1. Art and Diseño

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Factor VI11 Gene Inversions in Severe Hemophilia A: Results of an
International Consortium Study
By S.E. Antonarakis, J.P. Rossiter, M. Young, J. Horst, P. de Moerloose, S.S. Sommer, R.P. Ketterling,
H.H. Kazazian Jr, C. Negrier, C. Vinciguerra, J. Gitschier, M. Goossens, E. Girodon, N. Ghanem, F. Plassa,
J.M. Lavergne, M. Vidaud, J.M. Costa, Y . Laurian, S.-W. Lin, S.-R. Lin, M.-C. Shen, D. Lillicrap, S.A.M. Taylor,
S. Windsor, S.V. Valleix, K. Nafa, Y. Sultan, M . Delpech, C.L. Vnencak-Jones, J.A. Phillips 111, R.C.R. Ljung,
E. Koumbarelis, A. Gialeraki, T. Mandalaki, P.V. Jenkins, P.W. Collins, K.J. Pasi, A. Goodeve, I. Peake, F.E. Preston,
M . Schwartz, E. Scheibel, J. Ingerslev, D.N. Cooper, D.S.Millar, V.V. Kakkar, F. Giannelli, J.A. Naylor, E.F. Tizzano,
M. Baiget, M. Domenech, C. Altisent, J. Tusell, M. Beneyto, J.I. Lorenzo, C. Gaucher, C. Mazurier, K. Peerlinck,
G. Matthijs, J.J. Cassiman, J. Vermylen, P.G. Mori, M. Acquila, D. Caprino, and H. lnaba
Twenty-two molecular diagnostic laboratories from
14 countries participatedin a consortium studyto estimate the impact of Factor Vlll gene inversions in severe hemophilia A.
A total of2,093 patients with severehemophilia A were
studied; of those, 740 (35%) had a type 1 (distal) factor Vi11
inversion, and 140 (7%) showed a type 2 (proximal) inversion. In 25 cases, the molecular analysis showed additional
abnormal or polymorphic patterns. Ninety-eight percent of
532 mothers ofpatients with inversions were carriers ofthe
abnormal factorVlll gene; when onlymothers of nonfamilial
cases were studied, 9 de novo inversions in maternal germ
cells were observed among 225 cases(m 1 de novomaternal
origin of the inversion in 25mothersofsporadiccases).
When the maternal grandparental origin was examined,
the
inversions occurred de novo in male germ cells in 69 cases
and female germcells in l case.The presence of factor Vlll
inversions is not a major predisposing factor for
the development offactor VI11 inhibitors; however, slightly more patients with severe hemophilia A and factor Vlll inversions
develop inhibitors (130of 642 [20%1) than patientswith severe hemophilia A without inversions (131 of 821 [16%1).
0 1995 by The American Society of Hematology.
HEMOPHILIA
and many mutations have been ~haracterized.~-~
Most of the
mutations are private, ie, they have been found in one or only
a fewunrelatedfamilies.Recently,acommoninversion
of
factor VlII gene was identified in many unrelated patients with
severe hemophilia A.’,’ The inversion is mediatedby the presenceofthree copies of a particular DNA sequence (termed
sequence A)’”’; one copy is located within intron 22 of the
factor VIII gene’ and the other two are
4 0 0 kbtelomeric
to the first.’”’3 Unequal crossing-over between two ofthese
sequences results in inversion of a portion of the factor WI
gene (exons 1 to 22) so that no intact factor VTII protein is
produced. Cross-over between the most distal sequence A and
its IVS22 homolog results in a type
l inversion, apparently the
mostcommoninversional e~ent.7.’~
Cross-overbetweenthe
proximal extragenic sequenceA and its NS22 homolog results
in a type 2 inversion. Both of these inversions can easily be
diagnosed bySouthernblotanalysisusing
a probe from the
IVS22.7 It appears that there is also a polymorphism of the A
sequencesthatlieoutside
ofthe factor VIII gene, as some
individuals have more than two copies ofthe extragenic sequence A.14The Southern blot diagnostic patterns
of inversions
in thesepatients are different from thoseof types 1 and2,
termed here types 3A and 3B (Fig 1). In a recent study using
DNA polymorphismanalysistodeterminethe
origin of de
novo inversions, the origin was almost always ~aternal.’~
The
same study concluded that almost all mothers of patients with
severe hemophilia A that was caused by factor Wr inversion
were carriers of the in~ersion.’~
To determine the frequency of factor VI11 inversions in
populations of severe hemophilia A patients, we collected
data from a considerable number of hemophilia or molecular
genetic diagnostic laboratories; this paper describes the results of these consortium DNA diagnostic studies for factor
VI11 inversion mutations in hemophilia A.
