SEARCH

SEARCH BY CITATION

Keywords:

  • deletion;
  • F8;
  • gene rearrangement;
  • intron 22 inversion;
  • inverse shifting-PCR;
  • X chromosome

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Addendum
  8. Acknowledgements
  9. Disclosure of Conflict of Interest
  10. References
  11. Supporting Information

Summary.  Background:  Intron 22 inversion (Inv22) of the coagulation factor (F)VIII gene (F8) is a frequent cause of severe hemophilia A. In addition to Inv22, a variety of F8 mutations (1492 unique mutations) causing hemophilia A have been reported, of which 171 involve deletions of over 50 bp (HAMSTeRs database; http://hadb.org.uk/). However, only 10% of these large deletions have been fully characterized at the nucleotide level.

Patients and methods:  We investigated gene abnormalities in three unrelated severe hemophilia A patients with high titer FVIII inhibitors. They had previously been shown to carry large deletions of the F8, but the precise gene abnormalities remain to be elucidated.

Results:  Inverse shifting-PCR (IS-PCR) Inv22 diagnostic tests revealed that these patients carried either type I or II Inv22. However, they showed a wild-type (WT) pattern in the IS-PCR Inv22 complementary tests. We further analyzed their X chromosomes to account for the puzzling results, and found that they had different centromeric breakpoints in the Inv22 X chromosomes, adjacent to the palindromic regions containing int22h-2 or -3, and their spacer region, respectively. The connections appeared to be shifted towards the telomere of the WT F8 Xq28, resulting in a new telomere with an additional intact int22h copy.

Conclusions:  These gene rearrangements might result from double-strand breaks in the most distal regions of the long arms of the Inv22 X chromosomes, followed by DNA restorations using the WT F8 Xq28 by non-homologous end joining or break-induced replication; thus leading to large F8 deletions in severe hemophilia A patients.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Addendum
  8. Acknowledgements
  9. Disclosure of Conflict of Interest
  10. References
  11. Supporting Information

Hemophilia A (HA) (OMIM 306700), the most common severe coagulation disorder with an incidence of one in 5000 males worldwide, is caused by the absence or impaired activity of the coagulation factor (F)VIII resulting from various mutations of the FVIII gene (F8). This large gene, which has been mapped to the most distal region (Xq28) of the long arm of the X chromosome, comprises 26 exons spread over 186 kb.

An intron 22 inversion (Inv22) disrupting F8 at intron 22 is found in about half of the severe HA patients [1,2]. Inv22 results from homologous recombination between the int22h-1 region within the F8 locus and either int22h-2 (Inv22 type II) or int22h-3 (Inv22 type I), which lie approximately 400 kb distal to F8 [3]. Previously, int22h-2 and -3 were believed to be in opposite orientation to int22h-1. However, new sequence data show that only int22h-3 is in the opposite orientation to int22h-1, whereas int22h-2 is in the same orientation [4]. The palindromic arrangement of int22h-2 and -3 is now thought to permit an inversion polymorphism that allows int22h-2 to be in the telomeric arm of the palindrome and in opposite orientation to int22h-1 [5]. Intron 1 inversion (Inv1) is also a common mutation of HA with about 5% prevalence in severe HA [6]. It results from a homologous recombination between two nearly identical 1-kb sequences, int1h-1 and -2 in opposite orientations, lying in intron 1 of F8 and in a more telomeric region located about 140-kb upstream, respectively.

Although a genomic inversion normally does not result in gain or loss of DNA, unusual patterns observed in int22h-related inversions have led to the hypothesis of concomitant deletions [7–9], which can be associated with an increased risk of developing FVIII-inactivating antibodies (inhibitors). In a study of the correlation between a high incidence of inhibitors and gene defects producing a severe phenotype, approximately 41% of hemophiliacs carrying a large deletion in F8, whereas only 21% of patients with recurrent int22h-related inversions developed FVIII inhibitors [10]. A variety of F8 mutations (1492 unique mutations) causing HA have been reported, of which 171 involve deletions of over 50 bp (HAMSTeRs database; http://hadb.org.uk/). However, only 10% of these large deletions have been fully characterized at the nucleotide level.

In this study, we investigated gene abnormalities in three unrelated severe HA patients with high-titer inhibitors, and identified distinct X-chromosomal rearrangements with F8-intron 22 inversions. These gene rearrangements might result from double-strand breaks and repair of the DNA in the most distal regions of the long arms of the Inv22 X chromosomes, leading to Inv22-related large F8 deletions.

Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Addendum
  8. Acknowledgements
  9. Disclosure of Conflict of Interest
  10. References
  11. Supporting Information

Patients and DNA samples

Three unrelated Japanese patients (P1–P3) and P2 family members enrolled in this study that was approved by the Ethics Committee of the Nagoya University School of Medicine. The patients were affected by severe HA (FVIII: C < 1%) and anti-FVIII antibodies had developed after replacement therapy. The titer of inhibitors in Bethesda units and the clinical characteristics of the patients are given in Table 1. Some of the clinical features of the patients have been previously reported [11] and P1 was diagnosed with dwarfism. In the previous study [11], the patients have been shown to carry large deletions of the F8 exons 1–22 (P1 and P2) or 2–22 (P3), using Southern blot analysis. Genomic DNA samples were isolated from all patients after written informed consents were obtained; isolation was carried out from peripheral blood leukocytes using phenol extraction as described previously [12].

Table 1.   Clinical characteristics and results of F8 abnormalities of hemophilia A patients
PatientAgeSexFVIII:C (%)Inhibitor titer (BU mL−1)Gene abnormality
Deletion11Inv22
P117M< 1100–4000Exons 1–22Atypical
P225M< 1180–4000Exons 1–22Atypical
P316M< 124Exons 2–22Atypical

FVIII gene inversion detection

Relevant DNA nucleotide positions are indicated on GenBank accession NC_000023.10. A long range polymerase chain reaction (Long PCR) was performed using four primers (P, Q, A and B) as described previously by Liu et al. [13]. The PQ fragment (12 kb) was amplified from intact int22h-1 in a wild-type (WT) F8 male, the PB and AQ fragments (11 kb) were from int22h-1/-3 or -1/-2 and int22h-3/-1 or -2/-1 in Inv22 males, and the AB fragment (10 kb) was from non-recombined extragenic homologs. PCR was performed in 20-μL reaction volumes containing 50 ng of genomic DNA using 0.4 U of KOD FX DNA polymerase (Toyobo Co., Ltd., Osaka, Japan). Thermocycling involved 25 cycles of denaturation at 98 °C for 10 s and annealing/extension at 68 °C for 15 min; cycling was preceded by 94 °C for 2 min and followed by 7 min at 68 °C. PCR products were visualized after 3 h of electrophoresis at 50 V in a 0.6% agarose H (Nippon gene Co., Ltd., Tokyo, Japan) gel stained with 1 μg mL−1 ethidium bromide.

Inverse shifting-PCR (IS-PCR) was performed as described previously [14] with minor modifications. Genomic DNA (2 μg) was digested with 15 units of BclI according to the manufacturer’s instructions (New England Biolabs Japan, Inc., Tokyo, Japan) for 4 h in 50 μL. DNA fragments were circularized using Ligation high ver. 2 (Toyobo Co., Ltd.) in 8 μL at 16 °C for 1 h and recovered in 30 μL of 10 mm Tris with 1 mm EDTA, pH 8.0 (TE buffer) after ethanol precipitation. PCR was performed in reactions containing 2 μL of circularized DNA, in the presence of 0.5 μm of each primer, 0.4 U of KOD FX DNA polymerase and additional standard PCR reagents in a total volume of 20 μL. We designed modified primers for IS-PCR (Table S1). Thermocycling involved 34 cycles of denaturation at 98 °C for 10 s, primer annealing at 66 °C for 30 s and extension at 68 °C for 40 s; cycling was preceded by 94 °C for 2 min, each two cycles of 98 °C for 10 s and 68–74 °C for 35 s, followed by 1 min at 68 °C. PCR products were analyzed by electrophoresis on a 2% agarose gel stained with 1 μg mL−1 ethidium bromide.

PCR mapping to evaluate Xq28 deletions

To confirm and further assess the extent of the deletions, primer pairs for PCR mapping were designed to amplify fragments of 184–514 bp in the reference sequence (Table S2). PCR reactions were performed using rTaq DNA polymerase (Toyobo Co., Ltd.) or KOD FX DNA polymerase at an annealing temperature of 52–68 °C and products were analyzed on a 2% agarose gel. As the patients had only one X chromosome, we were able to assess the deletion by the absence of amplified products.

Identification of deletion breakpoints in X chromosomes

To amplify the unknown regions including the breakpoints, we performed inverse PCR using primer sets designed in opposite orientations in the respective known regions (Table S3). This procedure employs two thermal cycling reactions of nested PCR to amplify from a region of known sequence into an unknown area. First, we digested 100–200 ng of DNA with an appropriate restriction enzyme (SspI for P1, HindIII for P2, and BclI for P3) (New England Biolabs Japan, Inc., Roche Diagnostics K.K., Tokyo, Japan) at the appropriate temperature for each. DNA fragments were circularized using Ligation high ver. 2 and recovered in 5 μL of TE buffer after ethanol precipitation. PCR was performed in reactions containing 5 μL of circularized DNA in the presence of 0.5 μm of each primer, 0.4 U of KOD FX DNA polymerase and additional standard PCR reagents in a total volume of 20 μL.

