Stage-Specific Erythropoietic Regulation By Long Non-Coding RNAs (lncRNAs) in Patients with Monge's Disease

Abstract

Background: Approximately 20% of individuals living in Andean mountains suffer from Chronic Mountain Sickness, CMS, or Monge's disease. They manifest the disease in adulthood and often die because of excessive erythropoiesis (EE) (Hematocrit of >65%, averaging 72%, mostly occurring in males), an extreme phenotype that can often lead to myocardial infarction or stroke. The uniqueness of this Andean population is even more significant when we realize that there are individuals who live side by side at the same altitude as those with CMS but do not suffer from the same extreme phenotype (non-CMS). Methods: To understand the mechanistic basis of EE in these patients, we performed RNA seq of cells from both CMS and non-CMS populations. We further utilized an in-vitro human iPS-derived model system to study the functional role of genes at various erythroid stages. Results: Interestingly, we discovered a unique non-overlapping profile of differentially expressed long non-coding RNA (lncRNAs) between CMS and non-CMS cells. Among these lncRNAs, LINC00431 was significantly downregulated in non-CMS cells as compared to CMS in hypoxia (mimicking high altitude). We further validated the expression of LINC00431 and found it to be significantly downregulated under hypoxia in the non-CMS cells as determined by the qPCR (P<0.01). We hypothesized that this lncRNA plays a role in regulating EE response at high altitude. Knock-down of LINC00431in the CMS cells did not affect the early stages of erythroid proliferation (such BFU-E) but there was a large effect in the later erythroid stages such as the CFU-E and reticulocyte stages (CD71+, CD235a+ cells). Particularly, KD of LINC00431 resulted in a major reduction (from ~60% to ~7%, p<0.0001) of CD235a+ erythroid cells (Reticulocyte stage-a later erythroid stage). In contrast, we have also shown that the knock-down of LINC02228 has a large impact at the early erythropoietic stage BFU-E of the CMS cell reducing by 50% when we KD LINC00228. Conclusion: These results show a stage-specific erythropoietic regulation by lncRNAs in the CMS and non-CMS patients. LINC02228 plays a critical role at the BFU stage and LINC00431 plays an important role particularly at later erythroid stages (CD71+, CD235+ Reticulocyte stage). This stage specificity also suggests distinct mechanisms for each lncRNAs and their downstream targets.