HbS Homozygotes Research Articles

Biomarker Signatures of Sickle Cell Disease Severity

▪Introduction. Sickle cell disease (SCD) is characterized by a point mutation in the β-hemoglobin gene. Phenotypically and genetically, SCD is heterogeneous. Genetic variants have been identified that correlate with fetal hemoglobin (HbF) levels and some disease complications. Scores reflecting disease severity have been proposed to stratify patients by risk of complications and typically they include a combination of laboratory data and patient medical history. Except for the relationship of variants in HbF-modulating genes and α thalassemia with some aspects of disease severity, predicting patients at higher risk for complications using only genetic data has been difficult. Between a genetic variant and a physiologic outcome, or genotype and phenotype, are the proteins whose effects can be captured by laboratory measures. We propose a new approach to identify patients with various risks for common complications using hierarchical cluster analysis of standardized blood biomarkers coupled with a new method to identify statistically significant clusters.Methods and Results. We selected 17 uncorrelated blood biomarkers measured in 2,320 SCD patients with the common β-globin genotypes of this disorder from the Cooperative Study of Sickle Cell Disease (CSSCD). The biomarkers were selected based on significant correlation with age and sex and included 4 previously validated markers of hemolytic severity. We used hierarchical clustering to generate 17 clusters characterized by specific patterns of the biomarkers; the Figure shows an example. Eight of the 17 clusters included more than 40 patients, and we tested whether patients stratified in these 8 clusters differed in the distribution of β-globin genotypes, and risk of painful episodes, seizure, stroke, and mortality in longitudinally collected data. Compared with the largest cluster of 675 patients (Cluster 2) that is characterized by all 17 blood biomarkers following the average distribution, we identified a cluster of 437 patients (Cluster 3) with elevated HbF, hemoglobin, reduced bilirubin, LDH and other biomarkers who had reduced risk for stroke (hazard ratio HR=0.17, p=0.018) and mortality (HR=0.27, p=0.00045). Another cluster of 341 patients (Cluster 4) was characterized by elevated HbF, LDH and other markers of hemolysis and had reduced risk for number of seizures (OR=0.2, p=0.02) and pain severity (OR=0.59, p=0.00041). Cluster 3 was characterized by 47% HbSC disease patients and approximately 16% HbS homozygotes who, by their inclusion in this cluster, were likely to have milder disease. Cluster 4 was characterized by 79% HbS homozygotes and 18% HbS homozygotes with α thalassemia. Compared to patients in Cluster 2, patients with a signature represented by Cluster 3 had reduced hemolysis after 1 year and maintained a significantly higher HbF, while patients with a signature of Cluster 1 had increased hemolysis after 1 year and lower HbF.For independent replication, we implemented a Bayesian classification rule to predict the cluster membership of patients enrolled in the PUSH and WalkPHASST studies of SCD using only the blood biomarker data. We had 16 of the 17 biomarkers (missing uric acid) measured at enrollment in the PUSH study and 13 of the 17 biomarkers (missing eosinophils, lymphocytes, monocytes, and uric acid) in the WalkPHASST study. Patients in the 2 studies grouped into clusters with profiles of biomarkers similar to those discovered in the CSSCD. The predicted clusters also had a similar distribution of SCD genotypes as in CSSCD.Discussion and Conclusions. We identified a subset of patients with blood biomarker signatures associated with a better prognosis and hypothesize that the small number of HbS homozygotes with a positive prognosis characterized by reduced morbidity and mortality could carry rare protective variants. Identifying these variants could lead to the discovery of new therapeutic targets. These variants might be discovered in analysis of whole genome sequences available in the PUSH and WalkPHAAST study, and confirmed in CSSCD patients where GWAS data is available. Another advantage of this method is that commonly available laboratory data can be used to stratify patients by risk of complications in time for preventive therapy or for enrollment in clinical trials. [Display omitted] DisclosuresGordeuk:Emmaus Life Sciences: Consultancy.