For nested PCR, we designed two kinds of primers for each reaction (Table S3). PCR products were analyzed by electrophoresis on a 2% agarose gel stained with 1 μg mL−1 ethidium bromide. Inverse PCR products were purified using QIAEX II (Qiagen, Tokyo, Japan) and DNA sequencing was performed as described previously [15]. Screened sequences were analysed by NCBI blast (http://blast.ncbi.nlm.nij.gov/Blast.cgi).

We also designed multiplex PCR primers located on both sides of the junctions to amplify across the breakpoints of the rearrangements (Table S4). We performed PCRs for the respective genomic DNAs from the patients, the family members of P2 (his mother and grandmother) and the WT male controls, followed by DNA sequencing.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Addendum
  8. Acknowledgements
  9. Disclosure of Conflict of Interest
  10. References
  11. Supporting Information

Abnormal patterns in analyzes of F8 int22h-related inversions in severe HA patients

As the HA patients had a severe phenotype, we investigated whether they carried the common F8 int22h-related inversion (Inv22). Long PCR confirmed an Inv22 by yielding the expected 11-kb AQ product for int22h-2/-1 or -3/-1; however, the absence of an amplification product with primers P and B indicated a deletion of the int22h-1/-2 or -1/-3 counterpart (Fig. 1).

image

Figure 1.  Long PCR analysis for the F8 int22h-related inversion. (A) PCR products with primers PQ, PB, AQ and AB. The PQ fragment (12 kb) was amplified from intact int22h-1 in a wild-type (WT) F8 male, the PB and AQ fragments (11 kb) were from int22h-1/-3 or -1/-2 and int22h-3/-1 or -2/-1, respectively, in an Inv22 male (Inv22) and the AB fragment (10 kb) was from non-recombined extragenic homologs. All patients (P1–P3) showed AQ products indicating Inv22 on the centromeric side; however, they lacked PQ products indicating a deletion of the telomeric-side counterparts of Inv22. M, 1 kb DNA ladder marker; WT, wild-type control; Inv22, HA patient with Inv22; P1, patient 1; P2, patient 2; P3, patient 3. (B) A scheme of the expected structures of the wild-type, Inv22, or Del22 X chromosomes along with long PCR primer sites.

Download figure to PowerPoint

In the IS-PCR diagnostic test for Inv22, P1 and P2 showed a 341-bp fragment of the Inv22 type I pattern; whereas, P3 showed a 419-bp fragment of the Inv22 type II pattern (Fig. 2A). In the Inv22 complementary test, all patients showed the 489- and 411-bp fragments as the WT pattern (Fig. 2B). In the diagnostic test for Inv1, all patients lacked bands (Fig. 2C).

image

Figure 2.  Inverse shifting-PCR analysis of F8. (A) Inv22 diagnostic test: M, 100-bp DNA ladder marker; WT, wild-type control (498 bp); I, Inv22 type I control (341 bp); II, Inv22 type II control (419 bp); P1, patient 1; P2, patient 2; P3, patient 3. P1 and P2 show a 341-bp fragment as an Inv22 type I pattern, whereas P3 shows a 419-bp fragment as an Inv22 type II pattern. (B) Inv22 complementary test: WT, wild-type control (411 and 489 bp); I, Inv22 type I control (489 and 568 bp); II, Inv22 type II control (411 and 568 bp). All patients show 411 bp and 489 bp as the wild-type pattern. (C) Inv1 diagnostic test: WT, wild-type control (316 bp). The 281-bp fragment represents the Inv1 pattern. No patients show a band. The primer sequence data are given in Table S1.

Download figure to PowerPoint

PCR mapping to evaluate Xq28 deletions

PCRs using 16 different primer pairs (A–P in Table S2) were mapped to confirm and further assess the extent of the deletions, given that previous Southern blot analysis had shown large deletions of F8 in the patients [11]. In P1, primer pair K amplified a product but primer pair L did not, indicating that the centromeric breakpoint was located within the region defined by these two primer pair sets in the Inv22 type I X chromosome of the patient. Likewise, the centromeric ends of the deleted segments were localized between primer pairs M and F in the Inv22 type I X chromosome of P2, and O and P in the Inv22 type II X chromosome of P3, respectively (Fig. 3). The telomeric region products of primer pair J were amplified in all patients.

