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BRIEF REPORT
From the Service d'Hématologie Biologique,
Hôpital d'Enfants Armand Trousseau, Paris, France; Laboratoire
d'Hémostase et Thrombose. Unité INSERM U 143, Hôpital de Bicêtre, Le Kremlin Bicêtre, France;
Laboratoire de Biologie Moléculaire, Hôpital
Américain de Paris, Neuilly, France; Laboratoire
d'Hématologie, Hôpital Huriez, Lille, France; and
Laboratoire de Biochimie et Génétique Moléculaire,
Paris, France.
This report is of a 14-month-old girl affected with severe
hemophilia A. Both her parents had normal values for factor VIII activity, and von Willebrand disease type 2N was excluded. Karyotype analysis demonstrated no obvious alteration, and BclI
Southern blot did not reveal F8 gene inversions. Direct
sequencing of F8 gene exons revealed a frameshift-stop
mutation (Q565delC/ter566) in the heterozygous state in the proposita
only. F8 gene polymorphism analysis indicated that the
mutation must have occurred de novo in the paternal germline.
Furthermore, analysis of the pattern of X chromosome methylation at the
human androgen receptor gene locus demonstrated a skewed inactivation
of the derived maternal X chromosome from the lymphocytes of the
proband's DNA. Thus, the severe hemophilia A in the proposita results
from a de novo F8 gene frameshift-stop mutation on the
paternally derived X chromosome, associated with a nonrandom pattern of
inactivation of the maternally derived X chromosome.
(Blood. 2000;96:4373-4375) Hemophilia A is an X-linked bleeding disorder
caused by the deficiency of factor VIII (FVIII), a cofactor for the
activation of factor X by factor IXa. The incidence of the disease is
approximately 1 in 5000 males, and hemophilia A is classified as mild,
moderate, or severe, depending on the amount of residual factor VIII.
The gene encoding FVIII (F8) is located in band Xq28 and
spans 186 kilobase (kb).1 The molecular basis underlying
hemophilia A is now well characterized, and about half of severely
affected hemophiliacs have large genomic inversions resulting from a
hot-spot of recombination between a 9.5-kb region in intron 22 (int22h-1) and one of its 2 homologous extragenic copies
(int22h-2 and int22h-3).2,3 In the
remaining cases, hemophilia A is caused by a large number of different
point mutations that could be scattered throughout the 26 exons of the
F8 gene.4 Hemophilia A is transmitted by females who are denoted as carriers. These female carriers could have
normal or intermediate factor VIII activity (FVIIIC) levels depending
on the variable mosaicism of their somatic cells in which either the
normal X or the mutated X chromosome is active. Usually these females
are asymptomatic because X chromosome inactivation is random with an
approximately equal proportion of the 2 populations of somatic cells.
However, in rare instances, a nonrandom X chromosome inactivation could
result in a symptomatic female in whom the normal X chromosome is
predominantly inactive.
In the current report, we investigated a 14-month-old female proposita
with severe hemophilia A, and molecular data demonstrated that the
severe hemophilia A in this female patient resulted from the occurrence
of a de novo frameshift-stop mutation in the F8 gene on the
paternally derived X chromosome, associated with a nonrandom pattern of
inactivation of the maternally derived X chromosome.