A Candidate Trans-Acting Modulator of Fetal Hemoglobin Gene Expression in the Arab-Indian Haplotype of Sickle Cell Anemia

In the Arab-Indian (AI) β-globin gene (HBB) haplotype of sickle cell anemia, fetal hemoglobin (HbF) levels are higher and the disease phenotype milder than in African HBB haplotypes. To study the genetic basis of HbF regulation in the AI haplotype, whole genome sequencing (WGS) was done in 14 unrelated Saudi AI haplotype HbS homozygotes; 7 selected for low HbF (7.9±1.9%) and 7 for high HbF (18.5±3.2%). WGS was also done in 3 Indians with the AI haplotype (HbF 26.0±4.5%), 3 African Americans with the Benin haplotype selected because of the uncommonly high HbF for this haplotype (19.8±0.4%) and 1 African American homozygote with the Senegal haplotype (HbF 16.0%). Association of SNPs with HbF was tested using simple linear regression with an additive genetic model, and the Sequence Kernel Association Test using sliding windows of 5 SNPs. We found 465 SNPs that were significantly associated with HbF in a single SNP analysis (p≤10-5). Following annotation using SNPper and ENCODE RegulomeDB, PLINK was employed to remove all SNPs in high linkage disequilibrium (r2>0.8) ending with 128 SNPs for follow-up analysis. These prioritized variants were further investigated using functional evaluation tools such as SCAN, Hembase, ErythronDB and the results of WGS were followed by genome-wide SNP analysis, imputation of genotypes to 1000genomes, and targeted direct genotyping in the following cohorts with sickle cell anemia: 110 AI haplotype cases (HbF 18.0±7.0%); 71 Saudi Benin haplotype cases (HbF 11.4±4.1%); 44 Indian AI haplotype homozygotes (HbF 23.0±4.8%); 894 African Americans with diverse HBB haplotypes (HbF 5.2±5.6%). All cases were examined when the HbF levels had stabilized. None of the patients were using hydroxyurea when HbF was measured. Based on WGS, 2 SNPs, rs4527238 and rs35685045 (D'=1) in intron 9 of ANTXR1 (anthrax toxin receptor 1, 2p13.1) remained associated with HbF after correction for multiple comparisons. This result was replicated in the cohort of 110 additional AI haplotype Saudi patients but not in cohorts with other HBB haplotypes. The alternative alleles of these 2 SNPs had nearly equal frequencies in Saudis with the Saudi AI haplotype and lower frequencies in Indians, Saudi Benin haplotype and African Americans with sickle cell anemia. The T allele of rs4527238 and the C allele of rs35685045 are associated with high HbF. ANTXR1 SNPs explained 10% of the HbF variability compared with 8% for BCL11A; these genes had independent, additive effects on HbF and explained about 15% of HbF variability. RNA sequencing and gene expression microarray analysis at 3 time points during the erythroid differentiation of CD34+ and iPSCs isolated from the same subjects showed that only the long splice variant of ANTXR1 was expressed. Expression increased over time in CD34+ erythroid cells and decreased over time in iPSC-derived erythroid progenitors in a pattern similar to BCL11A expression, with increasing expression as CD34+ cells matured and HbF expression fell and declining expression as iPSCs differentiated and HbF expression increased. Immunofluorescence co-staining for ANTXR1 and HbF during the erythroid differentiation of sickle iPSCs showed that reduced expression of ANTXR1 was paralleled by increased expression of HbF. By GTEx analysis, eQTLs at rs4527238 and rs35685045 showed a trend for increased expression according to genotype with rs4527238 T/T having the lowest level of expression and T/C and C/C genotypes, higher expression levels (p= 0.08) and for rs35685045, C/C the lowest expression levels and C/T and T/T higher expression levels (p= 0.07); rs4527238 and rs35685045 had no effect on the expression of BCL11A, a repressor of HbF expression located at 2p16 (p=0.8). STRING 9.1 predicted protein interactions of ANTXR1 directly with LRP6, HDAC2, BCLX and BRAC1 with the highest level of confidence (0.95) with LRP6. The HbF phenotype in AI haplotype sickle cell anemia is unique and ANTXR1 is the first trans-acting element associated with HbF whose effects appear to be limited to the Saudi AI haplotype. ANTXR1 might have dual effects on HbF, indirectly affecting hematopoiesis via interactions with LRP6 and Wnt signaling, and directly modulating HBG expression through its effects on HDAC2. A dual effect on HbF expression has been suggested for MYB. Whether targeting ANTXR1 is a therapeutic option will require further studies. DisclosuresNo relevant conflicts of interest to declare.

