Felipe Lorenzo

Genetic Evidence for High-Altitude Adaptation in Tibet

No Genetic Vertigo Peoples living in high altitudes have adapted to their situation (see the Perspective by Storz). To identify gene regions that might have contributed to high-altitude adaptation in Tibetans, Simonson et al. (p. 72, published online 13 May) conducted a genome scan of nucleotide polymorphism comparing Tibetans, Han Chinese, and Japanese, while Yi et al. (p. 75) performed comparable analyses on the coding regions of all genes—their exomes. Both studies converged on a gene, endothelial Per-Arnt-Sim domain protein 1 (also known as hypoxia-inducible factor 2α), which has been linked to the regulation of red blood cell production. Other genes identified that were potentially under selection included adult and fetal hemoglobin and two functional candidate loci that were correlated with low hemoglobin concentration in Tibetans. Future detailed functional studies will now be required to examine the mechanistic underpinnings of physiological adaptation to high altitudes. A candidate gene approach reveals genes under selection in humans living at high altitudes. Tibetans have lived at very high altitudes for thousands of years, and they have a distinctive suite of physiological traits that enable them to tolerate environmental hypoxia. These phenotypes are clearly the result of adaptation to this environment, but their genetic basis remains unknown. We report genome-wide scans that reveal positive selection in several regions that contain genes whose products are likely involved in high-altitude adaptation. Positively selected haplotypes of EGLN1 and PPARA were significantly associated with the decreased hemoglobin phenotype that is unique to this highland population. Identification of these genes provides support for previously hypothesized mechanisms of high-altitude adaptation and illuminates the complexity of hypoxia-response pathways in humans.

Somatic HIF2A Gain-of-Function Mutations in Paraganglioma with Polycythemia

Hypoxia-inducible factors are transcription factors controlling energy, iron metabolism, erythropoiesis, and development. When these proteins are dysregulated, they contribute to tumorigenesis and cancer progression. However, mutations in genes encoding α subunits of hypoxia-inducible factors (HIF-α) have not previously been identified in any cancer. Here we report two novel somatic gain-of-function mutations in the gene encoding hypoxia-inducible factor 2α (HIF2A) in two patients, one presenting with paraganglioma and the other with paraganglioma and somatostatinoma, both of whom had polycythemia. The two mutations were associated with increased HIF-2α activity and increased protein half-life.

A genetic mechanism for Tibetan high-altitude adaptation

Tibetans do not exhibit increased hemoglobin concentration at high altitude. We describe a high-frequency missense mutation in the EGLN1 gene, which encodes prolyl hydroxylase 2 (PHD2), that contributes to this adaptive response. We show that a variant in EGLN1, c.[12C>G; 380G>C], contributes functionally to the Tibetan high-altitude phenotype. PHD2 triggers the degradation of hypoxia-inducible factors (HIFs), which mediate many physiological responses to hypoxia, including erythropoiesis. The PHD2 p.[Asp4Glu; Cys127Ser] variant exhibits a lower Km value for oxygen, suggesting that it promotes increased HIF degradation under hypoxic conditions. Whereas hypoxia stimulates the proliferation of wild-type erythroid progenitors, the proliferation of progenitors with the c.[12C>G; 380G>C] mutation in EGLN1 is significantly impaired under hypoxic culture conditions. We show that the c.[12C>G; 380G>C] mutation originated ∼8,000 years ago on the same haplotype previously associated with adaptation to high altitude. The c.[12C>G; 380G>C] mutation abrogates hypoxia-induced and HIF-mediated augmentation of erythropoiesis, which provides a molecular mechanism for the observed protection of Tibetans from polycythemia at high altitude.

