Exome Sequencing of Extreme Phenotypes Suggests Novel Candidate Genes As Modifiers of Leg Ulcer in Sickle Cell Anemia

Abstract Background: Despite resulting from homozygosity of a single mutation at position 6 of the beta-globin locus, the clinical aspects of sickle cell anemia (SCA) are very heterogeneous. Leg ulcer is one of the many resulting complications, exerting a quite negative impact in the quality of life of patients and is related to the severity of the disease. The pathogenesis of such complication is complex and still not well explained. Objective: Seeking for novel genes associated with leg ulcer in SCA, we proposed exome sequencing of extreme phenotypes in a sample of the Brazilian population. Methods: Our sample consisted of 40 unrelated patients with SCA from the Hematology and Hemotherapy Center of the University of Campinas (Unicamp), Campinas, SP, Brazil. The cohort was composed by 20 patients who didn't have leg ulcer, with ages varying from 40 to 61 years (it is unusual the appearance of this disorder at such age) and 20 patients with chronic leg ulcer. DNA was collected from peripheral blood leukocytes and submitted to exome sequencing. Capture and enrichment were performed with the Nextera Rapid Capture Exome kit (Illumina) and samples were loaded in the Illumina HiSeq2500. The bioinformatics process was based on the GATK Pipeline and manual filters in vcftools and excel. Briefly, we prioritized the variants according to some conditions, like score quality, depth, Hardy-Weinberg equilibrium and minor allele frequency greater than 0.1. Subsequently, through PLINK, we selected the variants with p value less than 0.05 from Fisher association test. The annotation was made through wAnnovar. We excluded the synonymous variants, remaining with 244 variants at this point. Finally, the deleterious variants were selected, in addition to those not classified by SIFT, PolyPhen2 and FATHMM. Twenty-one variants remained, all of them in genes beyond the usual set of a priori biological candidate genes for this phenotype. We picked up 6 variants with the lowest p values: rs4857302 (CRYBG3), rs3782489 (KRT77), rs11800462 (TNFRSF25), rs13428956 (FOXD4L1), rs201853154 (UBTFL1) and rs11454536 (VWDE). We suggest that these variants and their genes could potentially be related to the development of leg ulcer in SCA. However, they should all be validated in other populations for confirmation. Disclosures No relevant conflicts of interest to declare.

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Whole-exome sequencing indicates FLG2 variant associated with leg ulcers in Brazilian sickle cell anemia patients

Experimental Biology and Medicine ◽

10.1177/1535370219849592 ◽

2019 ◽

Vol 244 (11) ◽

pp. 932-939 ◽

Cited By ~ 1

Author(s):

Gabriela Queila de Carvalho-Siqueira ◽

Galina Ananina ◽

Bruno Batista de Souza ◽

Murilo Guimarães Borges ◽

Mirta Tomie Ito ◽

...

Keyword(s):

Quality Of Life ◽

Exome Sequencing ◽

Whole Exome Sequencing ◽

Sickle Cell ◽

Sickle Cell Anemia ◽

Leg Ulcer ◽

Leg Ulcers ◽

Whole Exome ◽

Extreme Phenotypes

Although sickle cell anemia results from homozygosity for a single mutation at position 7 of the β-globin chain, the clinical aspects of this condition are very heterogeneous. Complications include leg ulcers, which have a negative impact on patients’ quality of life and are related to the severity of the disease. Nevertheless, the complex pathogenesis of this complication has yet to be elucidated. To identify novel genes associated with leg ulcers in sickle cell anemia, we performed whole-exome sequencing of extreme phenotypes in a sample of Brazilian sickle cell anemia patients and validated our findings in another sample. Our discovery cohort consisted of 40 unrelated sickle cell anemia patients selected based on extreme phenotypes: 20 patients without leg ulcers, aged from 40 to 61 years, and 20 with chronic leg ulcers. DNA was extracted from peripheral blood leukocytes and used for whole-exome sequencing. After the bioinformatics analysis, eight variants were selected for validation by Sanger sequencing and TaqMan® genotyping in 293 sickle cell anemia patients (153 without leg ulcers) from two different locations in Brazil. After the validation, Fisher’s exact test revealed a statistically significant difference in a stop codon variant (rs12568784 G/T) in the FLG2 gene between the GT and GG genotypes ( P = 0.035). We highlight the importance of rs12568784 in leg ulcer development as this variant of the FLG2 gene results in impairment of the skin barrier, predisposing the individual to inflammation and infection. Additionally, we suggest that the remaining seven variants and the genes in which they occur could be strong candidates for leg ulcers in sickle cell anemia. Impact statement To our knowledge, the present study is the first to use whole-exome sequencing based on extreme phenotypes to identify new candidate genes associated with leg ulcers in sickle cell anemia patients. There are few studies about this complication; the pathogenesis remains complex and has yet to be fully elucidated. We identified interesting associations in genes never related with this complication to our knowledge, especially the variant in the FLG2 gene. The knowledge of variants related with leg ulcer in sickle cell anemia may lead to a better comprehension of the disease’s etiology, allowing prevention and early treatment options in risk genotypes while improving quality of life for these patients.

