Korean J Clin Lab Sci. 2019;51(3):323-328 https://doi.org/10.15324/kjcls.2019.51.3.323 Korean Society for Clinical Laboratory Science ORIGINAL ARTICLE Association between Tuberculosis Case and CD44 Gene Polymorphism Hee-Seon Lim, Sang-In Lee, Sangjung Park Department of Biomedical Laboratory Science, College of Life and Health Sciences, Hoseo University, Asan, Korea 결핵발병과 CD44 유전자다형성사이의연관성연구 임희선, 이상인, 박상정 호서대학교생명보건대학임상병리학과 ARTICLE INFO Received May 16, 2019 Revised June 4, 2019 Accepted June 13, 2019 Key words CD44 Genetic association study Mycobacterium tuberculosis SNP Tuberculosis ABSTRACT Tuberculosis, a chronic bacterial infection caused by Mycobacterium tuberculosis (MTB), differs in its status latency and activity because of the characteristics of MTB, immune status of the host, and genetic susceptibility. The host defense mechanism against MTB is caused mainly by interactions between macrophages, T cells, and dendritic cells. CD44 is expressed in activated T cells when infected with MTB and regulates lymphocyte migration. In addition, CD44 mediates leukocyte adhesion to the ECM and plays a role in attracting macrophages and CD4 + T cells to the lungs. Therefore, genetic polymorphism of the CD44 gene will inhibit the host cell immune mechanisms against MTB. This study examined whether the genetic polymorphism of the CD44 gene affects the susceptibility of tuberculosis. A total of 237 SNPs corresponding to the CD44 genes were analyzed using the genotype data of 443 tuberculosis cases and 3,228 healthy controls from the Korean Association Resource (KARE). Of these, 17 SNPs showed a significant association with the tuberculosis case. The most significant SNP was rs75137824 (OR=0.231, CI: 1.51 3.56, P=1.3 10 4 ). In addition, rs10488809, one of the 17 significant SNPs, is important for the tuberculosis outbreak can bind to the JUND and FOS transcription factors and can affect CD44 gene expression. This study suggests that polymorphism of the CD44 gene modulates the host susceptibility to tuberculosis in a variety of ways, resulting in differences in the status of tuberculosis. Copyright 2019 The Korean Society for Clinical Laboratory Science. All rights reserved. 