Molecular characterization of iranian patients with inherited coagulation factor VII deficiency

Coagulation factor VII (FVII) is a vitamin K-dependent serine protease that has a key role in the initiation of the coagulation cascade. This factor circulates in the blood at a concentration of 0.5 μg/mL and its severe reduction leads to rare autosomal recessive bleeding disorder, FVII deficiency [1,2]. The F7 gene maps to the long arm of chromosome 13 (13q34) and contains nine exons [1,3]. It is expressed as two major transcripts, A and B, each ~3.1 kb long, that encode polypeptides of 466 and 444 residues, respectively [4]. The pre proteins are synthesized with either 60 or 38 amino acid leader sequences that yield a unique mature single chain protein of 406 amino acids with a molecular weight of ~50 kDa. Exons 1a, 1b and a fraction of exon 2 encode the leader sequences, the rest of exon 2 and exons 3 to 8 encode the mature protein [5]. Upon blood vessel injury, tissue factor is exposed to the circulation and interacts with FVII and activates it by cleavage between Arg152 and Ile153. The activated FVII (FVIIa) consists of a 254-residue heavy chain that contains a catalytic domain and is linked by a disulfide bond to a 152-residue light chain that contains two epidermal growth factor-like (EGF-like) domains [6].

The most common FVII deficiency symptoms consist of soft tissue, mucocutaneous, joint and gastrointestinal hemorrhage. The hereditary form of the disease shows considerable phenotypic heterogeneity ranging from lethal to mild or even asymptomatic types. As a result, prevalence and incidence of FVII deficiency is not entirely determined [7]. Studies show that the incidence of clinically significant FVII deficiencies is estimated to be 1/500,000 [8]. However, the disease is more frequent where consanguineous marriages are commonplace [9]. The severity of hemorrhagic symptoms is not directly related to the level of plasma FVII coagulant activity [2,10]. In a study on 717 patients, Herrmann et al. [11] reported that about 60.0% of FVII deficient patients were asymptomatic who were diagnosed following an abnormal prothrombin time (PT) test. They also mentioned that the causative mutations responsible for FVII deficiency are spread throughout the F7 gene with no well-defined hot-spots. More than 250 mutations have been reported with FVII deficiency with missense mutations as the most frequent type. Exon 8 is the largest exon of the F7 gene that accommodates the highest number of mutations [12]. The most severe cases are either homozygous or compound heterozygous resulting in FVII:C levels less than 2.0% of normal. Occasionally, heterozygous carriers display hemorrhagic symptoms that can be severe in rare cases. Recently, a heterozygous patient was reported with severe spontaneous intra cranial bleeding. He was a previously healthy 19-year-old patient, without any reported hemorrhagic symptoms, even following tonsillectomy in childhood [13].

We here report on the characterization of the mutations in eight patients from eight unrelated families in Iran. We also intended to elucidate the effect of each mutation on their corresponding mRNA expression.

Patients. Table 3 shows clinical information and FVII activity levels of each patient. As shown, the severity of symptoms was not directly correlated with FVII activity levels. Chronic nosebleeds, easy bruising and bleeding from the gums were the most common symptoms. Patient 4 was the only asymptomatic patient in our study. He was diagnosed following pre surgery blood analysis. Patient 3 was a female patient who developed menorrhagia as the main clinical manifestation. Patient 2 and patient 6 were born from consanguineous marriages.

Table 3

Patient characteristics, symptoms and plasma FVII coagulation activity (FVII:C).

The F7 cDNA Analysis

Despite the low level of F7 transcript in peripheral blood cells, the extraction and amplification of F7 mRNA were successfully performed. We expected the amplification of alternative isoforms of F7 mRNA according to the presence or absence of exon 1b. Although the only observed mRNA isoform was the transcript lacking exon 1b.

Direct sequencing of PBMC-derived cDNA for A244V, S282K, H348Q and R304Q mutations revealed equal expression of wild-type and mutant allele transcripts. These variants were detected in the compound heterozygous and heterozygous patients. In addition, the cDNA analysis in homozygous patients indicated that mutationharboring transcripts, i.e., P303T, C91S and R304Q, were also detectable. In contrast, cDNA sequencing of the 64G>A heterozygote mutation showed the absence of the mutated allele transcript in patient 4. The sequencing chromatogram shown in Figure 1 presents 64G>A in the genomic DNA and its absence in the corresponding cDNA. Evaluation of transcript following the IVS7+7A>G variant that was detected in patient 8 showed a cDNA with normal pattern.

