KR100450001B1 - DNA marker associated with genetic obesity - Google Patents

Abstract

The invention From β ₂ - adrenergic receptor (β ₂ -adrenergic receptor), associated with mutations A → G ₄₆ a genotype of obesity due to the base sequence (46th base of SEQ ID NO: 1) present in the 16th codon of the gene DNA It is about a cover factor. The human β ₂ -adrenergic receptor gene is registered under NCBI Genebank Accession No. AF022955, the mutation resulting from the substitution of the G base with the 46th A base of the registered sequence. The present invention also provides a method of predicting the likelihood of developing obesity using the DNA marker.

Description

DNA marker associated with genetic obesity

The present invention relates to Korean specific DNA markers associated with genetic obesity, including single nucleotide variations (SNPs) in human β ₂ -adrenergic receptor gene sequences.

In Korea, the incidence and prevalence of obesity is gradually increasing due to the improvement of living standards and the spread of western diets due to industrialization and urbanization. Obesity has been concerned only with appearance in the past and has not paid much attention to the seriousness of the health problems of this disease. However, recent studies have reported that obesity is a dangerous disease that causes various adult diseases. It is recognized. Therefore, studies have been actively conducted to investigate the cause of obesity, and as a result, obesity is caused by an imbalance in the intake and use of energy, and pathophysiologically characterized by proliferation and hypertrophy of adipose tissue. Said. Obesity has also been found to be associated with essential hypertension, hyperlipidemia, diabetes and various other cardiovascular diseases.

The causes of obesity can be divided into genetic and environmental factors. Overeating, lack of exercise, and excessive stress are considered as major environmental factors, and genetic factors include leptin and leptin receptor ( leptin receptor), non-covalent protein (uncoupling protein), adiponectin (adiponectin), β ₃ - adrenergic receptor (β ₃ -adrenergic receptor), and β ₂ - adrenergic receptor (β ₂ -adrenergic receptor) of major genetic abnormalities obesity gene It is presumed to be a postmark.

Therefore, the present inventors conducted a study for finding Korean specific gene markers related to genetic obesity. As a result, the 46th A base of the sequence registered with NCBI Genebank No. AF022955 was identified as G base. Substituted DNA markers were found to be Korean specific DNA markers related to genetic obesity, and the present invention was completed.

Accordingly, an object of the present invention is to provide a DNA marker associated with obesity and a method for predicting obesity using the DNA marker.

1 shows genotype patterns of Nde I restriction fragments length polymorphism (RFLP) in the β ₂ -adrenergic receptor gene.

(In Figure 1, lanes 1, 5 and 7 are Arg / Arg homozygotes; lanes 2 and 3 are Arg / Gly heterozygotes; and lanes 4 and 6 represent Gly / Gly homozygotes.)

Figure 2 shows the adjusted residual of genotype in Nde I RFLP of β ₂ -adrenergic receptor gene.

To achieve the above object, the present invention is human β ₂ - and of the nucleotide sequence present in the 16th codon of the adrenergic receptor gene provides a DNA marker associated with the genotype of obesity due to mutations A → G _46. The human β ₂ -adrenergic receptor gene is registered under NCBI Genebank Accession No. AF022955, and the mutation is a result of the substitution of G base for the 46th A base of the registered sequence. This base mutation causes the codon # 16 of the human β ₂ -adrenergic receptor gene to change from Arg to Gly.

In addition, the present invention comprises the steps of 1) acquiring DNA from a human blood sample; 2) In the DNA sample, the base sequence including the 16th codon region of the amino acid sequence of the β ₂ -adrenergic receptor gene registered in NCBI Genebank (Registration No. AF203386), the base sequence of the codon region Amplifying by performing a PCR using a primer capable of amplifying the Nde I restriction enzyme cleavage site included; 3) cleaving the amplified DNA with Nde I restriction enzyme (SEQ ID NO: A → G ₄₆ ); And, 4) determining the genotype of the β ₂ -adrenergic receptor gene from the band size of the cleaved DNA fragment.

