Blood, 15 April 2002, Vol. 99, No. 8, pp. 3063-3065
BRIEF REPORT
Homozygous factor V splice site mutation associated with
severe factor V deficiency
Iris Schrijver,
Marion A. Koerper,
Carol D. Jones, and
James L. Zehnder
From the Department of Pathology, Stanford University
School of Medicine, and the Department of Pediatric Hematology,
University of California at San Francisco.
 |
Abstract |
We investigated a family whose proband has a severe bleeding
disorder and factor V antigenic and functional levels of 8% and less
than 1% of control values, respectively. Molecular analysis of the
factor V gene revealed a novel homozygous mutation in the last
nucleotide of exon 10. 1701G>T causes activation of a cryptic exonic
splice site in exon 10, which encodes part of the factor V heavy chain
(A2 domain). This leads to the deletion of 35 nucleotides and results
in a frameshift with a premature stop codon at amino acid position 498. The G1701 and corresponding Gln509 are conserved in murine, bovine, and
porcine factor V and in human factor VIII. Few factor V deficiency
mutations have been identified as yet. Several are present in
the heterozygous form in combination with factor V Leiden (Arg506Gln).
This is the first reported homozygous splice site mutation in a patient
with factor V deficiency.
(Blood. 2002;99:3063-3065)
© 2002 by The American Society of Hematology.
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Introduction |
Autosomal recessive factor V deficiency is a rare
coagulation defect, equally affecting both sexes and diagnosed in
multiple ethnic backgrounds.1 In most patients, the
phenotype is clinically benign and is characterized by mild bleeding
only.1-4 The factor V gene is located on chromosome
1q23,5 spans more than 80 kb, and contains 25 exons.6 The 5' untranslated sequence, a signal peptide,
and the factor V heavy chain (protein domains A1 and A2) are encoded by
exons 1 to 12. Exon 13 encodes the B domain, which is
posttranslationally removed during activation by thrombin. The light
chain (protein domains A3, C1, and C2) and the 3' untranslated region
are encompassed by exons 14 to 25.6 Few mutations
associated with factor V deficiency have been reported.7
We investigated the genetic basis of factor V deficiency in a patient
with umbilical bleeding at birth, gingival bleeding, and repeated
spontaneous central nervous system hemorrhage. She requires weekly
prophylactic fresh frozen plasma transfusions. The parents may be
distantly related. Both parents and the patient's siblings are
clinically unaffected.
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Study design |
Factor V activity and antigen assays
Factor V coagulant activity was determined in the proband and 5 family members (Figure 1) by a clotting
assay using an MLA 1200 instrument (Medical Laboratory Automation,
Pleasantville, NY). Factor V antigen concentrations were
investigated with a paired antibody enzyme-linked immunosorbent assay
as recommended by the manufacturer (Cedarlane Laboratories, Hornby,
BC, Canada).
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| Figure 1.
Pedigree of a Mexican family with exon 10 splice site
mutation: genotypes and factor V phenotypes.
The pedigree portrays 2 generations (I and II) in which all members are
numbered according to age (I-1 and I-2, II-1 to 5). FV act indicates
Factor V activity; FV ag, factor V antigen; het, heterozygous; n/a, not
available; WT, wild-type.
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Polymerase chain reaction amplification and sequencing
Genomic DNA was isolated from the index patient and 5 relatives.
Amplification of 24 factor V exons was achieved by the use of intronic
primers. Exon 13 was amplified in 7 overlapping segments because of its
large size. All primers and polymerase chain reaction (PCR) conditions
are available on www.stanford.edu/~zehnder.
After identification of the 1701G>T mutation, only RNA from the
proband was available for further studies. RNA was extracted from
peripheral blood mononuclear cells according to standard procedures.
Reverse transcription (RT) was carried out with random hexamer
priming.8 Primers for subsequent PCR were designed from
the factor V cDNA sequence9 to investigate illegitimate splicing. The forward primer in exon 9 (5'-GTGACCTTCTCGCCTTATGAA-3') and the reverse primer in exon 12 (5'-ACAGCCTGCTGTTCGATGT-3') generate
a 293-bp amplicon from the wild-type factor V cDNA
sequence.9 PCR products were purified directly after
amplification or through gel extraction after the excision of bands
from an agarose gel. These purifications were performed according to
the manufacturer's guidelines (Qiagen, Valencia, CA). Purified samples
were then sequenced with fluorescent dideoxy chain terminators (Applied Biosystems, Foster City, CA) on an ABI 377 sequencing instrument (Applied Biosystems). Sequence analyses were aided by GCG Sequence Analysis (www.gcg.com).
