Titin gene mutations are common in families with both peripartum cardiomyopathy and dilated cardiomyopathy

Transcription

1 7 Titin gene mutations are common in families with both peripartum cardiomyopathy and dilated cardiomyopathy Karin Y. van Spaendonck-Zwarts Anna Posafalvi Maarten P. van den Berg Denise Hilfiker-Kleiner Ilse A.E. Bollen Karen Sliwa Mariëlle Alders Rowida Almomani Irene M. van Langen Peter van der Meer Richard J. Sinke Jolanda van der Velden Dirk J. Van Veldhuisen J. Peter van Tintelen * Jan D.H. Jongbloed * * The last two authors contributed equally Submitted Spaendonck.indd :00

2 ABSTRACT Aims Peripartum cardiomyopathy (PPCM) can be an initial manifestation of familial dilated cardiomyopathy (DCM). We aimed to identify mutations in families that could underlie their PPCM and DCM. Methods and Results We collected 18 families with PPCM and DCM cases from various countries. We studied the clinical characteristics of the PPCM patients and affected relatives, and applied a targeted next-generation sequencing (NGS) approach to detect mutations in 48 genes known to be involved in inherited cardiomyopathies. We identified 4 pathogenic mutations in 4/18 families (22%): 3 in TTN and 1 in BAG3. In addition, we identified 6 variants of unknown clinical significance that are likely to be pathogenic in 6 other families (33%): 4 in TTN, 1 in TNNC1, and 1 in MYH7. Measurements of passive force in single cardiomyocytes and titin isoform composition potentially support an upgrade of one of the variants of unknown clinical significance in TTN to a pathogenic mutation. Only 2/20 PPCM cases in these families showed recovery of left ventricular function. Conclusion Targeted NGS shows that potentially causal mutations in cardiomyopathy-related genes are common in families with both PPCM and DCM. This supports the earlier finding that PPCM can be part of familial DCM. Our cohort is particularly characterised by a high proportion of TTN mutations and a low recovery rate in PPCM cases. 152 Spaendonck.indd :00

3 Chapter 7 INTRODUCTION Peripartum cardiomyopathy (PPCM) is an idiopathic cardiomyopathy presenting with heart failure secondary to left ventricular systolic dysfunction towards the end of pregnancy or in the first months following delivery, where no other cause of heart failure is found. The left ventricle may not be dilated but the ejection fraction is nearly always reduced below 45%. 1 According to this recent definition, the time frame is not strictly defined, in contrast to previous definitions. 2-4 The severity of PPCM is highly variable, ranging from complete recovery to rapid progression to end-stage heart failure. PPCM affects 1:300 to approximately 1:3000 pregnancies, with geographic hot spots of high incidence such as in Haiti and Nigeria. 4,5 The precise mechanisms that lead to PPCM are not fully known. Several risk factors and possible underlying pathological processes have received attention, such as abnormal autoimmune responses, apoptosis, and impaired cardiovascular microvasculature. 5,6 Recent work into the pathogenesis of PPCM has shown involvement of a cascade with oxidative stress, the prolactin-cleaving protease cathepsin D, and the nursing hormone prolactin, which may lead to a target for a disease-specific therapy, namely pharmacological blockade of prolactin by bromocriptine. 7-9 In addition, involvement of cardiac angiogenic imbalance may explain why PPCM is a disease seen in late pregnancy and why pre-eclampsia and multiple gestation are important risk factors. 10 PPCM is probably caused by a complex interaction of more than one pathogenic mechanism. The large variation in incidence and clinical characteristics may reflect the involvement of specific mechanisms, or combinations thereof, in certain subgroups of PPCM. We and others recently reported that PPCM can be an initial manifestation of familial dilated cardiomyopathy (DCM), 11,12 indicating that, at least in a subset of cases, genetic predisposition plays a role in the pathophysiology of pregnancy-associated heart failure. Accordingly, Haghikia et al. reported a positive family history for cardiomyopathy in 16.5% (19/115) of PPCM cases from a German PPCM cohort. 13 So far, eight cases with underlying mutations in DCM-related genes have been published 11,12,14,15 and several other cases with familial occurrences of PPCM and DCM, as well as familial clustering of PPCM, have been reported Here, we describe our extensive genetic analysis using next-generation sequencing (NGS) technology to identify potentially causal mutations in families with both PPCM and DCM from various parts of the world. METHODS Subjects and Clinical Evaluation We collected a cohort of families with cases of both PPCM and DCM from various parts of the world (the Netherlands, Germany, and South Africa) and studied their clinical characteristics by reviewing medical reports. The local institutional review committees approved the study, and all participants gave their informed consent. 153 Spaendonck.indd :00

