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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Division of Medical and Molecular Genetics,
Guy's, King's, and St Thomas' School of Medicine, London, United
Kingdom.
The messenger RNA (mRNA) from 5 of 69 patients with severe
hemophilia A did not support amplification of complementary DNA containing the first few exons of the factor VIII (F8)
gene but supported amplification of mRNA containing exon 1 of
F8 plus exons of the VBP1 gene. This chimeric
mRNA signals an inversion breaking intron 1 of the F8 gene.
Using an inversion patient, one deleted for F8 exons 1 to
6, and cosmids mapped 70 to 100 kb telomeric of the F8
gene, this study shows that this break strictly affects a sequence
(int1h-1) repeated (int1h-2) about 140 kb more
telomerically, between the C6.1A and
VBP1 genes. The 1041-base pair repeats differ at a single
nucleotide (although int1h-2 also showed one polymorphism) and are in opposite orientation. The results demonstrate that they
cause inversions by intrachromosome or intrachromatid homologous recombination. The genomic structure of the inversion region shows that
transcription traverses intergenic spaces to produce the 2 chimeric
mRNAs containing the F8 sequences and characteristic of the
inversion. This observation prompts the suggestion that nature may use
such extended transcription to test whether the addition of novel
domains from neighboring genes creates desirable new genes. A rapid
polymerase chain reaction test was developed for the inversion in both
patients and carriers. This has identified 10 inversions, affecting
F8 genes with 5 different haplotypes for the BclI, introns
13 and 22 VNTR polymorphism, among 209 unrelated families with severe
hemophilia A. This indicates a prevalence of 4.8% and frequent
recurrence of the inversion. This should result in absence of
F8, and one inversion patient is known to have inhibitors.
(Blood. 2002;99:168-174) Higuchi et al1,2 observed that
thorough screening of all the exons of the factor VIII (F8)
gene was efficient in detecting the mutations of patients with mild and
moderate hemophilia A and yet failed in 50% of patients with severe
disease. Traces of factor VIII messenger RNA (mRNA) from peripheral
lymphocytes soon revealed that this high failure rate was largely due
to mutations affecting internal regions of intron 22 of the
F8 gene.3 These mutations were later shown to
be inversions resulting from homologous intrachromatid or
intrachromosome (ie, intranemic) recombination between a 9503-base pair
(bp) sequence (int22h-1) in intron 22 of the
F8 gene and one or other of 2 inverted copies of this
sequence (int22h-2, int22h-3) located,
respectively, 500 and 600 kb more telomeric.4-6
The int22h-related inversions appeared to be sufficiently
frequent to account for the shortfall in mutation detection experienced using methods based on the screening of all exons of the F8
gene. However, analysis of factor VIII mRNA continued to detect further mutations that escape detection by this approach. Base substitutions deep inside introns were found that generate novel exons disrupting the
factor VIII coding sequence7 (R. D. Bagnall,
unpublished observations, January 2001). Moreover, an
inversion was identified that broke the F8 gene at intron 1 and resulted in the production of 2 chimeric mRNAs.8 One
of these mRNAs, presumably under the control of the factor VIII
promoter, contains the first exon of the F8 gene followed by
facultative exons and then exons 2 to 6 of a gene (VBP1)
coding for subunit 3 of prefoldin.9-11 The other mRNA,
transcribed under the control of the C6.1A promoter, contains all but
the last exon of the C6.1A gene plus a number of facultative
exons followed by exons 2 to 26 of the F8
gene.8
During the construction of a confidential United Kingdom database of
hemophilia A mutations and pedigrees, destined to optimize the genetic
service provided nationally for this disease, we have discovered that
the above inversion occurs repeatedly and accounts for about 5% of
severe cases of hemophilia A. We have also characterized the breakpoint
regions, elucidated the mode of origin of the inversions, and developed
a rapid polymerase chain reaction (PCR) procedure for the detection of
these mutations in both patients and carriers.
