<i>S100B</i> has pleiotropic effects on vaso‐occlusive manifestations in sickle cell disease
Authors: Xu Zhang, Binal N. Shah, Wei Zhang, Santosh L. Saraf, Mehdi Nouraie, Sergei Nekhai, Roberto F. Machado, Mark T. Gladwin, Victor R. Gordeuk
Journal: American Journal of Hematology (2019)

Acute pain crisis is the dominant manifestation of vaso-occlusion in sickle cell disease (SCD)1 and the major cause of morbidity. Frequent vaso-occlusive crises associate with progressive organ damage and bone infarction known as avascular necrosis (AVN). The molecular etiologies underlying the development of vaso-occlusive complications are not well understood. In this study, we assessed the genetic factors contributing to acute pain crisis and AVN, applying a prioritized analysis focusing on expression quantitative trait loci (eQTL) in SCD. The study involved 1162 patients from two cross-sectional cohorts and an additional 303 patients from two expression profiling cohorts. We found allelic heterogeneity in eQTL of S100B, encoding a damage-associated molecular pattern molecule.2 The A alleles of the tag single nucleotide polymorphisms (SNPs) of the eQTL, rs2154586 and rs2070435, both associated with higher S100B gene expression. The A allele of the former SNP associated with AVN whereas the A allele of the latter SNP with reduced rather than increased acute pain episodes. Serum S100B concentrations also correlated independently with AVN and with reduced acute pain episodes. Phenotype Definition: We examined pain episodes for which patients went to the ER or were admitted to hospital over 12 months prior to a baseline visit, in 650 Walk-PHaSST adolescent and adult patients and 484 PUSH pediatric patients (Table S1). To reflect the frequency of crisis naturally based on calendar year, quarter, and month while accommodating the distribution of count data, numbers of severe pain episodes in the past year were converted into four ordered groups defined as (a) no episodes, (b) 1-4 episodes, (c) 5-12 episodes, and (d) >12 episodes, herein referred to as pain frequency. Pain frequency had a strong association with history of acute chest syndrome, as expected,3 and showed a consistent trend of association with AVN, defined as symptomatic AVN at hip or shoulder confirmed by radiography (Figure S1). Older age associated with increased pain frequency (OR = 1.5, P = 6.5 × 10−5) and with AVN (OR = 4.3, P = 1.8 × 10−6). Indeed, 28 out of the 31 patients with AVN in the pediatric PUSH cohort were 12 years or older. eQTL Mapping: Genetic regulation of gene expression plays an important role in complex traits, as indicated by the enrichment of trait-associated SNPs in eQTL.4, 5 Such enrichment exhibits tissue-, disease-, and population-specificity.6 Gene expression in peripheral blood mononuclear cells (PBMCs) was markedly different between sickle cell anemia patients and healthy African Americans,7 suggesting its relevance in disease etiology. We mapped eQTL in a cohort of 61 adult patients from Howard University and the University of Chicago who were microarray-profiled for PBMC gene expression and genome-wide SNPs7 (Supplemental Methods). At a false discovery rate of 5%, 1004 independent SNPs, obtained by linkage disequilibrium (LD) clumping per gene expression trait (r2 > 0.3), herein referred to as expression quantitative trait nucleotides (eQTNs), were identified for 880 genes. Allelic Heterogeneity in eQTL of S100B Underlies Its Pleiotropic Effects on Vaso-Occlusive Complications: Among the eQTNs, 865 for 763 genes with MAF >0.1 and imputation quality r2 > 0.9 in Walk-PHaSST and PUSH were further analyzed. As the eQTL mapping was carried out genome-wide, the prioritized analysis represents a de novo approach for hypothesis generating. Pain frequency and AVN were regressed on allele dosage by the ordinal logistic model and the binary logistic model, respectively, adjusting for covariates of age, gender, hemoglobin β genotype severity, and population stratification. The A allele of an eQTN of S100B (rs2154586), located in its