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

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.

Download Full-text

Low-level laser therapy of leg ulcer in sickle cell anemia

Revista Brasileira de Hematologia e Hemoterapia ◽

10.5581/1516-8484.20120018 ◽

2012 ◽

Vol 34 (1) ◽

pp. 65-66 ◽

Cited By ~ 3

Author(s):

Claudia Regina Bonini-Domingos ◽

Flavia Mariana Valente

Keyword(s):

Laser Therapy ◽

Sickle Cell ◽

Sickle Cell Anemia ◽

Leg Ulcer ◽

Low Level Laser Therapy ◽

Low Level ◽

Low Level Laser ◽

Level Laser

Download Full-text

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.

Download Full-text

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.

Download Full-text

CHRONIC LEG ULCER IN DISEASES OF THE BLOOD

Blood ◽

10.1182/blood.v3.11.1283.1283 ◽

1948 ◽

Vol 3 (11) ◽

pp. 1283-1289 ◽

Cited By ~ 10

Author(s):

BENJAMIN R. GENDEL

Keyword(s):

Hemolytic Anemia ◽

Sickle Cell ◽

Sickle Cell Anemia ◽

Leg Ulcer ◽

Cirrhosis Of The Liver ◽

The Other

Abstract The literature pertaining to the association of chronic leg ulcer with diseases of the blood other than sickle cell anemia has been reviewed. Two patients with this association have been presented. One patient had a hemolytic anemia and the other a pancytopenia (secondary hypersplenism) in association with congestive splenomegaly due to cirrhosis of the liver. It is suggested that the association of leg ulcer with these various diseases of the blood is related in an unknown manner to either splenomegaly or hyperfunction of the spleen (hypersplenism).

Download Full-text

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

Blood ◽

10.1182/blood-2018-99-115192 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2351-2351

Author(s):

Gabriela Queila de Carvalho Siqueira ◽

Galina Ananina ◽

Murilo Guimarães Borges ◽

Bruno Batista de Souza ◽

Iscia Teresinha Lopes Cendes ◽

...

Keyword(s):

Exome Sequencing ◽

Candidate Genes ◽

Sickle Cell ◽

Sickle Cell Anemia ◽

Conflicts Of Interest ◽

Leg Ulcer ◽

Clinical Aspects ◽

P Value ◽

Single Mutation ◽

Extreme Phenotypes

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.

Download Full-text

A Phase 1, Dose-Escalation Study of Topical Sodium Nitrite in Patients with Sickle Cell Anemia and Leg Ulcers

Blood ◽

10.1182/blood.v120.21.86.86 ◽

2012 ◽

Vol 120 (21) ◽

pp. 86-86

Author(s):

Caterina P. Minniti ◽

Yuen Yi Hon ◽

Alexander Gorbach ◽

Kara M Delaney ◽

Dihua Xu ◽

...

Keyword(s):