A novel EPAS1/HIF2A germline mutation in a congenital polycythemia with paraganglioma

Congenital polycythemias have diverse etiologies, including mutations in the hypoxia sensing pathway. These include HIF2A at exon 12, VHL gene (Chuvash polycythemia), and PHD2 mutations, which in one family was also associated with recurrent pheochromocytoma/paraganglioma (PHEO/PGL). Over the past two decades, we have studied seven unrelated patients with sporadic congenital polycythemia who subsequently developed PHEO/PGL with, until now, no discernible molecular basis. We now report a polycythemic patient with a novel germline HIF2AF374Y (exon 9) mutation, inherited from his mother, who developed PHEO/PGL. We show that this is a gain-of-function mutation and demonstrate no loss-of-heterozygosity or additional somatic mutation of HIF2A in the tumor, indicating HIF2AF374Y may be predisposing rather than causative of PHEO/PGL. This report, in view of two other concomitantly reported PHEO/PGL patients with somatic mutations of HIF2A and polycythemia, underscores the PHEO/PGL-promoting potential of mutations of HIF2A that alone are not sufficient for PHEO/PGL development.

Genetic adaptation to extreme hypoxia: study of high-altitude pulmonary edema in a three-generation Han Chinese family.

Organismal response to hypoxia is essential for critical regulation of erythropoiesis, other physiological functions, and survival. There is evidence of individual variation in response to hypoxia as some but not all of the affected individuals develop polycythemia, and or pulmonary and cerebral edema. A significant population difference in response to hypoxia exist as many highland Tibetan, Ethiopian, and Andean natives developed adaptive mechanisms to extreme hypoxia. A proportion of non-adapted individuals exposed to high altitude develop pulmonary edema (HAPE), pulmonary hypertension, cerebral edema, and extreme polycythemia. The isolation of causative gene(s) responsible for HAPE and other extreme hypoxia complications would provide a rational basis for specific targeted therapy of HAPE, allow its targeted prevention for at-risk populations, and clarify the pathophysiology of other hypoxic maladaptations. The only suggested genetic linkage among unrelated individuals with HAPE has been with endothelial nitric oxide synthase (eNOS) gene. Here we describe a family with multiple members affected with HAPE in three generations. Families with multiple affected members with HAPE have not been described. We first ruled out linkage of HAPE with the eNOS gene. We then performed an analysis of the whole genome using high-density SNP arrays (Affymetrix v5.0) and, assuming a single gene causation of HAPE, ruled out linkage with 34 other candidate genes. Only the HIF2A haplotype was shared by individuals who exhibit the HAPE phenotype, and work on its possible causative role in HAPE is in progress. The small size of our family does not provide sufficient power for a conclusive analysis of linkage. We hope that collaboration with other investigators with access to more HAPE patients will lead to the identification of gene(s) responsible for HAPE and possibly other maladaptive hypoxic complications.

Ocular manifestations of hypoxia-inducible factor-2α paraganglioma-somatostatinoma-polycythemia syndrome.