Adrenaline receptors comprising the DNA markers of the present invention are involved in the supply of lipids, energy consumption and glycolysis, in particular β ₂ -adrenergic receptors play a major role in lipolysis in subcutaneous fat or other body fat stores. It is known to perform.

The β ₂ -adrenergic receptor gene is located at the q31-q32 position of chromosome 5 and is known to encode 413 amino acids (Kobika et al ., 1987a; Kobika et al ., 1987b). Three gene polymorphisms (RFLPs) have been known to date in this gene (Large et al ., 1997). Among them, the Arg → Gly ₁₆ polymorphism of the β ₂ -adrenergic receptor gene is known to cause downregulation of receptors for agonists, thus reducing the number of receptors. However, a study in Sweden (Large et al ., 1997) states that the Arg → Gly ₁₆ polymorphism has a very low risk of obesity of 1.3, with no statistical significance.

However, according to the results of the present invention, the Arg-> Gly ₁₆ polymorphism, in a study of Koreans, showed a high risk of obesity of 2.0, and this value has statistical significance ( p = 0.0003). Is showing.

Therefore, although Arg-> Gly ₁₆ polymorphism cannot be used as a marker for predicting genetic obesity in Westerners, in the present invention, Arg-> Gly ₁₆ polymorphism can be used as a Korean specific DNA marker. Revealed. Thus, the present invention provides Arg → Gly ₁₆ polymorphism of β ₂ -adrenergic receptor as a Korean specific DNA marker associated with obesity.

A mutation at the Nde I restriction enzyme position of the β ₂ -adrenergic receptor gene was found in a study by Reihsaus et al. (1983), but it was the first time in this study that the mutation had a significant association with obesity in Koreans. . Subjects with homozygotes (Gly / Gly) of mutant alleles were detected only in the obese group when compared to individuals with other genotypes. The mutation was known to not only change the amino acid of the gene, but also to reduce the number of receptors through downregulation of the receptor's agonists (Ligett, 1999).

However, according to the research results of the present invention, β₂Since this site in the adrenergic receptor gene may be related to the expression of the gene, a significant association between mutations in this gene and obesity is presumed to be the result of modification of gene expression by the mutation. That is, in Korea, there is an associative unbalance in the Gly allele itself, presumably causing obesity. Indeed, BMI values in individuals with Gly alleles A progressively rising gene dosage effect was identified in this study.

Thus, in Koreans the mutation can be used as a useful marker in the prediction of obesity associated with the Gly allele.

The mutation is a known mutation, but the process of detecting the DNA markers herein in the β ₂ -adrenergic receptor gene can be described as follows.

1. Subject

Eighty-six Korean blood samples were collected from outpatients at the Seoul Sanitation Hospital in Seoul, Korea. The group with body mass index (BMI) of 26 kg / m ² or more was selected as the obese group, and the group with body mass index (BMI) less than 26 kg / m ² was selected as the control group.

2. Blood Collection

To compare the frequency of Arg to Gly substitution of the 16th codon in the β ₂ -adrenergic receptor gene from 27 obese and 58 controls, approximately 3-5 ml of blood was collected and transferred to EDTA tubes, and then at 1,000 × g. Centrifugation separates the monocyte layer between the plasma and erythrocyte layers. DNA was isolated from the monocyte layer using Wizard kit (Promega, Co. Ltd.).

3. PCR reaction

The isolated DNA was PCR amplified by the method of reference, and PCR primers used the following sequence designed by Jia et al. (2000).

Sense sequence, F: 5'-AGCGCCTTCTTGCTGGCACCATAT-3 '

Antisense sequence, R: 5'-ACAGCACATCAATGGAAGTCC-3 '

The PCR reaction was carried out with M.J. Research thermocycler at 35 cycles for 1 minute at 94 ° C, 45 seconds at 62 ° C, and 1 minute at 72 ° C.

