A new in silico approach to investigate molecular aspects of factor IX missense causative mutations and their impact on the hemophilia B severity

Abstract

Factor IX (encoded by F9) is a protein in the coagulation process, where its lack or deficiency leads to hemophilia B. This condition has been much less studied than hemophilia A, especially in Latin America. We analyzed the structural and functional impact of 54 missense mutations (18 reported by us previously, and 36 other mutations from the Factor IX database) through molecular modeling approaches. To accomplish this task, we examine the electrostatic patterns, hydrophobicity/hydrophilicity, disulfide, and H‐bond differences of the Factor IX structures harboring the missense mutations found, correlating them with their clinical effects. The 54 mutated sequences were modeled and their physicochemical features were determined and used as input in clusterization tools. The electrostatic pattern seems to influence in disease severity, especially for mutations investigated in epidermal growth factors 1 and 2 (EGF1/2) domains. The combined use of all physicochemical information improved the clustering of structures associated to similar phenotypes, especially for mutations from GLA and EGF1–2 domains. The effect of mutations in the disease phenotype severity seems to be a complex interplay of molecular features, each one contributing to different impacts. This highlights that previous studies and tools analyzing individually single features for single mutations are missing elements that fulfill the whole picture.

F8/F9 variants in the population-based PedNet Registry cohort compared with locus-specific genetic databases of the European Association for Haemophilia and Allied Disorders and the Centers for Disease Control and Prevention Hemophilia A or Hemophilia B Mutation Project

“…For hemophilia B, variants in mild hemophilia B occurred more frequently in the EGF1 domain but less frequently in the pro-peptide and linker than in moderate hemophilia B. The impact of variants on the FVIII or FIX protein structure and consequently on the disease severity is a topic of current research [ [20] , [21] , [22] , [23] ] and evidence is emerging that phenotypic variation may also be related to the region where the variant occurs. [ 20 , 21 ] However, the role of the location of the variant warrants further exploration.…”

Clinical Implications of Discrepancy between One-Stage Clotting and Chromogenic Factor IX Activity in Hemophilia B

“…Genotyping helps to predict the phenotype, e.g., severe HB is associated with frameshift and non-sense variants, and structural changes; in addition, selected genetic variants express a homogeneous phenotype 19,20,21 . In the near future, improved in silico modeling could better predict genotype-phenotype relationships 22,23 . In cases where the hemophilia severity remains unclear, we suggest using the CSA or OSA assay that the center has most experience, as is supported by the World Federation of Haemophilia, the Nordic Haemophilia Council, and the British Committee for Standards in Haematology.…”

The Molecular Basis of FIX Deficiency in Hemophilia B

“…F9 mutations lead to FIX deficiency at multiple levels, including its gene structure, gene transcription, splicing, translation, posttranslational modifications, protein folding, and formation of functional complex [ 8 , 41 , 60 , 63 , 64 , 65 , 66 , 67 ]. In the F9 variants database, about 88% of patients carry point mutations, and only about 12% have deletions, insertions, duplications, or indels ( Table 1 and Table 2 ).…”

“…For hemophilia B patients with point mutations, their bleeding phenotypes vary from severe to mild, and there are multiple mechanisms causing FIX deficiency [ 8 , 60 , 66 , 67 ]. Generally, point mutations in the promoter region result in hemophilia B Leyden [ 9 ], those in the exons cause missense, nonsense, or silent mutations, and those in introns cause aberrant splicing [ 41 ].…”