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Recurrent Inversion with Concomitant Deletion and Insertion Events in The HUMAN MUTATION Mutation in Brief #977 (2007) Online MUTATION IN BRIEF Recurrent Inversion with Concomitant Deletion and Insertion Events in the Coagulation Factor VIII Gene Suggests a New Mechanism for X-Chromosomal Rearrangements Causing Hemophilia A Christiane Mühle1,2, Martin Zenker3, Nadia Chuzhanova4, and Holm Schneider1,2* 1Experimental Neonatology, Department of Pediatrics, Medical University of Innsbruck, Austria; 2Children’s Hospital, University of Erlangen-Nuernberg, Erlangen, Germany; 3Institute of Human Genetics, University of Erlangen-Nuernberg, Germany; 4Department of Biological Sciences, University of Central Lancashire, Preston, United Kingdom *Correspondence to Dr. Holm Schneider, Experimental Neonatology, Department of Pediatrics, Medical University of Innsbruck, Innrain 66, 6020 Innsbruck, Austria; Tel.: +43 512 50425732; Fax: +43 512 50424680; E-mail: [email protected] Grant sponsor: The study was supported by a grant from Wyeth Pharma GmbH (Germany) to CM and HS. Communicated by Stylianos E. Antonarakis 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. © 2007 Wiley-Liss, Inc. KEY WORDS: hemophilia A; F8; X-chromosome; breakpoints; inversion; deletion; insertion INTRODUCTION Hemophilia A (MIM# 306700), the most common severe coagulation disorder with an incidence of 1 in 5000 males, is caused by absence or impaired activity of clotting factor VIII (FVIII) resulting from various mutations of the FVIII gene (F8). This large gene, which has been mapped to the most distal band (Xq28) of the long arm of the X chromosome, comprises 26 exons spread over 186 kb. Approximately half of the hemophiliacs with less than 1 % FVIII activity carry a genomic inversion originating from a hot spot of intrachromosomal recombination Received 9 March 2007; accepted revised manuscript 17 May 2007. © 2007 WILEY-LISS, INC. DOI: 10.1002/humu.9506 2 Mühle et al. between a 9.5 kb region within F8 intron 22 (int22h1) and one of its two extragenic telomeric copies on the X- chromosome, int22h2 or int22h3 (Lakich et al., 1993). Disruption of F8 may also occur by recombination between a 1 kb portion of F8 intron 1 and its duplicated extragenic version (Bagnall et al., 2002). In the remaining cases, hemophilia A has been attributed to a broad spectrum of mostly private mutations scattered over the entire gene (Kemball-Cook et al., 1998). Although a genomic inversion normally does not result in gain or loss of DNA, unusual patterns in Southern blots or long-range PCR for the detection of int22h-related inversions have led to the hypothesis of concomitant deletions (Schroder et al., 1996; Andrikovics et al., 2003) which would be associated with an increased risk of developing FVIII-inactivating antibodies (inhibitors). Such antibodies represent a major obstacle to FVIII replacement therapy. They are found in approximately 41 % of hemophiliacs carrying a large deletion in F8, whereas only 21 % of patients with a recurrent int22h-related inversion develop FVIII-neutralizing antibodies (Oldenburg et al., 2004). However, DNA breakpoints of few large deletions in F8 have been determined exactly. Although some deletions have been associated with non-homologous recombination (Woods-Samuels et al., 1991), more recent reports demonstrated unequal homologous recombination between Alu-derived sequences to be responsible for deletions in the Alu-rich F8 (Vidal et al., 2002; Rossetti et al., 2004). None of any fully characterized deletion in this gene was combined with an int22h-related inversion. Here, we report the first case of a hemophiliac with a complex genomic rearrangement including a recurrent int22h-related inversion, a deletion of F8 exons 16-22 and an insertion of an MPP1 gene fragment. We propose a mechanism for a multi-step recombination event which is likely to have occurred within one generation during spermatogenesis and which led to severe hemophilia A with high-titer inhibitors resistant to repeated attempts of immune tolerance induction. MATERIAL AND METHODS Case report The index patient is the second child of Caucasian parents with no family history of bleeding disorders. Severe