image

Figure 3.  PCR Mapping. (A–P) PCR mapping products separated on agarose gel electrophoresis for the determination of intact or deleted regions on the Xq28 of the patients. The presence of a PCR product in the patient indicates that the primer sequences are located in the intact region, and the absence of a product indicates that the primer sequences are located in the deleted region. Primer pair locations are shown below and primer sequence data are given in Table S2. M, the 100 bp ladder; WT, wild-type control male DNA; P1, patient 1; P2, patient 2; P3, patient 3. (Q) Schematic summary of PCR-mapped locations on Xq28 (NC_000023.10, 154.0–154.8 Mb). A–P indicate sites of PCR primer pairs. Open circles indicate the PCR amplified regions, and closed circles indicate the PCR failure regions. Salmon pink boxes, F8; closed chevrons, intragenic int22h-1; dark gray and open chevrons within the arms of a large imperfect palindrome (light grey), int22h-2 and -3, respectively; orange and yellow chevrons, int1h-1 and -2. Chimeric int22h sequences are denoted as [/] e.g., int22h-1/-2 represents the chimera between int22h-1 and -2. Five genes between F8 and int22h-2, MTCP1 (a), BRCC3 (b), VBP1 (c), RAB39B (d), and CLIC2 (e) are shown as green boxes.

Download figure to PowerPoint

Identification of deletion breakpoints in X chromosomes

From the results of PCR mapping, we roughly determined the centromeric ends of the deletion regions in the X chromosomes of the patients. We then employed inverse PCR followed by DNA sequencing to identify the centromeric and telomeric breakpoints.

In the case of the Inv22 type I X chromosome of P1, SspI sites were present at 1.1 kb on the centromeric side and 0.8 kb on the telomeric side of the primer pair K for PCR mapping, respectively (Fig. 4). Inverse PCR of the SspI-digested DNA of P1 failed to amplify the deduced 1.8-kb fragment and instead amplified an abnormal 2.2-kb fragment. Sequence data of the abnormal 2.2-kb fragment revealed a breakpoint located at nt 154 426 072 (GenBank accession NC_000023.10), about 20 kb on the telomeric side from VBP1 on the Inv22 type I X chromosome. The adjacent telomere side sequences were located at nt 154 598 975 and nt 154 701 245 in the palindromic regions of int22h-2 and -3, respectively, in centromere direction in the Inv22 type I X chromosome; however, either one was in telomere direction in the WT F8 X chromosome. There were three bases of microhomology (CTT) at the junction (Fig. 5, P1).

image

Figure 4.  Locations of restriction enzyme sites and inverse PCR primers used to identify the breakpoint junctions. Values indicate the sizes of regions in kb, whereas circled numbers (1–3) show the binding sites of primers used for inverse PCR to identify the breakpoints. Open and closed circles indicate the PCR-mapped regions of successful and failed amplifications, respectively. We used SspI for P1, HindIII for P2 and BclI for P3 in inverse PCRs to determine the breakpoint junctions, respectively. Schematic symbols are as in Fig. 3Q. Primer sequences are shown in Table S3.

Download figure to PowerPoint

image

Figure 5.  DNA sequencing electropherograms of fragment breakpoint junctions. Sequence orientations are indicated by purple (centromeric side) and red (telomeric side) arrows. Breakpoint candidates are indicated by starburst symbols along with circled numbers. Schematic symbols are as in Fig. 3Q. (P1) The junction in Xq28 of P1 is located at nt 154 426 072 (the nucleotide position is with reference to the wild-type (WT) X chromosome: GenBank accession NC_000023.10), about 20 kb on the telomeric side from VBP1 on the Inv22 type I X chromosome. The adjacent telomere side sequences are located at nt 154 598 975 and nt 154 701 245 in the palindromic regions of int22h-2 and -3, respectively, in centromere direction in the Inv22 type I X chromosome. The CTT in the boxed sequence indicates an overlapping sequence common to both regions. (P2) The junction in Xq28 of P2 is located at nt 154 296 978 in MTCP1 of the Inv22 type I X chromosome. The adjacent telomere side sequences are located at nt 154 566 155 and nt 154 734 058 in the palindromic regions of int22h-2 and -3, respectively, in centromere direction in the Inv22 type I X chromosome. A cytosine residue common to both sequences is boxed. (P3) The junction in Xq28 of P3 is located at nt 154 241 636 in F8 intron 1, adjacent to nt 154 625 305 between the palindromic regions of int22h-2 and -3 proximally in the Inv22 type II X chromosome. There are no bases of microhomology at the junction. (WT Xq28 variants) Positions and directions of telomere side sequences of the breakpoints are plotted on the two WT F8 variants Xq28, int22h-123 and -132 (according to the Xq->Xtel orientation of the int22h-1, -2, and -3 sequences). Circled numbers correspond to the positions in the Inv22 X chromosomes of the patients. In the two WT variants Xq28, each adjacent telomere side sequence of the breakpoints found in the patients directed towards a telomere at least in one position.

