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ORIGINAL CONTRIBUTION
Sodium Channel Mutations and Susceptibility
to Heart Failure and Atrial Fibrillation
Timothy M. Olson, MD
Virginia V. Michels, MD
Jeffrey D. Ballew, MS
Sandra P. Reyna, MD
Margaret L. Karst, BA
Kathleen J. Herron, BA
Steven C. Horton, MD
Richard J. Rodeheffer, MD
Jeffrey L. Anderson, MD
D
ILATED CARDIOMYOPATHY
(DCM), an idiopathic form of
heart failure and the primary indication for cardiac
transplantation,1 has an incidence of
6.0 per 100000 person-years and prevalence of 36.5 per 100000 population.2
Dilated cardiomyopathy is familial in
more than 20% of cases,3 implicating genetic defects in single proteins in the disease pathogenesis. Mutations that impair excitation-contraction coupling,
which translates electrical excitation of
cell membranes into mechanical force
production via intracellular calcium,
have been demonstrated in DCM and in
other heritable cardiac disorders.
Although mutations in genes encoding cardiac ion channels are a molecular substrate for isolated ventricular4-8
and atrial9,10 arrhythmias, the majority of mutations identified in heritable
DCM disrupt structural proteins of the
cytoskeleton and contractile apparatus.11-14 Mutations in the gene for the
nuclear membrane protein lamin A/C,
by contrast, have pleiotropic noncardiac and cardiac manifestations including DCM, atrial fibrillation, and
conduction system disease.15,16 The
For editorial comment see p 491.
Context Dilated cardiomyopathy (DCM), a genetically heterogeneous disorder, causes
heart failure and rhythm disturbances. The majority of identified DCM genes encode
structural proteins of the contractile apparatus and cytoskeleton. Recently, genetic defects in calcium and potassium regulation have been discovered in patients with DCM,
implicating an alternative disease mechanism. The full spectrum of genetic defects in
DCM, however, has not been established.
Objectives To identify a novel gene for DCM at a previously mapped locus, define
the spectrum of mutations in this gene within a DCM cohort, and determine the frequency of DCM among relatives inheriting a mutation in this gene.
Design, Setting, and Participants Refined mapping of a DCM locus on chromosome 3p in a multigenerational family and mutation scanning in 156 unrelated probands with DCM, prospectively identified at the Mayo Clinic between 1987 and 2004.
Relatives underwent screening echocardiography and electrocardiography and DNA
sample procurement.
Main Outcome Measure Correlation of identified mutations with cardiac phenotype.
Results Refined locus mapping revealed SCN5A, encoding the cardiac sodium channel, as a candidate gene. Mutation scans identified a missense mutation (D1275N) that
cosegregated with an age-dependent, variably expressed phenotype of DCM, atrial fibrillation, impaired automaticity, and conduction delay. In the DCM cohort, additional
missense (T220I, R814W, D1595H) and truncation (2550-2551insTG) SCN5A mutations, segregating with cardiac disease or arising de novo, were discovered in unrelated
probands. Among individuals with an SCN5A mutation 27% had early features of DCM
(mean age at diagnosis, 20.3 years), 38% had DCM (mean age at diagnosis, 47.9 years),
and 43% had atrial fibrillation (mean age at diagnosis, 27.8 years).
Conclusions Heritable SCN5A defects are associated with susceptibility to earlyonset DCM and atrial fibrillation. Similar or even identical mutations may lead to heart
failure, arrhythmia, or both.
www.jama.com
JAMA. 2005;293:447-454
mechanisms by which lamin A/C mutations cause mechanical and/or electrical heart disease are unknown. Notwithstanding, a mechanistic link
between heart failure and arrhythmias
is further underscored by recent reports of genetic defects in cardiac ion
flux in DCM. Recently, altered intracellular calcium cycling due to phospholamban mutations was established
as a mechanism for familial DCM17;
also, genetic defects in the regulatory
subunit of the KATP channel that impair membrane potential adjustment in
©2005 American Medical Association. All rights reserved.
response to metabolic state were identified in patients with DCM.18
In 1986, we reported on a large
kindred with autosomal dominant
Author Affiliations: Division of Cardiovascular Diseases, Department of Internal Medicine (Drs Olson and
Rodeheffer, Mr Ballew, and Mss Karst and Herron),
Division of Pediatric Cardiology, Department of Pediatric and Adolescent Medicine (Dr Olson), and Department of Medical Genetics (Dr Michels), Mayo Clinic
College of Medicine, Rochester, Minn; and Cardiovascular Department (Drs Reyna, Horton, and Anderson), LDS Hospital, University of Utah, Salt Lake City.
Corresponding Author: Timothy M. Olson, MD, Mayo
Clinic, 200 First St SW, Rochester, MN 55905 (olson
[email protected]).
(Reprinted) JAMA, January 26, 2005—Vol 293, No. 4
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447
SODIUM CHANNEL MUTATIONS AND HEART FAILURE
arrhythmia, and impaired electrical
impulse initiation and propagation.
We report herein mutations in SCN5A
as a risk factor for heart failure
and atrial fibrillation, expanding the
spectrum of phenotypic anomalies
associated with sodium channel
defects.