Haplotypes, Sub-Haplotypes and Geographical Distribution in Omani Patients with Sickle Cell Disease

Despite the fact that patients homozygous for the sickle cell disease (SCD) mutation have an identical genotype, the severity of the disease can be extremely variable. The hemoglobin (Hb) S mutation has been described on five different haplotypes with different clinical expression. Identifying the genotypes, haplotypes and sub-haplotypes of the β gene cluster in Oman needs to be studied in more details to establish a correlation between the genotype/haplotype and phenotype diversity observed in SCD patients for prognostic purposes, accurate diagnosis and thus planning for the best tailored treatment. We have investigated 125 HbS homozygotes from different parts of Oman and determined their haplotypes and sub-haplotypes and correlated this to the hematological and clinical expression. We have found 11 haplotype combinations differently distributed in the country, with the Asian/Asian HbS haplotype being the most predominant. Sub-haplotypes was only found among patients with CAR/OmanI haplotype. As expected, the correlation between haplotypes, sub-haplotypes and disease severity was mainly associated with HbF expression. Our study on haplotype/phenotype correlation has shown which major haplotypes occur in the different regions of Oman. Furthermore, neither the haplotype or sub-haplotype nor the HbF alone appeared to be fully associable with the variable clinical phenotypes. External factors do occur and are associated with the expression of the disease.

Genetic modulation of HbF in Brazilians with HbSC disease and sickle cell anemia

Sickle cell anemia (homozygosity for HbS) and HbSC disease (compound heterozygosity for HbS and HbC) are the most common genotypes of sickle cell disease (SCD). Fetal hemoglobin (HbF), the major genetic modulator of SCD, interferes with deoxyHbS polymerization [1]. Patients with HbSC disease have about half the number of complications as do HbS homozygotes [1], however, their mean HbF levels were 2.4% compared with about 6% in patients with sickle cell anemia [2]. Single nucleotide polymorphisms (SNPs) in quantitative trait loci cis and trans to the β-globin gene cluster are associated with HbF. We analyzed the association of SNPs in BCL11A, (chromosome 2p), in the HBS1L-MYB intergenic region (HMIP) (chromosome 6q), and in the 5′ olfactory receptor (OR) gene cluster of chromosome 11p with HbF in Brazilian patients with SCD. 622 patients were studied. They were not treated with hydroxyurea and were aged ≥6 years. Hemoglobin was analyzed by HPLC. SNPs rs766432 and rs6732518 in BCL11A; rs35959442 and rs11759553 in HMIP; and rs4910755 and 7483122 located in OR51B5-OR51B6 were genotyped by ABI TaqMan Assays. The βS and βC globin haplotypes were investigated with PCR and restriction fragment length polymorphism techniques. Mean HbF was compared according to genotype using the additive genetic model adjusting for age, gender and HbS homozygotes versus HbSC. A dominant genetic model adjusting for age, gender and SCD genotype was used to test the association SNPs with HbF. We tested to see if there was an association between HbF and SNPs in the OR gene cluster region after adjusting for age, gender, SCD genotype and the HBB gene cluster haplotype (Central African Republic [CAR] or Bantu versus absence of a CAR haplotype). To determine if there was a synergistic effect between the SNPs or if the effect exerted on HbF was independent, we developed multivariable linear regression models adjusting for age, gender and SCD disease status and ran stepwise linear regression analyses where we added the most significant SNPs from BCL11A, HMIP one-at-a-time. Data analysis was performed using R version 2.14.1 and the cubic root transformation of HbF [3]. 392 patients were HbS homozygotes (HbF 7.4±4.4%) and 230 had HbSC disease (HbF 2.5±2.3%). The association between HbF and SNPs in the BCL11A, HMIP and the OR gene cluster for all patients combined and for HbS homozygotes and HbSC patients separately are shown in Table I a-c (on-line supplementary material). In the combined group, there is a statistically significant association between HbF levels and both BCL11A SNPs and with HMIP SNP rs11759553 after adjusting for age, gender and the SCD genotype. As the number of minor alleles increase, so does HbF level; in HbS homozygotes a similar result was found; in HbSC disease both SNPs in BCL11A and HMIP were associated with HbF. The association between HbF levels and SNPs in the OR genes, after adjusting fo r HBB haplotype was not significant (Table II, on-line supplementary material). In a stepwise linear regression analysis adding the SNPs to a multivariable linear regression model as described, after adjusting for age, gender and SCD genotype rs35959442 is still significantly associated with HbF indicating that its effect on HbF is independent of rs766432 (Table III, on-line supplementary material). We dichotomized the patients according to the HBB gene cluster haplotype. Brazilians with SCD have a higher prevalence of the CAR haplotype compared with African Americans. This haplotype is associated with lower levels of HbF than other haplotypes [4][5]. Sickle cell anemia patients were heterozygous or homozygous for the CAR haplotype chromosome or lacked a CAR haplotype chromosome; HbSC disease patients had or did not have a CAR haplotype chromosome. Most patients with a CAR haplotype had the minor allele for rs4910755 and rs7483122, both of which are located in the 5′ OR gene cluster of chromosome 11p and could be in linkage disequilibrium with SNPs characterizing this haplotype. HbSC disease patients usually have low HbF levels. Nevertheless, it is a milder condition than sickle cell anemia even though patients are more susceptible to clinical events related to high bloodviscosity. Although HbF in HbSC disease is lower than in HbS homozygotes, BCL11A and HMIP modulate HbF in both genotypes.

