Original article 211
Molecular characterization of female hemophilia A by
multiplex ligation-dependent probe amplification analysis
and X-chromosome inactivation study
Min-Jung Songa, Hee-Jin Kima,b, Ki-Young Yooc, In-Ae Parkd, Ki-O Leed,
Chang-Seok Kia and Sun-Hee Kima
Hemophilia A is an X-linked recessive bleeding disorder
caused by mutations in the F8 gene. Hemophilia A typically
occurs in male individuals, but female patients with
hemophilia A have rarely been reported. Here we describe
molecular characteristics of three unrelated female patients
with severe hemophilia A of Korean descent. Patient 1 was a
5-year-old girl and was found to be compound
heterozygous for intron 22 inversion inherited from her
father with hemophilia A and a large deletion mutation from
her mother. The large deletion detected by multiplex
ligation-dependent probe amplification involved the whole
F8 gene. Patient 2 was a 30-year-old woman and was
heterozygous for small duplication mutation in exon 14
(c.3275dupA; p.Asn1092LysfsX26). Patient 3 was a 16-year-
old girl and was heterozygous for intron 22 inversion. All
three patients showed nonrandom X-chromosome
inactivation status. The results underscore the need for a
meticulous search for another mutation in the maternally
Introduction
Hemophilia A is an X-linked recessive disorder, charac-
terized by partial or complete deficiency of coagulation
factor VIII (FVIII). The causative gene, F8, is located
on the long arm of chromosome X (Xq28) over 180 kb,
and consists of 26 exons. More than 900 mutations in
the coding and untranslated sequences of F8 have been
reported. The most common mutation is the intron
22 inversion mutation, inv(22), which accounts for
about 40% of patients with severe hemophilia A [1].
Due to X-linked recessive inheritance of hemophilia A,
families of hemophilia A typically have male patients
and female carriers who are usually asymptomatic
due to the presence of the other functional copy of
X-chromosome. Extremely rarely, however, female
patients with hemophilia A have been reported, and
the molecular genetic mechanisms in female hemophi-
lia A include one mutant allele and extreme X-chromo-
some inactivation (XCI) (predominant expression of the
mutated allele as a result of preferential inactivation of
the X-chromosome carrying the wild-type F8) [2]; two
mutant alleles of F8, either compound heterozygous or
homozygous [3,4]; numerical or structural abnormalities
of the X-chromosome, such as in Turner syndrome [5];
and one mutant and an XY genotype manifesting as a
female [6].
0957-5235 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins
Copyright Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
derived X-chromosome such as large-dosage mutations.
Blood Coagul Fibrinolysis 22:211214 2011 Wolters
Kluwer Health | Lippincott Williams & Wilkins.
Blood Coagulation and Fibrinolysis 2011, 22:211214
Keywords: F8, female hemophilia A, Korea, multiplex ligation-dependent
probe amplification, mutation, X-chromosome inactivation
a
Department of Laboratory Medicine and Genetics, Samsung Medical Center,
Sungkyunkwan University School of Medicine, bCardiac and Vascular Center,
Samsung Medical Center, cKorea Hemophilia Foundation and dSamsung
Biomedical Research Institute, Samsung Medical Center, Seoul, Korea
Correspondence to Hee-Jin Kim, MD, PhD, Department of Laboratory Medicine
and Genetics, Samsung Medical Center, Sungkyunkwan University School of
Medicine, 50 Ilwon-dong, Gangnam-gu, Seoul 135-710, Korea
Tel: +82 2 3410 2702; fax: +82 2 3410 2719; e-mail: heejinkim@skku.edu
Received 13 September 2010 Revised 1 December 2010
Accepted 21 December 2010
In this study, the authors investigated the molecular
characteristics of three unrelated female patients with
severe hemophilia A of Korean descent.
Methods
Participants
The study participants were three female patients with
severe hemophilia A (FVIII activity <1%) ascertained
