Emergence of clonal hematopoiesis in the majority of patients with acquired aplastic anemia

Abstract

Acquired aplastic anemia (aAA) is a nonmalignant disease caused by autoimmune destruction of early hematopoietic cells. Clonal hematopoiesis is a late complication, seen in 20-25% of older patients. We hypothesized that clonal hematopoiesis in aAA is a more general phenomenon, which can arise early in disease, even in younger patients. To evaluate clonal hematopoiesis in aAA, we used comparative whole exome sequencing of paired bone marrow and skin samples in 22 patients. We found somatic mutations in 16 patients (72.7%) with a median disease duration of 1 year; of these, 12 (66.7%) were patients with pediatric-onset aAA. Fifty-eight mutations in 51 unique genes were found primarily in pathways of immunity and transcriptional regulation. Most frequently mutated was PIGA, with seven mutations. Only two mutations were in genes recurrently mutated in myelodysplastic syndrome. Two patients had oligoclonal loss of the HLA alleles, linking immune escape to clone emergence. Two patients had activating mutations in key signaling pathways (STAT5B (p.N642H) and CAMK2G (p.T306M)). Our results suggest that clonal hematopoiesis in aAA is common, with two mechanisms emerging-immune escape and increased proliferation. Our findings expand conceptual understanding of this nonneoplastic blood disorder. Future prospective studies of clonal hematopoiesis in aAA will be critical for understanding outcomes and for designing personalized treatment strategies.

Keywords: Clonal hematopoiesis; MDS; aplastic anemia; bone marrow failure; myelodysplastic syndrome.

Figure 1. Clinical Characteristics Associated with Presence or Absence of Clonal Hematopoiesis in aAA

A. The distribution of the total number of identified somatic mutations per patient as it relates to the patients’ age and disease duration. For each patient, listed on the X-axis, the age is plotted as a vertical line, with the beginning of the line corresponding to age at diagnosis and an arrowhead depicting age at WES. The number above the line corresponds to the total number of somatic mutations. B. Association analysis of clinical characteristics with presence or absence of clonal hematopoiesis in aAA.

Figure 2. Acquired mutations in the bone marrow of aAA patients

A. IGV screenshots of WES of bone marrow(BM) and constitutional DNA (skin biopsy, SB) showing a g.40359729T>G (p.N642H) mutation in STAT5B gene present in the bone marrow but not in the paired constitutional DNA. B. Sanger sequencing chromatograph showing that the acquired g.40359729T>G (p.N642H) mutation is detected in the immunomagnetically sorted myeloid cell fraction of peripheral blood, PB (M), but is absent from the lymphocyte fraction, PB (L), and skin fibroblast DNA (SB); the chromatograph shows reverse complement sequence. C. IGV screenshots of WES of bone marrow (BM) and constitutional DNA (skin biopsy, SB) showing a g.75601956G>A (p.T306M) mutation in CAMK2G present in the bone marrow but not in the paired constitutional DNA. D. Sanger sequencing chromatograph validating that the acquired g.75601956G>A (p.T306M) mutation in CAMK2G is detected in the bone marrow (BM), but is absent in the skin fibroblast DNA (SB). E. A schematic of the STAT5B protein illustrating the location of previously reported activating mutations in the SH2 domain. F. A schematic of the CAMK2G protein showing the locations of regulatory phosphorylation sites.

Figure 3. Oligoclonal Loss of HLA class I alleles in aAA

A. SNP-A genotyping of patient 281.01 depicted as two scatter plots. The top plot shows B-allele Frequency (BAF, a relative frequency of the minor allele); the bottom plot shows Log R Ratio (LRR, a measure of normalized total signal intensity for both alleles). The chromosomal location is depicted on the X-axis. In a region with acquired CN-LOH, the copy number (indicated by the LRR) remains constant, while there is a decreased frequency of the heterozygous alleles (indicated by the change in the left part of the BAF plot). The location of the HLA-A gene is shown by the arrowhead. B) IGV screenshots of WES of bone marrow (BM) and constitutional DNA (skin biopsy, SB) showing the HLA-A g.29911127C>G (p.Tyr142X) mutation in the bone marrow but not in the paired constitutional DNA. C. Next generation sequencing of the HLA locus, showing the location of the nonsense HLA-A g.29911127C>G (p.Tyr142*) mutation (arrow) occurring in cis to the HLA-A*33:03:01 allele. D. SNP-A genotyping of patient 54.01. The top plot shows B-allele Frequency (BAF, a relative frequency of the minor allele); the bottom plot shows Log R Ratio (LRR, a measure of normalized total signal intensity for both alleles). In a region with acquired CN-LOH, the copy number (indicated by the LRR) remains constant, while there is a decreased frequency of the heterozygous alleles (indicated by the change in the left part of the BAF plot). The location of the HLA-B gene is shown by the arrowhead. E. IGV screenshots of WES of bone marrow (BM) and constitutional DNA (skin biopsy, SB) showing the frameshift HLA-A g.31323108delA (p.Leu294fs) mutation in the bone marrow but not in the paired constitutional DNA. F. Next generation sequencing of the HLA locus, showing the location of the HLA-B g.31323108delA (p.Leu294fs) mutation (arrow) occurring in cis to the HLA-B*14:02:01 allele. G. A model schematic depicting recurrent loss of HLA class I alleles in patients with aAA. Cytotoxic T lymphocytes (CTL) cause autoimmune depletion of hematopoietic stem and progenitor cells (HSPC) due auto-antigen presentation in the context of particular HLA class I alleles. Thus, cells with loss of particular HLA class I alleles either via an inactivating mutation, or by loss of heterozygosity favoring the opposite allele, lead to a growth advantage and resultant clonal expansion of the mutant hematopoietic cells. H. Table showing HLA typing results of the peripheral blood for patients 281.01 and 54.01.