4. Treatment of Nde I Restriction Enzyme

Complete the amplification reaction PCR product with 10 × buffer solution (10 mM Trisma base, pH 7.4, 50 mM sodium chloride, 0.1 mM EDTA, 1 mM DTT, 0.5 mg / ml bovine serum albumin, 50% glycerol), 10 units of Nde I restriction enzyme was used.

5. Electrophoresis

The PCR product, which completed the restriction enzyme reaction, was mixed with loading buffer (0.25% bromophenol blue, 30% glucose), and the sample was loaded on a 2% agarose gel (FMC Co.) in 0.5 X TBE buffer. Electrophoresis was performed on a horizontal electrophoresis apparatus at 8 W for about 1 hour at room temperature. After the electrophoresis, the gel was stained with ethidium bromide at a concentration of 0.5 mg / ml, and the band size was observed using a UV transilluminator.

6. Observation of the experimental results

DNA band morphology was observed as a result of electrophoresis.

If the bands of 304 and 21 bp are observed, the Gly allele is mutated at the restriction site, and if the band of 325 bp (304 bp + 21 bp) is present, the normal group without the Nd e I restriction site is present. Arg allele.

7. Biochemical Analysis

The body mass index BMI (kg / m ² ) was measured by dividing the weight measured in kg by the square of the height measured in meters. Total cholesterol (TC), triglyceride (TG) and high density lipoprotein cholesterol (HDL-cholesterol) concentrations were measured by enzymatic methods, and low density lipoprotein cholesterol (LDL-cholesterol) concentrations were measured by Friedwald et al. (1972). Calculated by the formula Blood and apolipoprotein AI concentrations were measured by the Turbidimetric Immuno Assay.

8. Statistical Analysis

Allele frequencies were calculated from genotype frequencies and deviations from Hardy-Weinberg equilibrium were analyzed by χ2-test. The genotype frequency and allele frequency between the obese group and the control group were analyzed by χ2-test, and when significant differences were found, the differences were analyzed by adjusted residuals. Polymorphism Information Content (PIC) was measured by the method of Bostein et al. (1980). Comparison of variables by genotype was performed with a parametric one-way ANOVA assay. As measures of relative risk for obesity, odds ratio, relative risk, sensitivity, specificity, positive predictive value, negative predictive value, and total predictive value were calculated. All statistical analyzes were performed by the SPSSWIN (version 8.0) computer program.

β ₂ Mutation of the 16th codon in the adrenergic receptor gene

The sense primer sequence for PCR amplifying the mutation of the 16th codon of the β ₂ -adrenergic receptor gene is 5'-AGCGCCTTCTTGCTGGCACCATAT-3 'and the antisense primer sequence is 5'-ACAGCACATCAATGGAAGTCC-3'. This mutation was first characterized by the work of Reihaus et al. (1993). After PCR amplification of this portion of the β ₂ -adrenergic receptor gene, the PCR product was analyzed by Nde I restriction enzyme. As shown in Figure 1, in the polymorphism pattern by the Nde I restriction enzyme of the β ₂ -adrenergic receptor gene, in the polymorphism in the presence of the Gly allele, bands of 304 bp and 21 bp were detected, when the Arg allele is present Only a band of 325 bp was detected.

The study sample was divided into the obese group and the control group, and the comparative study was performed using the χ2-test, and the P value was less than 0.05, indicating a significant difference between the two groups in the genotype frequency (Table 1), and the adjusted residual. As a result of investigating the cause of this difference, the Gly / Gly homozygote was a phenomenon only detected in the obese group (adjusted residual = 4.0) (FIG. 2).

Genotype (%) Allele (%) Arg / Arg Arg / Gly Arg / Arg Arg Gly H ¹ PIC ² Control 23 (40) 35 (60) 0 (0) 81 (70) 35 (30) 0.4214 0.3326 Obesity 9 (33) 11 (41) 7 (26) 29 (54) 25 (46) 0.4973 0.3735 χ2 16.541 4.1947 P 0.0003 0.0406 2.00 (1.03-3.88) ³ Release Bond 2. Polymorphic Information Diagram 3. The 95% confidence interval frequency is expressed as% in parentheses. There was a significant difference in genotype and allele frequencies between the obese and control groups.