Figure 1

Identification of underlying gene alterations and their expression changes can be a prerequisite for the proper evaluation and management of the FVII deficiency. In our study, we found three homozygous patients, two of them had consanguineous parents, while the third patient was an adopted child with no available records regarding his family of origin. We also characterized molecular changes in two compound heterozygous and three heterozygous patients.

Mutation detection in patient 2 revealed the 10824C>A homozygous substitution that causes the P303T defect in FVII protein. This mutation was previously detected in an Iranian patient [16]. It has been shown that residues P303, L305 and M306 are involved in tissue factor (TF) binding [17].

Another homozygous mutation was found in patient 5, C91S that was previously identified in an English patient [18]. Since C91 is engaged in disulfide bond formation, this mutation could be a basis of severe dysfunction of the enzyme. However, patient 5 did not exhibit severe complications such as hemarthrosis or gastrointestinal bleeding. Further studies are needed to explore the role played by different factors in determining the phenotypic variation of each mutation.

The highest prothrombin time (PT) and partial thromboplastin time (PTT) (34 and 44 min., respectively) were found in patient 6 homozygous for a rather frequent gene defect, the R304Q mutation. The R304Q mutation was first found in a heterozygous state in a patient with no clinical bleeding tendency by O’Brien et al. [19]. Variability in the degree of severity and phenotypic expression is commonly observed in patients with R304Q mutation. This mutation was found in symptomatic patients from Latin America, but the patients from Germany were asymptomatic while carrying R304Q [11]. This kind of variations in phenotypic outcome of a given genotype can be affected by environmental factors and genetic modifier loci. Previous studies revealed that the substitution R304Q had adverse effects on the enzyme’s activity and TF interaction [19]. We also detected the R304Q variation in Patient 8. He was identified as a compound heterozygote for R304Q as well as IVS7+7A>G alterations. The IVS7+7A>G is a common mutation reported in various populations [20,21]. It has been shown that IVS7+7A<G was responsible for the lowest relative FVII levels although they did not observe any changes in the mRNA structure [21]. We also detected the IVS7+7A>G transcript with a normal pattern in this patient.

Another compound heterozygous case in our study was patient 7. Both mutations in this patient were located on exon 8, S282R and H348R. The mutation S282R was reported previously by Peyvandi et al. [22] in a compound heterozygous Iranian patient, while the other defective allele remained “unknown.” H348R was previously reported in homozygous and compound heterozygous Indian patients [23,24].

As stated earlier, some individuals with heterozygous F7 mutations may show bleeding manifestations. In our study, two patients were heterozygous for A244V transition, which was associated with decreased FVII activity levels. A244V was previously reported by Tamary et al. [25] in Iranian-Jewish patients with an allele frequency of 1:40. They detected a heterozygous A244V substitution in 10 out of 23 symptomatic patients affected by this mutation [25].

The only asymptomatic patient that we tested was patient 4, a 54-year-old man diagnosed on pre anesthesia blood analysis. Further molecular investigation revealed a heterozygous genotype with the 64G>A missense mutation. This mutation was previously reported in a Turkish family [26]. The 64G>A is located at the last nucleotide of exon 1a and is known as V(–39)I or V(–17)I. As reported by Wulff and Herrmann [26], the homozygous form of this mutation could cause the severe manifestations such as postpartal (after birth) cephalic hematomas. It should be noted that in communities with a preference for consanguineous marriages, mutation detection of asymptomatic or mild cases could be of great importance in genetic counseling. The cDNA sequencing revealed no expression of mRNA carrying the 64A allele. This may be due to inefficient splicing or mRNA decay. However, the F7 cDNA sequencing in A244V, S282K, H348Q and R304Q implied that their transcripts were expressed at detectable levels. Therefore, it could be concluded that the reduction of FVII protein activity following these mutations is not related to the changes at mRNA level.

In conclusion, in the present study, we found eight different F7 gene mutations, four of them were the first to be reported from Iran. This report reinforces the genetic and phenotypic heterogeneity of FVII deficiency, and provides evidence regarding the expression of the pathogenic mutations at the mRNA level. We propose that the reduction of FVII protein activity subsequent to missense mutations does not reflect the degradation of mRNA. We also wanted to bring the mutation detection in asymptomatic patients to the attention of genetic counselors.