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BRIEF REPORT
From the Department of Human Genetics, University of
Würzburg, Biozentrum, Würzburg, Germany; the Department of
Experimental Haematology and Transfusion Medicine and the Department of
Clinical Biochemistry, University of Bonn, Bonn, Germany; and the Max
Planck Institut für Molekulare Genetik, Berlin, Germany.
The intron 22 inversion represents the most prevalent
factor VIII gene defect in severe hemophilia A, accounting
for about 40% of all mutations. It is hypothesized that the inversion
mutations occur almost exclusively in germ cells during meiotic cell
division by intrachromosomal recombination between 1 of 2 telomeric
copies of the Int22h region and its intragenic homologue. The
majority of inversion mutations originate in male germ cells, where the lack of bivalent formation may facilitate flipping of the telomeric end
of the single X chromosome. This is the first intron 22 inversion that presents as a somatic mosaicism in a female, affecting
only about 50% of lymphocyte and fibroblast cells of the proposita. Supposing a post-zygotic de novo mutation as the usual cause of somatic mosaicism, the finding would imply that the intron 22 inversion
mutation is not restricted to meiotic cell divisions but can also occur
during mitotic cell divisions, either in germ cell precursors or in
somatic cells.
(Blood. 2000;96:2905-2906) Hemophilia A is the most common severe
bleeding disorder in humans, affecting 1 in 5000 male births. The
disease is caused by a wide range of heterogeneous mutations in the
factor VIII gene and leads to a partial or total deficiency
of the factor VIII protein activity.1 In severe hemophilia
A the intron 22 inversion is the most prevalent mutation, accounting
for about 40%-50% of all mutations.2-4 It is widely
accepted that the inversion is caused by an intrachromosomal
recombination between a 9.6-kb sequence (Int22h region) within intron
22 of the factor VIII gene and 1 of 2 almost identical
copies located about 300 kb distal to the factor VIII gene
at the telomeric end of the X chromosome.5 Previous
studies have shown that the majority of the inversion mutations were of
paternal origin. The male:female ratio has been calculated to be
between 15:14 and 300:1.6 The strong bias
toward male origin of the inversion mutation can be explained by a
model suggesting that during male meiosis, the absence of a second X
chromosome facilitates flipping of the telomeric end, which is believed
to precede the intrachromosomal recombination. In contrast, during
female meiosis the pairing of the 2 homologous X chromosomes would
prevent this process. The suggested pathomechanism of the intron 22 inversion and its predominantly paternal origin has led to the general
belief that this mutation is almost exclusively of meiotic origin,
although there is no direct experimental evidence for this hypothesis. Herein, we report the first intron 22 inversion that presents in a
female as a somatic mosaicism, thus pointing to a mitotic origin of
this mutation during early embryogenesis.
Hemophilia A family
Southern blot analysis
The mutation in this family was identified by Southern blot
analysis in the severely affected hemophilia A patient as a typical distal intron 22 inversion (Figure 1,
L4). Subsequent testing of lymphocyte DNA from the mother (L2) revealed
much fainter signals for the bands that show the distal intron 22 inversion (20 kb and 17.5 kb) as compared to the pattern of a typical
heterozygote carrier of this mutation (L7). These results were
confirmed on several independent blots from different DNA samples.
Densitometric quantification of the signals in lymphocyte cells
revealed a reduced density of the inversion-specific bands to about
50% in contrast to the 100% observed in a typical female carrier
(Figure 2, L2 and L7). Consequently, only about 50% of the mother's
lymphocyte chromosomes are likely to carry the disease allele. Thus,
the distal intron 22 inversion presents as a somatic mosaicism in the
proposita. Numeric aberrations of the X chromosomes as a cause for the
uncommon Southern blot pattern were excluded by the presence of a
normal 46, XX karyotype in the proposita.
