Polyclonal somatic gene rescue via uniparental disomy confers multilineage hematopoietic potential in treatment-independent patients with DBA syndrome

Abstract Diamond-Blackfan anemia syndrome (DBAS) is an inherited bone marrow failure disorder typically caused by heterozygous mutations in ribosomal protein (RP) genes leading to erythropoietic failure. Some patients with DBAS experience spontaneous hematological improvement even in the setting of long-lasting dependency on steroids or transfusions to support erythropoiesis. We and others previously described single cases of hematopoietic recovery from anemia through uniparental disomy (UPD). Such UPD events result in the loss of the mutated allele and duplication of the wildtype allele. The frequency, clonal architecture, and lineage-specific effects of these somatic gene rescue (SGR) events remain largely unexplored. To address this, we developed a systematic approach by evaluating DBAS cohorts using bulk genomic methods (whole exome and targeted deep-sequencing, SNP array) to identify patients with potential SGR events. We also established a single-cell DNA and protein sequencing (scDNAseq) to characterize the UPD clonal architecture at cellular level. Our scDNAseq panel (Mission Bio) was designed to identify RP mutations and detect UPDs by targeting common single nucleotide variants across all ethnic backgrounds. For cell type determination, we assessed expression of 42 hematopoietic antigens using TotalSeq-D panel (BioLegend). We analyzed cohorts from St. Jude Children's Research Hospital (SJCRH) and collaborating institutions (cohort A: 37 patients) alongside the Japanese DBA registry (cohort B: 284 patients). Within cohort A, we found 5 cases with UPDs using upfront SNP array. In cohort B, we first assessed the variant allele frequency (VAF) of RP mutations by NGS and/or RP-gene copy by droplet digital PCR. Of 13 cases fulfilling screening criteria (reduced VAF or abnormal gene copy numbers in RP genes), 4 carried various UPDs encompassing the locus of causative RP gene. Focused analysis of UPD regions revealed that in 8/9 cases, multiple UPDs breakpoints were present with gradual origin centromeric to the RP gene, suggesting multiple independent rescue events. Presence of UPDs was associated with high rates of treatment-independence (7/9 cases had stable Hb). We next performed scDNA-seq on 8 treatment-independent patients, including 5 with known UPD and 3 without. These patients harbored mutations in RPS19 (n=4), RPS7 (n=2), RPL4 (n=1), and RPL15 (n=1). We genotyped 46,279 cells from bone marrow (BM) and peripheral blood (PB) samples with median=4,626 cells/sample (range=2,163–9,554 cells). In the 3 patients without UPD, all cells (100%) carried the germline RP gene mutation, while in the 5 cases with UPD, the proportion of cells retaining the mutation was expectedly reduced (median 34.7%, range=6.2–54.8%), reflecting selective expansion of wild-type cells. In the majority of UPD cases (4/5), we identified multiple UPD clones per patient (median=3), which were indistinguishable by prior bulk SNP array analysis. The presence of multiple UPD clones implies ongoing selective pressure favoring cells with corrected (wild-type) copy of the RP gene. Antigen profiling revealed that UPD clones showed distinct expression patterns compared to cells with RP mutations. In BM samples, some UPD clones showed enriched expression of the erythroid progenitor marker CD71, while maintaining broad multilineage potential. In peripheral blood samples, UPD clones exhibited variable expression of markers associated with both myeloid and erythroid lineages. This suggest that UPD clones may possess the capacity to contribute to multiple hematopoietic lineages, supporting multi-lineage hematopoietic recovery in setting of bone marrow failure in DBAS. Finally, in treatment-independent cases without UPD, no other potential reversion mutations in RP genes were identified, pointing to other yet unknown rescue mechanisms from ribosomal dysfunction. These findings demonstrate that that UPD rescue events are a recurrent mechanism of therapy-independence in DBAS patients. The polyclonal nature of this rescue mechanism (with multiple UPD clones expanding simultaneously across all hematopoietic lineages), provides robust correction of the underlying molecular defect.