The c.3780C > G (p.D1260E) change in the F8 gene, which predicts the amino acid substitution Asp1241Glu for mature factor VIII (FVIII), was, surprisingly, assigned as a novel missense mutation causing severe hemophilia A (HA) in two families by Repesse et al. [1]. However, this change was described in 1984 when the first sequences of the F8 gene were published [2]. It was subsequently reported to be a polymorphism during the search for mutations in HA patients [3] (HAMSTeRS database and it has been reported that the allele G was significantly associated with a lower FVIII activity [4,5]. However, the c.3780C > G change should not be considered the cause of severe HA, but rather that another undetected mutation may be present. This has implications for genetic counselling.

During a systematic search for mutations in the F8 gene among HA patients, we detected the above-mentioned polymorphism in 22 of 114 unrelated patients. Indeed, this change was over-represented among mild HA patients in whom the genetic analyses of the F8 gene failed to detect a pathological mutation. In addition, the c.3780C > G change was frequently associated with another polymorphism localized in intron 7 of the F8 gene (c.1009-27G > A). To further investigate this association, the respective frequencies of the two polymorphisms were compared in three different groups of unrelated individuals in a first approach. The normal control group comprised 59 males with FVIII activity ranging from 73 to 80 IU dL−1. The second group included 69 severe and moderate HA patients (SHA), and the third group was made up of 45 mild HA patients (MHA). These polymorphisms were analyzed by amplifying the corresponding fragments following the restriction analyses. DNA fragment analyses were performed in bis-acrylamide gels and silver stains. They were detected by sequencing in HA patients.

The Fisher’s exact test was carried out to evaluate whether these polymorphisms were in linkage disequilibrium. The results (P = 1.6 × 10−4, P = 1.8 × 10−5 and P = 1 × 10−8, respectively) confirmed that this was the case in all three groups, with a high degree of significance among the MHA patients. This finding is in agreement with the data of Viel et al. [5]. Thus, the two alleles occur together, their frequencies significantly differ from those predicted from individual allele frequencies and it is usual to find both the A and G nucleotides in the same allele.

In a second step, the frequencies of the AG haplotype (c.1009-27A/c.3780G) in the two patient groups, SHA and MHA, were compared with the frequency in the normal control group (NC) (see Table 1. No significant difference was found between the SHA group and the NC group, which indicates a homogenous population in both groups, and also suggests that these polymorphisms do not contribute to the disease. Conversely, the AG haplotype is clearly over-represented in the MHA group (P = 0.0028), and more specifically among those patients with an unknown further mutation (7 of 14 cases, 5 remain to be analyzed).

Table 1.   Genotypic frequencies of the two polymorphisms, c.1009-27A in intron 7 and c.3780G in exon 14, in the two patient groups, SHA and MHA vs. NC
 Number patientsAG haplotypeGC haplotypeGG haplotypeAC haplotypeP-value*
  1. *AG vs. non-AG haplotypes; Statistically significant.

Normal controls (NC)5945221 
Severe-moderate HA (SHA)69463201.00
Mild HA (MHA)451428210.0028

Other intragenic markers, such as the microsatellites in introns 13 and 22 [6], and the SNP in intron 18 [7], were analyzed. All the patients carrying the AG haplotype shared the same allele for these new markers, except for one patient with the causative mutation identified, c.5428C > T (p.Ser1791Pro), who showed just one repeat more in the two microsatellite alleles. This mutation cosegregated with the disease in the family and has been previously described (data base). It was associated with a mild HA phenotype with discrepant FVIII activity of about 20 IU dL−1 in a one-stage assay and 7 IU dL−1 when a chromogenic method was used. FVIII antigen was normal, confirming a functional deficiency.

An additional causative genetic alteration was detected in another patient sharing the same haplotype. The previously described missense mutation, c.5527G > A (p.Ala1824Pro), was identified in two patients from the family with FVIII levels of 25 IU dL−1.

In contrast, no mutation was found by sequencing the 26 exons, their flanking intronic region and the promoter region of the F8 gene in seven unrelated patients analyzed. FVIII activity measured by one-stage assay ranged from 23 IU dL−1 to 35 IU dL−1.

These findings suggest that the same genetic defect causing mild HA could arise in association with this haplotype, and could eventually be transmitted to these families, who are from the same Mediterranean area, after several generations. This pathological mutation must be located in the non-analyzed sequences, such as the interior of the introns, in the promoter region, or in the 3′ untranslated region. Alternatively, the causative mutation might go unnoticed by conventional sequencing methods, which occurred in a group of Italian mild HA patients carrying a duplication of exon 13 [8]. Further experiments are in progress in an attempt to not only find the causative mutation, including in the five families that have not yet been analyzed, but also to finally confirm the hypothesis of a founder haplotype associated with mild HA.


  1. Top of page
  2. Acknowledgements
  3. Disclosure of Conflict of Interests
  4. References

This work was partly supported by FIS grant PI020612 (Spain). We wish to thank M.A. Lerma for technical assistance, and H. Warburton for checking the English.

Disclosure of Conflict of Interests

  1. Top of page
  2. Acknowledgements
  3. Disclosure of Conflict of Interests
  4. References

The authors state that they have no conflict of interest.


  1. Top of page
  2. Acknowledgements
  3. Disclosure of Conflict of Interests
  4. References
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