African American Sickle Cell Disease Research Articles

Genetic diversity of CD14, CD28, CTLA-4 and ICOS gene promoter polymorphism in African and American sickle cell disease

Variable immune response to external stimuli remains a major concern in sickle cell disease (SCD), with such responses predicted to be contributors to disease pathogenesis. Elucidating the diversity of host genes contributing to immune response would assist to clarify differing outcomes among and between disease groups. We hypothesize that there is a significant interethnic diversity in the CD14 (rs2569190), CD28 (rs35593994), CTLA-4 (rs5742909) and ICOS (rs4404254) gene polymorphisms among and between SCD groups. We genotyped single nucleotide polymorphisms of the 4 loci among African and African American SCD and control groups and between SCD groups. In all, 375 individuals from Mali (145 SCD and 230 controls) and 700 DNA samples from the United States (321 SCD and 379 controls) were subjected to a PCR-RFLP assay. We found no intraethnic difference in genotypic and allelic frequencies of the 4 loci among Africans and African Americans, potentially significant in disease association studies, including a similar observation for interethnic frequencies of CD28, CTLA-4 and ICOS genes, but not CD14. The CD14 (rs2569190) gene promoter demonstrated a significant difference (p < 0.02) between African and African American SCD groups, with the mutant variant (−159 T/T) more frequent (p < 0.0002) in African American SCD (38.9% versus 26.2%). The higher frequency of CD14 mutants among African Americans without an accompanying defect in CD28, CTLA-4 and ICOS diversity possibly indicates a defective innate response, driven by CD14, is untethered to downstream T cell differentiation or effector function. Additionally, we show that CD28 (rs35593994) mutant variants have no impact on T cell differentiation, as the ICOS gene provides an alternative pathway to override this impairment. We conclude that in spite of the defect in CD14, T cell selection and differentiation is unimpeded and a robust adaptive immune response initiated.

Precision Medicine for Sickle Cell Disease through Whole Genome Sequencing

▪Although sickle cell disease (SCD) is a monogenic disorder, the severity and specific organ dysfunction and failure are strongly influenced by genetic modifiers. Rapid identification of all modifiers in patients and well-phenotyped cohorts will better define the impact of relevant variants on clinical status, inform disease biology, and identify new therapeutic strategies. We created the Sickle Genome Project (SGP), a whole genome sequencing (WGS) strategy, to define genomic variation and modifiers of SCD. We performed WGS on 871 African American SCD patients from St. Jude Children's Research Hospital who participated in the Sickle Cell Clinical Research and Intervention Program (SCCRIP, Hankins et al. Pediatr Blood Cancer, 2018) and Texas Children's Hospital Hematology Center (TCHC). We developed robust pipelines for accurate detection of single nucleotide polymorphisms (SNPs), identification of structural variants and data retrieval/sharing via the St. Jude Cloud platform (to be described elsewhere). Notable findings include:1) Confirmed associations of common genetic modifiers with SCD phenotypes, including levels of fetal hemoglobin (BCL11A, HBS1L-MYB, HBB), bilirubin (UGT1A1), and microalbuminuria (APOL1). Additional associations approaching genome-wide significance require further investigation, including replication in independent samples.2) Improved determination of the SCD modifier α-thalassemia. The most common α-thalassemia mutations in SCD are 3.7 kb or 4.2 kb deletions (-α3.7 and -α4.2 alleles), which arose from recombination between homologous HBA1 and HBA2 genes and are difficult to map using standard WGS reads. Three independent crossover events are described for -α3.7 and one for -α4.2 in SCD cohorts. We developed a novel approach to identify α-globin gene deletions by local de novo assembly of WGS data and coverage depth analysis. We identified 5 -α3.7 alleles (frequencies 0.77-32.12%) and 7 -α4.2 alleles (frequencies 0.19-5.77%). Collectively, the frequency of all -α alleles was 57%, reflecting at least 12 distinct recombination events, greatly exceeding previously published counts. These findings better define the evolution of α-globin genes to allow improved understanding of their regulation and influence on SCD.3) Characterization of β0-thalassemia alleles. Mutations in the extended β-globin locus influence SCD phenotypes. Five SGP patients had large β-globin (HBB) deletions associated with elevated fetal hemoglobin, which ameliorates symptoms of SCD. Twenty-three patients had HbSβ0-thalassemia, which reduces the severity of some SCD phenotypes. Overall, 48.6% (18/37) of patients clinically designated as HbSβ0 -thalassemia had no identified β-thalassemia mutation. Moreover, 4/680 patients (0.6%) designated HbSS were identified to be β0-thalassemia heterozygotes. The MCV, RBC and %HbA2 distributions overlapped substantially in correct vs. incorrect genotype assignments. Improved discrimination of HbSβ0 vs HbSS genotypes by WGS will better define associated phenotype differences to impact clinical care.4) Determination of a genetic variant linked to vaso-occlusive crisis (VOC). Previously, a single GWAS study linked rs3115229, located 63.7 kb 5′ upstream of the KIAA1109 gene, with VOC at borderline significance (P = 5.63 × 10−8) (Chaturvedi et al, Blood 130, 2017). Using WGS data for 327 SGP participants (HbSS or HbSβ0-thalassemia) enrolled in the SCCRIPP study, we found strong association (p = 7 x 10-5) between the onset of VOC and a 4-SNP diplotype within an adjacent LD block of the KIAA1109-TENR-IL2-IL21 region (chr4: 122.8Mb - 123.8Mb) which has been previously associated with numerous inflammatory disorders. We validated this association using imputed genome-wide array data in an independent group of SCD patients (Sleep and Asthma Cohort, n= 181 patients, p = 0.05) (Cohen et al, Ann Am Thorac Soc, 2016). This works provides confirmation that the region surrounding KIAA1109 is associated with pain crisis in SCD.Our studies provide new information on the genomic architecture of SCD patients and delineate a consolidated approach for future applications of precision medicine. [Display omitted] DisclosuresHankins:Novartis: Research Funding; Global Blood Therapeutics: Research Funding; NCQA: Consultancy; bluebird bio: Consultancy. Estepp:Global Blood Therapeutics: Consultancy, Research Funding; ASH Scholar: Research Funding; NHLBI: Research Funding; Daiichi Sankyo: Consultancy.

