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Prepublished online as a Blood First Edition Paper on June 21, 2002; DOI 10.1182/blood-2002-01-0277. This Article Abstract Full Text (PDF) All Versions of this Article: 2002-01-0277v1 100/8/3034    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Valleix, S. Articles by Negrier, C. Search for Related Content PubMed PubMed Citation Articles by Valleix, S. Articles by Negrier, C. Related Collections Hemostasis, Thrombosis, and Vascular Biology Brief Reports Clinical Trials and Observations Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  Blood, 15 October 2002, Vol. 100, No. 8, pp. 3034-3036 BRIEF REPORT Skewed X-chromosome inactivation in monochorionic diamniotic twin sisters results in severe and mild hemophilia A Sophie Valleix, Christine Vinciguerra, Jean-Maurice Lavergne, Marco Leuer, Marc Delpech, and Claude Negrier From the Faculte Cochin-Port Royal, Laboratoire de Biochimie et Genetique Moleculaire, Paris, France; the Hopital Edouard Herriot, Centre de Traitement de l'Hemophilie, Laboratoire d'Hemostase, Inserm U331, Lyon, France; the Hopital de Bicetre, Laboratoire d'Hemostase et Thrombose, Inserm U143, Le Kremlin Bicetre, France; the University of Bonn, Department of Clinical Biochemistry, Germany; and Biopsytec Analytik GmbH, Rheinbach, Germany.     Abstract Top Abstract Introduction Study design Results and discussion References This study describes the genetic mechanisms responsible for the de novo occurrence of severe and mild hemophilia A in monozygotic twin females. Both twins were found to carry a previously known factor VIII mutation (Tyr16Cys) in the heterozygous state which most probably arose in the paternal germ line. Both twins showed concordant skewing of X inactivation toward the maternally derived normal X chromosome, the most severely affected twin exhibiting a higher percentage of inactivation of the normal X chromosome. The degree of skewing of X inactivation closely correlated with both the coagulation parameters and the clinical phenotype of the twins. Since these twins were monochorionic, such results suggest that the twinning event in this case has occurred after the onset of the X-inactivation period. (Blood. 2002;100:3034-3036) © 2002 by The American Society of Hematology.     Introduction Top Abstract Introduction Study design Results and discussion References Hemophilia A is an X-linked recessive bleeding disorder caused by deficiency of factor VIII (FVIII) which occurs in 1/5000 male births.1 Clinical severity is inversely related to residual factor VIII activity (FVIII:C) such that patients with less than 1%, 1%-5%, and 5%-30% FVIII:C are classified as severe, moderate, or mild, respectively. The molecular basis underlying hemophilia A is well characterized, and about half of severely affected individuals have large genomic inversions disrupting the FVIII gene (F8).2-4 In the remaining cases, different heterogeneous point mutations, insertions, and deletions are found scattered throughout the 26 exons, as well as in the introns of F8.5 Hemophilia A is transmitted by heterozygous females denoted as carriers, who are generally asymptomatic since random X inactivation results in approximately equal proportions of somatic cells in which either the normal X or the mutated X chromosome is active.6 Even though the disease in females is extremely rare, a few cases have been documented, resulting from different pathophysiologic mechanisms.7-13 Here, we report monozygotic (MZ) twin girls both carrying the same F8 mutation which results in severe hemophilia A in one twin and mild phenotype in her co-twin. Although they exhibited a different clinical and biologic severity, they both showed concordant skewing of X inactivation toward the normal X chromosome. In this respect, they differed from some pairs of MZ twin females who typically showed a "mirror-image" pattern of X inactivation. As the twins were classified as monochorionic (MC) at birth, our data strongly support the hypothesis that the splitting event in MC-MZ twin pairs occurs after the onset of X-inactivation period.     