Genetic determinants of haemolysis in sickle cell anaemia

Jacqueline N Milton, Helen Rooks, Emma Drasar, Elizabeth L McCabe, Clinton T Baldwin, Efi Melista, Victor R Gordeuk, Mehdi Nouraie, Gregory R Kato, Caterina Minniti, James Taylor, Andrew Campbell, Lori Luchtman-Jones, Sohail Rana, Oswaldo Castro, Yingze Zhang, Swee Lay Thein, Paola Sebastiani, Mark T Gladwin, Walk-PHAAST Investigators, Martin H Steinberg, D B Badesch, R J Barst, O L Castro, J S R Gibbs, R E Girgis, M T Gladwin, J C Goldsmith, V R Gordeuk, K L Hassell, G J Kato, L Krishnamurti, S Lanzkron, J A Little, R F Machado, C R Morris, M Nouraie, O Onyekwere, E B Rosenzweig, V Sachdev, D E Schraufnagel, M A Waclawiw, R Woolson, N A Yovetich, Jacqueline N Milton, Helen Rooks, Emma Drasar, Elizabeth L McCabe, Clinton T Baldwin, Efi Melista, Victor R Gordeuk, Mehdi Nouraie, Gregory R Kato, Caterina Minniti, James Taylor, Andrew Campbell, Lori Luchtman-Jones, Sohail Rana, Oswaldo Castro, Yingze Zhang, Swee Lay Thein, Paola Sebastiani, Mark T Gladwin, Walk-PHAAST Investigators, Martin H Steinberg, D B Badesch, R J Barst, O L Castro, J S R Gibbs, R E Girgis, M T Gladwin, J C Goldsmith, V R Gordeuk, K L Hassell, G J Kato, L Krishnamurti, S Lanzkron, J A Little, R F Machado, C R Morris, M Nouraie, O Onyekwere, E B Rosenzweig, V Sachdev, D E Schraufnagel, M A Waclawiw, R Woolson, N A Yovetich

Abstract

Haemolytic anaemia is variable among patients with sickle cell anaemia and can be estimated by reticulocyte count, lactate dehydrogenase, aspartate aminotransferase and bilirubin levels. Using principal component analysis of these measurements we computed a haemolytic score that we used as a subphenotype in a genome-wide association study. We identified in one cohort and replicated in two additional cohorts the association of a single nucleotide polymorphism in NPRL3 (rs7203560; chr16p13·3) (P = 6·04 × 10(-07) ). This association was validated by targeted genotyping in a fourth independent cohort. The HBA1/HBA2 regulatory elements, hypersensitive sites (HS)-33, HS-40 and HS-48 are located in introns of NPRL3. Rs7203560 was in perfect linkage disequilibrium (LD) with rs9926112 (r(2) = 1) and in strong LD with rs7197554 (r(2) = 0·75) and rs13336641 (r(2) = 0·77); the latter is located between HS-33 and HS-40 sites and next to a CTCF binding site. The minor allele for rs7203560 was associated with the -∝(3·7) thalassaemia gene deletion. When adjusting for HbF and ∝ thalassaemia, the association of NPRL3 with the haemolytic score was significant (P = 0·00375) and remained significant when examining only cases without gene deletion∝ thalassaemia (P = 0·02463). Perhaps by independently down-regulating expression of the HBA1/HBA2 genes, variants of the HBA1/HBA2 gene regulatory loci, tagged by rs7203560, reduce haemolysis in sickle cell anaemia.

Conflict of interest statement

Conflict-of interest disclosure

The authors declare no competing financial interests.

© 2013 Blackwell Publishing Ltd.

Figures

Fig 1
Fig 1
Manhattan Plot of Haemolytic Score. Manhattan plot summarizing the results of the genome-wide association study of haemolytic score with minor allele frequency >005.
Fig 2
Fig 2
QQ plot from CSSCD for single nucleotide polymorphisms-with minor allele frequency >0·05.
Fig 3
Fig 3
Location of the α-globin regulatory elements (blue brackets) within introns of NPRL3 and the linkage disequilibrium patterns of rs7203560 according to HapMap. Rs7203560 is circled in yellow. Circled in red are single nucleotide polymorphisms (SNPs) for which it was possible to calculate linkage disequilibrium. The allele frequencies for the SNPs are as follows: 0139 for rs7203560, 0175 for rsl3336641, 0175 for rs9926112 and 0-219 for rs7197554.