BMI, Psychosocial Correlates, Pain and Activities of Daily Living in Sickle Cell Disease Patients

Pain, psychosocial issues and impaired activities of daily living (ADLs) are common in sickle cell disease (SCD). Improved longevity may be leading to increases in body mass index (BMI). We both describe an SCD clinic population and determine if BMI, psychosocial distress, and pain predict impairments in ADLs. Archival data from 252 adult African American SCD clinic patients were examined (ie gender, BMI, SCD type, psychosocial status via Symptom Checklist-90-Revised; pain via Short-form McGill Pain Questionnaire: Sensory Pain, Affective Pain (AP), Present Pain Intensity and ADLs. In total, 44% of the sample was overweight (BMI ≥ 25 kg/m2). Depression, anxiety, and Symptom Checklist-90-Revised Global Severity Index were in the clinical range. Patients with HbSβ+-thalassemia and hemoglobin SC disease had higher BMIs than those with HbSS (P < 0.001). AP and age-predicted impaired ADLs. In an OW/OB subsample, AP, age, and SCD severity predicted impaired ADLs. The consistent relationship of AP to ADL impairment suggests targeting AP via behavioral pain management may improve functioning.

Association of genetic variation in COMT gene with pain related to sickle cell disease in patients from the walk-PHaSST study.

BackgroundVaso-occlusive pain episodes (VOEs) are the hallmark of sickle cell disease (SCD), and our current understanding of disease biology, treatment, and psychological covariates does not adequately explain the variability of pain in SCD. Functional variants in catechol-O-methyltransferase (COMT) gene contribute to variability in pain perception, but their impact on pain perception in African American SCD patients is not well known.MethodsWe studied COMT single-nucleotide polymorphisms (SNPs) rs6269, rs4633, rs4818, rs4680, and rs165599 to determine their relationship to patient self-reported pain, the number of acute VOEs, and their impact on daily life and health care utilization in 438 hemoglobin SS patients who participated in the walk-PHaSST study.ResultsIn women, two risk SNPs (rs4633 and rs165599) and the corresponding haplotype (ATCAA) were associated with increased frequency of pain-related emergency room visit.ConclusionCOMT functional variants may predispose SCD patients to worse acute pain in women. The association of COMT variants with the intensity of self-reported acute pain warrants further genetic study of pain perception in SCD.

SAR1a promoter polymorphisms are not associated with fetal hemoglobin in patients with sickle cell disease from Cameroon

BackgroundReactivation of adult hemoglobin (HbF) is currently a dominant therapeutic approach to sickle cell disease (SCD). In this study, we have investigated among SCD patients from Cameroon, the association of HbF level and variants in the HU-inducible small guanosine triphosphate-binding protein, secretion-associated and RAS-related (SAR1a) protein, previously shown to be associated with HbF after HU treatment in African American SCD patients.ResultsOnly patients >5 years old were included; hemoglobin electrophoresis and a full blood count were conducted upon arrival at the hospital. RFLP-PCR was used to describe the HBB gene haplotypes and Gap PCR to investigate the 3.7 kb α-globin gene deletion. The iPLEX Gold Sequenom Mass Genotyping Array and cycle sequencing were used for the genotyping of four selected SNPs in SAR1a (rs2310991; rs4282891; rs76901216 and rs76901220). Genetic analysis was performed using an additive genetic model, under a generalized linear regression framework. 484 patients were studied. No associations were observed between any of the promoter variants and baseline HbF, clinical events or other hematological indices.ConclusionThe results of this study could be explained by possible population-specificity of some tagging genomic variants associated with HbF production and illustrated the complexity of replicating HbF-promoting variants association results across African populations.