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Leg Ulcers in Sickle Cell Anemia Are Associated with Laboratory Markers of Hemolysis and SNPs in KL and Genes of the TGF-β/BMP Pathway.

Blood ◽

10.1182/blood.v106.11.2317.2317 ◽

2005 ◽

Vol 106 (11) ◽

pp. 2317-2317

Author(s):

Vikki G. Nolan ◽

Adeboye H. Adewoye ◽

Clinton T. Baldwin ◽

Qian-Li Ma ◽

Diego F. Wyszynski ◽

...

Keyword(s):

Sickle Cell Disease ◽

Candidate Genes ◽

Sickle Cell ◽

Sickle Cell Anemia ◽

Receptor Activation ◽

Leg Ulcer ◽

Leg Ulcers ◽

Cell Disease ◽

P Values ◽

Bmp Pathway

Abstract Sickle cell disease patients have a high risk of developing leg ulcers. In the CSSCD database we found 378 patients with a confirmed history of leg ulcers and chose 920 patients without ulcers to serve as controls. α Thalassemia reduces hemolysis in sickle cell anemia which is consistent with our finding using an age-adjusted comparison, that sickle cell anemia-α thalassemia was more frequent among controls than cases (OR: 0.7, 95% CI: 0.5–0.9). This suggests that sickle cell anemia-α thalassemia patients are significantly less likely to have leg ulcers. Also, leg ulcer patients had lower hemoglobin levels, higher LDH, bilirubin, AST, reticulocyte and white blood cell count than controls. These differences were highly statistically significant (p < 0.001, except for AST, whose p was <0.005) and support the impression that cases have a higher rate of hemolysis than controls. Fetal hemoglobin was higher in controls compared with cases (p=0.002). The haplotype of the β-like globin gene cluster, creatinine and ALT were not associated with leg ulcers. Leg ulcers were associated with other clinical manifestations of sickle cell disease like ischemic stroke (OR:1.4, 95% CI: 0.9–2.0) and acute chest syndrome (OR:1.5, 95% CI:1.1–1.9). When analysis of laboratory data was restricted to the 759 patients (243 cases vs. 516 controls) that were also genotyped for SNPs in candidate genes, the results were similar. We studied 132 SNPs in 47 candidate genes for their association with leg ulcers. SNPs were studied by mass spectrometry in a screening phase and by using haplotype tagging (ht) SNPs and the ABI SNPlex for follow-up. Candidate genes included: mediators of inflammation; oxidant injury; NO biology; vasoregulation; cell-cell interaction; blood coagulation; hemostasis; growth factors; cytokines and receptors. The candidate genes having multiple SNPs associated with leg ulcers were KL (rs685417 and rs516306; p values <0.02), TEK (rs603085 and rs671084; p values <0.025), SMAD1 (rs1899784, rs10519733 and rs2068991; p values <0.05), and SARA1 (rs2271690 and rs870801; p values <0.04). KL directly or indirectly promotes endothelial NO production. The TEK receptor tyrosine kinase (TIE2) is expressed almost exclusively in endothelial cells, is involved in angiogenesis and is the ligand for angiopoietin-1 (ANG1). SMAD1 and SARA1 are members of the TGF-β/BMP pathway. The TGF-β receptor signals through the SMAD family of transcriptional regulators that are anchored to the cell membrane by factors like SARA (SMAD Anchor for Receptor Activation). This pathway modulates immunosuppression, cell migration, wound healing and angiogenesis, among its other functions. Hemolysis is likely to be an antecedent of certain vascular complications of sickle cell disease, like pulmonary hypertension This, and the current studies, suggest that there is a hemolysis-driven phenotype in sickle cell disease that now includes leg ulcers. While candidate genes that might modulate the rate of hemolysis have not yet been studied, we find that SNPs in genes that may be pathogenetically important in sickle vasculopathy are associated with the disease subphenotype of leg ulcer. Linking gene polymorphisms with disease subphenotypes, may eventually provide useful means of foretelling the likelihood of complications and allow better individualized treatment.