DCM that resembles the phenotype
in families later found to have lamin
A/C mutations.19 A decade later, we
mapped the disease locus to the short
arm of chromosome 3 by genetic linkage analysis.20 Thus, another protein,
yet to be identified, was implicatedin
the pathogenesis of heart failure,
METHODS
Patients
Probands with idiopathic DCM were
identified following referral to a cardiologist. Diagnostic criteria for idiopathic DCM were lack of an identifiable cause for myocardial dysfunction,
left ventricular dimensions greater than
Figure 1. Pedigree Structure and Clinical Features of Index Family With SCN5A Mutation
DC-19
Unaffected Male
Deceased Female
Major Phenotypic Traits
No Phenotypic Data
Short Tandem Repeat
DNA Markers
Minor Phenotypic Traits
Dilated Cardiomyopathy or
Congestive Heart Failure
Isolated Cardiomegaly or
Low Normal Function
Second-Degree Heart Block
or Complete Bundle-Branch Block
First-Degree Heart Block or
Incomplete Bundle-Branch Block
Sinus Node Dysfunction
Sinus Bradycardia
Atrial Fibrillation/Flutter
or Documented SVT
Symptoms of Tachycardia
N Asparagine
S Stroke
( ) Inferred Amino Acids
T Ventricular Tachycardia
SPT
15
15
25
28
62
24
( DD )
00
00
00
00
00
00
( DD )
40
50
40
50
20
20
( ND )
40
50
40
50
20
20
( ND )
00
00
00
00
00
00
( DD )
P
54
55
54
85
22
42
( DN )
04
05
04
05
02
03
( DN )
35
82
27
72
68
50
( DD )
SP
53
58
42
57
26
25
ND
40
50
40
50
20
20
( ND )
D3S3727 (30.7 Mb)
D3S2432 (32.1 Mb)
D3S1277 (34.6 Mb)
D3S1298 (38.0 Mb)
D3S3527 (39.3 Mb)
D3S3559 (42.7 Mb)
P Pacemaker
D Aspartic Acid
55
52
57
82
28
45
DD
Proband
10
60
50
20
40
70
( DD )
51
56
45
52
24
27
ND
43
58
42
57
26
25
ND
SP
40
30
20
60
50
10
( DD )
43
54
42
55
24
33
ND
P
P
44
35
24
65
52
13
DN
44
35
24
65
52
13
DN
70
60
50
20
30
70
( DD )
33
45
22
57
42
35
( DD )
14
15
24
25
62
22
DN
40
50
40
50
20
20
( ND )
25
26
25
37
17
41
DD
SP
43
55
42
57
22
33
ND
25
21
21
29
43
33
DD
43
55
42
57
22
35
ND
10
20
10
40
40
50
( DD )
P
20
10
30
10
30
40
( DD )
43
54
42
55
24
33
ND
43
54
42
55
24
33
ND
74
65
54
25
32
72
DN
42
52
42
53
21
24
ND
40
50
40
50
20
20
( ND )
56
76
22
27
44
45
DD
41
52
41
54
24
25
ND
SP
41
52
41
54
24
25
ND
41
52
41
54
24
25
ND
P
45
51
41
59
23
33
ND
24
15
34
15
32
43
DN
45
57
42
52
24
24
ND
Phenotypic traits are variably expressed, designated by shaded quadrants within pedigree symbols. Genotypes for closely spaced DNA markers are shown as numbers,
representing different lengths of short tandem repeat marker alleles that distinguish the paternally and maternally inherited chromosomal region. Markers are located
within the previously reported disease gene locus on chromosome 3p22-p25. The distance of each marker from the p-arm telomere is indicated by Mb (megabase). The
4 central markers (5-4-5-2) within the shaded chromosome segment define a haplotype, a group of alleles inherited as a unit, common to all family members with cardiac
disease. Recombination events indicate that the disease-causing gene resides between markers D3S3727 and D3S3559. SCN5A, located at 38.6-Mb, was investigated as
a candidate gene. A point mutation in SCN5A that alters a single amino acid, D1275N, was identified in all affected family members. Mutation-carrier status is indicated
by letter symbols for the amino acid at position 1275: aspartic acid (D) in the normal protein and asparagine (N) in the mutant protein. Amino acids shown in parentheses
were inferred, together with their corresponding haplotypes. SVT indicates supraventricular tachycardia.
448 JAMA, January 26, 2005—Vol 293, No. 4 (Reprinted)
©2005 American Medical Association. All rights reserved.
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SODIUM CHANNEL MUTATIONS AND HEART FAILURE
the 95th percentile indexed for body surface area, and left ventricular shortening fraction less than 28% and/or ejection fraction less than 50%, as determined
by echocardiography.3 Rhythm and conduction abnormalities were defined using established criteria.20 Probands and
their relatives who had enrolled in the
study provided written informed consent under protocols approved by the
LDS Hospital and/or Mayo Clinic institutional review board.
Medical records and electrocardiograms and echocardiography reports
were reviewed. Although some testing
of certain family members had been previously performed for clinical indications, most asymptomatic relatives underwent testing for the research study.
The clinical description of the multigenerational family used in this study,
designated as DC-19 (FIGURE 1), has
been reported.19,20 Phenotypic characterization was updated with additional clinical information and was expanded to include additional family
members. Genomic DNA was isolated
from peripheral white blood cells. To
avoid bias, phenotypic data were interpreted without knowledge of mutation status. Mutations in the known
DCM genes encoding cardiac actin,12
␣-tropomyosin,21 and metavinculin22
were previously excluded in our cohort of patients with DCM.
Genotyping and Refined Mapping
Short tandem repeat (STR) DNA markers were identified within the previously identified DCM locus on chromosome 3p in DC-19 (Figure 1). Gelbased genotyping was performed and
haplotypes (series of closely spaced
markers) were constructed as previously described.20 When possible, genotypes were inferred for individuals from
whom DNA samples were not available. Refined mapping was based solely
on recombination events in affected family members. (In our previous study
SCN5A was erroneously excluded as a
positional candidate gene based on a recombination event in an adult classified at the time as unaffected but who has
since developed heart disease.20)
Mutation Scanning
Primer pairs for exon-specific PCR
amplification of genomic DNA were
designed using OLIGO v6.51 Primer
Analysis Software (National Biosciences, Plymouth, Minn) and
WAVEMAKER version 4.0.32 software (Transgenomic, Omaha, Neb).
Primer pairs (n=31) were designed for
all 28 translated exons of SCN5A, including an alternatively spliced exon.
