System for Diagnosing of Avellino Corneal Dystrophy
The present invention relates to a system for diagnosing Avellino corneal dystrophy, and more particularly to a system for diagnosing Avellino corneal dystrophy, in which whether a sample is normal or Avellino corneal dystrophy is determined based on the ratio of the input first PGR amplification value and the second PGR amplification value. The system makes it possible to diagnosis Avellino corneal dystrophy in a simpler and accurate manner without being influenced by the doctor's skill. Particularly, the inventive system makes the overall process systematic, and thus provides accurate diagnosis. In addition, the system can also easily administer a number of test subjects.
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The present invention relates to a system for diagnosing Avellino corneal dystrophy, and more particularly to a system for diagnosing Avellino corneal dystrophy, in which whether a sample is normal or Avellino corneal dystrophy is determined based on the ratio of the input first PGR amplification value and the second PGR amplification value.
BACKGROUND ARTCorneal dystrophy is an autosomal dominant hereditary disease, which begins with an opacity in the center of cornea and gradually spreads and thus ends up vision loss as a patient gets older. It includes Avellino corneal dystrophy, Granular corneal dystrophy, lattice type I corneal dystrophy, Reis-bucklers corneal dystrophy, etc., and is caused by mutation of a gene coding βIG-H3 protein.
Heterozygous patients suffering from Avellino corneal dystrophy appear to have severe loss of vision as getting older and homozygous patients appear to have complete loss of vision since 6 years old. Avellino corneal dystrophy is a newly named disease in 1988, divided from generally called Granular corneal dystrophy because it was found to have genetic distinction. Also, it has been known to be the most common corneal dystrophy worldwide, 1/340 to 1/1000 of prevalence rate in Korea (the case of heterozygote) based on genetic analysis indicates that it is a common dystrophy (Holland, E. J. et al., Ophthalmology, 99:1564, 1992; Kennedy, S. M. et al., Br. J. Ophthalmol., 80:489, 1996; Dolmetsch, A. M. et al., Can. J. Ophthalmol., 31:29, 1996; Afshari, N. A. et al., Arch. Ophthalmol., 119:16, 2001;Stewart, H. S. Hum. Mutat., 14:126, 1999).
The present inventors have found that if a heterozygous Avellino corneal dystrophy patient has LASIK surgery, about 2 years later, opacity of cornea starts to develop aggressively and eventually .results in vision loss (Jun, R M et al., Ophthalmolgy, 111:463, 2004), Previously, eye surgery has been performed with an expectation that LASIK or Excimer Laser surgery would, get rid of vision blurriness of a patient suffering from corneal dystrophy. Also, even in Korea, approximately 3 hundred thousand cases of LASIK surgery have been performed, which leads to the assumption that 300 people lost their vision, based on 1/1000 of minimum estimation of heterozygous patients suffering from Avellino corneal dystrophy. Patients who have undergone LASIK surgery are mainly in their 20's and 30's carrying out productive activities; therefore, their vision loss causes serious troubles in both society and economics. In addition, after approval of LASIK surgery since the year 2000 in USA, Avellino corneal dystrophy patients who underwent LASIK surgery have been found to lose eye sight among African American, which infers that plenty of similar cases might be occurring throughout the world.
However, although accurate diagnosis of Avellino corneal dystrophy is required to prevent the progression of Avellino corneal dystrophy by LASIK surgery, the diagnosis of Avellino corneal dystrophy is just conducted by microscopic observation of corneal opacity, and thus often doctors miss latent symptoms of patients to perform LASIK surgery, which results in vision loss. Therefore, rapid and precise diagnosis of corneal dystrophy is desperately in need.
In addition, in conventional diagnosis, the results of diagnosis may vary depending on variables such as the skill of a doctor and the patient's condition. Thus, the development, of diagnostic methods having improved reliability and accuracy has been required.
