建立殺手細胞免疫球蛋白樣受體(KIR)基因鑑定平臺以及大腸直腸癌患者之KIR基因型分析

Abstract

殺手細胞免疫球蛋白樣受體 (KIRs) 表現於NK細胞表面能與第一類人類白血球抗原 (HLA class I) 結合並調控NK 細胞活化與抑制。本論文先利用PCR-SSP搭配毛細管電泳建立KIR基因的檢測平台。接著建立以探針捕獲為基礎的次世代定序方法應用在KIR基因型別鑑定。針對KIR的15個基因與2個偽基因的外顯子(exon)共設計233個不同的探針。利用購自國際組織相容協會(International Histocompatibility Working Group, IHWG)的56個DNA標準品進行測試，定序結果使用2016年發表之PING (Pushing Immunogenetics to the Next Generation)進行基因型及等位基因(allele)分析。結果顯示:於基因層級的分析除KIR2DL5A與KIR2DL5B，其餘的KIR基因正確率皆為100%。針對等位基因的分析，排除軟體無法分析的KIR2DS1、KIR2DS2及KIR3DP1， KIR等位基因的單一輸出結果正確率除KIR2DL4、KIR2DL5A、KIR2DL5B低於60%外，其餘KIR基因正確率皆超過80%，其中KIR2DL2、KIR2DS3、KIR2DS5與KIR3DS1的正確率達100%。最後NGS平臺所分析之56個國際標準品序列回貼至自主建立的KIR參考基因，結果顯示所設計的探針能夠完整捕獲KIR基因的外顯子區域，然而仍需要對探針進行縮減或選擇更具特異性的區域，以減少探針捕獲到內含子(intron)重複序列的區域，進而增加軟體過濾後的比例。 大腸直腸癌在世界癌症死亡率排名位於前三名，顯示需要更多研究了解此疾病。自然殺手細胞 (NK細胞) 於人體內監控與清除腫瘤細胞扮演重要角色。為了研究KIR基因型與大腸直腸癌的相關性，本論文第二部分以PCR-SSP平臺分析119例大腸直腸癌病患的KIR基因型，將之與2009年分析97例慈濟骨髓庫的KIR基因型進行比對。結果顯示:在大腸直腸癌(CRC)組別之KIR2DL5B和KIR3DL1頻率比慈濟組顯著較低( KIR2DL5B : P= 0.023, KIR3DL1 : P=0.041)。KIR2DS4F在CRC組別也有較低的趨勢但未達統計的顯著差異(P=0.069)。CRC組別之KIR2DL5A、KIR2DS1、KIR2DS5、KIR3DS1和KIR3DP1var的頻率皆較慈濟組高，但僅有KIR3DP1var (P=0.042)達顯著差異。此外在慈濟組有29種KIR基因型 (genotypes)，而CRC組別有23種KIR基因型，兩個組別總共包含42種KIR基因型。而慈濟組別與CRC組別的染色體單倍型比例相似，兩組之染色體屬於A單倍型(haplotype A) 與B單倍型(haplotype B) 的比例都接近3 : 1 (慈濟組haplotype A : haplotype B =76.3 : 23.7；CRC組haplotype A : haplotype B=75.9 : 24.1)；兩組之同型合子haplotype A/A的比例也相似，慈濟組為52.5%而CRC組為53.8%。針對KIR3DL1基因有無，於CRC組進行單變項臨床相關性分析與存活分析，皆未達統計顯著差異。再針對KIR2DS4F基因有無進行臨床相關性分析與存活分析，皆未達統計顯著差異。將CRC組別分成haplotype A/A組與haplotype B/X組進行分析，CRC屬於haplotype B/X組之KIR2DS4F基因頻率顯著低於慈濟組別(P=0.030) 。針對KIR2DS4F基因有無，於B單倍型CRC組別進行單變項臨床相關性分析與存活分析皆未達統計顯著差異；然而CRC病患不具KIR2DS4F基因其存活月數有較好的趨勢。 大腸直腸癌在世界癌症死亡率排名位於前三名，顯示需要更多研究了解此疾病。自然殺手細胞 (NK細胞) 於人體內監控與清除腫瘤細胞扮演重要角色。為了研究KIR基因型與大腸直腸癌的相關性，本論文第二部分以PCR-SSP平臺分析119例大腸直腸癌病患的KIR基因型，將之與2009年分析97例慈濟骨髓庫的KIR基因型進行比對。結果顯示:在大腸直腸癌(CRC)組別之KIR2DL5B和KIR3DL1頻率比慈濟組顯著較低( KIR2DL5B : P= 0.023, KIR3DL1 : P=0.041)。KIR2DS4F在CRC組別也有較低的趨勢但未達統計的顯著差異(P=0.069)。CRC組別之KIR2DL5A、KIR2DS1、KIR2DS5、KIR3DS1和KIR3DP1var的頻率皆較慈濟組高，但僅有KIR3DP1var (P=0.042)達顯著差異。此外在慈濟組有29種KIR基因型 (genotypes)，而CRC組別有23種KIR基因型，兩個組別總共包含42種KIR基因型。而慈濟組別與CRC組別的染色體單倍型比例相似，兩組之染色體屬於A單倍型(haplotype A) 與B單倍型(haplotype B) 的比例都接近3 : 1 (慈濟組haplotype A : haplotype B =76.3 : 23.7；CRC組haplotype A : haplotype B=75.9 : 24.1)；兩組之同型合子haplotype A/A的比例也相似，慈濟組為52.5%而CRC組為53.8%。針對KIR3DL1基因有無，於CRC組進行單變項臨床相關性分析與存活分析，皆未達統計顯著差異。再針對KIR2DS4F基因有無進行臨床相關性分析與存活分析，皆未達統計顯著差異。將CRC組別分成haplotype A/A組與haplotype B/X組進行分析，CRC屬於haplotype B/X組之KIR2DS4F基因頻率顯著低於慈濟組別(P=0.030) 。針對KIR2DS4F基因有無，於B單倍型CRC組別進行單變項臨床相關性分析與存活分析皆未達統計顯著差異；然而CRC病患不具KIR2DS4F基因其存活月數有較好的趨勢。