Heterozygous sequence variants in
PCR-amplified DNA fragments were
identified by denaturing highperformance liquid chromatography
(D-HPLC) heteroduplex analysis
(WAVE DNA Fragment Analysis System, Transgenomic), carried out according to the manufacturer’s instructions. Chromatographic elution profiles
of amplified fragments were compared against the wild-type homoduplex pattern; samples yielding anomalous traces (FIGURE 2A) were selected
for further analysis by sequencing. Control DNA samples were from a population-based sample (n = 500) with
normal electrocardiogram and echocardiogram results.
the conservation of amino acid residues that were altered by the identified missense mutations. The complete sequence of SCN5A was
submitted to the PredictProtein server
(http://cubic.bioc.columbia.edu
/predictprotein) yielding an alignment
of the protein sequence against other
known proteins in the database
(Figure 2C). Mutations were mapped on
a 2-dimensional schematic of SCN5A
(Figure 2D).23
Genotype-Phenotype Correlation
Correlation between clinical phenotype and SCN5A genotype was determined for unrelated probands in whom
an SCN5A mutation was identified, together with their relatives. Targeted sequencing of SCN5A was carried out to
determine whether the identified mutation was present in each proband’s
family members. The same markers on
chromosome 3p used for refined
mapping in DC-19 were used to construct haplotypes for these families
(FIGURE 3).
RESULTS
Phenotype of Index Family
Sequencing
Genomic DNA samples selected for sequencing were reamplified by PCR, and
products were treated with the PCR
Product Pre-sequencing Kit (USB Corp,
Cleveland, Ohio). The 2-bp insertion
mutant allele was isolated from the
wild-type allele following PCR on a 1⫻
MDE gel (FMC Bioproducts, Rockland, Md). Treated products were sequenced by the dye-terminator method
in a core facility, using an ABI PRISM
377 DNA Sequencer (Applied Biosystems, Foster City, Calif ). DNA sequence (Figure 2B) was viewed and
analyzed using the Sequencher computer program (Gene Codes Corporation, Ann Arbor, Mich). Numbering of
amino acids for SCN5A was based on
inclusion of an alternatively spliced residue, Q1077.
Protein Structure Analyses
By aligning protein homologs and
orthologs to SCN5A, we investigated
©2005 American Medical Association. All rights reserved.
Phenotypic data were updated for members of a large kindred (DC-19) who underwent cardiac evaluation since our
previous reports (Figure 1).19,20 A 44year-old woman had developed delayed-onset cardiac disease and was
found to have a genetic recombination event that provided critical information for refined mapping of the disease gene locus. At age 29 years with
symptoms of lightheadedness and palpitations, she had been classified as unaffected based on normal echocardiogram and electrocardiogram results.
Fifteen years later, she developed paroxysmal atrial fibrillation and, after cardioversion, had an underlying rhythm
of atrial standstill with junctional escape and incomplete right bundlebranch block.
Systematic evaluation of her family
revealed variable, age-dependent penetrance, which resulted in a spectrum
of disease manifestations including
DCM, diagnosed in 6 family members
(Reprinted) JAMA, January 26, 2005—Vol 293, No. 4
Downloaded from www.jama.com at Medical Library of the PLA, on August 21, 2007
449
SODIUM CHANNEL MUTATIONS AND HEART FAILURE
(mean age, 46 years; range, 29-55
years). Left ventricular ejection fraction (EF) ranged from 25% to 47%, and
4 of the 6 had symptomatic congestive
heart failure. Dilated cardiomyopathy
was typically preceded by atrial fibril-
lation, sinus node dysfunction, and conduction block. Cardiac histopathology, available for 4 family members,
Figure 2. Identification of SCN5A Mutations and Localization of Cardiac Sodium Channel Defects
DC-31
DC-96
DC-26
DC-19
DC-30
Exon 6
Exon 16
Exon 17
Exon 21
Exon 27
ACGCACCTTCC
A G C TG CGGGTC
C GTGT TCATC T
GGCTCGACTTC
T C T T C G AC T T
wt/mut
wt/mut
wt/mut
wt/mut
ACGCACCTTCC
T
AGCTGCGGGTC
T
GGCTCGACTTC
A
T C T T C G AC T T
C
A Heteroduplex Mutation
Detection Using DHPLC
Wild Type (wt)
Mutant (mut)
B DNA Sequence Analysis
at Mutation Site
Unaffected
Individuals
mut
Affected
Individuals
C Cardiac Sodium
Channel Protein
Sequences
T220I
human SCN5A-wt
human SCN5A-mut
human SCN1A
human SCN4A
human SCN7A
human SCN11A
mouse Scn5a
rat Scn5a
bovine Scn5a
electric eel Scn
fruit fly NaCP60E
S
.
.
.
P
L
.
.
.
.
A
A
.
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T
P
.
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L
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R
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Q
.
.
.
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.
.
T F
I .
. .
. .
A .
. .
. .
. .
. .
. .
. .
Insertion
fs851
R814W
DI/S4
Protein Region
C G TG TG T T C AT C T
R
.
.
.
.
.
.
.
.
.
.
DII/S4
V
.
.
.
T
.
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L
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.
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.
F
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F
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.
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L
.
R
.
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.
C
.
.
L L R
. .W
. . .
. . .
M. .
V . .
. . .
. . .
. . .
. . .
. . .
V
.
.
.
I
.
.
.
.
I
.
D1275N
DII/S5
F
.
.
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K
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L
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I
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F
V
.
.
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.
.
VF I FA
. CSS L
. . . . .
. . . . .
I . FS .
I . . . S
. . . . .
. . . . .
. . . . .
. . . . .
I . . . .
D1595H
DIII/S3
V V GMQ L F G K N Y S E L R
L WW A C S S L A R T T R S ∗
. . . . . . . . . S . KDC V
. . . . . . . . . S .K . CV
AF . . K . . . . . . E . F V
. . . . . . . . RS FNSQ K
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . Q .
L . . F . . . . . . .K . YV
. M . . . . . . . . . HD H K
AW
. .
. .
. .
G .
. .
. .
. .
. .
. .
. .
CW
. .
. .
. .
Y R
. C
. .
. .
. .
. .
. .
L
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.
.
DIV/S3
DF
N .
. .
. .
. .
. .
. .
. .
. .
. .
. .
L
.
.
.
V
I
.