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Longitudinal Analysis of Hydroxyurea and Fetal Hemoglobin in Adult Patients with Sickle Cell Anemia

Blood ◽

10.1182/blood-2021-153890 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 2054-2054

Author(s):

Aryeh Pelcovits ◽

Giancarlo Riotto ◽

Katie Cherenzia ◽

Patrick T. McGann ◽

John L Reagan

Keyword(s):

Sickle Cell ◽

Sickle Cell Anemia ◽

Conflicts Of Interest ◽

Surrogate Marker ◽

Fetal Hemoglobin ◽

P Value ◽

Significant Difference ◽

Dosing Strategies ◽

Over Time ◽

Painful Crisis

Abstract Introduction: Hydroxyurea (HU) was the first FDA-approved therapy for sickle cell anemia (SCA) and remains the most well-established disease-modifying therapy, reducing painful crisis and improving morbidity and mortality. HU improves outcomes through the upregulation of fetal hemoglobin (HbF). Dosing is highly variable with doses ranging from <10-35 mg/kg/day. Most dosing strategies escalate with a goal of mild myelosuppression, commonly defined using an absolute neutrophil count (ANC) between 2,000-4,000 and platelets >80,000. Macrocytosis is often used as a surrogate marker for compliance and effect. Despite its well-established effectiveness, clinical use remains limited with only 12% of adults with SCA taking HU. The reasons for this are multifactorial but include skepticism by both providers and patients regarding its effectiveness in the adult population. Some experts suggest up to 30% of adults are non-responders with no significant HbF change, and many believe that the effect of HU fades with time. Dosing strategies are usually quite conservative to prevent excessive myelosuppression, though dose is highly correlated with clinical effect and ultimate %HbF. There is very limited real-world data regarding long term effectiveness of HU in adults with SCA over time. Methods: We retrospectively analyzed the records of the 109 adults with SCA currently treated in our multidisciplinary sickle cell clinic. Data from 1/1/2011-6/30/2021 was collected, including genotype, HU prescription status, current and maximum laboratory values (HbF, MCV, ANC), and number of admissions for painful crisis. We performed Wilcoxon rank sum testing between pts prescribed and not prescribed HU and measures of HbF (peak, average and current) and number of admissions for painful crisis over the study period. Results: Among 106 pts (3 excluded from analysis, 2 for lack of data and 1 for post-transplant status), 79 are currently prescribed HU (75%). Among our 63 pts with HbSS genotype 58 are prescribed HU (92%). Average HbF over time for all pts prescribed HU was 11.9%. Average peak HbF was 18.1% and average current HbF is 12.4%. In the pts not prescribed HU HbF average, peak and current levels were 5.5%, 6.7%, and 5.0% respectively. HU prescription was significantly associated with increased HbF at peak, average, and current levels (p-value <0.001, Figure A). HU prescription was also significantly associated with decreased number of admissions for painful crisis (p-value 0.001). Among pts prescribed HU, MCV levels >100 at time of peak HbF were associated with higher peak levels than pts with MCV <100 (p-value <0.001, Figure B). Larger peak HU doses were also correlated with higher MCVs at the time of peak HbF levels (Figure C). Larger current HU doses were also significantly associated with higher current HbF levels (Figure, D). Doses >9.9m/kg were associated with significantly higher HbF levels however there was no significant difference between dose levels 10-19.9mg/kg and 20-29.9 or 20-29.9 and 30-39.9mg/kg (p-values 0.02, 0.68 and 0.84 respectively). Among pts prescribed HU, 34 obtained ANC levels <4000 at the times of peak HbF and 24 at the current dosing level (43% and 30% respectively). 24 pts obtained both MCV>100 and ANC <4000 at time of peak HbF and only 11 achieved both at current dosing levels (30% and 14% respectively). Conclusion: In our adult multidisciplinary sickle cell clinic prescribing rates are well above current HU usage for adults with SCA. Our data notes that elevated HbF levels can be maintained over long periods of time. HU prescription was significantly associated with increased HbF levels and reduced admissions for painful crisis. This was despite a majority of pts not meeting target prescribing levels of ANC <4000 and MCV >100. While higher peak HbF levels were significantly associated with higher HU doses, this was only true for doses above 9.9 mg/kg. Further investigation is needed to explore the factors contributing to suboptimal HbF and MCV response, including careful assessment with medication adherence and dose selection. Overall, these data illustrate the importance of dosing and suggest that one size dosing of HU for adults with SCA does not fit all. We hypothesize that there may be a role individualized dosing strategies in adults with SCA, incorporating factors such as pharmacokinetics and renal function to achieve the optimal dose for each patient. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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Propensity Score Matched Analysis of Risk for Malignancies in Sickle Cell Anemia

Blood ◽

10.1182/blood-2018-99-116423 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 5885-5885

Author(s):

Amber Afzal ◽

Nauman Siddiqui ◽

Amandeep Godara ◽

Zarmina Khan ◽

Hande H. Tuncer

Keyword(s):