Accordingly, the present inventors have made extensive efforts to provide a new system for diagnosing corneal dystrophies, including Avellino corneal dystrophy, which are required to be accurately diagnosed before vision enhancement surgery. As a result, the present inventors have found that, when a first PGR amplification value measured by adding to a sample DNA a primer pair capable of amplifying exon 4 of a transforming growth factor b-induced (TGFBI) gene and a probe capable of detecting a TGFBI gene containing no mutation in exon 4 is compared with a second PGR amplification value measured by adding to the sample DMA the primer pair capable of amplifying exon 4 of the TGFBI gene and a probe capable of detecting a TGFBI containing a mutation in exon 4, if the first PGR amplification value is 5-9 times the second PCR amplification value, the sample DNA is normal, and if the ratio of the first PCR amplification value to the second PCR amplification value is 1:1.5-1.5:1, the sample DNA is an Avellino corneal dystrophy heterozygote, and if the second PCR amplification value is 5-9 times the first PCR amplification value, the sample DNA is an Avellino corneal dystrophy homozygote. Based on this finding, the present invention has been completed.
DISCLOSURE OF INVENTION Technical ProblemIt is an object of the present invention to provide a new system for diagnosing Avellino corneal dystrophy, which can diagnose Avellino corneal dystrophy in a simpler and more skill.
Technical SolutionTo achieve the above object, the present invention provides a system for diagnosing Avellino corneal dystrophy, the system comprising:
(a) a client group comprising at least one client, which transmits information about a sample and a request for analysis through a network to a server comprising an input means to request a diagnostic service and receives and stores the data of determination of Avellino corneal dystrophy, which are transmitted from a server comprising an output means in response to the request;
(b) the input means for receiving the information about the sample, the .request for analysis, a first PGR amplification value measured by adding to a sample DNA a primer pair capable of amplifying exon 4 of a transforming growth factor b-induced (TGFBI) gene and a probe capable of detecting a TGFBI gene containing no mutation in exon 4, and a second PGR amplification value measured by adding to the sample DMA the primer pair capable of amplifying exon 4 of TGFBI gene and a probe capable of detecting a TGFBI gene containing a mutation in exon 4;
(c) an analysis means wherein when the first PGR amplification value is 5-9 times the second PGR amplification value, the sample DNA is determined to be normal, when the ratio of the first PGR amplification value to the second PGR amplification value is 1:1.5-1.5:1, the sample DNA is determined to be an Avellino corneal dystrophy heterozygote, and when the second PGR amplification value is 5-9 times the first PGR amplification value, the sample DNA is determined to be an Avellino corneal dystrophy homozygote; and
(d) the output means for outputting the results of determination from the analysis means.
The present invention also provides a computer readable medium or media comprising the above system for diagnosing Avellino corneal dystrophy.
The present invention also provides a computer comprising the above system for diagnosing Avellino corneal dystrophy.
The present invention also provides a method of diagnosing Avellino corneal dystrophy using either the above system, for diagnosing Avellino corneal dystrophy or the above computer readable medium or media.
Other features and embodiments of the present invention will be more apparent from the following detailed-descriptions and the appended claims.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Generally, the nomenclature used herein are well known and are commonly employed in the art.
The definition of main terms used in the detailed description of the invention is as follows.
As used herein, “Avellino corneal dystrophy” is also known as granular corneal dystrophy type II and is an autosomal dominant hereditary disease in which an opacity symptom occurs in the center of cornea. It is is a disease caused by genetic abnormality in which the sequence CGC in exon 4 of transforming a growth factor b-induced (TGFBI) gene is mutated to CAC so that the 124th amino acid arginine of BIGH3 protein is mutated to histidine (R124K).
As used herein, the term “heterozygote” refers to the case in which a mutation is present in only one gene of a pair of genes transmitted from parents. The term “Avellino corneal dystrophy heterozygote” refers to the case in which a mutation is present in only one of a pair of transforming growth factor b-induced (TGFBI) genes transmitted from parents.
As used herein, the term “homozygote” refers to the case in which a mutation is present in a pair of genes transmitted from parents. The term “Avellino corneal dystrophy homozygote” refers to the case in which a mutation is present in a pair of transforming growth factor b-induced (TGFBI) genes transmitted from parents.
As used herein, “a program” or “computer program” refers generally to a syntactic unit that conforms to the rules of a particular programming language and that is composed of declarations and statements or instructions, divisible into, “code segments” needed to solve or execute a certain function, task, or problem. A programming language is generally an artificial language for expressing programs.
As used herein, a “system” or a “computer system” generally refers to one or more computers, peripheral equipment, and software that perform data processing. A “user” or “system operator” in general includes a person, that uses a computer network accessed through a “user device” (e.g., a computer, a wireless device, etc.) for the purpose of data processing and information exchange. A “computer” is generally a functional unit that can perform substantial computations, including numerous arithmetic operations and logic operations without, human intervention.