4 PPCM was diagnosed when a patient had an idiopathic cardiomyopathy presenting with heart failure secondary to left ventricular systolic dysfunction towards the end of pregnancy or in the first months following delivery, where no other cause of heart failure was found. 1 DCM was diagnosed when a patient had both a reduced systolic function of the left ventricle (left ventricular systolic ejection fraction <0.45) and dilation of the left ventricle (left ventricular end-diastolic dimension >117% of the predicted value corrected for body surface area and age) and only after other identifiable causes like severe hypertension, coronary artery disease, and systemic disease had been excluded. 25 If only one of the two criteria was fulfilled, the patient was labeled with mild DCM. If the family history suggested DCM in a relative but there were no medical reports to confirm this, the relative was labeled as having possible DCM. Familial PPCM/DCM was diagnosed when there were 2 affected family members, at least one with PPCM and one with DCM or sudden cardiac death (SCD) 35 years. Targeted Next-Generation Sequencing of 48 Cardiomyopathy-Related Genes Genomic deoxyribonucleic acid (DNA) was extracted from blood samples obtained from all the available PPCM patients and their affected relatives. Targeted NGS was performed in one or two affected relatives in the selected families (these individuals are marked with an arrow in Figures 1 and 2). We developed a kit based on Agilent Sure Select Target Enrichment for mutation detection in 48 genes (all exonic and ± 20 bp of exon-flanking intronic sequences) known to be involved in inherited cardiomyopathies (ABCC9, ACTC1, ACTN2, ANKRD1, BAG3, CALR3, CRYAB, CSRP3/MLP, DES, DMD, DSC2, DSG2, DSP, EMD, GLA, JPH2, JUP, LAMA4, LAMP2, LMNA, MYBPC3, MYH6, MYH7, MYL2, MYL3, MYPN, MYOZ1, MYOZ2, PKP2, PLN, PRKAG2, PSEN1, PSEN2, RBM20, RYR2, SCN5A, SGCD, TAZ, TBX20, TCAP, TMEM43, TNNC1, TNNI3, TNNT2, TPM1, TTN, VCL, ZASP/LDB3). 26 Samples were prepared according to the manufacturer s protocols and multiplexed to an amount still permitting a theoretical coverage of 100 reads per targeted sequence/per patient. All samples were sequenced using 151 bp paired-end reads on an Illumina MiSeq sequencer and analyzed using the MiSeq Reporter pipeline and Nextgene software. 27 Eleven amplicons with low coverage were also analyzed by Sanger sequencing. Identified mutations were confirmed by Sanger sequencing. To study co-segregation, affected relatives were screened for carriership of the identified mutations by Sanger sequencing. Sanger Sequencing STAT3 Gene The STAT3 gene (all coding exons and flanking intronic sequences) was analysed by Sanger sequencing in PPCM patients of the collected families. 154 Spaendonck.indd :00

5 Chapter 7 Figure 1: Pedigrees of the Dutch families (NL1-11) Square symbols indicate men; circles, women; diamonds, unknown sex; and triangles, miscarriage. Blue symbols indicate a clinical diagnosis of PPCM; black symbols, (mild) DCM; grey symbols, possible DCM; orange symbols, sudden cardiac death (SCD). Diagonal lines through symbols indicate deceased; arrows indicate patients selected for targeted next-generation sequencing; and the number in a symbol indicates the number of individuals with this symbol (question mark if unknown). 155 Spaendonck.indd :00

6 Table 1. Clinical characteristics of confirmed PPCM cases Referred Family Patient for Diagnosis (age in yrs) Timing at Diagnosis NL1 II:6 HF PPCM (29) Just after delivery Cardiogenic NL1 III:4 PPCM (27) shock 3 days after delivery NL2 III:3 HF PPCM (26) Few days after delivery NL3 III:1 HF PPCM (33) 37th week of pregnancy NL4 III:2 HF PPCM (30) 3 months after delivery NL5 III:1 HF PPCM (33) 35th week of pregnancy NL6 III:2 HF PPCM (29) 2 months after delivery NL7 III:1 HF PPCM (23) Just after delivery NL8 III:3 HF PPCM (35) 2 weeks after delivery HF, respiratory NL9 III:1 insuf- PPCM (30) ficiency Just after delivery LVEF at Pregnancy Diagnosis LVEF at Follow-Up Cardiological remarks and outcome (age in yrs) P4 D (31) P1 20% D MOF (27) P4 LBBB, D asthma cardiale (26) P2 CS 25% 3 months 33% 21% 9 months no recovery 6 months 44%, P1 AI CS 23% 7 years 42% Thrombus LV apex, tachycardia P3 23% 6 months 23% AF (30), PVCs, VTs (46), D HF (51) P1 CS 29th week, 25% 8 years 10% eclampsia ICD/CRT (31), LVAD, VF, D cardiogenic shock (34) Thrombus LV apex, TIA, VT P2 18% (35), PM, HTX (37), normal LVEF (51) P1 CS, twin 30% pregnancy 6 months 55%, 3 years normal Pathology and other remarks Myocyte hypertrophy Dilated heart, myocyte hypertrophy, fibrosis Signs of acute myocarditis (EMB), suspicion of vasculitis New pregnancy, terminated (35) 156 Spaendonck.indd :00

7 Chapter 7 Chest pain, NL10 III:6 PPCM (36) coughing 3 weeks after delivery P2 CS 29th week, 20-30% HELLP 6 months 55%, 2 years 45%, 3 years 50-55% Dyspnea, NL11 III:1 PPCM (20) tachycardia Just after Poor delivery 4 months 30- Tachycardia 35% 1 month after SA1 II:5 PPCM (23) P2 22% No recovery delivery Screening, SA1 II:6 PPCM (22) P1 43% 24% No recovery asymptomatic SB 27 GER1 II:1 PPCM 20% weeks 6 months 37%, 2 years normal Full recovery with uneventful 2nd pregnancy 2 years later GER2 II:1 PPCM 25% ICD, HTX Suspicion of neurodermitis GER3 II:1 PPCM P1 25% 6 months 30% Subsequent pregnancy entered with 30% LVEF, VAD after 2nd pregnancy, no recovery BiVAD, no recovery, D after GER4 II:1 PPCM 25% 6 months 25% 2 years Graves disease, nicotin and drug abuse GER5 II:1 PPCM 25% 6 months 36%, >1 year 47% 3 months GER6 II:1 PPCM (33) <30% after delivery 6 months no recovery AF indicates atrial fibrillation; AI, artificial insemination; AT, atrial tachycardia; (Bi)(L)VAD, (bi)(left) ventricular assist device; CRT, cardiac resynchronization therapy; CS, caesarean section; D, death; EMB, endomyocardial biopsy; HELPP, hemolysis, elevated liver enzymes, low platelet count; HF, heart failure; HTX, heart transplantation; ICD, implantable cardiac defibrillator; LBBB, left bundle branch block; LV, left ventricle; LVEF, left ventricular ejection fraction; MOF, multiple organ failure; P, pregnancy; PM, pacemaker; PPCM, peripartum cardiomyopathy; PVC, premature ventricular contraction; RV, right ventricle; SB, still birth; TIA, transient ischemic attack; VF, ventricular fibrillation; VT, ventricular tachycardia. 157 Spaendonck.indd :00