Fetal hemoglobin in sickle cell anemia: Genetic studies of the Arab-Indian haplotype

Sickle cell anemia is common in the Middle East and India where the HbS gene is sometimes associated with the Arab-Indian (AI) β-globin gene (HBB) cluster haplotype. In this haplotype of sickle cell anemia, fetal hemoglobin (HbF) levels are 3-4 fold higher than those found in patients with HbS haplotypes of African origin. Little is known about the genetic elements that modulate HbF in AI haplotype patients. We therefore studied Saudi HbS homozygotes with the AI haplotype (mean HbF 19.2±7.0%, range 3.6 to 39.6%) and employed targeted genotyping of polymorphic sites to explore cis- and trans- acting elements associated with high HbF expression. We also described sequences which appear to be unique to the AI haplotype for which future functional studies are needed to further define their role in HbF modulation. All cases, regardless of HbF concentration, were homozygous for AI haplotype-specific elements cis to HBB. SNPs in BCL11A and HBS1L-MYB that were associated with HbF in other populations explained only 8.8% of the variation in HbF. KLF1 polymorphisms associated previously with high HbF were not present in the 44 patients tested. More than 90% of the HbF variance in sickle cell patients with the AI haplotype remains unexplained by the genetic loci that we studied. The dispersion of HbF levels among AI haplotype patients suggests that other genetic elements modulate the effects of the known cis- and trans-acting regulators. These regulatory elements, which remain to be discovered, might be specific in the Saudi and some other populations where HbF levels are especially high.

A functional promoter polymorphism of the δ‐globin gene is a specific marker of the Arab‐Indian haplotype

A functional promoter polymorphism of the δ‐globin gene is a specific marker of the Arab‐Indian haplotype

Sickle cell trait is not independently associated with susceptibility to end-stage renal disease in African Americans

Conflicting reports exist as to whether sickle cell trait is a risk factor for the progression of nephropathy. In order to determine whether African Americans with sickle cell trait are at increased risk for kidney disease, we assessed the genetic association between sickle cell trait and end-stage renal disease (ESRD). Hemoglobin S, non-muscle myosin heavy chain 9 (MYH9), and apolipoprotein L1 (APOL1) risk variants were genotyped in 3258 unrelated African Americans: 1085 with non-diabetic ESRD, 996 with type 2 diabetes-associated ESRD, and 1177 controls. Since APOL1 is strongly associated with ESRD in African Americans, interactions between APOL1 and MYH9 risk variants and hemoglobin S were assessed using case-only and case-control centered two-way logistic regression interaction analyses. The sickle cell trait genotype frequencies were 8.7% in non-diabetic ESRD, 7.1% in type 2 diabetes-ESRD, and 7.2% in controls. There was no age-, gender-, and admixture-adjusted significance for sickle cell trait association with non-diabetic ESRD (odds ratio 1.16); type 2 diabetes-ESRD (odds ratio 1.01); or all-cause ESRD (combined non-diabetic and type 2 diabetic-ESRD patients compared to the controls; odds ratio 1.05) in dominant models. In addition, no evidence of APOL1 or MYH9 interactions with sickle cell trait was detected. Hence, sickle cell trait is not associated with diabetic or non-diabetic ESRD in a large sample of African Americans.