A new syndrome of paraganglioma-somatostatinoma-polycythemia caused by somatic gain-of-function HIF2A mutations has been recently described.1,2 All patients presented with high erythropoietin. We report additional newly identified ocular manifestations of this syndrome in 4 HIF2A patients (Supplementary Table 1). Patients with this syndrome shared unique eye lesions clustered with new presentations of their tumors. IRB approval was received for this study. A 9-year-old female was referred for evaluation of an abdominal paraganglioma with polycythemia. On admission, we confirmed ocular abnormalities (Figure 1). A 32-year-old female with a Marfanoid habitus and paraganglioma-somatostatinomapolycythemia was previously reported in our initial study.1 A follow-up exam in 2013 revealed ocular abnormalities (Supplementary Figure 1). A 15-year-old male with a Marfanoid habitus was evaluated for the presence of right adrenal and pelvic paragangliomas associated with polycythemia from birth. At age 3 years, he was reported to have ocular abnormalities (Supplementary Figure 2). A 39-year-old female, who developed polycythemia at age 2, was referred to us for recurrent/multiple abdominal paragangliomas and a peripancreatic neuroendocrine tumor. Her eye exam was found to be abnormal (Supplementary Figure 3). The common finding in all these cases included bilateral dilated capillaries and fibrosis overlying the optic disc in both eyes. Two patients had exudation secondary to the optic disc abnormalities that resulted in macular edema and retinal hard exudate with visual acuity decrease, requiring anti-vascular endothelial growth factor agents. One patient developed peripheral retinal neovascularization that were flat, and some had dilated arterioles and venules. All patients had gain-of-function mutations in the EPAS1 gene (encoding HIF-2A peptide) detected in tumors but not in leukocyte DNA. Figure 1 A 9-year-old female was incidentally diagnosed at 18 months with bilateral disc edema, including dilated capillaries and fibrosis. She was asymptomatic until 8 years of age when she experienced bilateral macular edema with retinal hard exudates. She was ... Paraganglioma-somatostatinoma-polycythemia is a new syndrome, and the unique eye lesions clustered with the syndrome suggest that somatic gain-of-function HIF2A mutations play an important role in the pathogenesis of these eye lesions. Hypoxia-inducible factor (HIF) signaling is one of the critical regulation pathways for cells under hypoxia/pseudohypoxia to activate cellular responses. HIFs are composed of HIF-β and one of the three HIF-α (HIF-1α, HIF-2α, and HIF-3α) subunits.3 HIF-2α is expressed preferentially in neural crest cells, the endothelium, kidney, heart, lung, and gastrointestinal epithelium, and HIF-2α is the principal regulator of erythropoietin production. In humans, the retina develops from neural crest cells, through evagination from the diencephalon, and the lens originates from the surface of the ectoderm.4 HIF-2α alteration in a transgenic mouse model exhibits marked retinopathy.5 In addition, erythropoietin is known as a potent angiogenic stimulus in the retina. Therefore, a HIF2A mutation, along with high erythropoietin levels, may affect the eye, especially the retina, resulting in the ocular abnormalities among patients. In addition to the HIF2A mutation, the genes affecting the HIF signaling pathway such as von Hippel-Lindau, succinate dehydrogenase, and prolyl hydroxylase can have mutations found to be associated with neural crest cell tumors, paragangliomas/pheochromocytomas. Dysregulation of the HIF signaling pathway initiates an activation cascade of genes, many of them participating in angiogenesis, abnormal apoptosis, cell migration, and development, processes that can lead to tumorigenesis and may affect tissue-specific development of various organs or structures (as indicated by the presence of a Marfanoid habitus and brain abnormalities), including the eye and retina.3,5 As of the time of this report, over 20 patients have been described presenting with HIF2A mutations associated with paraganglioma and polycythemia. Our series of cases (4 out of 7 examined cases) with somatic HIF2A mutations clearly document the existence of an additional phenotype of this syndrome, i.e. ocular abnormalities in addition to paraganglioma, somatostatinoma, and polycythemia. Ophthalmologists and physicians of various subspecialties should recognize this new clinical entity. Referral to an ophthalmologist for screening of these patients for retinal abnormalities is advised.

Evolutionary history of Tibetans inferred from whole-genome sequencing.

The indigenous people of the Tibetan Plateau have been the subject of much recent interest because of their unique genetic adaptations to high altitude. Recent studies have demonstrated that the Tibetan EPAS1 haplotype is involved in high altitude-adaptation and originated in an archaic Denisovan-related population. We sequenced the whole-genomes of 27 Tibetans and conducted analyses to infer a detailed history of demography and natural selection of this population. We detected evidence of population structure between the ancestral Han and Tibetan subpopulations as early as 44 to 58 thousand years ago, but with high rates of gene flow until approximately 9 thousand years ago. The CMS test ranked EPAS1 and EGLN1 as the top two positive selection candidates, and in addition identified PTGIS, VDR, and KCTD12 as new candidate genes. The advantageous Tibetan EPAS1 haplotype shared many variants with the Denisovan genome, with an ancient gene tree divergence between the Tibetan and Denisovan haplotypes of about 1 million years ago. With the exception of EPAS1, we observed no evidence of positive selection on Denisovan-like haplotypes.

