Proteomic Expression Profile in Human Temporomandibular Joint Dysfunction

Temporomandibular joint dysfunction (TMD) is a multifactorial condition that impairs human’s health and quality of life. Its etiology is still a challenge due to its complex development and the great number of different conditions it comprises. One of the most common forms of TMD is anterior disc displacement without reduction (DDWoR) and other TMDs with distinct origins are condylar hyperplasia (CH) and mandibular dislocation (MD). Thus, the aim of this study is to identify the protein expression profile of synovial fluid and the temporomandibular joint disc of patients diagnosed with DDWoR, CH and MD. Synovial fluid and a fraction of the temporomandibular joint disc were collected from nine patients diagnosed with DDWoR (n = 3), CH (n = 4) and MD (n = 2). Samples were subjected to label-free nLC-MS/MS for proteomic data extraction, and then bioinformatics analysis were conducted for protein identification and functional annotation. The three TMD conditions showed different protein expression profiles, and novel proteins were identified in both synovial fluid and disc sample. TMD is a complex condition and the identification of the proteins expressed in the three different types of TMD may contribute to a better comprehension of how each pathology develops and evolutes, benefitting the patient with a focus–target treatment.


Introduction
Temporomandibular dysfunction (TMD) is a disorder of the masticatory system and it is characterized by pain, loss of function of one or both articulations, and impairment of the masticatory system. TMD impacts not only jaw function, but the life quality of affected patients, increasing their treatment costs and work absence [1]. According to the National Institute of Health [2], TMD management in the USA costs approximately 4 billion dollars per year. A diagnostic protocol developed for research named Research Diagnostic Criteria for TMD (RDC/TMD), classifies TMD as myalgia, arthralgia, condylar pathologies, disc displacement, osteoarthrosis, osteoarthritis, degenerative joint disease and subluxation [3]. TMD has a multifactorial etiology, the most common being trauma, psychological alterations, hormone, inflammatory diseases, parafunction, and genetics [1,4]. TMD usually requires a panorex, and depending on the TMD type, magnetic resonance imaging, scintigraphy and tomography, besides a thorough clinical evaluation [5,6].
Depending on the TMD type, it can be classified as condylar hyperplasia (CH), disc displacement without reduction (DDWoR) and mandibular dislocation (MD). DDWoR is the most common TMD disorder [7], and along with CH, its etiology's understanding is still unclear. MD is a condition that is probably caused by physical alterations [8], and since it is less likely to have hormone contribution, it is a good TMD condition to compare the results with the other pathologies. DDWoR is caused by an abnormal positional association between the disc and the condyle, where the disc is permanently anteriorly displaced Subjects did not present any of the following criteria: use of orthodontic appliances; chronic usage of anti-inflammatory drugs; history of diabetes, hepatitis, HIV infection; immunosuppressive chemotherapy; history of any disease known to compromise immune function; pregnancy or lactation; major jaw trauma; previous TMJ surgery; and previous steroid injection in the TMJ.
Subjects answered a personal medical history questionnaire and signed a consent form after being advised of the nature of the study. All patients were clinically examined by one experienced oral and maxillofacial surgeon. The clinical examination consisted of palpating the TMJ region, analyzing the occurrence of painful or limitation/excessiveness of mouth opening/closing, and the observation of facial asymmetry. Regarding complementary exams, all patients had a panorex and patients with disc displacement were submitted to a magnetic resonance image. The patients who were considered to be affected with disc displacement were treated surgically when they presented painful clinical signs of disc displacement after unsuccessful non-surgical treatment for at least 6 months [18]. Patients presenting pain related only to muscular spasms were not included in this research. Patients with condylar hyperplasia were diagnosed through clinical evaluation, panorex and when presenting a positive condylar growth in scintilography, a high condylectomy was indicated and performed [19]. Patients with recidivist mandibular dislocation (more than four episodes in six months) were treated with eminectomy [8].

