Tsewang Tashi

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.

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.

High altitude genetic adaptation in Tibetans: No role of increased hemoglobin–oxygen affinity

High altitude exerts selective evolutionary pressure primarily due to its hypoxic environment, resulting in multiple adaptive responses. High hemoglobin-oxygen affinity is postulated to be one such adaptive change, which has been reported in Sherpas of the Himalayas. Tibetans have lived on the Qinghai-Tibetan plateau for thousands of years and have developed unique phenotypes, such as protection from polycythemia which has been linked to PDH2 mutation, resulting in the downregulation of the HIF pathway. In order to see if Tibetans also developed high hemoglobin-oxygen affinity as a part of their genetic adaptation, we conducted this study assessing hemoglobin-oxygen affinity and their fetal hemoglobin levels in Tibetan subjects from 3 different altitudes. We found normal hemoglobin-oxygen affinity in all subjects, fetal hemoglobin levels were normal in all except one and no hemoglobin variants in any of the subjects. We conclude that increased hemoglobin-oxygen affinity or increased fetal hemoglobin are not adaptive phenotypes of the Tibetan highlanders.

Calreticulin mutated prefibrotic-stage myelofibrosis and PMF represent an independent clone from coexisting CLL.

To the editor: JAK2 V617F-positive myeloproliferative neoplasms (MPNs), especially polycythemia vera (PV), may occur concomitantly with chronic lymphocytic leukemia (CLL). This association has not been described with a variant of essential thrombocythemia (ET), prefibrotic-stage myelofibrosis (preF

Evolutionary selected Tibetan variants of HIF pathway and risk of lung cancer

Tibetans existed in high altitude for ~25 thousand years and have evolutionary selected unique haplotypes assumed to be beneficial to hypoxic adaptation. EGLN1/PHD2 and EPAS1/HIF-2α, both crucial components of hypoxia sensing, are the two best-established loci contributing to high altitude adaptation. The co-adapted Tibetan-specific haplotype encoding for PHD2:p.[D4E/C127S] promotes increased HIF degradation under hypoxic conditions. The Tibetan-specific 200 kb EPAS1 haplotype introgressed from an archaic human population related to Denisovans which underwent evolutionary decay; however, the functional variant(s) responsible for high-altitude adaptation at EPAS1/HIF-2α have not yet been identified. Since HIF modulates the behavior of cancer cells, we hypothesized that these Tibetan selected genomic variants may modify cancer risk predisposition. Here, we ascertained the frequencies of EGLN1D4E/C127S and EGLN1C127S variants and ten EPAS1/HIF-2α variants in lung cancer patients and controls in Nepal, whose population consists of people with Indo-Aryan origin and Tibetan-related Mongoloid origin. We observed a significant association between the selected Tibetan EGLN1/PHD2 haplotype and lung cancer (p=0.0012 for D4E, p=0.0002 for C127S), corresponding to a two-fold increase in lung cancer risk. We also observed a two-fold or greater increased risk for two of the ten EPAS1/HIF-2α variants, although the association was not significant after correcting for multiple comparisons (p=0.12). Although these data cannot address the role of these genetic variants on lung cancer initiation or progression, we conclude that some selected Tibetan variants are strongly associated with a modified risk of lung cancer.

Presence of polyclonal hematopoiesis in females with Ph-negative myeloproliferative neoplasms

Presence of polyclonal hematopoiesis in females with Ph-negative myeloproliferative neoplasms

Pegylated interferon Alfa-2a and hydroxyurea in polycythemia vera and essential thrombocythemia: differential cellular and molecular responses

