Mutations in the mitochondrial thioredoxin reductase gene TXNRD2 ...

European Heart Journal (2011) 32, 1121–1133
doi:10.1093/eurheartj/ehq507
Mutations in the mitochondrial thioredoxin
reductase gene TXNRD2 cause dilated
cardiomyopathy
BASIC SCIENCE
Dirk Sibbing 1† , Arne Pfeufer 2,3,4† , Tamara Perisic 5† , Alexander M. Mannes 5 ,
Karin Fritz-Wolf 6 , Sarah Unwin 1 , Moritz F. Sinner 7 , Christian Gieger 8 ,
Christian Johannes Gloeckner 9 , Heinz-Erich Wichmann 8,10,11 , Elisabeth Kremmer 12 ,
Zasie Schäfer 2,3 , Axel Walch 13 , Martin Hinterseer 7 , Michael Näbauer 7 , Stefan Kääb 7 ,
Adnan Kastrati 1 , Albert Schömig 1,14 , Thomas Meitinger 2,3 , Georg W. Bornkamm 5‡ ,
Marcus Conrad 5 * ‡} , and Nicolas von Beckerath 1,14 * ‡
1 2 3
Deutsches Herzzentrum München, Munich 80636, Germany; Institute of Human Genetics, Technische Universität München, Munich 81675, Germany; Institute of Human
Genetics, Helmholtz Zentrum München, German Research Center for Environmental Health, Neuherberg 85764, Germany; 4 Institute of Genetic Medicine, EURAC Academy,
Bozen 39100, Italy; 5 Institute of Clinical Molecular Biology and Tumor Genetics, Helmholtz Zentrum München, German Research Center for Environmental Health, Munich 81377,
Germany; 6 Max-Planck Institut für Medizinische Forschung, Heidelberg and Interdisciplinary Research Center, Justus-Liebig-University, Giessen, Germany; 7 Department of Medicine
I, University Hospital Grosshadern, Ludwig-Maximilians-Universität, Munich 81377, Germany; 8 Institute of Epidemiology, Helmholtz Zentrum München, German Research Center
for Environmental Health, Neuherberg 85764, Germany; 9 Department of Protein Science, Helmholtz Zentrum München, German Research Center for Environmental Health,
Neuherberg 85764, Germany; 10 Institute of Medical Informatics, Biometry and Epidemiology, Ludwig-Maximilians-Universität, Munich 81377, Germany; 11 Klinikum Grosshadern,
Munich 81377, Germany; 12 Institute of Molecular Immunology, Helmholtz Zentrum München, German Research Center for Environmental Health, Munich 81377, Germany;
13 14
Institute of Pathology, Helmholtz Zentrum München, German Research Center for Environmental Health, Neuherberg 85764, Germany; and 1. Medizinische Klinik, Technische
Universität München, Munich 81675, Germany
Received 29 July 2010; revised 29 November 2010; accepted 15 December 2010; online publish-ahead-of-print 18 January 2011
Aims Cardiac energy requirement is met to a large extent by oxidative phosphorylation in mitochondria that are highly
abundant in cardiac myocytes. Human mitochondrial thioredoxin reductase (TXNRD2) is a selenocysteine-containing
enzyme essential for mitochondrial oxygen radical scavenging. Cardiac-specific deletion of Txnrd2 in mice results
in dilated cardiomyopathy (DCM). The aim of this study was to investigate whether TXNRD2 mutations explain a
fraction of monogenic DCM cases.
.....................................................................................................................................................................................
Methods Sequencing and subsequent genotyping of TXNRD2 in patients diagnosed with DCM (n ¼ 227) and in DCM-free
and results (n ¼ 683) individuals from the general population sample KORA S4 was performed. The functional impact of
observed mutations on Txnrd2 function was tested in mouse fibroblasts. We identified two novel amino acid
residue-altering TXNRD2 mutations [175G . A (Ala59Thr) and 1124G . A (Gly375Arg)] in three heterozygous carriers
among 227 patients that were not observed in the 683 DCM-free individuals. Both DCM-associated mutations
result in amino acid substitutions of highly conserved residues in helices contributing to the flavin–adenine dinucleotide
(FAD)-binding domain of TXNRD2. Functional analysis of both mutations in Txnrd2
.....................................................................................................................................................................................
2/2 mouse fibroblasts
revealed that contrasting to wild-type (wt) Txnrd2, neither mutant did restore Txnrd2 function. Mutants even
impaired the survival of Txnrd2 wt cells under oxidative stress by a dominant-negative mechanism.
† These authors contributed equally to this work.
‡ These authors are last authors of this work.
* Corresponding author. Tel: +49(0) 2162/104 2220, Fax: +49(0) 2162/104 2370, Email: beckerath@dhm.mhn.de (N.B.); Tel: +49(0) 89 3187 4608, Fax: +49(0) 89 3187 4288,
marcus.conrad@helmholtz-muenchen.de (M.C.)
}
Present address. DZNE–Deutsches Zentrum für Neurodegenerative Erkrankungen and Institute of Developmental Genetics, Helmholtz Zentrum München, German Research
Center for Environmental Health, Neuherberg 85764, Germany
Published on behalf of the European Society of Cardiology. All rights reserved. & The Author 2011. For permissions please email: journals.permissions@oup.com.
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Conclusion For the first time, we describe mutations in DCM patients in a gene involved in the regulation of cellular redox state.
TXNRD2 mutations may explain a fraction of human DCM disease burden.
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Keywords Dilated cardiomyopathy † Genetics † TXNRD2
Introduction
Dilated cardiomyopathy (DCM) is a frequent cause of congestive
heart failure and is the most common diagnosis in patients undergoing
heart transplantation. According to systematic studies, famil-
1 – 3
ial transmission is observed in about 20–30% of DCM cases.
Rare mutations in more than 20 different disease genes, most of
them encoding for structural proteins of cardiomyocytes, have
been identified. 4 – 7 Mutations in known disease genes, though,
explain ,20% of inherited cases. 8
Thioredoxin reductases are essential components of the thioredoxin
system 9 and are therefore crucial for the control of cellular
redox balance. They are selenocysteine (Sec)-containing, homodimeric
flavoenzymes that maintain thioredoxins, small proteins that
catalyse redox reactions, in their reduced state using the reducing
power of NADPH. 10 Three mammalian thioredoxin reductases
exist; a cytosolic (TXNRD1), a mitochondrial (TXNRD2), and a
testis-specific thioredoxin reductase (TXNRD3). The Sec-residue,
encoded by a UGA codon, is the penultimate amino acid of the Cterminal
catalytic centre of all thioredoxin reductases and is essential
for enzyme activity. The premature termination of protein synthesis
at the UGA codon is prevented by a stem-loop-like
structure, the selenocysteine insertion sequence (SECIS) element
located in the 3 ′ UTR. 11,12 Cardiac energy requirement is met to
a large extent by oxidative phosphorylation in mitochondria that
are highly abundant in cardiac myocytes. 13 The mitochondrial
thioredoxin reductase (TXNRD2), along with mitochondrial thioredoxin
and peroxiredoxins III and V, is of paramount importance
for mitochondrial scavenging of reactive oxygen species (ROS). 9,10
It is well established that excessive ROS causes oxidative stress
and cell death. 14 On the other hand, compelling evidence has
established a role for ROS as modulators of intracellular signalling
cascades. 15 Beyond providing protection against ROS, thioredoxins
are known to inhibit or activate apoptotic signalling molecules like
apoptosis signal-regulating kinase 1 and Ras or transcription factors
like NF-kB. 16 We showed that glutathione (GSH) peroxidase 4,
along with GSH, senses and translates oxidative stress into a distinct
cell death signalling cascade involving the activation of 12/
15-lipoxygenase and apoptosis-inducing factor. 17
Recently, we generated and characterized transgenic mice deficient
for Txnrd2. 18 Ubiquitous inactivation resulted in embryonic death of
anaemic embryos exhibiting marked thinning of the ventricular heart
walls. Analysis of fibroblasts isolated from Txnrd2 2/2 embryos
revealed a critical role for Txnrd2 in the removal of toxic ROS
species. Heart-specific inactivation of Txnrd2 resulted in a phenotype
reminiscent of human DCM with dilatation of heart chambers and thinning
of ventricular walls and death shortly after birth. 18 This study set
out to search for and characterize rare TXNRD2 mutations associated
with DCM in humans.