MiR‐16 Mediated MYB Gene Silencing Induces Fetal Hemoglobin Expression

Sickle cell disease (SCD) is an inherited blood disorder caused by a point mutation in the β‐globin gene affecting about 100,000 people in the United States and millions worldwide. Individuals living with this disease suffer from hemolytic anemia, progressive organ damage, and acute painful episodes. Fetal hemoglobin (HbF), a major genetic modifier of clinical presentation and progression of SCD, is of great interest due to its ability to inhibit sickle hemoglobin (HbS) polymerization and reducing erythrocyte damage. Variation in HbF levels among sickle cell patients is highly heritable and has been linked to the presence of single nucleotide polymorphisms (SNPs) in several genetic regulatory regions. In this study, DNA isolated from two cohorts of African American SCD patients with high and low HbF levels were analyzed by PCR‐based SNP analysis in the HBB, BCL11A, and HBS1L‐MYB (HMIP) gene regions. The presence of SNP alleles was then correlated with their clinical phenotype data to identify those associated with increased levels of HbF. Three SNPs in the BCL11A and HBB gene regions were identified in the high HbF patient cohort that may contribute to milder phenotypes in African Americans. To complement the SNP studies, genome wide microRNA (miRNA) data was generated which showed increased miR‐16 levels associated with high HbF. Furthermore, MYB, the negative regulator of γ‐globin expression, is silenced by miR‐16 through binding the 3′‐untranslated region. RT‐qPCR and western blot were used to measure γ‐globin mRNA and HbF protein levels respectively after transfection of miR‐16 mimic into KU812 cells. Preliminary data revealed dose dependent miR‐16 mediated MYB suppression and γ‐globin activation. The discovery of regulatory regions that modulate HbF expression and miRNA mediated suppression of negative regulators of γ‐globin, provides expanded understanding of gene regulation, which can be used to define novel treatment options for individuals with SCD.Support or Funding InformationSponsored by the Medical College of Georgia Medical Scholars Program

Susceptibility to sickle cell co‐morbidities is regulated by single nucleotide polymorphisms of the TOLLIP gene

Infectious diseases, despite the availability of vaccines, remain a challenge for sickle cell disease (SCD) patients. This is exacerbated in developing countries with high disease rates, poor hospital infrastructures and preponderance of co‐morbidities, complicating SCD case management. Therefore, elucidating the role of pathogen recognition factors or signaling pathways in immune response is critical in clarifying genes regulating infectious co‐morbidities. We have shown that the dysregulated immune response observed in SCD is not attributable to dendritic cells (CD209 gene promoter polymorphism) or Toll‐like receptor (TLR‐4 polymorphisms). Though there is a significant difference in CD209 gene promoter polymorphism between groups, it does not explain infection overload in African but not African American SCD patients. Toll‐interacting protein (TOLLIP) regulates human TLR signaling pathways, and its polymorphisms have been associated with infectious diseases. To decipher whether TOLLIP polymorphisms affect immune response upstream of TLR‐4 signaling, we examined the genetic diversity of TOLLIP polymorphisms (rs5743899 and rs3750920) within and between control (230, 379 and 159 Africans, African Americans and Caucasians respectively) and disease groups (145 and 331 African and African Americans respectively), utilizing a polymerase chain reaction and restriction fragment length polymorphism assay. We found significant interethnic disparity in the genotypic frequency (15.2%, 21.6% and 2.5%) of rs5743899 mutant variant between Africans, African Americans and Caucasians respectively. In addition, we also found a significant difference for rs5743899 mutant variant between disease and control groups in Africa (22.7% versus 15.2%) but not in the United States (23.3% versus 21.6%), with no difference between sickle cell disease groups. This observation shows that Tollip gene rs5743899 mutant variant is a potential contributor to predisposing sickle cell patients but not control groups in Africa to infectious co‐morbidities, and may explain the high levels of circulating immune complexes in disease groups. This report reveals interethnic susceptibility to infectious co‐morbidities and how this contributes to immune response in sickle cell disease.Support or Funding InformationRIT Louis Stokes Alliance for Minority Participation Fellowship (NSF Award #25901‐03200‐S01) (SEA)Laboratory Support Fund, College of Health Sciences and Technology (BNT)

(258) CYP2C8 and CYP2C9 metabolic profiles and frequency of emergency department visits for sickle cell disease pain management

The CYP2C8 and CYP2C9 variant alleles play a significant role in NSAIDs analgesic effect and toxicity. NSAIDs are used to treat vasoocclusive pain at the prodromal stage in sickle cell disease (SCD) patients. We describe CYP2C8 and CYP2C9 genotypes and phenotypes and frequency of emergency department (ED) visits in an African American SCD patient cohort. DNA from 165 unrelated SCD patients (82 males/83 females, aged from 16 to 61 years) was genotyped for seven CYP2C8 (*1, *2, *3, *4, *5, *7 & *8) alleles and 15 CYP2C9 alleles (*1, *2,*3, *4, *5, *6, *8, *9, *10, *11, *12, *13, *15, *25 & *27) using the iPLEX® ADME PGx multiplexed panel.

