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In silico profiling of deleterious amino acid substitutions of potential pathological importance in haemophlia A and haemophlia B.

Doss C GP - J. Biomed. Sci. (2012)

Bottom Line: In this study, instead of current biochemical methods, the effects of deleterious amino acid substitutions in F8 and F9 gene upon protein structure and function were assayed by means of computational methods and information from the databases.Furthermore, we modeled mutant proteins and compared them with the native protein for analysis of protein structure stability.Overall, we found that I-Mutant which emphasizes support vector machine based method outperformed SIFT and PolyPhen in prediction of deleterious nsSNPs in both F8 and F9.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Bio Sciences and Technology, VIT University, Vellore, Tamil Nadu, India. georgecp77@yahoo.co.in

ABSTRACT

Background: In this study, instead of current biochemical methods, the effects of deleterious amino acid substitutions in F8 and F9 gene upon protein structure and function were assayed by means of computational methods and information from the databases. Deleterious substitutions of F8 and F9 are responsible for Haemophilia A and Haemophilia B which is the most common genetic disease of coagulation disorders in blood. Yet, distinguishing deleterious variants of F8 and F9 from the massive amount of nonfunctional variants that occur within a single genome is a significant challenge.

Methods: We performed an in silico analysis of deleterious mutations and their protein structure changes in order to analyze the correlation between mutation and disease. Deleterious nsSNPs were categorized based on empirical based and support vector machine based methods to predict the impact on protein functions. Furthermore, we modeled mutant proteins and compared them with the native protein for analysis of protein structure stability.

Results: Out of 510 nsSNPs in F8, 378 nsSNPs (74%) were predicted to be 'intolerant' by SIFT, 371 nsSNPs (73%) were predicted to be 'damaging' by PolyPhen and 445 nsSNPs (87%) as 'less stable' by I-Mutant2.0. In F9, 129 nsSNPs (78%) were predicted to be intolerant by SIFT, 131 nsSNPs (79%) were predicted to be damaging by PolyPhen and 150 nsSNPs (90%) as less stable by I-Mutant2.0. Overall, we found that I-Mutant which emphasizes support vector machine based method outperformed SIFT and PolyPhen in prediction of deleterious nsSNPs in both F8 and F9.

Conclusions: The models built in this work would be appropriate for predicting the deleterious amino acid substitutions and their functions in gene regulation which would be useful for further genotype-phenotype researches as well as the pharmacogenetics studies. These in silico tools, despite being helpful in providing information about the nature of mutations, may also function as a first-pass filter to determine the substitutions worth pursuing for further experimental research in other coagulation disorder causing genes.

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Related in: MedlinePlus

Structural representation of FVIII (2R7E) native and mutant proteins. a. Structure of FVIII native type protein (2R7E) in grey displaying the position of W274, W412, W2065 and W2232 in sphere shape (green color). b. Superimposed structure of native amino acid tryptophan in sphere shape (green color) with mutant amino acid cysteine (red color) at position 274 in ‘A’ chain of 2R7E. c. Superimposed structure of native amino acid tryptophan in sphere shape (green color) with mutant amino acid arginine (red color) at position 412 in ‘A’ chain of 2R7E. d. Superimposed structure of native amino acid tryptophan in sphere shape (green color) with mutant amino acid arginine (red color) at position 2065 in ‘B’ chain of 2R7E. e. Superimposed structure of native amino acid tryptophan in sphere shape (green color) with mutant amino acid arginine (red color) at position 22232 in ‘B’ chain of 2R7E.
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Figure 1: Structural representation of FVIII (2R7E) native and mutant proteins. a. Structure of FVIII native type protein (2R7E) in grey displaying the position of W274, W412, W2065 and W2232 in sphere shape (green color). b. Superimposed structure of native amino acid tryptophan in sphere shape (green color) with mutant amino acid cysteine (red color) at position 274 in ‘A’ chain of 2R7E. c. Superimposed structure of native amino acid tryptophan in sphere shape (green color) with mutant amino acid arginine (red color) at position 412 in ‘A’ chain of 2R7E. d. Superimposed structure of native amino acid tryptophan in sphere shape (green color) with mutant amino acid arginine (red color) at position 2065 in ‘B’ chain of 2R7E. e. Superimposed structure of native amino acid tryptophan in sphere shape (green color) with mutant amino acid arginine (red color) at position 22232 in ‘B’ chain of 2R7E.