서론 결핵 (Tuberculosis) 은 Mycobacterium tuberculosis (MTB) 에의한만성감염성질환으로, 매년 200만명의사람들이사망하는전세계의주요질병중하나이다 [1]. 결핵균 (MTB) 에감염된사람중대부분은증상이없는잠복성감염이고, 이중일부는임상증상을보이는활동성결핵으로진행된다. 이러한질병의진행차이는결핵균의특성차이일수도있지만, 결핵균에감 * Corresponding author: Sangjung Park Department of Biomedical Laboratory Science, College of Life and Health Sciences, Hoseo University, 20 Hoseo-ro 79beon-gil, Asan 31499, Korea E-mail: sangjung@hoseo.edu * ORCID: https://orcid.org/0000-0003-4240-4612 염한숙주의유전적감수성에의해일어날수도있다 [2]. 결핵발병률이매년증가하는경향을나타내기때문에, 결핵에대한숙주방어체계와관련한유전자에대하여규명할필요성이높아지고있다. 이에따라사람의면역학적인유전적차이가결핵발병에영향을줄수있다는연구가국내외에서진행되고있는실정이다 [3-8]. 결핵균에대한숙주의방어기전은결핵균이인체내로침입한후주로대식세포, T 세포, 수지상세포사이의상호작용에의해일어난다 [2]. 이상호작용은부착단백을통해 extra-cellular matrix (ECM) 에결합하는능력에의존하는백혈구의이동및백혈구의활성화를필요로한다 [3, 9]. CD44는염색체 11번내 CD44 유전자에암호화되어있는세포접착분자의히알루론산수용체계열의구성원이다. 염증부 pissn 1738-3544 eissn 2288-1662 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
324 Hee-Seon Lim, et al. CD44 Gene Polymorphism Affects Tuberculosis Case 위로향하는활성화된 T 세포는림프구이동을조절하는데선택적인역할을하는 CD44가발현된다 [3]. 또한 CD44는림프구를이동하는역할외에도 ECM에백혈구의부착을매개하여, 세포와세포사이의상호작용을중재할수있다 [10]. 최근연구에따르면, CD44 결핍 mouse에서결핵균에대항하는대식세포와 CD4 + T 세포의수가정상보다감소한다고보고했다. 또한이러한 CD44 결핍 mouse는폐에서 IFN- 생성이감소하고, 결핵균의성장조절을억제할수없었으며, 육아종이잘형성되지않아 mouse 생존율이저하되는등숙주세포가결핵균에대해저항할수있는기전이억제되는것이확인되었다 [10]. 즉, CD44는대식세포, CD4 + T 세포등을폐로불러모으는역할을하여, 생체내결핵균에대한방어면역작용을수행하는것으로알려져있다 [3, 10]. 따라서본연구는한국인유전체역학조사사업의일환으로구성되어있는코호트자료를활용하여, 결핵이발병했던환자군과건강대조군을분류하고, 두그룹간 CD44 유전자의유전적다형성 (genetic polymorphisms) 이결핵발병에영향을주었는지확인하고자유전적변이에대한관련성연구 (association study) 를시행하였다. 재료및방법 1. 연구대상자이번연구에서이용한한국인연구대상자는한국인유전체역학조사사업 (Korean Genome and Epidemiology Study; KoGES) 의일환인 Korean Association Resource (KARE) 자료를기반으로하였다 [11]. 이자료는질병관리본부인체자원은행에서분양을받아사용하였다 (KBN-2017-038). 연구에서사용한연구대상자의선별은이전연구와동일하게설정하였다 [12]. 즉, KARE의전체대상자 10,038명중 QC (Quality Control) 과정을통해분석기준에적합하지않은 1,198명을제외하여총 8,840명을연구대상자로선정하였다. 이중환자군은결핵진단을받은적이있는 443명을선별하였고, 건강대조군은결핵을포함한다른여러질환이없는건강한사람 3,228명을선별하였다. 환자군과건강대조군간의평균나이는각각 51.0 세, 51.6세로통계적으로유의한차이는없었다. 본연구에활용한유전정보는질병관리본부 (KNIH) 와호서대학교에서연구윤리승인을받은후수행하였다 (1041231-170418-HR-056-02). 2. 유전형분석과 SNP 선별본연구에서유전자영역의분석은 KARE 유전형자료를기반으로 CD44 유전자영역에서 MACH 1.0.16(4) 를사용하여 imputation SNP를추가적으로추출하였고, 이자료를바탕으로분석을진행하였다. 유전자영역은전사체양말단에서 5 kb 씩확장하여분석을진행하였다. 