KR100381902B1 - A glaucoma-coding DNA and protein - Google Patents

Abstract

The present invention relates to a glaucoma-coding novel DNA sequence, and more particularly, cytosine at the arginine (Arg) position 46 in the conventional glaucoma-coding DNA sequence is converted to thymine to stop codon. ), It relates to a glaucoma-encoding novel DNA sequence in which cytosine at threon position (Thr) 353 is converted to thymine and mutated to isoleucine (Ile). Therefore, according to the present invention, not only the pathogenesis and mechanism of new glaucoma can be understood, but also it is helpful to develop molecular genetic diagnostic kit in the future.

Description

A glaucoma-coding DNA and protein

The present invention relates to a glaucoma-coding novel DNA sequence, and more particularly, cytosine at the arginine (Arg) position 46 in the conventional glaucoma-coding DNA sequence is converted to thymine to stop codon. ), It relates to a glaucoma-encoding novel DNA sequence in which cytosine at threon position (Thr) 353 is converted to thymine and mutated to isoleucine (Ile).

In general, glaucoma is a disease in which intraocular pressure is significantly increased, and symptoms such as eye hardening, retinal atrophy, papillary depression, and blindness may occur. This type of glaucoma is the most common form of open angle glaucoma (OAG) after the age of 40, and the incidence is more frequent with age. In the early stages, there are many symptoms that are not noticed and the pathology is severe and can not be recovered. Although glaucoma is not always accompanied by ocular hypertension (OHT), many patients often have intraocular pressure (IOP) in excess of 21 mmHg. Eye drops may cause steroid-induced glaucoma with increased intraocular pressure in some patients.

The exact etiology for such glaucoma is not yet known, but it is reported that genetic factors play an important role in the pathogenesis of glaucoma. Recently, the TIGR gene, which has been identified as a cause of some glaucoma (JOAG, POAG), is expressed in trabecular meshwork cells and its expression is regulated by steroids. It is strongly suggested that intraocular pressure increases and the pathogenic mechanisms of glaucoma are related. Thus, understanding the biological function of TIGR genes is expected to contribute significantly to the study of intraocular pressure regulation as well as to the pathogenesis of glaucoma, and the necessity of studying the distribution and types of mutations of these TIGR genes is enormous. Was done.

Therefore, the inventors of the present invention completed the present invention by cloning the TIGR gene in genomic DNA of primary angular glaucoma (POAG) patients in Korea to investigate the distribution and type of the TIGR mutation as described above, and performing the mutation analysis by SSCP method. Was done.

After all, the main object of the present invention is to provide a glaucoma-encoding DNA of SEQ ID NO: 1.

It is also an object of the present invention to provide a glaucoma-encoding DNA comprising the DNA sequence of SEQ ID NO: 2.

Another object of the present invention is to provide a glaucoma-encoding protein comprising the amino acid sequence of SEQ ID NO: 3.

Finally, it is an object of the present invention to provide a glaucoma-encoding protein comprising the amino acid sequence of SEQ ID NO: 4.

Figure 1 shows a tree of patient families with mutations at amino acid position 46.

Figure 2 is an electrophoresis picture of the results of SSCP analysis for identifying mutations at amino acid position 46 and amino acid position 353.

Hereinafter, the present invention will be described in more detail.

(One). Glaucoma-coding DNA Isolation

After separating leukocytes from peripheral blood of a patient diagnosed with glaucoma in a hospital outpatient, whole genomic DNA was isolated using a DNA blood separation kit (BM, USA). In addition, blood was drawn from the patient's family in the same manner as above to isolate genomic DNA. And the pedigree of the family was confirmed and shown in FIG. Here, the white symbols are genetically and phenotypicly ill, the black symbols are glaucoma, the dots are genetically carriers, and the arrows are first-outpatients. More specifically, subject 3 is currently a patient with glaucoma disease, and 1, 2, and 4 are potential patients with glaucoma expression, although not yet expressed, all of them are family members of the same gene mutation (in this case, More than 46 DNA). On the other hand, as shown in the left figure of Figure 2, 3 is a case of homozygous that caused severe glaucoma in which all 46 DNA of the left and right pair of polypeptides of the chromosome is abnormal, and 1, 2, 4 are any of the left and right pair of polypeptides In the case of heterozygotes with only 46 or more DNAs, they are all likely to be glaucoma patients. On the other hand, No. 5 is normal for both left and right polypeptides, and there is no fear of glaucoma. On the other hand, the right figure of FIG. 2 is a case of another family, which is different from another 353 DNA unlike the case of the left figure of FIG. In this case, the sixth subject showed a heterozygote, which is a person having a possibility of expressing glaucoma as the case of one or more pairs of 353 DNAs in the left and right pairs, and the seventh indicates a normal person.

(2). Specific Glaucoma DNA Amplification

Using the genomic DNA isolated above as a template, specific sites were amplified using the following forward primers and reverse primers. At this time, PCR reaction was performed by adding 50ng of genomic DNA, 0.2mM of forward primer and reverse primer, and 0.19mM dNTP (dATP, dCTP, dGTP, and dTTP), 50mM KCl, 1.5mM MgCl2 and 10mM Tris-HCl. The total volume was set to 20 µl. Then, PCR was performed under each condition of the following Table 1.

