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Proc Natl Acad Sci U S A
Proc Natl Acad Sci U S A
PNAS
Proceedings of the National Academy of Sciences of the United States of America
0027-8424
1091-6490
National Academy of Sciences

202413726
10.1073/pnas.2413726121
replyReplybiochemBiochemistry407
42
Letters
Biological Sciences
Biochemistry
Reply to McGowan et al.: An intronic polymorphism associated with 2,3-bisphosphoglycerate levels in human red blood cells is linked to expression of RhCE blood groups
Earley Eric J. eearley@rti.org
a 1 https://orcid.org/0000-0001-6576-1319

D’Alessandro Angelo angelo.dalessandro@cuanschutz.edu
b 1 https://orcid.org/0000-0002-2258-6490

Kaestner Lars c d https://orcid.org/0000-0001-6796-9535

Page Grier P. e https://orcid.org/0000-0003-2582-3786

aDivision of Analytics, Research Triangle Institute International, Research Triangle Park, NC 27709
bDepartment of Biochemistry and Molecular Genetics, University of Colorado Denver Anschutz Medical Campus, Aurora, CO 80045
cTheoretical Medicine and Biosciences, Saarland University, Homburg D-66421, Germany
dDynamics of Fluids, Experimental Physics, Saarland University, Saarbrücken D-66421, Germany
eDivision of Analytics, Research Triangle Institute International, Atlanta, GA 30329
1To whom correspondence may be addressed. Email: eearley@rti.org or angelo.dalessandro@cuanschutz.edu.
22 8 2024
3 9 2024
22 8 2024
121 36 e2413726121Copyright © 2024 the Author(s). Published by PNAS.
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This article is distributed under Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND).

HHS | NIH | NHLBI | Division of Intramural Research (DIR) 100017540 R21HL150032 Angelo D'AlessandroGrier P Page HHS | NIH | NHLBI | Division of Intramural Research (DIR) 100017540 R01HL146442 Angelo D'AlessandroGrier P Page HHS | NIH | NHLBI | Division of Intramural Research (DIR) 100017540 R01HL149714 Angelo D'AlessandroGrier P Page HHS | NIH | NHLBI | Division of Intramural Research (DIR) 100017540 R01HL148151 Angelo D'AlessandroGrier P Page HHS | NIH | NHLBI | Division of Intramural Research (DIR) 100017540 75N2019D00033 Angelo D'AlessandroGrier P Page access-typefree
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pmcWe appreciate the thoughts shared by McGowan et al. (1) relating to our study of red blood cell metabolic changes in response to high altitude hypoxia and storage in over 13,000 blood donors from the Recipient Epidemiology and Donor Evaluation Study (REDS RBC Omics). In the original study (2), a strong genetic association was discovered between 2,3-bisphosphoglycerate (BPG) levels and SNPs in the ~300 kilobase region encompassing two genes coding Rh factor antigens, RHCE and RHD, and included 219 uniquely associated SNPs at genome-wide significance (P < 5 × 10−8). As McGowan et al. point out, the SNP with the lowest P-value in this region, rs636889, aka the “lead SNP,” is located intronic to RHCE, and it is not clear how it could impact RHCE gene expression or protein function. Indeed, the lead SNP in genetic association studies is often not the causative variant but is instead in tight linkage (i.e., in linkage disequilibrium or “LD”) with the true causative variant (3).

In the RHD gene region, five SNPs were significantly associated with BGP within the gene; however, all were intronic and had no clear functional impact predicted. Expanding to SNPs with modest but not genome-wide association strength (P < 10−5) an additional 34 SNPs were observed with all but one located in introns or downstream of RHD. One SNP, rs2257611, with predicted amino acid change of V/M at exon 8 was observed, but it was in low LD (R2 = 0.2) with the lead SNP for this region. Within RHCE, 79 SNPs were associated at genome-wide significance with BPG levels, including the SNPs linked to Rh C/c and E/e groups: rs586178 (P = 4.1 × 10−9) and rs61777615 (P = 3.2 × 10−11) in high LD with the lead SNP (R2 > 0.7), and rs609320 (P = 1.6 × 10−9) that was relatively unlinked (R2 = 0.1).

While antigen D status was measured in the REDS-III RBC Omics complete blood count data, the phenotype of Rh groups C/c and E/e was not measured, complicating our ability to assess associations with specific antigens. However, using genotype data for each of these variants as a proxy, a potential link between Rh group and BPG levels was discovered (Fig. 1A) with an ANOVA suggesting a significant difference in Rh genotype groups versus BPG [F(25) = 3.9; P = 2.4 × 10−8]. Tukey HSD test showed evidence of five different genotype groupings significantly different from one another (Fig. 1A). Population stratified GWAS of REDS-III (Fig. 1B) uncovered a stronger association signal in participants genetically similar to present day Europeans (N = 7,591) compared to participants genetically similar to present day Africans (N = 1,494), with participants similar to combined East and South Asians (N = 1,555) and admixed American (N = 975) with intermediate levels of associations, in agreement with the linkage pattern observed by McGowan et al. (1).

Fig. 1. (A) Boxplots of 2,3-bisphosphoglycerate levels versus groups of genotypes linked to Rh C/e and E/e for SNPs rs609320 [1], rs61777615 [2], and rs586178 [3]. Letter groups a, ab, abc, b, and c represent significantly different groups of genotypes according to Tukey HSD with boxplot colors added for ease of reference. (B) Detailed LocusZoom (4) view of population stratified GWAS results for four populations within REDS-III exhibiting genetic similarity to current-day Africans (AFR), combined East and South Asians (ASN), Europeans (EUR), and admixed Hispanic Americans (HIS).

In summary, we are grateful to McGowan et al. for their suggestion of investigating Rh group status, and the evidence within the REDS RBC Omics cohort does support the hypothesis of Rh group phenotypes mediating the association between the SNPs within the RHCE gene region and 2,3-bisphosphoglycerate levels. Future studies will need to test this link directly with Rh group phenotyping.

Author contributions

A.D., L.K., and G.P.P. designed research; E.J.E., A.D., and G.P.P. performed research; A.D., L.K., and G.P.P. contributed new reagents/analytic tools; E.J.E. and G.P.P. analyzed data; and E.J.E., A.D., and G.P.P. wrote the paper.

Competing interests

The authors declare no competing interest.
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1 E. McGowan, J. R. Storry, M. L. Olsson, An intronic polymorphism associated with 2,3-bisphosphoglycerate levels in human red blood cells is linked to expression of RHCE blood groups. Proc. Natl. Acad. Sci. U.S.A. 121 , e2412585121 (2024).39172775
2 D’Alessandro , Genetic polymorphisms and expression of Rhesus blood group RHCE are associated with 2,3-bisphosphoglycerate in humans at high altitude. Proc. Natl. Acad. Sci. U.S.A. 121 , e2315930120 (2024).38147558
3 Y. Adam, C. Samtal, J. T. Brandenburg, O. Falola, E. Adebiyi, Performing post-genome-wide association study analysis: Overview, challenges and recommendations. F1000Res 10 , 1002 (2021), 10.12688/f1000research.53962.1.35222990
4 A. P. Boughton , LocusZoom.js: Interactive and embeddable visualization of genetic association study results. Bioinformatics 37 , 3017–3018 (2021), 10.1093/bioinformatics/btab186.33734315