.
.
V
V
I
.
.
.
V
.
.
.
.
.
.
NS WN I
. . . . .
I G . . .
VG . . .
I A . . .
. G . . L
. . . . .
. . . . .
. . . . .
VG . . V
E P . . L
F
.
.
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D FV
H . .
. . .
. . .
. . M
. C .
. . .
. . .
. . .
. . A
. V .
D Cardiac Sodium
Channel (SCN5A)
S1 S2 S3 S4 S5
S6
Domain I
Domain II
Domain III
Domain IV
COO−
NH3+
A, Heteroduplex mutation scans of exons comprising the entire coding region of SCN5A were performed by denaturing high-performance liquid chromatography
(DHPLC). Heterozygous variation in DNA sequence was detected in exons 6, 16, 17, 21, and 27 for the 5 family probands in Figure 1 and Figure 3. In contrast to
normal exons generating single peaks on chromatographic profiles, exons harboring mutations had anomalous profiles characterized by 2 peaks. B, To determine if
detected variations were benign or pathogenic, genomic DNA sequencing was performed. In 4 of the exons (exons 6, 16, 21, and 27), mutations were discovered in
1 copy of the gene, resulting in amino acid substitutions. In the remaining exon (exon 17), insertion of 2 bases disrupts the coding sequence (only the mutant gene is
shown). C, Regions in the cardiac sodium channel protein altered by mutations were aligned with protein sequences of other human and nonhuman sodium channels.
The 4 missense mutations alter highly conserved amino acids as indicated by vertical boxes wherein identical amino acids are designated by dots. The frameshift mutation creates a series of 18 anomalous amino acids, shown by the horizontal box in the fs851 panel, and an early stop codon, designated by an asterisk. D, Twodimensional schematic of SCN5A, demonstrating the predicted transmembrane topology. SCN5A is a monomeric channel composed of 4 repeat domains (DI-DIV),
each with 5 homologous hydrophobic transmembrane segments (S1-S3, S5, and S6) and 1 positively charged voltage-sensing segment (S4).23 Mutations map to all 4
domains, altering residues within membrane-spanning segments; 2 mutations occur within S4 voltage-sensing segments. The insertion or frameshift mutation severely
truncates the protein by removing 13 of 24 transmembrane segments.
450 JAMA, January 26, 2005—Vol 293, No. 4 (Reprinted)
©2005 American Medical Association. All rights reserved.
Downloaded from www.jama.com at Medical Library of the PLA, on August 21, 2007
SODIUM CHANNEL MUTATIONS AND HEART FAILURE
revealed nonspecific degenerative
changes in 2 and no abnormalities in
2 others. Morbidities included congestive heart failure, thromboembolic
stroke attributed to atrial fibrillation
and/or atrial standstill, and bradyarrhythmias necessitating cardiac pace-
maker implantation. There was no history of sudden death and none had
corrected QT-interval prolongation or
ST-segment elevation, which are characteristic of SCN5A mutations in long
QT syndrome4 or Brugada syndrome,5
respectively.
Refined Mapping
Genotyping was performed with polymorphic DNA markers within the 3p
locus and haplotypes were constructed
(Figure 1). The disease-associated haplotype was inherited by 22 clinically affected family members and an unaf-
Figure 3. Pedigree Structures and Clinical Features of Additional Families With SCN5A Mutations
DC-30
DC-31
DC-26
S
Short Tandem Repeat
DNA Markers
D3S3727 (30.7 Mb)
D3S2432 (32.1 Mb)
D3S1277 (34.6 Mb)
D3S1298 (38.0 Mb)
D3S3527 (39.3 Mb)
D3S3559 (42.7 Mb)
80
50
70
60
70
40
(TT)
70
40
10
60
50
60
( I T)
70
40
10
60
50
60
( I T)
73
65
45
93
21
21
(HD)
(TT)
54
74
12
88
57
45
DD
62
73
44
54
52
55
96
55
55
32
65
26
( fs wt ) ( wt wt )
T
78
45
17
66
57
64
I T
60
40
50
70
50
60
(TT) (
70
40
10
60
50
60
I T)
35
57
52
38
17
15
DD
74
64
42
98
27
25
HD
70
60
40
90
20
20
(HD)
37
32
51
71
46
61
(DD)
35
57
52
38
17
15
DD
75
67
41
98
25
24
HD
47
42
63
15
15
44
71
75
55
57
56
95
43
45
52
22
34
66
( fs wt ) ( wt wt ) wt wt
23
44
25
65
52
56
wt wt
67
45
55
95
53
62
fs wt
PT
67
44
51
76
55
66
T I
Unaffected Male
73
63
45
97
24
26
HD
Deceased Female
Major Phenotypic Traits
No Phenotypic Data
77
62
41
91
26
21
HD
77
62
41
91
26
21
HD
73
63
45
97
24
26
HD
47
37
64
16
45
41
13
51
57
4
1
57
95
34
23
54
52
62
62
fs wt ( wt wt ) wt wt
Proband
DC-96
Minor Phenotypic Traits
Dilated Cardiomyopathy or
Congestive Heart Failure
Isolated Cardiomegaly or
Low Normal Function
Second-Degree Heart Block
or Complete Bundle-Branch Block
First-Degree Heart Block or
Incomplete Bundle-Branch Block
Sinus Node Dysfunction
Sinus Bradycardia
Atrial Fibrillation/Flutter
or Documented SVT
Symptoms of Tachycardia
T
Threonine
P Pacemaker
I
Isoleucine
D Aspartic Acid
S Stroke
T Ventricular Tachycardia
H Histidine
wt Wild Type (No Mutation)
R Arginine
fs Frameshift Mutation
W Tryptophan
( ) Inferred
44
11
71
54
43
25
wt wt
47
16
73
51
44
22
wt wt
35
63
35
75
21
52
RR
35
63
35
75
21
52
RR
4 5
6 3
5 5
2 5
5 1
1 2
RR
43
46
53
47
12
25
RR
T
Paternal
Haplotypes
Maternal
Haplotypes
43
46
53
47
12
25
45
63
55
25
51
12
35
63
35
75
21
52
RR
45
43
55
75
21
51
RR
35
63
35
75
21
52
RR
44
46
55
42
15
21
RR
34
66
35
42
15
21
RR
34
66
35
72
25
51
R/W
Haplotypes at the chromosome 3p locus where SCN5A is located are shown. Each shaded haplotype defines a chromosomal segment that harbors a mutant SCN5A gene.