Methods
DCM patients
From January 1996 to July 2004, we collected blood from 227 consecutive
patients with DCM at three participating centres in
Germany: Deutsches Herzzentrum München, 1. Medizinische Klinik,
Klinikum rechts der Isar, München, and Zentrum für Innere Medizin,
Klinikum Garmisch-Partenkirchen. Cardiac catheterization and echocardiography
was performed in all patients. Patients’ charts were
used as the main source for clinical information. Diagnosis of DCM
was based on the ‘Guidelines for the study of familial dilated cardiomyopathies’.
19 Patients were included if they had an ejection fraction
of the left ventricle (LV) of ,45% and an LV end-diastolic diameter
of .117% of the predicted value corrected for age and body
surface area according to the equation of Henry et al. 20 Patients
with coronary heart disease (.50% stenosis of at least one coronary
artery or a major branch), a history of severe systemic arterial hypertension
(arterial blood pressure .160/100 mmHg documented at
repeated measurements), myocarditis (suspected or confirmed), persistent
high-rate supraventricular arrhythmias, systemic disease, pericardial
disease, congenital heart disease, or cor pulmonale were
excluded. All patients had given written informed consent for participation
in the study. The investigation conforms to the principles outlined
in the Declaration of Helsinki and was approved by the
institutional Ethics Committee.
General population control sample
D. Sibbing et al.
Between 1999 and 2001, we conducted an epidemiological survey of
the general population living in or near the city of Augsburg,
Germany (KORA S4). 21 This was the fourth in a series of populationbased
surveys originating from our participation in the World Health
Organisation (WHO) Multinational MONItoring of trends and determinants
in CArdiovascular disease (MONICA) project. The study
population consisted of residents of German nationality born
between 1 July 1925 and 30 June 1975 and identified through the registration
office. A sample of 6640 subjects was drawn with 10 strata of
equal size according to gender and age. Following a pilot study of 100
individuals, 4261 individuals (66.8%) agreed to participate in the survey,
which were ethnic Germans with very few exceptions (.99.5%).
During 2002 and 2003, we reinvestigated a subsurvey of 880
persons specifically for cardiovascular diseases. Seven hundred and
two individuals from that subsurvey were selected as population-based
controls. In nineteen (2.7%) individuals, an ejection fraction of the LV
of ,45% was determined by echocardiography or symptoms/signs of
heart failure were observed. The remaining 683 individuals were used
as a DCM and congestive heart failure-free control sample. Blood
samples were drawn after informed consent had been obtained.
DNA extraction and DNA sequencing
Peripheral venous whole blood samples were collected using EDTAvials
(Sarstedt, Numbrecht, Germany). DNA was extracted from
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TXNRD2 and dilated cardiomyopathy 1123
these samples using the commercially available QIAamp DNA Blood
Mini Kit (Qiagen, Hilden, Germany).
A randomly selected subset of 96 patients of the entire DCM study
population was subjected to capillary Sanger sequencing. Primers,
flanking the 17 coding exons plus the 3 ′ SECIS (exon 18) element of
TXNRD2, were designed using the ExonPrimer software based on
Primer3 (http://primer3.sourceforge.net/) according to the human
genome build hg17. Amplifications were conducted following standard
polymerase chain reaction (PCR) protocol procedures. PCR products
were purified with both Exonuclease I and Shrimp alkaline phosphatase
(Fermentas, St Leon-Rot, Germany) to digest the remaining primers
and to reduce the remaining dNTPs. Subsequent cycle sequencing
was performed including the respective forward and reverse primers
and BigDye-Terminator 3.1 (Qiagen). Thereafter, products were
cleaned by DyeEx (Qiagen). Both forward and reverse strands were
sequenced for all 18 TXNRD2 exons in the population of 96 patients.
Sequencing was performed with an ABI 3730 Automated Sequencer
(Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s
recommendations. Results of sequencing were analysed with
Mutation Surveyor v3.00 (Softgenetics, State College, PA, USA).
Matrix-assisted laser desorption/
ionization-time-of-flight mass spectrometry
genotyping
All synonymous and non-synonymous exonic sequence variants identified
by direct sequencing in the population of 96 DCM patients were
subsequently genotyped in all DCM patients (n ¼ 227) and in the
KORA controls (n ¼ 683) using matrix-assisted laser desorption/
ionization-time-of-flight mass spectrometry (MALDI-TOF, Autoflex
HT, Sequenom, San Diego, CA, USA) in a 384-well format using the
standard protocols supplied by the manufacturer as described previously.
22 Genotype-analyses were performed with the Sequenom
MassARRAY Typer Analyzer software v3.4.0.6 (Sequenom).
Cloning of full-length wild-type Txnrd2,
generation of both Txnrd2 mutants, and
cloning into the lentiviral expression system
Vector pGEM-Teasy-Txnrd2 was a kind gift from Dr Antonio Miranda-
Vizuete (Universidad Pablo de Olavide, Sevilla, Spain). The vector contained
most parts of the 5 ′ region of murine Txnrd2 cDNA including
the mitochondrial leader sequence; however, it lacked the 3 ′ UTR
including the SECIS element. The missing sequence was amplified by
PCR from mouse liver cDNA 18 with the primers Oligo-SECIS-BglII-for
(5 ′ -TCTCAGAGATCTGAGAAGATGTGGATGGAAC-3 ′ ) and Oligo-
SECIS-rev (5 ′ -GTTTGAACCCCTGGCATTTCTAGAGCACT-3 ′ ). The
resulting 160 bp PCR product was purified, cloned into pDrive via PaeI
and BglII (Qiagen) and sequenced. The 3 ′ region of Txnrd2 was cloned
via PaeI and BglII in pGEM-T-Easy-Txnrd2 (4702 bp), yielding
pGEM-T-Easy-Txnrd2 [in the following referred to as wild-type (wt)].
