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Mutations Causing Coagulation Factor XI11 Subunit A Deficiency:
Characterization of the Mutant Proteins After Expression in Yeast
By Marjorie Coggan, Rohan Baker, Kris Miloszewski, Graeme Woodfield, and Philip Board
We identified the mutations causing factor Xlll A subunit
deficiency in two families. Two distinct mutationswere identified in the S family: the nonsense mutation Tyr 441-,stop
in exon 11, inherited throughthe paternal line, and the missense mutation Asn 60 Lys in exon 3, inherited through
the maternal line.Two members ofthe J family were heterozygous for the previously described type 3 A subunit. The
substitution giving rise to the type 3 variant was found to
60 --t Lys and Gly
be Gly 501 -+ Arg in exon12.TheAsn
501 .+ Arg mutations were constructed in cDNA clones and
expressed in yeast (Saccharomycescerewisiae AH22).Although mRNA could be detected, protein containing
the Asn
60 + Lys substitution couldnot be detected, suggesting extreme instability or susceptibility to proteolysis. A subunits
containing the Gly 501 Arg substitution were expressed
and found to be enzymatically activein fresh yeast lysates.
This variant has thermal instability and lost activity during
storage or purification.Gel filtration studies suggestedthat
the type 3 variant assembled as a dimer, as do normal A
subunits. The data suggest that the Gly 501 + Arg (Type 3
variant) wouldcause severe factorXlll deficiency if inherited
in the homozygous form oras a compound heterozygote
with another deleterious mutation.
0 1995 by The American Society of Hematology.
C
We have now identified the mutations in two additional
families with A subunit abnormalities. Two of the mutations
result in amino acid substitutions, and we have used sitedirected mutagenesis to engineer the expression of these
abnormal A subunits in yeast. The expression of recombinant
A subunits has allowed their independent characterization.
-+
OAGULATION factor XI11 is responsible for the formation of c-(y-glutamy1)-lysyl bonds between fibrin
chains during blood clotting. These crosslinks modify and
stabilize the clot structure and reduce its sensitivity to degradation by proteases. Factor XIII may also play a role in
wound healing and tissue repair processes. Genetically determined deficiency of factor XI11 gives rise to lifelong bleeding
diathesis that requires continual replacement therapy. In untreated cases, there is a high risk of intracranial bleeding
and death at an early age. Circulating plasma factor XI11 is
composed of two distinct subunits: the A subunit is responsible for the catalytic activity, and the B subunit appears to
be a carrier protein that protects the A subunits. In contrast,
intracellular factor XI11 in platelets and monocytes is composedonly of A subunits. The structure and function of
coagulation factor XI11 and its deficiencies have recently
been reviewed in detail.’
Although genetically determined deficiencies of either the
A or B subunit have been described, deficiency of the A
subunit is the most severe and the most common, being
reported in many populations throughout the wor1d.l4 Genetic deficiencies of either subunit are inherited in an autosomal recessive manner, as the A subunit has been mapped to
the short arm of chromosome 65 and the B subunit maps to
the long arm of chromosome
Recent advances in molecular biology have permitted the
determination of the precise mutations causing many genetic
diseases. However, few cases of factor XIII deficiency have
been studied using these techniques, and the cause of A
subunit deficiency has only been identified in a limited number of
These mutations include amino acid substitutions, frame shifts, and premature termination of translation.
Three A subunit mutations have been identified in Finland:
and it is notable that one of those mutations was found in
six of eight families studied. It is not yet clear how diverse
the mutations causing A subunit deficiency will be in other
ethnic groups. Information concerning the types of mutations
in different population groups and the relationship between
specific mutations and the severity of the disease is important
for genetic counseling and the provision of adequate replacement therapy. In addition, very little is currently known about
the residues involved in substrate binding and catalysis;
therefore, an analysis of mutations causing amino acid substitutions may give an indication of residues that are important for the maintenance of normal structure and function.
Blood, Vol 85,No 9 (May l), 1995: pp 2455-2460
-+
MATERIALS AND METHODS
Subjects
Family J. The father in this family was previously identified
because of the abnormal electrophoretic mobility of his plasma and
platelet A subunits. Although this subject has reduced factor XI11
activity in plasma, he is clinically normal. Initial studies suggested
that this subject was heterozygous for a variant, unstable A subunit
termed type 3.”