Download figure to PowerPoint

In the case of the Inv22 type I X chromosome of P2, HindIII sites were present at 0.8 kb on the centromeric side and 2.7 kb on the telomeric side of the primer pair M for PCR mapping, respectively (Fig. 4). Inverse PCR of the HindIII-digested DNA of P2 amplified a 2-kb fragment instead of the expected 3.3-kb fragment. The sequence of the abnormal 2-kb fragment contained a breakpoint located at nt 154 296 978 in MTCP1 in the Inv22 X chromosome. The adjacent telomere side sequences were located at nt 154 566 155 and nt 154 734 058 in the palindromic regions of int22h-2 and -3, respectively, in a centromere direction in the Inv22 type I X chromosome; however, either one was in telomere direction in the WT F8 X chromosome. There was one base of microhomology (C) at the junction (Fig. 5, P2).

In the case of the Inv22 type II X chromosome of P3, BclI sites were present at 3.3 kb on the centromeric side and 9.7 kb on the telomeric side of the primer pair O for PCR mapping. Inverse PCR of the BclI-digested DNA of P3 amplified a 5-kb fragment instead of the expected 12.6-kb fragment. The sequence of the abnormal 5-kb fragment contained a breakpoint located at nt 154 241 636 in F8 intron 1. The adjacent telomere side sequence was located at nt 154 625 305 between the palindromic regions of int22h-2 and -3 in centromere direction in the Inv22 type II X chromosome; however, it was in telomere direction in the WT F8 variant X chromosome, int22h-132 (according to the Xq->Xtel orientation of int22h-1, -2, and -3 sequences) [16]. There were no bases of microhomology at the junction (Fig. 5, P3).

All adjacent sequences of the breakpoints directed towards centromere in the Inv22 X chromosomes, P1 and P2 in type I, and P3 in type II, respectively. Whereas these sequences directed towards telomere in the WT F8 X chromosomes, P1 and P2 in int22h-123 or -132 variant, and P3 in int22h-132 variant, respectively (Fig. 5, bottom). The two WT Xq28 variants were as a result of a non-deleterious inversion polymorphism, which changed the relative positions and orientations of int22h-2 and -3 [3].

The putative breakpoint junctions were subsequently confirmed by the PCRs with primers located on both sides of the junctions. The patients (P1–P3) and the family members of P2 (his mother and grandmother) showed predicted PCR products, respectively, but the WT male control for each breakpoint did not (Fig. S1).

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Addendum
  8. Acknowledgements
  9. Disclosure of Conflict of Interest
  10. References
  11. Supporting Information

A variety of gene abnormalities, point mutations including nucleotide substitutions, small insertions/deletions and F8-related inversions causing HA have been reported. Large deletions in F8 account for approximately 5% of HA, and almost all cases of gross DNA rearrangements result in low levels of FVIII: C < 1%, corresponding to clinically severe disease. We have described complex X-chromosome rearrangements in three unrelated Japanese patients affected with severe HA. In a previous study [11], the patients were shown to carry large deletions of the F8 exons 1–22 (P1, P2) or 2–22 (P3), using Southern blot analysis; however, the precise gene abnormalities were not fully elucidated.

In the long PCR analyzes, all patients displayed expected AQ products resulting from centromeric recombination of the Inv22 X chromosome, but unexpectedly lacked PB products from its telomeric recombination. In the IS-PCR analyzes, Inv22 diagnostic tests showed that P1 and P2 had an Inv22 type I, whereas P3 had an Inv22 type II; however, they unexpectedly showed wild-type patterns in the complementary tests. These data indicated that the patients carried an inversion int22h copy on the centromeric side but not on the telomeric side, and thus could carry an intact int22h copy in the most telomeric region. Thus, abnormal rearrangements might be combined with Inv22 in the terminal end of the long arm of the X chromosomes of these patients.

We hypothesized that the patients carried Inv22 and large F8 deletions in the terminal region of the long arms of their X chromosomes. Accordingly, we investigated the X chromosomes of the patients by PCR mapping, inverse PCR and DNA sequencing to evaluate the deleted regions and their breakpoints. In PCR mapping, we could roughly estimate the locations of the centromeric ends of the deleted segments in the Inv22 X chromosome. Subsequent inverse PCR and DNA sequencing revealed their deletion breakpoints. The sequences of the telomeric ends of the breakpoints in P1 and P2 were found in palindromic arms of int22h-2 or -3, each allowing two possible positions, whereas the telomeric ends of the breakpoints in P3 were found between the palindromic regions of int22h-2 and -3. All adjacent sequences were directed towards the centromere in the Inv22 X chromosomes, resulting in loss of telomere structures. If the deletion took place after Inv22, the DNA restoration after double-strand breaks (DSBs) using a WT F8 Xq28 variant, int22h-123 or int22h-132, could make a breakpoint followed in matching direction towards a telomere as shown in Fig. 5 (bottom). It is well known that the telomere structure of the chromosome is essential for the cell to survive. Therefore, the DNA DSBs would be repaired with a WT X chromosome to regain a telomere structure, resulting in the presence of one more additional intact int22h-2 or -3 as detected in the IS-PCR (Fig. 2B). These complex abnormalities could be caused by repair mechanisms after DNA DSBs.

