Differential Comparative Genomic Hybridization Analysis of Normal and Glycosyl Phosphatidyl Inositol Deficient Clones in Paroxysmal Noctorunal Hemoglobinuria.

Various theories can explain the expansion of glycosyl phosphatidyl inositol anchored protein (GPI-AP)-deficient clones in PNH. The differential growth advantage of PIG-A mutant stem cell scould be due to immune escape in the context of an autoimmune attack on hematopoietic targets explaining the close clinical relationship to aplastic anemia. The causes of evolution of the PNH clone may be related to a less efficient immune recognition of PNH cells, their relative resistance to apoptosis or slower senescense. These changes may be related to additional mutations or genetic damage present in either normal or GPI-AP deficient hematopoietic clone. Using traditional karyotyping, chromosomal defects are not common in PNH but the limited resolution of this method may preclude detection of smaller aberrations. Due to these limitations, novel technologies which improve resolution and sensitivity are under development. In array-based comparative genomic hybridization (A-CGH), differentially labeled test and reference DNA samples are hybridized to genomic microarrays. Differences in sequence copy number between the samples are reflected in a shift of the fluorescence spectrum. The principle of A-CGH allows for detection of unbalanced chromosomal changes of the whole genome and its resolution is limited solely by the number of clones. In preliminary studies we found significantly decreased telomerase activity in normal vs. CD59−CD55− CD34+ cells derived from patients with PNH suggesting: 1) more accelerated telomere shortening in phenotypically normal hematopoietic cells derived from PNH patients, or 2) the possibility of chromosomal instability and acquisition of karyotypic defects should a critical telomere length be reached.

We hypothesized that higher resolution analysis of the karyotypic changes in PNH using CGH may result in detection of cryptic karyotypic abnormalities present in either the normal or GPI-AP deficient clone. We have analyzed a cohort of patients (N=6) with hemolytic PNH and major expansion of a GPI-AP deficient clone (50–95% of PNH granulocytes). All patients had normal cytogenetics by metaphase karyotyping. We have separated GPI-AP-deficient myeloid peripheral blood cells using CD55 and CD59 as well as CD2 and CD19 staining to exclude lymphocytes. Separated CD55−CD59− and CD55+CD59+ non lymphoid cells were sorted and, following DNA extraction, subjected to A-CGH analysis (Vysis microarrays containing 287 probes). In one patient, PNH cells appeared normal by A-CGH but the corresponding “normal” cells contained deletions of telomeric region of several chromosomes (2, 5, 16, 18, 19, 20qtel as well as 5, 11,19ptel). However, in the remaining patients telomeric deletions were present in both normal and PNH cells. There was no difference in the numbers of chromosomes affected and diverse chromosomal telomeric deletions were present. No other lesions were found. By comparison when A-CGH analysis was performed in 8 normal individuals telomeric deletions were only rarely found and only occasional gains of contiguous clones on chromosomes were detected.

Deletion of the telomeric portions of multiple chromosomes is compatible with accelerated global telomere loss in PNH. However, it appears that this phenomenon was not restricted to either normal or PNH cells. Our results are in agreement with previously described telomere shortening measured by FISH, flow cytometry or Southern blot analysis in PNH, a change not consistently restricted to normal cells.