Congenital Erythrocytosis Due to Novel CMTR1^K769L Mutation Has Augmented Hypoxia Signaling and Features of Both Primary and Secondary Erythrocytosis

Primary erythrocytoses (PE) include germline gain of function mutations of erythropoietin (EPO) receptor (EPOR) gene and polycythemia vera (PV), a clonal disease due to acquired somatic JAK2 mutations. Both these PEs have intrinsically abnormal early erythroid progenitors (BFU-E) characterized by their in vitro EPO hypersensitive growth (EPORmutations), or even EPO independent growth (the hallmark of PV); both have low plasma EPO (Chapter 58, Williams Hematology, 10e). Secondary erythrocytoses (SE) have intrinsically normal BFU-Es but are caused by increased circulating erythropoiesis stimulators, chiefly EPO. In contrast, congenital Chuvash erythrocytosis is characterized by augmentation of hypoxia inducible factors (HIFs) causing high EPO and hypersensitive BFU-E's EPO responses; thus, it has features of both PE and SE.

We report a 7-year-old normoxic girl with history of symptomatic erythrocytosis since birth of unknown etiology, with hemoglobin of 22 g/dL at birth, consistently elevated EPO at 50-75 mU/mL, and normal hemoglobin oxygen affinity. Her BFU-E grew without EPO yet no somatic PV defining mutations were found. A targeted analysis of previously described erythrocytoses genes was negative. Whole genome sequencing, performed under the auspices of Undiagnosed Disease Network, identified a heterozygous missense variant (p. Lys769Leu) of CMTR1(Cap Methyltransferase 1) gene, not listed in the Genome Aggregation Database (gnomAD). Cap methylation on mRNA protects mRNA from being degraded and promotes RNA translation by recruiting translation initiation factors (PMID:30312682). The variant was present in the proband but not in her asymptomatic mother (father was not available for the analysis). This variant was found in leukocytes and in nail DNA, indicating germline origin. Transcriptional X-chromosome inactivation assay (PMID:18641369) revealed polyclonal granulocytes and platelets, confirming that the erythrocytosis is caused by germline mutation, not by somatic mutation-driven clonal hematopoiesis.

Because of her high EPO, we hypothesized that CMTR1^K769L variant might cause high HIF activity. HIF-mediated gene regulation is tissue- and stage of differentiation-specific. Expression of HIF target genes SLC29A1, VEGFA, EDN1, and LDHA was upregulated in granulocytes while in platelets all target genes except SLC29A1 were upregulated. To test if CMTR1 regulates HIF transcriptional activity, 1) we correlated CMTR1 transcript levels with HIF target gene expression levels in granulocytes and platelets. CMTR1 transcript levels were positively correlated with expression levels of HIF target genes. 2) we tested if CMTR1 overexpression and knockdown alters HIF target gene expression. In erythroid cell lines (K562 and HEL), overexpressed CMTR1 increased SLC2A1 and VEGFA (HIF target genes transcripts) while knockdown of CMTR1 downregulated their expression. This demonstrates that CMTR1 positively regulates HIF transcriptional activity, suggesting that CMTR1^K769L accounts for increased plasma EPO and for EPO mediated feature of SE.

As erythroid progenitors express endogenous erythropoietin (PMID 9573013) which contributes to their differentiation and proliferation (PMID 10903342), we evaluated if CMTR1^K769L also promotes endogenous erythroid EPO accounting for EPO independent growth of CMTR1^K769L mutated BFU-Es and thus provides potential mechanism as a feature of PEfor the erythrocytosis. The proband's endogenous EPO transcript levels in in vitro expanded erythroid cells at day 4, when most of cells are erythroid progenitors, were 10 times higher than the mother's see Figure.

We present a new cause of erythrocytosis associated with the novel germline mutation of principal epigenetic modifier of RNA, CMTR1 (PMID:33922187), associated with high EPO (SE). Her BFU-E grew without extrinsic EPO (PE) likely due to previously unobserved stimulation of EPO production in erythroid progenitors.

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