한국인 인슐린 의존형 당뇨병환자에서 HLA DQA1 및 DQB1 유전자형의 분석

Abstract

[한글]

인슐린 의존형 당뇨병은 유전적 소인이 있는 사람에서 바이러스 감염 등의 환경적요인이 작용하여 췌장의 베타세포가 자가면역반응에 의하여 파괴되어 인슐린 결핍이 초래되는 질환으로 알려져 있다. 유전적 요인으로써 HLA class Ⅰ 항원과의 상관관계가 일찌기 보

고되었고, 이후 HLA class Ⅰ보다는 HLA class Ⅱ 항원과 밀접한 관계가 있는 것으로 밝혀졌다. 최근 분자유전학의 발전에 따라 HLA class Ⅱ 항원 중에서도 HLA DQ유전자의 중요성이 알려지면서, DQA1의 52번째 아미노산 위치의 아르기닌, DQB1의 57번째 아미노산 위치의 아스파르트산 등의 존재 유무에 따른 감수성의 차이 등이 여러 인종에서 보고되었다. HLA 유전자의 빈도는 인종 및 지역에 따라 차이가 있어 이들 인종간의 차이를 비교함으로써 인슐린 의존형 당뇨병의 감수성을 결정하는 유전자를 규명하는 것이 가능할 것으

로 생각되고 있다. 이에 본 연구에서는 중합효소연쇄반응을 이용한 제한효소 분절길이 다형성 방법, 대립유전자 특이 올리고뉴클레오티드탐침(allele specific oligonucleotide probe)을 이용한 점 블롯(dot-blot) 분석 방법 및 유전자 클로닝에 의한 DNA 서열 결정 방법을 사용하여 한국인 인슐린 의존형 당뇨병 환자와 정상인을 대상으로 HLA DR 표현형, DQA1 및 DQB1 유전자의 형별 분석을 시행하여 다음과 같은 결과를 얻었다.

1. 한국인 인슐린 의존형 당뇨병 환자에서 HLA DR3, DR4, DR3/4는 정상인보다 유의하게 증가되어 있었고, DR2, DR8은 감소되어 있었다.

2. HLA DQA1 유전자 중에서는 DQA1 (*)**0301 이 환자군에서 유의하게 증가되어 있었으며, DQA1 (*)**0101 ,(*)**0102 는 환자군에서 감소되어 있었다.

3. DQA1 유전자중 Arg52 양성/Arg52 양성 동형접합체(homozygote)의 빈도는 환자군에서 유의하게 높았으며, 환자군의 97%에서 적어도 1개 이상의 Arg52 양성인 유전자를 가지고 있는 것으로 나타났다.

4. HLA DQB1 유전자 중에서는 DQB1 (*)**0201 및 (*)**0303 이 환자군에서 유의하게 증가되어 있었고 (*)**0301 은 감소되어 있었다.

5. DQB1 유전자중 Asp57 양성인 유전자의 빈도는 환자군과 정상인 사이에 유의한 차이가 없었다.

6. DQA1-DQB1의 알파 베타 이형이합체(heterodimer)의 빈도는 환자군에서 DQA1 (*)**04 -DQB1 (*)**0201 , DQA1 (*)**301 -DQB1 (*)**0303 , DQA1 (*)**0301 -DQB1 (*)**0201 이 유의하게 증가되어 있었고, DQA1 (*)**0101 , 0102-DQB1 (*)**0604, DQA1 (*)**0101 ,

0102-DQB1 (*)**0302 는 유의하게 감소되어 있었다.