Study design Top Abstract Introduction Study design Results and discussion References Case report The family is presented in Figure 1. Coagulation studies showed FVIII:C of less than 1% for proposita II.2 and 12% for II.3 on repeated measurements, with normal VWF:Ag and VWF:RCo levels in both cases. None of the other family members (I.1, I.2, and II.1) exhibited hemophilia or had abnormal values of plasma FVIII:C and von Willebrand factor (VWF), suggesting an absence of familial inheritance for the disease. Binding of FVIII to VWF was found normal in both twins eliminating a type 2N von Willebrand disease (VWD).14 Patient II.2 suffered from repeated bleeding episodes, that is, hematomas and hemarthroses consistent with a severe hemophilia phenotype for which she was treated with FVIII infusions. The other twin only exhibited a minor hemophilia phenotype concordant with the plasma FVIII:C evaluation. At birth, these twins were documented to be diamniotic and monochorionic after examination of the fetal-placenta anatomy type. At 10 years of age, both were bright students. The study was initiated after an informed consent was obtained according to the Declaration of Helsinki and to the national regulation on genetic analysis. View larger version (19K): [in this window] [in a new window]   Figure 1. Identification of the Tyr16Cys mutation in the twins. The family pedigree is presented on the left side with the corresponding plasma FVIII activity for each member. Sequence chromatogram from twin II.1 is shown on the right side with the relevant wild-type and mutant partial sequences of the F8 gene noted under the chromatogram. The arrow points to the double peaks of A and G at the second nucleotide position of codon 16, demonstrating a nucleotide transition in the heterozygous state. FVIII binding assay to VWF FVIII binding to VWF assay was performed as previously described.14 Molecular F8 gene studies Genomic DNA was extracted from lymphocytes by standard procedures. X-chromosome inactivation at the HUMARA locus Assessment of X inactivation was performed using an HpaII polymerase chain reaction (PCR) assay for the X-linked human androgen receptor gene (HUMARA).15 Genomic DNA samples, one predigested with the methylation-sensitive enzyme HpaII (Boehringer Mannheim, Meylan, France) and the other without predigestion, were subjected to PCR amplification with specific fluorescent primers flanking both HpaII sites plus a highly polymorphic (CAG) repeat sequence in the first exon of the HUMARA. Only the methylated allele from the inactive X chromosome which resists HpaII cleavage was amplified. The PCR products were subjected to automated DNA sequencer analysis (model ABI 377, Applied Biosystems, Courtaboeuf, France) and the results analyzed by GeneScan software (Applied Biosystems). This assay was modified to facilitate quantitation of X inactivation at each allele, as described by Pegoraro et al.16 X-chromosome inactivation at the FMR-1 locus DNA samples were digested with EcoRI plus the methylation-sensitive enzyme, EagI, and were subjected to Southern blot analysis, as described.17 This assay, which can detect fragments containing the (CGG)n and the CpG island in the 5' untranslated region (UTR) of the FMR-1 gene, revealed the methylation status of this CpG island and also indicated possible abnormal expansion of the (CGG)n repeat.     Results and discussion Top Abstract Introduction Study design Results and discussion References In order to elucidate the genetic mechanisms responsible for hemophilia A in these MZ twin females, molecular investigations were carried out to characterize the F8 molecular defect. First, DNAs from both twins were subjected to BclI Southern blot analysis for F8 intron 22 inversions, and no altered migration pattern was observed (data not shown). All coding sequences and exon-intron boundaries of F8 were then screened by direct sequencing. Exon 1 from both twins was found to contain a single base-pair transition from A to G in codon 16, predicting the replacement of tyrosine (Tyr) by cysteine (Cys) in the factor VIII protein sequence (Figure 1). This alteration was previously reported to be associated with severe hemophilia A (http://europium.csc.mrc.ac.uk). No further F8 sequence alteration was found in the twins. The missense mutation was not detected in the twins' healthy sister or in the parents, suggesting