Polycythemia vera (PV) and essential thrombocythemia (ET) are chronic myeloproliferative neoplasms (MPNs) with high risk of thromboembolism and tendency to transform to myelofibrosis and acute leukemia. Pegylated interferon-α (PegINFa) is an effective treatment and unlike hydroxyurea (HU), it is reported to improve cytogenetic abnormalities, decrease JAK2V617F and CALR allelic burdens and achieve molecular remission [1–3]. We reported a return to polyclonal hematopoiesis in some females after PegINFa therapy [3], and unlike HU, PegINFa promoted proliferation and differentiation of PV and ET hematopoietic stem cells in vivo [4]. Cytokines, such as TNFα and interleukins are found to be elevated in MPN [5, 6] and are implicated as drivers of clonal expansion of JAK2V617F bearing cells [7]. Genome wide association studies have shown IL28 SNP and treatment with PegINFa were associated with sustained virologic response in hepatitis C [8] and suggested favorable hematological response in 20 PV/ET patients [9]. Given these observations, we analyzed the effect of PegINFa and HU treatment on molecular response, clonal hematopoiesis (females), TNFα transcript levels, and IL28B haplotype as correlated with PegINFa response. We included 82 patients diagnosed with high-risk PV (n= 45) and ET (n= 37) (per WHO 2008) treated from 2008 to March 2017. Mutational analysis for JAK2V617F and cMPL was performed by quantitative allele-specific-PCR [10], and CALR mutations by semi-quantitative fragment analysis [11]. In females, SNPs of 5 X-chromosome genes (G6PD, MPP1, FHL1, BTK and IDS) were genotyped and in heterozygotes transcriptional allelic frequencies of granulocytes were determined by quantitative allele-specific PCR. TaqMan Gene Expression Assays were used to determine TNFα and cytokine mRNAs and plasma levels of cytokines were measured. Relative TNFα expressions and JAK2V617F allelic burden were compared using paired ttest in GraphPad Prism (La Jolla, CA). As many of these patients participated in the MPD-RC trials the hematologic and clinical responses of these patients will be reported by the MPD-RC group. JAK2V617F mutation was detectable in 70 patients, CALR mutation in 11 ET, cMPL G1544T in one ET (Supplementary Table 1). In total 31 patients (27 PV and 4 ET) in the PegINFa group, and 19 patients (17 PV and 2 ET) in the HU group had JAK2V617F mutant allelic burden >10% at baseline and with at least one 6-month follow-up allelic burden value (Supplementary Figure 1). Overall molecular response rate was 14 (45%) in PegINFa group and 17 (77.2%) in the HU group. No complete molecular response rates were noted during the entire follow-up. Among 48 PegINFa treated patients, 29 had prior HU, and in this subset of HU refractory/intolerant patients, only 3 (16.6%) had molecular response. In all patients whose JAK2V617F allelic burden decreased to <5%, the allelic burden was retested using quantitative digital PCR, and their low These authors contributed equally: Tsewang Tashi, Sabina Swierczek, Soo Jin Kim.

Familial MPN Predisposition

Chronic myeloproliferative neoplasms (MPN) characteristically arise from a somatic mutation in the pluripotent hematopoietic stem cell, and most common recurring mutations are in the JAK2, CALR, and cMPL genes. However, these mutations are not founder mutations, but mainly drive the disease phenotype and a pre-existing germline predisposition has been long speculated, but has not been clearly defined to date. Genome-wide association studies in family clusters of MPN have identified a number of genetic variants that are associated with increased germline risk for developing clonal MPN. The strongest association discovered so far is the presence of JAK2 46/1 haplotype, and subsequently, many studies have found additional variants in other genes, most notably in TERT gene. However, these still account for a small fraction of familial MPN, and more in-depth studies including whole genome sequencing are needed to gain better insight into familial genetic predisposition of clonal MPNs.

A Genome-Wide Search for Greek and Jewish Admixture in the Kashmiri Population

The Kashmiri population is an ethno-linguistic group that resides in the Kashmir Valley in northern India. A longstanding hypothesis is that this population derives ancestry from Jewish and/or Greek sources. There is historical and archaeological evidence of ancient Greek presence in India and Kashmir. Further, some historical accounts suggest ancient Hebrew ancestry as well. To date, it has not been determined whether signatures of Greek or Jewish admixture can be detected in the Kashmiri population. Using genome-wide genotyping and admixture detection methods, we determined there are no significant or substantial signs of Greek or Jewish admixture in modern-day Kashmiris. The ancestry of Kashmiri Tibetans was also determined, which showed signs of admixture with populations from northern India and west Eurasia. These results contribute to our understanding of the existing population structure in northern India and its surrounding geographical areas.