Mutations in the mitochondrial thioredoxin reductase gene TXNRD2 ...

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$Heart failure with preserved ejection fraction - European Heart Journal$

1124 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 Downloaded from http://eurheartj.oxfordjournals.org/ by guest on June 7, 2013
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 Downloaded from http://eurheartj.oxfordjournals.org/ by guest on June 7, 2013
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Mutations in the mitochondrial thioredoxin reductase gene TXNRD2 ...

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