Transforming Growth Factor Beta Receptor 3 Haplotypes in Sickle Cell Disease Are Associated with Lipid Profile and Clinical Manifestations

Individuals with sickle cell disease (SCD) present both chronic and acute inflammatory events. The TGF-β pathway is known to play a role in immune response, angiogenesis, inflammation, hematopoiesis, vascular inflammation, and cell proliferation. Polymorphisms in the transforming growth factor-beta receptor 3 (TGFBR3) gene have been linked to several inflammatory diseases. This study investigated associations between two TGFBR3 haplotypes and classical laboratory parameters, as well as clinical manifestations, in SCD. We found that individuals with the GG haplotype presented higher levels of total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), triglycerides, non-HDL cholesterol, total proteins, and globulin than individuals with non-GG haplotypes. In addition, the GG haplotype was associated with a previous history of pneumonia. Individuals with the CGG haplotype presented increased plateletcrit, TC, LDL-C levels, and non-HDL cholesterol. The CCG haplotype was also associated with a previous history of pneumonia. Our findings suggest that individuals with the GG and CGG haplotypes of TGFBR3 present important alterations in lipid profile.


Introduction
Sickle cell disease (SCD) is an autosomal genetic disorder marked by a chronic inflammatory status with acute episodes. The acute condition occurs as a result of vaso-occlusion, persistent cycles of red blood cell sickling and unsickling, and hemolysis, leading to leukocyte and endothelial cell activation that induces the release of cytokines and adhesion molecules. By contrast, the chronic inflammatory status is the result of ischemic and reperfusion processes, which cause endothelial cell and vascular wall damage [1][2][3].
The clinical complications, which occur frequently in individuals with SCD, are classified as acute or chronic according to the age of affected individuals, yet are not restricted to any specific stage of life [4,5]. SCD individuals also present clinical manifestations including vaso-occlusive (VO) and painful crises, pneumonia, cholelithiasis, stroke, priapism, and chronic injury in a variety of organs [6]. One common acute complication in SCD is acute chest syndrome (ACS), characterized by cough, shortness of breath, and signs of hypoxemia that are difficult to distinguish from acute pneumonia [7]. Pulmonary complications in SCA are mostly associated with vascular impairment and vasoconstriction, leading to VO [8]. Among the clinical manifestations associated with hemolysis, cholelithiasis, which is related to gallstone formation and gallbladder obstruction, tends to increase in frequency with age [9]. Cholelithiasis occurs due to the accelerated rate of chronic erythrocyte destruction in individuals with SCD. Heme is released by hemolysis and becomes metabolized into bilirubin, which can form insoluble calcium bilirubinate in the bile and precipitate as pigments that form gallstones [10].
The immunological aspects of SCD have been widely studied among individuals with sickle cell anemia (SCA), with high levels of cytokines detected, including interleukin (IL) 4, 6, 8, 10, and tumor necrosis factor-alpha (TNF-a) [11]. In addition, transforming growth factor-beta (TGF-β) and IL-17 were also found to be associated with vascular activation and inflammation based on a direct association with arginase levels [12]. Differences were found in the levels of cytokines (IL1β, IL6, TNF-a, and TGF-β), lipid inflammatory mediators (LTB4 and PGE2), and modulators of vascular remodeling (MMP9 and TIMP1) between SCA individuals in steady-and crisis-state, permitting the characterization of these two groups using these parameters [13].
The TGF-β pathway is involved in several cellular processes, since signal transduction involves binding with transforming growth factor-(TGF-) beta receptors (TGFβRI, TGFβRII, or TGFβRIII), which activate mothers against decapentaplegic homolog (SMAD) proteins and other mediators. As TGF-β is known to activate several mediators, the TGF-β pathway is considered to play a role in immune response, angiogenesis, inflammation, hematopoiesis, vascular inflammation, and cell proliferation [12,14].
Considering the complex mechanisms underlying the pathogenesis of SCD, we sought to investigate associations between TGFBR3 haplotypes and classical laboratory parameters, as well as clinical manifestations.