Blood samples
Factor VIII mRNA amplification and
mutation analysis
Vectorette library construction Using the method of Riley et al,13 100 ng cosmid DNA was restricted with 10 U RsaI in a final volume of 20 µL for 12 hours, followed by heat inactivation at 65°C for 2 minutes. This digestion reaction (15 µL) was ligated to 4 µM of an RsaI vectorette cassette using 6 U DNA ligase at 4°C for 12 hours. The vectorette library was diluted with an equal volume of Tris (10 mM) plus EDTA (0.1 mM) buffer (pH 8) and stored at 20°C.
An aliquot of this library was used to amplify between an
int1h-specific primer and the vectorette-specific primer 224 (Table 1).
PCR amplification The PCR amplifications of factor VIII intron 1 regions and int1h repeats were performed on 100 ng genomic DNA or 10 ng cosmid DNA with 2.5 µL 10 × Amplitaq Reaction Buffer (Perkin-Elmer, Warrington, United Kingdom), 1.5 mM MgSO4, 200 ng of each oligonucleotide primer, 0.5 mM of each dNTP, 5% dimethyl sulfoxide, and 2.5 U Amplitaq DNA polymerase (Perkin-Elmer). Thirty cycles of PCR were carried out at 94°C for 30 seconds, 65°C for 30 seconds, and 72°C for 2 minutes. The primers used for PCR assays are shown in Table 1.Fluorescent sequencing Sequencing was performed on Genecleaned PCR products using the D-Rhodamine dye terminator kit according to the manufacturer's instructions (ABI Perkin-Elmer, Warrington, United Kingdom). Products were analyzed on an ABI 377 DNA sequencer using ABI Sequence Analysis software.Detection of int22h-related inversions These inversions were detected using a PCR-based method described by Liu et al,14 modified by reducing the concentrations of both primer A and B to 20 ng and performing the PCR annealing step at 67°C.
The intron 1 breaking inversion is a recurring cause of severe hemophilia A Our laboratory has received blood samples from 209 unrelated patients with severe disease in the course of constructing a United Kingdom confidential database of hemophilia A mutations and pedigrees. Ninety-four (45%) of these patients were found to have an intron 22 breaking inversion, whereas 115 patients, negative for these inversions, required full mutation screening. Analysis of factor VIII mRNA extracted from lymphocytes of 69 of these 115 patients showed that the mRNA of 5 patients did not support amplification of the segment containing exons 1 to 9, whereas the other segments containing the rest of the coding sequence readily amplified and contained no mutations. This was reminiscent of findings in 2 monozygotic twins with severe hemophilia A who were found to have an approximate 100-kb inversion breaking intron 1 of the F8 gene.8 Because this inversion leads to the production of a hybrid transcript that comprises exon 1 of the F8 gene and exons of the VBP1 gene, we used an RT-PCR reaction specific for this abnormal transcript and obtained a positive result in each of the above 5 patients (Figure 1). They therefore appear to have the intron 1 breaking inversion previously observed by Brinke et al.8 The families of the 5 patients were not related to each other or to the family of the monozygotic twins examined by Brinke et al,8 and analysis of the F8 gene haplotypes for variations at the BclI and intron 13 and 22 VNTR polymorphisms15-17 demonstrated that the intron 1 breaking inversion has occurred independently more than once because the 6 patients show 3 different haplotypes.
The intron 1 breaking inversion appears to be due to intranemic homologous recombination involving repeats in inverted orientation To discover the mode of origin of the above inversions, the region broken in intron 1 of the F8 gene was sought by subdividing the intron 1 sequence (GenBank accession no. AL390881) into 12 amplifiable overlapping segments of 2 to 3.5 kb (Figure 2). The PCR reactions for all 12 segments were successful in wild-type DNA but the DNA from one of the monozygotic twins with the intron 1 inversion (UKA29) consistently failed to support amplification of segment 9. This suggested that the 2 kb of segment 9 comprises an inversion junction. To locate this junction more precisely, segment 9 was divided into 4 adjacent sections (a-d, shown in Figure 2) but, surprisingly, PCR reactions for all 4 sections were successful in the DNA from UKA29. However, the 1.5-kb PCR stretching from segments a to c consistently failed, whereas 2 overlapping segments containing a + b and b + c consistently amplified (Figure 2). This suggested that the inversion break involved the a to c region in such a way that the sequences of the individual segments a, b, and c are not disrupted. This could have occurred if the inversions had arisen by homologous recombination within a repeated sequence, as previously proposed and demonstrated for the intron 22 inversions.4-6 This presupposes the existence of repeated sequences in inverted orientation spanning segment 9b as illustrated in Figure 3.
