Molecular Basis of Disease

The F9 gene is located on the long arm of the X chromosome (Xq27.1) and is 38 kb long with eight exons of varying lengths (25 to 1935bp). The resulting mRNA is approximately 3kb, of which the coding sequence is 1390 nucleotides. The intron/exon boundaries roughly correlate with the F9 domains and bear a high degree of homology to members of the vitamin K-dependent protein family (F7, F10, and protein C) (Figure 12-4). The F9 gene encodes a precursor protein of approximately 454 amino acids

-46 -19 1 40 85 415

P r e

pro

GLA

EGF

EGF

act

Catalytic

L i

L

Arginine Arginine 145 180

Arginine Arginine 145 180

Figure 12-4. Factor IX peptide showing domains. N, N-terminus; C, C- terminus; Pre, pre-propeptide; pro, propeptide; GLA, GLA domain; EGF, EGF domain, act, activation peptide.

consisting of a propeptide followed by a glutamic acid-rich (GLA) domain, two epidermal growth factor (EGF) domains, an activation peptide, and a catalytic domain.

F9 is activated to F9a when cleaved by F7a-TF and activated factor XI (F11a). Cleavage releases the activation peptide, resulting in a circulating light chain and heavy chain connected by a disulfide bond. Numerous posttrans-lational modifications are necessary for the normal function of F9a, including tyrosine sulfation, serine phos-phorylation, and 0- and N-linked glycosylation. The relatively small size of the F9 gene lends itself to detailed molecular analysis, which has been cataloged in an online database, Haemophilia B Mutation Database, which is updated regularly; the number system used below can be accessed at this Web site (http://www.kcl.ac.uk/ip/ petergreen/haemBdatabase.html).

Mutations in the F9 Gene

The majority of the F9 mutations are single base pair changes that result in missense, frameshift, or nonsense mutations. Short deletions (<30 nucleotides) account for approximately 7%, larger deletions approximately 3%, and insertions approximately 2% of mutations. Many of the single base pair changes occur at CpG doublets that are hotspots for mutation. However, a subset of repeated mutations is due to a founder effect, which typically results in mild disease. Mutations have been detected in all regions of the F9 gene including the poly(A) signal.

Missense mutations account for the majority of mutations that typically result in mild disease unless the mutations occur in residues critical for normal F9 function. Selected mutations in the promoter region of the F9 gene result in a unique phenotype, termed hemophilia B Leyden, which is characterized by severe disease at birth with progressive amelioration of severity through adolescence and puberty. However, some F9 promoter mutations (e.g., Brandenburg mutation at -26) result in lifelong severe disease. Nonsense mutations in the signal peptide and propeptide regions lead to severe HB; however, missense changes leading to retention of F9 within hepatic cells have been described (e.g., Ile30 and Ile19). Lack of cleavage of the propeptide leads to a dysfunctional F9 molecule (e.g., Arg4).

Mutations in the GLA domain disrupt y-carboxylation (posttranslational modification) that is important for normal F9a binding to collagen, activated platelets, and endothelial cells. Mutations in the EGF domain results in disruption of F9 binding to calcium that is essential to its procoagulant activity, as well as cofactor F8. Mutations in the catalytic domain typically disrupt the catalytic triad (His 221,Asp 269, and Ser 365) essential for F9a protease function.

An unusual F9 variant, due to mutation at Ala10, is characterized by normal baseline F9 activity. However, warfarin therapy results in a severe and disproportionate reduction in F9 activity (typically to <1%) and causes bleeding in patients being treated with warfarin who have an appar ently therapeutic international normalized ratio (INR). Indicative of such a situation is a disproportionate prolongation of the activated partial thromboplastin time (aPTT), which should be assessed by clotting factor assays and reveals the F9 deficiency.

Polymorphisms in the F9 Gene

Eight common polymorphisms have been described in different ethnic populations of European and African descent. These polymorphisms, however, are much less common in Asian and other populations. The most informative polymorphism documented in the Asian population is Hha I (allele frequency = 0.17); several recent reports have described additional polymorphic loci in these populations, facilitating molecular diagnosis of nonwhite carriers and patients with HB. A polymorphism within the F9 coding region, Ala148Thr, occurs within the activation peptide. This does not correlate with F9 activity or anti-genic levels. The Thr allele occurs with a frequency of 0.3 in the white population, but is much less frequent in the African American population (0.053 to 0.15) and Asian population (<0.01).

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