Materials and Methods
2.1. Subjects. One hundred seventy-five individuals with SCD (HbSS and HbSC genotypes) were seen at the Bahia Hemotherapy and Hematology Foundation between October 2016 and September 2017. The individuals, 83/175 (47.4%) of whom were female, had an average age of 14:46 ± 3:35 years and a median age of 14 years (interquartile range [IQR]: 12-17 years). All individuals with SCD were in steady-state, characterized by the absence of acute crisis in the three months prior to blood collection procedures. None of the patients were undergoing therapy with lipid-lowering agents, such as statins. All individuals or their legal guardians agreed to biological sample collection procedures and signed terms of informed consent. The present research protocol was approved by the Institutional Research Board of the São Rafael Hospital (HSR protocol number: 1400535) and was conducted in compliance with the Declaration of Helsinki (1964) and its subsequent amendments. Biochemical, hematological, genetic, and immunological analyses were performed at the Clinical Analyses Laboratory of the College of Pharmaceutical Sciences, Federal University of Bahia (LACTFAR-UFBA), and at the Laboratory of Genetic Investigation and Translational Hematology at the Gonçalo Moniz Institute-FIOCRUZ (LIGHT-IGM/FIOCRUZ).

Clinical
Manifestations. All legal guardians of the individuals with SCD were asked to complete a questionnaire containing information on clinical data regarding the occurrence of previous clinical manifestations. All information provided was confirmed by individual patient medical records.

Hematological and Biochemical
Parameters. Blood samples were collected by HEMOBA staff following a fasting period of no less than 12 hours.
Hematological parameters were determined using a Beckman Coulter LH 780 Hematology Analyzer (Beckman Coulter, Brea, California, USA), and hemoglobin profile was confirmed by high-performance liquid chromatography using an HPLC/Variant-II hemoglobin testing system (Bio-Rad, Hercules, California, USA).

3.3.
Association of TGFBR3 rs2765888, rs284875, and rs2038931 Polymorphisms with Laboratory Parameters and Clinical Manifestations in SCD. The dominant genetic model was employed in rs2765888 and rs2038931 polymorphisms, while the recessive genetic model was employed in rs284875 polymorphism to evaluate associations between alleles and laboratory biomarkers. With regard to TGFBR3 rs2765888, individuals with CC genotype presented higher mean corpuscular hemoglobin concentration (MCHC) (P = 0:039) and plateletcrit (P = 0:027) than those with T_ genotypes ( Table 2).
Regarding clinical manifestations, no significant association was found to rs2765888 and rs284875 polymorphisms, while the A_ genotypes of rs2038931 polymorphism was associated with a previous history of cholelithiasis (P = 0:038) ( Table 5).

Association of Clinical Manifestations with Laboratory
Parameters in SCD. Once the GG and CCG haplotypes were associated with a previous history of pneumonia and the non-GG haplotype was associated with a previous history of cholelithiasis, the association between these clinical manifestations and laboratory parameters was investigated. A comparison of the hematological, biochemical, and immunological parameters between the individuals with and without previous history of pneumonia revealed that individuals with previous history of pneumonia presented higher TC (P = 0:004), LDL-C (P = 0:025), non-HDL-C (P = 0:012), and CRP (P < 0:001), as well as decreased creatinine levels (P = 0:014) than those without previous history of pneumonia (Table 8).

Discussion
Although polymorphisms in the TGFBR3 gene, when evaluated individually, have been associated with several clinical manifestations in SCD [14, 20-24, 34, 35], Kim et al. (2010) found that, in asthmatic individuals, these polymorphisms are more informative when evaluated in the context of haplotype, versus on an individual basis [36]. Accordingly, this study evaluated associations between TGFBR3 haplotypes and laboratory markers, as well as clinical manifestations, in individuals with SCD.
Laboratory parameters of SCD individuals presented classical markers of hemolysis, anemia, increased leukocyte counts, as well as decreased HDL-C levels. These findings are consistent with previous reports describing SCD individuals [37][38][39][40].
In our study, six polymorphisms were evaluated; the polymorphisms rs7526590, rs284875, rs2038931, rs7526590, and rs284157 are intronic variants. Intronic variants might affect alternative splicing of the mRNA; however, until this    Mediators of Inflammation    Bold values indicate significance at P < 0:05.