Site-directed PCR mutagenesis using pGEM-T-Easy-Txnrd2 as a template
was performed to generate the Txnrd2-A59T and Txnrd2-G375R
mutant forms. Codons were chosen according to the highest murine
codon usage of the respective amino acid. The Ala codon (GCC) at
position 175 of Txnrd2 gene was replaced with Thr (ACC) using the
primer pair Oligo-Txnrd2-A59T-for (5 ′ -AGCGGGAATCGATTATAA
AGAT-3 ′ )/Oligo-Txnrd2-A59T-rev (5 ′ -GCCAGAAGCTTTCTTGCCT
TGATAGC-3 ′ ); the Gly codon (GGG) at position 375 was mutated
to Arg (AGG) with the primer pair Oligo-Txnrd2-G375R-for
5 ′ -GCTATCAAGGCAAGAAAGCTTCTGGC-3 ′ )/Oligo-Txnrd2-G375Rrev
(5 ′ -GCCAGAAGCTTTCTTGCCTTGATAGC-3 ′ ). Mutations
were verified by sequencing. All three forms were additionally
tagged by a novel N-terminal TAPe (tandem affinity purification tag
enhanced) 23 for detection of the tagged proteins with a FLAG-specific
antibody in immunocytochemistry.
The lentiviral expression vector 442L1 17 was used for efficient gene
transfer and expression of wt Txnrd2 and the two mutant variants in
mouse embryonic fibroblasts (MEFs). XhoI and EcoRI were used
for digestion of 442L1 (7870 bp) and pcDNA3-NTAPe-Txnrd2
(1992 bp). Two shorter fragments carrying the mitochondrial
NTAPe version of Txnrd2 were transferred into the backbone of
442L1, yielding the different NTAPe mitochondrial Txnrd2 forms. In
any of these vectors, the VENUS nuclear membrane anchor protein
in the bicistronic expression cassette was replaced by the puromycin
acetyltransferase gene from the plasmid 442L1-NTAPe-Txnrd1 using
BsrGI and SnaBI. This allowed stable expression of the various forms
under puromycin selection.
Isolation, maintenance of mouse embryonic
fibroblasts, and stable expression of the
different Txnrd2 forms in mouse embryonic
fibroblasts
Mouse embryonic fibroblasts were isolated from E12.5 Txnrd2 2/2 and
Txnrd2 +/+ embryos and cultured in DMEM supplemented with 10%
FCS, 1% glutamine, 50 U/mL penicillin G, and 50 mg/mL streptomycin
as described. For stable expression of wt Txnrd2, Txnrd2-A59T, and
Txnrd2-G375R in wt and knockout cells in a bicistronic manner, a thirdgeneration
lentiviral expression system was utilized as described. 17
Transduced cells were selected with puromycin (1 mg/mL) for at
least 3 weeks prior to the start of the experiment.
Immunoblotting and production of an
antibody directed against murine Txnrd2
Cell lysis, protein determination, SDS–PAGE, and protein transfer
were performed as described. 17 A peptide sequence next to the Cterminus
of Txnrd2 (VKLHISKRSGLEPTVTG) lacking the three Cterminal
amino acids Cys, Sec, and Gly was used to raise monoclonal
antibodies against Txnrd2 in rats. The peptide was obtained from
Peptide Specialty Laboratories (Heidelberg, Germany) and was
coupled to ovalbumin (OVA) or bovine serum albumin (BSA) at the
C-terminus (peptide-OVA/KLH).
Immunocytochemistry and confocal
microscopy
Mouse embryonic fibroblasts were seeded onto cover slips in six-well
cell culture dishes at ≏45 000 cells per well, cultured for 24 h in standard
DMEM and then fixed for 15 min in 2% PFA solution. Cells were
washed twice with PBS and then permeabilized by treatment with
0.15% Nonidet P-40 (NP-40) in PBS for 3 min. Unspecific antibody
binding was minimized by washing three times in PBS+ [PBS containing
1% (w/v) BSA, 0.15% (w/v) glycine]. After incubation of cells over night
with the primary antibody (FLAG; Sigma-Aldrich, Deisenhofen,
Germany; F1804 and Prx III; LabFrontier; LP-PA0030) at 48C in a
humidified chamber, cells were washed with PBS, treated twice with
PBS/NP-40 (0.15%), and transferred to PBS+. Cells were then incubated
with fluorophore-labelled secondary antibodies (anti-Mouse-
Alexa Fluor 568; A-10037 and anti-Rabbit-Alexa Fluor 488 A-21441;
Invitrogen, Karlsruhe, Germany) in the dark for 45 min. Cells were
then washed twice in PBS/NP-40 (0.15%) and twice in PBS. Finally,
DAPI solution (1:10 000 in PBS) was briefly added, and cells were
washed twice in PBS and mounted in mounting medium (Dako,
Glostrup, Denmark). Cover slips were sealed with nail polish to
prevent drying and they were kept overnight at 48C in the dark.
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Confocal microscopy was performed with a Leica DM IRBE microscope
(500 nm excitation and 550 nm emission), a Leica TCS SP2
scanner, and Leica Confocal software (Leica Microsystems GmbH,
Wetzlar, Germany). Pictures were processed by Adobe Photoshop
CS3. Processing included the removal of unspecific background and
addition of false colours (blue for DAPI, green for Alexa488, and red
for Alexa568) as well as the assembly of an overlay of a representative
optical section from each cell. Scale bars included represent 10 mm.
Treatment of mouse embryonic fibroblasts
with L-buthionine sulfoximine and
determination of cell viability
Cells were treated with increasing concentrations of L-buthionine sulfoximine
(BSO; Sigma-Aldrich). The cell number was determined 48 h
later by trypan blue exclusion as described previously. 18
Transmission electron microscopy
Cells were harvested and fixed in 2.5% glutaraldehyde in 0.1 M sodium
cacodylate buffer (pH 7.4; Electron Microscopy Sciences, Hatfield, PA,
USA) and embedded in epoxy resin (epon 812; Electron Microscopy
Sciences). Ultrathin sections were examined with an EM 10 CR transmission
electron microscope (Carl Zeiss, Inc.). For image acquisition, a
MegaView III camera system (Olympus) was used.
Statistical analyses
Statistical analysis of patients and controls was performed with the
STATA SE 8.0 Statistics/Data analysis package (StataCorp LP, TX,
USA). Genotypes were compared between patients and controls
with two-sided x 2 test. Statistical analysis of the cellular studies was
performed using SigmaStat 3.1 (Systat Software GmbH, Erkrath,
Germany). Within each panel, wt and knockout cells expressing
empty vector (mock), wt Txnrd2, and the different mutants were compared
using all pairwise multiple comparison procedures (Holm–Sidak
method). P-values ,0.05 were considered as significant.
Homology modelling
We modelled the structure of human TXNRD2 including FAD and
NADPH according to the crystal structure of human TXNRD1
(monomers C and D, 22) 24 and murine Txnrd2, 25 using the Swiss-
Model automated comparative protein modelling server (http://
swissmodel.expasy.org/).