8 Figure 2: Pedigrees of the South African (SA1) and German families (GER1-6) Square symbols indicate men; circles, women; diamonds, unknown sex. Blue symbols indicate clinical diagnosis of PPCM; black symbols, (mild) DCM; orange symbol, sudden cardiac death (SCD). Diagonal lines through symbols indicate deceased; arrows indicate patients selected for targeted next-generation sequencing; the number in a symbol indicates the number of individuals with this symbol (question mark if unknown); and SB indicates still birth. Classification of Identified Mutations The criteria used to classify mutations were published recently. 28 Briefly, we used a list of mutation-specific features based on in silico analysis using the mutation interpretation software Alamut (version 2.2.1). A score was given depending on the outcome of a prediction test for each feature (i.e. the PolyPhen-2 prediction tool). Then, depending on the total score and the presence/absence of the mutation in at least 300 ethnically matched control alleles (data obtained from the literature and/or available databases, e.g. EVS and or from our own control alleles), we classified mutations as: pathogenic, not pathogenic, or as a variant of unknown clinical significance (VUS; VUS1, unlikely to be pathogenic; VUS2, uncertain; VUS3, likely to be pathogenic). Co-segregation data and/or functional analysis were needed to classify a mutation as pathogenic. Functional Analysis of TTN mutation Passive force was measured in single membrane-permeabilized cardiomyocytes mechanically isolated from the heart tissue. 29,30 Titin isoform composition was analysed as described previously Spaendonck.indd :00

9 Chapter 7 RESULTS Clinical Characteristics: Low Rate of Full Recovery in PPCM Cases of Familial PPCM/DCM We collected 18 families with familial PPCM/DCM. These families originated from the Netherlands (n=11), Germany (n=6), and South Africa (n=1; black). Clinical data of the PPCM cases in these families are summarised in Table 1 and of all (likely) affected relatives in Supplemental Table S1. The pedigrees of all the families are shown in Figures 1 (NL1-11) and 2 (SA1 and GER1-6). In two families there were two cases of PPCM (NL1 and SA1). Eight families (NL1-7 and SA1) have been described previously. 11,31 The median age at diagnosis in PPCM patients was 29 years (n=15; range years), with mean parity 2 (n=13; range 1-4). PPCM diagnosis was postpartum in 12/14 patients. Only 2/20 PPCM patients showed a full recovery of left ventricular function, one of them even had an uneventful next pregnancy (NL9 III:1, LVEF still normal 3 years after diagnosis; and GER1 II:1, full recovery with uneventful second pregnancy two years later). Another PPCM patient showed recovery of left ventricular function, but only under treatment with a beta-blocker and ACE inhibitor (NL10 III:6). In addition to 20 confirmed PPCM patients in these families, five relatives show clinical characteristics suggestive for PPCM (NL4 II:2, GER1 I:1, GER3 I:1, GER4 I:1, GER5 I:1; Table S1). PPCM could not be confirmed because clinical data of these relatives was lacking. In addition, two relatives with DCM showed a decline of left ventricular function after delivery (NL2 IV:8 and SA1 II:3; Table S1). Targeted Next-Generation Sequencing: Potential Causal Mutations in Cardiomyopathy -Related Genes, in particular TTN, are Common in Familial PPCM/DCM Using our validated NGS approach, 27 a mean coverage of 220x per individual patient was reached and, on average, 98.5% of all targeted nucleotides were covered at least 20x. In 4/18 families (22%) pathogenic mutations in cardiomyopathy-related genes were identified (3 in TTN and 1 in BAG3). In addition, in 6 other families (33%) VUS3s were identified (4 in TTN, 1 in TNNC1, and 1 in MYH7). An overview of these mutations and VUS3s and the respective co-segregation analyses are shown in Table 2. All 7 TTN mutations/vus3s were located in the titin A-band, for which over-representation of mutations in DCM patients was reported previously.32 No potential mutations were identified in 8 families (NL2, NL5, NL7, NL8, SA1, GER2, GER3, and GER6). An overview of the 26 mutations that were not classified as potentially disease-causing (VUS1s and VUS2s) identified in the 18 families is shown in Supplemental Table S2. No STAT3 Mutations in PPCM Cases No STAT3 mutations were identified in 15 PPCM cases (DNA was available from 15/20 cases). 159 Spaendonck.indd :00

