Genetics of the hemophiliasINTRODUCTION
? The hemophilias are a group of related bleeding disorders that usually are inherited. Inherited bleeding disorders include abnormalities of coagulation factors as well as platelet function, the most common of which is von Willebrand disease. However, when the term "hemophilia" is used, it usually refers specifically to the following two disorders:
? ?Factor VIII deficiency (hemophilia A)
? Hemophilia A affects 1 in 5000 to 10,000 males; roughly 60 percent have severe disease, with factor VIII activity less than 1 percent of normal. Factor IX deficiency (hemophilia B)
? Hemophilia B affects 1 in 25,000 to 30,000 males; approximately one-half have mild to moderate disease, with factor IX activity greater than 1 percent of normal.
Severe factor VIII or factor IX deficiency leads to bleeding because of the role these factors play in the intrinsic pathway X-ase (ten-ase). The X-ase complex consists of activated factor IX (factor IXa) as the protease; activated factor VIII (factor VIIIa), calcium, and phospholipids as the cofactors; and factor X as the substrate (show figure 1) . GENETIC TRANSMISSION
? Hemophilia A and B are X-linked recessive disorders, which explains who is likely to bleed and the modes of genetic transmission . These hemophilias occur almost exclusively in a male having one defective copy of the relevant gene on his X chromosome (ie, he is hemizygous for the defect). Because the affected male will transmit a normal Y chromosome to all his sons and an abnormal X chromosome to all his daughters, his sons will not be affected and all of his daughters will be carriers (ie, they are heterozygous for the defect).
Although female carriers have one normal allele, they may experience bleeding symptoms similar to that seen in a patient with "mild" deficiency. Therefore, their care and symptoms should be carefully evaluated. The female carrier will transmit the disorder to one-half of her sons, and one-half of her daughters will be carriers.
However, some women have more symptomatic hemophilia [3,4]. Possible explanations include mating between an affected male and a female carrier, producing homozygous disease in one-half of their female offspring. Alternatively, loss of part or all of the normal X chromosome, as in Turner syndrome  or skewed inactivation of the normal X chromosome , may lead to symptomatic disease .
Approximately one-third of patients with hemophilia do not have a family history of the disease and appear to represent novel mutational events [6,7]. The large size of the factor VIII gene and the presence of "hot spots" within the gene are thought to predispose the gene to mutation.
FACTOR VIII GENE ? The factor VIII gene is one of the largest genes known, comprising about 0.1 percent of the X chromosome. The gene that encodes factor VIII is divided into 26 exons that span 186,000 base pairs [8-11]. Factor VIII contains several areas of internal homology, consisting of a heavy chain with A1 and A2 domains; a connecting region with a B domain; and a light chain with A3, C1, and C2 domains [10,11].
Some of these domains have specific functions. For example, different epitopes on the C2 domain are responsible for binding to the procoagulant phospholipid phosphatidylserine on activated platelets and endothelial cells, von Willebrand factor (which importantly slows the catabolism of factor VIII), factor Xa, and thrombin. Two domains contribute to the binding of factor IXa (A2 domain and the A1/A3-C1-C2 dimer).
Hemophilia A genes ? Examination of hemophilic genes has not demonstrated a uniform abnormality. Instead, numerous different mutations in the Factor VIII gene have been described:
? ?In one study of 200 affected genes, seven different mutations were demonstrated . Four of them were transpositions of single bases, of which three transformed a codon for arginine into a stop codon, which arrested factor VIII synthesis and resulted in truncated factor VIII and severe hemophilia. The fourth mutation resulted in substitution of a single amino acid and mild hemophilias. The other three mutations involved deletion of several thousand nucleotides and produced severe hemophilia .
? ?A 1994 hemophilia A database indicated that more than 25 percent of unique nucleotide substitutions were caused by C-to-T transition at CpG dinucleotides . The genetic defects of hemophilia A encompass deletions, insertions, and mutations throughout the gene. Approximately 5 percent have large (greater than 50 nucleotides) deletions in the gene.
? ?Approximately 40 percent of severe hemophilia A is caused by a major inversion of a section of the tip of the long arm of the X chromosome, one break point of which is situated within intron 22 of the gene. A consortium study, involving 22 laboratories from 14 countries, evaluated 2,093 patients with severe hemophilia A . Seven hundred and forty patents (35 percent) had a type 1 (distal) factor VIII inversion, and 140 (7 percent) showed a type 2 (proximal) inversion. In 25 cases, the molecular analysis showed additional abnormal or polymorphic patterns. This observation has had a major impact upon carrier detection.
? ?Approximately 5 percent of patients who have hemophilia A also have normal levels of a dysfunctional factor VIII protein and are termed cross-reacting material (CRM)-positive. The majority of genetic alterations that result in CRM-positive hemophilia A are missense mutations within the A2-domain. The A2 subunit is essential for FVIIIa activity, and in one study, several of these mutants were found to have defective interaction with factor IXa .
Inhibitory antibodies that develop in a proportion of patients with hemophilia A and B following replacement therapy are now known to correlate with factor VIII and factor IX mutation type and location [16,17]. This subject is discussed in detail separately. (See "Factor VIII and factor IX inhibitors in patients with hemophilia").
