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. 2005 May 15;105(10):3848-54.
doi: 10.1182/blood-2004-04-1472. Epub 2005 Feb 1.

PIG-A mutations in normal hematopoiesis

Rong Hu  1 , Galina L Mukhina, Steven Piantadosi, Jamie P Barber, Richard J Jones, Robert A Brodsky
Affiliations

PIG-A mutations in normal hematopoiesis

Rong Hu et al. Blood. .

Abstract

Paroxysmal nocturnal hemoglobinuria (PNH) is caused by phosphatidylinositol glycan-class A (PIG-A) mutations in hematopoietic stem cells (HSCs). PIG-A mutations have been found in granulocytes from most healthy individuals, suggesting that these spontaneous PIG-A mutations are important in the pathogenesis of PNH. It remains unclear if these PIG-A mutations have relevance to those found in PNH. We isolated CD34+ progenitors from 4 patients with PNH and 27 controls. The frequency of PIG-A mutant progenitors was determined by assaying for colony-forming cells (CFCs) in methylcellulose containing toxic doses of aerolysin (1 x 10(-9) M). Glycosylphosphatidylinositol (GPI)-anchored proteins serve as receptors for aerolysin; thus, PNH cells are resistant to aerolysin. The frequency of aerolysin resistant CFC was 14.7 +/- 4.0 x 10(-6) in the bone marrow of healthy donors and was 57.0 +/- 6.7 x 10(-6) from mobilized peripheral blood. DNA was extracted from individual day-14 aerolysin-resistant CFCs and the PIG-A gene was sequenced to determine clonality. Aerolysin-resistant CFCs from patients with PNH exhibited clonal PIG-A mutations. In contrast, PIG-A mutations in the CFCs from controls were polyclonal, and did not involve T cells. Our data confirm the finding that PIG-A mutations are relatively common in normal hematopoiesis; however, the finding suggests that these mutations occur in differentiated progenitors rather than HSCs.

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Figures

Figure 1.
Figure 1.
Schematic representation of the PIG-A gene and PCR primers. □ and ▪ represent coding and noncoding regions, respectively; arrows, PCR primer sites; and —, introns.
Figure 2.
Figure 2.
Flow cytometric analysis of pooled aerolysin-resistant colonies. Day-14 BFU-Es (top row) or CFU-GMs (bottom row) were washed from methycellulose-containing plates. BFU-Es were stained with FITC-conjugated anti–glycophorin-A and PE-conjugated anti-CD59. CFU-GMs were stained with PE-conjugated anti-CD15 and FLAER. Vertical and horizontal axes represent fluorescence intensity compared with isotypic controls.
Figure 3.
Figure 3.
Characterization of aerolysin resistant T-cell clones. (A) Flow cytometric analysis of pooled aerolysin-resistant T-cell clones. Day-16 colonies were washed and stained with FLAER. — represents expanded T-cell clones grown without aerolysin; –, expanded T-cell clones grown in the presence of aerolysin. (B) Mutation from CFU-GM/AE 9/7 lost MslI restriction site, which is CACAA′GGATG. Thus, MslI was used to screen aerolysin-enriched T-cell clones grown from the same donor. A total of 8 aerolysin-resistant T-cell clones were individually plucked and genomic DNA was extracted. A representative example is shown. Exon 4 of the PIG-A gene was amplified with the proofreading polymerase Pfu Ultra using the primers shown in Figure 1 and Table 1. The products were purified and digested with MslI. The PCR product from CFU-GM/AE 9/7 (lane P) which serves as a positive control were mixed in a 1:1 ratio with the normal allele, because the PIG-A gene is on the X chromosome and the X chromosome is randomly inactived in females. The TF1 cell line (lane C) serves as a normal control. Lane U represents uncut DNA from the T-cell clone and lane T shows the consequence of the Ms1I digest of DNA from a representative T-cell clone. M indicates molecular size markers.
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References

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