10 Table 2. Pathogenic mutations and VUSs that are likely to be pathogenic in 10/18 families Family Tested patient Gene Amino acid change Nucleotide change Classification Co-segregation Affected relatives carrier NL1 II:3 TTN p.arg27373* c.82117c>t Pathogenic Yes II:1, II:3, II:4, III:4, III:5, III:6 NL3 II:2 BAG3 p.gln340* c.1018c>t Pathogenic Yes II:2, III:1 NL4 III:2 TNNC1 p.gln50arg c.149t>c VUS3 Yes II:5, III:2, III:5, IV:1 NL6 II:3 TTN p.asn28726lysfs*3 c.86171_86174dupaaag VUS3 Yes II:1, II:3 NL9 III:1 TTN p.arg17599* c.52795c>t Pathogenic Yes III:1, III:5 NL10 II:6 TTN p.arg23956thrfs*9 c.71867_71876delgagttctgga Pathogenic Yes II:1, II:2, II:6, III:2, III:5, III:6 NL11 III:1 TTN p.ser27317lysfs*10 c.81949dupa VUS3 Yes II:1, III:1 GER1 II:1 TTN p.trp18357* c.55070g>a VUS3 Unknown No samples available GER4 II:1 TTN p.lys15664valfs*13 c.46990_46993delaagg VUS3 Unknown No samples available GER5 II:1 MYH7 p.arg1303gly c.3907c>g VUS3 Yes I:1, II:1 Nomenclature according to HGVS (Human Genome Variation Society) using the reference sequences: TTN (NM_ ; Q8WZ42-1), BAG3 (NM_ ), TNNC1 (NM_ ), MYH7 (NM_ ). VUS indicates variant of unknown clinical significance (VUS3, likely to be pathogenic; VUS2, uncertain). VUS2 p.arg279trp (c.835c>t) on same allele 160 Spaendonck.indd :00

11 Chapter 7 Functional and Protein Analyses Support the Pathogenicity of a Likely Pathogenic TTN Mutation Heart tissue from PPCM patient GER4 II:1 with a VUS3 in TTN was available for functional and protein analyses. Passive force was measured in single cardiomyocytes (n=4) at sarcomere lengths of 1.8 to 2.2 μm (see Figure 3). Our functional measurements of passive stiffness, which is largely based on titin composition in the heart, revealed a very low passive force development (1.0±0.3 kn/m 2 ) at a sarcomere length of 2.2 μm in the PPCM sample compared to previously reported values in control hearts (~2.5 kn/m 2 ). 29,30 Analysis of titin isoform composition showed a shift towards the more compliant N2BA isoform evident from a higher N2BA/N2B ratio (0.72±0.02; mean of triplo) in the PPCM heart compared to the previously reported ratio (0.39±0.05) in control hearts. 30 Figure 3: Force measurements in heart tissue of GER4 II:1 Single cardiomyocyte of the PPCM heart sample (A). Passive force development was measured at sarcomere lengths of 1.8, 2.0 and 2.2 μm. (B) DISCUSSION This is the first report of a comprehensive genetic analysis in a large series of cases with familial occurrences of PPCM and DCM. We identified pathogenic mutations in cardiomyopathy-related genes in 4/18 families (22%) and VUSs that are likely to be pathogenic in 6 other families (33%). These data support the earlier finding that PPCM can be part of familial DCM. 11,12 Cascade genetic screening can identify relatives at risk in those families in which an underlying mutation has been identified. Our data also specifically show a low recovery rate in our cohort (only 10%) compared to reports in other groups not selected for familial cases (recovery rates of around 25 to 50%), indicating that the presence of an underlying mutation or positive family history for cardiomyopathy in a patient with PPCM may be a prognostic factor for a low recovery rate. 161 Spaendonck.indd :00

12 The targeted NGS approach that we have developed provides high-throughput, rapid and affordable molecular analysis for cardiomyopathies. 27 As accurate annotation of mutations in cardiomyopathies is of the utmost importance, 37 we were extremely careful in classifying these. 28 Our study has several advantages: one is the inclusion of some large families, where co-segregation analysis added value to the classification of mutations. Another was the large number of genes we tested, including the large TTN gene, for which mutation analyses on a large scale were impossible before NGS became available, because exclusion of pathogenic mutations in 47 other candidate genes makes it more likely that the identified VUS3s have a pathogenic nature. Accordingly, the previously reported TNNC1 mutation is still the only potential genetic cause in family NL4. 11 And although the pathogenicity of truncating TTN mutations is still under debate due to these types of mutations being found in apparently healthy controls (up to 3%) and the general population, 32,38 the pathogenicity of TTN VUS3s identified in our families also becomes more likely after excluding pathogenic mutations in 47 other cardiomyopathy-related genes. Possible exclusion of mutations in other genes in patients carrying truncating TTN mutations was not explicitly addressed by Herman et al. 32 As expected, we identified several mutations in the majority of patients, however, we focused on the pathogenic mutations and VUS3s. Other identified mutations (VUS1s and VUS2s; see Table S2) might be benign genetic variations, but some may also contribute to the development of disease in these families. Some of these VUSs might even be independently pathogenic, but additional testing is needed to confirm this (this might be the case for two VUS2s in TTN (p.arg1408cys (c.4222c>t) in GER2, and p.glu2076gly (c.6227a>g) in GER6). Other possibilities are that these VUSs may act as modifiers, or that they are risk factors with a low penetrance. The great majority of pathogenic mutations and VUS3s (7/10) were in the TTN gene, which encodes the giant sarcomeric protein titin. It was recently reported that truncating mutations in TTN account for a significant portion (approximately 25%) of the genetic etiology in familial DCM. 32 The high yield of pathogenic mutations and VUS3s in TTN in our cohort of familial PPCM/DCM cases (39%; 7/18) suggests that TTN mutations are specifically related to PPCM. Changes in isoform expression and phosphorylation status of titin have been reported in acquired forms of heart failure (reviewed by Hildalgo and Granzier). 39 We were able to measure functional properties and titin isoform composition in heart tissue from one of the PPCM patients with a VUS3 in TTN. The passive force was twice as low as the value previously reported in control groups, and was associated with a shift towards the more compliant N2BA titin isoform. The shift towards more compliant N2BA has been reported in human heart failure. 30,40,41 Overall, our data from functional and protein analyses support the pathogenicity of this particular TTN mutation. We still classify this mutation as VUS3, however extended experience with these functional analyses might drive us to re-classify this VUS3 towards a pathogenic mutation. Recent studies indicated that titin phosphorylation is indirectly altered by increased oxidative stress 42 and, as such, may represent a likely pathomechanism in PPCM. Future studies will need to reveal the functional deficits induced by mutations in the TTN gene in relation to high oxidative stress, as present in PPCM. 162 Spaendonck.indd :00

