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.
All Science Journal Classification (ASJC) codes
- Immunology and Allergy
- chromosome X
- sex differences
- white blood cells