Co-Inheritance of Delta Thalassemia Might Contribute to the High Fetal Hemoglobin in Sickle Cell Anemia Patients with the Saudi-Indian Haplotype

Abstract 1056Most sickle cell anemia patients (HBB glu6val homozygotes) indigenous to the Eastern Province of Saudi Arabia have a fetal hemoglobin (HbF) level of about 20% that is associated with a mild clinical phenotype. Their HbS gene is on the Saudi-Indian (SI) haplotype characterized by an Xmn1 restriction site at position −158 5’ to HBG2 (rs7482144), a Hinc2 site 5’ to HBE (rs3834466) and other polymorphisms in the HBB gene-like cluster. However, the functional elements within the HBB gene-like cluster and elsewhere in the genome causing high HbF are yet to be determined. In a previous study we found that Saudi HbS homozygotes with the SI haplotype had a common region of autozygosity that spanned about 126 kb and included the complete HBB cluster. We have sequenced 13.6 kb in the HBD-HBG1 intergenic region (chr11:5255683-5269326, HG19), the region of the Corfu deletion. We found a SNP at position −68 bp 5’ to HBD (c.-118 C>T) only in individuals with a SI haplotype. This SNP was not present in dbSNP build 132 or the 1000 Genomes databases. No other mutation in HBD was identified. This same SNP was recently associated with δ thalassemia (Phylipsen et al. Int. J. Lab. Hematol. 2011, 33: 85–91). Homozygotes for the −68 HBD SNP, who were not on hydroxyurea, had a mean HbF of 23%, range 12.1%-33.4% and mean HbA2 of 2.1%, range 1.2%-3%. We did not find the −68 HBD SNP in 15 African Americans with sickle cell anemia selected because of their unusually high HbF (mean HbF 17.2%, range 11%-28.9%). Parents and sickle cell trait carriers from the families of Saudi Eastern Province patients were heterozygous for the −68 HBD mutation (mean HbF 1.6%, range 0–4.2% and mean HbA2 2.7%, range 2.4%-3.2%) and one normal sibling did not carry this mutation (HbF 0 and HbA2 2.9%). Thirty patients with sickle cell disease indigenous to the Southwestern Province of Saudi Arabia, with the HbS gene on an African origin haplotype, (mean HbF 15.5%, range 4.5%-23% and mean HbA2 2.9%, range 2.1%-3.4% in HbS homozygotes) and 13 normal Saudi controls were examined and none carried the −68 HBD mutation. We also sequenced HBD and its promoter in 8 Southwestern Province HbS homozygotes with a Benin haplotype, 4 with HbF <5% and 4 with HbF >15%, and none had the −68 HBD SNP or any other mutation in HBD. Inverse relationships between percent HbF level and percent HbA2 was seen in 3 groups of HbS homozygotes:1) SI haplotype homozygotes; 2) Benin haplotype homozygotes from the Southwestern Province; 3) African Americans with diverse HbS haplotypes. The increased HbF in sickle cell anemia with δ thalassemia might involve both post-translational and transcriptional mechanisms. Increased HbF levels might in part be due to the preferential post-translational assembly of αγ dimers compared with αδ and αβS dimers. When δ thalassemia reduces available δ-globin chains, this might further favor preferential binding of less positively charged γ-globin subunits to positively charged α-globin compared with more positively charged δ-globin subunits. Perhaps more importantly, at the transcriptional level, the −68 HBD δ+-thalassemia promoter mutation might favor the interactions of transcription complexes with HBG promoters. The stimulus of hemolytic anemia and expanded erythropoiesis might be required before the favorable genetic milieu for increased HbF production can be fully utilized with the achievement of clinically significant increases in HbF that modulates the course of disease. For example, increased HbF levels of only 3.3%-4.7% have been reported in some hematologically normal individuals with homozygous δ0 thalassemia (Ohta et al. Hemoglobin 1980, 4: 417–425). High HbF levels in SI haplotype HbS homozygotes might involve the interactions of one or more HBG regulatory regions linked to the HBB gene-like cluster, like the −68 HBD SNP, perhaps with trans acting elements. Although the −68 HBD δ+-thalassemia mutation is associated with the SI haplotype and high HbF, functional studies are needed to establish causation. Disclosures:No relevant conflicts of interest to declare.

Fetal hemoglobin in sickle cell anemia: Saudi patients from the Southwestern province have similar HBB haplotypes but higher HbF levels than African Americans

Patients with sickle cell disease (SCD) from the Southwestern (SW) Province of Saudi Arabia have variable fetal hemoglobin (HbF) levels and have HBB gene cluster haplotypes of African origin. We studied 77 patients, aged 17.7 ±10 (range 4-46) years (69% HbS homozygotes and 31% HbS-β 0 thalassemia), to determine the associations of known HbF quantitative trait loci (QTL) with HbF concentration. HBB gene cluster haplotypes were 74% Benin, 22% Bantu, and 4% others. Genotyping Single nucleotide polymorphism (SNPs) in BCL11A, HBS1L-MYB, and OR51B5/6 showed that BCL11A was the sole QTL associated with HbF level. We compared these findings with two studies of African American with SCD. After adjusting for the BCL11A genotype, Saudi cases from the SW Province had HbF levels almost twice that of African Americans (P < 0.0001). When we examined the genetic population structure of the African Americans and Saudi patients using genome-wide data, we found that African Americans were similar to Yoruban, Mandenka, and Bantu Africans while Saudi patients resembled Arab populations. The commonality of HBB haplotypes coupled with the genetic distance between these populations suggests that genetic modifiers remote from the HBB cluster or unknown environmental influences are likely to account for the higher HbF in these Saudi patients.