7 추정 가능한 HLA DR-DQA1-DQB1 일배체형(haplotype)의 빈도는 환자군에서 DR3-DQAl (*)**0301 -DQB1 (*)**0201 및 DR3-DQA1 (*)**04 -DQB1 (*)**0201 이 증가되어 있었다. 이상의 결과에서 한국인 인슐린 의존형 당뇨병 환자에서 HLA DR 표현형의 빈도는 코카시안과 비슷하였으나, DR3/4가 상대적으로 낮았고, 특히 동양인인 중국인, 일본인과도 차이가 있어 인종간에 차이가 있었다. HLA DQ 유전자형은 DQA1 (*)**0301 과 DQB1 (*)**0303 , DQB1 (*)**0201 이 유의하게 증가되어 있었고, DQA1 (*)**0101 , (*)**0102 와 DQB1 (*)*

*0301 은 유의한 감소를 보여 질병의 감수성에 기여할 것으로 생각되었으며, 이들 DQA1 및 DQB1 유전자 단독으로 보다는 알파 베타 이형이합체가 감수성을 결정하는데 중요한 역할을 할 것으로 사료되었다. 또한 병인적 또는 방어적인 유전자간의 상호작용 및 용량효과가 관찰되어 이러한 작용의 총화로써 질병에 대한 감수성이 결정 될 것으로 생각되었다.

HLA DQA1, DQB1 alleles associated with genetic susceptibility to insulin-dependent

diabetes mellitus in Korean

Mi Rim Kim

Department of Medical Science The Graduate School, Yonsei University

(Directed by Associate Professor Hyun Chul Lee)

Family and population studies have shown that at least one susceptibility locus

for insulin-de-pendent diabetes mellitus(IDDM) is located in the HLA (human

leucocyte antigen) class Ⅱ region. Transracial analysis provides a method of

distinguishing primary associations between IDDM and HLA calss Ⅱ alleles from

those secondary to linkage disequilibrium. This study was aimed to investigate the

HLA association with IDDM in Korean population.

DNA, amplified by polymerase chain reaction(PCR), was subjected to allele

specific oligonucleotide dot-blot analysis, restriction fragment length

polymorphism(RFLP) analysis and DNA sequencing.

The frequency of HLA DR3, DR4 and DR3/4 was significantly increased in the

diabetic patients(15/55[27.3%] vs control subjects, 8/76[10.5%], p<0.05, RR =3.1,

39/55[70.9%] vs. 23/76[30.3%], RR=5.3, p<0.01, 8/55[14.5%] vs.3/76[4.1%], RR=4.0,

p<0.05).The frequency of DR2 and DR8 was significantly decreased in the diabetic

patients(3/55[5.4%] vs. 14/76[18.4%], RR=0.3, p<0.05 and 5/55[9.1%] vs.

21/76[27.6%], RR=0.3, p<0.05).

The frequency of DOQA1 (*)**0301, which was positively associated with IDDM in

Caucasian and Japanese but not in Chinese, was significantly higher in the

patients(27/32[84.5%] vs. control subjects,24/39[61.5%], RR=3.4, p<0.05).

The frequency of DQA1 (*)**0101 ,(*)**0102 was significantly lower in the diabetic

Patients(9/32[28.1% vs. control subjects, 21/39[53.8], RR=0.3, p<0.05).

The frequency of DQB1 (*)**0201 , which was not associated with IDDM in Chinese,

was significantly higher in the diabetic patients(16/37[43.3% vs. control subjects,

5/36[13.9%], RR=6.2, p<0.005). The frequency of DQB1 (*)^^0303 was significantly

higher in the diabetic patients(16//37[43.2%0 vs.control subjects, 3/36[8.3%],

RR=8.4, p<0.001).

The frequency of the heterodimer DQA1 (*)**0301 -DQB1 (*)**0201 , DQA1 (*)**0301

-DQB1 (*)**0303 , DQA1 (*)**04 -DQB1 (*)**0201 was significantly increased and DQA1

(*)**0101 , 0102-DQB1 (*)**0302 was significantly decreased in diabetic patients.

The frequency of the deduced haptotype DR3-DQA1 (*)**0301 -DQB1 (*)**0201 and

DQA1 (*)**04 -DQB1 (*)**0201 was significantly increased in diabetic

patients(9/28[32.1%] vs. DR3-positive control subjects, O/l8[0.0%], p<0.01 and 7/28

vs. 0/18[0.0%], p<0.05).