Sickle Cell Disease ◽

Sickle Cell ◽

Sickle Cell Anemia ◽

Hematological Malignancies ◽

Conflicts Of Interest ◽

Hospitalized Patients ◽

P Value ◽

Cell Disease ◽

Protective Mechanisms ◽

Chi Square

Abstract BACKGROUND: The survival of sickle cell patients is limited as compared to the general population. The major morbidity and mortality comes from sickle cell complications including cardiovascular disease, renal failure, infections and thrombosis (Manci Br J Haematol 2013). However, literature is conflicting about risk for malignancies in this population and remains to be investigated by larger studies. Our retrospective study is aimed at studying the prevalence of different malignancies in a large cohort of hospitalized sickle cell patients in the year 2014 using the National Inpatient Sample (NIS). METHODS: Study Population: We identified hospitalized sickle cell patients in NIS using ICD-9 codes (282.5,282.6X -282.6X) for year 2014. We also identified the various malignancies through ICD-9 codes and studied the prevalence of individual malignancies in hospitalized sickle cell patients (SCD/cases) compared to a matched cohort of hospitalized patients with no sickle cell disease (No SCD/controls). Statistical Analysis: The 1:N Case-Control Matching Macro (Parsons LS et al SUGI 29) was used to match cases with controls with respect to potential confounders including age, race, gender in the ratio of 1:4. Chi Square was used to determine the difference in the proportions of individual malignancies between the two groups. P value <0.05 was considered statistically significant. All analyses were performed using SAS 9.4. RESULTS: We identified 117,405 hospitalized sickle cell patients in the NIS database who were matched to 469,620 controls (Table 1). A total of 1890 malignancies were found in sickle cell cohort. The prevalence of malignancies in hospitalized sickle cell patients was 1.6% as compared to 4.6% in controls (OR 0.34; range:0.30-0.38). The prevalence of all studied malignancies including hematological malignancies was lower in hospitalized sickle cell patients as compared to a matched population of hospitalized patients with no sickle cell disease (Table 2). CONCLUSION: Our study showed a lower prevalence of both solid tumors and hematological malignancies in patients with sickle cell disease as compared to matched cohorts. Future prospective cohort studies are needed to evaluate the risk of malignancies in sickle cell anemia and identify if any protective mechanisms exist against the development of malignancy in this patient population. Disclosures No relevant conflicts of interest to declare.

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Association of Single Nucleotide Polymorphisms in Klotho with Priapism in Sickle Cell Anemia.

Blood ◽

10.1182/blood.v104.11.1673.1673 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1673-1673

Author(s):

Vikki G. Nolan ◽

Clinton Baldwin ◽

Qianli Ma ◽

Diego F. Wyszynski ◽

John J. Farrell ◽

...

Keyword(s):