DSBs are potentially lethal lesions that occur spontaneously during normal cell metabolism or upon treatment of cells with DNA-damaging agents. There are two major mechanisms for repairing DSBs: non-homologous end joining (NHEJ) and homologous recombination (HR). NHEJ involves the religation of the two ends of the broken chromosome and can occur with high fidelity, or be accompanied by gain or loss of nucleotides at the junction [17,18]. NHEJ occurs intra- or inter-chromosomally and can lead to a small deletion, insertion, or indel or a large deletion or inversion in the first case and a translocation in the second case. HR relies on the presence of a homologous duplex as a template for repair of the broken chromosome [19]. Several sub-pathways of HR have been defined, including DSB repair, synthesis-dependent strand annealing and break-induced replication (BIR). BIR, which has been experimentally observed in yeast, might also be capable of causing complex rearrangements in humans leading to HA [20]. The microhomologies identified at the breakpoints of human complex rearrangements are not sufficiently long to be employed in classic BIR. Replication-based mechanisms can readily explain complex rearrangements and the microhomologies at breakpoints. However, breakpoint sequence analyzes of complex rearrangements also show in some cases an absence of microhomology, which could indicate NHEJ-specific nucleotide insertion or deletion [21].

These rearrangements might not occur simultaneously, because most instances of Inv22 occur during spermatogenesis [22], and a chromosome pair is needed to repair DSB. We deduced that in the studied families, first Inv22 occurred during spermatogenesis and then a DSB occurred on the maternal X chromosome with Inv22, followed by repair by NHEJ and/or BIR during oogenesis. Incidentally, P2’s mother and grandmother carried in the heterozygous state the same mutation as in P2 (Fig. S1B), suggesting that this rearrangement was inherited over the generations.

Several independent reports using Southern blot and long PCR analyzes have shown unusual patterns with both int22h-related inversion and deletion of F8 causing severe HA (‘rare inversion type’) [8,23,24], and we identified three such cases in our study. So far, we identified three such large deletions in 63 severe HA patients tested for Inv22 in our laboratory, these deletions could be missed using any of the standard Southern blot or PCR approaches to analyze the Inv22.

As one of the precedents for DNA repair, Sheen et al. [20] reported a complex mutational event resulting from DNA repair that incorporates BIR and serial replication slippage in a severe HA patient. That mutational event consisted of two adjacent complex deletion/insertions; one involving a deletion of the promoter and exon 1 of F8, and the other involving a large deletion/insertion that removes the entire coding sequence of the FUNDC2 gene. Moreover, they found a large duplication of four genes (TMEM185A, HSFX1, MAGEA9 and MAGEA11) in the latter deletion/insertion. They concluded that this complex genomic rearrangement was generated by two distinct, but linked, repair mechanisms in response to simultaneous DSB. Although the complex rearrangement by a combination of complex deletions and insertions was not found in our three cases, it is suggested that the combination of DNA DSB and a repair mechanism in the telomere region of the Inv22 X chromosome might be less rare than supposed.

Five genes between F8 and int22h-2 used as markers in this study are well characterized on Mendelian Inheritance in Man: MTCP1 (OMIM 300116), BRCC3 (OMIM 300617), VBP1 (OMIM 300133), RAB39B (OMIM 300774) and CLIC2 (OMIM 300138). In this study, P1 carried the largest deletion of about 270 kb including F8 exons 1–22, MTCP1 and BRCC3. Recently, Miskinyte et al. [25] reported that a loss of BRCC3 led to abnormal angiogenesis and was associated with X-linked moyamoya syndrome, which is characterized by the association of a moyamoya angiopathy, short stature and a stereotyped facial dysmorphism. They investigated families affected by X-linked syndromic moyamoya disorder, in which an overlapping deletion at Xq28 removed MTCP1 and BRCC3 and cosegregated with the affected phenotype. Very interestingly, P1 in our study had a facial dysmorphism and short stature, and was diagnosed with dwarfism. In view of the findings of Miskinyte et al., the short stature of P1 could be as a result of the deletion of BRCC3.