Case report
FVIII binding assay to vWF
Cytogenetic studies Karyotyping of blood lymphocytes from the proposita and her parents were performed, using standard and high-resolution procedures.Molecular F8 gene studies Genomic DNA was extracted from peripheral blood lymphocytes by a standard procedure and after informed consent of all the family members. BclI Southern blot was performed as previously reported, using the 0.9-kb fragment from plasmid p482.6 as a probe.2,3For denaturing gradient gel electrophoresis, the 26 exons and flanking intronic regions were amplified by polymerase chain reaction (PCR) with GC-clamped sense and anti-sense primers as previously reported.6 GC-clamped PCR products were loaded onto a 6% polyacrylamide gel containing a linear gradient of urea and formamide and were electrophoresed according to the conditions previously described.6 Analysis of the 2 microsatellite repeats in introns 13 and 22, the intron 18 BclI restriction fragment length polymorphism (RFLP), the XbaI RFLP in intron 22, and the TaqI variable tandem repeat polymorphism at the DXS52 locus were performed as previously described.7-10 X chromosome inactivation Analysis of X chromosome inactivation was performed as described elsewhere.11 Genomic DNA samples were digested with the methylation-sensitive enzyme HpaII (Boehringer Mannheim, Meylan, France) and were subjected to PCR amplification of the highly polymorphic CAG-repeat sequence in the first exon of the human androgen receptor gene (HUMARA), with specific fluorescent primers. The PCR products, both before and after HpaII digestion, were electrophoresed on an automated DNA sequencer (model ABI 377; Applied Biosystems, Warrington, United Kingdom) and were analyzed by GeneScan software (Applied Biosystems).Analysis of the X inactive specific transcript (XIST) promoter sequence DNA from the proposita and her mother were amplified, using the appropriate forward and reverse primers previously used.12,13 Each PCR product was purified and sequenced on both strands, using the Big Dye Terminator sequencing kit (Applied Biosystems) and was resolved on the automatic DNA sequencer. Nucleotide sequences were compared with the nucleotide sequence reported by Hendrich et al.12
Hemophilia A affects males, whereas females are generally spared.
However, there are a variety of potential genetic mechanisms that could
lead to phenotypic expression of very low FVIIIC levels in
females.14 These mechanisms involve, in some cases, the
gene of vWF, resulting either in a quantitative defect or in a
functional/structural defect of vWF that is essential for FVIII
transport and stability.15 However, in the majority of
cases, it is the gene coding for the factor VIII that is directly
altered. The genetic mechanisms characterized so far include (1)
abnormalities of the X chromosome in structure or in
number16,17; (2) homozygosity for a F8
mutation, mostly when consanguinity is present18,19; (3)
concomitant occurrence of 2 de novo F8
mutations20; and (4) most frequently, a selective inactivation of the normal X chromosome in a heterozygous
female.21 The female patient that we studied showed normal
levels of vWF and normal fixation of factor VIII to vWF, therefore
eliminating 2N vWD.15 High-resolution karyotype analysis
demonstrated a 46, XX karyotype without obvious structural
abnormalities. Polymorphism analysis in the family with several markers
close to and within the F8 gene confirmed that the female
proposita inherited 2 distinct X chromosomes from each of her parents
(Figure 1A). Subsequently the first and
the second mechanisms cited above were excluded as the underlying cause
of severe hemophilia A in this child. Therefore, the X chromosome
inactivation patterns in the peripheral blood of the patient and her
parents were analyzed at the HUMARA gene and were quantitated using a
fluorescent PCR assay previously described.11 The HUMARA
gene contains in its first exon a highly polymorphic CAG repeat that
allows the 2 X chromosomes of females informative at the locus to be
distinguished. Close to this CAG repeat are located 2 HpaII
sites that resist cleavage by methylation-sensitive restriction enzyme
HpaII on the methylated-inactive X chromosome, whereas these
sites are cleaved by HpaII on the unmethylated active X
chromosome.22 Therefore, PCR products after
HpaII digestion could be obtained only from the inactive X
chromosome. In a normal female in whom X inactivation is random, an
approximately equal portion of both the maternally and paternally
derived inactive X chromosomes remain undigested and both HUMARA CAG
repeat alleles will be amplified. Alternatively, if X chromosome
inactivation is nonrandom, only one allele corresponding to the
inactive X chromosome is expected to be amplified.