이 SNP의염색체상의위치는 UCSC Genome Browser on Human Feb. 2009 (GRCh37/ hg19) 를기준으로결정하였다. CD44 유전자영역에서 imputation은 HapMap database에서중국인 (Han Chinese Beijing) 과일본인 (Japanese in Tokyo) 의것을참고하여진행하였다 [13]. 또한, imputed SNP들중에서 minor allele frequency (MAF) 가 1% 미만이거나상관계수 (r 2 ) 가 0.5 미만인것은분석에서제외하였다. 이렇게선별된 CD44 유전자영역의 SNP 수는총 237개였다. 3. 상관성분석과통계분석대부분의통계분석에는 PLINK version 1.07 (http://pngu. mgh.harvard.edu/ purcell/plink) 과 PASW Statistics version 18.0 (SPSS Inc. Chicago, IL, USA) 을사용하였다. 환자군과건강대조군에대한유전적변이의상관성분석은로지스틱회귀분석을사용하였고, 이분석은 additive genetic model 을기반으로분석하였다. 그리고이러한회귀분석의시행에있어서나이, 지역, 성별을공변수로처리하여분석하였다. 분석한결과값에대한유의수준은 0.05 미만을기준으로설정하였다. 이결과들중 Bonferroni P-value의결과를알아보기위하여 adjust 분석을시행하였다. 높은유의수준을가진 SNP를대상으로 recombination rate (cm/mb) 과상관성을확인하기위하여 Locuszoom version 1.1 (http://csg.sph.umich.edu/locuszoom) 을사용하였다. 또한, CD44 유전자가단백질발현에어떠한영향을줄것인지알아보기위하여 RegulomeDB (http://www. regulomedb.org/index) database 를사용하였다. 결과 1. CD44 유전자의 SNP 선별과상관성분석결과 CD44 유전자에서선별한 237개의 SNP를대상으로로지스틱회귀분석을시행하였다. 그결과 237개의 SNP 중 17개의 SNP에서통계적으로유의한상관관계 (P<0.05) 를확인할수있었다 (Table 1). 가장높은유의수준을보이는 SNP 는 rs75137824 (P=0.0001304) 이다. 이 SNP의교차비 (Odds ratio) 는 2.31에
Korean J Clin Lab Sci. Vol. 51, No. 3, September 2019 325 Table 1. Results of SNP in the CD44 gene association analysis between controls and cases in tuberculosis patients based on KARE subjects No. SNP BP Function A1 MAF Cases (n=443) Control (n=3,228) OR (95% CI) Additive P-value 1 rs7111731 35184998 intronic T 0.113 0.082 1.43 (1.14 1.81) 0.0024 2 rs3794113 35189148 intronic G 0.105 0.079 1.37 (1.08 1.74) 0.0097 3 rs6484768 35190876 intronic G 0.114 0.142 0.78 (0.63 0.97) 0.0276 4 rs7937602 35191691 intronic A 0.121 0.094 1.30 (1.04 1.62) 0.0206 5 rs3829268 35191943 intronic C 0.121 0.094 1.30 (1.04 1.62) 0.0206 6 rs3794110 35192214 intronic A 0.121 0.094 1.30 (1.04 1.62) 0.0206 7 rs3794109 35192279 intronic G 0.121 0.094 1.30 (1.04 1.62) 0.0206 8 rs75803060 35195422 intronic C 0.116 0.086 1.39 (1.11 1.75) 0.0043 9 rs10488809 35196411 intronic T 0.116 0.086 1.40 (1.11 1.75) 0.0040 10 rs3794107 35198908 intronic A 0.116 0.086 1.40 (1.11 1.75) 0.0040 11 rs76393888 35201982 intronic T 0.116 0.086 1.40 (1.11 1.75) 0.0040 12 rs76829147 35204006 intronic T 0.116 0.086 1.40 (1.12 1.76) 0.0036 13 rs12278428 35207484 intronic A 0.116 0.088 1.36 (1.09 1.71) 0.0076 14 rs75137824* 35233778 