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- Patients and methods
- Note added in proof
The genetic defects of four Taiwanese patients with factor VII (FVII) deficiency were studied. FVII activity and antigen levels were < 1 u/dl and 125·7 u/dl (patient I), < 1 u/dl and < 1 u/dl (patient II), 3·4 u/dl and 5·9 u/dl (patient III), and 1·2 u/dl and 30·4 u/dl (patient IV) respectively. The 5′ flanking region, and all exons and junctions were amplified using polymerase chain reaction and sequenced. Patient I was homozygous for a 10824CA transversion with Pro303Thr mutation in exon 8. In patient II, a heterozygous transversion, 9007+1GT at the IVS6, a heterozygous decanucleotide insertion polymorphism at −323 (both mutations present in his father) and a heterozygous deletion, del TC (26–27) in exon 1A (originating from his mother) were identified. Patient III had a homozygous 10961TG transversion with His348Gln mutation in exon 8. Patient IV had a heterozygous 10902TG transversion with Cys329Gly mutation in exon 8 (transmitted to her second son) and a heterozygous decanucleotide insertion polymorphism at −323 (transmitted to her third son). All but one of the FVII gene mutations detected in the four patients have not been previously reported. In conclusion, four novel mutations of the FVII gene in Taiwanese, including two missense mutations in exon 8, one point mutation at the exon 6 splice site and one deletion in exon 1A, were identified.
Coagulation factor VII (FVII) is a vitamin K-dependent glycoprotein with a molecular weight of 53 kDa (Broze & Majerus, 1980). Upon vascular injury or following monocyte activation, tissue factor (TF) is exposed and forms a complex with FVII in a one-to-one stoichiometric reaction (Bach et al, 1986). The activation of FVII is effected by TF, forming activated FVII (FVIIa) (Nakagaki et al, 1991). The TF–FVIIa complex initiates blood coagulation and is capable of activating FIX and FX (Østerud & Rapaport, 1977).
Hereditary FVII deficiency was first reported by Alexander et al (1951). Since then, more than 200 cases have been described in the literature (Ragni et al, 1981; Tamary et al, 1996). FVII deficiency has an estimated prevalence of one per 300 000–500 000 in the general population (Caldwell et al, 1985; Mariani et al, 1998). It is transmitted as an autosomal recessive disorder. The gene has been fully characterized (O'Hara et al, 1987) and is located at the 13q34 chromosome (de Grouchy et al, 1984). The clinical picture is quite variable, consisting of asymptomatic patients as well as patients with severe to very severe bleeding tendencies (Mariani et al, 1998). The relationships between the clinical picture and FVII levels and the associated molecular genetic defects apparently lack consistency (Tuddenham et al, 1995; Cooper et al, 1997; Mariani et al, 1998). Plasma FVII levels vary significantly in the general population (Howard et al, 1994) and are known to be influenced by environmental factors (Scarabin et al, 1996) as well as genetic factors. Three polymorphisms within the factor VII gene are known to affect either the function or its level of expression, i.e. an ArgGln substitution at residue 353 (Green et al, 1991; Lane et al, 1992), a decanucleotide insertion polymorphism at −323 (Marchetti et al, 1993) and a variable number tandem repeat (VNTR) repeat in intron 7 (O'Hara & Grant et al, 1988; Bernardi et al, 1996). We studied four unrelated Taiwanese Chinese patients with hereditary factor VII deficiency and analysed their molecular genetic defects, which were unique and, thus, have not been reported previously.
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- Patients and methods
- Note added in proof
In the present study, two patients (patients I and II) with severe bleeding tendencies had FVII:C < u/dl and two patients (patients III and IV) with mild to moderate bleeding tendencies had FVII:C of 3·4 u/dl and 1·2 u/dl respectively. These levels are compatible with those in haemophilia. Nonetheless, FVII:C level is a relatively poor guide to the severity of bleeding in the patients with congenital FVII deficiency (Tuddenham et al, 1995; Cooper et al, 1997; Mariani et al, 1998). FVII deficiency was type 2 (cross-reacting material positive, CRM+) in patient I, type 1 (CRM−) in patient II and type 3 (Tuddenham et al, 1995) (CRMRed) in patients III and IV.
A summary of the molecular defects (Tuddenham et al, 1995; Cooper et al, 1997; Mariani et al, 1998) and other new mutations (James et al, 1997; Carew et al, 1998; Ozawa et al, 1998; Pinotti et al, 1998; Katsumi et al, 2000; Wulff & Herrmann, 2000) in hereditary factor VII deficiency has been published. All but one of the mutations of the FVII gene detected in our patients (Table I) were novel and have not been reported previously. These include two missense mutations in exon 8, 10824CA (Pro303Thr) and 10902TG (Cys329Gly), one exon 6 splice site mutation, IVS69007+1GT, and one delTC (26–27) in exon 1A. The decanucleotide insertion polymorphism at nucleotide −323, which was found in patients II and IV, has been shown to be associated with lower levels of plasma FVII:C and FVII:Ag in vivo (Sacchi et al, 1996), and reduced promoter activity by 33% in vitro (Pollak et al, 1996). Such polymorphic mutations may complicate the comparison of cases with the same mutation and could favour the laboratory detection of FVII deficiency (Mariani et al, 1998), particularly the heterozygous condition for other mutations, e.g., FVII:C and FVII:Ag were much reduced in patient IV compared with her second son.