A proof of this hypothesis required the definition of the repeats and their flanking sequences. The existence of a repeat sequence was established using the DNA from a United Kingdom hemophilia A patient with a deletion extending from the promoter region to intron 6 of the F8 gene. This DNA sustained the amplification of a 500-bp sequence identical to segment 9b. Moreover, this sequence was also amplified from 3 cosmids (183 B4, 21B11, 240D11) containing no segments of the F8 gene. These were obtained from a 49,XXXXX library18 and mapped to a region 70 to 100 kb telomeric to the F8 gene.19 The cosmids contained C6.1A gene sequences that were shown to be placed 5' of the second exon of the F8 gene in one of the hybrid mRNAs detected in the lymphocytes of UKA29.8 To determine the length of the repeat and identify its flanking
sequences, an RsaI vectorette library of cosmid 183B4 was prepared. This library supported PCR reactions driven by primer 9bF or
9bR paired with the vectorette primer 224. The 9b primers were oriented
outwardly and in opposite direction and were located in the 500 bp of
known repeated sequence. The reaction containing primers 9bF and 9bR,
respectively, yielded a 1-kb and an 0.8-kb product. The former
comprised 901 bp identical to nucleotide number 15 403 to 16 304 of
intron 1 of the F8 gene, except at nucleotide 16 271 where
C was found rather than A, plus 58 bp of novel sequence. The latter PCR
product was completely contained within the repeated sequence.
Therefore, the second boundary of the repeat and the adjacent flanking
sequence were sought by searching the GenBank database with the 500 bp
of known repeated sequence. This showed that the 500-bp sequence
bridged a gap between 2 independent sequence entries of 11.5 and 8.7 kb
(GenBank accession no. AC016977), which contained, respectively, 216 and 160 bp of the 500-bp repeat. With the help of these sequences, a
PCR was designed that amplified both wild-type genomic DNA and cosmid
183B4. This revealed that the repeated sequence extended for 1041 bp
and was flanked in the PCR product by unique sequences of 56 and 94 bp
(Figure 4). We call the 2 repeats
int1h (from intron 1 homology) and use -1 and -2 to specify,
respectively, the copy in the F8 gene and the one more
telomeric. A search of the current data on the human genome sequence
does not show any other copy of int1h.
The int1h repeat sequence (nucleotides 15 264-16 304 of intron 1) and flanking regions were searched for both sequences of possible biologic relevance and repetitive elements using NIX.20 No biologically significant sequences were identified within the repeat region; however, int1h-1 was flanked telomerically by a 316-bp LTR/ERVL (ERVL) repeat extending from intron 1 nucleotide 14 963 to 15 252 and on the other side by a 345-bp LTR/ERVL (LTR 16A) repeat extending from nucleotide 16 397 to 16 742. The int1h-2 repeat extended on the telomeric side into a 1026-bp LINE L2 repetitive element. According to the hypothesis illustrated in Figure 3, the int1h repeats of patient UKA29 should have recombined to yield repeats with novel arrangements of flanking sequence so that the repeat near exon 1 of the F8 gene should be flanked by the unique sequence centromeric of normal int1h-1 and the unique sequence centromeric to the normal int1h-2. Conversely, the repeat near to the C6.1A gene should be flanked by the sequences telomeric to normal int1h-1 and int1h-2. This was confirmed using the primer pairs 9aF + int1h-2R and int1h-2F + 9cR, because these yield no products in wild-type DNA while they amplify segments of 1.5 and 1.3 kb in DNA from UKA29, which show the arrangement of flanking sequences predicted above. This is in keeping with the hypothesis that the inversion of UKA29 results from homologous intranemic recombination within the int1h repeats. Location of int1h-2 and verification of its orientation Using information from the GenBank genome database, we constructed a continuous sequence extending from intron 6 of the F8 gene to the 3' untranslated region (UTR) of the C6.1A gene. This 143-kb sequence contains 6 overlapping sequence entries that were aligned using the Sequencher program. Unfortunately this sequence did not join up with the 20 kb contig comprising int1h-2. Therefore, to verify the orientation of int1h-2 we searched this 20-kb contig for the novel exon sequences found by Brinke et al8 in the chimeric mRNA of patient UKA29 containing all but the last exon of C6.1A and exons 2 to 26 of the F8 gene. Four of the 5 novel exons found spliced between the sequences of the 2 above genes were contained in the 20-kb contig. They were flanked by GT, AG splice consensuses and were in the order proposed by Brinke et al.8 They were separated by introns ranging in size from 440 to 3027 bp. The nucleotide analysis software, NIX,20 indicates that these exons may be part of an incompletely characterized gene encoding a protein homologous to the clathrin assembly protein. The novel 94-bp exon nearest to the C6.1A gene, called novel B, was not in the 20-kb contig but in a 6-kb sequence that should map to the gap between this contig and that of 143 kb. The order of the novel exons relative to the F8 and C6.1A genes indicates that int1h-2 is oriented with its first residue most centromeric, whereas int1h-1 has its first residue most telomeric. A map of this region showing the relevant features is shown in Figure 5.