Mentions: Single amino acid mutations can significantly alter the stability of a protein structure. So, the knowledge of a protein's three-dimensional (3D) structure is essential for a full understanding of its functionality. Mapping the deleterious nsSNPs into protein structure information was obtained from dbSNP and SAAPdb. Available X-ray crystallized structures for the FVIII and FIX protein in Protein Data Bank with PDB ID code 2R7E (3.70 Å), and 2WPH (1.5 Å). Mutation analysis was performed based on the results obtained from highest SIFT, and PolyPhen scores. It is noted that rs34371500 (W274C), VAR_028524 (W412R) in 'A' chain and rs28937299/rs137852455 (W2065R), and VAR_028712 (W2332R) in 'B' chain of PDB ID 2R7E, showed the highest deleterious score of 0.00 (SIFT) and damaging scores (PolyPhen) ranging from 3.318 to 3.543 respectively in FVIII. Similarly in FIX, VAR_006611 (W431R), and rs137852269 (W453R) showed the highest deleterious score of 0.00 (SIFT) and damaging scores (PolyPhen) of 4.434 and 4.632 respectively. For W431R and W453R mutation analysis was performed in the 'S' chain of the PDB ID 2WPH. The mutations for FVIII and FIX at their corresponding positions were performed by SWISS-PDB viewer independently to achieve modeled structures. Then, energy minimizations were performed by GROMACS 4.0.5 for the native type protein and the mutant type structures. Total energy and the RMSD values between the native (2R7E and 2WPH) and the mutant amino acids were calculated. Higher the RMSD value more will be the deviation between native and mutant type structures and which in turn changes their functional activity. In this analysis found that the total energy for the mutant proteins W274C, W412R, W2065R, and W2332R following energy minimization was -97899.13, -98142.42, -98013.21 and -97013.21 kJ/mol when compared to native protein (2R7E) energy -98911.33 kJ/mol. The RMSD values were calculated between the native and mutant amino acids and showed 2.74 Å in W274C, 2.78 Å in W412R, 2.85 Å in W2065R and 2.91 Å in W2332R. The superimposed structures of the native protein with the four mutant type proteins are shown in Figure 1a-d respectively. These figures were drawn using PyMOL54 release 0.99 [131]. Similarly, we checked the total energy for mutant type structure W431R and W453R were found to be -81428.83 and -81694.21 when compared to native energy of -84591.35 kJ/mol. The RMSD values were calculated between the native and mutant amino acids and showed 2.94 Å in W431R, and 3.18 Å in W453R. The superimposed structures of the native protein with the four mutant type proteins are shown in Figure 2a-c, respectively. In W412R, W2065R, W2332R W431R and W453R there is change in drift of charge from non-polar to polar residue Substitution of positively charged arginine in place of neutral tryphtophan may lead to disturbance in the interactions with other molecules or other parts of the proteins. These types of substitutions could introduce repulsive interactions between neighboring residues. Similarly we observed the potential effects of substitutions such as disruption of ligand binding site, disruption of annotated functional site, overpacking at buried site, contact with functional site, and hydrophobicity change at buried site in PolyPhen predictions. Solvent accessibilities and secondary structures of amino acid residues in the native and mutant proteins were analyzed by GETAREA and DSSP as shown in Table 2.


In silico profiling of deleterious amino acid substitutions of potential pathological importance in haemophlia A and haemophlia B.

Doss C GP - J. Biomed. Sci. (2012)