Family S. The parents are not known to be related and are of
European descent. Both have low plasma factor XI11 activity, as
measured by the dansylcadaverine-casein assay. Factor XIII A (FXIIIA) protein levels are low in both parents: mother, 36% and father,
68% of our standard normal pooled plasma (normal range, 90%
22%).The FXIIIA:FXIIIB protein ratio is 0.5 in the father and 0.35
in the mother (normal range, 1.O -C 0.26). These results suggest that
they are both heterozygous for A subunit deficiency. They have no
bleeding manifestations.
There are three daughters. One is normal and has never suffered
any bleeding manifestations. Her plasma factor XIII activity and A
protein levels are comparable with those in the parents, andher
FX1IIA:FXIIIB protein ratio is 0.64, so she is likely to be heterozygous. This daughter has not been included in the present study.
D.S. is a female born in 1959 who had severe bleeding from the
From theMolecular Genetics Group, John Curtin School ofMedical Research, Australian National University, Canberra, Australia;
the Academic Unit of Medicine, Depament of Clinical Medicine,
St. James’ University Hospital, Leeds, UK;and Auckland Regional
Blood Services, Auckland, New Zealand.
Submitted August 4, 1994; accepted December 14, 1994.
Address reprint requests to Philip Board, PhD, Molecular Genetics Group, John Curtin School of Medical Research, GPO Box 334,
Canberra ACT 2601, Australia.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
0 1995 by The American Society of Hematology,
oooS-4971/95/8.509-02$3.00/0
2455
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2456
umbilicus on separation of the cord 8 days after birth and had her
first blood transfusion. She was always covered in bruises as soon
as she started to walk and had numerous bleeding episodes from the
gums and mouth during teething. Minor injury to her left wrist at
the age of 4 years resulted in a huge hematoma that required several
weeks to resolve. After being hit on the head by a swing at the age
of 10 years, a hematoma on the forehead required 5 weeks toresolve.
D.S. suffered severe backache, probably due to bleeding into the
muscles in the region of the loin and pelvic girdle. She consequently
spent many months on orthopedic traction and could not walk for a
long time. Diagnosis of factor XI11 deficiency was made at the age
of 10 years. Plasma factor XI11 activity was zero with the dansylcadaverine-casein assay. No A-subunitprotein could be detected in
the plasma by immunoelectrophoresis. B-subunit protein levels were
47%of our standard normal pooled plasma. In 1972 D.S. started
prophylaxis with fresh frozen plasma given every 6 weeks. Since
1973. she has been treated with the Hoechst placental factor XI11
concentrate (Hoechst, Marburg, Germany). She has not had any
bleeding manifestations since and leads a normal active life. She
has gone through two pregnancies successfully and remains well on
regular prophylaxis with the factor XI11 concentrate.
L.S. is a female who was born in 1961. She bled from the umbilicus a few days after birth and was always covered in bruises as soon
as she started to walk. L.S. had severe bleeding from the gums after
she developed a dental abscess and had to be transfused with 2 U
of blood. At the age of 8 years, she had extensive and very painful
bruising of the chest, left loin area, and left hip region. This occurred
without any obvious trauma. She was given a 3-U blood transfusion,
and the bruising slowly resolved. Diagnosis of factor XI11 deficiency
was established when the patient was 8.5 years old. Plasma factor
XI11 activity was zero with the dansylcadaverine-casein assay. No
A-subunit protein could be detected in the plasma by immunoelectrophoresis. B-subunit protein levels were 48% of our standard normal
pooled plasma. In 1972, L.S. started regular prophylaxis with fresh
frozen plasma. In 1977, she commenced therapy with the Hoechst
factor XI11 concentrate. Bleeding manifestations are completely controlled. She has gone through three pregnancies successfully, leads
a normal and active life, and remains well on regular prophylaxis
with the factor XI11 concentrate.
