Changes of Clonality of Paroxysmal Nocturnal Hemoglobinuria (PNH) Clones during Clinical Courses in Patients with PNH.
Authors: Noji Hideyoshi , Kai Tatsuyuki , Okamoto Masatoshi , Ikeda Kazuhiko , Akutsu Kazuko , Nakamura-Shichishima Akiko , Shichishima Tsutomu 
Published: April 2020

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hematological disorder, in which almost all hematopoietic cells lack glycosylphosphatidylinositol (GPI)-anchored proteins due to phosphatidylinositol glycan-class A (PIG-A) gene abnormalities at the level of hematopoietic stem cells. It has been reported that several minor PNH clones in patients with aplastic anemia and myelodysplastic syndromes occur at random during the clinical course (Okamoto M et al, Leukemia, 2006), while one or more major PNH clones in patients with typical PNH continue to exist during their clinical courses (Nishimura J et al, Blood, 2002). To clarify how PNH clones in patients with PNH expand during their clinical courses, we examined sequence analyses of the PIG-A gene using peripheral blood granulocytes from PNH patients with high (&gt;10%; n=5) and low (&lt;10%; n=3) proportions of CD16− or CD59− granulocytes, which were determined by flow cytometry using CD16b or CD59 monoclonal antibody, respectively. A patient with over 1% of CD59− erythrocytes was judged to have PNH erythrocytes (Shichishima T et al, Br J Haematol, 1993). PNH clones were defined as GPI-negative hematopoietic cells with PIG-A mutations. Twenty to 40 bacterial clones of PCR or RT-PCR products of the PIG-A gene with GPI-negative or unsorted populations of peripheral blood granulocytes were analyzed by nucleotide sequencing. We examined sequence analyses of the PIG-A gene in peripheral blood granulocytes at least twice during the clinical course (the period of 1995–2007). Major PNH clones were decided by the Chi2 test or the Fisher’s exact probability test. In all of 5 PNH patients with high proportions of PNH clones, major PNH clones were found at least once and were common during their clinical courses. However, the PNH clones in 3 of 5 PNH patients were transiently major during their clinical courses. In 2 of 3 PNH patients with low proportions of PNH clones, there were common PIG-A mutations during their clinical courses, but were not major clones. The PNH clone from one of the 2 PNH patients acquired a new PIG-A mutation, which was also minor. Another patient of the 3 patients with low proportions of PNH clones had a major PNH clone at the time of the first examination, but disappeared at the time of the next analysis. In conclusion, our findings suggest that the clonality of PNH clones in PNH patients with high and low proportions of PNH clones could change during their clinical courses and that especially, the clonality of PNH clones in PNH patients with low proportions of PNH clones is not necessarily definitive.

PIG-A mutations in PNH patients with high and low proportions of PNH clones Case No. Common mutation Uncommon mutation Abbreviations: del , deletion; ND, not dectected; and ins, insertion PNH patients with high proportions of PNH clones Case 1 214 C to T, 5′ splicing site del G (Major) ND Case 2 698 G to A (Major), 1152 del C ND Case 3 548 G to A (Major) 982 G to T Case 4 ND 491 C to T Case 5 ND 407 A to G PNH patients with low proportions of PNH clones Case 5 ND 148 del G, 142 G to A (Major), 460 C to T Case 6 ND 11 G to C, 91 ins C, 916 del C Case 6 ND 67 del G, 72 del 13bp, 72 del 13bp/188 G to A, 358 G to A, 365 A to C, 952 del A