Results
Study populations
To identify disease-associated TXNRD2 mutations, we studied a
cohort of 227 DCM patients and 683 individuals from a general
population sample. The baseline characteristics of patients (n ¼
227) and the general population sample (n ¼ 683) are shown in
Table 1. The mean (+SD) ejection fraction in DCM patients was
26.9 + 9.5 vs. 65.5 + 11.9% in the general population sample.
Sequencing of all exons and the SECIS region of TXNRD2 in a
subset of the DCM patients (n ¼ 96) yielded seven synonymous
and seven non-synonymous TXNRD2 variants (Table 2). Genotype
distributions of non-synonymous variants in the DCM patients and
the general population sample were not significantly different
(Table 3).
D. Sibbing et al.
Table 1 Clinical characteristics of dilated
cardiomyopathy patients and control individuals from
the general population sample
DCM patients Controls
(n 5 227) (n 5 683)
................................................................................
Age (years) 59.7 + 12.8 57.4 + 12.4
Women 50 (22.0) 342 (50.8)
EF (%) 26.9 + 9.5 65.5 + 11.9
LVEDD (mm) 67.3 + 8.3 47.5 + 6.4
Family history for DCM
(+/2/unknown)
34/68/125
Mitral regurgitation 180 (79)
Left bundle branch block 54 (23.8)
Atrioventricular block 27 (11.9)
Previous pace maker
implantation
17 (7.5)
Previous ICD implantation 21 (9.3)
Previous heart
transplantation
13 (5.7)
Data are presented as mean + standard deviation or number of patients (%).
DCM, dilated cardiomyopathy; EF, ejection fraction; LVEDD, left ventricular
end-diastolic diameter; ICD, implantable cardioverter defibrillator.
Table 2 Genotype distributions of synonymous and
non-synonymous exonic TXNRD2 variants derived from
forward and reverse sequencing of 96 of the 227 dilated
cardiomyopathy patients
Nucleotide Exon Genotype distribution
................................................................................
175G . A 3 GG 95 AG 01 AA 00
177C . T 3 CC 20 CT 57 TT 19
196G . T 3 GG 41 GT 46 TT 09
762C . T 10 CC 94 CT 02 TT 00
816C . T 11 CC 94 CT 02 TT 00
858G . C 11 GG 95 GC 01 CC 00
895A . C 11 AA 56 AC 38 CC 02
933C . T 11 CC 95 CT 01 TT 00
1077C . T 12 CC 95 CT 01 TT 00
1101G . A 13 GG 95 AG 01 AA 00
1109C . T 13 CC 56 CT 37 TT 03
1124G . A 13 GG 95 AG 01 AA 00
1150G . A 13 GG 92 AG 04 AA 00
1206G . A 14 GG 65 AG 26 AA 05
Detected variants in n ¼ 96 DCM patients with nucleotide and exon position
within the TXNRD2 gene. Genotype distributions of the detected variants showed
no significant deviation from the Hardy–Weinberg equilibrium (P . 0.05 for all
variants). Genotypes are highlighted in bold letters in the table.
Mutations in dilated cardiomyopathy
cases
We identified two novel amino acid residue-altering TXNRD2
mutations [175G . A (Ala59Thr ¼ A59T) and 1124G . A
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TXNRD2 and dilated cardiomyopathy 1125
Table 3 Genotype distributions of non-synonymous TXNRD2 variants in dilated cardiomyopathy patients and controls
Nucleotide Amino acid Genotype distributions
...........................................................................................
P-value
DCM patients, n 5 227 (CR) Controls, n 5 683 (CR)
...............................................................................................................................................................................
175G . A Ala59Thr GG 222 AG 2 AA 0 (98.7%) GG 674 AG 0 AA 0 (98.6%) n.d.
1124G . A Gly375Arg GG 216 AG 1 AA 0 (95.6%) GG 670 AG 0 AA 0 (98.1%) n.d.
196G . T Ala66Ser 1
GG 101 GT 100 TT 26 (100%) GG 282 GT 314 TT 76 (98.4%) 0.64
858G . C Arg286Ser GG 224 GC 2 CC 0 (99.6%) GG 677 GC 2 CC 0 (99.4%) 0.26
895A . C Arg299Ser 2
AA 149 AC 70 CC 8 (100%) AA 450 AC 200 CC 26 (99.0%) 0.88
1109C . T Ile370Thr 3
CC 135 CT 81 TT 11 (100%) CC 353 CT 233 TT 34 (90.8%) 0.49
1150G . A Gly384Ser GG 216 GA 6 AA 0 (97.8%) GG 543 GA 5 AA 0 (80.2%) 0.06
Detected amino acid exchanging variants in the entire DCM population (n ¼ 227) and n ¼ 683 controls from a general population sample (KORA S4). CR, call rate of MALDI-TOF
genotyping. n.d., not determined. Genotype distributions of the detected variants showed no significant deviation from the Hardy–Weinberg equilibrium in the controls (P . 0.05
for all variants). RefSNP accession IDs (rs numbers) of previously described variants: 1 rs5748469, 2 rs5992495, 3 rs2073752. Genotypes are highlighted in bold letters in the table.
Figure 1 Identification of dilated cardiomyopathy-associated TXNRD2 mutations (marked with arrows). (A) Sequence electropherogram
from Patient 2, who was a heterozygous carrier of the 175G . A (Ala59Thr, A59T) mutation. He was also a heterozygous carrier of the synonymous
variant 177C . T (Ala59Ala) (asterisks). (B) Sequence electropherogram from Patient 3, who was a heterozygous carrier of the
1123G . A (Gly375Arg, G375R) mutation.
(Gly375Arg ¼ G375R)] in three heterozygous carriers of the 227
patients (3/227 ¼ 1.3%). Both A59T and G375R were not
observed in the general population KORA S4 sample. The first
mutation (A59T) results in a substitution of alanine by threonine
at residue 59 of the major splice isoform (isoform 1) of TXNRD2
and was identified twice (2/227 ¼ 0.88%) in the 227 DCM patients.
Figure 1A shows a sequence electropherogram of one of the A59T
mutation carriers. Both patients carrying this mutation were not
knowingly related. The second mutation (G375R) results in a substitution
of glycine by arginine at residue 375 of the major splice
isoform (isoform 1) of TXNRD2 and was found in one of the
227 DCM patients (1/227 ¼ 0.44%). Figure 1B shows a sequence
electropherogram of the G375R mutation carrier. Clinical characteristics
of the three patients with novel TXNRD2 mutations that
were only found in DCM patients are displayed in Table 4. We
obtained all available information about the three index patients
and their families by contacting the patients and their relatives by
phone and by scheduling visits for patients and relatives in our outpatient
clinic. In addition, genetic analyses in search of the mutation
carried by the index case in relatives were performed whenever
possible: Patient 1 (A59T mutation carrier) was childless and died
at the age of 68. The mother of Patient 1 died at the age of 75
due to congestive heart failure. The father had a negative history
for cardiovascular disease and died in World War II. The only
sister of Patient 1 had atrial fibrillation and died of sudden
cardiac death at the age of 69. No family members were available
for clinical assessment and genetic analyses. Patient 2 (A59T
mutation carrier) had an entirely negative family history of DCM
by hearsay. The patient was childless, had no siblings, and died at
the age of 65. No family members were available for clinical assessment
and genetic analyses. Patient 3 (G375R mutation carrier) was
childless as well and also had a negative family history for DCM. He
died at the age of 83. Two half-sisters and two daughters of the
half-sisters were available for clinical assessment including physical
examination, electrocardiogram, and ultrasound echocardiography.