Identijication of Mutations
Amplification of the 15 exons encoding the A subunit was performed by the polymerase chain reaction (PCR) with the primers
and conditions previously described in detaiL7 Amplifiedexons from
each subject were subjected to heteroduplex analysis on 8% and
10% polyacrylamide gel^.'^^'^ Those exons exhibiting the formation
of heteroduplexes were cloned in M13mp18 and sequenced by the
dideoxy chain termination method.I4 In each case, multiple clones
were sequenced to be sure of identifying the mutation in heterozygotes. Where a mutation was identified, it was confirmed by sequencing products from a separate amplification experiment and byexamination of family members.
Construction of Mutant cDNAs and Expression of
Recombinant A Subunits
A full-length F'XIIIA subunit cDNA'' was cloned in M13mp19,
and single-stranded DNA was used as a template for oligonucleotidedirected mutagenesis. Mutations detected in the coding sequence of
genomic DNA from patients with A-subunit deficiency were inserted
in cDNA clones. The mutagenesis was performed using a kit supplied by Amersham (Sydney, Australia) (RPN 1523). The oligonucleotides used to create the mutations are shown in Table 1. In each
case, an additional mutation was made to add or delete a closely
linked restriction site that allowed final confirmation that the correct
COGGAN ET AL
Table 1. Oligonucleotides Used For Mutagenesis
Exon 3
Normal
N60 K
Exon 11
+
Normal
G501
+
R
5"TGG GAC ACT AACAAG GTG GAC CAC CAC-3'
5"TGG GAC ACT A e AAG GTC GAG CAC CAC-3'
5"CTG ATG
TAC
GGA
GCT AAA AAG-3'
5"CTG ATG T A 1 AGA CGT AAAAAG-3'
A double underscore indicates the position of mutations to introduce amino acid substitutions. A single underscore indicates the position of mutations that introduce a Sal1 site in exon 3 and eliminate
an Rsa I site in exon 11.
construct was ultimately transferred to the yeast expression vector.
The inclusion of these additional diagnostic mutations did not change
the encoded amino acid sequence of the cDNA. The initial success
ofthe mutagenesis was confirmed by nucleotide sequencing. The
double-stranded replicative form ofthemutated
M13 clones was
subsequently purified, and fragments of cDNA containing mutations
were excised with appropriate restriction enzymes and ligated into
the A-subunit cDNA in a yeast expression vector, as shown schematically in Fig 1. Plasmid pRB334 contains the normal FXIIIA cDNA,
pN60K contains the FXIIIA cDNA with the N60K mutation, and
pG501R contains the FXIIIA cDNA with the G501R mutation.
In these constructs, the last (Gly76) codon of ubiquitin is followed
by a codon for the mature FXIIIA amino-terminal residue serine.
Cleavage of ubiquitin from ubiquitin-fusion proteins is accomplished
by endogenous ubiquitin-specific proteases" and, in this instance,
exposes serine at the N-terminus of FXIIIA. Expression of proteins
as fusions to ubiquitin has been reported to enhance their yield
in yeast, and cleavage of ubiquitin occurs very rapidly, perhaps
~otranslationally.'~.'~
Immunoblot analysis with FXIIIA antiserum
(Behringwerke AG, Marburg, Germany) detected a protein with the
expected molecular weight for FXIIIA, indicating that cleavage of
ubiquitin from the fusion protein had occurred (see Fig 3). No intact
fusion protein was observed.
Saccharomyces cerevisiae AH22 expressing recombinant A subunits from either pRB334, pN60K, or pG5OIR were lysed by vortexing with glass beadsM in a lysing solution containing 50 mmoI/
L HEPES pH 7.5, 150 mmol/L NaCl, 5 mmoVL EDTA, 1% Triton
X100, 0.1mmoVL phenylmethylsulphonyl fluoride, and S mmol/L
P-mercaptoethanol
Characterization of FXlllA
The transglutaminase activity of activated A subunits was determined by measuring the incorporation of I4C putrescine into dimethylcasein, as previously described." This assay contained 33 mmoV
L dithiothreitol to ensure that the active site cystein was not oxidized.
