys Phe Pro Gly Ile Arg Pro Tyr Leu             180 185 190 Ala Thr Leu Ala Gly Asn Phe Arg Met Pro Val Leu Arg Glu Tyr Leu         195 200 205 Met Ser Gly Gly Ile Cys Pro Val Ser Arg Asp Thr Ile Asp Tyr Leu     210 215 220 Leu Ser Lys Asn Gly Ser Gly Asn Ale Ile Ile Val Val Gly Gly 225 230 235 240 Ala Ala Glu Ser Leu Ser Ser Met Pro Gly Lys Asn Ala Val Thr Leu                 245 250 255 Arg Asn Arg Lys Gly Phe Val Lys Leu Ala Leu Arg His Gly Ala Asp             260 265 270 Leu Val Pro Ile Tyr Ser Phe Gly Glu Asn Glu Val Tyr Lys Gln Val         275 280 285 Ile Phe Glu Glu Gly Ser Trp Gly Arg Trp Val Gln Lys Lys Phe Gln     290 295 300 Lys Tyr Ile Gly Phe Ala Pro Cys Ile Phe His Gly Arg Gly Leu Phe 305 310 315 320 Ser Ser Asp Thr Trp Gly Leu Val Pro Thyr Ser Lys Pro Ile Thr Thr                 325 330 335 Val Val Gly Glu Pro Ile Thr Ile Pro Lys Leu Glu His Pro Thr Gln             340 345 350 Gln Asp Ile Asp Leu Tyr His Thr Met Tyr Met Glu Ala Leu Val Lys         355 360 365 Leu Phe Asp Lys His Lys Thr Lys Phe Gly Leu Pro Glu Thr Glu Val     370 375 380 Leu Glu Val Asn 385 <210> 3 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> DGAT2-Mut Forward primer <400> 3 cgggacacca tagaccattt gctttc 26 <210> 4 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> DGAT2-Mut Reverse primer <400> 4 gaaagcaaat ggtctatggt gtcccg 26 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> DGAT2-Forward primer <400> 5 ccatgaagac cctcatagcc 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> DGAT2-Reverse primer <400> 6 gctcagttca cctccaggac 20

Claims (13)

(Charcot-Marie-Toes disease) characterized by mutation in the 667th base from the adenine (A) base of the ATG translation initiation codon in the DGAT2 (diacylglycerol O-acyltransferase 2) gene represented by the nucleotide sequence of SEQ ID NO: The genetic cause of Marie-Tooth disease (CMT) is the DGAT2 mutation gene. 2. The DGAT2 mutant gene according to claim 1, wherein the mutation is a DGAT2 mutation gene in which the thymine is replaced with a cytosine at the 667th base from the adenine (A) base of the ATG translation initiation codon in the DGAT2 gene represented by the nucleotide sequence of SEQ ID NO: The DGAT2 mutant gene according to claim 1, wherein the mutant gene induces autosomal dominant axon type CMT characterized by early onset of CMT and slow disease progression. A DGAT2 mutant protein encoded from the DGAT2 mutant gene of claim 1. 5. The DGAT2 mutant protein of claim 4, wherein the mutant protein comprises a sequence in which the 223rd amino acid of the amino acid sequence of SEQ ID NO: 2 is substituted with histidine in tyrosine. A CMT diagnostic marker comprising the DGAT2 mutant gene of claim 1 or the protein of claim 4. A composition for diagnosing CMT comprising a DGAT2 mutant gene of claim 1 or an agent capable of detecting the expression of a protein encoded by the gene. 8. The CMT diagnosis composition according to claim 7, wherein the preparation comprises a primer set for specifically amplifying the DGAT2 mutation gene represented by SEQ ID NO: 3 and SEQ ID NO: 4. 8. The composition of claim 7, wherein said agent comprises an antibody specific for a protein encoded by a DGAT2 mutant gene. 10. A CMT diagnostic kit comprising a composition according to any one of claims 7 to 9. Isolating genomic DNA from a sample derived from an individual; And
Confirming the mutation at position 667 from the ATG translation initiation site of the DGAT2 gene represented by SEQ ID NO: 1 in the separated genomic DNA;
Gt; CMT &lt; / RTI &gt; 12. The method according to claim 11, wherein said mutation is the diagnosis of CMT characterized in that the thymine is replaced with cytosine at the 667th base from the adenine (A) base of the ATG translation initiation codon in the DGAT2 gene represented by the nucleotide sequence of SEQ ID NO: Early diagnosis before onset. Preparing a sample separated from an object; And
Contacting an antibody that specifically binds to the mutant protein of claim 4 with the sample to confirm expression of the protein;
Gt; CMT &lt; / RTI &gt;









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