13 Chapter 7 There may be genetic factors specific for PPCM development, for example a factor tentatively underlying the geographical hotspot of incidence in Haiti, and a locus near the PTHLH gene reported by Horne et al. 39 We only focused on the STAT3 gene as a possible specific genetic factor for PPCM. Because mice with cardiomyocyte-specific deletion of STAT3 develop PPCM, 7 STAT3 might also be involved in human PPCM but there are no human genetic data supporting this yet. STAT3 mutations are so far only known to cause hyper-ige syndrome. 40 In contrast to the PPCM cases, some women in our PPCM/DCM families went through several pregnancies without developing PPCM. We therefore hypothesized that STAT3 mutations in the PPCM cases of these families contributed to the development of PPCM, in addition to an underlying cardiomyopathy-related mutation. However, we found no STAT3 pathogenic mutations or VUSs in these PPCM cases, which was consistent with previous findings. 7 Exome sequencing of rare familial PPCM cases could lead to identifying novel genetic factors specific for PPCM. However, this approach is limited by the fact that familial PPCM cases with more than two affected relatives or with affected distant relatives are lacking. An alternative strategy could be to compare the data from exome sequencing on different PPCM cases in order to identify a shared genetic cause, but this might not lead to a result because the causal genetic factor may well be unique to each family. Limitations One limitation of our study is that it does not provide data on the frequency of familial disease in PPCM. Currently, we only have data from a German cohort reporting a positive family history for cardiomyopathy in 16.5% of PPCM cases, 13 but we hope to gain more information via the Peripartum Cardiomyopathy Registry of EURObservational Research Programme (www. eorp.org). (unpublished data, 2013, manuscript submitted to European Journal of Heart Failure) Another limitation is that retrieving information on larger deletions/duplications from NGS data is not possible yet, although software to enable such analysis is being developed. We may therefore have missed that type of mutation in our analyses. A further limitation is the difficulty of judging which TTN mutations are pathogenic, given the presence of truncating TTN mutations in the general population and reported truncating mutations that do not segregate with disease in DCM families. 30,36,41 In contrast to the latter observation, we were able to show co-segregation of truncating TTN mutations/vus3s in five of our families (NL1, NL6, NL9, NL10 and NL11; Table 2), and we have data from functional and protein analyses supporting the pathogenicity of one likely pathogenic TTN mutation (GER4 II:1). Additional functional studies on TTN mutations and collection of large families carrying these mutations are needed. Moreover, although our findings suggest a specific role for TTN mutations in families with PPCM and DCM, we do realise that the number of families studied is currently too small to definitely conclude this. Finally, we were lacking some clinical data, especially of cases that showed clinical characteristics suggestive of PPCM. 163 Spaendonck.indd :00

14 Conclusions and Practical Implications Potentially causal mutations in cardiomyopathy-related genes are common in families with both PPCM and DCM, in particular TTN mutations. The targeted next-generation sequencing approach we applied has been shown to be suitable for identifying such mutations. Functional studies as performed in the present study may provide a future tool to confirm pathogenicity of TTN mutations. Our results provide more support for the earlier finding that PPCM can be a manifestation of familial DCM. Cascade genetic screening can identify relatives at risk in those families in which an underlying mutation has been identified. Moreover, the presence of an underlying mutation or a positive family history for cardiomyopathy in a PPCM patient may be a prognostic factor for low recovery rate. Acknowledgements We thank all the patients who participated in this study; the Study Group on PPCM of the Heart Failure Association of the European Society of Cardiology; Birgit Sikkema-Raddatz for her help in validating and implementing the targeted enrichment kit; Ludolf Boven, Eddy de Boer and Lennart Johansson for technical assistance; Wies Lommen for assistance with functional analyses; Nicolaas de Jonge, cardiologist, for cardiac evaluation of family NL10; Wilma van der Roest, genetic counselor, for counseling some of the Dutch families; and Jackie Senior for editing this manuscript. Rowida Almomani was supported by the Netherlands Heart Foundation (grant 2010B164). 164 Spaendonck.indd :00