The qualitative evaluation of A-subunit expression in yeast extracts
and in fractions eluted from fast protein liquid chromatography
(FPLC) gel filtration columns was performed by observing the incorporation of monodansyl cadaverine into casein by a fluorescent spot
technique.''
The pH optimum was determined by evaluating activity in the
presence of buffers covering the pH range from 6 to11.5.*' The
stability of the mutant A subunits was evaluated by observing the
decay of activity during exposure to 55°C. Because preliminary studies indicated that the Gly 501 + Arg mutation lost activity during
storage and purification, the determination of pH optimum and heat
stability was performed in fresh yeast lysates.
To determine if the recombinant mutant A subunits formed a
normal dimeric complex, the elution volume of mutant A subunits
was compared with the elution volume of normal recombinant A
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2457
MUTATIONS IN FACTOR Xlll SUBUNIT A DEFICIENCY
pADH1
Ubiquitin
DOOR1
I
m111
s.011
>
FXlllA
I
\
\
I
I
\
‘U60
Q501
...............................................................................
Nata
1
2
I
F
3
4
I
L R Q Q S D T
73 7 4 7 5 76 1 a 3
1
S
Am
MO1
pQ501R
AAA
K6 0
pN60K
I
I
H tar
730 731
Fig 1. Schematic structure of plasmids expressing wild-type and mutant ubiquitin-FXIIIA fusions (pRB334,pNM)K, and pG501R). The
ubiquitin/FXIIIA regions are shown as open boxes and positioned downstream of an ADHl promoter (solid arrow). The coding regions are
not drawn to the
same scale. Dots indicate gaps in the sequence. Amino acids are given in the standard single letter code. Ter. termination
codon. Ubiquitin residues are numbered lthrough 76, and FXlllA residues are numbered 1through 731. The wild-type A subunit is expressed
by pRB334, and the locationsof the N60 and G501 codons mutated in plasmids pN6OK and pG501R, respectively, are shown below theFXlllA
coding region.
subunits on an FPLC Superose 12 column in IS0 mmol/L KCI,
20 mmol/L Tris/HCI pH 7.5. 1 mmol/L EDTA, and S mmol/L pmercaptoethanol. Because the activity of the GlyS01 -,Arg mutant
protein decreased during gel filtration. the presence of the protein
in eluted fractions was confirmed immunologically by spotting SpL samples onto nitrocellulose filters and detecting A-subunit protein with specific antiserum (Behringwerke).” Sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) of samples
was performed in 12.5% gels.”Nativeelectrophoresiswas
performed in agarose,” and after squash-blotting ontonitrocellulose,
the position of the A subunits was detected with antiserum to the A
subunit (Behringwerke), as previouslydescribed in detail.” Western
blots from SDS-PAGE were developed by similar methods.
RESULTS
Mutation Analysis
Family S. Heteroduplex analysis of the amplified FXIIIA exons from family S indicated the presence of sequence
differences in exons 3 and 11. These exons were cloned in
M13mp18 and sequenced. The mutation in exon 3 was found
to be a C + A transversion that alters codon 60 from AAC
to AAA. This mutation results in the substitution Asn60
Lys. The mutation in exon 11 was found tobe a C
A
transversion that alters codon 4 4 1 from TAC to TAA. This
mutation results in the substitution of Tyr 4 4 1 -t stop and
would prematurely terminate translation. The genetic transmission of these mutations was demonstrated in the family
as shown in Fig 2. The exon 3 mutation is clearly inherited
from the maternal parent, and the exon 11 mutation is inherited from the paternal parent.
Family J. Heteroduplex analysis indicated that exon 12
from the father contained a sequence abnormality. This exon
was cloned in M 13mpI8 and sequenced. The mutation in
exon 12 was found to be a G + A transition that changes
codon 501 from GGA to AGA. This mutation results in the
substitution Gly 501 + Arg. The genetic transmission of this
mutation was confirmed byan analysis of DNA from the
proband’s daughter (Fig 2).