that it had occurred de novo in the germ line of one parent. Both twins carried the mutation in the heterozygous state, indicating that the clinical expression of hemophilia A was not consecutive to homozygosity for the codon 16 mutation. Since the unbalanced X-chromosome inactivation process is currently recognized to be responsible for expression of X-linked recessive disorders in heterozygous females, we examined DNA methylation at the X-linked HUMARA locus. All 4 females (I.2, II.1, II.2, and II.3) were found to be informative as shown in Figure 2. The healthy sister (II.1) showed 2 amplification peaks corresponding to approximately an equal methylation of the maternal and paternal X alleles, thus reflecting a random X inactivation. Conversely, only one peak was observed for each twin, which corresponded to inactivation, or methylation, of the maternally derived allele, indicating a skewed X-chromosome inactivation (Figure 2). A quantitative evaluation of the degree of intrapair differences indicated that patient II.2 showed an "extremely skewed" profile (ratio 100:0), while the profile for patient II.3 was "skewed" (85:15), suggesting that a small proportion of her mother's F8 allele remained active. The difference in severity of the disease between the twins correlated with the degree of skewing of X inactivation. Although X-inactivation analysis could not be tested from liver where factor VIII is primarily synthesized, we hypothesized that both twins probably had similar X-inactivation patterns in the liver as suggested by plasma factor VIII values. Since both twins were clinically affected and both showed an inactivation of the maternal X chromosome, we concluded that Tyr16Cys mutation occurred on the paternal X chromosome. We also studied the methylation of the 5' region of the FMR-1 gene, and the results corroborated those found for the HUMARA locus (Figure 3). This assay, routinely used for diagnosis of the fragile X syndrome, revealed unexpectedly that both twins had inherited from their mother a premutated allele of 80-90 (CGG) repeats. Although this premutation is known to have a high risk of expanding further to a "full" mutation when it is transmitted by a female, the twins carried approximately the same number of copies as the mother. As seen in Figure 3, a band of 2.8 kb was found for the father (I.1), corresponding to an active unmethylated X chromosome (normal male pattern). The healthy sister showed a normal female pattern with random X inactivation since both bands of 2.8 kb and 5.2 kb were present in equal quantities, reflecting the unmethylated and the methylated alleles, respectively. The mother, conversely, showed 2 doubled bands at approximately 2.8 kb and 5.2 kb present in equal quantities. These bands corresponded to the normal and premutated FMR-1 alleles, both being present in the methylated and unmethylated states, typical of a carrier with random X inactivation. Both twins showed 4 bands, as did the mother, but the pattern was further complicated by the fact that X inactivation was skewed, leading to 2 weaker bands representing the methylated paternally derived X chromosome and the unmethylated maternally derived X chromosome carrying the triplet expansion. View larger version (22K): [in this window] [in a new window]   Figure 2. X-chromosome inactivation analysis. Results obtained at the HUMARA locus. The blue peaks and the black peaks correspond to PCR products before and after HpaII digestion, respectively. The length of the PCR fragments are noted on the top of each histogram, and the father had a 203-bp fragment in his only X allele, while the mother had both a 200-bp and a 206-bp fragment in her 2 alleles. The mother's 206-bp fragment was transmitted to the healthy daughter (II.1), while the mother's other X-chromosome containing the 200-bp fragment went to the twins (II.2, II.3). The 2 black peaks in approximately equal proportion are seen in X-chromosomes randomly inactivated from females I.2 and II.1, whereas only one black peak corresponding to the mother's 200-bp inactivated allele is observed in the twins (II.2 and II.3), indicating a skewed X-chromosome inactivation toward the maternally 200-bp-derived allele. View larger version (50K): [in this window] [in