Mediators of Inflammation
All P values obtained by the Chi-square, except for those with asterisk ( * ), for which the Fisher's exact test was used. 8 Mediators of Inflammation moment, there are no evidences of association between these polymorphisms and TGFBR3 function or TGFbeta signaling. We reinforce that all the selected SNPs were previously studied and associated with the occurrence of clinical manifestation in SCD [20][21][22][23][24]. With regard to rs1805110 polymorphism, this is a missense variant that causes a change in the polypeptide chain where a S ðSerÞ > F ðPheÞ. This polymorphism has been associated with Behçet's disease, susceptibility in HBV-related hepatocellular carcinoma, pulmonary emphysema, and aneurysm; however, it was not previously investigated in SCD [41][42][43][44].
Our results showed that individuals with the GG haplotype of TGFBR3 had higher levels of TC, LDL-C, triglycerides, and non-HDL-C than those with non-GG haplotypes. In addition, individuals with the CGG haplotype of TGFBR3 also presented lipid profile alterations, i.e., higher TC, LDL-C, and non-HDL-C levels than those with non-CGG haplotypes. Importantly, even statistically higher, serum lipid levels in the GG and CGG haplotypes were within the normal clinical range. The individual evaluation of polymorphisms showed that individuals with GG genotype of rs2038931 polymorphism presented increased VLDL-C, triglycerides, TC/HDL-C ratio, triglycerides/HDL-C ratio, and LDL-C/HDL-C ratio than those with A_ genotypes. Moreover, the CC genotype of rs2765888 polymorphism was also associated with increased triglycerides/HDL-C ratio. Indeed, when the TGFBR3 rs2765888, rs284875, and rs2038931 polymorphisms, which compose the haplotypes, were individually investigated, none of them seems to contribute more than the other polymorphism to the associations found by the haplotypes. Thus, the associations between the haplotypes and lipid parameters were equally modulated by the polymorphisms. Despite the rs2038931 polymorphism has been associated with lipid markers, they were different from those identified by haplotypes.
High total cholesterol, as well as LDL-C levels, triggers the process of atherosclerosis by stimulating cholesterol accumulation and promotes an inflammatory response in the artery wall [45]. The pathophysiological mechanisms present in atherosclerosis are analogous to those identified Carriers of the GG haplotype also had presented increased (e) total protein levels (P value obtained with t-testing) and (f) increased globulin levels (P value obtained by the Mann-Whitney U test).    Mediators of Inflammation  14 Mediators of Inflammation in SCA vasculopathy, marked by enhanced oxidative stress, low NO bioavailability in addition to endothelial dysfunction; however, it is important to emphasize that in SCD there is no formation of atheroma plaques [4,46]. Vasculopathy is responsible for several clinical manifestations in SCD, such as stroke, priapism, pulmonary hypertension, and leg ulcers [4]. Moreover, high triglyceride and non-HDL-C levels have also been associated to atherosclerosis. In the lipolysis of triglycerides, triglyceride-rich remnants are released in vessels contributing to increase inflammation, coagulation, and endothelial dysfunction [47]. Increased triglyceride levels were shown to be a potential risk factor for pulmonary hypertension, a frequent respiratory complication in individuals with SCD [29].