Because current knowledge of the human genome sequence does not indicate the size of the gaps between the 3 genomic sequences considered above, the precise distance between int1h-1 and int1h-2 cannot be determined, but the available data suggest a minimum of 136 kb. Similarity and conservation of int1h-1 and int1h-2 As mentioned earlier, only one difference was found between the int1h-1 sequences in GenBank AL390881 and the int1h-2 sequence from cosmid 183B4. Sequence variation in int1h was further investigated by screening the DNA of 43 United Kingdom men and 7 women using solid-phase fluorescent cleavage of mismatch.21 This showed that int1h-1 was identical in all 57 X chromosomes thus examined, whereas int1h-2 showed a polymorphism due to a C>G change at nucleotide 15 961 in 9 X chromosomes. In these 9 X chromosomes there are 2 nucleotide differences between int1h-1 and int1h-2.PCR test for the detection of the int1h-related inversion and prevalence of this mutation Because the inversion results in a reassortment of sequences flanking each int1h repeat, these flanking sequences were used to design 2 PCR reactions capable of detecting the inversion in patients and carriers. One reaction contains the primers specific for int1h-1 plus the primer specific for the sequence flanking int1h-2 on the centromeric side (9F, 9cR, int1h-2F); the other reaction contains the primers specific for int1h-2 plus that specific for the flanking sequence at the telomeric side of int1h-1 (int1h-2F, int1h-2R, 9F). The former reaction yields a 1.5-kb product from normal DNA and a 1.0-kb product from a patient with the inversion, whereas a female carrier's DNA yields both products. The latter reaction correspondingly yields 1, 1.5, 1 + 1.5-kb products from normal, inversion patient, and carrier DNA (Figure 6).
DNA from UKA29 and the other 5 patients with the inversion were tested with the above reactions and the expected results were obtained. Thus, we used this PCR diagnostic test on the 46 most recently referred severely affected patients whose hemophilia A mutation had not yet been sought. The test was positive in 4 patients so that a total of 10 int1h-related inversions was found to occur among the 209 unrelated patients with severe hemophilia A referred to our laboratory. This suggests a prevalence of 4.8% (95% CI = 2.4-8.7) in severe hemophilia A. The 9 inversion patients who could be analyzed with regard to haplotype for the BclI and intron 13 and 22 microsatellite polymorphisms of the F8 gene showed 5 different haplotypes. Among these patients one was reported to have developed inhibitors. Formal proof that the int1h-related inversions are due to intranemic homologous recombination All the results presented so far support the mode of origin for the int1h-related inversion proposed in Figure 3. However, formal proof of the hypothesis can only be obtained by sequencing the inversion breakpoint regions in patients. This was done for the 9 inversion patients whose DNA was available for this investigation. PCR products containing the inversion break regions were obtained as described above and sequenced. Int1h-1 and int1h-2 were found to be intact in 8 patients, whereas in one the A at nucleotide 16 271 of int1h-1 appears to have been converted to C as in int1h-2. Moreover, the repeats were precisely flanked on one side by the sequences present in wild-type DNA and on the other by the sequence normally flanking the alternative int1h repeat and expected to be part of the inverted DNA segment. These data formally prove that the int1h-related inversions are due to homologous intranemic recombination and because int1h-1 and int1h-2 differ at residue 16 271, the observation that the int1h-2-specific residue was associated with the wild-type int1h-2 flanking sequence indicates that the recombination breakpoint occurred between nucleotides 15 264 and 16 271 in 7 patients, and in 1 patient the presence of the rarer allele at the polymorphic site in int1h-2 further restricts the position of the recombination breakpoint to the segment between nt 15 264 and 15 961.