As used herein, a “computer readable medium” can be a hard disk, floppy disc, compact disc, magneto-optical disc, Random Access Memory, Read Only Memory or Flash Memory and the like, but is not limited thereto. In addition, the computer readable medium that is used in the present invention can be included in a single computer or distributed in a network. Herein, the network may be any conventional network system such as LAN (local area network) or WAN (wide-area network). A network or a server, which may be used in the present invention, may be in the form, of known network or server, such as World Wide Web application or World Wide Web server.
In one aspect, the present invention is directed to a system for diagnosing Avellino corneal dystrophy, the system comprising:
(a) a client group comprising at least one client, which transmits information about a sample and a request for analysis through a network to a server comprising an input means to request a diagnostic service and receives and stores the data of determination of Avellino corneal dystrophy, which are transmitted from a server comprising an output means in response to the request;
(b) the input means for receiving the information about the sample, the request for analysis, a first PGR amplification value measured by adding to a sample DNA a primer pair capable of amplifying exon 4 of a transforming growth factor b-induced (TGFBI) gene and a probe capable of detecting a TGFBI gene containing no mutation in exon 4, and a second PGR amplification value measured by adding to the sample DNA the primer pair capable of amplifying exon 4 of TGFBI gene and a probe capable of detecting a TGFBI gene containing a mutation in exon 4;
(c) an analysis means wherein when the first PGR amplification value is 5-9 times the second PGR amplification value, the sample DNA is determined to be normal, when the ratio of the first PGR amplification value to the second PCR amplification value is 1:1.5-1.5:1, the sample DNA is determined to be an Avellino corneal dystrophy heterozygote, and when the second PCR amplification value is 5-9 times the first PCR amplification value, the sample DNA is determined to be an Avellino corneal dystrophy homozygote; and
(d) the output means for outputting the results of determination from the analysis means.
Preferably, the system of the present invention may include a client group comprising at least one client, which transmits information about a sample and a request for analysis through a network to a server comprising an input means to request a diagnostic service and receives and stores the data of determination of Avellino corneal dystrophy, which are transmitted from a server comprising an output means in response to the request.
As information about the sample, one or more of the kind of sample, the client name, the client institution name, collection date, collection time, and contact may be input. The sample that is used herein may be one or more of an oral mucosa cell, blood, and a hair root. In addition, the request for analysis may include a written consent to genetic testing.
In addition, the absorbance at 260 nm and the absorbance at 280 nm of DNA of the sample can be input as data, and a first calculating means calculates the concentration and purity of the sample DNA based on the absorbance. Also, the system of the present invention may include a second calculating means that calculates the amounts of probes, a primer pair and distilled water in view of the total number of samples and a control group. Moreover, the calculated concentration and purity of the sample DNA may be used to determine the amount of the sample DNA.
The probes consist of a probe, which can detect a TGFBI gene containing no mutation in exon 4, and a probe which can detect a TGFBI gene containing a mutation in exon 4. The primer pair refers to a primer pair capable of amplifying exon 4 of the TGFBI gene. Meanwhile, as reagents for performing PCR, Taq polymerase, dNTP, MgCl2, PGR buffer and the like may be used.
The primer pair capable of amplifying exon 4 of the TGFBI gene is a primer pair capable of amplifying the position of codon 124 of the TGFBI gene and is preferably a primer pair represented by SEQ ID NOS: 1 and 2. In addition, the probe capable of detecting; the TGFBI gene containing no mutation in exon 4 is a probe capable of detecting a TGFBI gene containing “CGC” at codon 124 and is preferably a probe represented by SEQ ID NO: 3. In addition, the probe capable of detecting the TGFBI gene containing a mutation in exon 4 is a probe capable of detecting a TGFBI gene containing “CAC” at codon 124 and is preferably a probe represented by SEQ ID NO: 4.
