Blvrb Mutation Induces Thrombocytosis Via Redox Dysregulation in the Heme Degradation Pathway

Known genetic loci influencing blood cell production account for <10% of platelet and red blood cell variability, and thrombopoietin (Tpo)/c-MPL liganding is dispensable for definitive thrombopoiesis establishing that fundamentally important modifier loci remain unelucidated.  We completed RNASeq of highly-purified platelets from 7 essential thrombocythemia (ET) subjects [4 harbored the JAK2^V617F mutation and 3 were genotypically normal] and 5 healthy controls, and developed an iterative algorithm to identify 33 non-synonymous SNVs that are putatively linked to the ET phenotype.  Transcripts possessing these candidate SNVs were enriched on average in early-stage megakaryocyte/erythroid progenitors (MEPs), and became more restricted in terminally-differentiating megakaryocytes and erythroblasts.   Genotypic studies using an expanded ET cohort (N = 36), followed by statistical association analyses using controls from the 1000 Human Genomes Project and an independently-genotyped cohort of healthy controls (N = 208), established that 5 SNVs (excluding JAK2^V617F) were associated with ET.  A single mutation (BLVRB^S111L; p = 0.0006) retained its significance as a thrombocytosis risk allele using genotypic data from a secondary cohort with reactive (non-clonal) thrombocytosis (RT, N = 53), suggesting a function as an independent driver mutation of enhanced thrombopoiesis. The BLVRB (biliverdin IXβ reductase) gene functions downstream of heme oxygenase(s)-1 (inducible HMOX1) and -2 (constitutive HMOX2) within the heme degradation pathway to catalyze reduction of biliverdin (BV) tetrapyrrole(s) as an intermediary redox substrate in Bilirubin (BR) generation. Bacterially-expressed and purified recombinant BLVRB^S111L showed defective enzymatic activity [compared to BLVRB^WT (wild-type)] using flavin mononucleotide [flavin reductase (FR) activity; p <0.0001] and BV IXβ dimethyl esters (biliverdin reductase (BVR) activity; p <0.0001), the latter specifically generated by coupled heme oxidation as verdin-restricted BLVRB activity probes.  The loss-of-mutation BLVRB^S111L NAD(P)H-dependent redox coupling caused higher baseline ROS (reactive oxygen species) accumulation in lentivirus-transduced CD34⁺-derived induced pluripotent stem cells (iPSC/BLVRB^S111L), results sharply contrasting with ROS neutralization in iPSC/BLVRB^WT exposed to tert-butyl hydroperoxide (TBHP) as the oxidant stress source (p < 0.00001).  Disparate redox coupling/ROS handling in genetically-modified primary CD34⁺ multipotential progenitor cultures established disproportionate expansion of primitive CFU-GEMMs in CD34⁺/BLVRB^S111L (p = 0.001), and an absolute increase of BFU-E in CD34+/BLVRB^WT (p = 0.001).  Collagen-based progenitor cultures demonstrated a statistically-significant increase of CD41⁺ CFU-MKs in CD34⁺/BLVRB^S111L cells (p <0.01) with no increased CFU-MKs in CD34⁺/BLVRB^WT cells. Cumulative distribution plots of parallel Tpo-suspension cultures confirmed divergent ROS accumulation between MK/BLVRB^WT and MK/BLVRB^S111L, differences that were identifiable pre-terminal differentiation (Day 0, p = 0.0007), most pronounced at Day 5 corresponding to peak MK BLVRB expression across genotypes (p = 8 x 10^-7), and persistent at terminal differentiation (Day 10; p = 0.05).  Maximally disparate ROS accumulation (Day 5) corresponded to greatest size disparity and temporally-earlier (and sustained at Day 10) CD41 expression in MK/BLVRB^S111L (p = 0.03).  A bilineage (Tpo/Epo) differentiation model, designed to characterize erythroid/megakaryocyte (E/Meg) progenitor balance arising from common MEPs demonstrated no evidence for differential Mk (CD41⁺/Glycophorin A^-) lineage balance, suggesting that BLVRB^S111L ROS-promoting effects accelerate post-commitment expansion downstream of MEP lineage fate decisions.  These data provide the first evidence linking redox coupling to MK lineage fate and expansion in humans, either via activity as a flavin reductase, in partnered electron exchange with an unidentified protein, or as a verdin-regulated redox coupler regulated by heme degradation and isomer-restricted BV IXβ (or IXδ, IXγ) generation; in principle, development of BLVRB-specific redox inhibitors represent innovative approaches to selectively alter a regulatory pathway controlling MK lineage expansion and human platelet counts.