Tuberculosis remains the major health problem in the world, including Indonesia. The genetic studies of tuberculosis (TB) have been conducted in Javanese, a major population in the country. In this study, I started with describing the genetic affinity and comparison between the studied population and other Asian populations based on the polymorphisms of HLA class I and II genes. The possible association of HLA-A, -B and -DRB1 with TB was also analyzed both in primary TB and in recurrent TB patients. Another genetic study on TB was also done in this Javanese population by conducting a high-density association mapping of a candidate region for young-onset TB based on the result of a genome-wide linkage study in Thai (Mahasirimongkol et al., 2009). Thai is a Southeast Asian population with close genetic affinity with the Javanese (Indonesia) population. Two genes newly reported to be associated with TB especially for young-onset TB were found.

Human leukocyte antigen (HLA) genes are the most polymorphic genes in the human genome with the major role in the presentation of variety antigen peptides to lymphocyte T cells. The unique characteristics of HLA genes and their essential function in the immune system serve as the important candidate gene for disease susceptibility and make the genes as useful tool for population study. In the present study, the allele and haplotype frequencies of HLA-A, HLA-B and HLA-DRB1 were examined in 237 unrelated healthy western Javanese (Indonesia) by the high resolution PCR-Luminex method. A total of 18 A, 40 B and 20 DRB1 alleles were identified. The most frequent HLA-A, -B and -DRB1 alleles were HLA-A*24:07 (21.6%), HLA-B*15:02 (11.6%) and HLA-B*15:13 (11.2%) and DRB1*12:02 (37.8%), respectively. The most frequent 2-locus haplotypes were HLA-A*24:07-B*35:05 (7%) and HLA-B*15:13-DRB1*12:02 (9.2%), and for 3-locus haplotypes were HLA-A*34:01-B*15:21-DRB1*15:02:01 (4.6%), HLA-A*24:07-B*35:05-DRB1*12:02 (4.3%) and HLA-A*33:03:01-B*44:03:02-DRB1*07:01:01 (4.2%). HLA allele and haplotype frequencies in addition to phylogenetic tree and principal component analyses based on the 4-digit sequence level allele frequencies for HLA-A, HLA-B and HLA-DRB1 revealed the evidence of close genetic affinity between Western Javanese (Indonesia) with other Southern group of East Asian, especially Southeast Asian populations.

The association of HLA-A, -B, and -DRB1 alleles and haplotypes with TB was also studied to ascertain their role in susceptibility/resistance to primary and recurrent TB in 257 TB patients (216 primary and 41 recurrent TB patients) and 236 ethnically matched healthy controls in Western Javanese (Indonesia). HLA-B*40:06 was associated with primary TB (p=0.044, padj=ns), whereas HLA-B*18:02 was associated with recurrent TB (p=0.014, padj=0.024 for recurrent TB vs control, p=0.013, padj=0.016 for primary TB vs recurrent TB). Two other alleles: HLA-B*40:01 and HLA-DRB1*11:01 showed significant association with recurrent TB only in the comparison between recurrent TB and primary TB (p=0.015, padj=0.028 and p=0.008, padj=0.027, respectively). Except for HLA-B*40:06, those associations remained significant after adjustment for age and sex by logistic regression analysis, although they were disappeared after correction for multiple testing. The possible role of HLA-B*18:02 was further supported by the observation that the presence of HLA-B*18:02-DRB1*12:02 haplotype was associated with susceptibility to recurrent TB (p=0.014, OR=3.8 (1.18-12.27)). On the other hand, HLA-DRB1*12:02 in the absence of HLA-B*18:02 showed a significant association with resistance to recurrent TB (p= 8.2×10-4, OR= 0.32 (0.16-0.64)), suggesting that stronger susceptibility effect of HLA-B*18:02 masked the protective effect of HLA-DRB1*12:02.

