Hematopoiesis is not clonal in healthy elderly women

Abstract

Clonality assays, based on X-chromosome inactivation, discriminate active from inactive alleles. Skewing of X-chromosome allelic usage, based on preferential methylation of one of the HUMARA alleles, was reported as evidence of clonal hematopoiesis in approximately 30% of elderly women. Using a quantitative, transcriptionally based clonality assay, we reported X-chromosome-transcribed allelic ratio in blood cells of healthy women consistent with random X-inactivation of 8 embryonic hematopoietic stem cells. Furthermore, we did not detect clonal hematopoiesis in more than 200 healthy nonelderly women. In view of the susceptibility of aging hematopoietic stem cells to epigenetic dysregulation, we reinvestigated the issue of clonality in elderly women. Forty healthy women (ages 65-92 years; mean, 81.3 years) were tested by a novel, quantitative polymerase chain reaction (qPCR) transcriptional clonality assay. We did not detect clonal hematopoiesis in any of the tested subjects. We also tested DNA from the same granulocyte samples using the methylation-based HUMARA assay, and confirmed previous reports of approximately 30% extensively skewed or monoallelic methylation, in agreement with likely age-related deregulated methylation of the HUMARA gene locus. We conclude that the transcriptionally based X-chromosome clonality assays are suitable for evaluation of clonal hematopoiesis in elderly women.

Figure 1

Schematic diagram of X-chromosome clonality determination used here and in HUMARA assay. X-chromosome inactivation occurs early during embryogenesis. Hence, women are a mosaic of paternal or maternal active X chromosome (Step 1). Inactive X chromosome is represented by filled red circles. For the transcriptional clonality assay, a specific exonic polymorphism is selected and genotyped (Step 2a). Allele-specific expression is determined by real-time PCR using reverse-transcribed mRNA as described in “Novel transcriptional clonality assay” (Steps 3a and 4a). Resulting amplification curve is used to estimate the ΔCt and corresponding frequencies of each allele (Step 5a). In contrast, analysis at the HUMARA locus, shown methylated in the promoter region by filled red circles (Step 2b), is initiated by restriction digestion (scissors) of genomic DNA using a methylation-sensitive endonuclease (Step 3b). After restriction digestion, PCR amplification with primers flanking both the 5′ restriction digestion site and the 3′ end of the CAG tandem repeat sequence is performed (Step 4b). Hence, only intact, methylated, inactive X-chromosome DNA is amplified. Allele-specific PCR products can be distinguished from each other based on the number of tandem CAG repeats using agarose gel electrophoresis (Step 5b).

Figure 2

Linear regression analysis of the correlation between allelic expression ratios for 2 markers. PLT indicates platelets; GNC, granulocytes. Comparison between allelic expression ratios in the same individual, either between markers within the same lineage (A,B) or between lineages (C). Individuals informative for more than one marker were used for comparison of the expression ratios in platelets (A) or granulocytes (B). An identical analysis was performed for comparison of a single marker between platelet and granulocyte lineages, within the same individual (C). Excellent correlations were found between compared values obtained in platelets and granulocytes, with P value less than .001.