A, ONE OF THE most common coagulation disorders with an incidence of about 1 in 5,000 to
10,OOO males, is caused by mutations in the gene for coagulation factor VIII.’ This gene was cloned almost a decade agoz
From the Geneva UniversityMedical School and CantonalHospital, Geneva, Switzerland; Johns Hopkins University School of Medicine, Baltimore, MD: University OfMiinster, Germany: Mayo Clinic,
Rochester, MN; University of Pennsylvania, Philadelphia: Regional
Hemophilia Center, Lyon, France; University of Lund, Malmo, Sweden; INSERM U91, Hdpital Henri Mondor, Crkteil, France: INSERM U143, Hdpital de Bic2treand Hdpital Ame‘ricain, Paris,
France; National Taiwan University, Taipei, Taiwan; Queen ’S University, Kingston, Ontario, Canada; Hdpital Cochin, Paris, France;
Vanderbilt University, Nashville, TN; Laikon Hospital, Athens,
Greece; Tokyo Medical College, Japan; Royal Free Hospital and
School of Medicine, London, UK; Royal HallamshireHospital, Shef
jield, UK; Rigshospitalet, Copenhagen, Denmark: Skejby Hospital,
Arhus, Denmark; Thrombosis Research Institute, London, UK;
Guy’s Hospital, London, UK; HospitaldeSanPau,
Barcelona,
Spain: Hospital Val1d’Hebron, Barcelona, Spain; University Hospital La Fe, Valencia, Spain; Centre de Transfusion, Lille, France;
University of Leuven, Belgium: University of California, San Francisco; Gianninn Gaslini Institute, Genova, Italy.
Submitted February 24, 1995; accepted May 15, 1995.
Supported by grants from the National Institutes of Health, the
Swiss National Science Foundation, the Nationaaf Fonds voor Wetenschappelijk Onderzoek (Belgium), theSheffield University Research Stimulation grant (Sheffield, UK), the Swedish Medical Research Council, andthe
Fondo de Investigaciones Sanilarias
(Spain).
Address reprint requests to Stylianos E. Antonarakis, Division de
Ge‘nitique Medicale, Centre Medical Universitaire, l rueMichel
Servet, 1211 Gedve, Switzerland.
The publication costs of this article were defrayedin part by page
charge payment. This article must therefore beherebymarked
“advertisement” in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
0 1995 by The American Society of Hematology.
0006-4971/95/8606-0025$3.00/0
2206
MATERIALS AND METHODS
Each investigator contributed data to the first author’s laboratory
via a data submission questionnaire. Included were the number of
Blood, Vol 86, No 6 (September 15). 1995 pp 2206-2212
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2207
HEMOPHILIA A FACTOR Vlll INVERSIONS
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2208
ANTONARAKIS ET AL
WQe 2
Fig 1. Schematic representation of the mechanisms of the different
factor Vlll gene inversions
caused by intrachromosomalcrossing-over between
homologous sequences A. The mechanisms of types
1 and 2 are as proposed.' The mechanisms of inversions 3A and 38 are t w o of several alternatives to
explain the Southern blot patterns. The bottom of
the figure schematically show the results of Southern blot analysis in the different types of factor Vlll
inversions.
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HEMOPHILIA A FACTOR VI11 INVERSIONS
2209
Types of inversions of factor Vlll gene
in severe hemophiliaA
Fig 1. (Cont'd)
unrelated patients with severe hemophilia A (factor VI11 activity less
than I % of normal) studied by Southern blotting, the number of
patients with each type of inversion, whether any inversion patient
presented a mildhoderate phenotype, the number of mothers of
inversion factor VI11 patients who were carriers either by pedigree
or Southern blot analysis, the number of cases in which the origin
of a de novo mutation was found, and the number of patients with
severe hemophilia A with or without inversions that have developed
factor VI11 inhibitors (antifactor VI11 antibodies). The diagnosis of
the inversions in all laboratories was performed using Southern blot
analysis as described.' Genomic DNA from patients with hemophilia
A and members of their families was digested with Bcl I, the DNA
fragments were electrophoresed on 0.7% agarose gels for 24 hours
at SO V and transferred to nitrocellulose or nylon membranes. The
membranes were probed with the 0.9-kb EcoRUSsr I fragment from
plasmid p482.6' (American Type Culture Collection [ATCC] catalogue no. 57203). Earlier versions of the results from several laboratories have been published or presented in
RESULTS AND DISCUSSION
Twenty-two datasets from investigators in 14 countries are
included in the study (Table 1). A total of 2,093 patients with
severe hemophilia A have been studied by Southem blot for
the presence of a factor VIII inversion. Because some laboratories havealso studied patients withmildmoderate hemophilia
A,thetotalnumber
ofpatient..studiedwas2,560.