Linkage Disequilibrium ◽

Single Nucleotide Polymorphisms ◽

Candidate Genes ◽

Sickle Cell ◽

Sickle Cell Anemia ◽

Likelihood Method ◽

P Value ◽

Nucleotide Polymorphisms ◽

Genetic Modifiers ◽

Single Nucleotide

Abstract The phenotypic heterogeneity of sickle cell anemia (HbSS) is likely to be accounted for by multiple genetic modifiers. Priapism, a common vasoocclusive complication of HbSS, may reflect sickle vasculopathy. We hypothesized that the likelihood of developing priapism, and other vascular complications of sickle cell disease, may be influenced by genetic heterogeneity in genes that modulate inflammation, oxidant injury, nitric oxide (NO) biology, vasoregulation, cell-cell interaction and hemostasis. Accordingly, we studied patients with HbSS with or without coincident α thalassemia and examined the association of 129 single nucleotide polymorphisms (SNPs) in 44 candidate genes with priapism. One hundred forty-eight patients had at least one episode of priapism and were compared with 529 controls. Validated SNPs in the candidate genes were first selected from a public database. Genotypic counts were compared between cases and controls using multiple logistic regression. For each SNP, odds ratio (OR) and 95% confidence intervals (CI) were calculated. Pairwise linkage disequilibrium between each pair of SNP loci was evaluated by using a maximum likelihood method to infer phase for dual heterozygotes and was expressed as r2. In our first analysis, we considered a SNP to have an association with a phenotype when the p-value was equal to or less than 0.01, unless there was more than one SNP in a gene showing an association, when the p-value for significance was set at 0.05. When a SNP met these criteria, we further studied the gene with as many informative SNPs as possible. Haplotypes were reconstructed by using Bayesian methods as implemented in the PHASE package. A sliding window approach was used to assess evidence for association between haplotype and priapism. Patients with HbSS-α thalassemia were less likely to have priapism than patients with HbSS (p<0.05). Two SNPs in Klotho gene (KL), rs657049 and rs211239, were associated with priapism by genotypic analysis (OR: 1.74, 95% CI 1.03–2.96 and OR: 1.74, 95% CI: 1.16–2.63, respectively). Since it is likely that the control group contains patients who might ultimately develop priapism, this may be an underestimate of the true association. Seventeen SNPs were used to reconstruct the haplotype of KL gene for cases and controls separately. Haplotype association analysis was performed using sliding windows of 2, 3, and 4 SNPs separately, and p-values were calculated after 10,000 permutations. We found that haplotypes around rs211239 (5th SNP among 17 SNPs by physical location) were associated with priapism in different windows, (1) 2 SNPs: window 3-4 and 4-5, p: 0.001 and 0.01 respectively, (2) 3 SNPs: window 2-3-4, 4-5-6 and 6-7-8, p: 0.004, 0.01, and 0.05, respectively, (3) 4 SNPs: window 1-2-3-4, 3-4-5-6, and 4-5-6-7, p: 0.03, 0.002, 0.02, respectively. KL encodes a membrane protein that regulates many vascular functions, including endothelial NO release and a variant protein may alter NO biology. We conclude that polymorphisms in a limited number of genes, or in linkage disequilibrium with functionally important genes, may set an overall risk for some vasoocclusive complications of HbSS. Identifying potential genetic modifiers, like KL, will permit these genes to be evaluated in biological studies and for networks of genotype-phenotype interactions to be modeled.

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Up-Regulation of Mir-21 and Mir-130a and Serum Leptin Levels on Leg Ulcers Development in Sickle Cell Anemia

Blood ◽

10.1182/blood-2019-132043 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 975-975

Author(s):

Thais Helena Chaves Batista ◽

Rodrigo Marcionilo Santana ◽

Marcondes José de Vasconcelos Costa Sobreira ◽

Gabriela da Silva Arcanjo ◽

Diego Arruda Falcao ◽

...

Keyword(s):

Sickle Cell ◽

Sickle Cell Anemia ◽

Leg Ulcer ◽

Acute Chest Syndrome ◽

Control Group ◽

Blood Collection ◽

Leg Ulcers ◽

Serum Leptin ◽

Ulcer Group ◽

And Control

Introduction: Leg ulcers (LUs) are a cutaneous complication of sickle cell anemia (SCA), whose etiology is considered multifactorial. In the search for new candidates for modulators of SCA clinical events, recent evidence suggests the significant role of mechanisms related to post-transcriptional regulation, especially microRNAs (miRNAs). Thus, the analysis of miRNAs miR-21 and miR-130a differential expression in patients with SCA becomes an interesting approach, since both act in the regulation of several biological mechanisms related to the pathophysiology of LU, especially the tissue repair process. In addition, these miRNAs have already been related to the regulation of serum leptin levels, a strong angiogenic pleiotropic hormone that acts in the healing process of skin lesions. Therefore, the aim of the study was to investigate the influence of miR-21 and miR-130a and serum leptin levels on the development of LUs in SCA patients. Methods: After analyzing medical records, 60 SCA patients were selected. Patients who presented some of the main clinical manifestations that may have etiology due to the underlying disease (for example: osteonecrosis, stroke, priapism and acute chest syndrome) were not included. Patients with a history of LU were considered cases, and those who did not develop this complication (n=20), were considered control (median age: 26 years, range: 19-61, 50% males). The control group was called "HbSS-Control" and the case group was divided into two subgroups: Active leg ulcer group, composed of 19 patients with active LU at the time of blood collection (median age: 35 years, range: 24-56, 68% males), and healed leg ulcer group, composed of 21 patients with healed LU at the time of blood collection (median age: 34 years, range: 22-52, 43% males). In addition, it was analyzed a group of 10 donors with normal hemoglobin profile (median age: 25 years, range: 20-30, 50% males), identified as "HbAA-Control". Expression levels of miRNAs extracted from peripheral blood, using mirVanaTM PARIS Kit (Invitrogen™) were evaluated by RT-qPCR technique utilizing TaqMan® probes. Serum leptin levels of the patients were evaluated employing the ELISA method (Human Leptin ELISA Kit, Millipore®). Mann-Whitney and Kruskal-Wallis tests were applied to compare continuous variables. Results: Up-regulation of both miRNAs was observed in the active leg ulcer group in contrast to the healed leg ulcer (miR-21: P<0.0001, Figure 1A, Fold change [FC]=14,2; miR-130a: P=0.0004, FC=18,8, Figure 1B) and Control-HbSS groups (miR-21: P<0.0001, FC=34,4, Figure 1A; miR-130a: P=0.0006, FC=15,3, Figure 1B) and the HbAA-Control group (miR-21: P<0.0001, FC=5,8, Figure 1C; miR-130a: P=0.0009, FC=10,9, Figure 1D). However, there was no significant difference between the healed leg ulcer, HbSS-Control and HbAA-Control groups (miR-21: P=0.1829, Figure 1E; miR-130a: P=0.3537; Figure 1F). Furthermore, the active leg ulcer group had lower serum leptin levels when compared to the healed leg ulcer and Control-HbSS groups (P=0.0058; Figure 2A). The levels of leptin in the healed leg ulcer group did not differ from the Control-HbSS group (P=0.5929; Figure 2B). Conclusion: Our results demonstrated an inverse relation between the miRNAs miR-21 and miR-130a expression with serum leptin levels, suggesting that the up-regulation of these miRNAS may be related to the chronicity and healing of LUs in individuals with SCA through decreased of serum leptin levels. Disclosures No relevant conflicts of interest to declare.

