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

Abstract

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.

Phenotypic characterisation and associations of leg ulcers in adult sickle cell patients

“…This may suggest that the factors causing SLU is actually multiple and that a single pathogenetic pathway may not be at play in this condition. This has been the most popular view and deduction of several previous research, though the presence of some genetic variants, notably Klotho , TEK and FLG2 has been linked to this complication 12,23 . Also the fact SLU occurs repeatedly in some patients and never at all in other SCA patients over their lifetime indicates the persistence of a factor—genetic or environmental, which is unique to each individual patient.…”

LGALS3 +191A and +292C polymorphisms are associated with a reduction in serum gal-3 levels, but not with the clinical events of individuals with sickle cell anemia

“…When this occlusion is prolonged, it causes hemolysis, releasing inflammation mediators such as IL-6, IL-1b, TNF, IL-8, among other molecules capable of promoting the proliferation, recruitment, and adhesion of erythrocytes, leukocytes, and platelets to the endothelium. This promotes a reduction in blood flow, as a result, ischemia and damage to several organs may occur (Minniti et al, 2010;Senet et al, 2017;De Carvalho-Siqueira et al, 2019).…”

Up‐regulation of miR‐130a is related to leg ulcers in sickle cell anaemia

“…Although the aetiology of LUs is still not completely elucidated, there is a consensus that biological and environmental factors can contribute to the occurrence of this complication 1 . Furthermore, evidence suggests that there is a molecular heterogeneity that distinguishes patients according to the occurrence of LUs 2–4 . In this context, the overexpression of microRNA (miRNA) miR‐130a has been associated with chronic venous ulcers in diabetic patients and correlates with the deregulation of the re‐epithelisation processes, granulation tissue formation, and wound healing 5 .…”

“…Thais H. C. Batista 1 Rodrigo M. Santana 1 Marcondes J. Sobreira 1 Gabriela S. Arcanjo 1 Igor F. Domingos 1,2 Diego A. Pereira-Martins 1,3 Diego A. Falcão 1 Jéssica M. F. Oliveira 1 Jéssica V. G. F. Batista 1 Isabel Weinhӓuser…”

“…1 Furthermore, evidence suggests that there is a molecular heterogeneity that distinguishes patients according to the occurrence of LUs. [2][3][4] In this context, the overexpression of microRNA (miRNA) miR-130a has been associated with chronic venous ulcers in diabetic patients and correlates with the deregulation of the re-epithelisation processes, granulation tissue formation, and wound healing. 5 Additionally, this miRNA is involved in the regulation of the leptin receptor (LepR) 5 and previous studies revealed the role of leptin in the pathogenesis of skin diseases.…”