Clinical assessment, in particular LV size and function, was normal
in all four relatives. Genetic analyses showed that the four relatives
do not carry the G375R mutation. The discovery of two heterozygous
mutations in three DCM patients raised the questions
whether the mutations were functionally silent or important, i.e.
abolishing or impairing the function of the enzyme. In case the variants
were functionally important, the question would have to be
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Table 4 Characteristics of the three patients carrying dilated cardiomyopathy-associated TXNRD2 mutations
Patient TXNRD2 mutation Family history LVEDD EF (%) Coronary ECG Outcome
(gender)
of DCM (mm)
angiography
...............................................................................................................................................................................
Patient 1 (male) 175G . A (Ala59Thr) Positive 64 18 Normal Left bundle branch block Died at the age of 68
Patient 2 (male) 175G . A (Ala59Thr) Negative 66 25 Normal AV block 1st degree Died at the age of 65
Patient 3 (male) 1124G . A (Gly375Arg) Negative 68 27 Normal Right bundle branch
block
Died at the age of 83
DCM, dilated cardiomyopathy; EF, ejection fraction; LVEDD, left ventricular end-diastolic diameter; ECG, electrocardiogram.
answered, whether the mutations act in a dominant-negative
fashion, thus impairing the function of the heterozygous wt allele.
The protein structure of TXNRD2 and
the FAD-binding domain
To gain better insight into a possible adverse role of the mutations
for TXNRD2 function, we followed two complementary
approaches: structural modelling of TXNRD2 and integrated analysis
of the evolutionary conservation of FAD-binding domains in
thioredoxin reductases and GSH reductases (GR), which are evolutionary
highly related enzymes. Although the crystal structure of
murine Txnrd2 as well as rat and human Txnrd1 have been
resolved, 24 – 26 the molecular nature of the FAD-binding domain
has remained poorly defined in thioredoxin reductases. The FADbinding
domain is essential for enzyme function since the reducing
equivalents from NADPH are first transferred to FAD, from where
they are passed on to the N-terminal redox-reactive centre within
the same molecule and eventually to the Sec-containing C-terminal
catalytic site of the second monomer. 27 Close inspection of the
structure revealed that the human TXNRD2-binding pocket for
FAD is formed by the N-terminal parts of four helices (h1: 49–
61, h2: 92–106, h4: 228–240, and h6: 368–382), the N-terminally
adjacent amino acids, and eight non-contiguous, non-helical short
stretches of amino acids. The mutations A59T and G375R are
located in helices 1 and 6, respectively, that both contribute to
the formation of the FAD-binding pocket (Figure 2A).
As FAD binding is common to thioredoxin reductases and GR,
we reasoned that amino acids conserved among both types of
enzymes would participate in FAD binding. To address this, we
made two sequence alignments, one comparing human thioredoxin
reductase 2 with human GR (Figure 2B), and a second that
included 36 thioredoxin reductases (including 13 thioredoxin
reductase 2 genes) and 10 GR genes (see Supplementary material
online, Figure S1 and Table S1). Amino acids shared among all or
almost all enzymes across a large number of species were
defined and marked in the alignment of TXNRD2 and GR. The
overlay of the structural and evolutionary analysis clearly demonstrated
that the regions participating in FAD binding are evolutionary
highly conserved. We then compared the FAD-binding domain,
which had been previously determined for GR, 28 with the structural
information obtained from the analysis of the modelled
TXNRD2 structure. Notably, the FAD-binding domain of GR as
defined by Schulz et al. 28 is virtually identical to that of TXNRD2
as defined by inspection of the TXNRD2 structure (Figure 2B).
The four helices as well as the non-contiguous, non-helical
stretches of amino acids are highly conserved in evolution. In
GR, a fifth helix participates in the formation of the FAD-binding
pocket. This helix is somewhat disturbed in TXNRD2 (amino
acids 208–212), but the corresponding conserved residues contribute
to the binding pocket in a similar manner.
Implications of the mutations for
TXNRD2 protein structure
D. Sibbing et al.
G375R is located in the middle of helix 6. Not only is arginine
much larger than glycine, it is also a polar amino acid. Our
model indicates that the charged side chain points into the core
of the enzyme (Figure 2A). This makes it likely that the bulky
charged side chain disrupts the hydrophobic interaction with the
neighbouring helix 1 and thus destroys the FAD-binding pocket.
A59T (Figure 2A) is located at the end of helix 1 and is thus not
directly involved in FAD binding. Our sequence alignments (see
Supplementary material online, Figure S1) including 46 enzymes
revealed only alanine and valine at this position. Two explanations
may be given that are not mutually exclusive. First, an unpolar
amino acid at this position may be required in this region. In our
model, the side chain of threonine causes clashes with A55,
located on helix1 or with V65 in the adjacent b-sheet
(Figure 2A). Proper backfolding of this b-sheet towards FAD is
required for hydrogen bonding of D69 to the ribose of FAD (as
shown for E50 in GR 28 ) and for bringing helix 2 into the proper
position relative to the other helices forming the FAD-binding
pocket. Furthermore, the polar threonine may disturb the movement
of the second redox centre, which is located on the flexible
C-terminal part of the second subunit.
In addition, we took a close look at the position of the five nonsynonymous
variants that were identified in patients as well as in controls.
Four of them (A66S, R286S, S299R, and G384S) are located
more in peripheral regions of TXNRD2 facing the solvent and are
neither involved in FAD/NADPH binding nor enzymatic function
(Figure 2A). The fifth non-synonymous variant I370T is located at
the beginning of helix 6, but it points away from FAD and is thus
not involved in FAD binding. It points into a pocket formed by residues
P365, L367, M390, Y392, and V395 of one subunit and V494’ of
the other. All amino acids of this pocket are strictly conserved in
mouse Txnrd2, which harbours threonine at this position. This
shows that threonine is tolerated. Moreover, amino acid sequence
alignment of orthologous TXNRD2 sequences revealed that 8 of
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TXNRD2 and dilated cardiomyopathy 1127
Figure 2 The identified mutations localize to highly conserved amino acid residues in the FAD-binding domain of TXNRD2. (A) Overview of
one TXNRD2 molecule (grey). The surfaces of FAD and NADP are shown in yellow and blue, respectively. Amino acid variants [red (observed
in patients); blue and green (observed in patients and controls, not tolerated or tolerated according to SIFT analysis, respectively)] and relevant
amino acids (grey) are shown as a ball and stick model. N- and C-termini are marked by N (grey) and C (orange). Molecular graphics images
were produced using the UCSF Chimera package. (B) Alignment between human thioredoxin reductase 2 and human glutathione reductase.