by Korea Hemophilia Foundation (KHF). Patient 1 was
a 5-year-old girl who had experienced recurrent bleed-
ing episodes such as hematomas, muscle bleeding, and
hemarthrosis. Her father also had severe hemophilia A,
whereas her mother and relatives of maternal side had
no history of bleeding diathesis. Patient 2 was a 30-year-
old woman who had experienced recurrent episodes of
spontaneous musculo-skeletal and soft tissue bleeding
since childhood. Both of her parents were healthy;
however, her uncle of maternal side had died from
bleeding diathesis. Patient 3 was a 16-year-old girl
and had been diagnosed with severe hemophilia A
at the age of 2. She experienced numerous bleeding
episodes, including recurrent hemarthrosis (ankle and
knee). There was no family history of bleeding. Table 1
shows a summary of clinical and laboratory features of
the three patients.
DOI:10.1097/MBC.0b013e328343f873
 212 Blood Coagulation and Fibrinolysis 2011, Vol 22 No 3
Table 1 Clinical phenotype and laboratory results of female patients with severe hemophilia A
Age at F/Hx FVIII : C aPTT
Patients Age diagnosis of HA (%) (sec)
1 5 years 3 days <1 116
2 30 years 2 years
<1 53.5
3 16 years 2 years
<1 52
F/Hx, family history; HA, hemophilia A; MLPA, multiplex ligation-dependent probe amplification; ND, not done; XCI, X-chromosome inactivation.
Molecular genetic analysis
Molecular genetic tests were performed using genomic
DNA isolated from peripheral blood leukocytes,
after obtaining written informed consent. First, intron
22/intron 1 inversion mutations were screened by long
amplification-polymerase chain reaction (LA-PCR) with
three primers, P, Q and A [7]. Segment PQ (12 kb) is
produced from the nonrecombined F8 and segment AQ
(11 kb) is produced recombined F8 due to the inversion.
Female carriers, thus, produce PQ and AQ segments. To
detect point mutations, direct sequencing analysis was
performed on all 26 exons and flanking intronic regions of
F8 by the BigDye Terminator Cycle Sequencing Ready
Reaction kit on an ABI Prism 3130 Genetic Analyzer
(Applied Biosystems, Foster City, California, USA) using
primer pairs designed by the authors (available upon
request). Sequence variations were detected by aligning
with the reference sequence (NM_000132) using the
Sequencher program and were described according
to the nomenclature system by the Human Genome
Variation Society. A of the ATG translation initiation
codon and first methionine were numbered 1. To detect
large dosage mutations in F8, multiplex ligation-depen-
dent probe amplification (MLPA) analysis was performed
using the commercial kit SALSA P178 FVIII (MRC-
Holland, Amsterdam, the Netherlands). A peak ratio less
than 0.7 or greater than 1.4 was considered as deletion
or duplication, respectively. Conventional karyotype
analysis was performed by the standard culture procedure
and GTG-banding. Lastly, the XCI pattern was deter-
mined by PCR analysis of a polymorphic CAG repeat
in the first exon of the human androgen receptor
(HUMARA) gene. XCI was calculated as the ratio
between the intensities of the PCR products of two
alleles. A ratio of at least 80 : 20 was considered to indicate
the presence of extremely skewed XCI [8].
Results
Patient 1 was carrying inv(22) mutation, but in homo-
zygous not heterozygous status, identical to that observed
in her father with hemophilia A (Fig. 1a). MLPA analyses
revealed that patient 1 was heterozygous for a large
deletion mutation of F8 involving the whole F8 gene
(Fig. 1b). MLPA analyses on the DNA sample of her
mother with FVIII activity 122% showed that her mother
was a carrier of the large deletion mutation. Collectively,
patient 1 was compound heterozygous for inv(22) inher-
ited from her father with hemophilia A and a large
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VWF:Ag VWF:RCo Intron 22 Intron 1 Direct
(%) (%) inversion inversion sequencing MLPA XCI
ND ND

