European Journal of Cardiovascular Medicine

The troponin complex is a key effector of cardiac muscle contraction, regulating the interactions between actin and myosin filaments that generate force and movement. The troponin complex comprises three main subunits – troponin T (TnT), troponin C (TnC), and troponin I (TnI) each of    which plays a distinct role in the regulatory process‎1,‎2). TnT is the molecular anchor that binds the complex to tropomyosin and anchors it onto the actin filament. In humans, there are three isoforms of TnT that are expressed in a tissue-specific manner‎3) – TnT1, TnT2, and TnT3. TnT1 is expressed in slow skeletal muscle,

TnT2 in fast skeletal muscle and cardiac muscle, and TnT3 in fast skeletal muscle.(‎6). TnT2, encoded by the TNNT2 gene, is the predominant isoform expressed in the adult human heart, where it plays a critical role in regulating myocardial contraction and relaxation (‎7)., Mutations in TNNT2 have been implicated in various inherited cardiomyopathies, including hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), and restrictive cardiomyopathy (RCM). These mutations can impair TnT2 function and lead to alterations in the sarcomere structure and function, which can result in cardiac hypertrophy, fibrosis, and arrhythmias (‎8).

Figure 1: Troponin protein and its structural role in cardiac muscles contraction (‎1)

In addition to these cardiomyopathies, recent studies have suggested a potential role for TnT2 in certain syndromes, such as LEOPARD and Noonan syndrome. LEOPARD syndrome is an autosomal dominant disorder characterized by multiple lentigines, electrocardiogram abnormalities, ocular hypertelorism, pulmonary stenosis, abnormal genitalia, retardation of growth, and deafness. (‎9)

Mutations in several genes have been linked to this syndrome, including the TNNT2 gene and PTPN11 genes, which encodes for the protein tyrosine phosphatase. Interestingly, recent studies have identified Tnnt2 as a potential modifier gene that can modulate the clinical features of LEOPARD syndrome in individuals with PTPN11 mutations. For example, in a large cohort of patients with PTPN11 mutations, a potential association was found between the presence of a common TNNT2 variant  and  the  presence of hypertrophic cardiomyopathy, a common cardiac feature of LEOPARD syndrome ‎2‎10). These findings suggest that TNNT2 may play a role in modulating the development of cardiac manifestations of LEOPARD syndrome and Noonan syndrome by interacting with other disease-causing genes  for example {PTPN11} gene.( ‎9) Similarly, Noonan syndrome is another autosomal dominant disorder characterized by facial dysmorphism, short stature, cardiac abnormalities, and developmental delay. Mutations in several genes have been associated with this syndrome, including the PTPN11. Which was responsible  for about 45%-50 % of  Noonan cases.

Genetic modifiers defined as a genetic variants that can modify a phenotypic outcome of the primary disease causing  variant ,they can increase (defined as inhancers ) or decrease ( defined as suppressor) the severity of the disease but may not be disease –causing themselves ( ‎11‎12).

Recent studies have also implicated Tnnt2 as a potential modifier gene in Noonan syndrome, where its mutations have   been linked to cardiac dysfunctions and variable clinical outcomes. For example, in a study of a cohort of patients with Noonan  syndrome, Tnnt2 mutations were found to be associated with an increased risk of cardiac abnormalities accounting for 45%-50% of patients (‎13).

These findings suggest that Tnnt2 may modulate the clinical      expression of  Noonan syndrome and   Leopard syndrome by interacting with other disease-causing genes and modifying their effects on  cardiac function and cognitive development  (‎14).

Figure 2 :Two-dimensional echocardiography showed atrium (A) and atrioventricular valve (AV) with severe  regurgitation and large ventricular septal defect in noonan syndrome (‎15).

Figure 3: Two-dimensional echocardiographic showed apical left ventricular hypertrophy with in homogeneous echo lucent areas in the hypertrophic myocardium associated with leopard syndrome (8)

TABLE 1: Genetic Mutations Associated with Noonan and LEOPARD Syndromes

In this study, we aimed to investigate the impact of TnnT2 gene  mutations on the pathogenesis of Leopard Syndrome and Noonan Syndrome. We employed multiple computational tools to gain a comprehensive understanding of the structural and functional changes induced by these mutations.‎17).

Firstly, we have successfully used the Snap Gene Viewer program in our research methodology to view and detect gene mutations related to Leopard and Noonan syndrome. The program has provided us with an in-depth analysis of the genetic sequences and helped us identify the variations in the coding regions of the genes essential for diagnosing these syndromes(‎18)

We then used Discovery Studio Visualizer to obtain a three-dimensional structure of the TnnT2 protein and to edit molecular structures ,sequences,and sequence alignment which further more allowed us to understand the location of the mutations and their potential impacts on the protein's structure and function(‎19  ).

We then used Modellar and Phyre2 to predict changes in the protein's structure that are likely to be caused by mutations. These tools enabled us to examine how these mutations could affect the protein's ability to interact with other proteins within cells and to perform essential cellular      functions (‎20,‎21).