The alignment was performed using NCBI Blast. The numbering of amino acids is taken from the protein structures of mouse Txnrd2 (1ZDL)
and human glutathione reductase (3DJG). Amino acids shown in bold are conserved to more than 80% in 46 FAD- and NADPH-binding oxidoreductases
(36 thioredoxin reductases and 10 glutathione reductases) across a large number of species (see also Supplementary material online,
Figure S1). Helices in TXNRD2 are underlined in green and helices in glutathione reductases in yellow. Amino acids in close contact with FAD as
revealed by analysis of the modelled hTXNRD2 structure are shown in magenta. Corresponding amino acids forming the FAD-binding domain
of human glutathione reductase 28 are underlined in cyan. The binding pocket for FAD is formed by four conserved helices, amino acids Nterminally
adjacent to these helices, and eight non-helical, non-contiguous stretches of highly conserved amino acids. In glutathione reductases,
a fifth helix participates in the formation of the FAD-binding pocket. This helix is somewhat disturbed in TXNRD2 (amino acids 208–212), but
the corresponding conserved residues contribute to the binding pocket in a similar manner. The mutations G375R and A59T are shown as red
letters and the non-synonymous variants observed in patients as well as in controls in blue letters. Note that mutations G375 and A59T are
located in helices contributing to the formation of the FAD-binding pocket. Of the non-synonymous variants, only I370T is located in a helix
contributing to FAD binding, but threonine at this position is structurally tolerated and represents the evolutionary more ancient allele (see also
Supplementary material online, Figure S1, Figure S2, and Table S1).
11 species harbour threonine at this position, providing evidence
that threonine is the evolutionary older variant.
Taken together, G375 and A59 are highly conserved across a
wide range of species, whereas the five other non-synonymous
variants are conserved to a much lower degree in evolution and
can be predicted not to interfere with FAD binding (for a
summary, see Supplementary material online, Table S2).
Both mutations abolish the function
of Txnrd2
We reasoned that if the mutations were functionally silent and did
not impact on Txnrd2 function, they would be able to rescue the
phenotype of Txnrd2 2/2 cells in a manner similar to the wt Txnrd2
gene. To address this question experimentally, we cloned mouse
wt Txnrd2 and the two mutants Txnrd2-A59T and Txnrd2-G375R
into a bicistronic lentiviral vector and expressed them stably in
primary Txnrd2 2/2 MEFs. Immunoblotting of cellular lysates with a
Txnrd2-specific antibody showed that all three variants were
expressed in knockout MEFs, albeit, to varying extent that was
highly reproducible (Figure 3A). Double immunocytochemical staining
of these cells with a FLAG-specific antibody for the reconstituted
wt Txnrd2 and a peroxiredoxin III (Prx III)-specific antibody for mitochondria
29 revealed the expected mitochondrial localization of wt
Txnrd2 in Txnrd2 2/2 MEFs (Figure 3B). Likewise, localization of the
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1128
A59T mutant was mitochondrial in Txnrd2 2/2 MEFs as shown by
co-staining of Prx III and the TAPe-tagged mutant (Figure 3B). The
G375R mutant, however, was barely detectable immunocytochemically
in accordance with the data obtained by immunoblotting
(compare Figure 3A), rendering unequivocal assignment to a distinct
cellular compartment difficult. In Txnrd2 2/2 cells, this mutant also
appeared to be localized solely or predominantly in mitochondria
(Figure 3B). Next, we studied the function of both Txnrd2 mutants
D. Sibbing et al.
in Txnrd2 2/2 MEFs. To this end, we used an assay that is based on
the fact that Txnrd2 is indispensible for cell survival of fibroblasts
when the cells are depleted of GSH by treatment with the
g-glutamyl-cysteine-synthetase inhibitor BSO. 18 As illustrated in
Figure 3C, reconstitution of wt Txnrd2 in Txnrd2 2/2 cells fully
rescued cell death induced by GSH depletion. In contrast, cells
expressing the mutants A59T and G375R died at very low BSO concentrations
similarly to mock-transduced Txnrd2 2/2 cells
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TXNRD2 and dilated cardiomyopathy 1129
(Figure 3C). We conclude from this experiment that both mutations
abolish the function of Txnrd2.
Both mutants impair the function of
endogenous wild-type Txnrd2
Since Txnrd2 is enzymatically active as a homodimer and since all
three patients were heterozygous for the mutations, it was important
to see whether the mutant allele also impacts on the function
of the wt enzyme allele by a dominant-negative mechanism. To
address this question, the lentiviral vectors encoding wt as well
as the A59T and G375R Txnrd2 mutants were transduced into
Txnrd2 +/+ cells. Subcellular localization and cell survival in the
presence of BSO were monitored in the same manner as
described above for lentivirally transduced Txnrd2 2/2 cells.
Although wt Txnrd2 and the A59T mutant were expressed at
high level in Txnrd2 +/+ cells, the mutant G375R was barely detectable
by western blotting (Figure 4A) as well as immunofluorescent
staining (Figure 4B). Like in Txnrd2 2/2 cells, wt Txnrd2 and the
A59T mutant localized to mitochondria in Txnrd2 +/+ cells,
whereas the low expression of mutant G375R in Txnrd2 +/+ cells
precluded any conclusion regarding its subcellular localization
(Figure 4B). Although mock-transduced and wt Txnrd2-transduced
cells were resistant to GSH depletion over a wide range of BSO
concentrations, cell survival of A59T- and G375R-transduced
Txnrd2 +/+ cells was significantly impaired in the presence of
increasing BSO concentrations (Figure 4C). The fact that both
mutants had a similar impact on cell survival despite different
levels of expression suggests that mutant G375R is more toxic
and that a non-toxic threshold of expression is selected for
upon retroviral transduction. Notably, the introduction of the
mutant alleles into Txnrd2 +/2 cells would have been closer to
the in vivo situation in the patients. But, as it is impossible to
adjust the expression level to that of the endogenous Txnrd2
alleles in the patients in vivo (which has been unknown anyway),
it was of lesser importance whether one or two copies of the
wt allele have resided in the cells. Apparently, it is more difficult
to detect a dominant-negative effect in cells with two wt Txnrd2
copies left than in cells with one copy. Yet, the dominant-negative
effect was already visible in an unequivocal manner in cells harbouring
two wt alleles precluding the necessity of further breeding
and of establishing cell lines heterozygous for Txnrd2.
Ultrastructural analysis of cells expressing
the Txnrd2 mutants A59T and G375R
We then performed ultrastructural analyses to address the questions
(i) whether morphological differences in mitochondria can
be observed in knockout when compared with wt fibroblasts, similarly
to those observed in heart-specific Txnrd2 2/2 cardiomyocytes
ex vivo, 18 and (ii) whether the add-back of wt Txnrd2 or of
the different mutants would affect mitochondrial morphology
(Figure 5). We found that the size of mitochondria varied to a
greater extent in knockout (Figure 5B) when compared with wt
mitochondria (Figure 5A). Ultrastructural changes, ranging from
swelling and loss of cristae to deterioration of matrix and mitochondrial
membranes, were sometimes detectable in knockout
mitochondria (Figure 5B). The add-back of wt Txnrd2 and of the
different mutants, however, did not induce major changes in the
overall morphology of mitochondria (Figure 5A and B). From this,
we conclude that the functional parameters described in
Figures 3 and 4 are more sensitive and more readily quantifiable
than ultrastructural parameters.