Large deletion 92 : 8
49.6 52.4

c.3275dupA
100 : 0
57.8 77.2


100 : 0
deletion mutation from her carrier mother. The analysis
of XCI in patient 1 showed two distinct alleles: one allele
sized 278 inherited from her father and the other allele
286 from her mother (Fig. 2). Following HpaII digestion,
we observed that the allele 286 was mainly amplified in
the patient, indicating an extremely skewed XCI (92 : 8)
of the maternal X-chromosome harboring the large
deletion. The XCI in her mother showed complete
inactivation of allele 286 with large deletion (100 : 0).
Patient 2 was heterozygous for a small duplication
mutation in exon 14 of F8, leading to a premature
termination of the protein (c.3275dupA; p.Asn1092-
LysfsX26) (Fig. 1c). The XCI study showed completely
skewed pattern (100 : 0) (Fig. 2). Inv(22), inv(1), or large-
dosage mutations were all negative. Patient 3 was hetero-
zygous for inv(22) mutation (Fig. 1a). Inv(1), point or
dosage mutations were all negative. All three patients had
a normal female karyotype (46,XX).
Discussion
According to the 2009 Annual Report from KHF, the
number of unrelated female patients with severe hemo-
philia A was four, accounting for 0.27% of total hemo-
philia A. Among them, this study involved three patients
for thorough molecular genetic characterization. Patient 1
was compound heterozygous for two mutations, inv(22)
and large deletion mutations of F8. In typical X-linked
recessive diseases, symptomatic female patients usually
harbor the same mutation as in their fathers in hetero-
zygous state. Skewed XCI in addition to this heterozy-
gous mutation is sufficient to explain the phenotypic
expression of the disease in females. In patient 1, how-
ever, inv(22) mutation inherited from her father was
observed in homozygous status, which suggested follow-
ing two possibilities irrespective of the XCI status: truly
homozygous for inv(22) or heterozygous for inv(22) and a
large deletion mutation. Further investigation by invol-
ving dosage mutation analyses by using the MLPA tech-
nique demonstrated that the inv(22) was in pseudo-
homozygous status from the presence of a large deletion
mutation of the whole F8 gene inherited from her
asymptomatic mother (Fig. 1b). The mother of patient
1 showed completely skewed XCI on the X-chromosome
harboring the large deletion mutation, allele 286 (Fig. 2,
100 : 0). Indeed, she was asymptomatic with a normal
level of FVIII activity (122%). Since both mutations in
patient 1 are associated with severe type of hemophilia A
[9], the degree of XCI skewness might not have affected
 Genetics of female hemophilia A in Korea Song et al. 213

Fig. 1

Molecular genetic results of female patients with hemophilia A and the parents of patient 1. (a) Long amplification-polymerase chain reaction assay
for the detection of intron 22 inversion of the F8. Patient 1 and her father with homozygous intron 22 inversion were shown. Patient 2 and the
mother of patient 1 did not have the indicator band () for the inversion mutation, and patient 3 was heterozygous of the mutation. (b) Results from
multiplex ligation-dependent probe amplification analysis in patient 1 (left) and her mother (right), confirming large deletion involving the whole F8
gene. (c) Y-axis, peak ratio; X-axis, size (bps).

Fig. 2

X-chromosome inactivation analysis of the HUMURA locus. After digestion with HpaII, a PCR product is obtained from the inactive X-chromosome
only. The red asterisk in patient 1 indicates the maternal allele (286 bp).

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 214 Blood Coagulation and Fibrinolysis 2011, Vol 22 No 3
the clinical manifestation. If the mutation on the X-
chromosome with a residual activity is associated with
mild to moderate hemophilia A, however, 92% of the
skewness could result in milder clinical manifestation
[10,11].
Both patient 2 and patient 3 were heterozygous for a
known deleterious mutation (c.3275dupA; p.Asn1092-
LysfsX26) and inv(22), respectively. Both mutations have
been reported to be associated with severe phenotype of
hemophilia A (http://europium. csc.mrc.ac.uk), which
was considered to be due to completely skewed inacti-
vation of the other copy of X-chromosome.
This is the first study on the molecular characteriza-
tion of female hemophilia A in Korea. Thorough mole-
cular genetic evaluation is warranted in female hemo-
philia A for genotypephenotype correlations and
family counseling. In particular, a meticulous search
for a mutation in the maternally derived X-chromosome
is needed, and exon dosage analyses using the MLPA
technique could be a robust tool to detect large-dosage
mutation.
Acknowledgement
The study was supported by a grant from the Korea Food
and Drug Administration (KFDA).
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