Nature killer (NK) cells play important roles in surveillance and prevention of cancer initiation and development. Killer cell immunoglobulin-like receptors (KIRs) binding to HLA class I ligands can regulate the activation and inhibition of NK cells by signal transduction. To study KIR genes, we established a KIR genotyping platform by using a commercial PCR-SSP kit which can distinguish all of 17 KIR genes and the variants of KIR2DS4 and KIR3DP1. Second, we established probe capture-based next-generation sequencing platform of KIR genotyping, the KIR panel was consist of 233 different probes which were located in intron beside exons of KIR genes. We verified the probe design with 56 international histocompatibility working group (IHWG) standard DNAs and performed KIR genotype and allelotype analysis by Pushing Immunogenetics to the Next Generation (PING) software. The sequence coverage of the 56 IHWG standard samples concentrated at exon regions of 17 KIR genes. Comparing KIR gene result to allele frequency net database (AFND), the gene level accuracies of 17 KIR genes analyzed by PING were 100% except for KIR2DL5A and KIR2DL5B. We defined the KIR allelotype results as three different categories and then compared the single output results to AFND. The accuracies of single output results of all KIR genes by PING were larger than 60% except KIR2DL4, KIR2DL5A, and KIR2DL5B. We mapped the 56 standard DNA sequences to 17 KIR genes respectively and simultaneously. The mapping coverage patterns of all KIR genes are similar. However, the coverage patterns were better after filtration with PING KIR filter, which read depth patterns were more related to KIR genotypes and copy numbers. According to our results, the KIR probe panel was able to capture all exon of 17 KIR genes, however, the panel might have better efficiencies after the deletion of some probes. Colorectal cancer (CRC) is one of the most common types of human cancers. To study the CRC-associated KIR genes, 119 patients were recruited and DNA samples were prepared from their peripheral blood. KIR genotyping was performed by using a PCR-SSP platform we established. KIR gene frequencies and genotypes of the CRC group were compared with 97 Han Taiwanese from the Buddhist Tzu Chi Taiwan Marrow Donor Registry (Tzu Chi group) by the Chi-square test or Fisher’s exact test. The frequencies of KIR2DL5B and KIR3DL1 were significantly descended in the CRC patients compared to the Tzu Chi group (KIR2DL5B, P=0.023；KIR3DL1, P=0.041). The frequency of full-length KIR2DS4 alleles (KIR2DS4F) in the CRC patients was lower than that of the Tzu Chi group but not statistically significant yet(P=0.069). In contrast, KIR2DL5A, KIR2DS1, KIR2DS5, KIR3DS1, and KIR3DP1 variant with higher frequencies were observed in the CRC group, but only KIR3DP1 variant with statistical significance (P=0.042). In total, 23 genotypes were identified among the 119 CRC patients tested, and 29 genotypes among in the Tzu Chi group. The frequencies of individuals who were homozygous for group A haplotype (haplotype A/A) in the Tzu Chi group and the CRC group were 52.5% and 