Analysis of large structural changes of the factor VIII gene, involving intron 1 and 22, in severe hemophilia A.

Institute of Hematology and Immunology, National Medical Center, Diószegi út 64, Budapest H-1113, Hungary.
Haematologica (Impact Factor: 5.94). 08/2003; 88(7):778-84.
Source: PubMed

ABSTRACT Hemophilia A (HA), the deficiency of coagulation factor VIII (FVIII), is the most common, sex-linked inherited bleeding disorder. The disease is caused by FVIII gene intron 22 inversion in approximately 50% of the patients, and by intron 1 inversion in 5% of the patients with severe HA. Both inversions occur as a result of intrachromosomal recombination between homologous regions, in intron 1 or 22, and their extragenic copy located telomeric to the FVIII gene. The goal of the present study was to analyze the presence of large structural changes in the FVIII gene in patients with severe hemophilia A.
We studied 104 unrelated, severe HA-patients or obligate carriers for the presence of intron 22 and intron 1 inversions by Southern blotting, long-distance polymerase chain reaction (PCR), and simple PCR.
We found altered intron 22 restriction profiles by Southern analyses in 58 cases: 43 type 1, 11 type 2 inversions and 4 unusual patterns. Upon further examination of the last 4 cases, large deletions involving intron 22 were demonstrated in two cases. In the remaining two patients extra homologous regions were detected by Southern analysis, and long-distance PCR showed the presence of unaltered intra- and extragenic copies together with one inversion-affected copy, suggesting that an additional intronic fragment participated in the inversion process and was inserted in the genome. During screening for intron 1 inversion among 43 patients, who were intron 22 inversion negative, we identified only wild type individuals.
The relatively large proportion of unusual patterns further supports the observation that the structure of FVIII intron 22 represents a hot spot for large gene rearrangements with various mechanisms, while intron 1 inversion seems to be not common in Hungary.

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    ABSTRACT: The intron 22 inversion found in up to 50% of severe hemophilia A patients results from a recombination between three intron 22 homologous copies (int22h). This study evaluated the implication of these copies in the formation of extended duplications comprising exons 1-22 of the factor 8 (F8) gene and their association with hemophilia and mental retardation. Two hemophilic patients with moderate and severe phenotypes and a third nonhemophilic patient with developmental delay were studied. All exhibited a duplication of F8 gene exons 1-22 identified by multiplex ligation-dependent probe amplification along with abnormal patterns on Southern blotting and unexpected long-range PCR amplification. Breakpoint analysis using array comparative genomic hybridization was performed to delimit the extent of these rearrangements. These duplications were bounded on one side by the F8 intragenic int22h-1 repeat and on the other side by extragenic int22h-2 or int22h-3 copies. However, the simultaneous identification of a second duplication containing F8 gene exons 2-14 for the moderate patient and the classical intron 22 inversion for the severe patient are considered in this study as the genetic causal defects of hemophilia. This study shows that the well-known int22h copies are involved in extended duplications comprising F8 gene exons 1-22. These specific duplications are probably not responsible for hemophilia and intellectual disability, but should be carefully considered in genetic counseling, while continuing to investigate the causal mutation of hemophilia.European Journal of Human Genetics advance online publication, 9 January 2013; doi:10.1038/ejhg.2012.275.
    European journal of human genetics: EJHG 01/2013; · 3.56 Impact Factor
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    ABSTRACT: Recurrent int22h-related inversions in the coagulation factor VIII gene (F8) are the most common cause of severe hemophilia A. Such inversions have repeatedly been hypothesized to be associated with concomitant deletions that are responsible for an increased risk of immune responses against therapeutic exogenous factor VIII. However, exact DNA breakpoints have not yet been reported. In a patient with persistent factor VIII-inactivating antibodies, molecular analysis of F8 including Southern Blot, long-range PCR and primer walking techniques revealed a combination of an int22h2-related inversion, deletion of exons 16-22 and insertion of a duplicated part of the X-chromosomal MPP1 gene. This novel genomic rearrangement was also detectable in the patient's mother, but absent in both maternal grandparents. The genetic defect most likely originated from a complex X-chromosomal recombination event during spermatogenesis due to the formation of a DNA loop stabilized by Alu and LINE repeat elements. Elucidation of such combined mutations may allow early identification of patients at high risk of developing factor VIII-neutralizing antibodies and will help to understand the mechanisms behind gross chromosomal rearrangements causing hemophilia A and other diseases.
    Human Mutation 11/2007; 28(10):1045. · 5.21 Impact Factor
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    ABSTRACT: Hemophilia A is caused by the absence or impaired activity of clotting factor VIII (FVIII) resulting from various mutations of the FVIII gene (F8). Approximately half of the severely affected hemophiliacs carry a genomic inversion originating from a hot spot of intrachromosomal recombination between a 9.5 kb region within intron 22 (int22h1) and one of two extragenic copies on the X-chromosome, int22h2 or int22h3 [1], or from an analogous hot spot within the first intron [2]. In the remaining patients, hemophilia A has been attributed to a broad spectrum of mostly private mutations scattered over the entire gene comprising 186 kb and 26 exons [3]. In 5% of the cases of severe hemophilia A, multi-exon deletions in F8 resulting in complete absence of the FVIII protein predispose the patient to the development of FVIII-inactivating antibodies (inhibitors), which represent a major complication of FVIII replacement therapy. The type of F8 mutation is considered to be the most important risk factor for inhibitor formation. Whereas splice site and missense mutations are associated with a relatively low risk, approximately 21% of patients with the recurrent int22h-related inversion develop FVIII-neutralizing antibodies. Inhibitor prevalence is highest in hemophiliacs with large deletions in F8 reaching 88% in patients with deletions of exons encoding multiple domains [4]. However, only few DNA breakpoints of such large deletions have been characterized precisely so far, although their identification would facilitate molecular — including prenatal — diagnosis and carrier detection currently relying on sophisticated methods.
    12/2007: pages 130-137;

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