a new window]   Figure 3. Results of the southern blot assay at the FMR-1 locus. Lane numbers correspond to individuals in the pedigree and fragment sizes are indicated in kilobases at the side of the figure. The father (I.1) exhibits a normal pattern with a unique 2.8-kb band corresponding to his unmethylated active X-chromosome, whereas in normal females both bands of 2.8 kb and 5.2 kb are present and correspond to their active and inactive alleles (individual II.1). The mother (I.2) exhibits a pattern of 4 bands, suggestive of a premutated allele in the methylated and unmethylated states (bands of ~5.5 kb and 3 kb, respectively). Subjects I.2 and II.1 exhibit a random X-chromosome inactivation as indicated by an equal intensity of each fragment. The twins (II.2 and II.3) show a pattern of 4 bands like their mother but 2 bands are weaker (white arrowheads), indicating a skewed X-chromosome inactivation. MZ twins may be monochorionic (MC-MZ) or dichorionic (DC-MZ), depending on whether they develop in a single or 2 distinct chorionic sacs. DC-MZ twinning occurs prior to or around the onset of X inactivation and MC-MZ twinning occurs later.18 It might therefore be expected that a discordant X-inactivation pattern would more frequently be seen in DC-MZ twins than in MC-MZ twins. However, anecdotal examples of discordant female MC-MZ twins have been reported where the affected twin exhibited a selective inactivation of the normal X chromosome while her normal co-twin showed either random X inactivation or skewing toward the mutant X chromosome.19-22 Several theoretical explanations for such "mirror-image" inactivation have been proposed. It was initially suggested that random X inactivation followed by asymetric splitting of the inner cell mass would result in only one affected twin because she received a majority of cells in which the normal allele had been inactivated.20 More recently, Monteiro et al23 proposed that in fact MC-MZ twin pairs reflected a heterogeneous group slightly differing in the timing of the twinning event after the onset of X inactivation. MC-MZ pairs indeed split more or less closely after the onset of X inactivation, explaining the X inactivation differences observed between the twins.23 They demonstrated that for each round of replication after the onset of X inactivation, the probability of creating a difference in X inactivation decreased if a twinning event occurred. The genetic profile of MC-MZ twins described here is in accordance with this model. The preferential inactivation of the same X chromosome in both twins likely indicates that the twinning event occurred at a relatively later time than for MC-MZ twins who show discordant X inactivation. In conclusion, reports of such twins with a correct determination of the fetal-placenta anatomy type provide invaluable information to further understanding of X inactivation and twinning processes in humans.     Acknowledgments We would like to thank Dr Claudine Mazurier for the FVIII binding assay to VWF. We also wish to thank professors Cherif Beldjord and Laurent Richard for their technical help with the DNA methylation analysis at the FMR1 gene, and professor Noël Philippe who referred the twins and the family. We would like to express our gratitude to the members of this family for their participation in this study.     Footnotes Submitted January 31, 2002; accepted April 15, 2002. Prepublished online as Blood First Edition Paper, June 21, 2002; DOI 10.1182/blood-2002-01-0277. 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: Claude Negrier, Centre de traitement de l'hémophilie, Laboratoire d'Hémostase, Hôpital Edouard Herriot, Pavillon E, Place d'Arsonval, 69374 Lyon Cédex 08, France; e-mail: claude.negrier{at}chu-lyon.fr.     References Top Abstract Introduction Study design Results and discussion References 1. Gitschier J, Wood WI, Goralka JM, et al. Characterization of the human factor VIII. Nature. 1984;312:326-330[CrossRef][Medline] [Order article via Infotrieve]. 2. Lakich D, Kazazian HH, Antonarakis SE, et al. Inversions disrupting the factor VIII gene are a common cause of severe hemophilia A. Nature Genet. 1993;5:236-241[CrossRef][Medline] [Order article via Infotrieve]. 3. Naylor J, Brinke A, Hassock S, et al. 