Discussion
For the first time, we describe rare mutations in human TXNRD2 in
patients with DCM. Both identified mutations result in amino acid
substitutions in the highly conserved and functionally essential
FAD-binding domain of the enzyme. A major influence of
mutations located in FAD-binding domains on enzyme activity of
30 – 32
oxidoreductases has been repeatedly demonstrated.
Figure 3 Expression of wild-type (wt) Txnrd2 and the novel mutations (A59T and G375R) in Txnrd2 2/2 cells. Immunoblotting using a
Txnrd2-specific antibody (A) and confocal microscopy of immunocytochemical staining of transduced cells with an anti-Prx III-specific antibody
(Alexa488) for mitochondria, an anti-FLAG-specific antibody (Alexa568) for reconstituted wt Txnrd2 and the A59T and G375R mutants and a
nuclear counterstain with DAPI (B) revealed similar expression of wt Txnrd2 and A59T in both wild-type and knockout cells, whereas the
G375R mutant was only weakly expressed inTxnrd2 2/2 cells. (B) Wild-type Txnrd2 and the A59T mutant predominantly localized to mitochondria
[co-localization (yellow) of Prx III (green) and TAPe-tagged protein (red)] in knockout cells as shown in the false colour merge. The G375R
mutant was only weakly expressed [compare (A)], but appeared also to localize to mitochondria. Shown are optical sections acquired by confocal
microscopy of transduced cells stained with a nuclear DAPI counterstaining (blue), an anti-Prx III-specific antibody (Alexa488 shown in
green) and an anti-FLAG-specific antibody for TAPe-tagged Txnrd2 (Alexa568 shown in red). Co-localization is indicated by a yellow colour
arising from Prx III (green) and TAPe-tagged Txnrd2 (red) staining of knockout and wild-type cells as shown in the false colour merge. Mock ¼
empty virus-transduced cells. Scale bars, 10 mm. (C) The novel mutations did not restore the function of Txnrd2 in Txnrd2 2/2 fibroblasts under
oxidative stress. Both transduced mutants A59T and G375R failed to rescue cell death of Txnrd2 2/2 cells under glutathione depletion induced
by increasing L-buthionine sulfoximine concentrations. Txnrd2 2/2 cells transduced with the mutant forms were even more sensitive to glutathione
depletion than mock-transduced Txnrd2 2/2 cells. Cell numbers were determined by trypan blue exclusion 48 h after treatment with the
indicated L-buthionine sulfoximine concentrations. Shown are results of one out of three independent experiments with similar results. Cell
numbers are presented as relative to the number of mock-transfected Txnrd2 +/+ cells in the absence of L-buthionine sulfoximine. Bars represent
means and error bars standard deviations derived from cell counting of three different wells for each condition. P-values ,0.001
were considered as highly significant and are marked with two asterisks. Values between mock-transfected Txnrd2 +/+ cells (black bars) and
Txnrd2 2/2 cells (empty bars) treated with different concentrations of L-buthionine sulfoximine were always highly significant (not shown for
a simpler illustration).
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1130
D. Sibbing et al.
Figure 4 Expression of wild-type (wt) Txnrd2 and the novel mutations (A59T and G375R) in Txnrd2 +/+ cells. Immunoblotting using a
Txnrd2-specific antibody (A) and confocal microscopy of immunocytochemical staining of transduced cells with an anti-Prx III-specific antibody
(Alexa488) for mitochondria, an anti-FLAG-specific antibody (Alexa568) for reconstituted wild-type (wt) Txnrd2 and the A59T and G375R
mutants and a nuclear counterstain with DAPI (B) revealed comparable expression of wt Txnrd2 and A59T in wild-type cells, whereas the
G375R mutant was barely detectable in Txnrd2 +/+ cells. (B) Wild-type Txnrd2 and the A59T mutant predominantly localized to mitochondria
in Txnrd2 +/+ cells as shown in the false colour merge. The low expression of mutant G375R in Txnrd2 +/+ cells, however, precluded any conclusion
regarding its subcellular localization (for details regarding staining and illustration, see Figure 3 and the Methods section). Mock ¼ empty virustransduced
cells. Scale bars, 10 mm. (C) Cell survival of Txnrd2 +/+ cells transduced with A59T or G375R was impaired in response to increasing
L-buthionine sulfoximine concentrations, indicating that both forms harbour a dominant-negative function. The fact that both mutants had a
similar impact on cell survival despite different levels of expression suggests that mutant G375R is more toxic and that a non-toxic threshold of
expression is selected for upon retroviral transduction Bars represent means and error bars standard deviations derived from cell counting of
three different wells for each condition. P-values ,0.05 were considered as significant and are marked with an asterisk, and P-values ,0.001
were considered as highly significant and are marked with two asterisks (for experimental details and statistical analysis, see the legend of Figure 3).
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TXNRD2 and dilated cardiomyopathy 1131
Figure 5 Ultrastructural analysis of Txnrd2 +/+ and Txnrd2 2/2 cells expressing wt Txnrd2 and the different Txnrd2 mutants. (A) Empty virustransduced
(mock), wt Txnrd2-, and A59T- and G375R-transduced Txnrd2 +/+ mouse embryonic fibroblasts did not reveal major differences in
mitochondrial structures. (B) Although the submitochondrial structures (cristae) were less distinct in Txnrd2 2/2 mouse embryonic fibroblasts
compared with wild-type cells, the add-back of the different Txnrd2 variants did not grossly change the overall mitochondrial structure. The
scale bar represents 500 nm.
Functional analysis of the two identified mutants reconstructed in
murine fibroblasts revealed that both forms do not rescue
Txnrd2 2/2 cells from cell death induced by GSH depletion and
that they exerted a dominant-negative effect when expressed in
Txnrd2 +/+ cells.
Regarding a possible causative role of the mutations for DCM,
two questions had to be answered: first, are the mutations rare
polymorphisms that are functionally silent or do they abolish or
impair the function of Txnrd2? And second, as the mutations
were heterozygous in all three patients, do they exert a dominant-
negative function on the wt enzyme? Both questions raise fundamental
issues regarding the enzymatic function of Txnrd2 that is
shared among different cell types and tissues and should therefore
be independent of the cellular model system. Apparently, the first
question could only be addressed in Txnrd2 2/2 cells, thus precluding
the use of cardiomyocytes. We reasoned that if the mutant
alleles could rescue the Txnrd2 2/2 phenotype in a manner
similar to the wt allele, the mutations would be functionally
silent. Conversely, a functional significance of the mutations
could be disclosed if the mutant alleles were not able to rescue
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1132
the phenotype of Txnrd2 2/2 cells as the wt Txnrd2 gene does. The
phenotype of Txnrd2 2/2 cells is defined as high sensitivity to GSH
depletion, a phenotype that is highly sensitive and well quantifiable.