53.8%, respectively. Consequently, haplotype A (75.9%) outnumbered haplotype B (24.1%) in CRC group, and the same phenomenon (haplotype A and haplotype B, 76.3% and 23.7%, respectively) was observed in the Tzu Chi group. As for the clinical relevance of KIR gene polymorphism in CRC, the genotypes of KIR3DL1/S1 were not associated with clinical characteristics and overall survival of patients. Besides, the presence of KIR2DS4F did not show a clinical association, either. The frequency of KIR2DS4F in the CRC patients with haplotype B/X was significantly lower than that of the Tzu Chi group (P=0.03). Among CRC patients with 55 haplotype B/X, the presence of KIR2DS4F was not associated with overall survival. However, there was a trend that CRC patients without KIR2DS4F had longer survival time. Colorectal cancer (CRC) is one of the most common types of human cancers. To study the CRC-associated KIR genes, 119 patients were recruited and DNA samples were prepared from their peripheral blood. KIR genotyping was performed by using a PCR-SSP platform we established. KIR gene frequencies and genotypes of the CRC group were compared with 97 Han Taiwanese from the Buddhist Tzu Chi Taiwan Marrow Donor Registry (Tzu Chi group) by the Chi-square test or Fisher’s exact test. The frequencies of KIR2DL5B and KIR3DL1 were significantly descended in the CRC patients compared to the Tzu Chi group (KIR2DL5B, P=0.023；KIR3DL1, P=0.041). The frequency of full-length KIR2DS4 alleles (KIR2DS4F) in the CRC patients was lower than that of the Tzu Chi group but not statistically significant yet(P=0.069). In contrast, KIR2DL5A, KIR2DS1, KIR2DS5, KIR3DS1, and KIR3DP1 variant with higher frequencies were observed in the CRC group, but only KIR3DP1 variant with statistical significance (P=0.042). In total, 23 genotypes were identified among the 119 CRC patients tested, and 29 genotypes among in the Tzu Chi group. The frequencies of individuals who were homozygous for group A haplotype (haplotype A/A) in the Tzu Chi group and the CRC group were 52.5% and 53.8%, respectively. Consequently, haplotype A (75.9%) outnumbered haplotype B (24.1%) in CRC group, and the same phenomenon (haplotype A and haplotype B, 76.3% and 23.7%, respectively) was observed in the Tzu Chi group. As for the clinical relevance of KIR gene polymorphism in CRC, the genotypes of KIR3DL1/S1 were not associated with clinical characteristics and overall survival of patients. Besides, the presence of KIR2DS4F did not show a clinical association, either. The frequency of KIR2DS4F in the CRC patients with haplotype B/X was significantly lower than that of the Tzu Chi group (P=0.03). Among CRC patients with 55 haplotype B/X, the presence of KIR2DS4F was not associated with overall survival. However, there was a trend that CRC patients without KIR2DS4F had longer survival time.