Fetal Hemoglobin In Sickle Cell Anemia: Molecular Characterization of Saudi Patients From the Southwestern Province.

AbstractAbstract 1621Sickle cell disease (SCD) is common in the Eastern and Southwestern (SW) Provinces of Saudi Arabia. Patients from the SW Province have many complications of this disease but they have phenotypic differences from African Americans that include a high prevalence of splenomegaly, rare CNS disease and absence of leg ulcers. In contrast, Eastern Province patients have a milder phenotype that is related to a nearly uniformly high fetal hemoglobin (HbF) level that is associated with the Saudi-Indian haplotype of the HBB gene-like cluster. SW Province patients have variable HbF levels and do not have the Saudi-Indian haplotype. We studied patients from the SW Province to determine the associations of known HbF quantitative trait loci (QTL) with HbF concentration. Seventy-seven Saudi patients, aged ≥4 yrs, with SCD were studied, 53 HbS homozygotes and 24 with HbS-β0 thalassemia. Their age was 17.7±10 (range 4–46) yrs and 45% were on hydroxyurea. HBB gene cluster haplotypes were 58 (75%) Benin, 17 (22%) Bantu, 1(1.5%) Senegal/Bantu, and 1(1.5%) Senegal. HbF level was measured by capillary electrophoresis and we used the lowest HbF level after age 4 yrs, the age where HbF levels stabilized, for analysis. Genotyping was done by TaqMan SNP genotyping assays, and included SNPs in BCL11A (rs4671393, rs766432), HBS1L-MYB (rs28384513, rs9399137, rs4895441), and OR51B5/6 (rs5006884). Results are summarized in the Table. QTLs showed similar trend in their effects on HbF level when patients on hydroxyurea were excluded from analysis. BCL11A was the sole QTL associated with HbF level in Saudi patients from the SW Province whereas the HBS1L-MYB and OR51B5/6 loci had no effect. For comparison, we selected cases from 2 studies of African American with SCD. All were HbS homozygotes and none took hydroxyurea, and we compared them to Saudi patients who were HbS homozygotes and not on hydroxyurea (n=29). Compared with African Americans with similar HBB haplotypes, and after adjusting for the BCL11A genotype, Saudi cases from SW Province had HbF levels almost twice that of African Americans (p <0.0001). Given that Saudi and African American patients had a nearly identical distribution of HBB gene cluster haplotypes, we examined ancestral origin of Saudi and African American patients. Using a genome-wide set of SNPs, we performed a principal component analysis with the Saudi, the African Americans with SCD and African, Arab, Asian and European populations from the Human Genome Diversity Project. We estimated the Fst statistic between these populations which is defined as the proportion of genetic diversity due to allele frequency differences among populations and can be interpreted as the distance between populations. African American cases were in close proximity to Yoruban, Mandenka and Bantu populations while Saudi patients resembled Arab populations. African American patients were the farthest from Saudi patients as compared with Asian and European populations. The commonality of HBB haplotypes in these Saudi cases and African Americans, coupled with the genetic distance between these populations suggest that genetic modifiers remote from the HBB cluster or unknown environmental influences are likely to account for the higher HbF in these Saudi patients. Disclosures:No relevant conflicts of interest to declare.

