1. Hemoglobin concentration and reproductive fitness of Tibetan women in highland Nepal Cynthia M Beall, Ph.D. Case Western Reserve University Geoff Childs, Ph.D. Washington University Sienna Craig, Ph.D. Dartmouth College April 11, 2014

2. The take home message Challenge: Is a distinctive trait with heritable variation and functional significance under selection?

3. High altitude populations are natural experiments in evolution and adaptation. Courtesy of Brooke Thomas

4. A severe, unavoidable and graded stress

5. Genetic and molecular pathway for oxygen homeostasis is known. Hemoglobin concentration, gm/dL Sensor EGLN1 Transcription Factor Subunit EPAS1 Target Gene EPO

6. Different outcomes of the natural experiment

7. Hemoglobin concentration has functional significance. A cost of high hemoglobin concentration is poorer pregnancy outcome. Another cost is higher risk of Chronic Mountain Sickness. % with Chronic Mountain Sickness

8. Hb variance is heritable among Tibetans. 1.Heritability, h 2 =0.64 2.Tibetan allele frequency is divergent at SNP sites in oxygen homeostasis pathway. 3.Tibetans exhibit an altitude gradient in allele frequency at those SNP sites. 4.Hb associates with SNP sites in oxygen homeostasis pathway. http://genegeek.ca/2010/11/human- chromosomes-and-karyotype

9. Genome wide scan found allele - frequency differences between Tibetans and Han Chinese. Beall et al., 2010, PNAS ( HIF2α )

10. “Tibetan” alleles increase with altitude Xu S et al. Mol Biol Evol 2011;28:1003-1011

11. EPAS1 and EGLN1 associate with hemoglobin concentration

12. Is hemoglobin concentration under selection?

13. EPAS1 HYPOXIA Hypoxia-Inducible Factor, alpha subunit + HIF-beta HIF Hypoxia-Inducible Factor 2 Hypoxia-Inducible Factor, beta subunit HYPOXIA O2O2 O2O2 O2O2 O2O2 + O2O2 O2O2 Plausible, however there are other possibilities. Angiogenesis (VEGF) Cell Migration Cell Proliferation Cytoskeleton Formation Energy Metabolism (GLUT1) Erythrocytosis (EPO) Growth and Apoptosis Hormone Modulation Immune System (IL-6) Transcription Transport Vasomotor Regulation

14. Test the hemoglobin hypothesis

19. % O 2 saturation of hemoglobin decreased with altitude. r=-0.33, p< 0.001

20. Hemoglobin concentration - steady across altitude. Avg 13.9 gm/dL + 1.5(SD), n=943

21. ‘Continuously married’ sub-sample Whole sampleContinuously married subsample N1,011601 Age, years55 + 11 # Pregnancies5.8 + 2.96.5 + 2.8 # Livebirths5.3 + 2.86.2 + 2.6 # Children surviving to 15 years of age 3.7 + 2.2 (n=589)4.1 + 2.2 (n=350)

22. Many factors account for variance in the number of pregnancies – hb does not. 4.4 + 2.6, n=410 6.5 + 2.8, n=600 Hb

23. Conceptional and social factors account for variance in surviving children – hb does not. 3.0 + 2.0, n=239 4.1 + 2.2, n=350 Hb

24. The take home message A distinctive trait with heritable variation and functional significance may not be the trait under selection OR the study design requires rethinking.

25. Questions? cmb2@case.edu