intronic T 0.033 0.014 2.31 (1.51 3.56) 1.3 10 4 15 rs2295758* 35236125 intronic C 0.033 0.014 2.29 (1.49 3.52) 1.5 10 4 16 rs1547058* 35238578 intronic C 0.036 0.017 2.18 (1.45 3.28) 1.7 10 4 17 rs78573965 35239535 intronic G 0.043 0.024 1.79 (1.24 2.58) 0.0018 The SNP positions are based on the NCBI Build 37 human genome assembly. *Indicates SNPs are P<0.00294 (bonferroni correction). Abbreviations: A1, minor allele; BP, base pair; CI, confidence interval; MAF, minor allele frequency; OR, odds ratio; SNP, single nucleotide polymorphism. Figure 1. Regional plot of CD44 SNP on chromosome 11. Regional plots were based on hg 19 version ASN (Asian population). CD44 gene is divided into two regions based on the recombination rate. Around the rs75137824, which has the highest statistical significance, has a high r 2 SNP. The three SNPs that pass the Bonferroni P-value are three with high r 2, and they are found in the same region, indicating that SNPs are highly correlated. 유의수준 (95% Confidence interval) 은 1.51 3.56을나타냈다. 이 SNP의 minor allele frequency (MAF) 는건강대조군에서는 1.4% 의빈도를보여주고있으나, 결핵환자군에서는약 2 배높은것 (3.3%) 을확인할수있었다 (Table 1). 17개의유의한 SNP들중 16개의 SNP는교차비 (OR) 가 1보다높아결핵에대한감수성을증가시키는방향으로의영향을주는것을확인하였다. 나머지 1개의 SNP (rs6484768) 은교차 비 (OR) 가 0.78로결핵에대한감수성을감소시키는방향으로영향을주는것을확인하였다. 2. CD44 유전자 SNP의 regional plot 확인 Locuszoom 프로그램을사용하여 17개의 SNP을대상으로 recombination rate (cm/mb) 과 r 2 을나타내는 regional plot 을확인하였다 (Figure 1). CD44 유전자의 plot은 hg 19 version
326 Hee-Seon Lim, et al. CD44 Gene Polymorphism Affects Tuberculosis Case ASN (Asian population) 을기준으로하였다. 자주색다이아몬드는 regional plot의기준이되는 SNP으로, 상관분석결과가장높은유의수준을나타낸 rs75137824 이다. 그림에서나타나는파란선은 CD44 영역에대한 recombination rate을나타낸다. CD44 유전자영역중약 35.21 Mb에서약 25% 의 recombination rate을나타내는것을확인할수있었다. rs75137824와그주위의다른유의성있는 SNP들과의 r 2 값이높게나타낸것을통해이러한 SNP들간의연관성이높은것을확인할수있었다. Figure 1에서기준인 rs75137824를포함하여 r 2 값이가장높은 3개의 SNP은 Bonferroni P-value 를통과한것들로, 같은 recombination 영역안에속하는것을보아세개의 SNP 간연관성이높은것을알수있었다. 3. CD44 유전자의 SNP 가유전자와단백질발현에미치는영향 RegulomeDB (http://www.regulomedb.org/index) database를이용하여 CD44의유의한 17개 SNP들이어떻게유전자또는단백질발현에영향을미칠것인지확인해보았다 (Table 2). 그결과 rs3749113, rs3794110, rs10488809, rs76393888, rs76829147, rs75137824의 6개의 SNP에서의미있는 score가나타남을알수있었다. 