In 2 families, point mutations caused amino acid substitutions: D1595H in DC-30 and T220I in DC-31. In DC-26, the insertion of 2 bases in the mutant gene results in a
truncated protein that terminates in a string of 18 anomalous amino acids (fs851 [frameshift at amino acid 851]; Figure 2C). In DC-96, neither of the proband’s parents and
none of her 7 siblings had cardiac disease. The haplotypes she inherited from her father (dark blue) and mother (light purple) are also inherited by other siblings, yet she
is the only family member with a mutation in SCN5A. These findings indicate that the point mutation, R814W, arose as a spontaneous, or de novo, event on either the
paternal or maternal chromosome. SVT indicates supraventricular tachycardia.
©2005 American Medical Association. All rights reserved.
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451
SODIUM CHANNEL MUTATIONS AND HEART FAILURE
fected 10-year-old child (included to
permit inference of her deceased father’s haplotype). Genetic linkage analysis revealed odds of more than 100 000:1
against spurious linkage to this locus
(peak multipoint lod score, 5.8). Genetic recombination events defined a
critical region of 12 megabases, flanked
by markers D3S3727 and D3S3559.
SCN5A was selected as a positional candidate gene based on high cardiac expression and similarity of the family’s cardiac automaticity and conduction
abnormalities to other disorders caused
by SCN5A mutations.9,24
Mutation Identification
Mutation scanning of all translated exons of SCN5A was carried out by PCR
amplification of genomic DNA and denaturing high-performance liquid chromatography (D-HPLC) heteroduplex
analysis. An anomalous chromatographic tracing (Figure 2A) identified a
sequence variant in exon 21, present in
all family members with the diseaseassociated haplotype from whom a DNA
sample was available. Sequence analysis of the reamplified PCR fragment revealed a heterozygous point mutation,
G3823A, resulting in a D1275N substitution (Figure 2B). The mutation was not
present in 500 unrelated individuals
(1000 chromosomes) and it altered a
highly conserved amino acid in the
SCN5A DIII/S3 transmembrane domain (Figure 2C and 2D). Both DHPLC and sequence analysis of all remaining exons excluded additional
SCN5A mutations in the gene harboring the mutation.
Mutation Scans In DCM Cohort
Comprehensive mutation scans of
SCN5A were subsequently carried out
in 156 unrelated patients with idiopathic DCM. Distinct mutations in 4 individuals were discovered (Figure 2A
and 2B and Figure 3). Sequencing of the
exon harboring an SCN5A mutation
was then performed for relatives of each
proband to determine their mutationcarrier status. Haplotypes were also constructed in each family demonstrating
segregation of the mutation and the sur-
rounding chromosomal region or, in the
case of DC-96, showing that the mutation had arisen spontaneously in the
proband (Figure 3).
None of the probands and their relatives had prolonged QT intervals or STsegment elevation. To our knowledge,
none had been exposed to procainamide, which might have unmasked the
electrocardiographic patterns of Brugada syndrome. Individuals with atrial
fibrillation and DCM did not have rapid
ventricular heart rates, excluding tachycardia-induced DCM. Similarly, the few
family members with ventricular arrhythmia had nonsustained tachycardia based on Holter monitor or serial
electrocardiographic recordings or, in
1 case, exhibited persistent DCM after
medical control of tachycardia.
Genotype-Phenotype Correlation
In a 54-year-old man (DC-31) with
DCM (EF, 15%), atrial fibrillation, and
heart block, a heterozygous point mutation, C659T, was identified in exon 6
resulting in a T220I substitution. Coronary artery disease was excluded by angiography, and cardiac biopsy revealed
moderate myocyte hypertrophy and
marked interstitial fibrosis, consistent
with idiopathic DCM. No ventricular
tachycardia was observed on telemetry
monitoring during a 6-day hospitalization. He died 13 years later in severe congestive heart failure. The mutation was
also inherited by a female first cousin,
once removed, who at age 55 years was
diagnosed with DCM (EF, 10%) and incomplete bundle-branch block. Exercise testing revealed no evidence of ischemia or arrhythmia. She died 2 years
later, also in severe congestive heart failure. Other relatives were reported to
have enlarged hearts, but additional
clinical information and DNA samples
were unobtainable.
A second mutation was identified in
a 23-year-old woman (DC-96) with
sporadic DCM (EF, 30%), atrial flutter, and short runs of nonsustained ventricular tachycardia on 1 of 2 Holter
monitor recordings. Cardiac biopsy revealed mild to moderate myocellular
hypertrophy and mild interstitial fibro-
452 JAMA, January 26, 2005—Vol 293, No. 4 (Reprinted)
sis. Her parents and 7 siblings (aged
from 19-36 years ) had normal echocardiogram and electrocardiogram results. A heterozygous point mutation,
C2440T, was identified in exon 16 resulting in an R814W substitution. The
mutation arose de novo, because it was
absent in the patient’s parents and siblings and biological paternity and maternity were verified (Figure 3).
A third mutation was identified in a
man (DC-26) who at age 32 years developed DCM (EF, 35%) and monomorphic ventricular tachycardia (resolved on
medical therapy). Third-degree heart
block developed 7 years later, necessitating pacemaker implantation 7 years
later. A chest computed tomography scan
and stress echocardiography revealed no
evidence for either coronary artery or ischemic heart disease, respectively, and
a cardiac biopsy was normal. A 2-bp insertion in exon 17, 2550-2551insTG,
caused a frameshift at codon 851, resulting in a premature stop codon and truncation of the encoded protein. The mutation was inherited from his father,
diagnosed with DCM (EF, 30%), left
bundle-branch block, and monomorphic ventricular tachycardia at age 67
years, shortly before his death. The mutation was also present in his 63-yearold uncle who had sinus bradycardia,
first-degree heart block, and complete left
bundle-branch block. His paternal grandfather developed congestive heart failure at 50 years of age and died 6 years
later, but DNA was unavailable.