Preemptive Genotyping of CYP2C8 and CYP2C9 Allelic Variants Involved in NSAIDs Metabolism for Sickle Cell Disease Pain Management.

Interindividual variability in analgesic effects of nonsteroidal anti-inflammatory drugs prescribed for sickle cell disease (SCD) pain is attributed to polymorphisms in the CYP2C8 and CYP2C9 enzymes. We described CYP2C8 and CYP2C9 genotype/phenotype profiles and frequency of emergency department (ED) visits for pain management in an African American SCD patient cohort. DNA from 165 unrelated patients was genotyped for seven CYP2C8 and 15 CYP2C9 alleles using the iPLEX ADME PGx multiplexed panel. CYP2C8*1 (0.806),*2 (0.164), *3 (0.018), and *4 (0.012) alleles were identified. Genotype frequencies were distributed as homozygous wild type (66.7%), heterozygous (27.8%), and homozygous variant/compound heterozygous (5.4%), respectively. CYP2C9*1 (0.824), *2 (0.027), *3 (0.012), *5 (0.009), *6 (0.009), *8 (0.042), *9 (0.061), and *11(0.015) were observed with extensive (68.5%), intermediate (18.1%) and poor predicted metabolizers (0.6%), respectively. Fifty-two and 55 subjects, respectively had at least one variant CYP2C8 or CYP2C9 allele. Although the distribution of the CYP2C9 (p = 0.0515) phenotypes was marginally significantly in high and low ED users; some CYP2C8 and CYP2C9 allelic combinations observed in 15.2% (25) of the cohort are associated with higher risks for analgesic failure. CYP2C8 and CYP2C9 preemptive genotyping could potentially enable clinicians to identify patients with impaired metabolic phenotypes.

Genes Associated with Alloimmunization to Blood Group Antigens in Sickle Cell Disease

BACKGROUND: Alloimmunization to minor blood group antigens is more common in sickle cell disease (SCD) than in the general population. Causes include the high frequency of transfusion as well as phenotypic differences between the largely African-American SCD population and mostly Caucasian blood donors. Alloimmunization, as well as the risk of alloimmunization, results in higher costs as well as reduced availability of and delays in obtaining antigen-matched blood to meet clinical needs. We used a candidate gene approach to determine if specific genetic signatures were associated with alloimmunization and thus could identify patients at high risk for alloimmunization and therefore requiring prospective antigen matching for transfusion.METHODS: Patient data were previously collected through a multicenter study to identify genetic factors associated with SCD complications (Duke and UNC; DU) and through the Cooperative Study of SCD (CSSCD). All subjects were adults with HbSS genotype (DU mean yrs 34.9 ± 12.1, CSSCD mean yrs 29.1 ± 9.5, p < .0001). The DU data set was 43.7% male while the CSSCD data set was 37.1% male (p = .18). A genome-wide association study (GWAS) was conducted with the Illumina 610 BeadChips for ­­­390 individuals (215 DU, 175 CSSCD). Quality control processing of these data was published previously (Solovieff et al, 2010). Additionally, imputation of the DU data set was conducted using IMPUTE2 (Howie et al, 2009) and a global reference panel from 1000 Genomes in order to have consistent genotypes across both studies. Based on previous literature, we studied 255 SNPs in the HLA locus as well as 684 SNPs in 53 genes previously associated with immune responses, totaling 939 SNPs. Additive logistic regression was performed separately for the DU and CSSCD data sets with PLINK (Purcell et al, 2008), controlling for age at enrollment. Results from the two data sets were combined by meta-analysis, using the invariance weight method in METAL (Willer et al, 2010). False discovery rate (FDR) q-values were generated using PROC MULTTEST in SAS v9.4 (SAS Systems). Contingency tables were produced using SAS and association between candidate SNPs and specific antibodies was tested using PLINK.RESULTS: Alloimmunization rates were different in the DU and CSSCD cohorts (30.7% vs. 18.9%, respectively; p = .0075) but were not associated with age or number of transfusions > 0 in either cohort. The most significant genetic associations with alloimmunization (FDR q = .28) occurred for 46 SNPs in the HLA locus and 10 other genes (Table 1). Two of these SNPs were missense mutations in GZMB (granzyme B (granzyme 2, cytotoxic T-lymphocyte-associated serine esterase 1)), one of which (rs8192917) has previously been associated with acute kidney transplant rejection and acute GVHD after stem cell transplant. A missense mutation in TRIM31 (E3 ubiquitin-protein ligase) was also associated with alloimmunization (p = .01). TRIM31 regulates Src-induced anchorage-independent cell growth and has been implicated in antiretroviral defense, cancer and ADHD. Within the DU cohort, we conducted a sub-analysis among only antigen-negative individuals, in whom immunization rates were 15.8%, 14%, 4.5%, 10.2%, 11%, and 8.3% to E, C, K, S, Jkb and Fya, respectively. Regression analysis of all 939 SNPs identified nominally significant associations predicting specific antibody alloimmunization. A missense mutation in PRKCQ (rs2236379) was associated with both anti-E and –C alloimmunization. Additional missense mutations were identified as follows: in TGFBR3 and GZMB with anti-C; in CFH, andTLR6 with anti-K, and in TLR10 with anti-Jkb.CONCLUSIONS: We have identified several immune response genes that are also associated with increased risk of alloimmunization in SCD (nominal p ≤ .006, FDR q = .28). Some associations may arise from the prevalence of alloimmunization to specific antigens. For example, PRKCQ was associated with development of alloantibodies to both E and C antigens, which reside on the same nonglycosylated RH protein. Expanded studies may identify those patients who would benefit from intensive or specific antigen-matching transfusion protocols. TableGenes Associated with AlloimmunizationGene or Locus (ordered by descending p value)SNPs with FDR q = 0.28 SNPs with p ≤ .006GZMB3 (2 missense)3TGFBR242TLR311HLA locus262TGFBR332PRKCQ3CD801VAV21TRIM312 (1 missense)RNF391ZNRD11 DisclosuresNo relevant conflicts of interest to declare.