The novel C to A transversion at nucleotide 10824 leads to a missense mutation from Pro to Thr at codon 303 in patient I. The Pro 303 is hightly conserved among FVII, FIX, FX, FXI and various other serine proteases. The Pro303Thr mutation would conceivably have a significant impact on the protein structure. A mutation R304Q right next to Pro303 has been reported to cause a conformational change which then affects tissue factor binding (O'Brien et al, 1991). Therefore, it is probable that Pro303Thr mutation may also decrease FVII activation by tissue factor binding. This awaits further investigation. In contrast, a homozygous mutation of Pro343Ser of factor X, equivalent to Pro303Thr in FVII, showed normal levels of factor X antigen but reduced the activity to 4–9% of normal (Harold et al, 1991). The Pro342 in FX was shown to be a highly conserved residue orientated spatially close to both the cleavage site of the zymogen and the active site of the enzyme (Harold et al, 1991). Patient I, who is homozygous for this mutation, had normal antigenic levels of FVII without detectable coagulation activity. Both his parents, who are heterozygous for this mutation, also had low normal or nearly half of the normal mean FVII:C level. However, they had twofold higher levels of FVII:Ag. This suggests that Pro303Thr is a totally dysfunctional protein.
The dinucleotide deletion (TC) of position 26 and 27 caused a frameshift mutation in patient II. Sixteen amino acids after Leu(−53) are altered, followed by a TAA premature termination codon. This mutation should be null, consistent with the undetectable level of FVII:C and FVII:Ag in patient II and the fact that the heterozygous mother has low normal levels of FVII:C and FVII:Ag. As for the G to T change at position 9007+1 of 5′ junction in intron 6, it may disrupt the splicing signal. The intron 6 of FVII is a typical intron, compliant with the ‘GT–AG rule’. This mutation changed the ‘GT–AG’ into ‘TT–AG’. The splicing apparatus may utilize the next GT at 9007+40 as the surrogate signal or may read through and utilize the stop codon at 9007+199 in intron 6, resulting in a protein with extra amino acids or a truncated protein. Patient II's father has both the exon 6 splice site and the decanucleotide insertion mutations located on the same allele, causing slightly lower FVII:C and normal FVII:Ag levels. In this family, this polymorphism in the promoter region did not affect for their phenotypes.
The T to G transversion at nucleotide position 10961 in patient III led to a missense homozygous mutation from His to Gln at codon 348. Southern blot analysis showed normal results as expected, thus small or large deletion mutations in the FVII gene could be ruled out. This mutation was recently reported and was suggested to impair secretion of FVII proteins (Katsumi et al, 2000), which might cause low levels of FVII:C and FVII:Ag, as observed in patient III. The amino acid His348 is conserved among FVII, FIX and FX. In fact, a stretch of seven amino acids from codon 342–348 (GDSGGPH), including Ser344, one of the catalytic triads (Cooper et al, 1997), are completely conserved among human FVII, FIX, FX, mouse FVII, bovine FVII and avian FVII. Whether it may affect the catalytic activity of FVII proteins remains to be investigated. The heterozygous condition in one of patient III's sons caused very little decrease in FVII levels.
The Cys329 forms a disulphide bond with Cys310 (Banner et al, 1996). Both Cys are strictly conserved among various serine proteases. A mutation of Cys310Phe has been reported (Bernardi et al, 1994) and the molecular modelling of this mutant showed that the loop of Cys310–Cys329 was relaxed considerably. The Cys329Gly mutation detected in patient IV would no doubt disrupt the disulphide bond and has a dramatic effect on the protein structure. However, the overall structure and stability of the protein would probably be conserved by hydrophobic interaction, but function is probably impared, thus causing a type 2 or a type 3 deficiency (Tuddenham et al, 1995). Cys329Gly mutation in one allele plus one normal allele in the patient's second son may result in nearly half of the normal mean level of FVII:C (45 u/dl) and an almost twofold higher level of FVII:Ag (79 u/dl), while the heterozygous decanucleotide insertion polymorphism in the promoter region in the patient's third son may cause normal or slightly decreased levels of FVII:C and FVII:Ag. The overall effect of one mutant allele and one polymorphism allele may result in a type 3 deficiency, as noted in patient IV.
In conclusion, we found four novel mutations of the FVII gene in Taiwanese congenital factor VII-deficient patients. Two missense mutations located in exon 8, one IVS6, 9007+1GT mutation was at the exon 6 splice site, and one delTC (26–27) was located in exon 1A.