Sample Acquisition
During access to the TMJ to perform the needed surgery [20], a 21-gauge needle was inserted into the upper TMJ space, then 1 mL of saline was injected into the joint space, which was aspirated thereafter by a second adapted syringe. This procedure was repeated five times to obtain a synovial fluid sample as described previously by Alstergren [21]. For each type of surgery performed, TMJ disc recontouring and repositioning was needed [16], therefore, first the displaced disc was freed, repositioned and sutured to the latero-posterior side of the condyle with a Mitek bone-cleat. The suture was then placed between the posterior and intermediate bands, and recontouring the thickened disk with a scalpel was necessary (this posterior debrided cartilage constituted the disc sample). Synovial fluid was spun down at 300× g to remove debris, and stored at −80 • C until use or analysis, and the disc samples rinsed in phosphate-buffered saline (PBS), and either snap frozen in liquid nitrogen and stored at −80 • C.

Proteomic Analysis
The microcentrifuge tubes containing the synovial fluid and TMJ discs were removed from the −80 • C freezer, and after defrosting, the discs were cut into small pieces with the aid of sterile scissors, centrifuged, and the supernatants were collected and pooled according to each pathology group. The preparation of the samples for proteomic analysis was carried out as previously reported [22]. The analysis of the tryptic peptides was performed in the nanoACQUITY UPLC system (Waters, Milliford, CT, USA) coupled to the Xevo Q-TOF G2 mass spectrometer (MS) (Waters, Milliford, CT, USA). For this purpose, the UPLC nanoACQUITY system was equipped with a column of type HSS T3 (Acquity UPLC HSS T3 column 75 mm × 150 mm; 1.8 µm, Waters), previously balanced with 7% of the mobile phase B (100% ACN + 0.1% formic acid). The peptides were separated through a linear gradient of 7%-85% of the mobile phase B over 70 min with a flow of 0.35 µL/min and the column temperature maintained at 45 • C. The MS was operated in positive ion mode, with a 75 min data acquisition time. The obtained data were processed using ProteinLynx GlobalServer (PLGS) version 3.03 (Waters, Milliford, CT, USA). Protein identification was obtained using the ion counting algorithm incorporated into the software. The collected data were searched in the database of the species Homo sapiens downloaded from the catalog of the UniProt [23] in September of 2020. The identified proteins for the groups DDWoR, MD, and CH of synovial fluid and TMJ disc were classified and attributed by biological function, origin, and molecular interaction with the program Genemania [24]. The overlapping proteins between the groups were clustered by using an automatic Venn diagram generator.

Results
In this qualitative study, our aim was to explore, for the first time, a comparative analysis of the proteomic profile of three distinct TMD diseases. Although a statistical analysis was not performed, we were able to identify and describe the function of the proteins, including overlapping proteins between the investigated samples (DDWoR, MD and CH, and between both synovial fluid and disc samples).
In the synovial fluid samples, a total of 225 proteins (351 counting the repeated proteins in all groups) were successfully identified: 190 in the group DDWoR, 154 in the group MD and seven in the group CH. We also compared these three groups to identify shared or condition-specific proteins. We found 114 shared proteins between groups DDWoR and MD, and six proteins were shared by all groups (Table 2).
In the disc sample, 379 proteins were identified (697 counting the repeated proteins in all groups), with 235 proteins in group DDWoR, 196 in group MD and 266 in group CH. These three groups were also compared to identify shared or condition-specific proteins. There were nine shared proteins between groups DDWoR and MD, 28 shared proteins between groups DDWoR and CH, 17 shared proteins between groups MD and CH, and 132 shared proteins by all groups (Table 3).
Regarding the proteins in common in both synovial fluid and disc in the same sample groups, DDWoR presented two common proteins, MD presented three proteins, group CH had no protein in common, and the three groups together had six proteins in common (Table 4).
All synovial fluid and disc samples presented proteins involved in DNA repair, muscle and neural regeneration.
A selective pool of proteins was chosen to be studied according to the pathology group and protein function for synovial fluid and disc sample (Tables 5 and 6).
The synovial fluid sample presented the following proteins functions for each group (Table 5): the DDWoR group presented proteins involved in inflammatory process, apoptosis, hearing, interleukine-6 cascade, and protection against oxidative stress; the MD group showed proteins involved in inflammatory process, apoptosis, hearing, interleukine-6 cascade, protection against oxidative stress, and immune response; in the CH group, the expression of alcohol degradation protein (ADH1) was identified. The group comprising the pathologies DDWoR and MD were mainly involved in inflammatory process inhibition, bone resorption, chondrogenesis, bone and cartilage formation, osteoarthrosis, and neuropathic pain. No proteins were observed in the groups DDWoR and CH, and MD and CH. The proteins expressed in all three groups (DDWoR, MD and CH) were mainly implicated with muscle regeneration.           Table 3. Gene code and name of the proteins expressed in temporomandibular joint (TMJ) discs of all groups (DDWoR, MD, CH) and between the groups DDWoR and MD, DDWoR and CH, MD and CH and DDWoR, MD and CH.             Table 4. Proteins expressed in both synovial fluid and TMJ disc samples of each group.  Mediates signal transduction various cytokines including interleukin-1, transforming growth factor-beta, bone morphogenetic protein 2 and 4, Toll-like receptors, tumor necrosis factor receptor CD40 and B-cell receptor.

