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Blood, 1 November 2002, Vol. 100, No. 9, pp. 3432-3432
CORRESPONDENCE
To the editor:
Intron 1 factor VIII gene inversion in a population of Italian
hemophilia A patients
Bagnall and collegues1 report a technique to
investigate an inversion that disrupts the factor VIII
(F8) gene and that represents a frequent cause of
severe hemophilia A. This large genomic rearrangement, identified for
the first time by Brinke et al2 in 2 haemophilic
monozygotic twins, affects intron 1 of the F8 gene.
Bagnall et al1 demonstrated that this inversion of intron
1 derives from a homologous recombination between 2 nearly identical
1041-base pair (bp) sequences, int1h-1 and
int1h-2, in opposite orientation, positioned
respectively in intron 1 of the gene and in a more telomeric region,
140 kilobases (kb) downstream, between the C6.1A and
VBPI genes. This rearrangement results in the
production of 2 chimeric mRNAs; the first, possibly under the control
of the F8 gene promoter, contains the first exon of the F8
gene, followed by some exons of VBP1. The other chimeric mRNA, under control of the C6.1A gene promoter, contains almost all of
the coding region of the C6.1A gene followed by part of intron 1 and exon 2 to 26 of the F8 gene. We have examined 28 patients with severe to moderate hemophilia
A (21 with severe, 2 with moderately severe, and 5 with moderate hemophilia A phenotype). All of the patients were investigated for the
inversion of intron 223-4; 8 of them are characterized by
the presence of this common mutation. The other affected males that
were negative for the inversion of intron 22 were screened for the
inversion of intron 1; this mutation occurs repeatedly and seems to
have a prevalence of 5% in patients with severe disease in the United
Kingdom.1 We have performed an analysis directly on genomic DNA extracted
with a QIAamp DNA blood mini-kit (QIAGEN, Hilden, Germany) under polymerase chain reaction (PCR) conditions suggested
by Bagnall et al.1 We performed 2 amplifications. One
reaction contained 3 different primers: 9F and 9cR, specific for the
int1h-1 region, and int1h-2F, which is specific for
the region flanking int1h-2. The other reaction contained 2 primers, int1h-2F and int1h-2R, which are specific for the
amplification of the int1h-2 region, and a third primer, 9F,
which flanks the telomeric side of the int1h-1
region. These PCRs are able to differentiate wild type from the patients with inversion and the patients with inversion from carriers. The amplified segments were readily separated on a 1.5%
agarose gel. One patient out of 20 with severe disease tested
positive for intron 1 inversion (Figure 1).
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| Figure 1.
Amplification of int1h-1 and int1h-2 regions.
Regions were amplified using 9F, 9cR, and Int1h-2F primers (lanes
1,2), and int1-2F, int1h-2R, and 9F primers (lanes
3,4). Lanes 1 and 3 show a male with inversion; lanes 2 and 4 show a wild-type male. MVI indicates DNA molecular weight marker VI
(Boehringer Mannheim, Mannheim, Germany). The dimensions of the
amplified fragments are approximately 1500 bp and 1000 bp.
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These observations demonstrate, in accordance with Bagnall et al, that
this approach is a rapid and efficient method for the individuation of
intron 1 inversion and it should be routinely performed in patients
with severe hemophilia A.
Federica Riccardi, Annarita Tagliaferri, Cesare Manotti, Corrado Pattacini, and Tauro Maria Neri
Correspondence: Federica Riccardi, Laboratory of Molecular
Genetics and Diagnostic Biotechnology, Hospital of Parma, Via Gramsci,
14, 43100 Parma, Italy; e-mail:
federica_riccardi{at}libero.it
References
1.
Bagnall D, Waseem N, Green PM, Giannelli F.
Recurrent inversion breaking intron 1 of the factor VIII gene is a frequent cause of severe haemophilia A.
Blood.
2002;99:168-174[Abstract/Free Full Text].
2.
Brinke A, Tagliavacca L, Naylor J, Green P, Giangrande P, Giannelli F.
Two chimeric 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 Gen.
1996;5:1945-1951[Abstract/Free Full Text].
3.
Lakich D, Kazazian HH Jr, 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].
4.
Liu Q, Nozari G, Sommer SS.
Single-tube polymerase chain reaction for rapid diagnosis of the inversion hotspot of mutation in hemophilia A [letter].
Blood.
1998;92:1458-1459[Free Full Text].
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Recurrent inversion breaking intron 1 of the factor VIII gene is a frequent cause of severe hemophilia A
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