Clonal Hematopoiesis and Genetic Mutations in Individual Patients with Acquired Bone Marrow Failure Diseases

Clonal hematopoiesis (CH) is characterized by clonal expansion of hematopoietic stem/progenitor cells with the capability of multilineage differentiation and can be detected through the biomarker of genetic mutations. Bone marrow failure (BMF) diseases including idiopathic cytopenia of undetermined significance (ICUS), aplastic anemia (AA), paroxysmal nocturnal hemoglobinuria (PNH) and myelodysplasia syndrome (MDS) are reported to partially coexist with CH. However, till now, the CH and genetic mutations in acquired BMF diseases are still not very clear. Therefore, we designed a panel that contains more than 500 candidate genes to perform a targeted sequencing of bone marrow cells isolated from 48 BMF patients, taking their mucosal cells as controls. In total, 75 somatic mutations and two germline mutations (PTCH1 c.1177G>A, CHEK2 c.1240C>T) were profiled in 30 patients, including 5 ICUS (also termed CCUS (clonal cytopenia of undetermined significance)), 14 AA, 3 PNH and 7 MDS patients. 67.53% missense mutations, 10.39% nonsense mutations, 10.39% frameshift mutations, 1.30% splicing mutations, 7.79% cds-del mutations, 2.60% CNV (copy number variations) can be found in our sequenced mutations. In comparison, the genetic mutation burden is relatively lower in AA and ICUS but higher in PNH and MDS, suggesting that the extent of CH possibly correlated with disease progression. Genetic mutations in our assay also implicated insightful views in the pathogenesis of BMF diseases. We can functionally divide genetic mutations into several types such as signaling transduction (21.6%), transcriptional regulation (19.6%), epigenetic regulation (15.5%), cell cycle regulation (7.2%), DNA repair (3.1%), cohesion complex (2.1%), immunity (3.1%), RNA splicing (4.1%) and others (3.1%). Specifically, we found a serial of novel genetic mutations. For example, the mutations in extracellular membrane receptors such as Notch (Notch1 c.4759A>C , Notch2 c.4819C>T, Notch3 c.3592A>C), CXCR4(c.598C>T), IGF1(c.341C>T), MPL(c.611C>T) suggest bone marrow environment is possibly associated with BMF diseases. In detail, Notch, IGF1, CXCR4 and MPL-mediated signaling are critical for HSC niche regulation, suggesting niche signals serve as a promoting factor for acquired BMF pathogenesis, which is consistent with previous reports. Also, we found the immunity-related mutations in HLA-A(c.750_751delGG) and CD58(c.475T>G), which are closely related with specific T cell recognition, implicated th