(275) CYP2C8 allele and genotype frequencies in adult Sickle Cell Disease Patients: implications for NSAIDs metabolism

Nonsteroidal anti-inflammatory drugs (NSAIDs) used to treat prodromal signs of vasoocclusive pain in patients with sickle cell disease (SCD) are metabolized partly by the CYP2C8 enzyme. We determined the frequency of CYP2C8 alleles for the first time in adult African American SCD patients. Genomic DNA was isolated from blood samples of 165 patients aged between 18 and 61 years. Genotyping of seven CYP2C8 alleles (*1, *2, *3, *4, *5, *7 and *8) was performed using the iPlex ADME PGx panel and analyzed by iPlex Data Analysis Software. The CYP2C8*5,*7, and *8 alleles were not observed in our sample. The wild type *1 allele frequency was 0.806. The most common variant detected was the CYP2C8* 2 (0.164). The combined frequency of the *2, *3 and *4 variants was 0.194. The effects of CYP2C8*4 allele on substrate pharmacokinetics are unclear. In vitro data suggest that variant CYP2C8*2 and *3 alleles result in decreased metabolism of CYP2C8 substrates. However, in vivo data on the phenotypic consequences of these alleles have yielded contradictory results, problematizing assignment of extensive and intermediate predicted metabolic phenotypes (EM and IM) for some allelic combinations. We detected seven genotypes in our subjects as follows:*1/*1 (66.7%; EM), *1/*2 (24.2%; EM-IM), *1/*3 (2.4%; EM-IM), *1/*4 (1.2%; EM), *2/*2 (3.0%; IM), *2/*3 (1.2%; IM), and *1/*4 (1.2%;EM-IM). Our subjects had a disproportionately higher frequency of the CYP2C8*2 and *3 alleles in comparison to available CYP2C8 data for African Americans (0.182 vs. 0.110). This significant finding is similar to recent report that SCD patients had slightly higher frequency of the CYP2D6 gene deletion allele compared to healthy African Americans. Larger clinical studies should compare the frequency of impaired function alleles of CYP450 metabolic enzymes in SCD patients with other healthy African American groups to determine consequences on NSAIDs therapy and SCD pain phenotypes.

Association of Genetic Variation in the Catechol-O-Methyl Transferase Gene with Pain and Six Minute Walk Distance in Sickle Cell Anemia Patients From the Walk-PHaSST Study