이중가장높은 2a score의결과를나타낸 SNP는 rs10488809였다. 2a score가나타내는것은해당 SNP가전사인자결합반응에영향을줄수있을뿐만아니라, DNase peak 차이가있다는것을의미한다. 또한, 이곳은 FOS와 JUND 전사인자가결합하는영역일뿐아니라, GATA-2,3 의 motif로작용하는위치로서유전자발현에영향을미칠수있다는것을확인하였다. 이중 rs75137824는 Bonferroni P-value에서유의한결과를나타낸 3개의 SNP 중하나이다. 이 SNP의 score는 3a로전사인자결합반응과 DNase peak 차이도나타남을확인할수있 다. 나머지두 SNP 은상대적으로의미가낮은 5 score 를나타냈다. 고찰이번연구는 CD44 유전자 SNP들과결핵환자군과건강대조군의로지스틱회귀분석을통해통계적유의성을분석하고, 이러한유전적변이와결핵의발생간의상관관계를알아보았다. 그결과 CD44 유전자영역중 17개 SNP에서결핵발병과높은통계적유의성이있음을확인하였다. 이 SNP 중 16개의 SNP 는건강대조군에비해결핵환자군에서 MAF가높아교차비 (OR) 가 1 이상이고, 하나의 SNP은결핵환자군의 MAF가낮아교차비 (OR) 가 1 이하로확인되었다. 이결과는 CD44 유전자의유전적변이가결핵발병에영향을미칠수있음을의미한다. 또한로지스틱회귀분석의통계적유의수준을전체 237개의 SNP 수로나눈값인 Bonferroni 유의수준 (P<0.00294, 0.05/237 SNPs) 을확인하였다. 그결과, 17개의통계적으로유의한 SNP 중 3개의 SNP에해당하는 P-value가 Bonferroni 유의수준을통과하였다. 또한 CD44 유전자영역에해당하는전체 237개의 SNP에서 regional plot 을확인해보았을때 Bonferroni 유의수준을통과한 3개의 SNP 간 r 2 이높고 (3개의 SNP 중 rs75137824 가기준 ) 같은 recombination 영역안에속한것을확인할수있었다. 이러한결과들을통해 CD44 유전자영역에유전적변이가결핵상태와유의한상관관계가있다는것을확인할수있다. 통계적유의성이높은 17개의 SNP에대한 regulome DB 결과를확인한결과 6개의 SNP에서의미있는 score를갖는것을확인할수있었다. 가장높은 score (2a) 를갖는 rs10488809는 GATA-2,3 의 motif로작용하는위치이다. GATA-3 은 CD4 + T 세포가 Th1 또는 Th2의세포계통으로발달하는데영향을주는전사인자이다 [14]. 또한, rs10488809는 JUND와 FOS 전사 Table 2. Results of the RegulomeDB in CD44 SNP No. SNP BP A1 A2 Regulome DB score TFBS DNase Proteins bound Motifs 2 rs3794113 35189148 G T 3a + + CEBPB, EP300 Elf3, Srf 6 rs3794110 35192214 A G 2b + + POLR2A, EP300, GATA3 HOXA13 9 rs10488809 35196411 T A 2a + + EP300, FOS, CEBPB, JUND etc GATA-2, GATA-3 11 rs76393888 35201982 T C 3a + + POLR2A E2F7 12 rs76829147 35204006 T C 2b + + NFIC, SPI1 HNF3A, SPDEF, FOXD3, HNF3A 14 rs75137824 35233778 T A 3a + + SPI1, SMARCA4 Mtf1 The SNP positions are based on the NCBI Build 37 human genome assembly. Abbreviations: SNP, single nucleotide polymorphism; BP, base pair; A1, minor allele; A2, major allele; TFBS, transcription factor binding site; +, affect; G, Genomic; I, Imputation.
