Molecular Basis of Disease

The F8 gene is located on the long arm of the X chromosome (Xq28), is 186kilobases (kb) in length, and has 26 relatively short exons, ranging from 69 to 262 base pairs (bp),

Figure 12-1. The coagulation cascade. a, activated factor; Ca2+, calcium; PL, phospholipid. (Reprinted with permission from Davie EW, Fujikawa K, Kisiel W.The coagulation cascade:initiation, maintenance and regulation. Biochemistry. 1991;30:10363. Copyright 1991 American Chemical Society.)

and two long exons, exon 14 (3106bp) and exon 26 (958bp) (Figure 12-2). The resulting messenger RNA (mRNA) is approximately 9kb, of which the coding sequence is 7053 nucleotides. The intron/exon boundaries roughly correlate with the F8 protein domains. The introns are large (14 to 23 kb), with intron 22 being that largest (32kb). A CpG island in intron 22 acts as a bidirectional promoter for two additional genes. The first, termed F8-associated gene A (F8A or INT22H1), is an intronless gene approximately 2 kb long within intron 22 of F8 and is transcribed in the opposite direction from F8. The second, F8-associ-ated gene B (F8B), is 2.5 kb long and transcribed in the same direction as F8. The F8A and F8B transcripts originate within 122 bases of each other, and the functions of their potential protein products are unknown. The F8A sequence is replicated at least twice, approximately 500kb telomeric to the F8 gene and close to the tip of the X chromosome, termed INT22H2 (proximal) and INT22H3 (distal). INT22H2 and INT22H3 are about 100kb apart and transcribed in the same direction as the F8 gene. These three homologous repeats are involved in the intron 22 inversion mutation, which is a frequent rearrangement of the F8 gene resulting in severe HA. F8 is predominantly expressed in the liver. The putative promoter region is located 300 nucleotides 5' of the gene, and although a TATA box is not essential for transcription, liver-enriched transcription factors (e.g., HNF1, NFkB, C/EBPa, and C/EBPb) interact with the F8 promoter region. The F8 gene encodes a precursor protein of 2351 amino acids consisting of a 19 amino acid leader peptide followed by 2332 amino acids in the mature protein. The mature protein has q29 x chromosome q29 x chromosome

F8A F8B (Int22h-1)

F8A F8B (Int22h-1)

Figure 12-2. The factor VIII gene. G6PD, glucose 6 phosphate dehydrogenase; kb, kilobases. (Reprinted with permission from Kazazian HH, Tuddenham EGD, Stylianos EA. Hemophilia A: deficiency of coagulation factor VIII. In: Scriver CR, Beaudet AL, Valle D, et al., eds. The Metabolic and Molecular Bases of Inherited Disease. Copyright 2001 McGraw-Hill.)

several homologous domains termed A1, A2, B, A3, C1, and C2.

Proteolytic cleavage, by F2a or F10a, in the presence of phospholipid surfaces results in activation of F8 to F8a (Figure 12-3). Although cleavages at 740 or 1721 have no effect on coagulant activity, the cleavages at 372 or 1689 are important for F8 procoagulant activity. Cleavage at 1689 releases F8 from VWF, permitting F8 interaction with phospholipids and platelets. Missense mutations affecting these cleavage sites have been found in patients with HA and result in cross-reacting material positive (CRM+)-HA with normal levels of F8 antigen but low activity (1% to 7%). Although missense mutations have been found in the A2 domain in HA, and the importance of the A2 domain to F8 coagulant activity has been confirmed by in vitro studies, the exact role of the A2 subunit remains unknown.

The B domain is cleaved during proteolytic activation. Since B-domain-deleted F8 molecules are expressed at 5-to 10-fold higher levels than non-B-domain-deleted F8,the B domain may have a role in intracellular processing or

\

If,,

A1

a1

A2 a2

B a3

A3 C1 C2

Figure 12-3. F8 peptide showing domains (A1 to C1),cleavage sites (arrows) for thrombin (FIIa), activated factor IX (FIXa), activated factor X (FXa), and activated protein C (APC). Amino acids are numbered in parentheses. The heavy chain cleavage site (A1-a1-A2-a2) is linked to the light chain cleavage site (a3-A3-C1-C2) via the B domain.(Reprinted with permission from Pruthi RK, Nichols WL. Autoimmune factor VIII inhibitors. Curr Opin Hematol 6:314. Copyright 1999 Lippincott Williams and Wilkins.)

secretion of F8 or both. Mutations in this region have been reported in HA.