Abstract 1075▪▪This icon denotes a clinically relevant abstract Background:Pain crisis is the hallmark of sickle cell disease (SCD) but varies in frequency and severity. Variation in pain sensitivity and perception among individuals is partially attributed to genetic factors. Catechol-O-methyltransferase (COMT) regulates neurotransmission and plays an important role in the perception of pain sensation. Three major COMT haplotypes are associated with COMT activity and correlate with high, average and low pain sensitivity (HPS, APS, and LPS) in Caucasians. However, the role of COMT genetic variants in SCD pain has not been explored. Methods:This study utilizes a cohort of African American patients with hemoglobin SS from the walk-PHaSST study (n=438). Pain phenotypes were obtained at the screening visit including description of pain, presence of chronic and acute pain, and associated treatments. Acute pain crises were classified as a) mild (may or may not require pain medicine and did not prevent normal daily activity), b) moderate (required medications and caused significant changes in daily activities, c) severe (patient went to the ER but was not admitted and d) extremely severe (required hospital admission). Patients also reported the number of each type of pain in the week, month and year prior to screening.Five single nucleotide polymorphisms (SNPs), rs6269, rs4633, rs4818, rs4680, and rs165599, of the COMT gene were genotyped. Haplotypes were constructed based on the SNP genotypes. Single SNP and haplotype associations with pain phenotypes and 6-minute walk distances (6MWD) were analyzed. Chi-square test and ANOVA or multinomial logistic regression analysis were used for categorical and continuous variables, respectively. Results:Of the 438 patients, the median age was 35 years (12 to 69 years) and 52% were females. Acute pain crisis was reported for 50%, 72%, and 91% of the subjects in the week, month, and year prior to screening. Over 36% of the patients reported chronic pain. The median intensity of acute and chronic pain on a scale of 1–10 was 8 and 6, respectively. Severe and extremely severe pain in the year prior to screening was not affected by age and gender. However, the self-reported use of hydroxyurea (HU) treatment was associated with pain episodes in the year prior to screening (p=0.0001). The least number of episodes was reported by patients who were never received HU, while the most number of episodes was reported by patients who were previously on HU. Subjects who reported current HU use had intermediate pain frequencies compared to the other two groups.The genotype frequencies of rs6269, rs4633, rs4818, rs4680 and rs165599 were in Hardy–Weinberg equilibrium (p<0.1) with minor allele frequency of 37.4%, 30.5%, 19.4%, 28.6% and 28.1%, respectively. Associations were identified for rs6269 and rs4633 with moderate pain in the week (p=0.0261) and month (p=0.0290) prior to screening, respectively. Chronic pain was associated with rs165599, and less patients experienced chronic pain for the GG genotype compared to AA or AG genotype carriers (34% vs 40%, p=0.0152). Differences were detected in chronic pain rate for the genotypes of rs6269 (p=0.0097) and rs4680 (p= 0.0719).Nine haplotypes with frequencies of >3% accounted for 97.6% of all haplotypes in this cohort, six of which corresponded to HPS, LPS and APS haplotypes described in Caucasians. The GCCGG haplotype that was not common in Caucasians had an 18.8% frequency in this African American SCD cohort. Haplotype ACCGA (HPS) was associated with severe pain in the month prior to screening (p=0.0330) and haplotype GCGGA (LPS) was associated with mild pain in the year prior to screening (p=0.0302). The degree of acute pain rate was associated with haplotype GCGGG (LPS) (p=0.0251).Although association between SNPs and 6MWD was not detected, haplotype ATCGG was significantly associated with the 6MWD. Twenty-nine percent of the patients with zero copies of this haplotype (114/399) walked >=500 meters while only 9% (3/33) of the subjects with this haplotypes were able to walk >=500 meters (p=0.0267). Conclusions:These data demonstrate that COMT variants are associated with pain phenotypes in SCD. Unique haplotypes and haplotype distributions were observed for the African American population. Further study of COMT and other genetic factors associated with pain in SCD may provide insights into the molecular pathobiology associated with SCD pain and ultimately lead to better SCD management. Disclosures:Barst:Actelion and Pfizer.: Consultancy. Hassell:NIH: Research Funding.

The Association of Optimism and Perceived Discrimination With Health Care Utilization in Adults With Sickle Cell Disease

We evaluated the effect of perceived discrimination, optimism, and their interaction on health care utilization among African American adults with sickle cell disease (SCD). Measures of optimism and perceived discrimination were obtained in 49 African American SCD patients. Multiple regression analyses controlling for sex and age tested effects of optimism and perceived discrimination on the number of emergency department visits (ED) and number and duration of hospitalizations over the past year. A perceived discrimination-optimism interaction was associated with number of emergency departments visits (b = .29, p = .052), number of hospitalizations (b = .36, p = .019), and duration of hospitalizations (b = .30, p = .045) such that those with high perceived discrimination/high optimism had the greatest health care utilization. African American SCD patients with high perceived discrimination/high optimism had greater health care utilization than patients who reported either low perceived discrimination or low optimism. This study suggests that patient personality and coping styles should be considered when evaluating the effects of stress on SCD-related outcomes.