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[Abstract/Free Full Text]. 4. 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[Abstract/Free Full Text]. 5. Kemball-Cook G, Tuddenham EGD, Wacey AI. The factor VIII structure and mutation resource site: HAMSTeRS version 4. Nucleic Acids Res. 1998;26:216-219[Abstract/Free Full Text]. 6. Puck JM, Willard HF. X inactivation in females with X-linked disease. N Engl J Med. 1998;338:325-328[Free Full Text]. 7. 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[Medline] [Order article via Infotrieve]. 8. Mori PG, Pasino M, Vadala CR, et al. Haemophilia A in a 46, X, i(Xq) female. Br J Haematol. 1979;43:143-147[Medline] [Order article via Infotrieve]. 9. Coleman R, Genet SA, Harper JI, et al. Interaction of incontinentia pigmenti and factor VIII mutations in a female with biased X inactivation, resulting in hemophilia. J Med Genet. 1993;30:497-500[Abstract/Free Full Text]. 10. 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[CrossRef][Medline] [Order article via Infotrieve]. 11. Windsor S, Lyng A, Taylor SAM. 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]. 12. Seeler RA, Vnencak-Jones CL, Bassett LM, et al. Severe haemophilia A in a female: a compound heterozygote with nonrandom X-inactivation. Haemophilia. 1999;5:445-449[CrossRef][Medline] [Order article via Infotrieve]. 13. Favier R, Lavergne JM, Costa JM, et al. Unbalanced X-chromosome inactivation with a novel FVIII gene mutation resulting in severe hemophilia A in a female. Blood. 2000;96:4373-4375[Abstract/Free Full Text]. 14. Mazurier C, Gaucher C, Jorieux S, et al. 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]. 15. Allen RC, Zoghi HY, Moseley AB, et al. 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. 1992;51:1229-1239[Medline] [Order article via Infotrieve]. 16. 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]. 17. Rousseau F, Heitz D, Biancalana V, et al. Direct diagnosis by DNA anlysis of the fragile X syndrome of mental retardation. N Engl J Med. 1991;325:1673-1681[Abstract]. 18. Puck JM. The time of twinning: more insights from X inactivation. Am J Hum Genet. 1998;63:327-328[CrossRef][Medline] [Order article via Infotrieve]. 19. Pena SDJ, Karpati G, Carpenter S, et al. The clinical consequences of X chromosome inactivation: Duchenne muscular dystrophy in one of monozygotic twins. J Neuro Sci. 1987;79:337-344. 20. Lupski JR, Garcia CA, Zogbi HY, et al. Discordance of muscular dystrophy in monozygotic female twins: evidence supporting asymetric splitting of the inner cell mass in a manifesting carrier of Duchenne dystrophy. Am J Med Genet. 1993;40:354-364[CrossRef]. 21. Jørgensen AL, Philip J, Raskind WH, et al. Different patterns of X inactivation in MZ twins discordant for red-green color vision deficiency. Am J Hum Genet. 1992;51:291-298[Medline] [Order article via Infotrieve]. 22. Winchester B, Young E, Geddes S, et al. Female twin with Hunter disease due to nonrandom inactivation of the X-chromosome: a consequence of twinning. Am J Med Genet. 1992;44:834-838[CrossRef][Medline] [Order article via Infotrieve]. 23. Monteiro J, Derom C, Vlietinck R, et al. Commitment X inactivation precedes the twinning event in monochorionic MZ twins. Am J Hum Genet. 1998;63:339-346[CrossRef][Medline] [Order article via Infotrieve]. © 2002 by The American Society of Hematology.   CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this? This article has been cited by other articles: T. E. Hickey, R. S. Legro, and R. J. Norman Epigenetic Modification of the X Chromosome Influences Susceptibility to Polycystic Ovary Syndrome J. Clin. Endocrinol. Metab., July 1, 2006; 91(7): 2789 - 2791. [Abstract] [Full Text] [PDF] 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] This Article Abstract Full Text (PDF) All Versions of this Article: 2002-01-0277v1 100/8/3034    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Valleix, S. Articles by Negrier, C. Search for Related Content PubMed PubMed Citation Articles by Valleix, S. Articles by Negrier, C. Related Collections Hemostasis, Thrombosis, and Vascular Biology Brief Reports Clinical Trials and Observations Social Bookmarking                   What's this?

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