Body measurements and intermediate phenotypes of subjects with and without mutations were compared (Table 2). As a result, in the body mass index value, as the Gly allele increased, the body mass index value showed a gene dosage effect (gene dosage effect). This phenomenon is caused by the mutation of A to G causes a change in gene expression, this change in gene expression affects the body mass index value, it is judged to be the result of increasing the risk of obesity.

variable genotype Arg / Arg (Wed) Arg / Gly Gly / Gly Age (years) 58.9 ± 9.6 ⁸ (32) 55.3 ± 8.9 (46) 60.3 ± 12.1 (7) ^a BMI (kg / m2) ¹ 23.9 ± 3.6 (32) 24.0 ± 2.4 (46) 26.7 ± 0.6 (7) Tg (mg / dl) ² 111.8 ± 54.7 (27) 143.5 ± 103.7 (37) 129.0 ± 15.2 (4) TC (mg / dl) ³ 155.1 ± 34.6 (27) 150.8 ± 34.6 (37) 156.3 ± 35.3 (4) LDL-Chol (mg / dl) ⁴ 105.5 ± 33.4 (27) 94.7 ± 37.6 (37) 108.7 ± 30.7 (4) HDL-Chol (mg / dl) ⁵ 27.3 ± 7.3 (27) 28.2 ± 8.6 (37) 21.8 ± 8.3 (4) Apolipoprotein AI (mg / dl) ⁴ 62.8 ± 15.1 (5) 81.3 ± 33.1 (9) 100.9 ± 18.7 (3) ^a Significant difference (one-way ANOVA test, P = 0.048). Body Mass Index: Obesity Index 2. Triglycerides 3. Total cholesterol 4. LDL-cholesterol 5. HDL-cholesterol 6. Apolipoprotein AI7. Number of individuals 8. Values are mean ± SD (standard deviation).

Several relative risk measures were calculated to determine how predictable the Arg → Gly mutation at the 16th codon of the β ₂ -adrenergic receptor gene can predict the onset of obesity (Table 3). As a result, the person with the Gly allele showed about a twofold increase in risk compared to the person without the allele.

Marker Crossover Relative risk responsiveness Specificity Forecast positivity voice gun Gly allele 2.00 1.53 0.46 0.70 0.42 0.74 0.62

As described above, the Arg → Gly mutation occurring in the 16 codon of the β ₂ -adrenergic receptor gene is significantly associated with obesity. This means that people with the Gly allele are about twice as likely as those who are not likely to become obese. The risk, since the P value of the chi-square test results in the results of Table 1 is 0.0003, it can be seen that the statistical significance is very high.

Therefore, it can be confirmed that the mutation of the A base 46 to the G base in the β ₂ -adrenergic receptor gene can be used as a Korean specific marker in predicting genetic obesity.

Using such DNA markers, a method for predicting the possibility of future obesity can be carried out as follows.

1. Blood Collection

For the general public who want to know about the possibility of developing obesity due to genetic abnormalities, about 3 ~ 5 ml of blood is collected and transferred to EDTA tube, and then centrifuged at 1,000 × g for the mononuclear layer between plasma and erythrocyte layer. Separate. Then, DNA is isolated from the Wizard kit (Promega, Co. Ltd.).

2. PCR reaction

The isolated DNA is amplified by PCR by the method of reference, and PCR primers use the following sequence designed by Jia (2000) as follows.

Sense sequence, F: 5'-AGCGCCTTCTTGCTGGCACCATAT-3 '

Antisense sequence, R: 5'-ACAGCACATCAATGGAAGTCC-3 '

The PCR reaction was performed with M.J. Research thermocycler at 35 cycles for 1 minute at 94 degrees, 45 seconds at 62 degrees, and 1 minute at 72 degrees.