RH genotyping in a sickle cell disease patient contributing to hematopoietic stem cell transplantation donor selection and management

AbstractAfrican individuals harbor molecular RH variants, which permit alloantibody formation to high-prevalence Rh antigens after transfusions. Genotyping identifies such RH variants, which are often missed by serologic blood group typing. Comprehensive molecular blood group analysis using 3 genotyping platforms, nucleotide sequencing, and serologic evaluation was performed on a 7-year-old African male with sickle cell disease who developed an “e-like” antibody shortly after initiating monthly red blood cell (RBC) transfusions for silent stroke. Genotyping of the RH variant predicted a severe shortage of compatible RBCs for long-term transfusion support, which contributed to the decision for hematopoetic stem cell transplantation. RH genotyping confirmed the RH variant in the human leukocyte antigen–matched sibling donor. The patient's (C)ces type 1 haplotype occurs in up to 11% of African American sickle cell disease patients; however, haplotype-matched RBCs were serologically incompatible. This case documents that blood unit selection should be based on genotype rather than one matching haplotype.

Primary stroke prevention for sickle cell disease in north-east Italy: the role of ethnic issues in establishing a Transcranial Doppler screening program

BackgroundStroke is a serious complication of sickle cell disease (SCD) in children. Transcranic Doppler (TCD) is a well-established predictor of future cerebrovascular symptoms: a blood flow velocity >200 cm/sec in the Middle Cerebral Artery (MCA) correlates with a high risk of stroke in cohorts of African-american HbS/HbS patients. In North-East Italy the recent increase in SCD patients is mainly due to immigration from Africa. A comprehensive care program for children with SCD was established in our Center since 2004, but a wide and routine screening for Primary stroke prevention needs to be developed.MethodsIn order to verify the feasibility of TCD and Transcranial color coded Sonography (TCCS) screening in our setting and the applicability of international reference values of blood velocities to our population of African immigrants with HbS/HbS SCD, we performed TCD and TCCD in 12 HbS/HbS African children and two groups of age-matched controls of Caucasian and African origin respectively. TCD and TCCS were performed on the same day of the scheduled routine hematologic visit after parental education.ResultsAll parents accepted to perform the sonography to their children. TCD and TCCD were performed in all patients and an adequate temporal window could be obtained in all of them. Pulsatility index and depth values in both the MCA and the Basilar Artery (BA) were similar at TCD and TCCS evaluation in the three groups while time-average maximum velocities (TAMM), peak systolic velocity and diastolic velocity in the MCA and BA were higher in the patients' group on both TCD and TCCS evaluation. African and Caucasian healthy controls had similar lower values.ConclusionOur preliminary data set the base to further evaluate the implementation of a primary stroke prevention program in our setting of HbS/HbS African immigrants and HbS/beta thalassemia Italians. Parental education-preferably in the native language- on stroke risk and prevention in SCD increases compliance and should be a necessary part of the program. Ethnic background does not seem to influence TCD velocity and internationally accepted reference values already validated in African-American SCD pediatric patients can be used, but long prospective trials are needed to verify their efficacy in defining stroke risk in our setting.

Defects in Postabsorptive Plasma Homeostasis of Fatty Acids in Sickle Cell Disease

The chronic hemolytic anemia experienced by sickle cell disease (SCD) patients leads to adverse effects on oxygen transport by the blood and to a decrease in oxygen availability for peripheral tissues. Limited tissue oxygen availability has the potential to modify events of intracellular metabolism and, thus, alter lipid homeostasis. The impact of SCD on plasma fatty acid homeostasis was determined in 8 African American SCD patients and in 6 healthy African American control subjects under postabsorptive conditions and during a 3-hour IV infusion of a nutrient solution containing lipid, glucose, and amino acids. SCD patients had higher fasting levels of plasma nonesterified fatty acids (NEFA), triglycerides, and phospholipids than healthy controls. Similarly, SCD patients had higher fasting levels of fatty acids in plasma triglycerides and phospholipids than healthy controls. Infusion of nutrients resulted in equivalent plasma NEFA profiles, total NEFA, and triglycerides in SCD patients and controls. However, the plasma phospholipid concentrations and fatty acid composition of plasma triglycerides and phospholipids were significantly higher in SCD patients; in particular, plasma pools of oleic acid were consistently increased in SCD. Plasma free oleic acid levels were elevated basally, leading to increased oleic acid content in triglycerides and phospholipids both post absorptively and during nutrient infusion. There is an underlying defect in lipid metabolism associated with SCD best manifested during the fasting state. This abnormality in lipid homeostasis has the potential to alter red blood cell (RBC) membrane fluidity and function in SCD patients.

Association of T-786C eNOS gene polymorphism with increased susceptibility to acute chest syndrome in females with sickle cell disease.

Acute chest syndrome (ACS) is a life-threatening complication of sickle cell disease (SCD). A retrospective study was performed to evaluate the role of endothelial nitric oxide synthase (eNOS) gene polymorphisms (E298D and T-786C) in African-American SCD patients. The D298 allele showed no association; the C-786 allele showed a statistically significant association (P = 0.0061) in female ACS cases. Multiple logistic regression analysis showed that relative risk of ACS was 8.695 (P = 0.0076, 95% confidence interval 1.761-42.920) for female carriers of C-786. eNOS T-786C is a gender-specific genetic modifier that is associated with increased susceptibility to ACS in female SCD patients.