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Objective Measurement of Leg Ulcer Area in Sickle Cell Anemia Patients Using Infrared Photography

10.21611/qirt.2012.338 ◽

2012 ◽

Author(s):

N. Malik ◽

C.P. Minniti ◽

J. Maivelett ◽

A. Koroulakis ◽

K. Delaney ◽

...

Keyword(s):

Sickle Cell ◽

Sickle Cell Anemia ◽

Objective Measurement ◽

Leg Ulcer ◽

Infrared Photography ◽

Ulcer Area

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Assessment of Bone Marrow Function in Sickle Cell Anaemia Patients Using Corrected Reticulocyte Counts

Blood ◽

10.1182/blood.v126.23.4581.4581 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4581-4581

Author(s):

Titilola S. Akingbola ◽

Chinedu Anthony Ezekekwu ◽

Joseph Yaria ◽

Santosh L. Saraf ◽

Lewis L. Hsu ◽

...

Keyword(s):

Bone Marrow ◽

Steady State ◽

Sickle Cell ◽

Sickle Cell Anemia ◽

Reticulocyte Count ◽

P Value ◽

Production Index ◽

Risk Of Death ◽

Increased Risk ◽

Reticulocyte Production

Abstract Introduction: Chronic hemolysis occurs in sickle cell anemia as a result of recurrent sickling and other abnormalities of the red blood cells including eryptosis. Exuberant reticulocytosis is anticipated to partially compensate for the resultant anemia. Sickle cell anemia patients may also have aplastic crisis, bone marrow (BM) infarction and erythropoietin deficiency which could lead to reticulocytopenia despite the anemia. High degree of reticulocytosis among asymptomatic infants with sickle cell anemia has been associated with an increased risk of death or stroke during childhood. Assessment of BM function in sickle cell anemia is important due to potential complications associated with both under-activity and hyperactivity. This study aimed at evaluating the erythropoietic function of the BM in steady state sickle cell anemia using corrected reticulocyte counts. Methods: This study was carried out at the hematology clinic in the University College Hospital, Ibadan. HbSS patients in steady state were recruited from the hematology clinic. Local ethical committee approval was obtained and all participants gave written informed consent. Patients with M. tuberculosis, Hepatitis B, HIV and P. falciparum infection were excluded. Peripheral blood samples were analyzed using Sysmex Ki-X21 for complete blood count (CBC) and standard point of care for serum electrolytes and liver function tests. The glomerular filtration rates were calculated using the Cockcroft-Gault formula. Reticulocyte counts were determined manually using fresh samples from K2 EDTA bottles and methylene blue stain. Two drops of stain were mixed with two to four volumes of anticoagulated blood and incubated at 37ºC for 15 minutes. Afterwards, the cells were re-suspended and blood films were made. Corrected reticulocyte count and reticulocyte production index were calculated. Participants were categorized according to corrected reticulocyte counts of greater than or less than 2.5%. Univariate and multivariate analyses were performed to determine variables associated with corrected reticulocyte count <2.5%. Results: 92 HbSS patients were recruited with a mean (SD) age of 19.6 (5.8) years. There was no correlation between age and eGFR (p-value: 0.227). Median (range) reticulocyte count, corrected reticulocyte count and reticulocyte production index were 5.5 (0.5 - 29.9), 3.3 (0.1 - 17.1) and 1.7 (0.2 - 8.6) respectively. 40 (43.5%) patients had corrected reticulocyte count <2.5% and 52 (56.5%) had a corrected count >2.5%. Those corrected reticulocyte count <2.5% were older (p: 0.013), taller (p: 0.041) and had higher aspartate transaminase (AST) levels (p: 0.006) than those with corrected counts >2.5% (Table 1). CBC parameters were not different when compared between both groups. Results of multivariate logistic regression analysis carried out showed that only AST was independently linked with corrected reticulocyte count <2.5% (R2: 0.172, p-value: 0.001) (Table 2). Table 1. Factors Associated with Low Reticulocyte Count Corrected count<2.5% Corrected count>2.5% p-Value Age (Mean, SD) 21.4 (6.3) 18.4 (5.0) 0.013 Gender (N, %) Male 22 (55.0) 28 (53.8) 0.912 Female 18 (45.0) 24 (46.2) Height (Mean, SD) 1.6 (0.1) 1.5 (0.1) 0.041 BMI (Mean, SD) 18.7 (3.1) 18.7 (3.0) 0.753 GFR (Mean, SD) 64.3 (37.7) 66.4 (29.3) 0.453 Bilirubin (Mean, SD) 1.7 (1.1) 1.9 (2.6) 0.674 AST (Mean, SD) 22.5 (13.5) 14.5 (6.6) 0.006 ALT (Mean, SD) 13.4 (7.7) 14.4 (11.1) 0.876 Table 2. Independent Predictors of Corrected Reticulocyte Count <2.5% or 95% CI p-Value Age 1.08 0.97 - 1.21 0.169 Height 19.8 0.11 - 366.10 0.259 AST 1.10 1.04 - 1.17 0.002 Hemoglobin 1.00 0.97 - 1.02 0.872 R2: 0.172, p: 0.001 Conclusion: Despite corrected reticulocyte count <2.5% in about half of the patients, there were similar hematological parameters and eGFR in both groups of patients. AST is a marker of hemolysis and low ALT rules out hepatic involvement. Since only 17.2% of the variability in BM response as assessed by corrected reticulocyte count could be accounted for by variables included in this study, there is a need to further evaluate the BM function of sickle cell patients to establish the causes of corrected reticulocyte count <2.5% in the setting of anemia, having ruled out erythropoietin as well as iron, folate or cobalamin deficiencies. This will aid the development of a functional algorithm for the individualized management of sickle cell disease patients with anemia. Disclosures No relevant conflicts of interest to declare.