+
Expression of Mutant FXIIIA Subunits
The mutations that cause amino acid substitutions in exons
3 and 12 were engineered in the FXIIIA cDNA and incorporatedin the yeast expression vector pRB334 as shown in
Fig 1. The level of factor XI11 activity in crude lysates of
fresh yeast cultures expressing these mutant subunits is
shown in Table 2. No activity could be detected in the sample
Family S
Family J
expressing the exon 3 Am60 + Lys mutation. In contrast,
substantial activity was detected in cultures expressing A
subunit with the exon 12 Gly501 + Arg substitution. However, it was noted that the activity expressed by this mutation
diminished during storage of the sample compared with identically stored wild-type extracts.
I
I
Immunoblots of extracts fractionated by SDS-PAGE
showed that abundant amounts of A subunit were expressed
D.S &j
LS.
+
r!l
Tyr441
I
Asn6O
+ stop
Lys
1
0
Gly501
Normal
--f
Arg
Table 2. Transglutaminase Activity of Fresh Yeast Lysates
Expressing Normal or Mutant FXlllA Subunits
Sample
N
Mean Activity ? SD
(prnolhlrng)
--
4
8
4
38.8 -c 0.94
44.3 2 4.34
0
Normal
Fig 2. The genetic transmission of particular A-subunit mutations
Gly 501 Arg
is shown in each family. The symbols for male (0)and female (0)
Asn6O Lvs
are hatched according t o the alleles present.
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COGGAN ET AL
2458
1 2 3 4 5 6 7 8 , 9 1 0
kD
200
a comparison of thenormalandGlyS01
Arg enzyme
activities at different pH levels showed a pH optimum of
8.5 in each case and essentially identical activity/pH profiles
(data not shown).
We also considered the possibility that the Gly SO1 Arg
substitution may affect the formation of homodimers. Gel
filtration studies on an FPLC Superose 12 column indicated
that the recombinant normal and Gly S01 Arg-substituted
enzymes are eluted in identical volumes (data not shown).
This result indicates that the variant has a dimeric structure
identical to that of the normal enzyme.
+
+
97
+
69
DISCUSSION
30
21 .S
Fig 3. Expression ofnormal and mutant FXlllA subunits in S cerevisiaeAH22cultures. Human platelet extracts orlysatesofyeast
carrying plasmids expressing A subunits were subjected to SDSPAGE, blotted onto nitrocellulose, and developed with antiserum to
human FXlllA subunit. Lane 1: human platelet extract; lanes 2 and 8:
yeast containing pRB334 (normal); lanes 3, 4, 6, and 7: yeast containing pG50lR; lane 5: molecular size markers; lane 9 yeast containing pN6OK; lane 10: yeast strain AH22.
in cultures containing thewild-type cDNA (pRB334) and
cultures expressing the exon 12 GlyS01 + Arg substitution
(Fig 3). In each case, the recombinant A subunits were the
same size as A subunits derived from platelets. In some
experiments, there appeared to bea trace of A subunit detectable in the cultures with the exon 3 Am60 + Lys substitution; however, this is not visible in Fig 3. To determine if
FXIIIA mRNA was transcribed from the construct with the
As1160
Lys substitution, we performed a Northern blot
experiment. As shown in Fig 4, FXIIIA mRNA of the same
size and approximate quantity was present in the yeast expressing the normal A subunit (pRB334) or either of the two
mutated forms (pGSOIR. pN60K). These data indicate that
the C A mutation causing the Asn60 -t Lys substitution
does not destabilize the mRNA and suggest that the newly
synthesized proteinisvery unstable and/or the subject of
increased proteolytic degradation.
The present studies have identified three mutations in the
FXIIIA subunit genes in two families. In family S, both
affected children have a severe deficiency of A subunit as a
result of inheriting a mutation causing premature termination
of translation at codon 4 4 1 from the paternal parent and an
amino acid substitution (Asn 60 Lys) from the maternal
parent. It has previously been very difficult, if not impossible, to characterize abnormal A subunits in deficient patients.