In this family, both the proband and her mother were informative at the HUMARA locus, as shown in Figure 1B, after PCR amplification of the HUMARA CAG repeat before HpaII digestion. As shown in Figure 1B, 2 distinct alleles were observed in the female proposita (II.1), one allele (186) inherited from her mother and the allele 198 derived from her father. Following HpaII digestion, we observed that only one allele (186) was amplified in the patient II.1, indicating a skewed X inactivation. This allele represents the inactivated X chromosome and corresponds to the maternally derived allele. The patient showed a degree of skewing consistent with the designation of "extremely skewed" with a ratio greater than 95:5.11 X-inactivation analysis of this family demonstrated that the female patient carried only one active paternally derived X chromosome. In conclusion, analysis of methylation at the HUMARA locus from peripheral blood cells suggests that skewed X inactivation is the mechanism underlying hemophilia A in this girl, although the liver, where factor VIII is primarily synthesized, could not be tested. Because the proband's father was not hemophiliac, it suggests that the mutation in the F8 gene occurred de novo on the paternal active X chromosome of the female patient. A high-resolution karyotype from the proband and her parents did not reveal any chromosomal abnormalities that might explain the pattern of skewed X chromosome inactivation. To identify the F8 gene mutation, BclI Southern blot analysis for the F8 intron 22 inversion, which is commonly found in severe hemophiliacs, was performed in the patient and her parents.2,3 The results showed that the proband and both her parents exhibited a normal BclI pattern, demonstrating the absence of F8 gene inversions (data not shown). Therefore, we screened all the coding sequences and exon-intron boundaries of the F8 gene by denaturing gradient gel electrophoresis and direct sequencing.6 We characterized by direct sequencing of exon 11 a frameshift-stop mutation in the heterozygous state from the proposita (Figure 1C). This mutation corresponds to a frameshift 1-base pair (bp) deletion that led to a premature termination codon at position 566 (Q565delC/ter566) that is expected to generate an unstable transcript that is degraded. Therefore, this molecular pathology is consistent with a severe hemophilia A phenotype observed in the female proposita. This mutation was detected neither in the proband's father nor in the proband's maternal leukocytes' DNA, suggesting that this mutation probably occurred de novo in the father's germline (Figure 1D). Three mechanisms are currently recognized for an unbalanced pattern of X chromosome inactivation in chromosomally normal females: a stochastic fluctuation in the embryonic progenitor cells, a postinactivation selection of cells bearing a mutation that affects cell survival in a particular tissue, and finally a defect in the X-inactivation process itself affecting the correct expression of the XIST gene or the spreading of the inactivation signal.14 For instance, 2 unrelated families have been documented in which females carried a mutation in the XIST promoter that segregated with a skewed X chromosome inactivation.23 Subsequently, we sequenced the minimal XIST promoter region in the female proposita, but no change in the sequence of this region was observed (data not shown). In conclusion, the severe hemophilia A in this female results from the occurrence of a de novo frameshift-stop mutation in the F8 gene on the paternally derived X chromosome, associated with a nonrandom pattern of inactivation of the maternally derived X chromosome. This study further illustrates the value of X chromosome inactivation analysis to explain the phenotypic expression of an X-linked disorder in heterozygous female carriers, such as hemophilia A. Furthermore the report of such clinical cases, which are unusual, are of utmost importance for the understanding of the X chromosome inactivation process in humans.
Submitted July 13, 2000; accepted August 29, 2000.
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: Marc Delpech, Laboratoire de Biochimie et Génétique Moléculaire, 123 boulevard du Port-Royal, 75014 Paris, France; e-mail: delpech{at}icgm.cochin.inserm.fr.
1. Gitschier J, Wood WI, Goralka JM, et al. Characterization of the human factor VIII. Nature. 1984;312:326-330[Medline] [Order article via Infotrieve]. 2. Lakich D, Kazazian HH, Antonarakis SE, Gitschier J. Inversions disrupting the factor VIII gene are a common cause of severe hemophilia A. Nat Genet. 1993;5:236-239[Medline] [Order article via Infotrieve].
3.
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
4.
Antonarakis SE, Rossiter JP, Young M, et al.
A consortium of 65 international consortium study.
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1995;86:2206-2212 5. Mazurier C, Gaucher C, Jorieux S, Parquet-Gernez A, Goudemand M. Evidence for a von Willebrand factor defect in factor VIII binding in three members of a family previously misdiagnosed mild haemophilia A and haemophilia A carriers: consequences for therapy and genetic counselling. Br J Haematol. 1990;76:372-379[Medline] [Order article via Infotrieve].
6.
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.