Primer Pair for Amplifying Codon 124 of TGFBI Gene
Probe for detecting TGFBI gene containing “CGC” at cod on 124
Probe for detecting TGFBI gene containing “CAC” at codon 124
The inventive system for diagnosing Avellino corneal dystrophy may farther comprise a PGR device which outputs, based on the calculated amounts of the probes, the primer pair and distilled water, a first PGR amplification value, measured by adding to the sample DNA the primer pair capable of amplifying exon 4 of the transforming growth factor b-induced gene and the probe capable of the TGFBI gene containing no mutation in exon 4, and a second PGR amplification value measured by adding to the sample DNA the primer pair capable of amplifying exon 4 of the TGFBI gene and the probe capable of detecting the TGFBI containing a mutation in exon 4. The PGR device may comprise a real-time PCR program set up therein.
For rapid processing, the primer pair capable of amplifying exon 4 of the TGFBI gene, the probe capable of detecting the TGFBI gene containing no mutation in exon 4, and the probe capable of detecting the TGFBI gene containing a mutation in exon 4 are simultaneously added to the sample DNA to perform PGR, so that the first PCR amplification value and the second PCR amplification value can be simultaneously obtained.
The output first PCR amplification value and second PCR amplification value are input into the input means. When the first amplification value is determined to be 5-9 times the second PCR amplification value by an analysis means, the sample is determined to be normal, and when the ratio of the first PCR amplification value to the second PCR amplification value is 1:1.5-1.5:1, the sample is determined to be an Avellino corneal dystrophy heterozygote. Also, when the second PCR amplification value is 5-9 times the first PCR amplification value, the sample is determined to be an Avellino corneal dystrophy homozygote. Preferably, when the first PCR amplification value is 7-8 times the second PCR amplification value, the sample is determined to be normal, and when the second PCR amplification value is 7-8 times the first PCR amplification value, the sample is determined to be an Avellino corneal dystrophy homozygote.
The system of the present, invention may further comprise a display means that provides a two-dimensional allelic discrimination plot in which the first PCR amplification value and the second PCR amplification value are plotted along two axes. In the two-dimensional allelic discrimination plot, the first PCR amplification value is plotted along the X-axis, and the second PCR amplification value is plotted along the Y-axis. When the PCR amplification values are located at the right lower portion of the plot, the sample is determined to be normal, and when the PCR amplification values are located at the left upper-portion of the plot, the sample is determined to be an Avellino corneal dystrophy homozygote.
EXAMPLESHereinafter, the present invention will be described in further detail with reference to examples. It will be obvious to a person having ordinary skill in the art that these examples are illustrative purposes only and are not to be construed to limit the scope of the present invention.
Example 1 Example of Diagnosis of Avellino Corneal Dystrophy 1-1: Input of Information about Sample and Request for AnalysisA client group consisting of an individual client or a medical institution in which samples are collected from individual clients transmits information about a sample and a request for analysis to the system's operator server.
Herein, as the information on the sample, the kind of sample, the client's name, the institution's name, collection date, collection time, contact and so on are input, and the kind of sample is selected from whole blood, oral epidermis, and hair roots according the collected form. In addition, before the request for analysis is prepared, a written consent to genetic testing is prepared.
Information about an online request displayed on the system operator's server is as shown in
The system for diagnosing Avellino corneal dystrophy according to the present invention includes a first calculating means for calculating the concentration and purity of sample DNA.
A Genios instrument (TECAN, Austria) capable of measuring the absorbance at 260 nm and the absorbance at 280 nm of sample DAN may be connected to the system of the present invention. The XFluor4 program (TECAN, Austria) is executed in the system operator's apparatus (computer) connected to the instrument to measure the absorbance and calculate the concentration and purity of DNA.
Specifically, in ‘XFluor4->Edit->Measurement parameter’, ‘Absorbance’ is selected. Then, in ‘Meas.params’, the absorption wavelength is set at 260 nm, and ‘Number of reads’ is set at about 2, after which the OK button is clicked. Then, when ‘XFluor4->Start measurement’ is clicked, the absorbance at 260 nm is measured.
Meanwhile, in ‘XFluor4->Edit->Meas.params’, the absorption wavelength is set at 280 nm, and ‘Number of reads’ is set at about 2, after which the OK button is clicked. Then, when ‘XFluor4->Start measurement’ is clicked, the absorbance at 280 nm is measured.