As stated by Brinke et al,8 the intron 1 breaking inversion causes severe hemophilia A because the coding sequence for coagulation factor VIII, although transcribed, lies downstream of the translation stop codon of the C6.1A gene and is separated from the sequence coding for the factor VIII signal peptide. Thus, even if the chimeric mRNAs discovered in the patient's peripheral lymphocytes were sufficiently stable and produced in the liver in adequate amounts, no factor VIII protein should be present in the patient's blood unless unexpected internal translation of the mRNA occurred. This suggests that the patient should be predisposed to the inhibitor complication. Among the 10 patients we have identified, 1 is known to have developed inhibitors. Now a larger series of patients with this inversion is required better to estimate their empirical risk of developing inhibitors. Our results demonstrate that Brinke et al8,10 were right in saying that the inversion breakpoint external to the F8 gene does not disrupt either the C6.1A or the VBP1 gene (Figure 5) even though sequences of these genes are found spliced into mRNA with, respectively, exons 2 to 26 and exon 1 of the F8 gene. A number of facultative exons separated the factor VIII exons from those of the C6.1A and VBP1 genes in the chimeric mRNA of the inversion patients. We find that the order of the facultative exons proposed by Brinke et al8 on the basis of the structure of chimeric mRNA amplified from the lymphocytes of UKA29 agrees with the information we could obtain on their genomic location and we also find that these exons are flanked by conventional splice signals. In addition, we show that int1h-2 is at least 16 kb downstream and telomeric to the polyadenylation site of the C6.1A gene and about 37 kb upstream and centromeric to the major transcription start site of the VBP1 gene. Therefore, our patients are expected to produce normal C6.1A and VBP1 mRNA as well as the chimeric mRNA mentioned above. The observation that in the inversion patients the intact transcription units of the C6.1A and VBP1 genes were also used to produce chimeric mRNA suggests that rearrangements may provide the opportunity to explore the value of gene products acquiring novel domains from new neighbors without loss of the original functional units. This is supported by several reports of intergenic splicing in humans.22-26 The results of Naylor et al6 and those presented above
demonstrate that the same mechanism The int22h-related inversions originate mainly in the male germline26 and it was suggested that monosomy of the X chromosome may favor folding of the X chromosome onto itself and hence the occurrence of intrachromatid recombination at male meiosis. A similar male bias may characterize the int1h-related inversion, but more families with this inversion are required to verify this expectation. Internemic recombination between int1h-1 and int1h-2, that is, recombination between these repeats from sister chromatids or homologous chromatids and chromosomes, would result in dicentric chromosomes and acentric fragments and hence should not lead to viable embryos. The inversions causing severe hemophilia A are part of a varied group of rearrangements involving duplicons in the human genome. Ji et al27 suggest that this group of abnormalities causes diseases with a combined incidence of 0.7 to 1/1000 births. However, the inversions causing hemophilia A dramatically demonstrate how some mutations may seriously disrupt gene function and yet go undetected by strategies directed to the screening of exons in genomic sequences. Thus, the relevance of duplicon-related rearrangements to human disease may well be higher than suggested by Ji et al.27 The analysis of mRNA has allowed the detection of common inversions causing hemophilia A. This approach should be equally effective in other X-linked disorders. Once the inversions are identified, it is possible to develop rapid diagnostic tests based on the analysis of genomic DNA as we have illustrated for the intron 1 breaking inversion. This test will now allow the rapid identification of patients with severe hemophilia due to the int1h-related inversions and will become a very useful addition to the methods available to provide genetic service in hemophilia A. In addition, it will serve to identify a further group of patients who may be at high risk of developing inhibitors.