Gain-of-function EGLN1 prolyl hydroxylase (PHD2 D4E:C127S) in combination with EPAS1 (HIF-2α) polymorphism lowers hemoglobin concentration in Tibetan highlanders

Tibetans have lived at high altitude for generations and are thought to be genetically adapted to hypoxic environments. Most are protected from hypoxia-induced polycythemia, and a haplotype of EPAS1, encoding hypoxia-inducible factor (HIF-2α), has been associated with lower hemoglobin levels. We earlier reported a Tibetan-specific EGLN1 haplotype encoding PHD2 which abrogates HIF augmentation in hypoxia. We genotyped 347 Tibetan individuals from varying altitudes for both the Tibetan-specific EGLN1 haplotype and 10 candidate SNPs in the EPAS1 haplotype and correlated their association with hemoglobin levels. The effect of the EGLN1 haplotype on hemoglobin exhibited age dependency at low altitude, while at higher altitudes, it showed a trend to lower hemoglobin levels in the presence of the Tibetan-selected EPAS1 rs142764723 C/C allele. The observed gene-environment and gene-gene interactions and the moderate effect of the EGLN1 and EPAS1 haplotypes on hemoglobin indicate that other modifiers exist. It remains to be determined whether a blunting of erythropoiesis or other physiological consequences of HIF downregulation are the primary drivers of these genetic adaptations among Tibetans.Key messageMost Tibetans are protected from polycythemia while living in high altitude.An EGLN1 co-adapted haplotype, EGLN1 c.12C>G, c.380G>C is uniquely Tibetan.The Tibetan EPAS1 haplotype has introgressed from the Denisovan genome.While EGLN1 and EPAS1 genotypes lower Hb, this study indicates additional Hb modifiers.

RUNX1 and NF-E2 upregulation is not specific for MPNs but is seen in polycythemic disorders with augmented HIF signaling

Overexpression of transcription factors runt-related transcription factor 1 (RUNX1) and nuclear factor, erythroid-derived 2 (NF-E2) was reported in granulocytes of patients with polycythemia vera and other myeloproliferative neoplasms (MPNs). Further, a transgenic mouse overexpressing the NF-E2 transgene was reported to be a model of MPN. We hypothesized that increased transcripts of RUNX1 and NF-E2 might characterize other polycythemic states with primary polycythemic features, that is, those with exaggerated erythropoiesis due to augmented erythropoietin (EPO) sensitivity. We tested the expression of RUNX1 and NF-E2 in polycythemic patients of diverse phenotypes and molecular causes. We report that RUNX1 and NF-E2 overexpression is not specific for MPN; these transcripts were also significantly elevated in polycythemias with augmented hypoxia-inducible factor activity whose erythroid progenitors were hypersensitive to EPO. RUNX1 and NF-E2 overexpression was not detected in patients with EPO receptor (EPOR) gain-of-function, suggesting distinct mechanisms by which erythroid progenitors in polycythemias with defects of hypoxia sensing and EPOR mutations exert their EPO hypersensitivity.

Molecular basis of two novel mutations found in type I methemoglobinemia

Congenital methemoglobinemia due to NADH-cytochrome b5 reductase 3 (CYB5R3) deficiency is an autosomal recessive disorder that occurs sporadically worldwide, although endemic clusters of this disorder have been identified in certain ethnic groups. It is present as two distinct phenotypes, type I and type II. Type I methemoglobinemia is characterized by CYB5R3 enzyme deficiency restricted to erythrocytes and is associated with benign cyanosis. The less frequent type II methemoglobinemia is associated with generalized CYB5R3 deficiency affecting all cells and is lethal in early infancy. Here we describe the molecular basis of type I methemoglobinemia due to CYB5R3 deficiency in four patients from three distinct ethnic backgrounds, Asian Indian, Mexican and Greek. The CYB5R3 gene of three probands with type I methemoglobinemia and their relatives were sequenced revealing several putative causative mutations; in one subject multiple mutations were present. Two novel mutations, S54R and F157C, were identified and the previously described A179T, V253M mutations were also identified. All these point mutations mapped to the NADH binding domain and or the FAD binding domain. Each has the potential to sterically hinder cofactor binding causing instability of the CYB5R3 protein. Wild-type CYB5R3, as well as two of these novel mutations, S54R and F157C, was amplified, cloned, and purified recombinant peptide obtained. Kinetic and thermodynamic studies of these proteins show that the above mutations lead to decreased thermal stability.