In conclusion, we identified three distinct gene rearrangements in the most distal region of the long arm of the Inv22 X chromosome that appeared to result from DSB-BIR (and/or -NHEJ) DNA repair, leading to large F8 deletions in severe HA patients with high responding FVIII inhibitors. Elucidation of such complex gene rearrangements will help to understand the molecular mechanism behind not only gross X chromosomal rearrangements causing severe HA, but also genomic rearrangements causing other genetic diseases.

Addendum

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Addendum
  8. Acknowledgements
  9. Disclosure of Conflict of Interest
  10. References
  11. Supporting Information

J. Fujita and Y. Miyawaki contributed equally to this work, sharing first authorship, and designed and performed the research, analyzed the data and drafted the manuscript; A. Suzuki, A. Maki, E. Okuyama and M. Murata performed experiments, analyzed the data and contributed analytic methodology; A. Takagi and T. Murate contributed analytic methodology and analyzed the data; T. Matsushita and N. Suzuki developed the project, collected and analyzed the clinical data; H. Saito supervised the project and edited the manuscript; and T. Kojima designed the project, analyzed data and wrote the manuscript.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Addendum
  8. Acknowledgements
  9. Disclosure of Conflict of Interest
  10. References
  11. Supporting Information

We wish to thank C. Wakamatsu for her excellent technical assistance. This study was supported in part by grants-in-aid from the Japanese Ministry of Education, Culture, Sports, Science, and Technology (2259524) (T.K.), the Japanese Ministry of Health, Labour and Welfare (Research on Measures for Intractable Diseases) (T.K.) and the Baxter Hemophilia Foundation (Y.M.). The authors would like to thank Enago for the English language review.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Addendum
  8. Acknowledgements
  9. Disclosure of Conflict of Interest
  10. References
  11. Supporting Information
  • 1
    Lakich D, Kazazian HH, Antonarakis SE, Gitschier J. Inversions disrupting the factor VIII gene are a common cause of severe haemophilia A. Nat Genet 1993; 5: 23641.
  • 2
    Naylor J, Brinke A, Hassock S, Green PM, Giannelli F. Characteristic mRNA abnormality found in half the patients with severe haemophilia A is due to large DNA inversions. Hum Mol Genet 1993; 2: 17738.
  • 3
    Naylor JA, Buck D, Green P, Williamson H, Bentley D, Gianneill F. Investigation of the factor VIII intron 22 repeated region (int22h) and the associated inversion junctions. Hum Mol Genet 1995; 4: 121724.
  • 4
    Bagnall RD, Giannelli F, Green PM. Polymorphism and hemophilia A causing inversions in distal Xq28: a complex picture. J Thromb Haemost 2005; 3: 25989.
  • 5
    Bagnall RD, Giannelli F, Green PM. Int22h-related inversions causing hemophilia A: a novel insight into their origin and a new more discriminant PCR test for their detection. J Thromb Haemost 2006; 4: 5918.
  • 6
    Bagnall RD, Waseem N, Green PM, Giannelli F. Recurrent inversion breaking intron 1 of the factor VIII gene is a frequent cause of severe hemophilia A. Blood 2002; 99: 16874.
  • 7
    Schroder W, Wehnert M, Herrmann F. Intron 22 of factor VIII gene – a hot spot for structural aberrations causing severe hemophilia A [letter]. Blood 1996; 87: 30678.
  • 8
    Andrikovics H, Klein I, Bors A, Nemes L, Marosi A, Varadi A, Tordai A. Analysis of large structural changes of the factor VIII gene, involving intron 1 and 22, in severe hemophilia A. Haematologica 2003; 88: 77884.
  • 9
    Abou-Elew H, Ahmed H, Raslan H, Abdelwahab M, Hammoud R, Mokhtar D, Arnaout H. Genotyping of intron 22-related rearrangements of F8 by inverse-shifting PCR in Egyptian hemophilia A patients. Ann Hematol 2011; 90: 57984.
  • 10
    Oldenburg J, Pavlova A. Genetic risk factors for inhibitors to factors VIII and IX. Haemophilia 2006; 12: 1522.
  • 11
    Sugihara T, Takahashi I, Kojima T, Okamoto Y, Yamamoto K, Kamiya T, Matsushita T, Saito H. Identification of plasma antibody epitopes and gene abnormalities in Japanese hemophilia A patients with factor VIII inhibitor. Nagoya J Med Sci 2000; 63: 2539.
  • 12
    Kojima T, Tanimoto M, Kamiya T, Obata Y, Takahashi T, Ohno R, Kurachi K, Saito H. Possible absence of common polymorphisms in coagulation factor IX gene in Japanese subjects. Blood 1987; 69: 34952.
  • 13
    Liu Q, Nozari G, Sommer SS. Single-tube polymerase chain reaction for rapid diagnosis of the inversion hotspot of mutation in hemophilia A. Blood 1998; 92: 14589.
  • 14
    Rossetti L, Radic C, Larripa I, De Brasi C. Developing a new generation of tests for genotyping hemophilia-causative rearrangements involving int22h and int1h hotspots in the factor VIII gene. J Thromb Haemost 2008; 6: 8306.
  • 15
    Tsukahara A, Yamada T, Takagi A, Murate T, Matsushita T, Saito H, Kojima T. Compound heterozygosity for two novel mutations in a severe factor XI deficiency. Am J Hematol 2003; 73: 27984.
  • 16
    Rossetti L, Radic C, Abelleyro M, Larripa I, De Brasi C. Eighteen years of molecular genotyping the hemophilia inversion hotspot: from southern blot to inverse shifting-PCR. Int J Mol Sci 2011; 12: 7271785.
  • 17
    Daley JM, Palmbos PL, Wu D, Wilson TE. Nonhomologous end joining in yeast. Annu Rev Genet 2005; 39: 43151.
  • 18
    Lieber MR, Ma Y, Pannicke U, Schwarz K. The mechanism of vertebrate nonhomologous DNA end joining and its role in V(D)J recombination. DNA Repair 2004; 3: 81726.
  • 19
    Symington L. Role of RAD52 epistasis group genes in homologous recombination and double-strand break repair. Microbiol Mol Biol Rev 2002; 66: 63070.
  • 20
    Sheen CR, Jewell UR, Morris CM, Brennan SO, Férec C, George PM, Smith MP, Chen J-M. Double complex mutations involving F8 and FUNDC2 caused by distinct break-induced replication. Hum Mutat 2007; 28: 1198206.
  • 21
    Lieber MR. The Mechanism of Human Nonhomologous DNA End Joining. J Biol Chem 2008; 283: 15.
  • 22
    Rossiter JP, Young M, Kimberland ML, Hutter P, Ketterling RP, Gitschier J, Horst J, Morris MA, Schaid DJ, de Moerloose P, Sommer SS, Kazazian HH, Antonarakis SE. Factor VIII gene inversions causing severe hemophilia A originate almost exclusively in male germ cells. Hum Mol Genet 1994; 3: 10359.
  • 23
    Mühle C, Schulz-Drost S, Khrenov AV, Saenko EL, Klinge J, Schneider H. Epitope mapping of polyclonal clotting factor VIII-inhibitory antibodies using phage display. Thromb Haemost 2004; 91: 61925.
  • 24
    Mühle C, Zenker M, Chuzhanova N, Schneider H. 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. Hum Mutat 2007; 28: 1045.
    Direct Link:
  • 25
    Miskinyte S, Butler MG, Hervé D, Sarret C, Nicolino M, Petralia JD, Bergametti F, Arnould M, Pham VN, Gore AV, Spengos K, Gazal S, Woimant F, Steinberg GK, Weinstein BM, Tournier-Lasserve E. Loss of BRCC3 deubiquitinating enzyme leads to abnormal angiogenesis and is associated with syndromic moyamoya. Am J Hum Genet 2011; 88: 71828.