Table 1
shows the numberof patients with inversions ofthe factorVm
gene. The DNA analysis showed 740 unrelated patients with
a type 1 factor VIII inversion(35%ofpatientswithsevere
hemophilia A studied, or 82% of the patients with inversions).
This percentage varied from 25%
of severe hemophiliacs in
group 4 to 47% in thepatientsof group 2. There were 140
patients with a type 2 inversion of factor VITI, ie, 7% of those
with severe hemophilia A or 15% of patients with inversions.
This percentage varied from0% of severe hemophilia Ain the
collection of patients of group 22 to 19% in those from group
20. Thus, the total number of unrelated patients with severe
hemophilia A andeithertype 1 or type 2factor Vm gene
inversion was 880 of 2,093, or 42%. This varied from 28%in
the sample of group 4 to 53% in the sample of group 2. The
overall frequency of 42% does not differ significantly from that
reported in the original studies of Lakich et al? Naylor et al?
&d Rossiter et al.?' Thus, the search for inversions of factor
VIII should be considered as the first DNA diagnostic option
in a patient or a family with severe hemophilia A.
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ANTONARAKIS ET
2210
Table 2. Hemophilia A, F8 Inversions Consortium-Origin of F8 Inversions
Group
Mother
Carrier
54 1
53
2
17
18
4
18
519
(l
19
28
6
28
7
9
8 (0.98) 87
49
10
2
1 1 (1.00)
65
49
43 19
12 (0.98)
13
7
14 (0.82) 18
17
15
28
16
15
17
(0.94)
17
18
17
16 19
(1.00)
20
7
49
21
22
7
(0.98)
532
Total
523
Mother
Tested
Mother Mother
Carrier
Tested
Mother
Novo
(% Carrier)
De No-FH
No-FH
Familial
0.98)
1
( 1 .OO)
.OO)
9
89
2
65
44
7
22
17
28
16
17
17
7
49
15
7
MGF
De Novo Novo
22
7
6
MGM
De
1
19
(1.00)
9
9
( 1 .OO)
2
2
(1.00)
14
5
8
2
10
2
3
3
2
1
3
6
4
7
2
( 1 .OO)
( 1 .OO)
1 1 17
17
( 1 .OO)
17
17
1
7
1
7
3
3
1
(1.00)
( 1 .OO)
15
(1.00)
34
4
A total of 25 patients had Southern blot patterns that differed both from normal and from type 1 or type 2 inversions,
representing 1% of patients with severe hemophilia A analyzed. Ten of the 25 rare patterns belong to the type 3A or
3B categories (5 and 5, respectively); in the remaining 15
cases, the molecular basis of the Southern blot results has
not yet been elucidated. These 15 cases may represent polymorphic (normal) variants or deletionshearrangements that
involve intron 22 of the factor VI11 gene.
Results were also provided on the number of carrier mothers of patients with factor VI11 inversion severe hemophilia
A (Table 2). Based on family history and/or Southern blot
analysis, 523 of 532 (98%) mothers were carriers of the
inversion. This result confirms the earlier observation14 that
almost all mothers of patients with severe hemophilia A
that was caused by factor VIII inversions are carriers of the
abnormal factor VI11 gene.
A total of 255 of the 520 mothers were members of pedigrees with positive family history of hemophilia A, and
therefore, the mothers were obligate carriers. There were
225 cases of inversion hemophilia A without family history
in which the mothers were tested for carrier status by Southern blot analysis. In the 225 mothers, 216 (96%) were carriers of the factor VI11 inversions (Table 2). Therefore, it
seems that in sporadic cases of hemophilia A, there is 1 new
mutation in the mother’s germ cells (a mother who is not a
carrier) in every 25 cases. The data on the origin of de novo
factor VI11 inversions from the laboratory of group 14 in
Malmo, Sweden, differ from these of all the other datasets.
The inversion originated in 4 of 14 mothers of patients with
sporadic, severe hemophilia A with factor VI11 inversion.
These data have been carefully checked on a second sample
from the respective families.
1
9
9
69
1
The origin of certain de novo inversions of factor VI11 in
the grandparental generation has been studied by Southern
blotting and by analysis of DNA polymorphisms within or
near the factor VI11 gene. The data collected on the origin
of inversion mutations do not represent a thorough study of
all cases; it is rather an opportunistic study based on the
availability of DNA samples from the necessary family
members. The de novo factor VI11 inversion originated in
the male germ cells (maternal grandfather) of 69 cases and
in the female germ cells of one case (maternal grandmother).
