Table 1. Missense and nonsense mutations in the F8 gene identified in Portuguese families with haemophilia A.
Exon
Family/Patient
Identification
Nucleotide
Change
Type of Mutation Codon
/ Recurrency
Change
Exon 2
c
c.206T>C
c
Non-CpG
transition
F1503/0417
c.206T>C
Exon 3
F524
c.292G>A
Exon 4
F277
c.491G>A
F370
c.515G>T
F4181
F245
c.545A>C
c.553A>G
.
F3291
Exon 7
F2845
b
b
F1503/0271
Exon 9
Exon
10
f
Inhibitors
Comments
Familial; steric clashes, misfolding and secretion/stability defect
L50P
<1
4.2
Severe
Neg.
L50P
<1
†
Conservatio
n
LVVVV–
g
References
Present study
3.4
Severe
Neg.
E79K
2.4
3.1
Moderate
Neg.
De novo; maternal origin; disrupts a salt bridge with residue R3
EEEEE–
(21)
G145D
1
ND
Severe
Neg.
De novo; coil residue buried in A1 domain and secretion/stability defect
GGGGG-
(21)
C153F*
1.8
1.9
Moderate
Neg.
CCCCC–
Present study
D163A*
K166E*
4
3
ND
ND
Moderate
Moderate
Neg.
Neg.
DDDDDKKKKR-
Present study
Present study
c.556G>T
Transversion
Non-CpG
transition
Transversion
Familial; disrupts the disulphide bridge with C179 and secretion/stability
defect
Familial; loss of A1/A3 interdomain H-bonds
Familial; core residue close to the A1/A3 interface with steric clashes
D167Y
<1
1.6
Moderate
Neg.
Sporadic; loss of H-bonds with steric clashes and secretion/stability defect
Transversion
H281P*
1
ND
Severe
Neg.
F215
c.902G>A
R282H
7
ND
Moderate
Neg.
RRRRR–
(22)
F1202
F2531
c.902G>A
c.902G>A
CpG transition/
Recurrent
Recurrent
Recurrent
Familial; disrupts a H-bond with S524 and probably decreased FVIIIa
stability
Familial; decreased FVIIIa stability
DDDDDHHHHH–
Present study
c.899A>C
R282H
h
R282H
4
5
ND
80
Moderate
Moderate
Neg.
Neg.
Familial
Familial
F80 Lisboa2
c.1043G>C
Transversion
C329S
2.6
3.2
Neg.
Familial; intracellular retention and increased rate of A2 subunit dissociation
CCCCC–
(10,18)
F401
c.1063C>T
CpG transition
R336X
2
ND
Neg.
Sporadic; premature termination of translation
RRRQQ-
Present study
F2970
c.1349A>G
Y431C*
10
ND
Neg.
YYYYY-
Present study
F35
c.1349A>G
YYYYY-
Present study
a
c.1492G>A
Non-CpG
transition
Non-CpG
transition
CpG transition
Severe to
moderate
Severe to
moderate
Mild
GGGGG–
(6,14)
a
a
c.1492G>A
c.1492G>A
a
c.1589A>G
YYYYY–
Present study
a
c.1589A>G
c.1589A>G
c.1589A>G
c.1589A>G
c.1589A>G
c.1589A>G
c.1589A>G
c.1636C>T
c.1636C>T
c.1648C>T
c.1648C>T
RRRRR–
Present
Present
Present
Present
Present
Present
Present
(24)
RRRRR–
(24,25)
F140
F177 Porto1
F1573
Exon
11
Severity
Non-CpG
transition
Non-CpG
transition
Transversion
b
Exon 8
FVIII:C FVIII:Ag
(%)
(%)
F805
F1090
F1224
a
F2825
a
F2864
a
F3775
a
F3844
a
F3905
a
F803
a
F2043
b
F346
a
F486
a
Non-CpG
transition
CpG transition
CpG transition
Present study
Y431C*
9.7
8
Mild
Neg.
Sporadic; disrupts a H-bond with R439 and probably illegitimate disulphide
bonding
Familial
G479R
13.4
22
Mild
Neg.
Sporadic; core residue, steric clash with R531
G479R
G479R
15.8
10.0
31.6
NA
Mild
Mild
Familial
Familial
Y511C*
11.8
9
Moderate
Neg.