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Genetic Polymorphisms Associated with Fetal Hemoglobin Response to Hydroxyurea in Patients with Sickle Cell Anemia.

Blood ◽

10.1182/blood.v104.11.108.108 ◽

2004 ◽

Vol 104 (11) ◽

pp. 108-108 ◽

Cited By ~ 2

Author(s):

Diego F. Wyszynski ◽

Clinton T. Baldwin ◽

Mario Cleves ◽

John J. Farrell ◽

Alice Bisbee ◽

...

Keyword(s):

Cytochrome P450 ◽

Candidate Genes ◽

Sickle Cell ◽

Sickle Cell Anemia ◽

Globin Gene ◽

Linear Regression Analysis ◽

Fetal Hemoglobin ◽

Computer Application ◽

Average Increase ◽

Genetic Traits

Abstract Hydroxyurea (HU) is an effective treatment for most patients with symptomatic sickle cell anemia, yet the fetal hemoglobin (HbF) response to treatment is variable. A capacity to predict an individual’s HbF response to HU would aid the selection of patients for treatment and reduce toxicity from unfruitful dose escalation. Unfortunately, this is presently not possible. We hypothesized that HbF levels and the HbF response to HU are regulated as complex genetic traits and previously showed that 12 single nucleotide polymorphisms (SNPs), associated with a 20% to 30% difference in baseline HbF concentrations, were found in the introns of 4 genes, PDE7B, MAP7, MAP3K5 and PEX7, spanning the genomic region from 136.1 Mb to 137.5 Mb on chromosome 6q (Cell Mol Biol 50:23, 2004). To begin to define the genetic predictors of the HbF response to (HU), we examined SNPs in candidate genes and genetic loci in 214 patients with sickle cell anemia whose HbF levels were available before HU treatment was started and after these patients reached a stable dose of this drug. Forty-six candidate genes were chosen because of their possible role in HbF regulation and HU metabolism and 226 SNPs in these genes were examined by mass spectrometry. A computer application developed in STATA was used to carry out multiple linear regression analysis with simultaneous adjustment for age, sex and the α- and β-globin gene cluster haplotypes for each SNP and combinations of nearby SNPs. Dominant, codominant and recessive models for modulating HbF expression were tested. In this QTL analysis, SNPs in a member of the cytochrome P450 family (CYP2C9), in aquaporin 9 (AQP9) and in the chromosome 6q qtl described above were significantly associated with the HbF response to HU. The effect of genotype on the magnitude of HbF response to HU was examined for selected SNPs in AQP9 and CYP2C9. In AQP9, AA was associated with an average increase of 6% in HbF compared with GG (rs1867380; OR 6.6, p<0.001). In CYP2C9, AG was associated with an average increase of 3% and GG with an average increase of 11% (rs2209331; OR 1.6 and 7.5, p=0.05, 0.000). An effect was also noted for the 6q qtl. We also treated the increase in HbF as a discretized variable, comparing individuals in the lower two quartiles of HbF response with individuals in the top quartile of HbF response to HU (Blood 89:1078, 1997). These same genotypes were more common in good HU responders than in poor responders (p<0.05). CYP2C9 (10q24) encodes a member of the cytochrome P450 superfamily of enzymes, monooxygenases catalyzing many reactions involved in drug metabolism, plays some role in the metabolism of HU derivatives. AQP9 (15q22.1–22.1), belongs to a family of water-selective membrane channels and stimulates urea transport, permitting passage of many uncharged solutes. These results begin to define the pattern of genetic heterogeneity that may be used ultimately to predict a patient’s HbF response to HU. As multiple genes are very likely to play roles in this response, the interactions and predictive value of their polymorphisms will need to be modeled with methods that account for simultaneous associations.