Primer Sequence for PCR and PCR and SSCP Conditions area Primer sequence Annealing Temperature (℃) PCR product size (bp) SSCP temperature (℃) 88-281 5'-TGTGCACGTTGCTGCAGC-3'5'-ATGGATGACTGACATGGCC-3 ' 56 204 10 1014-1202 5'-ATACTGCCTAGGCCACTGG-3'5'-CAATGTCCGTGTAGCCACC-3 ' 62 189 14 1296-1493 5'-CTGGAACTCGAACAAACCTGG-3'5'-CATGCTGCTGTACTTATAGCG-3 ' 60 198 8 -83-281 5-'TGGCCACCTCTGTCTTCC-3'5'-ATGGATGACTGACATGGCC-3 ' 60 384 177-int1A 5'-AGGAAGGCCAATGACCAG-3'5'-TAGGAGAAAGGGCAGGGGAGGC-3 ' 62 593 int1B-795 5'-AACATAGTCAATCCTTGGGCC-3'5'-CGGTGTCTCCCTCTCCACT-3 ' 56 170 796-1316 5'-GATGTGGAGGACTAGTTTGG-3'5'-CCAGGTTTGTTCGAGTTCCAG-3 ' 56 521 1296-3'UTR 5'-CTGGAACTCGAACAAACCTGG-3'5'-GCTTGGAGGCTTTTCACATC-3 ' 60 283

(3). Mutation Identification

As a method of identifying mutations, a cold-single strand conformational polymorphism (SSCP) method using an isotope was used. That is, the PCR product amplified in the above process was denatured using MMH (SERVA, Germany), and then 100V, 10 minutes, and 300V, 2 hours on a 20% polyacrylamide TBE gel (NOVEX, USA). After separation by electrophoresis at the temperature of Table 1, the gel was stained with EtBr to confirm the DNA band (Fig. 2). In addition, compared to the band form of the control group, the site where the mutation occurred was identified and separated from the gel.

(4). DNA sequence identification

In the above process, the band separated and estimated to be a mutation site was used as a template and again amplified by PCR. This PCR product was then linked to a T-easy vector and then transformed into competent cells and incubated for one day on a liquid medium at 37 ° C. Thereafter, 10 μg of plasmid DNA containing the PCR product was obtained using a Promega Plasmid DNA purification kit, and sequencing was performed using the Promega Plasmid DNA purification kit. As a result, glaucoma-encoding DNA was found in the normal subjects, while DNAs 46 and 353 were all normal (subjects 5 and 7 of FIG. 2) and in the subjects in lanes 1 to 4 of the left side of FIGS. 1 and 2. Cytosine at the arginine (Arg) position 46 in the sequence is converted to thymine, which causes abnormal stop codons. In the case of the subject in lane 6 of the right figure of FIG. 2, threonine 353 ( Cytosine at the Thr position was converted to thymine, and it was confirmed that the mutation occurred to isoleucine (Ile). The ALF expression DNA sequencing kit (Pharmacia, USA) using a primer labeled with a fluorescent material was used. The DNA sequence of the attached SEQ ID NO: 1 is described from position 5 (tgt) as part of the DNA sequence of homosapiens. DNA sequence and cytosine ( c ga) at position 46 arginine (Arg) is converted to thymine ( t ga) (the 124th base in SEQ ID NO: 1), the protein sequence of SEQ ID NO: 3 It is also the protein sequence from position 5 (Cys), defined by the amino acid expressed by, and since 46 is also a stop codon, only up to 45 glycine (Gly) appears in SEQ ID NO. Meanwhile, the DNA sequence of SEQ ID NO: 2 is a DNA sequence described from position 317 (ata) as another part of the DNA sequence of Homo sapiens, and cytosine (a c a) of threonine (Thr) position 353 is thymine. isobutane is converted to (a t a) It can be seen that a mutation has occurred to Ile (the 110th base in SEQ ID NO: 2), and the protein sequence of SEQ ID NO: 4 is also a protein from position 317 (Ile) defined by the amino acid expressed by SEQ ID NO: 2 As a sequence, it can be also seen that 373 is mutated to isoleucine (Ile) (the 37th base in SEQ ID NO: 4).

As described above, the present invention relates to a glaucoma-coding novel DNA sequence. More specifically, 46 arginine (Arg) in the conventional glaucoma-coding DNA sequence is a stop codon, 353 threonine ( Thr) relates to a glaucoma-encoding novel DNA sequence that has been mutated to isoleucine (Ile). Therefore, according to the present invention, not only the pathogenesis and mechanism of new glaucoma can be understood, but also it is helpful to develop molecular genetic diagnostic kit in the future.

Claims (4)

Glaucoma-encoding DNA comprising the DNA sequence of SEQ ID NO: 1 Glaucoma-encoding DNA comprising the DNA sequence of SEQ ID NO: 2 Glaucoma-encoding protein comprising the amino acid sequence of SEQ ID NO: 3 Glaucoma-encoding protein comprising the amino acid sequence of SEQ ID NO: 4