SERPINC1
Serpin Family C Member 1 This protein inhibits thrombin and it regulates the blood coagulation cascade.

ALDH1L1
Aldehyde Dehydrogenase 1 Family Member L1 Associated with decreased apoptosis, increased cell motility, and cancer progression.

C4A
Complement C4A (Rodgers Blood Group) An antimicrobial peptide and a mediator of local inflammation.

HPX Hemopexin
Acute phase protein that transports heme from the plasma to the liver and may be involved in protecting cells from oxidative stress.

IFT122
Intraflagellar Transport 122 Involved in cell cycle progression, signal transduction, apoptosis, and gene regulation.

MYO6
Myosin VI This protein maintains the structural integrity of inner ear hair cells and mutations in this gene cause hearing loss.

PRDX1 Peroxiredoxin 1
Has an antioxidant protective role in cells and may contribute to the antiviral activity of CD8(+) T-cells.

SERPINH1
Serpin Family H Member 1 Plays a role in collagen biosynthesis as a collagen-specific molecular chaperone.

SMPD3 Sphingomyelin Phosphodiesterase 3
Mediates cellular functions, such as apoptosis and growth arrest.

ADH1
Alcohol Dehydrogenase Subunit Alpha Catalyzes the oxidation of alcohols to aldehydes.

ANXA1
Annexin A1 Inhibits phospholipase A2 and has anti-inflammatory activity. Functions as an autocrine factor which heightens osteoclast formation and bone resorption.

ASPN Asporin
Regulate chondrogenesis by inhibiting transforming growth factor-beta 1-induced gene expression in cartilage. May induce collagen mineralization.

BGN Biglycan
Plays a role in bone growth, muscle development and regeneration, and collagen fibril assembly in multiple tissues. This protein may also regulate inflammation and innate immunity.

CILP Cartilage Intermediate Layer Protein
This protein is present in the cartilage intermediate layer protein (CILP), which increases in early osteoarthrosis cartilage.

CLU Clusterin
Under stress conditions can be found in the cell cytosol.
May be involved in cell death, tumor progression, and neurodegenerative disorders COMP Thrombospondin-5 Present in rheumatoid arthritis, is a noncollagenous extracellular matrix protein.

DCN Decorin
Has a stimulatory effect on autophagy and inflammation and an inhibitory effect on angiogenesis and tumorigenesis.

FMOD Fibromodulin
May also regulate TGF-beta activities by sequestering TGF-beta into the extracellular matrix.

FN1 Fibronectin 1
Fibronectin is involved in cell adhesion and migration processes including embryogenesis, wound healing, blood coagulation, host defense.

DDWoR and CH
x x x

MD and CH
x x x

ENO2
Enolase 2 Found in mature neurons and cells of neuronal origin.

ENO3 Enolase 3
May play a role in muscle development and regeneration.

AKAP13
A-kinase anchor protein 13 Regulation of apoptotic process.

ERAS GTPase ERas
Tumor-like growth properties of embryonic stem cells.

PTPN7
Tyrosine-protein phosphatase non-receptor type 7 Regulation of T and B-lymphocyte development and signal transduction.

MFGE8 Milk Fat Globule EGF And Factor V/VIII Domain Containing
Promotes phagocytosis of apoptotic cells. This protein has also been implicated in wound healing, autoimmune disease, and cancer.