Characterization of HbF Decline In Compound Heterozygotes for HbS and Deletional Hereditary Persistence of Fetal Hemoglobin

AbstractAbstract 1626In gene deletion hereditary persistence of fetal hemoglobin (HPFH) affecting people of African descent, more than 80 kb of DNA is deleted including the δ- and β-globin genes and downstream sequences. Compound heterozygotes for sickle hemoglobin (HbS) and HPFH have high fetal hemoglobin (HbF) levels and have few if any sickle cell disease-related complications. Among patients with sickle cell disease who were referred for hemoglobin diagnostic testing, 28 compound heterozygotes for HbS and HPFH (6 HPFH type 1 and 22 HPFH type 2) were identified. The diagnosis of HbS and HPFH 1 and 2 were confirmed by DNA-based specific gap-PCR tests. We characterized factors associated with HbF and its changes over time to better understand the modulation of HbF in this population. Laboratory information were obtained through chart review and/or extracted from information provided at the time of hemoglobin diagnostic testing. Data regarding age at which HbF was measured allowed us to study the change in HbF over time. For comparison, we analyzed data collected through the Cooperative Study of Sickle Cell Disease (CSSCD) containing over 3000 HbF measurements at different ages in HbS homozygotes (HbSS) or HbS-β0 thalassemia patients. Statistical analyses were performed using Stata/SE11.1 software. In 28 HbS-HPFH cases, 62 HbF values at different ages ranging from the newborn period to age 20 years were available for analysis. In these patients, HbF was 50% to 90% during infancy and declined steeply within the first few years of life, stabilizing between ages 3 to 5 years. Mean HbF at age 5 or older was 31 ± 2%. Mean hemoglobin concentration was 13 ± 1g/dL and mean MCV was 75 ± 6 fL. Age was log-transformed for all analyses. In univariate and multivariate regression analyses, HbF was significantly associated with age, hemoglobin concentration, and MCV (P<0.001). There was no relationship with gender or genotype of HPFH. There was a strong inverse correlation between HbF and age (r=-0.9, P<0.001). Likewise, in patients with HbSS or HbS-β0 thalassemia from the CSSCD database, HbF was significantly associated with age, hemoglobin concentration, and MCV. In the CSSCD population alone, HbF was also associated with female gender. Patients with compound heterozygosity for HbS and HPFH have a logarithmic age related decline in HbF with marked decline during the first few years of life and plateauing at a HbF of approximately 30% in adulthood. Although these patients maintained a higher HbF after early childhood, their HbF is subjected to a similar age-related decline as seen in patients with other genotypes of sickle cell disease. Future studies aimed at understanding age related regulation and distribution of HbF in these and other patients may offer insight into mechanisms that can halt decline of HbF and ameliorate sickle cell disease. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

The Frequency of Glucose-6-Phosphate Dehydrogenase Phenotypes and Sickle Cell Genes in Al-Qatif Oasis

This study was conducted on a selected population of Al-Qatif in Eastern Saudi Arabia to determine the gene frequencies of glucose-6-phosphate dehydrogenase (G-6-PD) deficiency and sickle cell (HbS) genes and to study the extent of interaction between the two genes. A total of 960 blood samples collected from Saudi males (515) and females (445) attending the outpatient clinics and hospitals for minor illnesses were subjected to electrophoresis for separation of hemoglobin types and G-6-PD phenotyping and for spectrophotometric determination of G-6-PD activity. The prevalence of HbS heterozygotes was 25.9%, Hb S/b_-thalassemia was 1.563% and HbS homozygotes was 2.917%. The overall gene frequency of HbS was 0.1666. Severe glucose-6-phosphate dehydrogenase deficiency was encountered in a large percentage of the population with a frequency of 0.392 for G-6-PD Mediterranean and 0.0058 for G-6-PD-A- in the male population and 0.2020 and 0.0112 for G-6-PD Mediterranean and G-6-PD-A- in the female population respectively. Partial deficiency was encountered at a frequency of 0.0272 and 0.0697 in the male and female populations respectively. G-6-PD deficiency caused by G-6-PD Mediterranean occurred at a higher frequency in individuals with normal hemoglobin (Hb AA) (0.414 and 0.217 in males and females respectively) compared to the HbS heterozygotes (0.338 and 0.168 in males and females respectively) and HbS homozygotes and HbS bo-thalassemia cases (0.3125 and 0.1852 in males and females respectively). Compared to all areas of Saudi Arabia, Al-Qatif had the highest gene frequencies for HbS and G-6-PD deficiency genes.

Conjunctival vessel abnormalities in sickle cell diseases: the influence of age and genotype

Conjunctival vessels were examined with a slit-lamp and the abnormalities were graded in 77 patients with sickle cell diseases, including 48 with sickle cell anaemia, 17 with sickle cell beta (0)-thalassaemia and 12 with HbS/HbC disease. The study demonstrated the occurrence of similar conjunctival vessel abnormalities for the three sickle cell diseases which are, however, more severe in HbS homozygotes than in the two other genotypes. An increase of the severity of the vascular abnormalities with the increase of patient age was also observed for HbS homozygotes. The data suggest that the conjunctival lesions accumulate and increase in severity in a similar fashion as other tissue damages observed in sickle cell diseases.