The difficulties include the occurrence of normal A subunits
in the plasma of patients provided with replacement therapy,
the probable existence of compound heterozygotes who express two different mutant forms of A subunit, and the extremely low levels of residual A subunit in patients, such as
the two children in family S. Some of these problems can
now be circumvented by the expression of recombinant A
subunits in microbial systems. We have previously demonstrated the expression of functional FXIIIA in Escherichia
coli"; however, the yields from this procedure are not as
great as those obtained in yeast.'" Therefore, we have used
a yeast expression system that can produce significant quan+
+
1234 5
+
Clmracterization of the GIy.501
+
Arg Mutant Subunit
Although fresh cultures expressing A subunit with the
GlyS01 + Arg substitution have high catalytic activity, attempts topurify this protein were frustrated by a loss of
activity during storage andpurification procedures. This
instability is clearly shown in Fig S. Heating the crude extracts at 55°C results in a rapid total loss of activity in the
Gly501 Arg sample compared with the normal enzyme,
which is only slightly decreased over the same time period.
The GlyS01 Arg substitution results in a charge change
of + 1. This change confers a slower native electrophoretic
mobility on the recombinant isoenzyme (Fig 6), as was originally observed in theproband."We
considered that the
charge change may directly influence the active site, and this
may be reflected in a change in the pH optimum. However,
- 28s
-18s
+
+
Fig 4. Northern blot of RNA from yeast cultures containing plasmids expressing normal and mutated A subunits. Lanel,S cerevisiae
AH22; lane 2, AH22 containing pRB334; lane 3,AH22 containing
pN6OK; lanes 4 and 5, AH22 containing pGSO1R. The positions of
the 18s and 28s ribosomal RNA were determined on the ethidium
bromide-stainedagarose gel. RNA (10 p g per lane) was loaded, and
the blot was hybridized with a '2P-labeled FXlllA subunit cDNA.
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MUTATIONSIN FACTOR XI11 SUBUNIT A DEFICIENCY
2459
tities of A subunit that has beenmutatedto
include the
individual substitutions detected in patients. In the case of
family S, the Asn60 lays mutation results in the expression
of a protein that is extremely unstable and/or is the subject
of rapid proteolytic degradation. This conclusion is supported by the observation that mRNA encoding this mutation
isreadily detectable in yeast containing the recombinant
construct. Thus. in this particular case and despite the use
of yeast expression technology, we were unable to obtain
sufficient protein to allow any characterization.
In contrast, the expression of A subunit with the GIy501 +
Arg substitution detected in family J has allowed more extensive characterization of the protein. Members of this family
with the mutation are heterozygotes and do not show any
clinical symptoms. We originally detected individuals with
this variant because of its slow native electrophoretic mobility in agarose gels.” The variant subunit was termed type 3,
and its slow electrophoretic mobility suggested that it may
result from an amino acid substitution that increased the net
charge on the protein. This observation has been confirmed,
as the Gly + Arg substitution gives a charge change of + 1
and the electrophoretic mobility of the recombinant mutant
enzyme is electrophoretically slow.
Our earlier studies of this variant suggested that the mutant
subunit was unstable and had an elevated pH optimum.”
The recombinant Gly501 + Argmutant A subunit shows
marked instability on storage and under heat treatment; how-
1
2
3
+
“ I \
0
30
Minutes
15
45
Fig 5. Heat stability of normal and variant A subunit. Lysates of
fresh cultureswere heated at 55°C for varying lengths of time before
the transglutaminase activity was determined. Open circles represent yeast containing pRB334 (normal), and the solid circles represent yeast containing pG501R expressing the type 3 variant A subunit. Each point is the mean of two experiments.
f
Fig 6. Electrophoretic separation of recombinant FXlllA
subunits expressed in S cerevisiae. Lysates of yeastcarrying
plasmids expressing A subunits
were subjected to electrophoresis in agarose gel, blotted onto
nitrocellulose,
and
developed
with specific antiserum. Lanes 1
and 2, AH22 containing pG501R
(type 3); lane 3, AH22 containing
pRB334 (normal).
ever, we found no evidence for a shift in its pH optimum.
This difference from our previous results may reflectthe
different origins of the material studied in each experiment.