OGN Osteoglycin
Induces ectopic bone formation in conjunction with transforming growth factor beta and may regulate osteoblast differentiation. The disc sample presented the following protein functions for each group (Table 6): the DDWoR group expressed proteins involved in inflammatory process, neurogenesis, cartilage formation, extracellular matrix degradation, oxidative stress and apoptosis. The MD group presented proteins related to apoptosis, vascular growth, inflammatory inhibitors, immunologic factors and epithelial growth, and the CH group showed protein expression implicated in apoptosis, apoptosis inhibition, oxidative stress, bone formation, chondroitin, bone and cartilage formation. The group with DDWoR and MD samples had proteins involved in inflammatory process; the group with DDWoR and CH samples showed proteins with collagen formation and wound healing functions; the group with MD and CH was involved in wound healing; and the group containing DDWoR, MD and CH samples was involved with inflammatory cascade modulation, osteoclastogenesis, chondrogenesis, apoptosis, bone formation, vascular and tissue repair, antioxidative activity.
There were proteins identified in both synovial fluid and TMJ disc samples, however, some of them in different pathology groups (Table 7). Table 7. Name and function of expressed proteins in common between synovial fluid and TMJ disc sample, and the groups in each protein was expressed.  DDWoR, MD and CH DDWoR and MD Different types of collagen were identified in discs of the MD group, CH group, DDWoR and CH group, and in the group with all pathologies together (DDWoR, MD and CH). Besides the known collagen type I present in TMJ discs, collagen type IV, VI, XII and XIV were also identified ( Table 8). Table 8. Types of collagen identified in each TMJ disc group. All shared and group-specific proteins are indicated in a Venn diagram for the synovial fluid ( Figure 1) and disc samples (Figure 2). Different types of collagen were identified in discs of the MD group, CH group, DDWoR and CH group, and in the group with all pathologies together (DDWoR, MD and CH). Besides the known collagen type I present in TMJ discs, collagen type IV, VI, XII and XIV were also identified ( Table 8).