Pos, Type
1
Neg.
Y511C*
Y511C*
Y511C*
Y511C*
Y511C*
Y511C*
Y511C*
R527W
R527W
R531C
R531C
10
9
5
15
13
15
17.5
e
12
e
NA
15
e
9.5
10
8
ND
11
7
ND
9
NA
NA
21
11
Mild
Mild
Mild
Mild
Mild
Mild
Mild
Mild
NA
Mild
Mild
Neg.
Neg.
Neg.
Neg.
Neg.
Neg.
Neg.
Neg.
NA
Neg.
NA
Familial; steric clashes and probably illegitimate disulphide with secretion
defect
Familial
Familial
Sporadic
Familial
Sporadic
Familial
Familial
Familial; defect in interaction with FIXa
Sporadic; obligate carrier tested
Sporadic; increased rate of A2 subunit dissociation
Sporadic
Present study
study
study
study
study
study
study
study
23
a
F2324
F2972
F1565
F1093
c.1648C>T
c.1648C>T
c.1649G>A
c.1702G>A
F1669
c.1804C>T
Recurrent
CpG transition
Non-CpG
transition
CpG transition
Exon
12
Exon
13
Exon
14
Exon
15
Mild
Mild
Mild
Severe
Neg.
Neg.
Neg.
Neg.
Sporadic
Familial
Familial; increased rate of A2 subunit dissociation
Familial; steric clashes with severe misfolding and lack of secretion
GGGGG–
(24,25)
Present study
<1
<1
Severe
Neg.
Sporadic; maternal somatic mosaicism; premature termination of translation
RQRQR–
Present study
F463
c.1834C>T
CpG transition
F2558
c.1933C>T
CpG transition
R593C
12.5
ND
Mild
NA
Sporadic, intracellular accumulation
<1
NA
Severe
Familial; premature termination of translation
7
NA
Mild
Pos, Type
2
NA
RRRRR–
QQQEE–
(26)
Q626X
F184
c.2044G>T
Transversion
V663F
F4240
c.2150G>A
CpG transition
R698Q*
63
Mild
Neg.
Familial; mild functional defect and enhanced A2 domain dissociation
VVVVV–
RRRRRR
Present study
44
F816
F1853
b
F199
b
F400
b
F2624
F2399
c.2167G>A
c.3031A>T
c.5122C>T
c.5122C>T
c.5122C>T
c.5144G>C
CpG transition
Transversion
CpG transition
Recurrent
Recurrent
Transversion
A704T
K992X*
R1689C
R1689C
R1689C
R1696P*
12.5
<1
4.3
5
9
3
ND
<1
60%
ND
115
NA
Mild
Severe
Moderate
Moderate
Moderate
Moderate
Neg.
Neg.
Neg.
Neg.
Neg.
Neg.
AAAAAA
KKKKSL
RRRRRR
study
study
study
study
study
study
A1701D*
R1721M*
<1
2
1
1.2
Severe
Severe
Neg.
Neg.
Familial; impaired interaction with FIXa
Sporadic; premature termination of translation
Familial; thrombin activation site
Familial; thrombin activation site
Fporadic; thrombin activation site
Familial; obligate carrier tested; abolish a H-bond with D1769, misfolding
and secretion/stability defect
Familial; steric clashes with severe misfolding and secretion/stability defect
Sporadic; probably affects splicing, FXa cleavage site
F1378
F3162
c.5159C>A
c.5219G>T
Transversion
Transversion
a
c.5275G>A
D1740N*
3
30
Moderate
Neg.
D1740N*
5
20
Moderate
1
F436
Non-CpG
transition
c.5275G>A Non-CpG
transition
c.5321A>G Non-CpG
transition
c.5363A>G Non-CpG
transition
c.58788C>T CpG transition
F194Porto2
F70
c.5900G>A
c.5953C>T
F1390
F569
a
F2843
F2739
Exon
21
Exon
23
Exon
24
R583X
i
32
32
e
33
1
e
Familial; carrier tested;
Neg.
Sporadic;
DDDDDD
Present study
Severe
Neg.