And we also used Swiss-Model which can be utilized to  predict various aspects of protein biology including disease-causing mutations and interactions with other proteins ‎21(‎21,‎22)

Finally, we used polymol to analyze potential drug- target interactions. This tool helped identify potential drug targets and suggest potential drug candidates for the treatment of these syndromes (‎23)

1-Snap gene analyzer  we utilized the Snap Gene Viewer program to explore and analyze genes associated with Leopard and Noonan syndrome. We were particularly interested in identifying any mutations or variations in these genes that could explain the underlying causes of these conditions. By using the Snap Gene Viewer, we were able to visualize and annotate the DNA sequences for these genes, and compare them to reference sequences to identify any discrepancies. Through our analysis, we discovered several significant genetic variations that have been linked to both Leopard and Noonan syndrome, shedding new light on the genetic basis of these complex disorders (‎18‎24).

Figure 4 : utilizing TNNT2 gene showing red line areas of potential mutations and overlapping modifying regions (‎25).

Figure 5 : : utilizing PTPN11 gene showing red line areas of potential mutations and overlapping modifying regions.(‎26)

2-Discovery Studio Visualizer is a software tool used for visualization and analysis of molecular structures in three dimensions. In the context of our study, we used Discovery Studio Visualizer to obtain the three- dimensional structure of the TNNT2 protein.After loading the protein structure into the software, we were able to examine its overall structure and identify the specific locations of mutations associated with both Leopard Syndrome and Noonan Syndrome. By visualizing the mutations in the protein structure, we were able to assess their location and their potential impact on the protein's structure and function (‎19)

Additionally, Discovery Studio Visualizer allowed us to perform molecular docking studies to investigate potential interactions between the TNNT2 protein and other molecules. Using the software's docking tools, we were able to simulate the binding interactions between the protein and         various compounds, enabling us to understand the potential efficacy of these compounds to bind to the protein and inhibit its function (‎27)

Overall, the use of Discovery Studio Visualizer allowed us to obtain a comprehensive understanding of the structure and function of the TNNT2 protein in the context of disease- associated mutations. The software's visualization and docking tools were essential in enabling us to study the potential impact of mutations and to evaluate the efficacy of potential drug candida (28)

Figure 6 : structural and  molecular view {at the N-terminal }  of TNNT2 protein using discovery studio visualizer software (‎6,‎23).

3-Modeller and phyre2 is a software tools used for  comparative protein structure modeling. Comparative modeling is a method used to predict the 3D structure of a target protein based on the availability of a known protein structural template from a homologous protein ‎21). Modeller uses this method to create a protein model for a specific amino acid sequence, by predicting the three-dimensional structure of that protein based on its similarity to other proteins for which experimental structures are available.‎30)

Phyre2, on the other hand, is a web-based protein structure prediction tool that uses various algorithms, including both comparative modeling and ab initio modeling,to predict the 3D structure of a protein from its amino acid sequence. Phyre2 takes a protein sequence as input and predicts the structure using multiple algorithms, including homology modeling, fold recognition, and ab initio structure prediction.

In our study, we used both  Modeller and Phyre2 to predict   the three-dimensional structure of the TNNT2 protein. We used Modeller to generate a comparative model of the protein using available structures of other related proteins as templates, while Phyre2 was used to generate an ab initio model (‎31)

The ab initio mode is  a computational approach that predicts protein structure based on first principles and fundamental physical  laws. In the context of protein structure prediction, "ab initio" refers to the fact that the model does not rely on any prior knowledge or templates from experimentally determined protein structure which relies to predict a structure without using a known template structure (‎32).

Figure 7: predicted protein structure using phyre 2 software.

Using both tools allowed us to generate multiple models of the protein structure, which we subsequently evaluated based on various criteria, including model quality scores and agreement with available biochemical data. By analyzing the predicted protein structures, we were able to gain insight into the potential impacts of mutations on the protein's structure and function, which can help guide the development of targeted therapies for related diseases.

Figure 8 :different type of predicted proteins associated with potential  mutations in their structural amino acid sequence (‎34).

Table 2 : suspected amino acid mutants that affect the defected TNNT2 protein 

4-Swiss-Model Using Swiss-Model, we generated multiple homology models of human TNNT2 protein based on the X-ray crystal structure of human PDB ID: c5goxb, c1j1eB, c6f1tX) as a template (Error! Reference source not found.). The resulting models had a QMEAN score {Q or Qmean is the global model quality estimation score. It provides an assessment of the reliability of a given protein model by comparing it with a large number of experimentally determined protein structures in the Protein

Data Bank (PDB). } of 0.82,0.21 indicating good quality. Qmean can be used as a measure of the accuracy of a protein model and to compare different models of the same protein. Structural analysis of the model revealed that the N- terminal region of TNNT2 contains a cytoplasmic extension that interacts with other components of the thin filament in the sarcomere. This region also contains several disease-causing mutations (‎36‎37).