Molecular Analysis of Factor VIII and Factor IX Genes in Hemophilia Patients: Identification of Novel Mutations and Molecular Dynamics Studies

Abstract

BackgroundHemophilias A and B are X-linked bleeding disorders caused by mutations in the factor VIII and factor IX genes, respectively. Our objective was to identify the spectrum of mutations of the factor VIII and factor IX genes in Saudi Arabian population and determine the genotype and phenotype correlations by molecular dynamics (MD) simulation.MethodsFor genotyping, blood samples from Saudi Arabian patients were collected, and the genomic DNA was amplified, and then sequenced by Sanger method. For molecular simulations, we have used softwares such as CHARMM (Chemistry at Harvard Macromolecular Mechanics; http://www.charmm-gui.org) and GROMACS. In addition, the secondary structure was determined based on the solvent accessibility for the confirmation of the protein stability at the site of mutation.ResultsSix mutations (three novel and three known) were identified in factor VIII gene, and six mutations (one novel and five known) were identified in factor IX gene. The factor VIII novel mutations identified were c.99G>T, p. (W33C) in exon 1, c.2138 DelA, p. (N713Tfs*9) in eon14, also a novel mutation at splicing acceptor site of exon 23 c.6430 - 1G>A. In factor IX, we found a novel mutation c.855G>C, p. (E285D) in exon 8. These novel mutations were not reported in any factor VIII or factor IX databases previously. The deleterious effects of these novel mutations were confirmed by PolyPhen2 and SIFT programs.ConclusionThe protein functional and structural studies and the models built in this work would be appropriate for predicting the effects of deleterious amino acid substitutions causing these genetic disorders. These findings are useful for genetic counseling in the case of consanguineous marriages which is more common in the Saudi Arabia.

Identification of six novel factor viii gene variants using next generation sequencing and molecular dynamics simulation

“…PCR amplification and capillary sequencing. Amplification of the F8 gene exons was done as previously described (Al-Allaf et al, 2017a;Al-Allaf, 2017b). In summary, the gene was sequenced with 50 ng of genomic DNA as template in a 20 µl reaction mixture using 0.4 µl HotStarTaq plus DNA Polymerase (Qiagen, Hilden, Germany), 2 µl 10 × PCR buffer, 2 µl 25 mM MgCl 2 , 0.4 µl 10 mM dNTPs, 2 µl of 10 µM forward and reverse primers; descriptions of the primers used for PCR amplification and sequencing have been reported previously (Vidal et al, 2001).…”

Genotype Hemophilia Screening Program Identified 2 Novel Variants Including a Novel Variant (c.5816-2A > G) Causing a Pathogenic Variant of the Factor 8 Gene

“…Hemophilia A (HA) and hemophilia B (HB) (OMIM: 306700 and 306900, respectively) are X-linked recessive bleeding disorders that are caused by the inheritance of genetic variants affecting F8 and F9 genes, those encoding coagulation factors VIII and IX with a consequent deficiency or dysfunction for its relevant factor. 1 The F8 gene sited at the end of the long arm of the X chromosome, at Xq28, is extremely large and structurally complex (186 932 bp; nearly 180-kb length containing 26 exons), while the F9 gene placed more toward the centromere, at Xq27.6, is considerably smaller and structurally simpler (nearly 34-kb length containing only 8 exons). 2 HA is more prevalent than HB worldwide.…”

HindIII and MseI in the Biomolecular Profile of Hemophilia in Cameroon: A Primer Study

“…Both plasma-derived (pd) and recombinant clotting factor concentrates are suitable for these different strategies of hemophilia management [18]. Nowadays, many studies are being carried out on polymorphisms within the genes coding for factors VIII and IX [19] [20] [21]. Both factor VIII and IX genes contain two types of polymorphisms.…”