References

    1. Akinsheye I, Alsultan A, Solovieff N, Ngo D, Baldwin CT, Sebastiani P, Chui DH, Steinberg MH. Fetal hemoglobin in sickle cell anemia. Blood. 2011;118:19–27.
    1. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. Journal of molecular biology. 1990;215:403–410.
    1. Barrett JC, Fry B, Mailer JDMJ, Daly MJ. Haploview: analysis and visualization of LD and haplotype maps. Bioinformatics. 2005;21:263–265.
    1. Cannan RK. Proposal for a certified standard for use in hemoglobinometry; second and final report. The Journal of laboratory and clinical medicine. 1958;52:471–476.
    1. De Ceulaer K, Higgs DR, Weatherall DJ, Hayes RJ, Serjeant BE, Serjeant GR. Alpha-Thalassemia reduces the hemolytic rate in homozygous sickle-cell disease [letter] New England Journal of Medicine. 1983;309:189–190.
    1. Dham N, Ensing G, Minniti C, Campbell A, Arteta M, Rana S, Darbari D, Nouraie M, Onyekwere O, Lasota M, Kato GJ, Gladwin MT, Castro O, Gordeuk V, Sable C. Prospective echocardiography assessment of pulmonary hypertension and its potential etiologies in children with sickle cell disease. American Journal of Cardiology. 2009;104:713–720.
    1. Dozy AM, Kan YW, Embury SH, Mentzer WC, Wang WC, Lubin B, Davis JR, Jr, Koenig HM. α-Globin gene organisation in blacks precludes the severe form of α-thalas-saemia. Nature. 1979;280:605–607.
    1. Ganesh SK, Zakai NA, van Rooij FJ, Soranzo N, Smith AV, Nails MA, Chen MH, Kottgen A, Glazer NL, Dehghan A, Kuhnel B, Aspelund T, Yang Q, Tanaka T, Jaffe A, Bis JC, Verwoert GC, Teumer A, Fox CS, Guralnik JM, Ehret GB, Rice K, Felix JF, Rendon A, Eiriksdottir G, Levy D, Patel KV, Boerwinkle E, Rotter JI, Hofrnan A, Sambrook JG, Hernandez DG, Zheng G, Bandinelli S, Singleton AB, Coresh J, Lum-ley T, Uitterlinden AG, Vangils JM, Launer LJ, Cupples LA, Oostra BA, Zwaginga JJ, Ouwehand WH, Thein SL, Meisinger C, Deloukas P, Nauck M, Spector TD, Gieger C, Gudnason V, van Duijn CM, Psaty BM, Ferrucci L, Chakravarti A, Greinacher A, O’Donnell CJ, Witteman JC, Furth S, Cushman M, Harris TB, Lin JP. Multiple loci influence erythrocyte phenotypes in the CHARGE Consortium. Nature Genetics. 2009;41:1191–1198.
    1. Gaston M, Rosse WF. The cooperative study of sickle cell disease: review of study design and objectives. The American Journal of Pediatric Hematology-Oncology. 1982;4:197–201.
    1. Gladwin MT, Crawford JH, Patel RP. The biochemistry of nitric oxide, nitrite, and hemoglobin: role in blood flow regulation. Free Radical Biology and Medicine. 2004;36:707–717.
    1. Gladwin MT, Kanias T, Kim-Shapiro DB. Hemolysis and cell-free hemoglobin drive an intrinsic mechanism for human disease. The Journal of Clinical Investigation. 2012;122:1205–1208.
    1. Gordeuk VR, Campbell A, Rana S, Nouraie M, Niu X, Minniti CP, Sable C, Darbari D, Dham N, Onyekwere O, Ammosova T, Nekhai S, Kato GJ, Gladwin MT, Castro OL. Relationship of erythropoietin, fetal hemoglobin, and hydroxyurea treatment to tricuspid regurgitation velocity in children with sickle cell disease. Blood. 2009;114:4639–4644.