Structural representation of FVIII (2R7E) native and mutant proteins. a. Structure of FVIII native type protein (2R7E) in grey displaying the position of W274, W412, W2065 and W2232 in sphere shape (green color). b. Superimposed structure of native amino acid tryptophan in sphere shape (green color) with mutant amino acid cysteine (red color) at position 274 in ‘A’ chain of 2R7E. c. Superimposed structure of native amino acid tryptophan in sphere shape (green color) with mutant amino acid arginine (red color) at position 412 in ‘A’ chain of 2R7E. d. Superimposed structure of native amino acid tryptophan in sphere shape (green color) with mutant amino acid arginine (red color) at position 2065 in ‘B’ chain of 2R7E. e. Superimposed structure of native amino acid tryptophan in sphere shape (green color) with mutant amino acid arginine (red color) at position 22232 in ‘B’ chain of 2R7E.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 1: Structural representation of FVIII (2R7E) native and mutant proteins. a. Structure of FVIII native type protein (2R7E) in grey displaying the position of W274, W412, W2065 and W2232 in sphere shape (green color). b. Superimposed structure of native amino acid tryptophan in sphere shape (green color) with mutant amino acid cysteine (red color) at position 274 in ‘A’ chain of 2R7E. c. Superimposed structure of native amino acid tryptophan in sphere shape (green color) with mutant amino acid arginine (red color) at position 412 in ‘A’ chain of 2R7E. d. Superimposed structure of native amino acid tryptophan in sphere shape (green color) with mutant amino acid arginine (red color) at position 2065 in ‘B’ chain of 2R7E. e. Superimposed structure of native amino acid tryptophan in sphere shape (green color) with mutant amino acid arginine (red color) at position 22232 in ‘B’ chain of 2R7E.
Mentions: Single amino acid mutations can significantly alter the stability of a protein structure. So, the knowledge of a protein's three-dimensional (3D) structure is essential for a full understanding of its functionality. Mapping the deleterious nsSNPs into protein structure information was obtained from dbSNP and SAAPdb. Available X-ray crystallized structures for the FVIII and FIX protein in Protein Data Bank with PDB ID code 2R7E (3.70 Å), and 2WPH (1.5 Å). Mutation analysis was performed based on the results obtained from highest SIFT, and PolyPhen scores. It is noted that rs34371500 (W274C), VAR_028524 (W412R) in 'A' chain and rs28937299/rs137852455 (W2065R), and VAR_028712 (W2332R) in 'B' chain of PDB ID 2R7E, showed the highest deleterious score of 0.00 (SIFT) and damaging scores (PolyPhen) ranging from 3.318 to 3.543 respectively in FVIII. Similarly in FIX, VAR_006611 (W431R), and rs137852269 (W453R) showed the highest deleterious score of 0.00 (SIFT) and damaging scores (PolyPhen) of 4.434 and 4.632 respectively. For W431R and W453R mutation analysis was performed in the 'S' chain of the PDB ID 2WPH. The mutations for FVIII and FIX at their corresponding positions were performed by SWISS-PDB viewer independently to achieve modeled structures. Then, energy minimizations were performed by GROMACS 4.0.5 for the native type protein and the mutant type structures. Total energy and the RMSD values between the native (2R7E and 2WPH) and the mutant amino acids were calculated. Higher the RMSD value more will be the deviation between native and mutant type structures and which in turn changes their functional activity. In this analysis found that the total energy for the mutant proteins W274C, W412R, W2065R, and W2332R following energy minimization was -97899.13, -98142.42, -98013.21 and -97013.21 kJ/mol when compared to native protein (2R7E) energy -98911.33 kJ/mol. The RMSD values were calculated between the native and mutant amino acids and showed 2.74 Å in W274C, 2.78 Å in W412R, 2.85 Å in W2065R and 2.91 Å in W2332R. The superimposed structures of the native protein with the four mutant type proteins are shown in Figure 1a-d respectively. These figures were drawn using PyMOL54 release 0.99 [131]. Similarly, we checked the total energy for mutant type structure W431R and W453R were found to be -81428.83 and -81694.21 when compared to native energy of -84591.35 kJ/mol. The RMSD values were calculated between the native and mutant amino acids and showed 2.94 Å in W431R, and 3.18 Å in W453R. The superimposed structures of the native protein with the four mutant type proteins are shown in Figure 2a-c, respectively. In W412R, W2065R, W2332R W431R and W453R there is change in drift of charge from non-polar to polar residue Substitution of positively charged arginine in place of neutral tryphtophan may lead to disturbance in the interactions with other molecules or other parts of the proteins. These types of substitutions could introduce repulsive interactions between neighboring residues. Similarly we observed the potential effects of substitutions such as disruption of ligand binding site, disruption of annotated functional site, overpacking at buried site, contact with functional site, and hydrophobicity change at buried site in PolyPhen predictions. Solvent accessibilities and secondary structures of amino acid residues in the native and mutant proteins were analyzed by GETAREA and DSSP as shown in Table 2.

Bottom Line: In this study, instead of current biochemical methods, the effects of deleterious amino acid substitutions in F8 and F9 gene upon protein structure and function were assayed by means of computational methods and information from the databases.Furthermore, we modeled mutant proteins and compared them with the native protein for analysis of protein structure stability.Overall, we found that I-Mutant which emphasizes support vector machine based method outperformed SIFT and PolyPhen in prediction of deleterious nsSNPs in both F8 and F9.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Bio Sciences and Technology, VIT University, Vellore, Tamil Nadu, India. georgecp77@yahoo.co.in

ABSTRACT

Background: In this study, instead of current biochemical methods, the effects of deleterious amino acid substitutions in F8 and F9 gene upon protein structure and function were assayed by means of computational methods and information from the databases. Deleterious substitutions of F8 and F9 are responsible for Haemophilia A and Haemophilia B which is the most common genetic disease of coagulation disorders in blood. Yet, distinguishing deleterious variants of F8 and F9 from the massive amount of nonfunctional variants that occur within a single genome is a significant challenge.

Methods: We performed an in silico analysis of deleterious mutations and their protein structure changes in order to analyze the correlation between mutation and disease. Deleterious nsSNPs were categorized based on empirical based and support vector machine based methods to predict the impact on protein functions. Furthermore, we modeled mutant proteins and compared them with the native protein for analysis of protein structure stability.

Results: Out of 510 nsSNPs in F8, 378 nsSNPs (74%) were predicted to be 'intolerant' by SIFT, 371 nsSNPs (73%) were predicted to be 'damaging' by PolyPhen and 445 nsSNPs (87%) as 'less stable' by I-Mutant2.0. In F9, 129 nsSNPs (78%) were predicted to be intolerant by SIFT, 131 nsSNPs (79%) were predicted to be damaging by PolyPhen and 150 nsSNPs (90%) as less stable by I-Mutant2.0. Overall, we found that I-Mutant which emphasizes support vector machine based method outperformed SIFT and PolyPhen in prediction of deleterious nsSNPs in both F8 and F9.

Conclusions: The models built in this work would be appropriate for predicting the deleterious amino acid substitutions and their functions in gene regulation which would be useful for further genotype-phenotype researches as well as the pharmacogenetics studies. These in silico tools, despite being helpful in providing information about the nature of mutations, may also function as a first-pass filter to determine the substitutions worth pursuing for further experimental research in other coagulation disorder causing genes.

Show MeSH
Related in: MedlinePlus