4. Treatment of Nde I Restriction Enzyme

Complete the amplification reaction PCR product with 10 × buffer solution (10 mM Trisma base, pH 7.4, 50 mM sodium chloride, 0.1 mM EDTA, 1 mM DTT, 0.5 mg / ml bovine serum albumin, 50% glycerol), Treat with Nde I restriction enzyme equivalent to 10 units.

5. Electrophoresis

The PCR product, complete with the restriction enzyme reaction, is mixed with loading buffer (0.25% bromophenol blue, 30% glucose) and the sample is loaded onto a 2% agarose gel (FMC Co.) in 0.5 X TBE buffer. Electrophoresis is performed in a horizontal electrophoresis apparatus at 8 W for about 1 hour at room temperature. After electrophoresis, the gel was stained with ethidium bromide at a concentration of 0.5 mg / ml, and the band size was observed using a UV transilluminator.

6. Observation of the experimental results

Observe the DNA band morphology resulting from electrophoresis.

If only 304 bp and 21 bp bands are observed, the mutant Gly allele is mutated at the restriction site, and if a band of 325 bp (304 bp + 21 bp) is present, Arg alleles in the normal group without the Nd e I restriction site It is a gene. At this time, if a GlyGly genotype with only the Gly allele is detected, the person may be judged to have a relatively high risk of obesity compared to a person without this genotype.

The association between the 16th codon mutation of the β ₂ -adrenergic receptor gene and obesity has been used to predict the possibility of obesity. In other words, the present inventors have identified Korean specific DNA markers related to obesity first identified in Koreans, and used the DNA markers to prevent future obesity risk in general people with genetic predisposition in terms of preventive medicine. By predicting, there is an effect of preventing obesity, which causes adult diseases such as diabetes and essential hypertension, through improvement of diet and exercise.

references

Bostein, D., White, RL, Skolnick, M. and Davis, RW (1980) Construction of a genetic linkage map in man using restriction fragment length polymorphism. Am. J. Hum. Genet. 32 : 314-331.

Friedwald, WT, Levy, RI And Fredrickson, DS (1972) Estimation of the concentration of low-density lipoprotein cholesterol in plasma, without use of the preparative ultracentrifuge. Clin. Chem. 18 : 499-502.

3.Jia, H., Sharma, P., Hopper, R., Dickerson, C., Lloyd, DD and Brown, MJ β ₂ -adrenergic receptor gene polymorphisms and blood pressure variations in East Anglian Caucasians. J. Hypertens. 18 : 687-693, 2000.

Kobilka, BK, Dixon, RA, Frielle, T., Dohlman, HG, Bolanowski, MA, Sigal, IS, Yang-Feng, TL, Francke, U., Caron, MG and Lefkowitz, RJ cDNA for the human beta2 -adrenergic receptor: a protein with multiple membrane-spanning domains and encoded by a gene whose chromosomal location is shared with that of the receptor for platelet-derived growth factor. Proc. Natl. Acad. Sci. USA. 84 : 46-50, 1987a.

Kobilka, BK, Frielle, T., Dohlman, HG, Bolanowski, MA, Dixon, RA, Keller, P., Caron, MG and Lefkowitz, RJ Delineation of the intronless nature of the genes for the human and hamster beta2- adrenergic receptor and their putative promoter regions. J. Biol. Chem. 262 : 7321-7327, 1987b.

6.Large, V., Hellstro, L., Reynisdottir, S., Lonqvist, F., Eriksson, P., Lannfelt, L. and Arner, P. Human β ₂ -adrenoceptor gene polymorphisms are highly frequent in obesity and associate with altered adipocyte beta-2 adrenoceptor function. J. Clin. Invest. 100 : 3005-3013, 1997.

7. Liggett, SB Molecular and genetic basis of β ₂ -adrenergic receptor function. J. Allergy Clin. Immunol. 104 : S42-S46, 1999.

