Takedatsu H, Taylor K D, Mei L, McGovern D P B, Landers C J, Gonsky R, Cong Y, Vasiliauskas E A, Ippoliti A, Elson C O, Rotter J I, Targan S R
Division of Gastroenterology, Inflammatory Bowel Disease Center, Immunobiology Research Institute, Cedars-Sinai Medical Center, Los Angeles, CA 90048, USA.
Gut. 2009 Jan;58(1):60-7. doi: 10.1136/gut.2008.156422. Epub 2008 Oct 2.
Genetics studies of the serum expression of antibodies to microbial antigens may yield important clues to the pathogenesis of Crohn's disease. Our aim was to conduct a linkage study using expression of anti-CBir1, anti-I2, anti-OmpC and ASCA as quantitative traits.
Expression of antibodies to microbial antigens was measured by enzyme-linked immunosorbant assay (ELISA) and a standard approximately 10 cM whole genome microsatellite study was conducted. Single nucleotide polymorphism genotyping was performed using either Illumina or TaqMan MGB technology. Nuclear factor Kappa B (NF-kappaB) activation in cells from Epstein-Barr virus (EBV)-transformed cell lines was assessed using an electrophoretic mobility shift assay and protein was measured using ELISA and western blotting.
Evidence for linkage to anti-CBir1 expression was detected on human chromosome 4 (logarithm of odds (LOD) 1.82 at 91 cM). We therefore directly proceeded to test the association of haplotypes in NFKB1, a candidate gene. One haplotype, H1, was associated with anti-CBir1 (p = 0.003) and another, H3, was associated with ASCA (p = 0.023). Using cell lines from Crohn's disease patients with either H1 or H3, NF-kappaB activation and NF-kappaB p105 and p50 production were significantly lower for patients with H1 compared to patients with H3.
These results suggest that NFKB1 haplotypes induce dysregulation of innate immune responses by altering NF-kappaB expression. The results also show the use of EBV-transformed lymphoblastoid cell lines to conduct phenotypic studies of genetic variation.
对微生物抗原抗体血清表达的遗传学研究可能为克罗恩病的发病机制提供重要线索。我们的目的是利用抗CBir1、抗I2、抗OmpC和抗酿酒酵母抗体（ASCA）的表达作为数量性状进行连锁研究。
采用酶联免疫吸附测定（ELISA）检测微生物抗原抗体的表达，并进行标准的约10厘摩（cM）全基因组微卫星研究。使用Illumina或TaqMan MGB技术进行单核苷酸多态性基因分型。采用电泳迁移率变动分析评估来自爱泼斯坦-巴尔病毒（EBV）转化细胞系的细胞中核因子κB（NF-κB）的激活情况，并使用ELISA和蛋白质印迹法测量蛋白质。
在人类4号染色体上检测到与抗CBir1表达连锁的证据（在91 cM处优势对数（LOD）为1.82）。因此，我们直接对候选基因NFKB1中的单倍型关联进行检测。一种单倍型H1与抗CBir1相关（p = 0.003），另一种单倍型H3与抗酿酒酵母抗体（ASCA）相关（p = 0.023）。使用来自携带H1或H3的克罗恩病患者的细胞系，与携带H3的患者相比，携带H1的患者的NF-κB激活以及NF-κB p105和p50的产生显著降低。
这些结果表明，NFKB1单倍型通过改变NF-κB表达诱导先天性免疫反应失调。结果还显示了利用EBV转化的淋巴母细胞系进行遗传变异的表型研究。