Trabecular and integral bone density in adults with sickle cell disease.

Sickle cell disease (SCD) is a hereditary disorder of hemoglobin synthesis that can affect the skeletal system owing to accelerated hematopoiesis and/or bone infarction. Additionally, several studies have suggested that a low bone mass is associated with SCD, partly because of adverse effects on growth and development. The few previous studies of bone mineral density (BMD) in these patients have utilized dual-photon or dual-energy x-ray absorptiometric (DXA) techniques, which may have limited value in this population because it cannot correct for differences in bone size. We undertook a study of BMD in the forearm of patients with sickle cell disease using peripheral quantitative computed tomography (QCT), which provides a measure of volumetric bone density for the trabecular bone component as well as cortical and trabecular bone together. We studied 32 African-American SCD patients with no known history of bone infarction in the wrist, and compared them with data from healthy African-American volunteers. We found a 13% lower integral (cortical and trabecular) BMD in the SCD patients (p=0.001), but no difference in trabecular BMD (p=0.40 for males, 0.32 for females). We hypothesize that the maintenance of trabecular BMD in the wrist may be related to the persistence of metabolically active red marrow in this region in adults with SCD.

Protein turnover and energy expenditure increase during exogenous nutrient availability in sickle cell disease

In the present study, energy expenditure (EE) and rates of whole-body protein, glucose, and lipid metabolism were assessed in 8 African American sickle cell disease (SCD) patients and in 6 healthy African American control subjects during the infusion of amino acids, glucose, and lipid. Whole-body protein, glucose, and lipid kinetics were estimated by using L-[1-(13)C]leucine, D-[6,6-(2)H2]glucose, and [(2)H5]glycerol, respectively. After a 2-h tracer equilibration period and a 0.5-h basal period, nutrients were administered intravenously for 3 h with 16% of the energy as protein, 52% as carbohydrate, and 32% as fat. Breath and blood were collected during the last 30 min of nutrient infusion and EE was measured by indirect calorimetry. EE was 14% greater (P < or = 0.05) in SCD patients [145.0 +/- 3.5 kJ x kg fat-free mass (FFM)(-1) x d(-1)] than in control subjects (126.8 +/- 3.8 kJ x kg FFM(-1) x d(-1)). Whole-body protein breakdown (4.4 +/- 0.4 compared with 3.1 +/- 0.1 mg x kg FFM(-1) x min(-1), P < or = 0.05) and protein synthesis (4.6 +/- 0.4 compared with 3.2 +/- 0.1 g x kg FFM(-1) x min(-1), P < or = 0.05) were 42% and 44% greater, respectively, in the SCD patients than in control subjects, but whole-body amino acid oxidation (0.90 +/- 0.05 compared with 1.03 +/- 0.09 mg x kg FFM(-1) x min(-1)) was not significantly different between the 2 groups. Whole-body glucose and lipid kinetics did not differ significantly between the groups. EE increased in SCD patients during exogenous nutrient availability, and the additional energy required for the accelerated rates of whole-body protein breakdown and synthesis made a significant contribution to the increase in EE. These metabolic aberrations may increase the dietary energy and protein requirements of SCD patients.

Alterations in basal nutrient metabolism increase resting energy expenditure in sickle cell disease.

Basal rates of whole body protein, glucose, and lipid metabolism and resting energy expenditure (REE) were measured in eight African-American sickle cell disease (SCD) patients and in six African-American controls. Catheters were placed 1) in an antecubital vein for stable isotope infusion and 2) in a heated hand vein for arterialized venous blood. Breath and blood were collected during the last 30 min of the 2.5-h study, and REE was measured by indirect calorimetry. REE [128 +/- 5 vs. 111 +/- 1 kJ.kg fat-free mass (FFM)-1.day-1; P < 0.05 vs. controls] was 15% greater in the SCD patients. Whole body protein breakdown (5.0 +/- 0.3 vs. 3.8 +/- 0.2 mg.kg FFM-1.min-1; P < 0.05 vs. controls) and protein synthesis (4.4 +/- 0.3 vs. 3.2 +/- 0.2 mg.kg FFM-1.min-1; P < 0.05 vs. controls) were 32 and 38% greater, respectively, in the SCD patients, but whole body amino acid oxidation was similar (0.58 +/- 0.03 vs. 0.66 +/- 0.03 mg.kg FFM-1.min-1). Measures of whole body glucose and lipid metabolism were not significantly different between the groups. The additional energy required for the greater rates of whole body protein breakdown and synthesis caused by SCD contributes significantly to the observed increase in REE, suggesting that dietary energy and protein requirements are enhanced in SCD patients.