In our earlier experiments, we measured activity in plasma
from heterozygotes expressing the type 1 and type 3
(Gly501 -+ Arg) subunits. In these samples, therewasno
evidence for type 3 homodimers, and the activity attributable
to the type 3 subunit was probably contributed by the type
3 subunit in a heterodimeric combination with type 1 subunits. In the present investigation, we are studying a homodimer, andit is possible that the previously observed difference
in the pH optimum results from the interaction of dissimilar
subunits. Alternatively, as the original experiments were performed with plasma that contained B subunits, a differential
interaction between the Gly 501 + Lys A subunits and the
B subunits cannot be excluded. The possibility that different
posttranslational modifications could also influence the pH
response should also be considered.
These studies suggest that although heterozygotes in family J are unaffected by this mutation, it would cause clinical
deficiency in the homozygous form or in combination with
another deleterious mutation. Several different mutations
causing FXIIIA deficiency have been described in this and
previous st~dies.”“’Clearly, insufficient numbers of cases
have been studied to determine the degree of genetic heterogeneity in this disease. Considerably more cases will need to
be studied in different racial groups to determine if particular
mutations are common in specific populations, as clearly
occurs in Finland.”
Little is known about the structure/function relationships
of the A-subunit protein and its interaction with the B subunit. The expression of mutants in yeast and characterization
of A subunits initially identified in deficient patients have
the potential to identify areas of theA-subunit molecule
involved in particular functions and can be used
to gain a
greater understanding of structure/function relationships.
REFERENCES
1. Board PG, Losowsky MS. Miloszewski KJA: Factor XIII: In-
heritedandacquired deficiency. Blood Rev 7:229, 1993
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
COGGAN ET AL
2460
2. Duckert F, Jung E, Shmerling DH: A hitherto undescribed
congenital haemorrhagic diathesis probably due to fibrin stabilising factor deficiency. Thromb Diathesis Haemorrhagica 5 : 179,
1960
3. Lorand L, Losowsky MS, Miloszewski KJA:Human factor
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245
4. Saito M, Asakura H, Yoshida T, Ito K, Okafuji K, Matsuda
T: A familial factor XI11 subunit B deficiency. Br J Haematol74290,
I990
5. Board PG, Webb GC, McKee J, Ichinose A: Localization of
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6. Webb G, Coggan CM, Ichinose A, Board PG: Localization of
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bands lq31-32.1 and restriction fragment polymorphism of the locus.
Hum Genet 81:157, 1989
7. Board P, Coggan M, Miloszewski K: Identification of a point
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exon 111. J Clin Invest 90:315, 1992
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nonsense mutation (Argl”
deficiency. Br J Haematol 85:769, 1993
11. Castle S, Board PG, Anderson R A M : Genetic heterogeneity
of factor XI11 deficiency: First description of unstable subunits. Br
J Haematol 48:337, 1981
12. Cavanaugh JA, Easteal S, Simons LA, Thomas DW, Serjeantson SW: FH-Sydney 1 and2-two
novel frameshift mutations in
exon 10 ofthelow density lipoprotein receptor gene detected by
heteroduplex formation. Hum Mutation 4:276, 1994
13. White MB, Carvalho M, Derse D, O’Brien SJ, Dean M: De+
tecting single base substitutions as heteroduplex polymorphisms.
Genomics 12:301, 1992
14. Sanger F, Nicklen S, Coulson AR: DNA sequencing with
chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463, 1977
15.Board PG, Pierce K, Coggan M: Expression of functional
coagulation factor XI11 in Eschericia coli. Thromb Haemost 63:235,
1990
16. Takagi T, Doolittle RF: Amino acid sequence studies of factor
XI11 and the peptide released during its activation by thrombin.
Biochemistry 13:750, 1974
17. Baker RT, Tobias JW, Varshavsky A: Ubiquitin-specific proteases of Saccharomyces cerevisiae. Cloning of UBP2 and UBP3,
and functional analysis ofthe UBP gene family. J BiolChem
267:23364, 1992
18. Ecker DJ, Stadel JM, Butt TR, Marsh JA, Monia BP, Powers
DA, Gorman JA, Clark PE, Warren F, Shatzman A, Crooke ST:
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From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
1995 85: 2455-2460
Mutations causing coagulation factor XIII subunit A deficiency:
characterization of the mutant proteins after expression in yeast
M Coggan, R Baker, K Miloszewski, G Woodfield and P Board
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