VWF-bound F8 is protected from inactivation by activated protein C (APC). The putative VWF binding region of F8 is thought to be at the N-terminus of the light chain of F8 and in the C2 domain. The binding site for F9a has been localized to the A2 domain and regions of the light chain. In addition, the binding site for F10 is localized to the C-terminus of the A1 domain. Binding to phospholipids, which are important for F10 activation by F9a and F8a, occurs in the C1 and C2 domains of the light chain F8. No deleterious mutations in HA have been identified in the inactivation cleavage sites of F8.

Mutations in the F8 Gene

Reported mutations and polymorphisms are cataloged in an international database, the Haemophilia A Mutation, Structure, Test and Resource Site, available on the Internet and updated periodically: http://europium.csc.mrc.ac.uk/ WebPages/Main/main.htm. References for the amino acid numbering system used below can be accessed at this Web site.

Currently, no mutations have been found in the F8 promoter region. Approximately 40% of HA patients with severe disease have an inversion at the tip of the X chromosome that disrupts the F8 gene.2 Homologous recombination can occur when the F8A gene (INT22H1) in intron 22 recombines with one of the two homologous regions (INT22H2 or INT22H3) telomeric to the F8 gene probably as a result of folding over of the tip of the X chromosome. Upon unfolding, exons 1 to 22 are inverted and placed about 500kb upstream of exons 23 to 26 and oriented in the opposite direction. Depending on which repeat F8A pairs with, the inversion may be termed type I (distal) or type II (proximal). Rarely type III inversion mutations occur in patients with a third extragenic copy of F8A. Given that the majority of inversions originate in male meiosis, almost all mothers of patients with the inversion mutation are carriers. An additional inversion of exon 1 of the F8 gene also occurs and affects up to 5% of patients with severe HA.3

Given the large size of the F8 gene, deletions are common and account for about 5% of characterized mutations. Typically these result in severe disease with <1% F8 activity. However, specific deletions of 156bp in exon 22 or 294bp in exons 23 and 24 are associated with moderate disease, likely due to in-frame splicing of exon 21 to exon 23, or exon 22 to exon 25. Patients with large deletions are susceptible to formation of F8 inhibitor (antibodies) in response to therapy with F8 concentrates. In an analysis of the HA database, up to 40% of patients with deletions develop F8 inhibitors, whereas up to 60% of patients with single base pair changes resulting in nonsense mutations and 15% of patients with single base pair changes resulting in missense mutations develop inhibitors.4

The remaining patients typically have single base pair changes (resulting in missense, frameshift, or splice junction mutations), insertions, or duplications. Single base pair changes that result in missense mutations are spread throughout the F8 gene. Although the structure-function relationships of some of the missense mutations are known or can be deduced (e.g., alteration of the VWF binding site or thrombin cleavage site), the structural consequences of most missense mutations remain undefined.

Although HA predominantly affects males, some female carriers have reduced F8 levels and may have clinically significant bleeding. The molecular basis of symptomatic females includes lyonization of the normal X chromosome and Turner syndrome (XO karyotype),where the dominant mutant F8 gene is responsible for production of the abnormal F8. Rarely, mating of a carrier female with an affected male, an X-autosome translocation involving a breakpoint within the F8 gene, and uniparental isodisomy have been implicated.

Polymorphisms in the F8 Gene

Polymorphisms present within the F8 gene (intragenic) or outside the F8 gene (extragenic) have been used to assign haplotypes (combinations of polymorphisms) for linkage analysis. The putative defective F8 gene can be tracked with polymorphisms that are closely linked to the gene. The carrier frequency of such polymorphisms varies depending on the ethnicity of the study population and needs to be considered when studying patients of diverse ethnic origin. A complete listing of polymorphisms is available in the F8 mutation database (http://europium.csc.mrc.ac.uk/ WebPages/Main/main.htm).

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