Because the proportion of mosaic cells may vary in different tissues, fibroblasts were taken from the proposita by skin biopsy and cultured until the cell growth was sufficient for DNA extraction. The autoradiogram was of lower quality, probably due to some residual RNA content. Nevertheless, faint bands deriving from the inversion clearly indicated the presence of a mosaicism in fibroblasts (Figure 1, L3). The higher density of the normal signals, compared to the mosaic lymphocytes (Figure 1, L2), suggested that fewer fibroblasts than lymphocytes carry the distal intron 22 inversion. Densitometric quantification confirmed this interpretation (Figure 2, L3). Because a considerable proportion of cells carry the mutation in lymphocytes and additionally in fibroblasts, it must be assumed that the somatic mosaicism developed within the very first days of embryogenesis. It is therefore likely that tissues deriving from other cell lineages may also carry the mutation. For germ cells the presence of the inversion mutation is demonstrated by the hemophilic son. For liver cells the normal factor VIII levels of greater than 100% may argue for the presence of a mosaicism, although different degrees of lyonization of the 2 X chromosomes should also be considered. Somatic mosaicisms usually are caused by de novo mutations in early embryogenesis. However, the finding in the proposita may also be the result of a human chimerism caused by the fusion of dizygotic twins, of which one is a carrier of the inversion and the other is not.7,8 Somatic mosaicism of the intron 22 inversion caused by a post-zygotic de novo mutation would imply that this mutation is not, as suggested before, exclusively restricted to meiotic cell divisions, but it may also occur during mitotic cell divisions either in germ cells or in somatic cells. Because the Southern blot technique is not very sensitive for the detection of mosaic mutations, with a presumptive detection limit of 10%-20% mutated cells, mosaicisms involving the inversion mutation may occur with unknown frequency. Therefore, genetic counseling of apparent de novo cases should take into account the possibility of somatic and germline mosaicism of the intron 22 inversion mutation.
Submitted February 7, 2000; accepted June 14, 2000.
Supported by a grant (J.O.) from the Stiftung Hämotherapie-Forschung, Königsberg, Germany, and grants Ol 21/18-1 and Ol 100/1-2 from the Deutsche Forschungsgemeinschaft, Bonn, Germany.
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: Johannes Oldenburg, Institute of Human Genetics, Biozentrum, Am Hubland, 97074 Würzburg, Germany; e-mail: j.oldenburg{at}biozentrum.uni-wuerzburg.de.
1.
Kemball-Cook G, Tuddenham EGD, Wacey AI.
The factor VIII structure and mutation resource sit: HAMSTeRS version 4.
Nucleic Acids Res.
1998;26:216-219 2. Naylor JA, Green PM, Rizza CR, Giannelli F. Factor VIII gene explains all cases of haemophilia A. Lancet. 1992;340:1066-1067[Medline] [Order article via Infotrieve].
3.
Antonarakis SE, Rossiter JP, Young M, et al.
Factor VIII gene inversions in severe hemophilia A: results of an international consortium study.
Blood.
1995;86:2206-2012 4. Becker J, Schwaab R, Möller-Taube A, et al. Characterization of the factor VIII defect in 147 patients with sporadic hemophilia A: family studies indicate a mutation type dependent sex ratio of mutation frequencies. Am J Hum Genet. 1996;58:657670. 5. Lakich D, Kazazian HH, Antonarakis SE, Gitchier J. Inversions disrupting the factor VIII gene are a common cause of severe haemophilia A. Nat Genet. 1993;5:236-241[Medline] [Order article via Infotrieve].
6.
Rossiter JP, Young M, Kimberland ML, et al.
Fac-tor VIII gene inversion causing severe hemophilia A originate almost exclusively in male germ cells.
Hum Mol Genet.
1994;3:1035-1039 7. Benirschke K. The biology of the twinning process: how placentation influences outcome. Semin Perinatol. 1995;19:342-350[Medline] [Order article via Infotrieve]. 8. Sanchez JM, Goldschmidt E. Fetal chimerism or fetal mosaicism? Prenat Diagn. 1990;10:548-549[Medline] [Order article via Infotrieve].
© 2000 by The American Society of Hematology.
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  L. C. Rossetti, C. P. Radic, I. B. Larripa, and C. D. De Brasi Genotyping the Hemophilia Inversion Hotspot by Use of Inverse PCR Clin. Chem., July 1, 2005; 51(7): 1154 - 1158. [Abstract] [Full Text] [PDF]
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Abstract
Introduction