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17 Chapter 7 30 Borbely A, Falcao-Pires I, van Heerebeek L, Hamdani N, Edes I, Gavina C, Leite-Moreira AF, Bronzwaer JGF, Papp Z, van der Velden J, Stienen GJM, Paulus WJ. Hypophosphorylation of the stiff N2B titin isoform raises cardiomyocyte resting tension in failing human myocardium. Circ Res. 2009;104: Tibazarwa K, Sliwa K, Wonkam A, Mayosi BM. Peripartum cardiomyopathy and familial dilated cardiomyopathy: a tale of two cases. Cardiovasc J of Afr. 2013; 24:e4-e7. 32 Herman DS, Lam L, Taylor MR, Wang L, Teekakirikul P, Christodoulou D, Conner L, DePalma SR, McDonough B, Sparks E, Teodorescu DL, Cirino AL, Banner NR, Pennell DJ, Graw S, Merlo M, Di Lenarda A, Sinagra G, Bos JM, Ackerman MJ, Mitchell RN, Murry CE, Lakdawala NK, Ho CY, Barton PJ, Cook SA, Mestroni L, Seidman JG, Seidman CE. Truncations of titin causing dilated cardiomyopathy. N Engl J Med. 2012;366: Blauwet LA, Libhaber E, Forster O, Tibazarwa K, Mebazaa A, Hilfiker-Kleiner D, Sliwa K. Predictors of outcome in 176 South African patients with peripartum cardiomyopathy. Heart. 2013;99: Goland S, Bitar F, Modi K, Safirstein J, Ro A, Mirocha J, Khatri N, Elakayam U. Evaluation of the clinical relevance of baseline left ventricular ejection fraction as a predictor of recovery or persistence of severe dysfunction in women in the United States with peripartum cardiomyopathy. J Card Fail. 2011;17: Fett JD, Christie LG, Carraway RD, Murphy JG. Five-year prospective study of the incidence and prognosis of peripartum cardiomyopathy at a single institution. Mayo Clin Proc. 2005;80: Duran N, Günes H, Duran I, Biteker M, Ozkan M. Predictors of prognosis in patients with peripartum cardiomyopathy. Int J Gynaecol Obstet. 2008;101: Norton N, Robertson PD, Rieder MJ, Züchner S, Rampersaud E, Martin E, Li D, Nickerson DA, Hershberger RE; National Heart, Lung and Blood Institute GO Exome Sequencing Project. Evaluating pathogenicity of rare variants from dilated cardiomyopathy in the exome era. Circ Cardiovasc Genet. 2012;5: Golbus JR, Puckelwartz MJ,Fahrenbach JP, Dellefave-Castillo LM, Wolfgeher D, McNally EM. Population-based variation in cardiomyopathy genes. Circ Cardiovasc Genet. 2012;5: Hidalgo C, Granzier H. Tuning the molecular giant titin through phosphorylation: Role in health and disease. Trends Cardiovasc Med. 2013;23: Makarenko I, Opitz CA, Leake MC, Kulke M, Gwathmey JK, del Monte F, Hajjar RJ, Linke WA. Passive stiffness changes caused by upregulation of compliant titin isoforms in human dilated cardiomyopathy hearts. Circ Res. 2004;95: Nagueh SF, Shah G, Wu Y, Torre-Amione G, King NM, Lahmers S, Witt CC, Becker K, Labeit S, Granzier HL. Altered titin expression, myocardial stiffness, and left ventricular function in patients with dilated cardiomyopathy. Circulation. 2004;110: van Heerebeek L, Hamdani N, Falcão-Pires I, Leite-Moreira AF, Begieneman MP, Bronzwaer JG, van der Velden J, Stienen GJ, Laarman GJ, Somsen A, Verheugt FW, Niessen HW, Paulus WJ. Low myocardial protein kinase G activity in heart failure with preserved ejection fraction. Circulation. 2012; 126: Horne BD, Rasmusson KD, Alharethi R, Budge D, Brunisholz KD, Metz T, Carlquist JF, Connolly JJ, Porter TF, Lappé DL, Muhlestein JB, Silver R, Stehlik J, Park JJ, May HT, Bair TL, Anderson JL, Renlund DG, Kfoury AG. Genome-wide significance and replication of the chromosome 12p11.22 locus near the PTHLH gene for peripartum cardiomyopathy. Circ Cardiovasc Genet. 2011;4: Spaendonck.indd :00

18 44 Minegishi Y, Saito M, Tsuchiya S, Tsuge I, Takada H, Hara T, Kawamura N, Ariga T, Pasic S, Stojkovic O, Metin A, Karasuyama H. Dominant-negative mutations in the DNA-binding domain of STAT3 cause hyper-ige syndrome. Nature. 2007;448: Norton N, Li D, Rampersaud E, Morales A, Martin ER, Zuchner S, Guo S, Gonzalez M, Hedges DJ, Robertson PD, Krumm N, Nickerson DA, Hershberger RE. Exome sequencing and genome-wide linkage in 17 families illustrates the complex contribution of TTN truncating variants to dilated cardiomyopathy. Circ Cardiovasc Genet. 2013;6: Spaendonck.indd :00

