Titin Gene Mutations and Cardiomyopathies
Titin Gene Mutations and Cardiomyopathies
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 the participants gave their informed consent.
Peripartum cardiomyopathy 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. Dilated cardiomyopathy 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 such as severe hypertension, coronary artery disease, and systemic disease had been excluded. If only one of the two criteria was fulfilled, the patient was labelled with 'mild DCM'. If the family history suggested DCM in a relative but there were no medical reports to confirm this, the relative was labelled 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 ≤35 years.
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).
(Enlarge Image)
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 peripartum cardiomyopathy; black symbols, (mild) dilated cardiomyopathy; grey symbols, possible dilated cardiomyopathy; orange symbols, sudden cardiac death. 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).
(Enlarge Image)
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 peripartum cardiomyopathy; black symbols, (mild) dilated cardiomyopathy; orange symbol, sudden cardiac death. 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.
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). 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 analysed using the MiSeq Reporter pipeline and Nextgene software. Eleven amplicons with low coverage were also analysed 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.
The STAT3 gene (all coding exons and flanking intronic sequences) was analysed by Sanger sequencing in PPCM patients of the collected families.
The criteria used to classify mutations were published recently. 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. http://evs.gs.washington.edu/EVS and http://www.nlgenome.nl, 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.
Passive force was measured in single membrane-permeabilized cardiomyocytes mechanically isolated from the heart tissue. Titin isoform composition was analysed, as described previously.
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 the participants gave their informed consent.
Peripartum cardiomyopathy 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. Dilated cardiomyopathy 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 such as severe hypertension, coronary artery disease, and systemic disease had been excluded. If only one of the two criteria was fulfilled, the patient was labelled with 'mild DCM'. If the family history suggested DCM in a relative but there were no medical reports to confirm this, the relative was labelled 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 ≤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).
(Enlarge Image)
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 peripartum cardiomyopathy; black symbols, (mild) dilated cardiomyopathy; grey symbols, possible dilated cardiomyopathy; orange symbols, sudden cardiac death. 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).
(Enlarge Image)
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 peripartum cardiomyopathy; black symbols, (mild) dilated cardiomyopathy; orange symbol, sudden cardiac death. 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.
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). 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 analysed using the MiSeq Reporter pipeline and Nextgene software. Eleven amplicons with low coverage were also analysed 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.
Classification of Identified Mutations
The criteria used to classify mutations were published recently. 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. http://evs.gs.washington.edu/EVS and http://www.nlgenome.nl, 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. Titin isoform composition was analysed, as described previously.
