Department of Surgery, Rutgers-New Jersey Medical School, 185 South Orange Ave., MSB G-578, Newark, NJ, 07103, USA.
Inflammation. 2020 Feb;43(1):370-381. doi: 10.1007/s10753-019-01127-6.
Random X chromosome (ChrX) inactivation and consequent cellular mosaicism for the active ChrXs in white blood cells (WBCs) is unique to females and may contribute to sex-biased modulation of the innate immune response. Polymorphic differences between the two parental ChrXs may result in ChrX skewing of circulating WBCs (ChrX inactivation-ratio (XCI) > 3) driven by differences in stem cell selection and activity in the bone marrow or WBC trafficking at the periphery. Independent of the mechanism, ChrX skewing may result in genotype-phenotype discrepancies. This study aimed to develop an allele-specific assay and test its applicability in clinical samples to determine the "direction" of ChrX skewing in the variant IRAK1 haplotype, a common X-linked polymorphism with major clinical impacts. Because alternative splice variants of IRAK1 are also produced in the region surrounding the critical single-nucleotide polymorphism (SNP, rs1059703), we also tested the abundance of alternative splice variant IRAK1 transcripts. The expression of splice variants IRAK1-B and IRAK1-C was about 30 and 50% of the full-length (IRAK1-A) in WBCs from healthy subjects (n = 53). IRAK1-A, B, and C showed about 30% lower expression level in males (n = 25) than females (n = 28). By contrast, the expression levels of IRAK1-A, B, and C were not affected by the variant IRAK1 haplotype in either sex. Allele-specific primers generated WT and variant-IRAK1 amplicons with high selectivity, and on average produced about half the expression levels of each transcript in heterozygous IRAK1-mosaic females. Because injury was shown to induce de novo ChrX skewing of WBCs, we tested the directional XCI ratio changes in WBC in a sample of trauma patients heterozygous for the variant IRAK1 haplotype (n = 18). Using the allele-specific assay in combination with the DNA methylation status at the polymorphic HUMARA locus, we found that at admission, about 60% the patients presented XCI ratios skewed toward WBCs with active ChrXs carrying the variant-IRAK1 similar to healthy controls. De novo, trauma-induced XCI ratio changes showed increased extravasation of the more abundant mosaic WBC subset without reversal in the direction of ChrX skewing during the injury course.
随机 X 染色体（ChrX）失活以及随之而来的白细胞（WBC）中活性 ChrX 的细胞嵌合是女性所特有的，可能导致先天免疫反应的性别偏向调节。来自两个亲本 ChrX 的多态性差异可能导致由骨髓中干细胞选择和活性或外周血 WBC 转运的差异驱动的循环 WBC 中 ChrX 倾斜（ChrX 失活比（XCI）>3）。无论机制如何，ChrX 倾斜都可能导致基因型-表型差异。本研究旨在开发一种等位基因特异性检测，并在临床样本中测试其适用性，以确定变体 IRAK1 单倍型中 ChrX 倾斜的“方向”，该变体 IRAK1 单倍型是一种具有重大临床影响的常见 X 连锁多态性。由于 IRAK1 的可变剪接变体也在关键单核苷酸多态性（SNP，rs1059703）周围的区域产生，我们还测试了替代剪接变体 IRAK1 转录本的丰度。在来自健康受试者的 WBC 中（n = 53），剪接变体 IRAK1-B 和 IRAK1-C 的表达约为全长（IRAK1-A）的 30%和 50%。在男性（n = 25）中，IRAK1-A、B 和 C 的表达水平比女性（n = 28）低约 30%。相比之下，变体 IRAK1 单倍型在两性中均不影响 IRAK1-A、B 和 C 的表达水平。等位基因特异性引物产生了具有高选择性的 WT 和变体 IRAK1 扩增子，并且在杂合 IRAK1 嵌合体女性中，每个转录本的表达水平平均产生约一半。由于损伤已被证明会诱导 WBC 中新的 ChrX 倾斜，我们在一组携带变体 IRAK1 单倍型的创伤患者（n = 18）中测试了 WBC 中 ChrX 失活比的定向变化。使用等位基因特异性检测结合多态性 HUMARA 基因座处的 DNA 甲基化状态，我们发现入院时，约 60%的患者表现出 WBC 中 ChrX 失活比向携带变体 IRAK1 的活性 ChrX 倾斜，与健康对照组相似。在受伤过程中，新出现的创伤诱导的 ChrX 失活比变化显示出更多丰富的嵌合性 WBC 亚群的额外渗透，而 ChrX 倾斜的方向没有逆转。