Familial; severe steric clashes with misfolding and secretion/stability defect
HHHHHH
Present study
Familial; abolish a H-bond with R1696, misfolding and secretion/stability
defect
Familial; premature termination of translation
DDDDDD
Present study
RRRRRR
Present study
Sporadic; intracellular retention and increased rate of A2 subunit dissociation
Familial; premature termination of translation
GGGGGG
RRRRRR
(10)
(11)
Familial; disrupt a salt bridge with residue D1828 and of a H-bond with
K661
Familial; probably illegitimate disulphide bonding with secretion/stability
defect
Sporadic; premature termination of translation
RRRRRR
(21)
WWWWW-
Present study
RRQRR-
Present study
RRRRRQQQQQ-
(29)
5
8.2
Moderate
Neg.
R1941X
<1
ND
Severe
CpG transition
CpG transition
G1948D
R1966X
7.4
<1
48.7
<1
Moderate to mild
Severe
c.5954G>A
CpG transition
h
R1966Q
14
35
Mild
Pos, Type
1
Neg.
Pos, Type
1
Neg.
F185
c.6243G>C
Transversion
W2062C*
<1
<1
Severe
F2611
c. 6497C>T
CpG transition
R2147X
1.2
1
Severe
Pos, Type
2
Neg.
F1656
c. 6506G>A
CpG transition
R2150H
5
1
Mild
Neg.
Sporadic; implicated in vWF interaction
a
c.6222C>G
Transversion
Q2189E
23.5
ND
Mild
Neg.
Sporadic; disrupt a H-bond and presumably mild secretion/stability defect
a
c.6222C>G
c.6222C>G
c.6222C>G
c.6222C>G
c.6682C>T
Transversion
Transversion
Transversion
Transversion
CpG transition
Q2189E
Q2189E
Q2189E
Q2189E
j
R2209X
27.6
28
20
23
<1
29
ND
ND
ND
ND
Mild
Mild
Mild
Mild
Severe
Neg.
Neg.
Neg.
Neg.
Pos.
transitory
Sporadic
Sporadic
Sporadic
Sporadic
Sporadic; premature termination of translation
F2676
F3605
a
F3606
a
F4136
F3848
study
Sporadic; new glycosylation site with interference in its interaction with FIXa
D1769G*
a
Present
(27)
Present
Present
Present
Present
Present
Present
Present study
Present study
<2
F430
RRRRRR
Present study
AAAAAA
RRRRRT
DDDDDD
H1755R*
F1203
Exon
18
R531C
R531C
R531H
G549S*
e
13
ND
84
2.7
b
RRRRR-
Present study
Present study
Present
Present
Present
Present
Present
study
study
study
study
study
24
Exon
25
Exon
26
F739
c.6793C>A
Transversion
Q2246K*
c
c.6910G>A
Non-CpG
transition
G2285R*
c
c.6910G>A
c.6910G>A
c.6910G>A
c.6910G>A
c.6910G>A
c.6910G>A
c.6910G>A
G2285R*
G2285R*
G2285R*
G2285R*
G2285R*
G2285R*
G2285R*
F181/0736
F181/0150
F181/2068
a
F574
d
F211
d
F258
d
F284
a
F2495
c
10
4
Mild
Neg.
Familial; refolding associated with moderate secretion/stability defect
QQQQQ-
Present study
2.4
1
Moderate
Neg.
Familial; severe steric clashes with misfolding and secretion/stability defect
GGGGG-
Present study
3
3
2.4
7
3.5
4.3
4.2
ND
ND
1
3.7
5.5
2.5
2.8
Moderate
Moderate
Moderate
Moderate to mild
Moderate to mild
Moderate to mild
Moderate
Neg.
Neg .
Neg.
Neg.
Neg.
Neg.
Neg.