The necessity to assess the function of Txnrd2 quantitatively in a
homogeneous population of cells precluded the use of primary
cells. Given these experimental constraints, we considered the
question whether conclusions drawn from fibroblasts might be
irrelevant or relevant for cardiomyocytes. At least two points
suggest that a phenotype observed in fibroblasts will also be relevant
for cardiomyocytes. First, cardiomyocytes are extremely
dependent on energy production by mitochondria, probably
more so than fibroblasts. Findings made in fibroblasts are therefore
likely to impact also on cardiomyocytes. Secondly, fibroblasts
express Txnrd1 as well as Txnrd2, whereas cardiomyocytes only
show low expression of Txnrd1 33 and depend on Txnrd2 for
reduction of thioredoxin. 18 If there is some redundancy between
the thioredoxin-1 and thioredoxin-2 system, which is suggested
by the phenotype of mouse knockouts in both systems, cardiomyocytes
should be functionally affected by mutations at least as
severely as fibroblasts.
We did not reconstruct or functionally test any of the five nonsynonymous
variants identified in both patients and controls to
rule out a major pathological effect by them. Structural considerations
combined with evolutionary studies on 46 aligned FADbinding
oxidoreductases (Figure 2A and B; see Supplementary
material online, Figure S1) including 13 Txnrd2 genes revealed
that A59 and G375 are located in two helices contributing to
the formation of the FAD-binding pocket. The mutations A59T
and G375R are predicted not to be tolerated (SIFT; see Supplementary
material online, Table S3) and to disrupt the formation
of the FAD-binding pocket. G375 is conserved in all FAD-binding
oxidoreductases across evolution that we have studied. In all but
two oxidoreductase genes, A59 is conserved and only two Drosophila
thioredoxin reductases harbour valine as another hydrophobic
amino acid at this position. Both mutations A59T and
G375R stand out in that they are unique across all in silico analyses
performed which discriminates them from the other five variants
and supports their causality (see Supplementary material online,
Table S3). None of the five non-synonymous variants observed
in patients and controls are conserved in evolution. Four of
them are located at the surface of the molecule and are thus irrelevant
for NADPH/FAD binding and enzymatic activity. The fifth
I370T is located in the same helix as G375R, but the side chain
points away from FAD. Most importantly, T370 is present at this
position in mouse Txnrd2 used for modelling human TXNRD2,
and sequence alignments have furthermore provided conclusive
evidence that threonine at this position is the evolutionary
ancient allele.
In summary, two mechanisms are likely to account for the
observed phenotype in the human DCM patients: first, increased
oxidative stress due to reduced expression of fully functional
TXNRD2. Even though heterozygosity of Txnrd2 (Txnrd2 +/2 )or
of mitochondrial thioredoxin (Txn2 +/2 ) has no gross effect on
mouse development or maturation, 18,34 gene dosage may contribute
to the capacity to cope with oxidative stress under challenge.
Secondly, increased oxidative stress due to a dominant-negative
effect of the toxic mutants impairing the function of the wt
protein that is plausible because thioredoxin reductases act as
homodimers. 27 All three patients were heterozygous carriers of
the two TXNRD2 mutations. Although having severely impaired
LV function when diagnosed, they died at rather advanced age.
This suggests that heterozygous carriage of the mutations has
resulted in a moderate phenotype and accumulation of cardiac
tissue damage during life that has long been compensated.
Similar to previous observations for other already established
DCM causing mutations in different genes, 4 – 7 TXNRD2 mutations
described here explain only a small fraction of overall disease
burden. As TXNRD2 is a Sec-containing enzyme, the presence
of selenium is essential for proper enzyme function. In this
context, TXNRD2 in addition to GPX1 may represent the link
between selenium deficiency and Keshan’s disease, an endemic cardiomyopathy
prevalent in China. 18,35,36
Limitations
The following limitations of our study must be acknowledged: due
to the fact that mostly rare mutations in more than 20 different
disease genes have been described in inherited cases of DCM,
we are unable to fully exclude the presence of already known
mutations in our study cohort. In addition, due to their advanced
age in none of the three patients with novel amino acid
residue-altering TXNRD2 mutations, parents were available for
study. Thus, the question of an inherited vs. a de novo mutation
and cosegregation within the family cannot be resolved in either
of them. The fact that we observed only three TXNRD2 mutation
carrying patients precludes to reliably describing a TXNRD2
mutant DCM subphenotype. The answer to this question will
have to await the identification of further TXNRD2 mutation carrying
patients in other cohorts and the present study may provide
the rationale for further investigations. Experimental limitations of
our study include the fact that cardiomyocytes would have been
the optimal model system. Yet, there are experimental constraints
regarding the use of cardiomyocytes and therefore we had to
choose the MEFs as the experimental system.
Conclusions
Our data underline the essential role of TXNRD2 for mitochondrial
redox homeostasis in cardiomyocytes and normal heart function
18 and point to a novel pathophysiological mechanism for heart
failure: failure of the cellular mechanisms that counterbalance the
production of ROS. For the first time, we describe mutations in
DCM patients in a gene involved in the regulation of cellular
redox state. TXNRD2 mutations may explain a fraction of
human DCM disease burden and further studies are needed to
corroborate the present results.
Supplementary material
Supplementary material is available at European Heart Journal
online.
Acknowledgements
D. Sibbing et al.
We thank Dr Antonio Miranda-Vizuete (Universidad Pablo de
Olavide, Sevilla, Spain) for providing the plasmid encoding the
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TXNRD2 and dilated cardiomyopathy 1133
murine Txnrd2 cDNA and Luise Jennen for excellent technical
assistance. We are most grateful to Stefan Rahlfs for his suggestion
to include glutathione reductases in the definition of the FADbinding
domain in TXNRD2.
Funding
This work has been supported by a grant from the Technische Universität
München (KKF 69-04) to N.B. and A.P. as well as by grants from
the German Research Foundation (DFG) Priority Programme 1087
‘Selenoproteins’ to G.W.B. and M.C. The KORA research platform
was initiated and financed by the Helmholtz Center Munich, German
Research Center for Environmental Health, which is funded by the
German Federal Ministry of Education and Research and by the
State of Bavaria. The work of KORA is supported by the German
Federal Ministry of Education and Research (BMBF) in the context of
the German National Genome Research Network (NGFN-2 and
NGFN-plus). Our research was also supported within the Munich
Center of Health Sciences (MC Health) as part of LMUinnovativ.
Additional funding was obtained by A.P. from the German National
Genome Research Network NGFN 01GR0803 and the BMBF
German Federal Ministry of Research BMBF 01EZ0874, T.M. German
National Genome Research Network NGFN and S.K. from German
National Genome Research Network NGFN BMBF 01GS0838;
Leducq Foundation 07-CVD 03, LMU Excellence Initiative. The sponsors
had no role in the design and conduct of the study; collection,
management, analysis, and interpretation of the data; or preparation,
review, or approval of the manuscript.
Conflict of interest: none declared.
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