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Addendum
  8. Acknowledgements
  9. Disclosure of Conflict of Interest
  10. References
  11. Supporting Information

Figure S1. Rearrangement specific PCR of the patients and the family members of P2. In the patients, PCR products used multiplex primers were showed two bands, in contrast one band in the wild-type. (A) PCR for the breakpoint junction of P1. (B) Breakpoint junction of P2, his mother and his grandmother. (C) Breakpoint junction of P3. Primer sequences are listed in Table S4. M, 100 bp DNA ladder marker; WT, wild-type control; P1–P3, Patient 1–3; Mo, P2’s mother; GMo, P2’s grandmother. (D–F) Schematic summaries of rearrangement specific PCR locations of Xq28. Upper; patient model, lower; wild-type (int22h-123 variant for P1 and P2, int22h-132 variant for P3). Arrows indicate the sites of PCR primer pairs. Two PCR bands with green-blue and red-blue primer pairs were amplified in the patients, but only one band with green-blue primer pair was amplified in the wild-type.

Table S1. (A) IS-PCR primers. (B) Expected product size in each reaction.

Table S2. PCR mapping primers.

Table S3. Inverse PCR and sequencing primers.

Table S4. Breakpoint specific primers.

FilenameFormatSizeDescription
JTH_4897_sm_FigS1-TableS1-S4.pdf120KSupporting info item
JTH_4897_sm_FigS1a-c.tiff1520KSupporting info item
JTH_4897_sm_FigS1d-f.tiff1520KSupporting info item

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.