The frequency of Arg52 positive allele homozygotes was significantly increased in

diabetic patients but the frequency of Asp57 positive allele was not different from

control subjects. The distribution of HLA DR phenotype of Korean IDDM patients was

similar to Caucasian IDDM patients except for relatively low frequency of HLA DR3/4

and was slightly different from other Oriental populations. HLA DQAl (*)**0301 ,

DQB1 (*)**0303 , DQB1 (*)**0201 were increased and DQA1 (*)**0101 , DQA1 (*)**0102

, DQB1 (*)**0301 were decreased in Korean IDDM patients. It seems likely that there

exists dose effect among these susceptible and Protective alleles. And also the

role of heterodimer which is consisted of these alleles may be important to

determine susceptibility to IDDM.

[영문]

Family and population studies have shown that at least one susceptibility locus for insulin-de-pendent diabetes mellitus(IDDM) is located in the HLA (human leucocyte antigen) class Ⅱ region. Transracial analysis provides a method of

distinguishing primary associations between IDDM and HLA calss Ⅱ alleles from those secondary to linkage disequilibrium. This study was aimed to investigate the HLA association with IDDM in Korean population.

DNA, amplified by polymerase chain reaction(PCR), was subjected to allele specific oligonucleotide dot-blot analysis, restriction fragment length polymorphism(RFLP) analysis and DNA sequencing.

The frequency of HLA DR3, DR4 and DR3/4 was significantly increased in the diabetic patients(15/55[27.3%] vs control subjects, 8/76[10.5%], p<0.05, RR =3.1, 39/55[70.9%] vs. 23/76[30.3%], RR=5.3, p<0.01, 8/55[14.5%] vs.3/76[4.1%], RR=4.0,

p<0.05).The frequency of DR2 and DR8 was significantly decreased in the diabetic patients(3/55[5.4%] vs. 14/76[18.4%], RR=0.3, p<0.05 and 5/55[9.1%] vs. 21/76[27.6%], RR=0.3, p<0.05).

The frequency of DOQA1 (*)**0301, which was positively associated with IDDM in Caucasian and Japanese but not in Chinese, was significantly higher in the patients(27/32[84.5%] vs. control subjects,24/39[61.5%], RR=3.4, p<0.05).

The frequency of DQA1 (*)**0101 ,(*)**0102 was significantly lower in the diabetic Patients(9/32[28.1% vs. control subjects, 21/39[53.8], RR=0.3, p<0.05).

The frequency of DQB1 (*)**0201 , which was not associated with IDDM in Chinese, was significantly higher in the diabetic patients(16/37[43.3% vs. control subjects, 5/36[13.9%], RR=6.2, p<0.005). The frequency of DQB1 (*)^^0303 was significantly

higher in the diabetic patients(16//37[43.2%0 vs.control subjects, 3/36[8.3%], RR=8.4, p<0.001).

The frequency of the heterodimer DQA1 (*)**0301 -DQB1 (*)**0201 , DQA1 (*)**0301 -DQB1 (*)**0303 , DQA1 (*)**04 -DQB1 (*)**0201 was significantly increased and DQA1 (*)**0101 , 0102-DQB1 (*)**0302 was significantly decreased in diabetic patients.

The frequency of the deduced haptotype DR3-DQA1 (*)**0301 -DQB1 (*)**0201 and DQA1 (*)**04 -DQB1 (*)**0201 was significantly increased in diabetic patients(9/28[32.1%] vs. DR3-positive control subjects, O/l8[0.0%], p<0.01 and 7/28 vs. 0/18[0.0%], p<0.05).

The frequency of Arg52 positive allele homozygotes was significantly increased in diabetic patients but the frequency of Asp57 positive allele was not different from control subjects. The distribution of HLA DR phenotype of Korean IDDM patients was

similar to Caucasian IDDM patients except for relatively low frequency of HLA DR3/4 and was slightly different from other Oriental populations. HLA DQAl (*)**0301 , DQB1 (*)**0303 , DQB1 (*)**0201 were increased and DQA1 (*)**0101 , DQA1 (*)**0102

, DQB1 (*)**0301 were decreased in Korean IDDM patients. It seems likely that there exists dose effect among these susceptible and Protective alleles. And also the role of heterodimer which is consisted of these alleles may be important to determine susceptibility to IDDM.