? Deficiency of factors VIII and V ? An uncommon cause of factor VIII deficiency is a mutation in the LMAN1 gene, which causes a rare autosomal recessive disease with combined deficiency of factor VIII and factor V. This gene encodes a protein that may act as a molecular chaperone of these and other secreted proteins from the endoplasmic reticulum to the Golgi apparatus [18-20]. The disease is associated with a moderate bleeding tendency, with plasma levels of 5 to 30 percent of normal for both factors.
In other kindreds, the combined deficiency is associated with mutations in a gene on chromosome 2 called multiple coagulation factor deficiency 2 gene (MCFD2) . MCFD2 encodes a protein that forms a Ca(++)-dependent stoichiometric complex with LMAN1 and acts as a cofactor in the intracellular trafficking of factors V and VIII .
FACTOR IX GENE ? The gene encoding factor IX is also located near the terminus of the long arm of the X chromosome. This 34 kb gene contains eight exons and seven introns; its protein product is one of the vitamin K dependent proteins. As such, it is the largest gene of this family and the only one present on the X chromosome. Further, the number of exons and splice junction types are highly conserved in homologous vitamin K dependent proteins.
Hemophilia B genes ? Hemophilia B is a markedly heterogeneous disorder with a wide range of plasma levels of factor IX and a variety of specific gene defects . The original factor IX deficient subject, Mr. Christmas, was a severely deficient patient whose mutation was cysteine206serine .
Most affected families show a unique mutation. For example, one study of 70 unrelated patients in the Rhone Alps in France with hemophilia B found 2 complete gene deletions in patients with antifactor IX inhibitor, 6 small insertions/deletions, and 62 point mutations . Two of these nucleotide substitutions were detected in 21 patients (30 percent), suggesting the existence of a local founder effect. Of the mutations, 13 were previously undescribed.
The hemophilia database lists, in an easily accessible form, all known factor IX mutations caused by small changes (base substitutions and short additions and/or deletions of <30 bp) identified in hemophilia B patients . The 9th database includes 1918 patient entries with 689 unique molecular events (www.kcl.ac.uk/ip/petergreen/haemBdatabase.html
). Mutations have been detected in all regions of the Factor IX gene except the poly (A) site. Molecular events include 425 different amino substitutions and 143 short deletions and/or additions. Point mutations, the most common defect, can be associated with mild and severe hemophilia B. However, this database may not be completely representative of the disease for the following reasons:
? ?The database over-represents defects leading to severe disease, as these are more likely to be analyzed and reported.
? ?Founder effects contribute to over-representations at some specific sites.
? ?Double mutations may be underrepresented because of lack of complete gene screening.
Deletions of the gene and some point mutations may be associated with absence of the factor IX antigen, leading to inhibitor formation, with an incidence of approximately 3 percent.
? Leyden phenotype ? In one form, mutations at -20 and -26 in the factor IX promoter region impair transcription by disrupting the binding site for the liver-enriched transcription factor 3333. The -26, but not the -20 mutation, also disrupts an androgen-responsive element, which overlaps the LF-A1/HNF4 site. For this reason, patients with the -20 mutation improve after puberty (The Leyden phenotype); patients with the -26 mutation (Hemophilia B Brandenburg) do not improve .
? Dysfunctional protein mutants ? Similar to those families with hemophilia A, some families have antigen-positive (cross-reacting material positive, CRM+) defects, with clinical severity ranging from mild to severe. These patients have antigenic levels of factor IX that are near normal, but they have much lower factor IX activity levels. Approximately one-third of cases fall within this group. Mutations have been described that affect post-translational protein processing, gamma carboxylation and lipid binding, EGF domain function, zymogen activation, and substrate recognition and/or enzymatic activity.
? ?Defects in post-translational processing prevent cleavage of the 18-amino-acid propeptide of factor IX in factor IX Cambridge and factor IX Oxford [26,27].
? ?In factor IX Chapel Hill, the first factor IX mutation characterized at the molecular level, activation of a zymogen is impaired because of the mutation of an arginine preceding a sessile bond .
? ?Many of these families display point mutations affecting various regions of the gene, ranging from the Gla region to defects in the 180 to 182 and 390 to 397 trypsin-like (catalytic) domain. In Factor IX Vancouver, for example, the codon for isoleucine 397 (ATA) is changed to a threonine codon (ACA) . Hydrogen bonding between the side chain hydroxyl group of threonine 397 and the carbonyl oxygen of tryptophan 385 reduces the ability of Factor IX Vancouver to bind factor X in a configuration favoring catalysis.
? ?Hemophilia Bm is a variant of hemophilia B that results in a dysfunctional protein, characterized by a prolonged prothrombin time when ox brain but not human brain is used as the source of thromboplastin. The Bm phenotype is associated with mutations at residue 180,181, or 182 near the amino terminus of the heavy chain and at residue 311, 364, 368, 390, 396, or 397 near the beta cleavage site of factor IX [30,31]. Most have clinically severe disease.
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