Clonal stability of blood cell lineages indicated by X-chromosomal transcriptional polymorphism.

Abstract

SummaryThe idea that stem cells oscillate between a state of activity and dormancy, thereby giving rise to differentiating progeny either randomly or in orderly clonal succession, has important implications for understanding normal hematopoiesis and blood cell dyscrasias. The degree of clonal stability in individuals also has practical implications for the evaluation of clonal lymphomyeloproliferative diseases. To evaluate the clonality pattern of the different types of blood cells as a function of time we have validated the applicability, sensitivity, and reproducibility of a thermostable ligase reaction to detect transcripts of the G6PD allele on the active X-chromosome in normal heterozygous females. While the ratio of the two X-chromosome-derived allelic transcripts varied widely among hemopoietic and nonhemopoietic tissues in a given individual, this allelic ratio was virtually identical in all types of mature myeloid and lymphoid cells. Longitudinal studies indicated constancy of the G6PD allelic ratio in blood cells over a 912-d period of observation in healthy females. The individual variability observed in this allelic ratio suggests that the progeny of a relatively small number of original embryonic hemopoietic stem cells, approximately eight, contribute to the sustained production of all types of blood cells in healthy individuals.

Mutation of JAK2 in the myeloproliferative disorders: timing, clonality studies, cytogenetic associations, and role in leukemic transformation

“…First, several groups have demonstrated small differences in XCIPs from T cells and granulocytes in healthy subjects. 24,29,32,33 We show here that these differences result in wide confidence limits for the calculated proportion of V617F Ϫ cells that are apparently clonally derived. Importantly, this variability is systematically biased toward overestimating the proportion of clonally derived granulocytes.…”

The relative quiescence of hematopoietic stem cells in nonhuman primates

“…This slow cycling behavior of baboon PHSCs is in agreement with the data generated by other researchers in other large animal models, including cats and rhesus macaques, and in humans. 13,19,[44][45][46][47][48] In particular, on the basis of measurements of telomere shortening in humans, Rufer et al 44 have previously reported that a rapid expansion of stem cells occurs early in life, followed by a marked decrease in the rate of stem cell divisions in the years that follow. They concluded that 15 to 30 stem cell divisions occur during the first half-year of life followed by less than 1 stem cell division per year in following years.…”

Rapid determination of clonality by detection of two closely-linked X chromosome exonic polymorphisms using allele-specific PCR.

“…tional products (10,11). We have verified the reproducibility, sensitivity, and quantitative nature of rtPCR-LDR (10)(11)(12) This ASPCR assay is a modification of the previously reported ASPCR (13), PCR-ASOH (14) and PCR amplification of specific alleles (PASA) (15). The utility of PASA for this type of application is limited because the mismatched pairing which occurs at the 3 Ј end of the primer-template duplex (i.e., T:C, T:G, T:T) is amplified by Taq polymerase as efficiently as the perfectly complementary primer-template duplexes.…”

“…These assays are limited by racial specificity, lack of quantitation, and inapplicability to nonnucleated cells such as platelets and reticulocytes. We recently developed a clonality assay based on transcriptional polymorphisms of two ubiquitously expressed X chromosome genes; G6PD (C/T at 1311) and palmitoylated membrane protein, p55 (G/T at cDNA 358) (10,11,12). The assay uses reverse transcription polymerase chain reaction followed by ligase detection reaction (rtPCR-LDR) 1 (10,11,12) to distinguish the transcriptionally active X chromosome.…”