Splenic Function in Sickle-Cell Diseases

Studies of splenic function were carried out on patients with sickle-cell diseases by the measurement of the clearance of autologous heat-damaged 99mTc-labelled erythrocytes from circulation and into the spleen, the spleen area by a scintillation scanning, the enumeration of pitted erythrocytes by direct-interference microscopy, and the percentage of irreversibly sickled cells (ISC) and of cells with Howell-Jolly bodies. All measurements were performed in seven HbS homozygotes, 10 patients with sickle cell beta(0)-thalassaemia (S/beta(0)-thalassaemia), three patients with sickle-cell disease (SC), four AS heterozygotes and 17 controls. Three different patterns of splenic function were observed among the 20 patients with symptomatic sickle-cell diseases: six patients had enlarged hyperactive spleens, four had enlarged hypoactive spleens, and in 10 patients no splenic activity was detected. The percentage of ISC was higher in sickle-cell anaemia than in S/beta (0)-thalassaemia and very low in SC patients. These results would suggest that the spleen goes through similar successive functional stages in the sickle-cell diseases, namely enlargement in the early years of life, which is followed by hypoactivity and finally atrophy. This evolution seems to be faster in sickle-cell anaemia than in S/beta(0)-thalassaemia and SC disease.

Proportion of glycosylated hemoglobin in erythrocytes of a person with hemoglobins A, S, and G Philadelphia.

The apparent proportion of column-chromatographically measured glycosylated hemoglobin in erythrocytes from an individual with hemoglobins A, S, and G was only 2.3% because the slow glycosylated variant hemoglobins were retarded in the column. In contrast, the value for glycosylated hemoglobin was 7.8% by a new cellulose acetate electrophoretic method that includes use of dextran sulfate buffer. The erythrocyte metalloprotein, carbonic anhydrase B, was shown to co-migrate with glycosylated hemoglobin by this technique, however. Thus carbonic anhydrase B, HbF, and HbA all have weak attraction for negative charges at acid pH. We believe that carbonic anhydrase B should contribute significantly (at least 10-20% absorbance) to the HbA1 by this electrophoretic method. We conclude that microcolumn chromatography should remain the method of choice for HbA1 determination, that subject-based reference intervals should be used for HbS or HbC heterozygotes, and that electrophoretic HbA1 methods should be reserved for use with both HbS and HbC homozygotes and HbSC disease.

On the Levels of Hemoglobins F and A2 in Sickle-cell Anemia and Some Related Disorders

Results of analyses of Hb-A2 and/or Hb-F levels in more than 300 Negro subjects with sickle-cell anemia, the two forms of Hb-S- β - thalassemia, Hb-S trait, Hb-SC disease, the Hb-S hereditary persistence of Hb-F (HPFH) condition, /3-thalassemia trait, and the HPFH heterozygosity are presented. Ages of the patients ranged from 1 to 70 years; several patients were studied repeatedly over long periods. Previously published data indicate that the levels of Hb-A2 and Hb-F in normal individuals range from 2.0 to 3.0% and from 0.2 to 1.0%, respectively. In subjects with a sickle-cell trait the level of Hb-A2 is slightly higher but the level of Hb-F does not differ from that of normal adults. Conclusions from recent observations are: (1) Sickle-cell anemia. The level of Hb-A2 is slightly higher than in normal adults and that of Hb-F ranges from 1 to 20%. No significant difference between the Hb-F levels of male and female patients of different ages has been noted. The level of Hb-F tends to remain constant in subjects 5 years old and older. (2) Hb-S- β - thalassemia. The level of Hb-A2 in either Hb-S- β +-Th or Hb-S- β °-Th is elevated to the same extent as in β -thalassemia heterozygotes. In a few cases a Hb-A2 level that did not differ from that seen in many Hb-S homozygotes was observed. The level of Hb-F in the two conditions does not differ from that observed in sickle-cell anemia. Definite diagnosis of Hb-S- β °-Th requires careful hematologic evaluation and family studies. The presence of small amounts of Hb-A aids in the diagnosis of the Hb-S- β +-Th condition. (3) Hb-S-HPFH. The level of Hb-A2 is decreased and that of Hb-F usually above 20%. These observations, together with findings indicating minimal hematologic abnormalities and an equal distribution of Hb-F within the erythrocytes, make the diagnosis of this condition possible.