The Tibetan near-complete pangenome (He et al. 2025 bioRxiv preprint) identified 224 Tibetan-enriched structural variants (TESVs), five statistical associations between specific structural variants and physiological traits, an unexplained enrichment of glutamatergic and GABAergic synapse genes among TESV-associated genes, and a Tibetan-elevated copy-number variant (VNTR) in intron 8 of the dopamine transporter gene SLC6A3 — none of which has been experimentally validated as causal. The open problem is to establish causal mechanisms: for each of the five SV–trait associations, whether the structural variant is causal and through what molecular mechanism; whether and how the enriched neuronal-signaling pathways participate in hypoxic adaptation; and what the functional consequences of the Tibetan-elevated SLC6A3 intronic VNTR are. No follow-up study has tested any of these claims to date; the nearest experimental work concerns the classic EPAS1 locus, not the novel TESVs.

Background

Populations native to the Tibetan Plateau live above 3,500 m, where the partial pressure of oxygen is roughly 60% of its sea-level value, yet they maintain near sea-level hemoglobin concentrations and avoid the excessive red-blood-cell production (polycythemia) that afflicts lowland newcomers. Genome-wide scans have attributed this adaptation mainly to regulatory variants in two genes of the cellular hypoxia-response pathway: EPAS1, which encodes a subunit of the hypoxia-inducible factor (HIF) transcription complex and whose Tibetan adaptive haplotype was inherited through interbreeding with archaic Denisovans, and EGLN1, which encodes an enzyme that marks HIF subunits for degradation. Almost all known adaptive signals are non-coding, and only a handful have been experimentally validated — most notably a pleiotropic, hypoxia-sensitive EPAS1 enhancer (ENH5) that Tibetan adaptive alleles disrupt, characterized with reporter assays in cultured cells by Gray et al. (2022) and later in enhancer-knockout mice (Thornburg et al. 2026); a functional EPAS1/HIF2A missense variant was likewise validated in Andean highlanders (Lawrence et al. 2024).

Short-read genome-wide association studies are largely blind to structural variation (SV: insertions, deletions, inversions, and copy-number changes of at least 50 bp) and to highly repetitive regions. He et al. (2025, bioRxiv preprint) therefore built a near-complete, telomere-to-telomere-quality (i.e., gapless or near-gapless across repetitive DNA) pangenome — a graph representation of many individual genomes that captures variation a single linear reference cannot — from 70 Tibetan and 204 Han Chinese haploid assemblies. They identified 224 Tibetan-enriched structural variants (TESVs: SVs with allele frequency at least 0.2 higher in Tibetans than in Han Chinese), 207 of them novel, together with 287 associated genes. Regression analyses in 1,164 individuals associated five SVs with physiological traits: a 316-bp insertion within the RNA gene LOC124906001 on chromosome 2 with lower red-blood-cell count; a 60-bp insertion in an enhancer of PIK3C3 (a lipid kinase involved in autophagy and vesicle trafficking) with lower diastolic blood pressure; a 758-bp deletion near GP5/ATP13A3 with lower platelet distribution width; a 55-bp deletion in BDNF-AS (an antisense regulatory transcript of the brain-derived neurotrophic factor gene) with increased interventricular septum thickness; and a 309-bp insertion in LOC105376496 with higher lymphocyte count. These are statistical associations only; breakpoint-level definitions of all 224 TESVs, including these five, are given in Supplementary Table 18 of the preprint.

Two further observations lack mechanistic accounts. First, Gene Ontology (GO) enrichment analysis — testing whether genes near a variant set are overrepresented among annotated functional categories — found three functional categories enriched among TESV-associated genes: glutamatergic synapse (false-discovery rate 0.001), plasma membrane (0.006), and GABAergic synapse (0.011), an unexplained hint that neural pathways contribute to Tibetan hypoxia adaptation; the co-enrichment of the generic, non-neural plasma-membrane category is directly relevant to how specific the neural signal is. Second, SLC6A3, the gene encoding the dopamine transporter DAT (also called DAT1), carries a hypervariable variable number tandem repeat (VNTR: a locus where a short DNA motif repeats a variable number of times; here a 38-bp motif in intron 8) with elevated copy number in Tibetans; this VNTR was identified in a separate tandem-repeat analysis rather than among the 224 TESVs, and the preprint states that the mechanism driving this population-stratified pattern remains unknown and that further studies are required to clarify whether it reflects high-altitude adaptation. Prior functional work on SLC6A3 VNTRs includes Guindalini et al. (2006), who reported that a VNTR in intron 8 (a 30-bp-motif "Int8 VNTR") reduced expression in reporter-gene constructs and was associated with cocaine dependence in a Brazilian cohort — the study the preprint cites for its intron-8 locus — alongside extensive association work on the well-studied 40-bp repeat in the 3' untranslated region; the hypervariable 38-bp intron-8 repeat was characterized only recently (Apsley et al. 2023), and the functional consequences of its Tibetan-elevated copy number have not been tested. The preprint's own limitations section states that experimental assays — including reporter constructs, genome editing, and molecular phenotyping under hypoxic conditions — are essential to establish causal mechanisms for the TESV–phenotype associations.

Problem Statement

The problem is to establish the causal mechanisms — where they exist — by which the Tibetan pangenome's candidate variants contribute to high-altitude hypoxia adaptation, and concerns three open threads from the same preprint: the causal status of the five SV–trait associations, and two further open mechanistic threads raised in its results and discussion:

(1) For each of the five reported Tibetan-enriched SV–trait associations (the 316-bp LOC124906001 insertion on chromosome 2 and red-blood-cell count; the 60-bp PIK3C3 enhancer insertion and diastolic blood pressure; the 758-bp deletion near GP5/ATP13A3 and platelet distribution width; the 55-bp BDNF-AS deletion and interventricular septum thickness; the 309-bp LOC105376496 insertion and lymphocyte count), determine whether the structural variant causally affects the associated trait and identify the molecular mechanism (for example, altered regulatory activity or target-gene expression) if it does, or refute the causal interpretation if it does not.

