Proaerolysin Multi Color Flow-Fish Reveals Shortened Telomere Length in GPI− as Compared to GPI+ Granulocytes from Patients with Paroxysmal Nocturnal Hemoglobinuria (PNH).

Objective: Replication-dependent telomere shortening can result in cellular senescence or genetic instability. Telomere length both reflects and limits the replicative potential of normal somatic cells. Shortened telomeres have been linked to disease stage, degree of cytopenia as well as to response to treatment in patients with bone marrow failure syndromes. Paroxysmal nocturnal haemoglobinuria (PNH) is caused by a somatic mutation in the X-linked PIG-A gene resulting in a deficiency of GPI-linked proteins on the cell surface. Pancytopenia in PNH is thought to result from an auto-immune mediated depletion of the GPI+ stem cell compartment. In the current study, we aimed to develop a methodology that allows the selective analysis of telomere length in granulocytes from patients with PNH dependent on GPI expression. The aim was to find out whether replicative stress posed on the residual GPI− HSC is reflected in shortened telomere length compared to their GPI+ counterparts.

Methods: The telomere length of GPI+ and GPI− granulocytes of 12 patients with PNH and 22 healthy individuals was analysed. For this purpose, we developed Proearolysine Multi-color flow FISH which is based on the crosslinking of a fluorescence-labeled non-toxic derivative of the bacterial toxine Aerolysine (that selectively binds to the GPI anchor of cells) in combination with fluorescence in situ hybridization and flow cytometry.

Results: We found significantly (p<0.05) shortened telomeres in GPI− granulocytes (6.68±0.3 telomere fluorescence unit (TFU), n=12) compared to age-matched healthy individuals (mean±S.E.: 7.73±0.8 TFU, n=22), but no significant shortening in GPI+ (7.06±0.3 TFU, n=12) granulocytes from PNH patients. Telomere length in GPI− granulocytes was found to be significantly shorter as compared to their GPI+ counterparts (deltaTEL: 0.38±0.1, p=0.002). These results were confirmed by Multi-color flow FISH using an anti-CD59 antibodies.

Conclusion: Autoimmune-mediated damage to the GPI+ HSC compartment leads to compensatory hyperproliferation of predominantly residual GPI− HSC. In line with this hypothesis, coexisting GPI-negative HSC clones showed even more accelerated telomere shortening as compared to their GPI+ counterparts. Potentially, replicative exhaustion of individual HSC clones could contribute to disease progression in PNH. Whether acquired genetic instability due to progressive telomere shortening also accounts for an increased incidence of secondary clonal disorders will need to be evaluated in future prospective studies.