case_images: PMC10822943_01
case_images: PNH clones before and after syngeneic BMT. (A) GPI (-) granulocytes and erythrocytes 6 months before (Upper left) and 37 months after BMT (Upper right). Deep-targeted sequencing of GPI (-) granulocytes sorted from the same blood samples revealed three different identical PIGA mutations (Lower). (B) Changes in the LDH level and the percentage of GPI (-) cells before and after syngeneic BMT. BMT, bone marrow transplantation; Chr, chromosome; GPI, glycosylphosphatidylinositol; LDH, lactate dehydrogenase, PNH, paroxysmal nocturnal hemoglobinuria; VAF, variant allele frequency.
case_images: fimmu-14-1329403-g001.jpg
case_images: PMC10822943_01_fimmu-14-1329403-g001.jpg
case_images: f1
case_images: Flow cytometry analysis of peripheral blood showed that 71.9% and 15.3% of the granulocytes and erythrocytes were GPI (-) ( Figure 1A ). Deep-targeted sequencing covering 390 different genes of GPI (-) granulocytes revealed three different PIGA mutations (p.I69fs, variant allele frequency (VAF) 24.2%; p.T192P, VAF 5.8%; p.V300fs, VAF 5.1%;  Figure 1A ) and no other mutations ( Table 2 ). However, the percentages of GPI (-) granulocytes and erythrocytes remained almost the same thereafter ( Figure 1B ). The percentages of GPI (-) granulocytes and erythrocytes were 0.04% and 0.05%, respectively ( Figure 1B ). Deep sequencing of the sorted GPI (-) granulocytes using amplified PIGA exons revealed the same three PIGA mutations at proportions similar to those before transplantation (p.I69fs, VAF 36.1%; p.T192P, VAF 3.7%; p.V300fs, VAF 8.6%;  Figure 1A ) in the small PNH clones that persisted after transplantation.
case_images: Copy number alteration of sorted granulocytes. Copy number alteration analysis of flowcytometric-sorted granulocytes from peripheral blood six months before bone marrow transplantation. Red arrows indicate the deletion of chromosome 13q in the wild-type granulocytes (upper), while it is not evident in the GPI (-) granulocytes (lower). CN, copy number; PNH, paroxysmal nocturnal hemoglobinuria; SNP, single nucleotide polymorphism.
case_images: fimmu-14-1329403-g002.jpg
case_images: PMC10822943_01_fimmu-14-1329403-g002.jpg
case_images: f2
case_images: Copy number alteration analysis of flow cytometric-sorted granulocytes revealed del(13q) in wild-type granulocytes but not in GPI (-) granulocytes ( Figure 2 ).