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Genetic Polymorphisms Associated with Priapism in Sickle Cell Disease.

Blood ◽

10.1182/blood.v108.11.789.789 ◽

2006 ◽

Vol 108 (11) ◽

pp. 789-789

Author(s):

Laine Elliott ◽

Allison E. Ashley-Koch ◽

Jude Jonassaint ◽

Jennifer Price ◽

Jason Galloway ◽

...

Keyword(s):

Genetic Variation ◽

Linkage Disequilibrium ◽

Sickle Cell Disease ◽

Candidate Genes ◽

Sickle Cell ◽

P Value ◽

Cell Disease ◽

P Values ◽

History Of ◽

Male Patients

Abstract Priapism, a painful and prolonged erection, has been reported to occur in 30–45% of male patients with sickle cell disease (SCD). However, little is known about the pathological processes and genetic risk factors that contribute to the occurrence of priapism. The identification of genetic variables that are associated with priapism may therefore help define both critical pathophysiologic mechanisms not otherwise apparent, as well as patients at increased risk. We examined genetic variation in our sample of 199 unrelated, adult (>18 years), male patients with Hb SS and Hb Sβ0-thalassemia, 83 (42%) of whom reported a history of priapism. Candidate genes for association with priapism were identified based on their involvement in adhesion, coagulation, inflammation, and cell signaling. Additionally, we examined genes involved in NO biology (NOS2, NOS3, SOD1, SLC4A1). Finally, we also examined polymorphisms in the KLOTHO gene, which has previously been associated with priapism. We examined a total of 389 SNPs in 48 candidate genes. Except for the gene encoding the β2 adrenergic receptor, SNP genotyping was performed by TaqMan, using Assays-on-Demand or Assays-by-Design genotyping products (Applied Biosystems). Allele tests were used to detect genetic associations with priapism. Strong evidence of association was found for SNP rs7526590 in the transforming growth factor-β receptor, type III (TGFBR3) gene (p=.00058), SNP rs10244884 in the aquaporin (AQP1) gene (p=.00068), and SNP rs3768780 in the integrin αV (ITGAV) gene (p=0.00090). A second ITGAV SNP (rs3768778), in linkage disequilibrium (r2=.59) with the first, also showed association with priapism (p=.00888). The A1 subunit of coagulation factor XIII (F13A1) had four SNPs (hcv1860621, rs1032045, rs1674074, rs381061) with p-values less than 0.010 (p-values = 0.00156, 0.00415, 0.00648, and 0.00712, respectively). The linkage disequilibrium among these F13A1 SNPs is negligible (r2 <.15). We also adjusted for multiple testing using the Benjamini-Hochberg procedure (significance threshold <.10). SNP rs7526590 in TGFBR3, SNP rs10244884 in AQP1 and SNP rs3768780 in ITGAV each had a false discovery rate (FDR) p-value of .09834. SNP rs1674074 in F13A1 had an FDR p-value of .12733. The other SNPs in F13A1 had large FDR p-values, close to .30. We did not detect an association between priapism and genetic variation in the Klotho gene, as was previously reported by Nolan et al. (2005). Specifically, SNPs rs2249358, rs211234 and rs211239 showed a virtually identical distribution of genotypes for individuals with and without a history of priapism. However, our population is not identical to the previous study, which included patients as young as 10 years old. In conclusion, our data support the hypothesis that genetic variation is associated with risk for priapism among males with SCD and suggest that genes involved in the TGFβ pathway, coagulation, cell adhesion and cell hydration pathways may be important.

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