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Results and discussion |
Factor V activity levels were determined in the proband, her
mother, and 4 siblings (Figure 1). Factor V antigen levels were assayed
in the same persons except sibling II-1. Only the proband (II-4) has
severely reduced levels: less than 1% activity (normal range,
60%-120% of the control value) and 8% of factor V antigen compared
with the standard. Siblings II-3 and II-5 do not appear to be factor V
deficient. Siblings II-1 and II-2 and their mother, I-2, have
intermediate levels, consistent with heterozygous factor V deficiency
(Figure 1). In the proband, we identified 2 novel homozygous genetic
alterations and 17 previously described, clinically innocent
polymorphisms (Whitehead cSNP database,
http://waldo.wi.mit.edu/cvar_snps/).6,9,10
The patient's severe phenotype, together with the markedly reduced
factor V antigenic and activity levels, point to a type 1 deficiency.
One of the 2 novel sequence variants is a single nucleotide variant
(4300C>T),9 which is inconsistent with type 1 factor V
deficiency because it is located in exon 13. The entire protein domain
(B) encoded by this exon is removed during factor V activation. Thus,
unless the mutation interfered with activation, it would not be
expected to result in a deficiency phenotype. Additionally, the
4300C>T mutation is located downstream from the homozygous splice site
mutation and consequent premature stop codon in this family. Therefore,
the most plausible disease causing mutation is a G>T transversion at
position 17019 in the exon 10 splice site (GenBank
accession number, AY046060). We postulated that this mutation could
induce altered RNA splicing. The genotypes in this family are
compatible with this hypothesis: the patient's mother I-2 and sister
II-2 are heterozygous at position 1701, whereas her brother II-5 is
homozygous wild type. To demonstrate use of another splice site and to
determine the sequence of the consequent transcript, RNA studies were
performed. The RT-PCR product of the patient is smaller than that of
normal controls (Figure 2A). No
normal-sized band was detected. Sequence analysis of the proband's
amplified cDNA revealed deletion of the last 35 bases of exon 10 (Figure 2B). The cryptic splice site activation results in a shift of
the reading frame. This frameshift ultimately introduces a premature
stop codon wherein the first nucleotide of amino acid 498 in exon 10 (U) fuses to the first 2 nucleotides of exon 11 (AG) (Figure 2B).
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| Figure 2.
mRNA analysis of proband with activation of cryptic exon 10 splicing
from 1701G>T.
(A) Reverse transcription-PCR products of factor V cDNA spanning exon
10 and including part of exons 9 and 11. The expected wild-type product
in the control (Ctr) measures 293 bp, whereas the patient (Pt) has a
35-bp deletion and a product size of only 258 bp. Neg indicates reagent
control in RT-PCR; Ld, ladder. (B) Sequence tracing
of the affected area in the proband's mutant cDNA and protein product,
compared with the wild-type factor V cDNA from the same region. The
sequence shown in bold reflects the 3' exon 10 segment, which is
illegitimately spliced out in the patient.
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The normal exon 
intron (donor) splice site consensus sequence is
[C/A]AGgt[a/g]agt,11 where capital letters
indicate exonic nucleotides and lowercase letters correspond to the
sequence in the intron. The bold g and t nucleotides at the beginning
of the intron are nearly invariant. As in the donor splice site of factor V exon 10,9 the last exonic nucleotide is a G in
78% of 5' splice sites.12 Guanine replacements at
positions +1 and +5 in an intron, or at the last base of the preceding
exon, are predicted to significantly reduce base pairing stability
between the splice site and the complementary region of the U1 small
nuclear (sn)RNA.12 This attachment forms the first step in
the assembly of the spliceosome.13
An estimated 15% of the point mutations that cause human disease
change the conventional splicing pattern of the primary RNA transcript.14 Skipping the preceding exon is the most
frequently observed consequence, but illegitimate splice sites may also
be used.15 Use of such a cryptic splice site may be
preferred over exon skipping if the site is located in the direct
vicinity of the expected splice site. It must also be sufficiently
similar to the splice site consensus sequence to compete successfully with the affected splice site.12 In our patient, the
sequenced mRNA product indicates the use of such an illegitimate splice site.
In conclusion, the observed 35-bp deletion in the patient's cDNA
causes a reading-frame shift that predicts a truncated factor V
molecule, missing the distal part of the A2 domain and the B, A3, C1,
and C2 domains. This finding provides a compelling explanation for the
deficiency phenotype in this family and is the first report of a
homozygous splice site mutation in the factor V gene resulting in
severe factor V deficiency and bleeding.
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Acknowledgments |
We thank Dr Uta Francke for helpful discussion and advice and Dr
Samuel Parks for technical assistance.
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Footnotes |
Submitted July 19, 2001; accepted December 4, 2001.
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: James L. Zehnder, Department of Pathology,
L235, Stanford University Medical Center, Stanford, CA 94305; e-mail:
zehnder{at}stanford.edu.
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Abstract
Introduction