At OD260=1C the concentration of double-stranded DNA corresponds to about 50 ng/ul. Thus, the concentration of DNA is calculated by subtracting a blank value from the value at 260 nm and multiplying the subtracted value by 50. Also, after the absorbance at 260 nm and 280 nm has been measured, the purity of DNA can be determined based on the ratio of both (OD260/OD280). Generally, when the ratio of OD260/OD280 is between 1.8 and 2.0, the DNA is determined to have good purity.
The system for diagnosing Avellino corneal dystrophy according to the present invention comprises a second calculating means for calculating the amount of sample DMA for PCR amplification and a PCR device.
The amounts of probes, a primer pair and distilled water were calculated before preparation by adding UP the number of samples and a control normal (NN) group, an Avellino corneal dystrophy heterozygote (HN), an Avellino corneal dystrophy homozygote (HH) and a negative control (NTC), and then adding 0.2 μl to reduce the loss. The amount of sample DNA was determined based on the calculated concentration and purity of the sample DNA.
A primer pair for amplifying a mutation region (i.e., codon 124 position) of exon 4 of the TGFBI gene, a probe binding to a normal gene fragment having a mutation (i.e., a probe for detecting a TGFBI gene containing “CGC” at codon 124), and a probe binding to a gene fragment containing a mutation (i.e., a probe for detecting a TGFBI gene containing “CAC” at codon 124) are as follows. Herein, the probe binding to the normal gene fragment having no mutation was labeled with VIC, and the probe binding to the gene fragment having a mutation was labeled with FAM. In addition, a minor groove binder (MGB) was attached to facilitate binding to a complementary gene fragment.
Primer pair for amplifying codon 124 position of TGFBI gene
Probe for detecting TGFEu gene containing “CGC” at codon 124
Probe for detecting TGFBI gene containing “CAC” at codon 124
Then, to obtain PCR values from the PCR device (Applied Biosystems), a real-time PCR program is executed. Specifically, when step-one software V2.0 in the RT-PCR device is double-clicked and ‘Advanced setup’ is clicked, ‘Experiment Properties’ appears. Then, ‘Step One™ Instrument (48 wells), Genotyping’ is selected. In ‘Plate Setup’ under the ‘Experiment, Properties’ line, ‘ACD probe’ is clicked for ‘SNP Assay’. Line 1 of column A on a screen showing a 48-well plate is designated as ACD1, line 2 of column A is designated as ACD 2, and in this manner, 48 wells are all designated. The number of cycles in ‘Run Method’ is changed
In a PCR reaction, a master mix consisting of 0.0625 μl of primers, 5 μl of probes, 2.9375 μl of distilled water and 2 μl of sample DNA was used. Experimental conditions were set up as follows.
Temperature Conditions
Pre-read: 60° C. for 30 sec;
Holding: 95° C. for 10 min;
Cyclying: 36 cycles, each consisting of 95° C. for 3-15 sec and 60° C. for 30-60 sec; and
Post-read: 60° C. for 30 sec.
1-4: Determination of Avellino Corneal Dystrophy Based on PGR Amplification ValuesThe VID positive value (first PGR amplification value measured by adding a primer pair for amplifying exon 4 of TGFBI gene and a probe binding to a normal gene fragment) and the FAM positive value (second PGR amplification value measured by adding a primer pair for amplifying exon 4 of TGFBI gene and a probe binding to a mutant gene fragment) in Example 1-3 were expressed in an allelic discrimination plot as shown in
When the first PGR amplification value is determined to be 5-9 times the second PGR amplification value by the analysis means of the inventive system, the sample DNA is determined to be normal. When the ratio of the first PGR amplification value to the second PGR amplification value is 1:1.5-1.5:1, the sample DNA is determined to be a Avellino corneal dystrophy heterozygote, and when the second PCR amplification value is 5-9 times the first PCR amplification value, the sample DNA is determined to be an Avellino corneal dystrophy homozygote. Whether the sample is normal is expressed as a color on the left of
The results of determination in Example 1-4 are output on a screen as shown in
As described above, the system for diagnosing Avellino corneal dystrophy according to the present invention is a novel diagnostic system that can diagnose Avellino corneal dystrophy in a simpler and more accurate manner without being influenced by the doctor's skill. Particularly, it makes the overall process systematic, and thus provides accurate diagnosis. In addition, the system can also easily administer a number of test subjects.
Although the present invention has been described in detail with reference to the specific features, it will be apparent to those skilled in the art that this description is only for a preferred embodiment and does not limit the scope of the present invention. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.