    1. Harteveld CL, Muglia M, Passarino G, Kiel-man MF, Bernini LF. Genetic polymorphism of the major regulatory element HS-40 upstream of the human alpha-globin gene cluster. British Journal of Haematology. 2002;119:848–854.
    1. Hebbel RP. Reconstructing sickle cell disease: a data-based analysis of the “hyperhemoly-sis paradigm” for pulmonary hypertension from the perspective of evidence-based medicine. American Journal of Hematology. 2011;86:123–154.
    1. Hughes JR, Cheng JF, Ventress N, Prabhakar S, Clark K, Anguita E, De Gobbi M, de Jong P, Rubin E, Higgs DR. Annotation of cis-regulatory elements by identification, subclassification, and functional assessment of multispecies conserved sequences. Proceedings of the National Academy of Sciences of the United States of America. 2005;102:9830–9835.
    1. Kamatani Y, Matsuda K, Okada Y, Kubo M, Hosono N, Daigo Y, Nakamura Y, Kamatani N. Genome-wide association study of hematological and biochemical traits in a Japanese population. Nature Genetics. 2010;42:210–215.
    1. Kato GJ, Gladwin MT, Steinberg MH. Deconstructing sickle cell disease: reappraisal of the role of hemolysis in the development of clinical subphenotypes. Blood Reviews. 2007;21:37–47.
    1. Kowalczyk MS, Hughes JR, Babbs C, Sanchez-Pulido L, Szumska D, Sharpe JA, Sloane-Stanley JA, Morriss-Kay GM, Smoot LB, Roberts AE, Watkins H, Bhattacharya S, Gibbons RJ, Ponting CP, Wood WG, Higgs DR. Nprl3 is required for normal development of the cardiovascular system. Mammalian Genome: Official Journal of the International Mammalian Genome Society. 2012;7–8:404–415.
    1. Liu YT, Old JM, Miles K, Fisher CA, Weatherall DJ, Clegg JB. Rapid detection of à-thalassaemia deletions and à-globin gene triplication by multiplex polymerase chain reactions. British Journal of Haematology. 2000;108:295–299.
    1. Machado RF, Barst RJ, Yovetich NA, Hassell KL, Kato GJ, Gordeuk VR, Gibbs JS, Little JA, Schraufhagel DE, Krishnamurti L, Girgis RE, Morris CR, Rosenzweig EB, Badesch DB, Lanzkron S, Onyekwere O, Castro OL, Sachdev V, Waclawiw MA, Woolson R, Goldsmith JC, Gladwin MT. Hospitalization for pain in patients with sickle cell disease treated with sildenafil for elevated TRV and low exercise capacity. Blood. 2011;118:855–864.
    1. Maurano MT, Humbert R, Rynes E, Thurman RE, Haugen E, Wang H, Reynolds AP, Sandstrom R, Qu H, Brody J, Shafer A, Neri F, Lee F, Kutyavin T, Stehling-Sun S, Johnson AK, Canfield TK, Giste E, Diegel M, Bates D, Hansen RS, Neph S, Sabo PJ, Heimfeld S, Raubitschek A, Ziegler S, Cotsapas C, Sotoodehnia N, Glass I, Sunyaev SR, Kaul R, Stamatoyannopoulos JA. Systematic localization of common disease-associated variation in regulatory DNA. Science. 2012;337:1190–1195.
    1. Mayor C, Brudno M, Schwartz JR, Poliakov A, Rubin EM, Frazer KA, Pachter LS, Dubchak I. VISTA: visualizing global DNA sequence alignments of arbitrary length. Bioinformatics. 2000;16:1046–1047.
    1. Milton JN, Sebastiani P, Solovieff N, Hartley SW, Bhatnagar P, Arking DE, Dworkis DA, Casella JF, Barron-Casella E, Bean CJ, Hooper WC, DeBaun MR, Garrett ME, Soldano K, Telen MJ, Ashley-Koch A, Gladwin MT, Baldwin CT, Steinberg MH, Klings ES. A genome-wide association study of total bilirubin and cholelithiasis risk in sickle cell anemia. PLoS ONE. 2012;7:e34741.
