Mutational spectrum of F8 gene and prothrombotic gene variants in haemophilia A patients from Southern Italy

Abstract

Haemophilia A (HA) is an X-linked recessive haemorrhagic disorder caused by a deficiency of coagulation factor VIII. Disease causative mutations are heterogeneous and spread all over the F8 gene sequence, with the exception of intron 22 inversion occurring in about 50% of severe patients. To define the specific mutational repertoire and mutation detection rate, we analysed F8 gene, by polymerase chain reaction and direct sequencing, in 128 unrelated patients from Southern Italy with severe (n = 108), moderate (n = 9) or mild (n = 11) HA. We identified 120 mutations, including 64 cases of intron 22 inversion (53.3%), three of intron 1 inversion (2.5%), one large deletion (0,8%) and 52 point mutations (43.3%). In particular, 19/120 were novel and 7/52 point mutations (13.5%) occurred at CpG sites. We also investigated eight prothrombotic genetic variants in a subgroup of 74 severe HA patients to evaluate their possible protective effect on the severity of clinical expression. Methylenetetrahydrofolate reductase A1298C and plasminogen activator inhibitor-1 4G variants recurred more frequently in HA patients with a less-severe phenotype. Clinical impact of these findings needs large-scale studies to further define the role of these prothrombotic variants as possible modifier factors of HA phenotype.

Prothrombotic risk mutations and polymorphisms in patients with hemophilia A – a preliminary study / Polimorfismele și mutațiile cu risc protrombotic la pacienții cu hemofilie A - studiu preliminar

“…(24,25) There is raising evidence about the role of prothrombotic risk mutations or polymorphisms in modulating clinical phenotype of hemophilia, mitigating its expression and serving consequently as a marker for risk evaluation for bleeding, that could be important for a personalized therapy approach. (3)(4)(5) The frequency of F V, F II and PAI-1 genes alterations in our group of hemophilia patients is significantly higher than in normal population. Therefore, considering the uneven distribution in different ethnic groups and geographical regions, more extended studies are worthwhile to be performed, with a better design, with the inclusion of a larger number of patients and finally with the use of a control group with age-and sex -matched persons from our geographical region and from our ethnic group; an impact analyze of the investigated polymorphisms and mutations on the clinical outcomes of hemophilia are pending.…”

“…A gratifying aspect is the successful identification of the intimate nature of the bio-molecular substrate of a huge number of diseases, which allows to answer questions related to risk: significance of various mutations, genotype-phenotype correlation or heterogeneous clinical and biological expression of the same mutation (1)(2)(3)(4). These are issues which have largely been debated in the frame of hemophilia A.…”

“…The question to be raised: is it a singular accidental situation or is it an evolutionary protective reaction, occurred over the centuries with the purpose of mitigating the hemorrhagic risk? (1)(2)(3)(4)(5) In order to try a reply to this question, special attention is focused on the main alterations, polymorphisms and mutations, more evidently connected to thromboembolic events: -factor V with the substitution of adenine (A) for guanine (G) at nucleotide position 1691 (G1691A) guanine: GG -wild type without harming effects; GA -heterozygous and AA homozygous factor V Leiden (FVL) (6,7) -prothrombin with the mutation of guanine (G) to adenine (A) transition at nucleotide position 20210 generating: GG -wild type (normal), GA -heterozygous and AA -homozygous type (7) -MTHFR structural changes with nucleotide at position 677 in the gene , generating two possibilities, cytosine (C) or thymine (T): 677CC represents the "normal" or "wild type" genotype, 677TT -the homozygous with mild MTHFR deficiency and 677CT -the heterozygote pattern with almost a normal activity (8,9) -PAI-1 gene mapped on chromosome 7 (q21.3-q22) with 4G/5G polymorphism (deletion/insertion of guanine in position 675 of the PAI-1 gene promoter) and three possible outcomes: homozygous genotype with normal or wild type -5G/5G and heterozygous genotype -4G/5G or homozygous genotype -4G/4G, both with abnormal activity (10,11). Aiming at evaluating the frequency of these mutations in persons with hemophilia (PwH), we started this single-center, analytical cross-sectional study supported by the Laboratory of Molecular Biology, Munster (Germany).…”

Prenatal diagnosis of haemophilia: our experience of 44 cases

“…Therefore, gene scanning procedures like DHPLC [5] or gene sequencing must be carried out so that the family mutation (or the haplotype) is known at the time of PD. In this context, also the following factors are essential for PD: 1) carrier diagnosis (and cascade screening) of all consanguineous women once a new severe haemophilic patient is identified, and again the multidisciplinary counselling plays a relevant role in promoting this activity; and 2) construction of a database of mutations of all known severe haemophilic patients in each region, which will include novel pathogenic mutations [7,10,22].…”

“…Intron 1 inversion is tested by two PCRs with four primers differently combined, followed by agarose gel electrophoresis [1]. In patients negative to IVS22/IVS1, starting from 2003, we scanned the entire coding sequence of the gene by DNA sequencing, using universal primers that amplify all 26 gene exons under the same conditions [10]. The amplicons were analysed on both strands by automated sequencing with the 373A apparatus (Applied Biosystem, USA).…”