We are grateful to the hemophilia A patients and the hemophilia centers in the United Kingdom that have collaborated in the construction of the United Kingdom database of hemophilia A mutations and pedigrees. In particular, we are grateful to Dr E. A. Chalmers, Dr P. W. Collins, Dr B. T. Colvin, Professor P. L. F. Giangrande, Dr M. Laffan, Professor C. A. Lee, Dr G. L. Scott, and Dr R. F. Stevens, whose centers have referred patients and carriers with the int1h-related inversions to us. We acknowledge the secretarial help of Adrienne Knight.
Submitted June 5, 2001; accepted August 27, 2001.
Supported by Medical Research Council grant G9500698 Mb and the Guy's and St Thomas' Charitable Foundation.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Francesco Giannelli, Division of Medical and Molecular Genetics, GKT School of Medicine, 8th Fl Guy's Hospital Tower, London Bridge, London SE1 9RT, United Kingdom; e-mail: adrienne.knight{at}kcl.ac.uk.
1.
Higuchi M, Antonarakis SE, Kasch L, et al.
Molecular characterization of mild-to-moderate hemophilia A: detection of the mutation in 25 of 29 patients by denaturing gradient gel electrophoresis.
Proc Natl Acad Sci U S A.
1999;88:8307-8311
2.
Higuchi M, Kazazian Jr HH, Kasch L, et al.
Molecular characterization of severe hemophilia A suggests that about half the mutations are not within the coding regions and splice junctions of the factor VIII gene.
Proc Natl Acad Sci U S A.
1991;88:7405-7409 3. Naylor JA, Green PM, Rizza CR, Giannelli F. Factor VIII gene explains all cases of haemophilia A. Lancet. 1992;340:1066-1067[CrossRef][Medline] [Order article via Infotrieve]. 4. Lakich D, Kazazian Jr HH, Antonarakis SE, Gitschier J. Inversions disrupting the factor VIII gene are a common cause of severe haemophilia A. Nat Genet. 1993;5:236-241[CrossRef][Medline] [Order article via Infotrieve].
5.
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;2:1773-1778
6.
Naylor JA, Buck D, Green P, Williamson H, Bentley D, Giannelli F.
Investigation of the factor VIII intron 22 repeated region (int22h) and the associated inversion junctions.
Hum Mol Genet.
1995;4:1217-1224 7. Bagnall RD, Waseem NH, Green PM, Colvin B, Lee C, Giannelli F. Creation of a novel donor splice site in intron 1 of the factor VIII gene leads to activation of a 191 bp cryptic exon in two haemophilia A patients. Br J Haematol. 1999;107:766-771[CrossRef][Medline] [Order article via Infotrieve].
8.
Brinke A, Tagliavacca L, Naylor J, Green P, Giangrande P, Giannelli F.
Two chimaeric transcription units result from an inversion breaking intron 1 of the factor VIII gene and a region reportedly affected by reciprocal translocations in T-cell leukaemia.
Hum Mol Genet.
1996;5:1945-1951
9.
Tsuchiya H, Iseda T, Hino O.
Identification of a novel protein (VBP-1) binding to the von Hippel-Lindau (VHL) tumor suppressor gene product.
Cancer Res.
1996;56:2881-2885 10. Brinke A, Green PM, Giannelli F. Characterization of the gene (VBP1) and transcript for the von Hippel-Lindau binding protein and isolation of the highly conserved murine homologue. Genomics. 1997;45:105-112[CrossRef][Medline] [Order article via Infotrieve]. 11. Vainberg IE, Lewis SA, Rommelaere H, et al. Prefoldin, a chaperone that delivers unfolded proteins to cytosolic chaperonin. Cell. 1998;93:863-873[CrossRef][Medline] [Order article via Infotrieve]. 12. Waseem NH, Bagnall R, Green PM, et al. Start of UK confidential haemophilia a database: analysis of 142 patients by solid phase fluorescent chemical cleavage of mismatch. Thromb Haemost. 1999;81:900-905[Medline] [Order article via Infotrieve].