A fourth mutation was discovered in
a 7-year-old boy (DC-30) with sinus bradycardia, normal contractile function,
and left ventricular dilation, an early
manifestation of DCM.25,26 A heterozygous point mutation, G4783C, was identified in exon 27 resulting in a D1595H
substitution. Analysis of DNA extracted from postmortem paraffinembedded tissue confirmed presence of
the mutation in his brother, who died at
34 years of age with an autopsy diagnosis of cardiomyopathy and only mild
coronary artery disease. The mutation
was also identified in 2 of his siblings
(aged 17 and 24 years) and a paternal
uncle (aged 45 years), all of whom had
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SODIUM CHANNEL MUTATIONS AND HEART FAILURE
sinus bradycardia, and a paternal aunt
(aged 53 years) with borderline left atrial
enlargement. His father, an obligate mutation carrier, had atrial fibrillation and
died at 49 years of age from pulmonary
embolism following a surgical procedure. His paternal grandfather, a presumed mutation carrier, developed congestive heart failure at 70 years of age and
died from a stroke 8 years later. Each
identified mutation was absent in 500 unrelated individuals and altered highly
conserved residues in transmembrane
domains of SCN5A (Figure 2C and
2D).23 Mutation scans identified no additional mutations in the coding region
of SCN5A in probands with a defined
mutation.
In our study, 37 individuals in 5 families with available clinical data carried an
SCN5A mutation. Isolated cardiomegaly and/or low-normal systolic function, considered early markers of
DCM,25,26 were present in 27% of mutation carriers, with a mean age of 20.3
years at diagnosis. DCM was present in
38% of carriers, with mean age at diagnosis similar to that reported for patients
with familial and sporadic forms of DCM
(47.9 vs 45.2 years).3 Collectively, 65%
of carriers had a cardiomyopathy phenotype. Atrial fibrillation was documented in 43% of individuals with an
SCN5A mutation (mean age at diagnosis, 27.8 years). Sex was not observed to
influence phenotypic expression.
COMMENT
Mutations in SCN5A have been previously discovered in 6 cardiac rhythm disorders: long QT syndrome,4 Brugada
syndrome,5 idiopathic ventricular fibrillation,6 nonfamilial arrhythmia susceptibility,7 conduction system disease,24 and
sick sinus syndrome.9 These conditions
are characterized by ventricular tachyarrhythmia, heart block, or atrial bradyarrhythmia. We identified SCN5A mutations associated with atrial fibrillation,
providing further evidence that ventricular and atrial tachyarrhythmias can be allelic disorders.10,27
Susceptibility to myopathic heart disease in patients with inherited and de
novo heterozygous SCN5A mutations,
now established in this report, may have
gone unrecognized because of inherent selection bias in previous studies. Individuals and families with identified
SCN5A mutations were usually studied based on diagnosis of an idiopathic
rhythm disorder, with coexisting structural heart disease normally used as an
exclusionary criterion.5,6,10,23 Moreover, studies have not typically reported cardiac dimensions and function in patients with rhythm disorders
caused by SCN5A mutations.4,9,24 Systematic screening for SCN5A mutations in a DCM cohort has not been previously reported.
Our genetic data coupled with existing knowledge provide compelling evidence and a physiologic rationale for
SCN5A dysfunction conferring risk for
heart failure in DCM. First, intracellular sodium homeostasis is vital for myocellular function,28 including regulation of pH and intracellular calcium, the
key mediator of excitation-contraction
coupling. Indeed, high fidelity beat-tobeat regulation of intracellular calcium
flux is essential for normal cardiac function, and altered calcium cycling due to
phospholamban mutations has been established recently as a mechanism for familial DCM.17 We speculate that reduced sodium entry into myocytes could
lead to compensatory increases in activities of the sodium-calcium exchanger (opposite the effect of digitalis) and sodium-hydrogen exchanger,
resulting in decreased intracellular calcium and pH, respectively,28,29 and thus
impairing calcium-mediated myocellular force production and/or mitochondrial energy production. Second, sodium channel agonists increase cardiac
contractility,30 whereas certain class 1A
and 1C antiarrhythmic drugs that block
sodium channels can depress contractile function.31 Third, mutations in the
homologous skeletal muscle sodium
channel gene, SCN4A, cause a muscle
disorder characterized by episodic weakness and, in some patients, progressive
degenerative myopathy analogous to cardiac muscle disease in DCM.32 Fourth,
2 isolated cases of cardiomyopathy have
been reported recently in small fami-
©2005 American Medical Association. All rights reserved.
lies with rhythm disorders who had heterozygous33 or compound heterozygous34 mutations in SCN5A.
Each of our identified mutations would
be predicted to cause loss of function of
the cardiac sodium channel, based on
previous reports of identical or similar
mutations. The same T220I and D1275N
substitutions were reported as recessive
alleles in sick sinus syndrome9 and familial atrial standstill,35 respectively. The de
novo R814W substitution here identified in DII/S4 is exactly analogous in position to an R225W substitution in DI/S4,34
identified in 2 siblings with compound
heterozygosity (R225W and W156X
mutations) and conduction system disease; one had cardiomyopathy on
autopsy. The D1595H substitution is
similar to a previously reported D1595N
substitution in autosomal dominant conduction system disease.36 A frameshift
and truncation mutation (V1397X) distal to the frameshift we identified was
reported in Brugada syndrome.5 Collectively, in vitro biophysical analyses of
these 5 mutations, identical or similar to
those reported herein, resulted in loss of
channel function with reduced sodium
current.