last intron, associated with higher S100B expression (β = 0.40, P = 1.6 × 10−6) (Figure 1A) and with AVN in meta-analysis (OR = 2.0, nominal P = 1.4 × 10−5, Bonferroni-corrected P = .012) (Table S2, Figure 1B). The result is consistent with an inflammation promoting effect of S100B at localized high dose.8 There was no eQTN reaching a Bonferroni-corrected P = .05 for pain frequency association. The two eQTNs having the most significant associations with pain frequency were an eQTN of DIP2A (rs4347941, C allele OR = 0.71, nominal P = 1.6 × 10−4) and another eQTN of S100B (rs2070435, A allele OR = 0.73, nominal P = 5.7 × 10−4) (Table S2, Figure 1C). These two eQTNs were in LD (r2 = 0.92, D′ = 0.99). As gene expression levels of S100B and the nearby DIP2A were correlated (ρ = 0.63), this genetic locus, located downstream of S100B and within DIP2A, appears to regulate both genes. The A allele of rs2070435 not only associated with higher S100B expression (β = 0.34, P = 3.1 × 10−7) (Figure 1A) but also with higher DIP2A expression (β = 0.14, P = 2.6 × 10−9). The association with reduced nociceptive pain9 may be related to the functions of S100B and DIP2A in stimulating growth of peripheral nerve axons10 and reducing neuronal apoptosis after ischemia.11 In regional associations (Figure 1D,E), the SNPs with the greatest significance for AVN (rs376461336, OR = 2.1, nominal P = 2.2 × 10−6) and pain frequency (rs2096507, OR = 0.69, nominal P = 4.5 × 10−5) were in LD with rs2154586 (r2 = 0.84, D′ = 1) and rs2070435 (r2 = 0.83, D′ = 0.98), respectively. The two eQTNs of S100B, located in two consecutive LD blocks (Figure S2), individually explained 35% (rs2154586) and 39% (rs2070435) and jointly explained 57% of S100B expression variation. The largely non-overlapping effects of the eQTNs on S100B expression reflect only weak LD (r2 = 0.043, D′ = 0.42) and imply that the two SNPs tag distinct regulatory sites, which may underlie the pleiotropic effects of S100B expression on AVN and pain frequency. Serum S100B Correlated with AVN and Reduced Acute Pain Episodes: We used data of 242 adult patients from the University of Illinois at Chicago (UIC) cohort (Table S1) to replicate the findings. The two S100B eQTNs were validated for PBMC expression association in 159 patients with data available (β = 0.50, P = 4.8 × 10−18 for rs2154586; β = 0.35, P = 1.2 × 10−13 for rs2070435). AVN in UIC was determined by radiography regardless of the presence of symptoms; pain episodes were defined as episodes for which patients went to acute care center or the ER or were admitted to hospital in 1 year. AVN and pain episodes were analyzed by the binary logistic model and the negative binomial model, respectively, adjusting for the same covariates as in the discovery phase. Our initial replication effort did not show statistically significant association of rs2154586 with AVN (OR = 1.1, P = .7) or rs2070435 with pain episodes (eβ = 0.85, P = .2) (Table S3), probably due to the small sample size, discrepancy in outcome definition, and variation in local LD structure. Further studies, involving substantially more patients, will be necessary. We further examined S100B protein level by enzyme-linked immunosorbent assay, from 51 UIC patients with variable S100B expression (the top and bottom quartiles of expression level) and with available serum samples. Serum S100B concentration strongly correlated with array gene expression (ρ = 0.70, P = 3.6 × 10−8) (Figure S3A). Higher level of serum S100B showed a stronger association with the A allele of rs2154586 (the AVN associated eQTN, β = 0.71, P = 6.6 × 10−5) than with the A allele of rs2070435 (the pain associated eQTN, β = 0.39, P = .013). Similar patterns were observed for their linked SNPs (Table S3). Furthermore, higher serum S100B concentration correlated independently with AVN (β = 0.68, P = .018) and with fewer acute pain episodes (β = −0.041, P = .0082) (Figures S3B, S3C) in multivariate analysis. S100B eQTL Associated with Venous Thromboembolism in General African American Population: To assess the effects of the S100B eQTNs under non-sickle cell conditions, we examined their associations with venous thromboembolism (VTE) in general African Americans. VTE shares certain pathogenetic features with sickle vaso-occlusion.12, 13 Under non-sickle conditions, VTE prevalence is higher in patients with idiopathic osteonecrosis than in the general population.14 We analyzed VTE, defined as having a blood clot in leg or lung as told by a doctor, among African Americans from Atherosclerosis Risk in Communities, Jackson Heart Study, and Cardiovascular Health Study in dbGaP. The A allele of rs2154586, which associated with AVN in SCD, associated with VTE in the general African-Americans (OR = 1.4, P = .0067) (Table S4). This work is supported in part by grants R01 HL079912-04, 2 R25-HL03679-08, and 1P30HL107253 (V.R.G.); 1P50HL118006 (S.N.); R01HL111656 and R01HL127342 (R.F.M.); K23HL125984 (S.L.S); UL1TR000050 (UIC Center for Clinical and Translational Science). We thank UI Health Biorepository for preparing serum for ELISA experiment. The Atherosclerosis Risk in Communities Study is carried out as a collaborative study supported by National Heart, Lung, and Blood Institute contracts (HHSN268201100005C, HHSN268201100006C, HHSN268201100007C, HHSN268201100008C, HHSN268201100009C, HHSN268201100010C, HHSN268201100011C, and HHSN268201100012C). The authors thank the staff and participants of the ARIC study for their important contributions. Funding for GENEVA was provided by National Human Genome Research Institute grant U01HG004402 (E. Boerwinkle). The Jackson Heart Study is supported by contracts HHSN268201300046C, HHSN268201300047C, HHSN268201300048C, HHSN268201300049C, HHSN268201300050C from the National Heart, Lung, and Blood Institute and the National Institute on Minority Health and Health Disparities. Phenotype data have been collected as described in PMID: 16320381 and at https://www.jacksonheartstudy.org/jhsinfo/. Authorized access to genotype data may be obtained through accession numbers phs000286.v4.p1 (JHS parent study), phs000402 (HeartGO_JHS), phs000498 (JHS_AllelicSpectrum_Seq), and phs000499 (JHS_CARe). Funding for CARe genotyping was provided by NHLBI Contract N01-HC-65226. The Cardiovascular Health Study research reported in this article was supported by contract numbers N01-HC85079, N01-HC-85080, N01-HC-85081, N01-HC-85082, N01-HC-85083, N01-HC85084, N01-HC-85085, N01-HC-85086, N01-HC-35129, N01 HC-15103, N01 HC55222, N01-HC-75150, N01-HC-45133, N01-HC-85239 and HHSN268201200036C; grant numbers U01 HL080295 from the National Heart, Lung, and Blood Institute and R01 AG-023629 from the National Institute on Aging, with additional contribution from the National Institute of Neurological Disorders and Stroke. A full list of principal CHS investigators and institutions can be found at https://chs-nhlbi.org/pi. This manuscript was not prepared in collaboration with CHS investigators and does not necessarily reflect the opinions or views of CHS, or the NHLBI. Support for the Cardiovascular Health Study Whole Genome Study was provided by NHLBI grant HL087652. Additional support for infrastructure was provided by HL105756 and additional genotyping among the African-American cohort was supported in part by HL085251. The DNA handling and genotyping at Cedars-Sinai Medical Center was supported in part by National Center for Research Resources grant UL1RR033176, now at the National Center for Advancing Translational Technologies CTSI grant UL1TR000124; in addition to the National Institute of Diabetes and Digestive and Kidney Diseases grant DK063491 to the Southern California Diabetes Endocrinology Research Center. The authors declare no competing financial interests. X.Z., V.R.G. conceived and designed study; X.Z., W.Z., V.R.G. analyzed data. B.N.S. Performed ELISA; X.Z., W.Z., S.L.S., M.N., G.M., S.N., M.T.G., R.F.M., and V.R.G. contributed material and tools; X.Z. and V.R.G. wrote paper with all authors providing critical comments. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.