A number of studies were carried out to include molecular insights of haemophilia A and B.
It was found in a study that haemophilia b. is caused by more than 300 mutants are, in these complete and partial deletions, rare insertions, and point mutations are involved. The latter may reduce transcription, RNA processing and translation or cause of amino acid changes. Eighty-four residues are involved in the 105 presumed detrimental amino acid substitutions reported so far and these are usually conserved in the factor IX homologues. In addition mutations result in the development of antibodies against the therapeutic factor IX. Hotspots of mutations have been identified and are usually associated with CpG sequences (12).
Sommer et al. studied mutation rates per base-pair and after careful consideration and biases, predicted about 76 de novo mutations per generation per individual resulting in 0.3 deleterious changes.
The male-to-female sex ratio of mutation varies with type of mutation. There is evidence for a maternal age effect and a non-CpG G: C to A: T transitions. They found that the IX mutation pattern is similar to geographically, racially and ethnically diverse human populations (22). It was found in a study that most of the mutations occur from endogenous processes rather than the environmental mutagens and the factor IX protein is composed mostly of two classes of amino acids: critical residues in which all single-base missense changes will be protein function disrupted, and “spacer” residues in which the residue of the residue is unimportant but the peptide bond is required to register in critical residues (21).By using the direct sequencing method of genomic amplification, underlying pattern of mutation were studied and the mutation rates per base pair per generation was calculated; for transitions (27×10-10), transversions (4.1 x 10-10), and deletions (0.9 x 10-10) for total mutation rate of 32 x 10-10. The proportion of transitions at non-cpg nucleotides is elevated sevenfold over that expected if one base substitution was as second as possible.
At the dinucleotide CpG, transitions are elevated 24-fold relative to transitions at other sites (4). In Canada, a laboratory was established to create a mutation database of Canadian in 2000. It collected information about haemophilia from about 10 years from 2000 to 2011. In 1,177 patients the factor VIII gene was observed and factor IX gene in 267 patients . The mutation detection rates for HA and HB were 91% and 94% respectively. 380 different F8 mutations were identified: inversions of intron 22 and intron 1, 229 missense, 45 nonsense, eight deletions, 70 frameshifts, 25 splice sites, and one compound mutation.
A total 125 different F9 mutations were identified: 80 missense, 12 frameshift, 12 splice site, nine nonsense and seven promoter mutations, three large deletions, and two compound mutations, both missense and nonsense changes. The Canadian F8 and F9 mutation database reflects the allelic heterogeneity of HA and HB (14). Recombinant DNA techniques have been employed to analyze and disrupt the normal factor. Knowledge of the primary sequence of factor IX allowed identification of the specific defect in the factor IX Chapel Hill variant. Analysis of general factor IX genomic clones has determined that the 35 kb gene is composed of eight coding exons and seven intervening sequences. Sequence analysis of the CRM + variables will determine the mutations of interrupting the normal interactions of factor IX.
Southern analysis of CRM-variants has revealed that in some cases IX gene deletions are gross factor. Such deletions have been employed for carrier deletion in some families (15). Three hemophilia B patients with anti-factor IX antibodies who had no detectable gross deletion of the factor IX gene by Southern blotting analysis were investigated at the molecular level. Three different types of novel single base substitutions and a 2-base-pair nucleotide deletion were identified. Patient HB-5 had two point mutations in its factor IX gene. Patient HB-6 had a point mutation (G-to-A) in the splice acceptor site, which changed the normal splicing of the last intron G. A small two-nucleotide deletion in exon III was Detected in HB-7 and found Frameshifted amino acids and terminated by a stop codon. These results suggest that not only the gross gene deletion factor of the IX gene but also the point mutations or small nucleotide deletion that may cause interruptions of coding information to mature mature protein synthesis to predict the anti-factor IX inhibitors in patients with hemophilia B (23).
The single nucleotide primer extension method was used to detect haemophilia by denaturing polyacrylamide gel electrophoresis and autoradiography. As predicted by the Watson-Crick base-pair rule, in the wild type only the normal base, in an affected member only the mutant base, and in the carriers both the normal and the mutant base in the primer. Thus it was found in the SNP extension method that the base immediately 3 ‘to the template-bound primer is one of those mutant in altered, since in this way a single base by the preliminary extension of an extended molecule characteristic of either the mutant or the wild type (11).It was found in 2016 that mutations thatcause early translation termination account for 50% of inherited disorders.Transcripts bearing aberrant termination codons are most likely and areidentified by an evolutionarily conserved posttranscriptional pathway known asnonsense-mediated mRNA decay (NMD). There are many pieces of evidence of decayamong coagulation factor genes. However, the hemophilia gene (F8) does not seemto be exposed to NMD. Since the F8 gene is located on the X chromosomes, aconnection between X-linked traits and mRNA decay could be assumed (18).
Multiple mutations in the same gene within haemophilia have been reported. Incase of haemophilia A, out of 2740 entries, ten are double mutants. Among the2891 patient entries in the Haemophilia B mutation database, there are 34double mutants and one triple mutant (19).
The formation of inhibitorswhich inhibit the function offactors VIII and IX is a bigchallenge for the treatment of patients with congenital haemophilia. Geneticfactors are involved in the formation ofantibodies (7). Drugs that have a negative influence on blood clotting, such asNSAIDs, can lead to life-threatening bleeding in haemophilia patients (9).Current treatments for haemophilia consist of injections with plasma-derived orrecombinant clotting factors. These do not constitute a cure and patients arestill at risk of bleeding. Significant progress has recently been made in thedevelopment of gene therapy for hemophilia.
This has been mainly due to thetechnical improvements of existing vector systems and the development of newgene delivery methods. Therapeutic and sometimes physiological levels of FVIIIand FIX could be achieved in FVIII and FIX-deficient mice and hemophilic dogsusing different types of viral vectors. However, in addition to the inductionof neutralizing antibodies; viral promoter inactivation often results. A numberof gene therapy phase I clinical trials have been started in patients sufferingfrom acute Hemophilia A or B.
The results of extensive pre-clinical studies andpreliminary clinical data are encouraging. Successful gene therapy forhemophilia will become a reality in future (24).