13.
Riley J, Butler R, Ogilvie D, et al.
A novel, rapid method for the isolation of terminal sequences from yeast artificial chromosome (YAC) clones.
Nucleic Acids Res.
1990;18:2887-2890
14.
Liu Q, Nozari G, Sommer SS.
Single-tube polymerase chain reaction for rapid diagnosis of the inversion hotspot of mutation in haemophilia A.
Blood.
1998;92:1458-1459 15. Gitschier J, Drayna D, Tuddenham EGD, et al. Genetic mapping and diagnosis of haemophilia A achieved through a BclI polymorphism in the factor VIII gene. Nature. 1985;314:738-740[CrossRef][Medline] [Order article via Infotrieve]. 16. Lalloz MRA, McVey JH, Pattinson JK, Tuddenham EGD. Haemophilia A diagnosis by analysis of a hypervariable dinucleotide repeat within the factor VIII gene. Lancet. 1991;338:207-211[CrossRef][Medline] [Order article via Infotrieve]. 17. Lalloz MRA, Schwaab R, McVey JH, Michaelides K, Tuddenham EGD. Haemophilia A diagnosis by simultaneous analysis of two variable dinucleotide tandem repeats within the factor VIII gene. Br J Haematol. 1994;86:804-809[Medline] [Order article via Infotrieve]. 18. Holland J, Coffey AJ, Giannelli F, Bentley DR. Vertical integration of cosmid and YAC resources for interval mapping on the X-chromosome. Genomics. 1993;15:297-304[CrossRef][Medline] [Order article via Infotrieve]. 19. Hassock S. Physical and transcriptional mapping in the distal Xq28 region of the human X chromosome [doctoral thesis]. London United Kingdom: University of London; 2000. 20. Available at: http://www.hgmp.mrc.ac.uk/Registered/Webapp/nix/ NIX is a WWW tool to view the results of running many DNA analysis programs on a DNA sequence. 21. Rowley G, Saad S, Giannelli F, Green PM. Ultrarapid mutation detection by multiplex, solid-phase chemical cleavage. Genomics. 1995;30:574-582[CrossRef][Medline] [Order article via Infotrieve].
22.
Fears S, Mathieu C, Zeleznik-Le N, et al.
Intergenic splicing of MDS1 and EVI1 occurs in normal tissues as well as in myeloid leukemia and produces a new member of the PR domain family.
Proc Natl Acad Sci U S A.
1996;93:1642-1647
23.
Magrangeas F, Pitiot G, Dubois S, et al.
Cotranscription and intergenic splicing of human galactose-1-phosphate uridylyltransferase and interleukin-11 receptor alpha-chain genes generate a fusion mRNA in normal cells: implication for the production of multidomain proteins during evolution.
J Biol Chem.
1998;273:16005-16010 24. Finta C, Zaphiropoulos PG. The human CYP2C locus: a prototype for intergenic and exon repetition splicing events. Genomics. 2000;63:433-438[CrossRef][Medline] [Order article via Infotrieve].
25.
Communi D, Suarez-Huerta N, Dussossoy D, Savi P, Boeynaems JM.
Cotranscription and intergenic splicing of human P2Y11 and SSF1 genes.
J Biol Chem.
2001;276:16561-16566
26.
Antonarakis SE, Rossiter JP, Young M, et al.
Factor VIII gene inversions in severe hemophilia A: results of an international consortium study.
Blood.
1995;86:2206-2212
27.
Ji Y, Eichler EE, Schwartz S, Nicholls RD.
Structure of chromosomal duplicons and their role in mediating human genomic disorders.
Genome Res.
2000;10:597-610 28. Brinke AJM. Transcriptional consequences of large DNA inversions in the FVIII gene [doctoral thesis]. London United Kingdom: University of London; 1999.
© 2002 by The American Society of Hematology.