Korean J Clin Lab Sci. Vol. 51, No. 3, September 2019 327 인자의결합부위로이러한 Jun-Jun 및 Jun-Fos 단백질이량체는 activatior protein-1 (AP-1) 전사인자를구성한다 [15]. AP-1은 2가지수준으로세포침입을조절한다. 첫째로, AP-1 은 matrix metalloproteases (MMPS) 및접착단백질과같은세포침입에직접적으로필요한유전자의발현을조절한다. 두번째로, AP-1은세포내신호전달단백질의발현을조절함으로써새로운유전자산물의발현을정확히조절한다 [16]. 결과적으로 JUND 단백질이결합하는영역에 rs10488809와같은유전적다형성이생긴다면 JUND 단백질과의결합효율이낮아져서 CD44 유전자발현에영향을미칠가능성을내포하고있다. 결핵균 (MTB) 은숙주세포내에서생존하고숙주의면역기전에대해저항할수있는주요한병원균이다 [17]. 이에대해숙주의방어작용중 T 세포가매개하는세포성면역반응은인간의결핵균감염을통제하는데중요하다 [18]. 백혈구는혈액으로부터백혈구와내피세포사이의상보적인리간드의상호작용을통해염증부위로유출된다. T 세포의활성화는히알루론산에대한결합을증가시키고, CD44 매개 1차부착을가능하게한다 [19]. CD44는 transmembrane glycoprotein이며, ECM 성분인히알루론산의주요세포표면수용체이다. 즉, CD44는활성화된 T 세포표면에발현되어염증부위로이동하는것에관여하여숙주의세포성면역반응을유도한다 [3, 20]. 이전연구들에서 CD44 유전자발현이결핵균증식을억제하는것으로밝혀졌다 [3, 21]. 이러한 CD44 의발현기전은 Mitogen-activated protein kinases (MAPK) pathway를통해서발현된다고알려져있으며, MAPK pathway는결핵균감염에대항하기위한대식세포의기전에서세포자멸사억제및 IFN- 형성에중요한신호전달경로로알려져있다 [22, 23]. 이러한기존연구뿐만아니라최근의연구에서는다재내성결핵환자와건강대조군사이에 CD44와연관되어있는 mir- 4433b-5p의발현의차이가발생한다는보고도있다 [24]. 따라서 CD44 유전자에유전적다형성이생긴다면, 활성화된 T 세포가폐로이동하는기전에영향을미칠것이고, 결과적으로결핵균에대응하는숙주의세포성면역반응이억제될것이다. 결과적으로, CD44 유전자의유전적다형성은결핵균에대항하는숙주의유전적감수성에영향을미칠수있음을알수있다. 결핵이잠복성과활동성으로진행정도의차이가나타나는것은결핵균의특성, 숙주의면역상태, 유전적감수성의차이에의해나타난다. 이전연구에서는결핵발병이결핵균의특성과숙주의면역상태에영향을받는다고이야기하였다 [11, 25]. 본연구에서는결핵발병에영향을미치는면역기전과관련한숙주의유전적다형성이결핵발병에관한감수성에영향을미칠 수있음을제시하고있다. 이러한결과를통해결핵에유전적감수성이있는환자에대한연구와관리및치료가가능할것이라고생각한다. 요약결핵균에의한만성세균성감염인결핵은결핵균의특성, 숙주의면역상태와유전적감수성의차이에의해잠복성과활동성으로의진행정도에차이가있다. 결핵균에대한숙주방어기전은주로대식세포, T 세포및수지상세포사이의상호작용에기인한다. CD44는결핵균에감염되면활성 T 세포에서발현되며림프구이동을조절한다. 또한 CD44는 ECM에대한백혈구의부착을매개하여대식세포, CD4+ T cell 등을폐로불러모으는역할을한다. 따라서, CD44 유전자의다형성은결핵균에대한숙주세포의면역기전저하를유발할수있다. 이연구의목적은 CD44 유전자의유전자다형성이결핵의감수성에영향을미치는지조사하는것이다. 결핵균과 CD44의연관성에대하여대한한국협회자원의 443명의 cases와 3228명의 control을이용하여 CD44 유전자의 237개의 SNP를분석하였다. 이중 17 개의 SNP가결핵과통계적으로유의한관련성을보였다. 가장유의성있는 SNP 는 rs75137824였다 (OR=0.231, CI: 1.51 3.56, P=1.3 10 4 ). 또한결핵발병에유의성이있는 SNP중 rs10488809의경우는전사인자 JUND 및 FOS에결합하는부위로써 CD44 유전자발현에영향을줄수있는것으로확인할수있었다. 이러한결과는결핵발병이 CD44 발현차이에의한숙주면역반응에차이에의해서감수성의차이가있을수있음을나타낼수있다. 이번연구결과는결핵균감염에대한숙주면역의유전적차이가결핵진행정도의차이를유발할수있다는유전적배경에대한기반을마련해줄수있을것이라고기대한다. Acknowledgements: This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by Ministry of Sciences, ICT & Future Planning (2017R1C1B5016589). This study was conducted with bioresources from National Biobank of Korea, the Centers for Disease Control and Prevent ion, Republic of Korea (KBN-2017-038). Conflict of interest: None Author s information (Position): Lim HS, Undergraduate student; Lee SI, Graduate student; Park S, Professor.
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