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 that clonal hematopoiesis and evolution to malignancies can be partially attributed to immune evasion possibly. The mutation ERBB4 (c.3424C>T), a membrane receptor which binds to and is activated by nerve cell released neuregulins, implicated that nerve fiber might involve in BMF. Additionally, we also found novel mutations in epigenetic factors such as KDM5C (c.265delA) and CHD3 (cds-del c.222GCC [7>6]) in PNH and MDS patients. Interestingly, LRP1B (c.13771G>A) that plays an important role in lipid metabolism was found in PNH patient firstly. These gene mutations provide clues for potential biomarkers for BMF diagnosis and prognosis and need more functional investigations.

To systemically observe the clonal hematopoiesis at individual patient-level, we delineated the mutational composition in 30 individual BMF patients. We found that 56.7% patients have more than two mutations (eg. MDS 85.71%, PNH 100%, CCUS 60% and AA 35.7%), suggesting that multiple gene mutations-driven clonal expansion constitutes a complex mutational composition and their interaction possibly influences disease progression. At individual patient-level, mutational composition better promotes our understanding on BMF pathogenesis and clinical diagnosis. For example, an MDS patient bearing several mutations in ASXL (non-sense mutation, c.3187C>T, VAF: 18.2%), DNMT3A (missense mutation, c.2644C>T, VAF: 3.01%) and RUNX1 (missense, c.602G>A, VAF: 2.08%) suggested a complex pathological process and poor prognosis. Therefore, mutational composition structure can be revealed in individual acquired BMF patients, promoting precise diagnosis and prognosis.

In conclusion, our study not only found a serial of novel genetic mutations that need further investigations, but also provided potential clues for the identification of biomarkers in acquired BMF diseases.

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