8.Reihaus, E., Innis, M., Maclntyre, N. and Liggett, SB Mutations in the gene encoding for the beta ₂ -adrenergic receptor in normal and asthmatic subjects. Am. J. Respir. Cell Mol. Biol. 8 : 334-339, 1993.

<110> Seoulin Bioscience Institute <120> DNA marker associated with genetic obesity <130> PA01105 <160> 3 <170> KopatentIn 1.71 <210> 1 <211> 1242 <212> DNA <213> Homo sapiens <400> 1 atggggcaac ccgggaacgg cagcgccttc ttgctggcac ccaatagaag ccatgcgccg 60 gaccacgacg tcacgcagca aagggacgag gtgtgggtga tgggcatggg catcgtcatg 120 tctctcatcg tcctggccat cgtgtttggc aatgtgctgg tcatcacagc cattgccaag 180 ttcgagcgtc tgcagacggt caccaactac ttcatcactt cactggcctg tgctgatctg 240 gtcatgggcc tggcagtggt gccctttggg gccgcccata ttcttatgaa aatgtggact 300 tttggcaact tctggtgcga gttttggact tccattgatg tgctgtgcgt cacggccagc 360 attgagaccc tgtgcgtgat cgcagtggat cgctactttg ccattacttc acctttcaag 420 taccagagcc tgctgaccaa gaataaggcc cgggtgatca ttctgatggt gtggattgtg 480 tcaggcctta cctccttctt gcccattcag atgcactggt accgggccac ccaccaggaa 540 gccatcaact gctatgccaa tgagacctgc tgtgacttct tcacgaacca agctatgtt 600 attgcc gtgtc cttctacgtt cccctggtga tcatggtctt cgtctactcc 660 agggtctttc aggaggccaa aaggcagctc cagaagattg acaaatctga gggccgcttc 720 catgtccaga accttagcca ggtggagcag gatgggcgga cggggcatgg actccgcaga 780 tcttccaagt tctgcttgaa ggagcacaaa gccctcaaga cgttaggcat catcatgggc 840 actttcaccc tctgctggct gcccttcttc atcgttaaca ttgtgcatgt gatccaggat 900 aacctcatcc gtaaggaagt ttacatcctc ctaaattgga taggctatgt caattctggt 960 ttcaatcccc ttatctactg ccggagccca gatttcagga ttgccttcca ggagcttctg 1020 tgcctgcgca ggtcttcttt gaaggcctat gggaatggct actccagcaa cggcaacaca 1080 ggggagcaga gtggatatca cgtggaacag gagaaagaaa ataaactgct gtgtgaagac 1140 ctcccaggca cggaagactt tgtgggccat caaggtactg tgcctagcga taacattgat 1200 tcaatag sequence for PCR amplication of beta-2-adrenergic receptor includin g 16th codon mutation <400> 2 agcgccttct tgctggcacc atat 24 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Antisense primer sequence for PCR amplication of beta-2-adrenergic receptor including 16th codon mutation <400> 3 acagcacatc aatggaagtc c 21

Claims (3)

A Korean specific obesity related DNA marker characterized in that the 46th base of the human β ₂ -adrenergic receptor gene set forth in SEQ ID NO: 1 is a mutated genotype substituted with A to G. The method of claim 1, wherein the genotype is Nde I restriction enzyme recognition site is not present Arg allele with Nde I restriction enzyme recognition Korean specific obesity-related DNA marker, characterized in that it comprises the Gly allele that region is present . 1) obtaining DNA from a human blood sample; 2) Amplified Nde I restriction enzyme cleavage site formed by including the 16th codon region of the amino acid sequence of the human β ₂ -adrenergic receptor gene of SEQ ID NO: 1, including the base sequence of the codon region Amplifying by performing PCR using primers of SEQ ID NO: 2 and SEQ ID NO: 3; 3) cleaving the amplified DNA with Nde I restriction enzyme; And 4) A method for detecting the DNA marker according to claim 1, comprising determining the genotype of the β ₂ -adrenergic receptor gene from the band size of the cleaved DNA fragment.