Adverse Events ◽

Surface Area ◽

Sickle Cell ◽

Sickle Cell Anemia ◽

Sodium Nitrite ◽

Dose Escalation ◽

Phase 1 ◽

Leg Ulcer ◽

Leg Ulcers ◽

Plasma Nitrite

Abstract Abstract 86 Background: Leg ulcers are a common and debilitating complication of sickle cell anemia and other hemolytic disorders. Despite many advances in the care of SCD, there is still a clinical need for a well-tolerated, safe and effective therapy. Sodium nitrite may function as a reservoir for local delivery of nitric oxide, whose vasodilating, angiogenic, and antimicrobicidal activities make it an attractive novel therapy for chronic wounds. We describe the safety data and preliminary efficacy data of topical sodium nitrite in adults with sickle cell anemia. Design and Methods: This is a phase 1 dose-escalation, single institution, study of topical sodium nitrite (ClinicalTrials.gov NCT01316796). Eligibility criteria: > 18 years old, have a leg ulcer of > 4 weeks duration, between 2.5 and 100 cm2 in size, and not acutely infected. Patients were screened with medical history, physical exam and laboratory tests. Sodium nitrite cream was applied twice a week for 4 weeks on one leg ulcer (“study ulcer”) in four cohorts of three subjects each with escalating concentrations in each cohort of 0.5., 1, 1.5 and 2%. Safety, tolerability and pharmacokinetic data of plasma nitrite and nitrate were obtained during the first 48 hours of drug exposure and then weekly for four weeks. Leg ulcer healing was a secondary endpoint and was assessed by calculating the change in surface area from visible light photographs of the study leg ulcer obtained at week 1, 3, and end of study, using a digital camera at a distance of 0.25 – 0.5 m from the ulcer. Borders were traced using ENVI software and ulcer size was calculated by converting the pixel area of the ulcer to cm2 using a calibrated square for reference. Pain at the ulcer site was assessed both with a Brief Pain Inventory and Visual Analog Pain Scale at predetermined time points. Results: Sixteen subjects were screened and 12 enrolled. There were no serious adverse events associated with the drug. Possibly related, grade 1; adverse events occurred in 10 of the subjects, more frequently in the cohorts with the highest concentration, which included non-clinically significant decrease in diastolic blood pressure in two subjects of cohort 4. Adverse events resolved without clinical intervention. Methemoglobin levels did not exceed pre-established safety thresholds (max of 4.1 % in one subject in cohort 3). Tolerability was excellent, with short-lived stinging at the site of application reported by two subjects. Pharmacokinetics of plasma nitrite and nitrate indicated minimal systemic absorption of topically applied sodium nitrite (median plasma nitrite AUC: 0.311 (0.169–0.659) umol*h/L/umol nitrite dose), with high interpatient variability. There was no evidence of plasma nitrite and nitrate and methemoglobin accumulation during the 4- week study trial. All but one subject experienced a decrease in leg ulcer surface area, fig 1, (pretreatment 4.65 vs. post-treatment 2.78 cm2, p <.001) and improvement in pain scores (pretreatment 4.87 vs. 2.91, p=.024 ), which often preceded the decrease in size. Healing was most marked in cohort 4, the one treated with the highest strength of sodium nitrite cream, with 2 of 3 subjects experiencing complete closure of the ulcer. Conclusions: On the basis of these safety, pharmacokinetic and tolerability data, and promising efficacy results, topical sodium nitrite warrants further clinical evaluation in patients with sickle cell disease or other hemolytic disorders and leg ulcers. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

The orientation of sickle hemoglobin fibers within fascicles

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100104066 ◽

1988 ◽

Vol 46 ◽

pp. 400-401

Author(s):

Christopher A. Miller ◽

Bridget Carragher ◽

William A. McDade ◽

Robert Josephs

Keyword(s):

Sickle Cell ◽

Sickle Cell Anemia ◽

Relative Orientation ◽

Unit Cell ◽

Red Cell ◽

High Ph ◽

Sickle Hemoglobin ◽

Polar Crystal ◽

Helical Configuration

Highly ordered bundles of deoxyhemoglobin S (HbS) fibers, termed fascicles, are intermediates in the high pH crystallization pathway of HbS. These fibers consist of 7 Wishner-Love double strands in a helical configuration. Since each double strand has a polarity, the odd number of double strands in the fiber imparts a net polarity to the structure. HbS crystals have a unit cell containing two double strands, one of each polarity, resulting in a net polarity of zero. Therefore a rearrangement of the double strands must occur to form a non-polar crystal from the polar fibers. To determine the role of fascicles as an intermediate in the crystallization pathway it is important to understand the relative orientation of fibers within fascicles. Furthermore, an understanding of fascicle structure may have implications for the design of potential sickling inhibitors, since it is bundles of fibers which cause the red cell distortion responsible for the vaso-occlusive complications characteristic of sickle cell anemia.

Download Full-text