    1. Minniti CP, Sable C, Campbell A, Rana S, Ensing G, Dham N, Onyekwere O, Nouraie M, Kato GJ, Gladwin MT, Castro OL, Gordeuk VR. Elevated tricuspid regurgitant jet velocity in children and adolescents with sickle cell disease: association with hemolysis and hemoglobin oxygen desaturation. Haematologica. 2009;94:340–347.
    1. Morris CR, Kato GJ, Poljakovic M, Wang X, Blackwelder WC, Sachdev V, Hazen SL, Vichinsky EP, Morris SM, Gladwin MT. Dysregulated arginine metabolism, hemolysis-associated pulmonary hypertension and mortality in sickle cell disease. Journal of the American Medical Association. 2005;294:81–90.
    1. Nouraie M, Lee JS, Zhang Y, Kanias T, Zhao X, Xiong Z, Oriss TB, Zeng Q, Kato GJ, Gibbs JS, Hildesheim ME, Sachdev V, Barst R, Machado R, Hassell KL, Little JA, Schraufhagel DE, Krishnamurti L, Novelli E, Girgis RE, Morris C, Rosenzweig EB, Badesch DB, Lanzkron S, Castro O, Goldsmith JC, Gordeuk V, Gladwin MT. The relationship between the severity of hemolysis, clinical manifestations and risk of death in 415 patients with sickle cell anemia in the US and Europe. Haematologica. 2012 (Epub ahead of print).
    1. Phillips JE, Corces VG. CTCF: master weaver of the genome. Cell. 2009;137:1194–1211.
    1. Purcell S, Neale B, Todd-Brown K, Thomas L, Ferreira MA, Bender D, Mailer J, Sklar P, de Bakker PI, Daly MJ, Sham PC. PLINK: a tool set for whole-genome association and population-based linkage analyses. American Journal of Human Genetics. 2007;81:559–575.
    1. Reiter CD, Wang X, Tanus-Santos JE, Hogg N, Cannon RO, III, Schechter AN, Gladwin MT. Cell-free hemoglobin limits nitric oxide bioavailability in sickle-cell disease. Nature Medicine. 2002;8:1383–1389.
    1. Ribeiro DM, Zaccariotto TR, Santos MN, Costa FF, Sonati MF. Influence of the polymorphisms of the alpha-major regulatory element HS-40 on in vitro gene expression. Brazilian Journal of Medical and Biological Research. 2009;42:783–786.
    1. Rother RP, Bell L, Hillmen P, Gladwin MT. The clinical sequelae of intravascular hemolysis and extracellular plasma hemoglobin: a novel mechanism of human disease. Journal of the American Medical Association. 2005;293:1653–1662.
    1. Solovieff N, Milton JN, Hartley SW, Sherva R, Sebastiani P, Dworkis DA, Klings ES, Farrer LA, Garrett ME, Shley-Koch A, Telen MJ, Fucharoen S, Ha SY, Li CK, Chui DH, Baldwin CT, Steinberg MH. Fetal hemoglobin in sickle cell anemia: genome-wide association studies suggest a regulatory region in the 5' olfactory receptor gene cluster. Blood. 2010;115:1815–1822.
    1. Steinberg MH, Embury SH. Alpha-thalassemia in blacks: genetic and clinical aspects and interactions with the sickle hemoglobin gene. [Review] Blood. 1986;68:985–990.
    1. Steinberg MH, Sebastiani P. Genetic modifiers of sickle cell disease. American Journal of Hematology. 2012;87:824–826.
    1. Taylor JG, Nolan VG, Mendelsohn L, Kato GJ, Gladwin MT, Steinberg MH. Chronic hyper-hemolysis in sickle cell anemia: association of vascular complications and mortality with infrequent vasoocclusive pain. PLoS ONE. 2008;3:e2095.

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