Haemophilia A and haemophilia B: molecular insights

Abstract

This review focuses on selected areas that should interest both the scientist and the clinician alike: polymorphisms within the factor VIII and factor IX genes, their linkage, and their ethnic variation; a general assessment of mutations within both genes and a detailed inspection of the molecular pathology of certain mutations to illustrate the diverse cause-effect relations that exist; a summary of current knowledge on molecular aspects of inhibitor production; and an introduction to the new areas of factor VIII and factor IX catabolism. An appendix defining various terms encountered in the molecular genetics of the haemophilias is included, together with an appendix providing accession numbers and locus identification links for accessing gene and sequence information in the international nucleic acid databases.

Figure 1

Schematic showing the intrinsic and extrinsic pathways of the coagulation cascade leading to fibrin formation. A deficiency or dysfunction of coagulation factor VIII or factor IX compromises the activation of factor X, the ensuing reactions are inefficient and haemophilia results.

Figure 2

(A) Genomic organisation of the human factor VIII gene. Exon and intron sizes are given in table 1 ▶. (B) Enlargement of a portion of intron 22 of the factor VIII gene to show the relative locations and orientations of the CG island, the intragenic gene F8A, the 5` exon of the putative gene F8B, and the extent of the int22h-1 homologue, which is duplicated towards the telomere of Xq. (C) Orientation of the factor VIII gene within Xq28 and location and orientation of int22h homologues.

Figure 3

(A) Factor VIII mRNA showing the extent and location of the open reading frame. (B) The newly synthesised factor VIII protein molecule comprising a pre-sequence of 19 amino acids and a mature peptide of 2332 amino acids (total length, 2351 amino acids). A1–3, B, C1, and C2 represent domains assigned according to homologies within the protein (the boundaries are somewhat arbitrary). The arginine residues signalling the sites for proteolytic activation are arrowed. (C) Activated factor VIII comprising a heterotrimer in which the dimeric N-terminal heavy chain is held together with the monomeric C-terminal light chain by a metal ion bridge (Ca²⁺).

Figure 4

(A) Genomic organisation of the human factor IX gene. Exon and intron sizes are given in table 2 ▶. (B) Factor IX mRNA showing the relative size and location of the open reading frame. (C) The newly synthesised factor IX protein molecule comprising a pre- and pro-sequence (27 and 19 amino acids, respectively) and a mature peptide of 415 amino acids (total length, 461 amino acids). (D) Activated factor IX comprising an N-terminal light chain and a C-terminal heavy chain held together by a disulphide bridge between cysteine resides 132 and 279. GLA, “GLA” domain, in which 12 glutamic acid residues undergo post-translational γ-carboxylation by a vitamin K dependent carboxylase; EGF, epidermal growth factor-like domain; act, activation peptide released after proteolytic activation at arginine 145 and arginine 180; catalytic, the serine protease domain.

Figure 5

Some of the known polymorphisms in the human genes for (A) factor VIII and (B) factor IX. Source references: factor VIII gene intron 7 G/A, intron 13 (CA)n, intron 18 BclI, intron 19 HindIII, intron 22 XbaI A, intron 22 MspI A, intron 22 (CA)n, intron 25 BglI, 3` MspI, factor IX gene 5` −793 G/A, 5` BamHI, 5` MseI, intron 1 DdeI, intron 3 XmnI, intron 3 BamHI, intron 4 TaqI, intron 4 MspI, exon 6 MnlI, exon 8 (RY)n, and 3` HhaI.

Figure 6

Genotyping the intron 22 XbaI A restriction fragment length polymorphism (RFLP) using long distance PCR (LD-PCR). A primer specific for factor VIII gene sequence (1A or 2A) is used in conjunction with a primer that flanks the XbaI A site and that hybridises within the int22h homologue (1B or 2B, respectively). The resulting LD-PCR product (∼ 7 kb) contains XbaI A (asterisk) and can be genotyped by XbaI digestion. The analysis is specific for XbaI A because the sequences to which primers 1A or 2A hybridise do not occur at the extragenic int22h loci. Primers 1B or 2B can hybridise, but in the absence of primers 1A and 2A amplification cannot take place.

Figure 7

One approach to polymorphism screening using DNA chips. Multiplex PCR could be used to generate locus specific fragments for hybridisation to the chip or, conceivably, fragmented, labelled genomic DNA could be hybridised directly, without amplification.

Figure 8

The intron 22 inversion of the factor VIII gene. (A) The normal configuration of the factor VIII gene and int22h homologues. (B) The distal or the proximal int22h homologue aligns with the intragenic int22h homologue (distal shown here). Homologous recombination takes place. (C) Linearisation of the X chromosome gives a completely disrupted factor VIII gene: exons 1 to 22 are displaced towards the telomere and are orientated in a direction opposite to their normal orientation. A contiguous factor VIII gene transcript running from exon 1 to 26 is no longer possible and severe haemophilia A results.

Figure 9

Detection of the intron 22 inversion of the factor VIII gene using long distance PCR (LD-PCR). Primers P and Q hybridise specifically to sequences within the factor VIII gene and flank int22h-1. Primers A and B hybridise specifically to sequences flanking int22h-2 and int22h-3. The normal factor VIII gene therefore gives the LD-PCR products P + Q (12 kb) and A + B (10 kb). The intron 22 inversion results in the hybridisation site for primer Q being displaced towards the telomere, whereas the hybridisation site for primer B from the distal or proximal int22h homologue (depending on which was involved in the homologous recombination leading to the inversion, distal shown here) is brought into context with primer P. The inverted gene therefore gives the LD-PCR products P + B (11 kb), A + B (10 kb), and Q + A (11 kb). An A + B product is always obtained because one copy of the int22h homologues is unaffected by the inversion.

Figure 10

Arginine residues encoded by CGA within (A) the factor VIII and (B) the factor IX protein sequences. Nonsense mutations are brought about by C to T transition within the CG site of these codons. These are recurrent gene defects within haemophilia populations.

Figure 11

Mutations as a result of CG transitions at the codons encoding the activation cleavage site arginine residues of (A) factor VIII and (B) factor IX.

Figure 12

The promoter region of the human factor IX gene showing the androgen response element (ARE) and the regions implicated in binding LF-A1/HNF4, C/EBP. Coordinates are relative to the transcription start site (+1). Haemophilia B Leyden mutations (closed arrows) lie outside the ARE but within the LF-A1/HNF4 and C/EBP regions implicated in constitutive expression. In contrast, the promoter mutation G-26C (haemophilia B Brandenburg, open arrow) causes lifelong haemophilia B and lies within both the ARE and the −9 to −40 region associated with constitutive expression.