Haemophilia is a genetic
disease, mostly inherited in nature that reduces the body’s ability to make
blood clots. An almost astonishing
diversity of mutations has since been characterized in the coagulation factor
VIII gene in haemophilia A and the coagulation factor IX gene in haemophilia B.
factor 8 and factor 9 proteins circulate as inactive precursors which are
activated when haemostatic challenge arises, through the intrinsic or extrinsic
routes of the coagulation pathway. No enzyme activity yet seen for factor VIII
which is a protein cofactor. Factor IX is a serine protease with an absolute
requirement for factor VIII as a cofactor. On activation, limiting factor VIII
and factor IX form an active complex ‘the tenase complex’ which activates
factor X, which then results in the deposition of fibrin, which is the
structural polymer of the blood clot. Activation of factor X is compromised by
a deficiency of either factor VIII or factor IX, so that the following steps of
the coagulation pathway are also compromised and fibrin deposition becomes
inefficient or non-existent. Thus, the main biochemical reasoning under the
haemophilias is an impairment of the activity of the tenase complex, caused by
a deficiency of coagulation factor VIII cofactor activity (haemophilia A) or
coagulation factor IX enzyme activity (haemophilia B) (5).Hemophilia A is X
linked genetic disease and the most common coagulation disorder with an incidence
of about 1-2 in 10,000 males and its reason is the mutation of factor VIII
coagulation gene (3). The factor VIII gene
has been cloned in 1984 and has a large number of mutations that cause
hemophilia have been identified (20). It has been observed that almost half the severe cases of
haemophilia A are due to frequently occurring inversions caused by homologous
intra-chromosome recombination between repeated sequences 9.5 kb long. Of the three
repeats one is in intron 22 of the factor VIII genes and two are 400-500 kb
more telomeric. They are 99.8% similar to each other (1). A homologous
recombination mechanism is proposed for the inversion between an intragenic
copy of the F8A gene and either the distal (80% of the inversion) or the
proximal copy (20%). Both of these inversions lead to severe HemA because no F8
is produced and can be easily diagnosed by Southern blot analysis (3).
Haemophilia A results in joint and muscle blood extravasation, easy injury and bleeding from wounds for a long period of time. Blood loss from minor cuts and abrasions is not
excessive. Patients will be having severe hemophilia if factor VIII activity is
less than 1%, while the remaining will have mild to moderate disease with
factor VIII levels of 1 to 20%. Nearly all patients with severe disease have no
protein VIII in their plasma (20).Hemophilia B is a
deficiency of coagulation factor which is observed in cases of reduced levels
or an absence of factor IX (2). Factor 9 is a coagulation factor serine
protease that is encoded by a 34 kb gene located on chromosome 10 (25).
Haemophilus B occurs when a sequence change in the factor 9 gene disrupts one
or more of the functional domains of factor 9. Mutations occur at multiple
sites in the factor IX gene (6). It has been also observed that Amino acid
substitutions at or near the site of activation may cause inactivation of
clotting factor 9 and results in decreased activity. For maximum interaction of
factor 9 with its cofactors and substrates, release of the activation peptide
is necessary. If there are abnormalities in the calcium binding region, which
could be Gla independent or dependent clotting activity will be decreased (15).
Multiple mutations in the same gene in a Haemophilia family are being noted.
But the genetic diagnosis is the major challenge in patients with multiple
mutations particularly in developing countries wherein the entire gene is not
being sequenced (19).
Prenatal diagnosis of haemophilia followed by potential genetic counselling and risk assessment of potential carrier and subsequent support during the diagnostic process may be effective for haemophilia treatment. Chorionic villus sampling is the most widely used method but amniotic fluid; fetal blood and pre-implantation genetic diagnostics can also be used in prenatal diagnosis of haemophilia A or B (16). One of the main complications of haemophilia treatment is the formation of inhibiting antibodies that inactivate FVIII or FIX (9). The exogenous factors of the inhibitors inhibiting the appearance of inhibitors VIII (FVIII) and IX (FIX) continue to be a major challenge in patients with congenital haemophilia (7). In severe haemophilia A and B, mutations that result in an absent or reduced FVIII / FIX protein are associated with a 20-80% risk of inhibitor formation (8). Improved understanding of these interactions may lead to the development of preventive measures to minimize inhibitor formation. Currently treatment for hemophilia involves the injection of either the recombinant or plasma-derived clotting factor concentrates which is ironically improved the life expectancy of patients. They have a drawback; Regular transfusions caused HIV or Hepatitis C in hemophiliac patients (10). Clotting factor concentrates are very expensive, so there is a need of cost effective treatment (13). AAV mediated gene transfer holds the effect of the effective treatment of haemophilia (17).