PF335 MUTATION SPECTRUM OF F8 GENE IN HEMOPHILIA A PATIENTS FROM RUSSIA
Authors: O. Pshenichnikova, V. Salomashkina, V. Surin, E. Likhacheva, N. Zozulya, F. Perina
Journal: HemaSphere (2019)

Background: Congenital hemophilia A (HA) is an inherited recessive X‐linked bleeding disorder caused by a wide spectrum of mutations in the F8 gene and affected 1 in 5000 males. Its high degree of mutational heterogeneity complicates the carrier and prenatal diagnoses for genetic counseling but offers a wealth of data on the relationship between structure and function of FVIII. Aims: The aim of this study was to describe mutation spectrum in Russian patients with HA and to improve significantly genetic carriage and prenatal diagnostics of this disease in our country. Study results are especially important for families with sporadic form of HA (i.e. in families with the first reported patient), because in these cases only genetic analysis can give a reliable carriage status for proband's mothers. Methods: We conducted molecular genetic analysis for 239 unrelated patients with different forms of HA, recruited from 1990 to 2019 from different regions of Russia. Genomic DNA was isolated from peripheral blood using phenol–chloroform extraction. All patients with severe HA were examined for intron 22 (LD‐PCR method, modified from Liu et al., 1998 and Bagnall et al, 2006) and intron 1 (Bagnall et al., 2002) inversions. For patients without inversions we sequenced all functionally significant F8 gene fragments by Sanger. Results: We found inversion 22 in 130 unrelated patients with severe hemophilia A (68%, 17 patients with inhibitor). In 11 patients we found inversion 1 (5.8%, 1 patient with inhibitor), 3 patients had abnormal inversion 1 with additional deletion or duplication of nearby regions. In other 98 patients we detected 79 different mutations (12 patients with inhibitor), 40 of which had never been reported before in the international databases. Among them prevailed missense mutations (37), there were also nonsense (15), frameshift (13, 3 of them were indels), splicing mutations (9), large deletions (4) and inframe deletion (1). In three patients we found two mutations simultaneously. Five patients were women suffered from HA. One of them carried two mutations in F8 gene, others had only one mutation (one carried a missense mutation, while others had inversion 22), but we found for them evidence for an asymmetric inactivation of X‐chromosome using HUMARA test. Summary/Conclusion: All mutations detected in this study were distributed uniformly along the FVIII gene. As expected, significant part of missense and nonsense mutations occurred in CpG hot spot dinucleotides (21 out 52). We found two other mutation hot spots located in oligoA tracts of exon 14: p.Asn1460Lysfs ∗ 1 and p.Ile1213Phefs ∗ 5 detected in four and six unrelated patients, respectively. During our work we revealed a new mutation p.His634Arg found in 7 patients with mild HA (F8 = 10–30%) originated from Sverdlovsk region of Russia. We constructed four‐loci haplotypes for those patients and for 10 other patients with HA from the same region carrying other different mutations. All patients with this mutation shared the same unique haplotype, while all other patients had different haplotypes, which indicates founder effect for p.His634Arg substitution. For two large deletions (del Ex6 and del Ex6–12) we determined breakpoints, which were in both cases located within or nearby Alu‐repeats. In our cases HA in women mostly developed as a result of an asymmetric inactivation of X‐chromosome. For one woman with this inactivation we detected missense mutation led to a late manifestation of the disease (at 59 years old), which was at first diagnosed as acquired HA.