Type of Collagen Identified in Each Group
All shared and group-specific proteins are indicated in a Venn diagram for the synovial fluid ( Figure 1) and disc samples (Figure 2).  The interactions between the proteins were analyzed with Genemania ( https://genemania.org -September 5 th 2020), and its genetic network pointed out distinct protein cascades that might be modulating each pathology through the synovial fluid and disc Different types of collagen were identified in discs of the MD group, CH group, DDWoR and CH group, and in the group with all pathologies together (DDWoR, MD and CH). Besides the known collagen type I present in TMJ discs, collagen type IV, VI, XII and XIV were also identified ( Table 8).
All shared and group-specific proteins are indicated in a Venn diagram for the synovial fluid ( Figure 1) and disc samples (Figure 2).  The interactions between the proteins were analyzed with Genemania ( https://genemania.org -September 5 th 2020), and its genetic network pointed out distinct protein cascades that might be modulating each pathology through the synovial fluid and disc The interactions between the proteins were analyzed with Genemania (https:// genemania.org-accessed on 5 September 2020), and its genetic network pointed out distinct protein cascades that might be modulating each pathology through the synovial fluid and disc samples. The physical and genetic interactions, co-expression and pathway of the proteins are shown in Figures 3 and 4. layer protein (CILP), osteoglycin (OGN), transforming growth factor beta induc (TGFBI) involved in bone and cartilage formation, fibronectin 1 (FN1), lumican (LUM and tenascin XB (TNXB) involved in tissue repair, and neurofilament medium (NEFM and thrombospondin 4 (THBS4) involved in neuropathic pain were included in the n The DDWoR and CH group, and MD and CH group had no protein to be analyzed. T group with the three pathologies (DDWoR, MD and CH) showed an interaction of enola 2 (ENO2) and 3 (ENO3), involved in muscle regeneration ( Figure 3E).    The disc sample presented the following protein interactions in Genemania ( Figure  4): group DDWoR ( Figure 4A) presented mainly the matrix metalloproteinase protein (MMP) family (1,2,3,6,8,10,13,15,16), integrin subunit alpha 6 (ITGA6) and phospholipase A2 group VII (PLA2G7) that are involved in inflammatory cascade. Additionally, thrombospondin 3 (THBS3) and 4 (THBS4) involved in tissue remodeling, and THADA armadillo repeat containing (THADA) involved in apoptosis were present. In the MD group ( Figure 4B), A-kinase anchor protein 13 (AKAP13), Erbin (ERBIN) and uroplakin-3a (UPK3A) involved in apoptosis, collagen alpha-1(IV) chain (COL4A1) and GTPase Eras (ERAS) involved in disc matrix constitution, and liprin-alpha-1 (PPFIA1) and (PPFIA2) 2 responsible for cell interactions were identified in the Genemania network. In the CH group ( Figure 4C), the present proteins were ADAM metallopeptidase domain 10 (ADAM10), that regulates apoptosis, collagen type I alpha 2 chain (COL1A2) and serpin family H member 1 (SERPINH1) involved in collagen formation, actinin alpha 4 (ACTN4), PDZ Additionally, LIM domain 4 (PDLIM4), transthyretin (TTR) and protein tyrosine phosphatase non-receptor type 13 (PTPN13) involved in apoptosis, hormone modulation and bone formation. In the group of DDWoR and MD ( Figure 4D), the complement C4A (C4A) and complement C4B (C4B) proteins that mediates the inflammatory process were identified. In the DDWoR and CH group ( Figure 4E), mainly the proteins aggrecan (ACAN), collagen type I alpha 1 chain (COL1A1) and collagen type IV alpha 6 chain (COL4A6) that constitutes disc matrix, and periostin (POSTN) involved in wound healing were identified. In the MD and CH group ( Figure 4F), keratin 6A (KRT6A) involved in wound healing was identified. Additionally, in the group with all three pathologies (DDWoR, MD and CH) the proteins that interacted were annexin A1 (ANXA1), complement C3 (C3) and tenascin C (TNC) involved in inflammatory cascade modulation, annexin A2 (ANXA2) and transforming growth factor beta induced (TGFBI) involved in osteoclastogenesis, asporin (ASPN), biglycan (BGN), collagen type VI alpha 1 chain (COL6A1), osteoglycin (OGN) and vimentin (VIM) involved in chondrogenesis and osteogenesis, amyloid P component (APCS) and complement C3 (C3) in apoptosis and lumican (LUM) involved in tissue repair ( Figure 4G). The main proteins with important functions and networks that were identified in the synovial fluid sample were analyzed for each group (Figure 3). A brief description of these findings are: in the DDWoR group ( Figure 3A) alpha-2-macroglobulin (A2M) involved in inflammatory process, amyloid P component (APCS) involved with apoptosis and complement factor H (CFH) that modulates inflammatory cascade were highlighted in the Genemania interaction figure; in the MD group ( Figure 3B), hemopexin (HPX) involved in protection against oxidative stress was present; in the CH group ( Figure 3C), alcohol dehydrogenase subunit alpha (ADH1) that is responsible for alcohol degradation and interacts with growth hormone receptor (GHR) was present. In the group of DDWoR and MD ( Figure 3D), annexin A1 (ANXA1), decorin (DCN), and immunoglobulin heavy constant gamma 1 (IGHG1) involved in inflammatory process, annexin A2 (ANXA2) involved with bone resorption, asporin (ASPN), biglycan (BGN), cartilage intermediate layer protein (CILP), osteoglycin (OGN), transforming growth factor beta induced (TGFBI) involved in bone and cartilage formation, fibronectin 1 (FN1), lumican (LUM) and tenascin XB (TNXB) involved in tissue repair, and neurofilament medium (NEFM) and thrombospondin 4 (THBS4) involved in neuropathic pain were included in the net. The DDWoR and CH group, and MD and CH group had no protein to be analyzed. The group with the three pathologies (DDWoR, MD and CH) showed an interaction of enolase 2 (ENO2) and 3 (ENO3), involved in muscle regeneration ( Figure 3E).

