Balkan Journal of Medical Genetics

This paper presents the first comprehensive study of the molecular characterization of the fVIII gene in hemophilia A patients from the Republic of Macedonia. A total of 50 unrelated hemophilia A patients were screened for molecular defects in the fVIII gene. The presence of an inversion in intron 22, gross gene deletions/insertions and point mutations in the whole coding regions of the fVIII gene, with the exception of exon 14 (only the 5’ and 3’ ends of exon 14 were screened), were analyzed. The molecular defects causing hemophilia A were determined in 34 out of 50 (68%) of the patients. The lower percentage of characterized mutations is most likely due to the low sensitivity of the screening method used, especially SSCP. Also mutations could reside in regions that were not analyzed in this study [exon 14, promoter region or the 3’ untranslated region (3’UTR)].

Inversion in Intron 22. The most frequent molecular defect causing hemophilia A is the inversion in intron 22 of the fVIII gene, found in 23-35% of all patients studied, or 41-57% of patients with a severe form of the disease. The inversion in intron 22 was investigated in different countries and ethnic groups (Table 3). There were no differences in incidence among the different countries, while the slight variations of obtained results are, as a result of different numbers of patients, included in the study. The obtained results of the incidence of intron 22 in the Republic of Macedonia was slightly higher than the reported frequency among severe hemophilia A patients worldwide [6]. This might be due to the high percentage of inversions found in hemophilia A patients of Albanian ethnic origin (Table 1), where consanguinity and a high birth rate are common. The percentage of inversions found among the Macedonian Slavic population (24.1%) is in accordance with the frequency of inversions found in the Slavic population [16], and is not different than the world-defined frequency of this molecular defect [6].

Eighteen inversions were type 1, when a distal copy of intron 22 is involved in a crossover event, while one patient had a rare type of inversion, type 3A, when an additional extragenic copy of int22h is involved in the recombination. None of the hemophilia A patients from the Republic of Macedonia had the inversion type 2, where the proximal extragenic copy of int22h is involved. According to the Hemophilia A International Consortium Study [6], the percentage of proximal inversion varies from 0% to 19% in different groups of hemophilia A patients. Mothers of 15 patients with an inversion, included in this study, were found to be heterozygotes for the inversion. This finding correlates well with the previous observation that the inversion occurs predominantly in male germ cell meiosis, and all mothers of patients with an inversion are carriers of the abnormal fVIII gene [17].

None of the hemophilia A patients from the Republic of Macedonia who were included in this study had gross gene deletions or insertions in the fVIII gene as a cause of the disease.

Nucleotide Substitutions. All the other molecular defects determined in our group of hemophilia A patients were single base substitutions. Nucleotide substitutions were found in 15 hemophilia A patients with different clinical severity, mostly being found in one family only, with the exception of a C®T change in codon 2159 found in four (8%) of the patients studied.

Two nucleotide changes found in severe hemophilia A patients, lead to nonsense mutations (GAA®TAA, exon 7 codon 272, and CGA®TGA, exon 14, codon 1696) [18, 19], while the others are missense mutations [ATG®AGG (Met®Arg), exon 1, codon 19; GCT®CCT (Ala®Pro), exon 3, codon 78; TAT®TGT (Tyr®Cys), exon 10, codon 473; CGG®TGG (Arg®Trp), codon 527, exon 11; GCC®ACC (Ala®Tyr), exon 14, codon 704; CGC®TGC (Arg®Cys), exon 14, codon 1689; CGC®TGC (Arg® Cys), exon 23, codon 2159; TGC®GGC (Cys®Gly), exon 24, codon 2174, and TAT®GAT (Tyr®Asp), exon 25, codon 2256] [19-22]. Eight missense mutations were found in a CpG dinucleotide, resulting in methylation in cytosine and conversion of CG to TG or CA. These agree with the general incidence of point mutations in the CpG dinucleotide (38%) in human diseases [23]. Small deletions/insertions, as well as nucleotide substitutions between exon/intron boundaries, were not found among the patients studied.

Novel Missense Mutations. Four novel missense mutations were found for the first time in hemophilia A patients from the Republic of Macedonia [24]. The Met-19®Arg change in the start codon destroyed the normal translation of the gene in an Albanian boy with a severe form of the disease (Fig. 2A) [3]. The next probable initiation codon would be out of frame, 100 bp downstream. This initiation codon produces a truncated fVIII protein since the termination codon appears in the frame after five codons. Until now, only one mutation in the start codon Met-19®Ile has been reported [25], in a severe hemophilia A patient with fVIII:C less than 1%.

Molecular characterization of the fVIII gene in a 17-year-old Albanian boy with severe hemophilia A, revealed a novel missense mutation GCT®CCT (Ala®Pro) at codon 78 of the fVIII gene (Fig. 2B). Ala78 is a conserved amino acid in proteins known to be homologous to the fVIII (human factor V and ceruloplasmine) gene [13]. Until now, no other substitution at this position (Ala78) has been described. The proline substitution of alanine would most probably lead to a distortion of the third shift of the A1 domain, that could easily have a disruptive influence on the molecule [26].

A novel missense mutation, TGC®GGC (Cys®Gly) at codon 2174 in exon 24, was characterized in a moderate-to-mild hemophilia A patient and his uncle who were of ethnic Macedonian origin (Fig. 2C). The substitution of Cys2174®Gly destroys the disulfide bound between Cys2174 and Cys2326 within the C2 domain [27]. The C2 domain, at positions 2303-2332, was shown to be involved in fVIII interaction with phospholipid. Therefore, the destruction of the disulfide bridge might affect the conformation of the phospholipid binding site in the C2 domain, or destabilize the structure of fVIII.

The fourth novel missense mutation, determined in a patient with a mild form of hemophilia A, was a TAT® GAT or Tyr®Asp substitution at codon 2256 of exon 25 (Fig. 2D). This amino acid substitution destroys normal binding of activated fVIII to the thrombocytes in the C2 domain phospholipid binding site [28].

Prenatal Diagnosis. As a result of the molecular characterization of hemophilia A in the Republic of Macedonia, six prenatal diagnoses have been performed. In three families, a direct search for molecular defects was performed (two families with the inversion type 1 and one family with the Met-19®Arg nucleotide substitution). In three families prenatal diagnoses were indirectly performed, with an informative BclI polymorphic marker in intron 18.

Male fetuses were identified in five pregnancies. Consequently, two affected fetuses were identified, while in three families, healthy, unaffected male fetuses were diagnosed. All obtained results were confirmed after birth.

In conclusion, after molecular characterization of fVIII gene, direct genetic counseling, i.e., offering accurate carrier detection and prenatal diagnosis is possible for 68% of families with hemophilia A in the Republic of Macedonia. As a result of these studies, six prenatal diagnoses were performed. Also, the novel mutations identified in this study have contributed to the understanding of the molecular pathology of hemophilia A.

Figure 2. Four novel mutations determined in hemophilia A patients from the Republic of Macedonia. A) SSCP analysis of exon 1 identifying an abnormal electrophoretic pattern in a patient (lanes 3 and 4) and his mother (lanes 5 and 6), and sequence analysis determining the ATG®AGG change at codon 19. B) SSCP analysis of exon 3 identifying a GCT®CCT, or Ala78®Pro change, in patient (P) and his mother (M), confirming her as a carrier of the mutation; N: normal control. C) SSCP analysis of exon 24 with a mobility shift in the patient (M1) and his uncle (M2); N: normal control. Direct sequencing identified the TGC®GGC change in codon 2174. D) SSCP analysis and sequencing of exon 25 in a patient with a mild form of hemophilia A with a TAT®GAT change in codon 2256.