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  S. Lacroix-Desmazes, J. D. Dimitrov, Y. Repesse, C. L. Eckhardt, P. W. Kamphuisen, K. Fijnvandraat, G. Yang, L. Yao, Z. Lu, F. Peyvandi, et al. Inhibitors of Factor VIII in Hemophilia N. Engl. J. Med., July 16, 2009; 361(3): 308 - 310. [Full Text] [PDF]
|
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K. R. Viel, A. Ameri, T. C. Abshire, R. V. Iyer, R. G. Watts, C. Lutcher, C. Channell, S. A. Cole, K. M. Fernstrom, S. Nakaya, et al. Inhibitors of Factor VIII in Black Patients with Hemophilia N. Engl. J. Med., April 16, 2009; 360(16): 1618 - 1627. [Abstract] [Full Text] [PDF]
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 P. Casana, N. Cabrera, A. R. Cid, S. Haya, M. Beneyto, C. Espinos, V. Cortina, M. A. Dasi, and J. A. Aznar Severe and moderate hemophilia A: identification of 38 new genetic alterations Haematologica, July 1, 2008; 93(7): 1091 - 1094. [Abstract] [Full Text] [PDF]
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M. Margaglione, G. Castaman, M. Morfini, A. Rocino, E. Santagostino, G. Tagariello, A. R. Tagliaferri, E. Zanon, M. P. Bicocchi, G. Castaldo, et al. The Italian AICE-Genetics hemophilia A database: results and correlation with clinical phenotype Haematologica, May 1, 2008; 93(5): 722 - 728. [Abstract] [Full Text] [PDF]
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L. C. Rossetti, C. P. Radic, M. Candela, R. P. Bianco, M. de Tezanos Pinto, A. Goodeve, I. B. Larripa, and C. D. De Brasi Sixteen novel hemophilia A causative mutations in the first Argentinian series of severe molecular defects Haematologica, June 1, 2007; 92(6): 842 - 845. [Abstract] [Full Text] [PDF]
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 V. Bansal, A. Bashir, and V. Bafna Evidence for large inversion polymorphisms in the human genome from HapMap data Genome Res., February 1, 2007; 17(2): 219 - 230. [Abstract] [Full Text] [PDF]
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 J. Goudemand, C. Rothschild, V. Demiguel, C. Vinciguerrat, T. Lambert, H. Chambost, A. Borel-Derlon, S. Claeyssens, Y. Laurian, T. Calvez, et al. Influence of the type of factor VIII concentrate on the incidence of factor VIII inhibitors in previously untreated patients with severe hemophilia A Blood, January 1, 2006; 107(1): 46 - 51. [Abstract] [Full Text] [PDF]
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 R. K. Pruthi Hemophilia: A Practical Approach to Genetic Testing Mayo Clin. Proc., November 1, 2005; 80(11): 1485 - 1499. [Abstract] [PDF]
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P. D. James, S. Raut, G. E. Rivard, M.-C. Poon, M. Warner, S. McKenna, J. Leggo, and D. Lillicrap Aminoglycoside suppression of nonsense mutations in severe hemophilia Blood, November 1, 2005; 106(9): 3043 - 3048. [Abstract] [Full Text] [PDF]
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R. D. Bagnall, K. L. Ayres, P. M. Green, and F. Giannelli Gene conversion and evolution of Xq28 duplicons involved in recurring inversions causing severe hemophilia A Genome Res., February 1, 2005; 15(2): 214 - 223. [Abstract] [Full Text] [PDF]
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 C. S. Manno Management of Bleeding Disorders in Children Hematology, January 1, 2005; 2005(1): 416 - 422. [Abstract] [Full Text] [PDF]
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F. Riccardi, A. Tagliaferri, C. Manotti, C. Pattacini, and T. M. Neri Intron 1 factor VIII gene inversion in a population of Italian hemophilia A patients Blood, October 16, 2002; 100(9): 3432 - 3432. [Full Text] [PDF]
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P. M. Mannucci Ham-Wasserman Lecture : Hemophilia and Related Bleeding Disorders: A Story of Dismay and Success Hematology, January 1, 2002; 2002(1): 1 - 9. [Abstract] [Full Text]
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Top
Abstract
Introduction
homologous, intranemic
recombination