Prevalence of inversions in introns 1 and 22 of the factor VIII gene and inhibitors in patients from southern Brazil

Objectives: The development of antibodies (inhibitors) against exogenous factors is the main complication in the treatment of hemophilia. Both genetic and non-genetic factors are related to inhibitor development. Among the genetic factors, the type of mutation that caused the disease is one of the most important. The objectives of the present study were to establish the prevalence of inversions in introns 1 and 22 of the factor VIII gene in patients with severe hemophilia A, correlating these with inhibitor development, and to compare the results with data from the literature. Method: Unrelated severe hemophilia A patients were analyzed for the presence of inversions in intron 1 ( n = 77) and intron 22 ( n = 39) by polymerase chain reaction (PCR). Detection of the inhibitor was performed by the mixing test and its quantification was performed by the Bethesda method. Results: The prevalence of inversions in introns 1 and 22 was 2.6% and 41%, respectively. No patient with inversions in intron 1 had inhibitors, whereas 26.3% of patients with inversions in intron 22 developed inhibitors. Conclusion: Due to the small number of patients with inversions in intron 1, it was not possible to perform a statistical test for the correlation with risk of inhibitor development. Inversions in intron 22 of the factor VIII gene were not associated with an increased risk of inhibitor development in the analyzed samples ( p = 1).

introDuCtion Hemophilia treatment involves the intravenous replacement of the deficient factor. The formation of inhibitors [polyclonal antibodies of the immunoglobulin class G (IgG)] against the exogenous factor is the most serious complication of replacement therapy in patients with hemophilia (1) . The affected patients stop responding to infusion of the deficient factor and exhibit hemorrhagic episodes that are difficult to control.
Between 10% and 30% of patients with hemophilia A develop inhibitors to treatment, while the incidence among patients with hemophilia B is much lower, about 1%-5% (2) . The production of inhibitors is influenced by genetic as well as non-genetic factors (3,4) . The major genetic factors are the type of mutation that caused the disease and the genetic susceptibility of cell surface molecules involved in the immune response, such as major histocompatibility complex molecules, T-lymphocyte receptors, and cytokine receptors, as well as several immunomodulatory molecules (5) .
Mutations associated with the highest risk of inhibitor development are those that prevent the synthesis of endogenous protein (null mutations) and are associated with the severe disease phenotype. These are the large deletions, nonsense mutations, and (for hemophilia A) intron 1 and 22 inversion mutations (5,6) .
It is estimated that more than 40% of severe cases of hemophilia A are caused by inversions in the gene encoding factor VIII (7) .

oBjECtivES
This study aimed to establish the prevalence of inversions in introns 1 and 22 of the gene encoding factor VIII in a sample of patients with hemophilia A from the state of Paraná, Brazil, correlating these mutations with the presence of inhibitors against factor VIII and its quantification in the same population.

MEthoD
The subjects who participated in the study had hemophilia A and were enrolled in the Hematology and Hemotherapy Center of the state of Paraná. Patients from different families were generally selected because hemophiliac individuals from the same family exhibit the same disease-causing mutation. This was an observational, analytical, and cross-sectional experimental study.
Inhibitors were detected through the mixing test. The Bethesda method was used for quantifying inhibitors and classifying samples as low/high response; that is, when inhibitors were persistently quantified at ≤ 5 Bethesda units (BU)/ml or above 5 BU/ml in response to exogenous factor exposure, respectively. This classification correlates with the clinical severity, since patients with ≤ 5 BU/ml usually respond to an increase in the dose of the infused factor, whereas those with more than 5 UB/ml require the use of a bypass agent to control their bleeding episodes (2) .
Polymerase chain reaction (PCR) amplification using reverse transcription was used to identify the inversion in intron 22 (8) ; and fast PCR, to identify the inversion in intron 1 (9) .
The prevalence frequencies obtained in this study were compared with those from articles appearing in PubMed over the last 10 years, by selecting studies with the largest number of participants. The chi-square frequency test (χ 2 ) was applied with a 5% α, and random Monte Carlo simulations were performed when the assumptions of the frequency test were not met. The tests were performed through the R platform. The estimated difference between categorical variables was performed using Fisher's exact test, with a minimum level of significance of 5%.
The research was conducted in accordance with the 2008 Revised Declaration of Helsinki and was approved by the Research Ethics Committee. Patients signed a free and informed consent form prior to their participation.

rESuLtS anD DiSCuSSion
A total of 45 patients with severe hemophilia A were tested for intron 22 inversion, of which 39 were unrelated; 84 participants were tested for intron 1 inversion, of which 77 were unrelated. The prevalence for intron 22 inversion among unrelated individuals was 41% and did not exhibit a significant difference (χ 2 = 1.13; p = 0.3) (10) . The prevalence for intron 1 inversion among unrelated patients was 2.6% and also did not show a significant difference (χ 2 = 0.05; p = 1.18) (11) . Leiria et al. (2009) (12) studied 107 patients with hemophilia A also in southern Brazil, but from the state of Rio Grande do Sul, and reported a prevalence of 46% and 3% for inversions in introns 22 and 1, respectively.
The prevalence of inhibitors in related and non-related patients with the inversion in intron 22 was 26.3% and did not show a significant difference (χ 2 = 0.01; p = 0.9) (10) . However, a significant difference (χ 2 = 26; p < 0.01) was observed among patients with an inversion in intron 1 (11) .
Although not used in this study, applications for the development of genograms, such as the Genogram Development and Management System (GDMS), can facilitate the classification in related and unrelated patients and make the collection of unrelated samples more effective, because in the presence of multiple affected family members, the adequate collection of family history is a laborious task (13) .
Regarding the behavior of inhibitors in the 45 patients tested for intron 22 inversion, the prevalence of inhibitors in patients with and without inversion (19 and 26 patients, respectively) was very similar: 26.3% (n = 5) and 26.9% (n = 7), respectively. Thus, no difference was observed in the intron 22 inversion distribution according to the presence of inhibitor (p = 1) (Figure 1), nor with respect to the degree of response (p = 0.41) (Figure 2). The two patients with an inversion in intron 1 showed no inhibitor.
The causes underlying inhibitor development remain elusive with respect to the various possible etiological factors.

ConCLuSion
Inversion in intron 22 of the gene encoding factor VIII was not associated with a higher risk of inhibitor development in the sample analyzed, whereas the small number of patients with the intron 1 inversion did not allow the application of a statistical test for determining its association with the risk of inhibitor development.

aCknowLEDgMEntS
We would like to thank Prof. Dr. Margareth Castro Ozelo, of the State University of Campinas, and Prof. Dr. Johannes Oldenburg, of the Universität Würzburg, for the molecular analyses and the authorization for publication of results.