Mediators of Inflammation
Non-HDL-C is a cholesterol carried by apolipoprotein B, including those carried by LDL-C and VLDL-C, and is considered a useful marker of atherosclerosis [48]. Previous studies performed in individuals with cardiovascular disease reported high non-HDL-C levels [49,50]; however, no significant alterations in non-HDL-C levels were found in SCD individuals [32].
TC, LDL-C, triglycerides, and non-HDL-C are markers of the lipid metabolism associated with inflammation related to vascular response [45,46,48]. The TGF-β pathway has been associated with vascular inflammatory responses in several diseases. In many cases, cholesterol uptake and trafficking are also responsible for vessel wall modifications, which can contribute to inflammation [51,52]. A previous study reported that TGF-β signaling regulates lipid metabolism through the induction of lipogenesis genes resulting in increased triglyceride synthesis and lipid accumulation. TGF-β signaling activates SMAD proteins, triggering the activation of these pathways. The inhibition of these pathways suppresses changes in gene expression associated with lipid metabolism [53]. Therefore, the identification of markers, which may modulate the endothelial inflammatory response in SCD is relevant to monitor the clinical course and may contribute to understand the disease pathophysiology.
TGFBRIII, encoded by the TGFBR3 gene, plays an important role in regulating and mediating the signal transduction of TGF-β [54]. In the literature, polymorphisms in TGFBR3 have been associated with inflammatory diseases other than SCD, such as Marfan syndrome, bladder cancer, and Behçet's disease [14,[16][17][18]. Marfan syndrome presents as clinical complications in the cardiovascular, skeletal, pulmonary, ocular, and nervous system; its physiopathology includes alterations in extracellular matrix deposition in vessels, similar to findings reported in SCD [16]. Behçet's disease is characterized by multisystemic vasculitis with marked inflammatory lesions in the central nervous system, skin, joints, orogenital mucosa, and eyes; analogous to SCD, the inflammatory component is the main physiopathological feature of this disease [18].
Individuals with the GG haplotype presented high levels of total proteins and globulin, which is similar to previous reports in individuals with SCD compared to controls [55,56]. This hyperproteinemia arises from hyperglobulinemia, which is known to occur in SCD individuals as a result of erythrocyte destruction during sickling [57]. Thus, it is expected that individuals with the GG haplotype would present more prominent hemolysis than those with non-GG haplotypes. High levels of total protein were also identified in individuals with GG genotype of rs2038931 polymorphism when compared to A_ genotypes.
With regard to clinical manifestations, the haplotypes have showed to be more informative than the polymorphisms individually analyzed; both the GG and CGG haplotypes of gene TGFBR3 were associated with the occurrence of pneumonia. In a large cohort of SCD individuals, pneumonia was one of the leading causes of hospitalization, second only to sickle cell crisis [58]. A previous study performed in individuals with SCA, the majority of whom presented Streptococcus pneumoniae infection, identified that a polymorphism in TGFBR3 was associated with increased susceptibility to bacteremia [34]. Streptococcus pneumoniae infection is one of the main causes of pneumonia in individuals with SCD [34]. It is important to note that all individuals in this study were immunized against pneumococcal disease; therefore, it is possible that these children could have been undergoing a process of autosplenectomy, which consequently increases susceptibility to serious infections [59].
Other respiratory complications, such as asthma and bronchopulmonary dysplasia, have also been associated with TGFBR3 in the literature [36,60]. Similarities exist in the pathogenesis of asthma, bronchopulmonary dysplasia, and SCD, which is marked by inflammation and the activation of several cytokines, including TGF-β [36,60].
We found that individuals with a previous history of pneumonia presented increased TC, LDL-C, non-HDL-C, CRP levels, and decreased creatinine. A previous study performed in individuals with SCD identified that the occurrence of pneumonia is related to increased frequency of other respiratory complications, such as acute chest syndrome (ACS), arising from fat embolism and pathogenic infection [61]. In a cohort without SCD, high cholesterol levels were associated with death related to a miscellaneous of respiratory diseases in a large study involving adults [62].
Serum CRP levels are clinically used to differentiate pneumonia from other acute respiratory infections [63]. In SCD, community-acquired pneumonia triggers inflammatory response and lung injury [64]. Alterations in CRP levels and other immunological markers were found in SCA individuals with lung dysfunction deriving from ACS [65]. High creatinine levels were also described as a severity marker of community-acquired pneumonia; however, in our results, the individuals with SCD and previous history of pneumonia presented lower creatinine levels [66].
We also found that non-GG haplotypes were associated with the occurrence of cholelithiasis, one of the most frequent SCD complications that occurs in 26-58% of patients. In addition, an individual with a previous history of cholelithiasis presented more prominent hemolysis than those without history. This is often associated with increased bilirubin levels, and consequently with hemolysis, and has been described as part of the hemolysis-endothelial dysfunction subphenotype [4,67,68]. Cholelithiasis, a clinical manifestation of SCD related to chronic hemolysis, triggers bilirubin 15 Mediators of Inflammation production, leading to the formation of gallstones [69]. The upregulation of the TGF-β pathway was previously detected in the gallbladders of individuals with cholelithiasis [70]. Moreover, an investigation of gene expression in patients undergoing cholecystectomy found significant expression of TGF-β receptor (TGFβR) I and TGFβRII during chronic cholelithiasis in comparison to acute cholelithiasis. However, no significant increase in TGFβRIII expression was found [71].
Altogether, our results suggest that TGFBR3 haplotypes seem to be related to inflammation and the occurrence of pneumonia. Inflammation is a physiopathological mechanism present in SCD, highlighting the relevance investigating novel biomarkers of disease severity in the clinical management of individuals with SCD. To the best of our knowledge, the present study is the first attempt to demonstrate associations between TGFBR3 haplotypes and hematological and biochemical parameters, as well as clinical manifestations in SCD.

Conclusion
Collectively, the present findings suggest that individuals with the GG and CGG haplotypes of TGFBR3 present significant lipid profile alterations and could be associated with the occurrence of pneumonia, while the non-GG haplotypes was associated with the occurrence of cholelithiasis. Further studies are essential to evaluate TGFBR3 haplotypes as prognostic markers and identify possible therapeutic targets in SCD individuals.

Data Availability
All relevant data used to support the findings of this study are included within the article and the supplementary information file.

Disclosure
The sponsors of this study, who played no role in gathering, analyzing, or interpreting the data presented herein, are public organizations whose role is to support science in general.