Finally, the high-density association mapping of a candidate region for young-onset TB in chromosome 20p13 was performed in the present study in 275 cases (155 young-onset, 120 late-onset) and 250 control of Javanese (Indonesia). Of 135 analyzed SNPs, two SNPs located in the OXT and AVP gene region showed significant association event after correction for multiple testing (rs6084265(T): pallele=7.19×10-6, OR=2.00 (1.48-2.72) and rs2770381(C): pallele=4.58×10-5, OR= 1.83(1.37-2.45)) in young-onset TB. Moreover, the two-locus haplotype analysis of rs2770381-rs6084265 showed higher significant association with the disease (global phaplotype=2.68×10-8). In contrast, significant association was not observed in late-onset TB group. These results suggest that the association observed in rs6084265 and rs2770381 could be due to other primary SNP in strong LD with it. Oxytocin and arginine vasopressin are neuroendocrine hormones that involve in the regulation of the hypothalamic-pituitary-adrenal (HPA) axis. HPA response through glucocorticoids modulates the immune/inflammatory reaction. It was hypothesized that the variation in the OXT-AVP gene region mediated by rs2770381(A)-rs6084265(C) haplotype increase the activity of the gene, consequently activate the HPA-axis and subsequently increase the glucocorticoid production and lead to the shift of immunological balance from T helper (Th) 1 to Th2 that decrease the capability of individuals to respond mycobacterium infection and confer the susceptibility to young-onset TB. This finding is novel and I believe that this work would contribute to better understanding of TB etiology and better strategy to combat the disease. Further study to confirm this finding in other Asian and non Asian populations would be of great important to evaluate the global role of the genes.

本研究では、インドネシア国で最大の民族集団であるジャワ人を対象とした結核に対する宿主感受性の遺伝学的研究を行った。関連解析に先立ち、この集団と他のアジア系集団との間の遺伝的類似性を、ヒト白血球抗原(HLA)の遺伝子多型を用いて解析した。関連解析では、候補遺伝子アプローチと他の東南アジア民族集団で報告された連鎖解析の結果に基づいた領域の高密度関連解析の二つのアプローチを用いた。これらから、以下の知見を得た。

1. ジャワ人(インドネシア)と他のアジア系集団との間の遺伝的類似性を高密度PCR-Luminex法によって、HLA-A、HLA-B、HLA-DRB1のアレル頻度及び2つもしくは3つのHLA遺伝子座のハプロタイプ頻度を用いて検討した。HLAのアレル頻度とハプロタイプ頻度から、更に系藤樹と主成分分析の結果、ジャワ人(インドネシア)は、他の東アジア系集団の南部のグループ、特に東南アジア系集団と遺伝的類似性が存在することを示した。

2. ジャワ人(インドネシア)の結核患者群257名(新規診断患者216名、再発患者41名)と対照群263名を用いた関連解析では、HLA-A、HLA-B、HLA-DRB1のアレル及びハプロタイプと結核の間に弱い関連があった。このうち、最も強い関連は、結核再発患者においてHLA-B*18:02の単一アレルの関連解析及び二因子分析で観察された。二因子分析では、HLA-B*18:02-HLA-DRB1*12:02ハプロタイプの存在が、結核再発の感受性と関連していた。一方、HLA-B*18:02の非存在かでは、DRB1*12:02アレルは、結核再発に抵抗性と有意な関連を示した。このことは、HLA-B*18:02アレルの強い感受性効果が、DRB1*12:02アレルの抵抗性効果をマスクしていると示唆された。

3. 最後に、ジャワ人(インドネシア)の患者群275名(若年発症患者155名、晩発発症患者120名)と対照群250名を用いた結核弱年発症候補領域である20p13-12.3領域の高密度関連解析では、Oxytocin (OXT) 遺伝子とArginine Vasopressin (AVP) 遺伝子領域に位置する2つのSNPが、多重検定補正後も若年発症群で有意な関連を示した。2つのSNPから構成される2遺伝子座ハプロタイプ解析では、結核とより強い関連を示した。一方、晩発発症患者群では有意な関連は示さなかった。このことは、OXT-AVP遺伝子領域の変異が、遺伝子の活性を上昇させ、海馬-下垂体-副腎軸(HPA-axis)を活性化させる。その結果、糖質コルチコイド産生を増加させるとともに、Th1細胞からTh2細胞へ免疫学的バランスをシフトさせることで、個人の結核菌感染への反応能の低下を招き、若年時の結核感受性に影響を与えることが示唆された。

以上、本論文で得られた遺伝学的研究の成果は、インドネシア国における今後の遺伝学的研究にとって重要であり、更に、高密度関連解析では、有意な関連を示す遺伝子を新たに見いだしその結果、更なる機能解析研究が必要であるものの結核発症の病因の理解に貢献し、その克服につながる治療戦略の端緒を示すことが出来た。従って本論文は学位の授与に値するものと考えられる。