The mechanisms by which similar or
identical loss of function mutations in
SCN5A lead to variable expression of
heart disease remain unknown. Our data,
together with previous reports, do not
support mutation-specific genotypephenotype correlations that predict either
electrical or myopathic phenotypes. Indeed, it has been shown that the same
mutation can cause either long QT syndrome, Brugada syndrome, or conduction abnormalities within the same family37 and that subtle in vitro changes in
channel function can be associated with
severe cardiac phenotypes.38 Extremes of
heart rate have been proposed as a potential factor in variable phenotypic expression,38 raising the possibility that the
bradycardia observed in many of our patients may have influenced development of DCM. In mice, heterozygous deletion of Scn5a leads to conduction
system disease and ventricular arrhythmias, as observed in humans, at 8 to 10
weeks.39 If analogous to humans with
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453
SODIUM CHANNEL MUTATIONS AND HEART FAILURE
SCN5A mutations, who develop DCM as
middle-aged adults, ventricular dysfunction would not be expected to occur until much later in the 2-year lifespan of
these mice. Targeted disruption of both
Scn5a alleles causes severe defects in cardiac morphogenesis and embryonic lethality, precluding postnatal assessment of cardiac phenotype. Further
mechanistic insight into the myopathic
phenotype in humans associated with
SCN5A mutations will require expression of these mutations in living animal
models. The in vivo effects of SCN5A mutations leading to heart failure in DCM
are likely modulated by additional genetic, environmental, and stochastic factors within the heterogeneous milieu of
the cardiac myocyte.
Our findings uncover a novel molecular defect in DCM and further implicate
impaired ion homeostasis in the pathogenesis of heart failure.17,18 Together with
LMNA15,16 and ABCC9,18 SCN5A may be
a candidate for comprehensive genetic
testing in the future, targeting patients
with a syndrome of DCM and rhythm
or conduction disturbances. Like patients
with Brugada syndrome, avoidance of
sodium channel–blocking drugs in
patients with DCM and SCN5A mutations may prove to be prudent. Our findings provide a rationale for longitudinal studies of patients with isolated
rhythm disorders caused by loss of
SCN5A function, to determine whether
they are also at risk for DCM.
Author Contributions: Dr Olson had full access to all
of the data in the study and takes responsibility for
the integrity of the data and the accuracy of the data
analysis.
Study concept and design: Olson, Ballew, Karst,
Anderson.
Acquisition of data: Olson, Michels, Ballew, Reyna,
Karst, Herron, Horton, Rodeheffer, Anderson.
Analysis and interpretation of data: Olson, Ballew,
Karst, Anderson.
Drafting of the manuscript: Olson, Ballew, Reyna, Karst,
Herron, Horton, Anderson.
Critical revision of the manuscript for important intellectual content: Olson, Michels, Ballew, Reyna,
Rodeheffer.
Obtained funding: Olson.
Administrative, technical, or material support: Olson,
Michels, Ballew, Reyna, Karst, Herron, Horton,
Anderson.
Study supervision: Olson, Anderson.
Funding/Support: This work was supported by grant
R01 HL71225 from the National Heart, Lung, and Blood
Institute, National Institutes of Health; Miami Heart Research Institute; Marriott Program for Heart Disease Research; Siragusa Foundation; Mayo-Dubai Healthcare
City Research Project; and the Mayo Foundation (Dr
Olson).
Role of the Sponsors: The sponsors had no role in the
design and conduct of the study, in the collection, management, analysis, and interpretation of the data, or in
the preparation, review, or approval of the manuscript.
REFERENCES
1. Manolio TA, Baughman KL, Rodeheffer RJ, et al.
Prevalence and etiology of idiopathic dilated cardiomyopathy (summary of a National Heart, Lung, and
Blood Institute workshop). Am J Cardiol. 1992;69:
1458-1466.
2. Codd MB, Sugrue DD, Gersh BJ, Melton LJ III. Epidemiology of idiopathic dilated and hypertrophic cardiomyopathy: a population-based study in Olmsted
County, Minnesota, 1975-1984. Circulation. 1989;80:
564-572.
3. Michels VV, Moll PP, Miller FA, et al. The frequency of familial dilated cardiomyopathy in a series
of patients with idiopathic dilated cardiomyopathy.
N Engl J Med. 1992;326:77-82.
4. Wang Q, Shen J, Splawski I, et al. SCN5A mutations associated with an inherited cardiac arrhythmia, long QT syndrome. Cell. 1995;80:805-811.
5. Chen Q, Kirsch GE, Zhang D, et al. Genetic basis
and molecular mechanism for idiopathic ventricular
fibrillation. Nature. 1998;392:293-296.
6. Akai J, Makita N, Sakurada H, et al. A novel SCN5A
mutation associated with idiopathic ventricular fibrillation without typical ECG findings of Brugada
syndrome. FEBS Lett. 2000;479:29-34.
7. Splawski I, Timothy KW, Tateyama M, et al. Variant of SCN5A sodium channel implicated in risk of cardiac arrhythmia. Science. 2002;297:1333-1336.
8. Splawski I, Shen J, Timothy KW, et al. Spectrum
of mutations in long-QT syndrome genes. KVLQT1,
HERG, SCN5A, KCNE1, KCNE2. Circulation. 2000;102:
1178-1185.
9. Benson DW, Wang DW, Dyment M, et al. Congenital sick sinus syndrome caused by recessive mutations in the cardiac sodium channel gene (SCN5A).
J Clin Invest. 2003;112:1019-1028.
10. Chen Y-H, Xu S-J, Bendahhou S, et al. KCNQ1
gain-of-function mutation in familial atrial fibrillation.
Science. 2003;299:251-254.
11. Towbin JA, Hejtmancik JF, Brink P, et al. Xlinked dilated cardiomyopathy: molecular genetic evidence of linkage to the Duchenne muscular dystrophy (dystrophin) gene at the Xp21 locus. Circulation.
1993;87:1854-1865.
12. Olson TM, Michels VV, Thibodeau SN, Tai YS,
Keating MT. Actin mutations in dilated cardiomyopathy, a heritable form of heart failure. Science. 1998;
280:750-752.