Familial
Familial
Familial
Familial
Familial
Familial
Familial
Nucleotide +1 corresponds to position 172, i.e., the A of the first Met codon, of the sequence with GeneBank Accession No. NM_000132. The nomenclature system for the mutations is
according to den Dunnen and Antonarakis (20). Recurrency: i.e. independent occurrence in the Portuguese population established by haplotype analysis. †Single letter amino-acid code of the
index residue in FVIII of six species: Homo sapiens, Sus scrofa, Canis familiaris, Mus musculus, Rattus norvegicus, Gallus gallus (partial sequence). Novel mutations are indicated by *. NAnot available. ND- not done. a Related by haplotype; bUnrelated by haplotype; c Patients from the same family; dBelong to the same kindred. e Mutation with one stage/two-stage activity
discrepancy. fType 1, high responder and Type 2 low responder. gReference regarding the comments. hMutation segregating with the polymorphic FVIII-1241E variant. i Mutation segregating
with the polymorphic FVIII-2238V variant. jMutation segregating with the silent polymorphism c.2118A>G at codon L687 and with the polymorphic FVIII1310C variant. kFound in
association with the recently reported vWF polymorphism c.2771G>A, R924Q (37).
25
Table 2. Insertions and deletions in the F8 gene identified in Portuguese families with haemophilia A.
Exon
Exon 2
Exon 3
Exon 13
Exon 14
Patient
Identificatio
n
F916
F196
F1206
F4143
F1205
a
F300
a
F674
a
F2550
F1822
F876
a1
F466
Nucleotide Change
c
DNA sequence
Codon Change
FVIII:C FVIII:A
(%)
g (%)
Severity
Inhibitors
c.205_206delCT
c.305_306insCTGAGATTTATGTTCTTA
c.2100_2105delATGGA
c.2593delG
c.2614_2615insGAGT
c.2945_2946insA
c.2945_2946insA
c.2945_2946insA
from IVS13 -1341 to c.3291
c.3302_3303delAG
c.3637delA
L50fsX12*
V84fsX
M682fsX26*
D846fsX12*
S853fsX9*
V964fsX7
V964fsX7
V964fsX7
Large deletion*
E1082fsX15*
I1194fsX4
<1
1
<1
<1
<1
5
<1
<1
1
<1
<1
<1
ND
ND
1.3
ND
ND
ND
ND
1
ND
NA
Severe
Severe
Severe
Severe
Severe
Moderate
Moderate
Severe
Severe
Severe
Severe
Neg.
Neg.
Neg.
Sporadic; African origin
Sporadic
Familial
Pos. transitory Sporadic
Neg.
De novo; maternal origin
Neg.
Familial
Neg.
Familial
Neg.
Familial
Neg.
Sporadic; 4044bp deletion
Neg.
Familial; carrier tested
NA
Sporadic
Present study
Present study
Present study
Present study
Present study
Present study
Present study
Present study
Present study
Present study
Present study
a1
c.3637delA
ND
Moderate
Neg.
De novo; maternal origin
Present study
a2
c.3637delA
ND
Severe
Pos. type
F2614
c.3637delA
Moderate
Neg.
De novo; maternal origin; African
origin
Familial
Present study
a2
b
F2719
c.3637delA
ND
Severe
Neg.
Familial
Present study
G1272fsX28
H1415fsX5*
N1441fsX1
e
N1441fsX1
N1441fsX1
N1441fsX1
V1479fsX78*
R1548fsX21*
T1590fsX3
f
R1696del*
<1
<1
1
<1
<1
<1
1
<1
<1
1
ND
<1
<1
<1
NA
1.2
ND
ND
ND ?
12
Neg.
Neg.
Neg.
Neg.
Neg.
Neg.
Neg.
Neg.
Neg.
Pos. type 1
Familial
Familial.