(2) Determine whether the overrepresentation of glutamatergic and GABAergic synapse genes among TESV-associated genes reflects a genuine neural component of Tibetan hypoxic adaptation — a determination that, whether positive or negative, must rest on evidence bearing on the involvement of specific Tibetan-enriched variants or their genes in hypoxia-related neural phenotypes, not on re-analysis of the enrichment statistic alone — and, if so, provide a mechanistic account connecting specific Tibetan-enriched variants to a defined hypoxia-related neural phenotype.

(3) Determine the functional consequences of the Tibetan-elevated copy number of the SLC6A3 intron-8 variable number tandem repeat for dopamine transporter expression or function, including under hypoxic conditions, so as to clarify whether this population-stratified pattern reflects high-altitude adaptation, as the preprint asks.

The three threads are distinct open threads of the same preprint, not counterparts of a single statistical association: the synapse-pathway enrichment and the SLC6A3 intron-8 VNTR stand as open questions in their own right alongside the five SV–trait associations.

References

  1. Yaoxi He, Kai Liu, Leyan Mao, Feifei Zhou, Jiaxu Song, Xuebin Qi, Dongya Wu, Junmin Han, Lanting Fu, Jiangguo Li, Yu Zhang, Chaoying Cui, Ouzhuluobu, Guojie Zhang, Yafei Mao, Bing Su. Tibetan near-complete pangenome reveals complex variants underlying high-altitude adaptation. bioRxiv preprint, posted 2025-12-21. DOI: 10.64898/2025.12.16.694547. https://www.biorxiv.org/content/10.64898/2025.12.16.694547v1
  2. Olivia A. Gray, Jennifer Yoo, Débora R. Sobreira, Jordan Jousma, David Witonsky, Noboru J. Sakabe, Ying-Jie Peng, Nanduri R. Prabhakar, Yun Fang, Marcelo A. Nobréga, Anna Di Rienzo. A pleiotropic hypoxia-sensitive EPAS1 enhancer is disrupted by adaptive alleles in Tibetans. Science Advances 8, eade1942 (2022). DOI: 10.1126/sciadv.ade1942. https://www.science.org/doi/10.1126/sciadv.ade1942
  3. Alexis G. Thornburg, Soo-Young Park, Li Zhang, Ivy Aneas, Débora R. Sobreira, Isabella M. Salamone, Noboru J. Sakabe, Kathryn M. Farris, Zachary T. Weber, Olivia A. Gray, Jennifer Yoo, Hae Kyung Im, Anna Di Rienzo, Marcelo A. Nóbrega. A pleiotropic EPAS1 enhancer mediating Tibetan adaptation to hypoxia is active in adipocytes. bioRxiv preprint, posted 2026-06-15. DOI: 10.64898/2026.06.11.730447. https://www.biorxiv.org/content/10.64898/2026.06.11.730447v1
  4. Elijah S. Lawrence, Wanjun Gu, Ryan J. Bohlender, Cecilia Anza-Ramirez, Amy M. Cole, James J. Yu, Hao Hu, Erica C. Heinrich, Katie A. O'Brien, Carlos A. Vasquez, Quinn T. Cowan, Patrick T. Bruck, Kysha Mercader, Mona Alotaibi, Tao Long, James E. Hall, Esteban A. Moya, Marco A. Bauk, Jennifer J. Reeves, Mitchell C. Kong, Rany M. Salem, Gustavo Vizcardo-Galindo, Jose-Luis Macarlupu, Rómulo Figueroa-Mujíca, Daniela Bermudez, Noemi Corante, Eduardo Gaio, Keolu P. Fox, Veikko Salomaa, Aki S. Havulinna, Andrew J. Murray, Atul Malhotra, Frank L. Powel, Mohit Jain, Alexis C. Komor, Gianpiero L. Cavalleri, Chad D. Huff, Francisco C. Villafuerte, Tatum S. Simonson. Functional EPAS1/HIF2A missense variant is associated with hematocrit in Andean highlanders. Science Advances 10, eadj5661 (2024). DOI: 10.1126/sciadv.adj5661. https://www.science.org/doi/10.1126/sciadv.adj5661
  5. Abner T. Apsley, Emma R. Domico, Max A. Verbiest, Carly A. Brogan, Evan R. Buck, Andrew J. Burich, Kathleen M. Cardone, Wesley J. Stone, Maria Anisimova, David J. Vandenbergh. A novel hypervariable variable number tandem repeat in the dopamine transporter gene (SLC6A3). Life Science Alliance 6, e202201677 (2023). DOI: 10.26508/lsa.202201677. https://doi.org/10.26508/lsa.202201677 (PMID 36754567)
  6. Camila Guindalini, Mark Howard, Kate Haddley, Ronaldo Laranjeira, David Collier, Nik Ammar, Ian Craig, Colin O'Gara, Vivian J. Bubb, Tiffany Greenwood, John Kelsoe, Phil Asherson, Robin M. Murray, Adauto Castelo, John P. Quinn, Homero Vallada, Gerome Breen. A dopamine transporter gene functional variant associated with cocaine abuse in a Brazilian sample. Proceedings of the National Academy of Sciences USA 103, 4552–4557 (2006). DOI: 10.1073/pnas.0504789103. https://doi.org/10.1073/pnas.0504789103 (PMID 16537431)