13. Schonberger J, Seidman C. Many roads lead to a
broken heart: the genetics of dilated cardiomyopathy.
Am J Hum Genet. 2001;69:249-260.
14. Towbin JA, Bowles NE. The failing heart. Nature.
2002;415:227-233.
15. Fatkin D, MacRae C, Sasaki T, et al. Missense mutations in the rod domain of the lamin A/C gene as
causes of dilated cardiomyopathy and conductionsystem disease. N Engl J Med. 1999;341:1715-1724.
16. Taylor MR, Fain PR, Sinagra G, et al. Natural history of dilated cardiomyopathy due to lamin A/C gene
mutations. J Am Coll Cardiol. 2003;41:771-780.
17. Schmitt JP, Kamisago M, Asahi M, et al. Dilated
cardiomyopathy and heart failure caused by a mutation in phospholamban. Science. 2003;299:1410-1413.
18. Bienengraeber M, Olson TM, Selivanov VA, et al.
ABCC9 mutations identified in human dilated cardiomyopathy disrupt catalytic KATP channel gating. Nat
Genet. 2004;36:382-387.
19. Greenlee PR, Anderson JL, Lutz JR, Lindsay AE,
Hagan AD. Familial automaticity-conduction disorder with associated cardiomyopathy. West J Med.
1986;144:33-41.
20. Olson TM, Keating MT. Mapping a cardiomyop-
454 JAMA, January 26, 2005—Vol 293, No. 4 (Reprinted)
athy locus to chromosome 3p22-p25. J Clin Invest.
1996;97:528-532.
21. Olson TM, Kishimoto NY, Whitby FG, Michels VV.
Mutations that alter the surface charge of alphatropomyosin are associated with dilated
cardiomyopathy. J Mol Cell Cardiol. 2001;33:723-732.
22. Olson TM, Illenberger S, Kishimoto NY, Huttelmaier S, Keating MT, Jockusch BM. Metavinculin mutations alter actin interaction in dilated cardiomyopathy.
Circulation. 2002;105:431-437.
23. Tan HL, Bezzina CR, Smits JPP, Verkerk AO, Wilde
AAM. Genetic control of sodium channel function. Cardiovasc Res. 2003;57:961-973.
24. Probst V, Hoorntje TM, Hulsbeek M, et al. Cardiac conduction defects associate with mutations in
SCN5A. Nat Genet. 1999;23:20-21.
25. Baig MK, Goldman JH, Caforio ALP, Coonar AS,
Keeling PJ, McKenna WJ. Familial dilated cardiomyopathy: cardiac abnormalities are common in asymptomatic relatives and may represent early disease.
J Am Coll Cardiol. 1998;31:195-201.
26. Michels VV, Olson TM, Miller FA, Ballman KV,
Rosales G, Driscoll DJ. Frequency of development of
idiopathic dilated cardiomyopathy among relatives of
patients with idiopathic dilated cardiomyopathy. Am
J Cardiol. 2003;91:1389-1392.
27. Wang Q, Curran ME, Splawski I, et al. Positional
cloning of a novel potassium channel gene: KVLQT1
mutations cause cardiac arrhythmias. Nat Genet. 1996;
12:17-23.
28. Pieske B, Houser SR, Hasenfuss G, Bers DM. Sodium and the heart: a hidden key factor in cardiac
regulation. Cardiovasc Res. 2003;57:871-872.
29. Bers DM, Barry WH, Despa S. Intracellular Na+
regulation in cardiac myocytes. Cardiovasc Res. 2003;
57:897-912.
30. Flesch M, Erdmann E. Na+ channel activators as
positive inotropic agents for the treatment of chronic
heart failure. Cardiovasc Drugs Ther. 2001;15:379-386.
31. Zipes DP. Management of cardiac arrhythmias:
pharmacological, electrical, and surgical techniques.
In: Braunwald E, ed. Heart Disease: A Textbook of Cardiovascular Medicine. 5th ed. Philadelphia, Pa: WB
Saunders Co; 1997:597-610.
32. Plassart E, Reboul J, Rime CS, et al. Mutations in
the muscle sodium channel gene (SCN4A) in 13 French
families with hyperkalemic periodic paralysis and paramyotonia congenita: phenotype to genotype correlations and demonstration of the predominance of two
mutations. Eur J Hum Genet. 1994;2:110-124.
33. Chen S, Chung MK, Martin D, Rozich R, Tchou
PJ, Wang Q. SNP S1103Y in the cardiac sodium channel gene SCN5A is associated with cardiac arrhythmias and sudden death in a white family. J Med Genet.
2002;39:913-915.
34. Bezzina CR, Rook MB, Groenewegen WA, et al.
Compound heterozygosity for mutations (W156X and
R225W) in SCN5A associated with severe cardiac conduction disturbances and degenerative changes in the
conduction system. Circ Res. 2003;92:159-168.
35. Groenewegen WA, Firouzi M, Bezzina CR, et al.
A cardiac sodium channel mutation cosegregates with
a rare connexin40 genotype in familial atrial standstill.
Circ Res. 2003;92:14-22.
36. Wang DW, Viswanathan PC, Balser JR, George
AL, Benson DW. Clinical, genetic, and biophysical characterization of SCN5A mutations associated with atrioventricular conduction block. Circulation. 2002;105:
341-346.
37. Grant AO, Carboni MP, Neplioueva V, et al. Long
QT syndrome, Brugada syndrome, and conduction system disease are linked to a single sodium channel
mutation. J Clin Invest. 2002;110:1201-1209.
38. Viswanathan PC, Balser JR. Inherited sodium channelopathies: a continuum of channel dysfunction.
Trends Cardiovasc Med. 2004;14:28-35.
39. Papadatos GA, Wallerstein PM, Head CE, et al.
Slowed conduction and ventricular tachycardia after
targeted disruption of the cardiac sodium channel gene
Scn5a. Proc Natl Acad Sci U S A. 2002;99:6210-6215.
©2005 American Medical Association. All rights reserved.
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