hemophilia A was diagnosed at the age of three months subsequent to intramuscular vaccination. FVIII activity was found to be below 1 % with VWF:Ag of 90 % and VWF:RCo of 56 % and no abnormal changes in multimer analysis. Following treatment with recombinant FVIII, the child developed inhibitors within 40 exposure days, which were quantified using the Nijmegen modification of the Bethesda assay (Verbruggen et al., 1995). Two attempts of immune tolerance induction under different regimes and clotting factor concentrates failed. Inhibitor levels rose temporarily above 2000 BU/ml before a third trial with simultaneous immunosuppression finally lowered the antibody concentration to <2 BU/ml. The polyclonal FVIII-specific antibodies were found to bind to both the heavy and the light chain of FVIII with main epitopes in the domains A1, A2 and C1 (Muhle et al., 2004). The patient’s sister showed a FVIII activity of 10 % with VWF:Ag of 69 % and VWF:RCo of 28 % suggesting a carrier status, whereas the FVIII levels of the patient’s mother were within the normal range. The family gave informed consent to participate in the study in accordance with the Declaration of Helsinki. DNA analysis Genomic DNA was extracted from peripheral blood lymphocytes and investigated for int22h-related inversions by Southern Blot (Lakich et al., 1993) and long-range PCR (Liu et al., 1998) employing two additional PCR primers with 5’-G/C-extensions, F8int22_A2 (5’-GCCTGCATTTCCCATCAAAATGCTAACATTGTTTTTCA- 3’) and F8int22_A3 (5’-GTTACGGGCCTTGCTGCTTTGTCCAGTTTCAGG-3’), to distinguish between recombinations with int22h3 and int22h2 (type 1 and type 2 inversion, respectively). This primer set differs from the recently published novel set for a discriminating long-range PCR (Bagnall et al., 2006). Routine PCR analysis (Oldenburg et al., 2001) was carried out to demonstrate the presence or absence of F8 exons. Further PCR amplifications of intronic regions for breakpoint localization, across the breakpoint and for haplotyping were performed according to standard PCR protocols based on the F8 sequence from the UCSC Human Genome Browser Project (genome.ucsc.edu, NCBI Build 36.1; primer sequences and conditions obtainable from the authors). The genomic DNA insert in the patient’s F8 was identified using the DNA Walking SpeedUp Premix Kit (Seegene, BioCat, Germany). DNA sequencing was performed with the BigDye Terminator sequencing mix (Applied BiosystemsSpecific) on an automated capillary ABI PRISM 310 sequencer. Paternity of the maternal grandfather was verified using the PowerPlex 16 system (Promega, Mannheim, Germany). Combined Inversion, Deletion and Insertion in F8 3 Karyotyping of blood lymphocytes was done by routine procedures. Interphase nuclei and metaphase chromosome spreads were investigated by fluorescence in situ-hybridization (FISH) with a F8-specific probe from the BAC clone RP11-671F22. RESULTS AND DISCUSSION In the index patient, routine Southern blot analysis to detect int22h-related inversions in F8 resulted in an unusual pattern. While bands of 16.0 kb and 15.5 kb representing the int22h3 and the hybrid int22h2/1 fragment, respectively, were easily detected, the 20.0 kb band typical for the inversion (Lakich et al., 1993) was missing. Long-range PCR confirmed an int22h2-related inversion by yielding the expected A2Q product for int22h2/1, however, lack of an amplification product with primers P and B (Fig. 1A) indicated a deletion within the int22h1/2 counterpart. The newly designed primers A2 and A3 allowed here a PCR-based distinction between the frequent type 1 and the less common type 2 inversion involving int22h3 and int22h2, respectively, which is routinely done by more laborious and material-consuming Southern blot analysis (Lakich et al., 1993). Attempts to amplify F8 exons 16 to 22 by PCR failed consistently, although other exons of this gene were readily detected. PCR analysis of regions within introns 15 and 22 was performed to determine the extent of the deletion (Fig. 1B), but the presence of large sequence homology regions flanking both int22h2 and int22h3 as a palindrome (Fig. 2) precluded the exact localization of the distal breakpoint. Because
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