1991;88:8307-8311 7. Oberlé I, Camerino G, Heilig R, et al. Genetic screening for hemophilia A (classic hemophilia) with a polymorphic DNA probe. N Engl J Med. 1985;312:682-686[Abstract]. 8. Windsor S, Taylor SAM, Lillicrap D. Multiplex analysis of two intragenic microsatellite repeat polymorphisms in the genetic diagnosis of hemophilia A. Br J Haematol. 1994;86:810-815[Medline] [Order article via Infotrieve]. 9. Antonarakis SE, Waber PG, Kitter SD. Hemophilia A. Detection of molecular defects and of carriers by DNA analysis. N Engl J Med. 1985;313:842-848[Abstract].
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A new polymorphism in the factor VIII gene for prenatal diagnosis of hemophilia A.
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1986;14:4535-4542 11. Pegoraro E, Schimke RN, Arahata K, et al. Detection of new paternal dystrophin gene mutations in isolated cases of dystrophinopathy in females. Am J Hum Genet. 1994;54:989-1003[Medline] [Order article via Infotrieve].
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Hendrich BD, Plenge RM, Willard HF.
Identification and characterization of the human XIST gene promoter: implications for models of X chromosome inactivation.
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1997;25:2661-2671 13. Plenge RM, Hendrich BD, Schwartz C, et al. A promoter mutation in the XIST gene in two unrelated families with skewed X-chromosome inactivation. Nat Genet. 1997;17:353-356[Medline] [Order article via Infotrieve].
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Puck JM, Willard HF.
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1998;338:325-328 15. Mazurier C. Von Willebrand disease masquerading as haemophilia A. Thromb Haemost. 1992;67:391-396[Medline] [Order article via Infotrieve]. 16. Migeon BR, McGinniss MJ, Antonarakis SE, et al. Severe hemophilia A in a female by cryptic translocation: order and orientation of factor VIII within Xq28. Genomics. 1993;16:20-25[Medline] [Order article via Infotrieve]. 17. Gilchrist GS, Hammond D, Melnyk J. Hemophilia A in a phenotypically normal female with XX/XO mosaicism. N Engl J Med. 1965;273:1402-1406. 18. Morita H, Kagami M, Ebata Y, Yoshimura H. The occurrence of homozygous hemophilia in the female. Acta Haemat. 1971;45:112-119[Medline] [Order article via Infotrieve]. 19. Pietu G, Thomas-Maison N, Sie P, Larrieu M, Meyer D. Hemophilia A in a female: study of a family using intragenic and extragenic restriction site polymorphisms. Thromb Haemost. 1988;60:102-106[Medline] [Order article via Infotrieve]. 20. Windsor S, Lyng A, Taylor SAM, Ewenstein BM, Neufeld E, Lillicrap D. Severe hemophilia A in a female resulting from two de novo factor VIII mutations. Br J Haematol. 1995;90:906-909[Medline] [Order article via Infotrieve]. 21. Mannucci PM, Coppola R, Lombardi R, Papa M, De Baisi R. Direct proof of extreme lyonisation as a cause of low factor VIII levels in female. Thromb Haemost. 1978;39:544-545[Medline] [Order article via Infotrieve]. 22. Allen RC, Zoghi HY, Moseley AB, Rosenblatt HM, Belmont JW. Methylation of HpaII and HhaI sites near the polymorphic CAG repeat in the human androgen-receptor gene correlates with X chromosome inactivation. Am J Hum Genet. 1991;51:1229-1239. 23. Naumora AK, Plenge RM, Bird LM, et al. Heritability of X chromosome-inactivation phenotype in a large family. Am J Hum Genet. 1996;58:1111-1119[Medline] [Order article via Infotrieve].
© 2000 by The American Society of Hematology.
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  P. Dhar, S. Abramovitz, D. DiMichele, C. B. Gibb, and F. Gadalla Management of pregnancy in a patient with severe haemophilia A Br. J. Anaesth., September 1, 2003; 91(3): 432 - 435. [Abstract] [Full Text] [PDF]
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 S. Valleix, C. Vinciguerra, J.-M. Lavergne, M. Leuer, M. Delpech, and C. Negrier Skewed X-chromosome inactivation in monochorionic diamniotic twin sisters results in severe and mild hemophilia A Blood, September 26, 2002; 100(8): 3034 - 3036. [Abstract] [Full Text] [PDF]
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Abstract
Introduction