These results include the data of Rossiter et all4of 20 cases of
grandparental origin. The grandmaternal origin from group 4
was obtained using an extragenic polymorphic marker
(DXS52) for which the error rate in this determination is
1% to 5%. These data also concur with the earlier conclusion
that the overwhelming majority of de novo mutations occur
in male germ ~ e l 1 s .Because
l~
the mean paternal age was not
advanced in the study of Rossiter et al,I4 the hypothesis is
that most de novo inversions occur in male meiosis.
The presence of inhibitors in patients with severe hemophilia A with or without inversions is also reported by the
majority of the groups. The inhibitor titres variedwidely
among the groups. The frequency of inhibitors in the two
categories of patients also varied widely among the different
groups (Table 3). A total of 130 patients of 642 studied
(20%) with factor VIII inversions had developed inhibitors.
Similarly, 131 patients of 821 studied (16%) with severe
hemophilia A without factor VI11 inversions had developed
inhibitors. The difference is marginally statistically significant (x2 = 4.53; df = 1; P = .033). The odds ratio for
developing inhibitors in severe hemophilia A according to
the presence or absence of factor VI11 inversions is 1.34
(95% confidence interval, 1.02 to 1.75). Therefore, we con-
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2211
HEMOPHILIA A FACTOR Vlll INVERSIONS
Table 3. Hemophilia A, FS Inversions ConmtiumDevelopment of F8 lnhbitors
Group
Inv
1
2
3
4
6
7
9
10
11
12
13
14
15
17
18
19
20
21
22
Total
Inh
With
Inv
Total With
5
3
2
2
14
6
2
3
24
1
9
6
10
6
0
3
9
16
9
130
17
15
69
10
38
22
10
34
90
31
37
22
38
31
51
48
21
43
15
642
Inh
Without
(% Inh)
Inv
Inv
(0.29)
(0.20)
(0.03)
(0.20)
(0.37)
(0.27)
(0.20)
(0.09)
(0.27)
(0.03)
(0.24)
10.27)
(0.26)
(0.19)
(0.00)
(0.06)
(0.43)
10.37)
(0.60)
(0.20)
Total Without
l% Inh)
5
3
3
14
15
95
(0.36)
(0.20)
(0.03)
12
16
42
22
(0.29)
10.73)
4
20
5
8
5
19
1
3
7
6
9
5
131
68
107
35
64
27
81
36
53
62
26
59
15
82 1
(0.06)
(0.19)
(0.14)
(0.13)
(0.19)
(0.23)
(0.03)
(0.06)
(0.11)
(0.23)
(0.15)
(0.33)
(0.16)
Abbreviations: Inh, inhibitors; Inv, inversion.
clude that the presence of factor VI11 gene inversion is not a
major predisposing factor for the development of inhibitors.
The results presented here suffer from the limitations of
studies based on questionnaires. The Southern blot analyses
were performed in different laboratories with slightly different methodologies; no exchange of samples was done to
perform interlaboratory quality assessment. In addition, in
the data reported on inhibitors, no attempt was made to
distinguish high versus low titer, or transient versus longterm inhibitors.
In summary, the results from the analysis of 2,093 severe
hemophilia A patients in the consortium study of factor WI
inversions indicate that (1) the type 1 inversion accounts for
35% and the type 2 inversion accounts for 7% of cases with
severe hemophilia A , (2) in about 1% of cases of severe
hemophilia A, rare Southern blot patterns are found (types
3A and 3B and other variants); (3) because of inversion,
=98% of mothers of severe hemophilia A patients are carriers of the mutant factor VIII gene and, among mothers of
sporadic cases, the frequency of carriers is 96%; (4) the
majority of the de novo inversion mutation occurs in male
germ cells; and (5) the presence of factor VIII inversions is
not a major predisposing factor for the development of factor
VIII inhibitors.
ACKNOWLEDGMENT
We thank H. Hanabusa, T. Nagao, M.A. Moms, P. Hutter for
their contributions to the data collection.
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1995
4. Higuchi M,Antonarakis SE, Kasch L, Oldenburg J, EconomowPetersen E, Olek K, Inaba H, Kazazian HH: Towards a complete characterization of mild to moderate hemophilia A: Detection
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mutations are not within the coding region and splice junctions of
the factor VI11 gene. Proc Natl Acad Sci USA 88:7405, 1991
6. Naylor JA, Green PM, Rizza CR, Gianelli F Analysis of factor
WLI mRNA reveals defects in every one of 28 hemophilia A patients.
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1995 86: 2206-2212
Factor VIII gene inversions in severe hemophilia A: results of an
international consortium study
SE Antonarakis, JP Rossiter, M Young, J Horst, P de Moerloose, SS Sommer, RP Ketterling, HH
Jr Kazazian, C Negrier and C Vinciguerra
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