De novo; maternal grandfather
Familial
De novo; maternal origin
De novo; maternal origin
Sporadic
Sporadic
Sporadic
Familial; in-frame deletion
Present study
Present study
(6)
Present study
Present study
Present study
Present study
Present study
Present study
Present study
P1761fsX4*
E1970del*
M2199fsX18*
<1
40
<1
<1
46
1
Severe
Severe
Severe
Severe
Severe
Severe
Severe
Severe
Severe
Severe to
moderate
Severe
Mild
Severe
Pos, Type 2
Sporadic
Familial; in-frame deletion
Sporadic
Present study
Present study
Present study
F1374
F143
b
F24 Porto3
a
F61
b
F379
a
F1376
F566
F1636
F1398
F2844
c.3870_3871insA
c.4293_4297delGAGAA
c.4379_4380insA
c.4379_4380insA
c.4379_4380insA
c.4379_4380insA
c.4482delG
c.4691_4692insTTTC
c.4825_4826insA
c.5142_5144delACG
ACT CTG TTT
GAT AC^A TGT
TCG ATG GAA
GGG GAC ATG
CCT GAG T^CA
GGA AAA A^AT
GGA AAA A^AT
GGA AAA A^AT
d
ctctcaga- CAGAAA
AAA GAG GGC
GAA AAA AAA
ATT
GAA AAA AAA
ATT
GAA AAA AAA
ATT
GAA AAA AAA
ATT
GAA AAA AAA
ATT
AA^A GGG GAG
AAC TCT TCT
AAA A^AT AAC
AAA A^AT AAC
AAA A^AT AAC
AAA A^AT AAC
ACT GTT CTC*
CCC TTT C^TG
GAA AAA ^ACA
AACA CGA C
F4302
F2215
F2448
c.5337delG
c.5965_5967delGAG
c.6652_6653delAT
TG GGG CCA*
A GAG GAG T
AAT ATG TTT
F1207
F2217
Exon 15
Exon 18
Exon 24
I1194fsX4
I1194fsX4
1.8
<1
I1194fsX4
1.6
I1194fsX4
1
1
Neg
Neg
Comments
References
Present study
For mutations in tandem repeats the position of the last unchanged 3’ nucleotide is indicated. Novel mutations are indicated by *. a Related by haplotype; bUnrelated by haplotype;
c
Deleted nucleotides are in bold; the position of insertions are indicated by ^; slipped nucleotides are underlined. dIntronic sequence is in lower case while exonic nucleotides are
in upper case. e Combined FVII/FVIII deficiency. fMutation segregating with the silent polymorphism c.3864A>C at codon S1269.
26
Table 3. Mutations in the splice site regions of the F8 gene identified in Portuguese families with haemophilia A.
Intron
Nucleotide
Change
IVS4-2A>G
IVS6+1G>A*
DNA
sequence
tttcag GG
AG gtatgt
CVN
Intron 4
Intron 6
Patient
Identification
F62 Lisboa1
F2551
0.955
0.887
Intron 11
Intron 24
F2533
F2672
IVS11+1G>A*
IVS24-1G>A
AG gtgagt
cctcag GT
0.967
0.952
CVM
FVIII:A
g (%)
1.3
1
Severity
0.716
0.705
FVIII:C
(%)
1.7
1
0.785
0.714
1
<1
1
<1
Inhibitors
Comments
References
Severe
Neg.
Moderate to mild Neg.
Familial; in-frame skipping of exon 5 and 4-5
NA; in-frame skipping of exons 4 to 7
(18)
Present study
Severe
Severe
Sporadic; in-frame skipping of exons 10 and 11
Familial; in-frame skipping of exon 25 with or without exon
19
Present study
Present study;
(33)
Pos. type 1
Neg.
Novel mutations are indicated by *. M utated nucleotide is in bold. CVN wild-type splice site consensus value; CVM mutated splice site consensus
value.
27
Table 4. Portuguese haemophilia A families without an obvious causative F8 mutation and screened for mutations in the FVIIIbinding region of vWF.
Patient
Identification
F1103
F1377
F3543
c
F1705
Exon
d
19
F2832
a
_
14
_
_
F8
Nucleotide
Codon
Change
Change
No identified F8 mutation
a
c.3780C>G
D1241E
No identified F8 mutation
No identified F8 mutation
e
c.6003T>C
Exon
_
_
_
19
V1982V
19
b
vWF
Nucleotide Change
No type 2N vWF mutation
No type 2N vWF mutation
No type 2N vWF mutation
[c.2446C>T]+[c.2446C>T
]
[c.2446C>T]+[?]
c
FVIII:Ag
(%)
ND
ND
ND
6
vWF:Ag
(%)
ND
Severe
NDE
70
Severity
a
Inhibitors
Comments
Mild
Familial
Moderate
Moderate
Neg
R816W
FVIII:C
(%)
9
1
2.5
4
Neg
Neg
Familial
Familial
Sporadic
Familial
R816W
ND
_
_
Mild
_
Sporadic
Codon
Change
b
Pos, Type 1
d
Polymorphic FVIII-1241E variant. Type 1, high responder. Female patient. Daughter of a haemophilia A patient with FVIII:C of 6%, not
available for the study. e Apparently silent mutation c.6003T>C at codon V1982.
28