Claims
1. A system for diagnosing Avellino corneal dystrophy, the system comprising:
- (a) a client group comprising at least one client, which transmits information about a sample and a request for analysis through a network to a server comprising an input means to request a diagnostic service and receives and stores the data of determination of Avellino corneal dystrophy, which are transmitted from a server comprising an output means in response to the request;
- (b) the input means for receiving the information about the sample, the request for analysis, a first PCR amplification value measured by adding to a sample DNA a primer pair capable of amplifying exon 4 of a transforming growth factor b-induced (TGFBI) gene and a probe capable of detecting a TGFBI gene containing no mutation in exon 4, and a second PCR amplification value measured by adding to the sample DNA the primer pair capable of amplifying exon 4 of TGFBI gene and a probe capable of detecting a TGFBI gene containing a mutation in exon 4;
- (c) an analysis means wherein when the first PCR amplification value is 5-9 times the second PCR amplification value, the sample DNA is determined, to be normal, when the ratio of the first PGR amplification value to the second PGR amplification value is 1:1.5-1.5:1, the sample DNA is determined to be an Avellino corneal dystrophy heterozygote, and when the second. PGR amplification value is 5-9 times the first PGR amplification value, the sample DNA is determined to be an Avellino corneal dystrophy homozygote; and
- (d) the output means for outputting the results of determination from the analysis means.
2. The system for diagnosing Avellino corneal dystrophy of claim 1, further comprising a display means that provides a two-dimensional allelic discrimination plot in which the first PGR amplification value and the second PGR amplification value are plotted along two axes.
3. The system for diagnosing Avellino corneal dystrophy of claim 1, wherein the first PGR amplification value and the second PGR amplification value are simultaneously measured by adding to the sample DNA the primer pair capable of amplifying exon 4 of the TGFBI gene, the probe capable of detecting the TGFBI gene containing no mutation in exon 4, and the probe capable of detecting the TGFBI gene containing a mutation in exon 4 to perform PGR.
4. The system for diagnosing Avellino corneal dystrophy of claim 1, wherein the information about the sample is one or more of the kind of sample, the client name, the client institution name, collection date, collection time, and contact.
5. The system for diagnosing Avellino corneal dystrophy of claim 4, wherein the sample is any one of an oral mucosa cell, blood, and a hair root.
6. The system for diagnosing Avellino corneal dystrophy of claim 1, wherein the request for analysis comprises a written consent to genetic testing.
7. The system for diagnosing Avellino corneal dystrophy of claim 1, further comprising a PGR device that outputs the PGR amplification values,
8. The system for diagnosing Avellino corneal dystrophy of claim 1, further comprising a first calculating means that calculates the concentration and purity of the sample DNA by inputting the absorbance at 260 nm and the absorbance at 280 nm of the sample DNA, and a second calculating means that calculates the amounts of probes, the primer pair and distilled water.
9. The system for diagnosing Avellino corneal dystrophy of claim 1, wherein the primer pair capable of amplifying exon 4 of the TGFBI gene is a primer pair represented by SEQ ID NOS: 1 and 2.
10. The system for diagnosing Avellino corneal dystrophy of claim 1, wherein the probe capable of detecting the TGFBI gene containing no mutation in exon 4 is a probe capable of detecting a TGFBI gene containing “CGC” at codon 124.
11. The system for diagnosing Avellino corneal dystrophy of claim 10, wherein the probe capable of detecting a TGFBI gene containing “CGC” at codon 124 is a probe represented by SEQ ID NO: 3.
12. The system for diagnosing Avellino corneal dystrophy of claim 1, wherein the probe capable of detecting the TGFBI gene containing a mutation in exon 4 is a probe capable of detecting a TGFBI gene containing “CAC” at codon 124.
13. The system, for diagnosing Avellino corneal dystrophy of claim 12, wherein the probe capable of detecting a TGFBI gene containing “CAC” at codon 124 is a probe represented by SEQ ID NO: 4.
Type: Application
Filed: Apr 6, 2018
Publication Date: Sep 6, 2018
Applicant: Avellino Co., Ltd. (Gangnam-gu)
Inventors: Gene Lee (Gyeonggi-do), Jung Kuk Yun (Seoul)
Application Number: 15/947,463