Discussion
The different types of TMD may jeopardize patients' quality of life, masticatory function and have a great impact on health expenses. The identification of its multifactorial etiological components will enhance the employment of specific treatments, diminishing the hazard it causes in the TMJ. Therefore, the identification of the proteins expressed on each pathology group of this study (DDWoR, MD, and CH) might elucidate the cascades involved in the progression and severity of each TMD, leading to an assertive handling of TMD.
A total of 225 proteins were identified in the synovial fluid sample, and 379 in the TMJ disc sample (Table 2). It is important to highlight that the synovial fluid sample is very complex to obtain, therefore some proteins might not have been identified due to the technique that advocates the dilution of the synovial fluid. Nevertheless, the sample was collected according to worldwide employed standard methods previously described by other research groups [21,25]. Additionally, even though few proteins' expression might not have been observed, the expression of new proteins were identified for each pathology group, which enriches the global analysis of this study.
In our analysis, we found that all proteins expressed in the DDWoR group (synovial fluid and disc sample) (Tables 2 and 3) presented many proteins related to inflammatory process (MMP-3, -10, -27 in the disc sample) and apoptosis (mitogen-activated protein kinase 7-MAP3K7) and THADA in synovial fluid). Only the MMP-3 protein was previously associated with TMD [26,27]. These are proteins that highly impact the degeneration process in the TMJ of patients with DDWoR [26,28]. In the MD group, ERBIN protein was found in the disc sample, and it modulates TGFB, which was previously associated with TMJ degeneration [29]. Additionally, unprecedented proteins were seen in the synovial fluid associated with apoptosis (aldehyde dehydrogenase 1 family member L-ALDH1L1) and protection against oxidative stress (HPX), which probably helps diminish the mechanical overload consequences of the dislocation in the TMJ. Regarding CH proteins in the synovial fluid sample, ADH1 catalyzes the oxidation of alcohols to aldehydes, but as seen in Genemania ( Figure 3C), it interacts with GHR, which might be involved with the condylar overgrowth. In a previous study, GHR has been injected in rabbits' TMJ to increase cartilage thickness [30], but it has not been studied as a possible etiology of condylar overgrowth yet.
Additionally, we also found a set of proteins to be common in both synovial fluid and disc samples (Table 4) in the groups DDWoR (chromodomain-helicase-DNA-binding protein 8 and myosin light chain 6B), MD (filamin A and liprin-alpha-1), and in the three groups (enolase 1, 2, 3, myosin heavy chain 16, ribosomal protein L7 like 1 and component of the shield in complex). These proteins were involved in cell matrix adhesion, cellular motor protein, reorganization of cytoskeleton, muscle development and regeneration. Additionally, another group of proteins were identified in both synovial fluid and disc samples (Table 7), being prevalent in all groups of disc samples. In the DDWoR and MD groups of synovial fluid samples, proteins implicated in apoptosis, inflammatory process, bone formation and resorption, chondrogenesis, wound healing, tissue repair and protection against oxidative stress were found. CH disc samples and MD synovial fluid samples presented, as common proteins, HPX (protection against oxidative stress) and SERPINC1 (biosynthetic pathway of collagen).
LUM is associated with the regulation of collagen fibers and with cell migration. In this study, LUM was present in all disc samples, and it has been pointed out to be elevated when the disc is under stress, as it enhances tissue repair [31]. Ulmner [32] reported that higher levels of LUM in synovial tissue might diminish TMD surgical success. On the other hand, TNC was present in all disc samples and in DDWoR and MD synovial fluid sample, being an important protein in wound healing [33].
Temporomandibular joint discs are fibrocartilaginous discs composed mainly by collagen, glycosaminoglycan and proteoglycans [34]. Studies in human adults and fetuses showed the expression of mainly collagen type I and III in TMJ discs, with type I collagen observed in the posterior band of the articular disc and collagen type III on the inferior surface of the articular disc [35,36]. Moreover, collagen type II synthesis was expressed on the external layer of the TMJ disc [37]. In this study, collagen type IV was identified in MD and CH samples (Table 8), and a previous study observed the presence of collagen type IV in the middle part of fetuses' TMJ disc, indicating the development of blood vessels [38]. The TMJ disc is an avascular tissue, although under stress it may undergo metaplasia, forming a vascularized fibrous tissue. Collagen type VII was present in all samples, and along with collagen type IV, it has chondroprotective effects against inflammation [39]. Collagen type XII and XIV were present in the disc samples of this study, which have never been identified in this region before in humans. A study identified collagen type XII only in bovine disc samples, which helps maintain collagen type I integrity [40]. Nevertheless, collagen type XIV was also observed in all TMJ disc samples, and it plays an essential structural role in the integrity of collagen type I, mechanical properties, organization, and shape of articular cartilage, which has never been described in the TMJ disc before [41]. This is important information to understand the composition's strength and weakness of the TMJ disc.

Conclusions
In conclusion, many proteins were identified for the first time in the TMJ disc and synovial fluid of the groups DDWoR, MD and CH, leading to the enlightenment of each pathology's etiology, modulation and progression. Further studies with a greater sample are necessary to evaluate other proteins that might be present in these pathologies as well.