19 Chapter 7 SUPPLEMENTAL MATERIAL Table S2. Overview of mutations classified as VUS1 or VUS2 Family Tested patient Gene Amino acid change Nucleotide change Classification NL2 III:2 TTN p.glu10855dup c.32562_32564dupaga VUS1 NL4 III:2 LAMA4 p.met1202val c.3604t>c VUS1 NL4 III:2 PRKAG2 3 UTR c.*2c>t VUS1 NL4 III:2 TTN p.ala9135pro c.27403g>c VUS1 NL5 II:1 PKP2 p.asp26asn c.76c>t VUS1 NL6 II:3 DMD p.asn2713ser c.8138t>c VUS2 NL6 II:3 RYR2 p.ile2721thr c.8162t>c VUS2 NL6 II:3 TTN p.glu21080lys c.63238g>a VUS1 NL7 II:2, III:1 MYBPC3 p.ala833thr c.2497g>a VUS2 NL7 II:2, III:1 TMEM43 p.arg312trp c.934c>t VUS1 NL7 II:2, III:1 TTN p.glu10855dup c.32562_32564dupaga VUS1 NL8 III:3 RBM20 p.ser637asn c.1910g>a VUS2 NL10 II:6 TTN p.arg279trp c.835c>t VUS2 NL10 II:6 TTN p.pro17045ala c.51133c>g VUS2 NL11 III:1 TTN p.lys4401glu c.13201a>g VUS1 SA1 II:5 MYPN p.ser774tyr c.2321c>a VUS2 SA1 II:5 PKP2 p.val842ile c.2524c>t VUS1 SA1 II:5 TTN p.ser1400thr c.4199g>c VUS1 SA1 II:5 TTN p.glu18378lys c.55132g>a VUS1 SA1 II:5 TTN p.val32108met c.96322g>a VUS2 SA1 II:5 TTN p.arg33402cys c c>t VUS2 GER2 II:1 PKP2 p.ile531ser c.1592t>g VUS1 GER2 II:1 TTN p.arg1408cys c.4222c>t VUS2 GER5 II:1 TTN p.glu15076asp c.45228g>c VUS1 GER5 II:1 TTN p.ile17461thr c.52382t>c VUS2 GER6 II:1 TTN p.glu2076gly c.6227a>g VUS2 Nomenclature according to HGVS (Human Genome Variation Society) using the reference sequences: TTN (NM_ ; Q8WZ42-1), LAMA4 (NM_ ), PRKAG2 (NM_ ), PKP2 (NM_ ), DMD (NM_ ), RYR2 (NM_ , MYBPC3 (NM_ ), TMEM43 (NM_ ), RBM20 (NM_ ), MYPN (NM_ ). VUS indicates variant of unknown clinical significance (VUS1, unlikely to be pathogenic; VUS2, uncertain). II:2 and III:1 were both analyzed; only shared mutations were investigated further (analyzed in silico) pathogenic mutation on same allele (p.arg23956thrfs*9 (c.71867_71876delgagttctgga)) 169 Spaendonck.indd :00

20 Table S1. Clinical characteristics of PPCM cases and all affected (or likely affected) relatives Family Patient M/F Referred for Diagnosis (age in yrs) Timing at diagnosis LVEF at Diagnosis LVEF at Follow-Up Cardiological remarks and outcome (age in yrs) NL1 II:1 M DCM (83) 44% PVCs, VTs (70) NL1 II:3 F Dyspnea DCM (61) 23% 1 year 41%, 12 years 20-25% AVB1, PVCs, VTs, ICD (61) NL1 II:4 M DCM (61) 32% PVCs, VTs (61), AF (70) NL1 II:5 F Died SCD (54) NL1 II:6 F HF PPCM (29) Just after delivery P4 D (31) NL1 III:4 F Pregnancy Screening Screening Cardiogenic shock PPCM (27) 3 days after delivery P1 20% D MOF (27) NL1 III:5 M Dyspnea, chest pain DCM (48) 24% PVCs, VTs (48), ICD (58), D HF (59) NL1 III:6 M Screening DCM (48) 40% 4 years 40% PVCs, VTs (48), ICD (50), appropriate ICD shock (53) NL1 III:8 M Died SIDS NL2 I:2 F Possible DCM D HF (60) NL2 II:2 F Died SCD (27) NL2 II:3 M DCM D (63) NL2 III:2 F HF DCM (41) 25% 9 years 18% LBBB, VTs (41), ICD (51), intramyocardial stem cell implantation (53), HTX, D DIC (54) Pathology and other remarks Myocyte hypertrophy Myocyte hypertrophy, interstitial fibrosis 170 Spaendonck.indd :00

21 Chapter 7 NL2 III:3 F HF PPCM (26) Few days after delivery P4 LBBB, D asthma cardiale (26) NL2 IV:2 F Mild DCM (25) 45-50% 6 years 53% NL2 IV:4 M Screening Screening, palpitations DCM (22) 37-49% 7 years 40-45% PVCs (15), VTs (25) NL2 IV:5 M Screening DCM (20) 40% 5 years 35-40% NL2 IV:6 F DCM (28) 30-40% 8 years 45% PVCs NL2 IV:8 F DCM (35) 10 weeks after P5, but 4 years earlier already abnormal contraction LV with preserved LVEF 43% NL2 IV:9 F Possible DCM (21) NL3 I:2 F Died SCD (25) NL3 I:3 M Died SCD (57) NL3 II:2 M Screening Screening Screening Screening, fatique, palpitations DCM (57) NL3 III:1 F HF PPCM (33) 37th week of pregnancy P2 CS 25% 3 months 33% NL4 II:1 F Died DCM (54) D (54) NL4 II:2 F Died SCD (26) Just after delivery P2 Dilated heart, myocyte hypertrophy, fibrosis Dilated heart, myocyte hypertrophy, hyperchromatic nuclei Rheumatic disease 171 Spaendonck.indd :00

22 Table S1. Continued Family Patient M/F Referred for Diagnosis (age in yrs) Timing at diagnosis Pregnancy LVEF at Diagnosis LVEF at Follow-Up Cardiological remarks and outcome (age in yrs) NL4 II:4 F Screening Mild DCM (63) NL4 II:5 F Screening Mild DCM (62) LBBB NL4 III:2 F HF PPCM (30) 3 months after delivery 21% 9 months no recovery NL4 III:5 F Screening, palpitations Possible DCM (46) 50% NL4 IV:1 F Heart murmur Mild DCM (16 months) 7 years stable NL5 II:1 F HF DCM (50) 8 years 45% Thrombus heart (50) NL5 II:4 F Screening Possible DCM (53) 4 years 50% PVCs (54), AF (56) NL5 III:1 F HF PPCM (33) 35th week of pregnancy P1 AI CS 23% 6 months 44%, 7 years 42% Thrombus LV apex, tachycardia NL6 II:1 M AF DCM (74) AF NL6 II:3 M Dyspnea DCM (70) AF NL6 III:2 F HF PPCM (29) 2 months after delivery P3 23% 6 months 23% AF (30), PVCs, VTs (46), D HF (51) NL7 II:2 M Screening DCM (61) 20% 2 years 40% Also RV dysfunction Pathology and other remarks TIA and LE due to thrombus heart, PAD (50) Signs of acute myocarditis (EMB), suspicion of vasculitis Chemotherapy (5FU/LV), colon carcinoma (50) 172 Spaendonck.indd :00

