A Single Mutation in the SENP1 Region Regulates the Excessive Erythropoiesis in the Andes

Background: Approximately 20% of individuals living in Peruvian mountains suffer from debilitating Monge’s disease or Chronic Mountain Sickness (CMS). 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 often leads 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). We have shown a critical role of SENP1 (a gene we obtained from differential SNP analysis throughout whole genomes) in regulating EE response in the highlanders. In order to understand the mechanism(s)that upregulates SENP1 levels in CMS, we applied bioinformatics approaches and CRISPR/Cas9-mediated genomic editing to decipher causal SNPs in the SENP1 region.

Methods: We performed Whole Genome Analysis and applied bioinformatics methods iSAFE (integrated Selection of Allele Favored by Evolution) and CADD (Combined Annotation Dependent Depletion) Score to identify causal SNPs in the selected region of SENP1 gene that could lead to the pathology. We further utilized an in-vitro human iPS-derived model system and CRISPR approach to switch alleles and study their functional role in regulating the EE response.

Results: By applying iSAFE and CADD score we were able to shortlist 7 causal SNPs: rs10783232, rs7959755, rs17122612, rs72644843, rs11609399, rs11613781 and rs60629297. Among them, rs7959755 and rs10783232 were the top ranked (Rank 1 and 2) SNPs. We successfully generated and validated A to G switch in the rs10783232 in the non-CMS cells using CRISPR. We further tested the impact of this switch on function of the allele and observed a significant increase in SENP1 mRNA levels (p<0.05) as well doubling of BFU-e colonies (p<0.05) in the non-CMS cells as compared to the control non-CMS cells. We also tested the effect of allele switch (G to A) for the lower ranked (Rank 44) SNP rs11613781 and did not observe a significant effect on SENP1 levels or BFU colony formation.

Conclusion: These results demonstrate that rs10783232 plays a critical role in regulating SENP1 mRNA levels under hypoxia as well as the EE in the Andean highlanders.