23 Chapter 7 NL7 III:1 F HF PPCM (23) Just after delivery P1 CS 29th week, eclampsia 25% 8 years 10% ICD/CRT (31), LVAD, VF, D cardiogenic shock (34) NL8 II:3 M DCM (50) HTX, D (58) NL8 II:4 M Collapse Possible DCM (72) 35% Collapse, VT, MI, coronary sclerosis, PTCA, ICD (72), AF (79) NL8 III:3 F HF PPCM (35) 2 weeks after delivery P2 18% Thrombus LV apex, TIA, VT (35), PM, HTX (37), normal LVEF (51) NL8 IV:1 F DCM (16) 23% PVCs, ICD (17), MI, PTCA (18), HTX (19) NL9 II:3 F Dyspnea DCM (42) 40% D (44) NL9 II:5 M Dyspnea DCM (42) 21% MI (40), PVCs, LBBB, severe IV conduction delay, PM (50), HTX waiting list, D (52) NL9 III:1 F HF, respiratory insufficiency PPCM (30) Just after delivery P1 CS, twin pregnancy 30% 6 months 55%, 3 years normal NL9 III:5 F VF DCM (25) 40-45% VF, VT, ICD (25) NL10 II:1 F DCM (58) AF (62), D (78) NL10 II:2 M DCM HTX (48), PVCs (62), normal LVEF (73) NL10 II:6 M DCM (47) <30% PACs (47), LA hemiblock (53), HTX, D (66) NL10 III:2 F DCM NL10 III:5 M Mild DCM (28) 40% Aortic bifurcation prothesis (59) Raynaud s phenomenon (17) New pregnancy, terminated (35) 173 Spaendonck.indd :00

24 Table S1. Continued Family Patient M/F Referred for Diagnosis (age in yrs) Timing at diagnosis Pregnancy LVEF at Diagnosis LVEF at Follow-Up Cardiological remarks and outcome (age in yrs) NL10 III:6 F Chest pain, coughing PPCM (36) 3 weeks after delivery P2 CS 29th week, HELLP 20-30% 6 months 55%, 2 years 45%, 3 years 50-55% NL11 II:1 F HF DCM (46) 31% 10 years 56% PVCs (46), AT, VT, ICD (51) NL11 III:1 F Dyspnea, tachycardia PPCM (20) Just after delivery 26% 4 months 30-35% Tachycardia SA1 II:1 M Palpitations, near fainting DCM (39) 45% SA1 II:3 F HF DCM (26) Worsening HF after 2nd delivery, D (30) SA1 II:5 F PPCM (23) 1 month after delivery P2 22% No recovery SA1 II:6 F Screening, asymptomatic PPCM (22) P1 43% 24% No recovery GER1 I:1 F PPCM or DCM GER1 II:1 F PPCM SB 27 weeks 20% 6 months 37%, 2 years normal Full recovery with uneventful 2nd pregnancy 2 years later GER2 I:1 F DCM GER2 II:1 F PPCM 25% ICD, HTX GER3 I:1 F PPCM or DCM Died soon after delivery D Pathology and other remarks No signs of myocarditis (EMB) CVA (29) Suspicion of neurodermitis 174 Spaendonck.indd :00

25 Chapter 7 GER3 II:1 F PPCM P1 25% 6 months 30% Subsequent pregnancy entered with 30% LVEF, VAD after 2nd pregnancy, no recovery GER4 I:1 F SCD or PPCM (<35) Died after delivery P2 GER4 II:1 F PPCM 25% 6 months 25% BiVAD, no recovery, D after 2 years Graves disease, nicotin and drug abuse GER5 I:1 F PPCM or myocarditis GER5 II:1 F PPCM 25% 6 months 36%, >1 year 47% GER5 II:2 F Arrhythmias (unspecified) Echocardiogram advised GER6 I:1 M SCD (28) GER6 II:1 F PPCM (33) 3 months after delivery <30% 6 months no recovery Confirmed PPCM cases are displayed in bold; affected cases with clinical characteristics suggestive for PPCM are displayed in bold and italic. AF indicates atrial fibrillation; AI, artificial insemination; AVB, atrioventricular block; (Bi)(L)VAD, (bi)(left) ventricular assist device; CRT, cardiac resynchronization therapy; CS, caesarean section; CVA, cerebral vascular accident; D, death; DCM, dilated cardiomyopathy; DIC, diffuse intravascular coagulation; EMB, endomyocardial biopsy; F, female; HELPP, hemolysis, elevated liver enzymes, low platelet count; HF, heart failure; HTX, heart transplantation; ICD, implantable cardiac defibrillator; IV, intraventricular; LA, left anterior; LBBB, left bundle branch block; LE, lung embolism; LV, left ventricle; LVEF, left ventricular ejection fraction; M, male; MI, myocardial infarction; MOF, multiple organ failure; P, pregnancy; PAC, premature atrial contraction; PAD, peripheral arterial disease; PM, pacemaker; PPCM, peripartum cardiomyopathy; PTCA, percutaneous transluminal coronary angioplasty; PVC, premature ventricular contraction; SB, still birth; SCD, sudden cardiac death; SIDS, sudden infant death syndrome; TIA, transient ischemic attack; VF, ventricular fibrillation; VT, ventricular tachycardia. 175 Spaendonck.indd :00

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