Duanxiang Li

Genome-wide studies of copy number variation and exome sequencing identify rare variants in BAG3 as a cause of dilated cardiomyopathy.

Dilated cardiomyopathy commonly causes heart failure and is the most frequent precipitating cause of heart transplantation. Familial dilated cardiomyopathy has been shown to be caused by rare variant mutations in more than 30 genes but only ~35% of its genetic cause has been identified, principally by using linkage-based or candidate gene discovery approaches. In a multigenerational family with autosomal dominant transmission, we employed whole-exome sequencing in a proband and three of his affected family members, and genome-wide copy number variation in the proband and his affected father and unaffected mother. Exome sequencing identified 428 single point variants resulting in missense, nonsense, or splice site changes. Genome-wide copy number analysis identified 51 insertion deletions and 440 copy number variants > 1 kb. Of these, a 8733 bp deletion, encompassing exon 4 of the heat shock protein cochaperone BCL2-associated athanogene 3 (BAG3), was found in seven affected family members and was absent in 355 controls. To establish the relevance of variants in this protein class in genetic DCM, we sequenced the coding exons in BAG3 in 311 other unrelated DCM probands and identified one frameshift, two nonsense, and four missense rare variants absent in 355 control DNAs, four of which were familial and segregated with disease. Knockdown of bag3 in a zebrafish model recapitulated DCM and heart failure. We conclude that new comprehensive genomic approaches have identified rare variants in BAG3 as causative of DCM.

Cardiac magnetic resonance imaging of myocardial contrast uptake and blood flow in patients affected with idiopathic or familial dilated cardiomyopathy

Idiopathic dilated cardiomyopathy (IDC) is characterized by left ventricular (LV) enlargement with systolic dysfunction, other causes excluded. When inherited, it represents familial dilated cardiomyopathy (FDC). We hypothesized that IDC or FDC would show with cardiac magnetic resonance (CMR) increased myocardial accumulation of gadolinium contrast at steady state and decreased baseline myocardial blood flow (MBF) due to structural alterations of the extracellular matrix compared with normal myocardium. CMR was performed in nine persons affected with IDC/FDC. Healthy controls came from the general population (n = 6) or were unaffected family members of FDC patients (n = 3) without signs or symptoms of IDC/FDC or any structural cardiac abnormalities. The myocardial partition coefficient for gadolinium contrast (lambda(Gd)) was determined by T1 measurements. LV shape and function and MBF were assessed by standard CMR methods. lambda(Gd) was elevated in IDC/FDC patients vs. healthy controls (lambda(Gd) = 0.56 +/- 0.15 vs. 0.41 +/- 0.06; P = 0.002), and correlated with LV enlargement (r = 0.61 for lambda(Gd) vs. end-diastolic volume indexed by height; P < 0.01) and with ejection fraction (r = -0.80; P < 0.001). The extracellular volume fraction was higher in IDC patients than in healthy controls (0.31 +/- 0.05 vs. 0.24 +/- 0.03; P = 0.002). Resting MBF was lower in IDC patients (0.64 +/- 0.13 vs. 0.91 +/- 0.22; P = 0.01) than unaffected controls and correlated with both the partition coefficient (r = -0.57; P = 0.012) and the extracellular volume fraction (r = -0.56; P = 0.019). The expansion of the extracellular space correlated with reduced MBF and ventricular dilation. Expansion of the extracellular matrix may be a key contributor to contractile dysfunction in IDC patients.

Coding sequence rare variants identified in MYBPC3, MYH6, TPM1, TNNC1, and TNNI3 from 312 patients with familial or idiopathic dilated cardiomyopathy.

Background—Rare variants in >30 genes have been shown to cause idiopathic or familial dilated cardiomyopathy (DCM), but the frequency of genetic causation remains poorly understood. We have previously resequenced 9 genes in a cohort of idiopathic or familial DCM probands for rare variants, and now we report resequencing results for 5 more genes with established relationships to DCM. Methods and Results—Blood samples were collected, and DNA specimens were prepared from 312 patients, 181 with familial DCM and 131 with idiopathic DCM. Genomic DNA underwent bidirectional sequencing, and DNA of additional family members underwent analysis when a rare variant was identified. We identified rare variants in 34 probands (10.9% overall), including 29 unique protein-altering rare variants and 2 splicing variants that were absent in 246 control subjects (492 chromosomes). These variants were 12 MYBPC3 (myosin-binding protein C) in 13 (4.2%) probands, 8 MYH6 (α-myosin heavy chain) in 10 (3.2%), 6 TPM1 (tropomyosin) in 6 (1.9%), 4 TNNC1 (cardiac troponin C) in 4 (1.3%), and 1 TNNI3 (cardiac troponin I) in 2 (0.6%). Variants were classified as likely or possibly disease causing in 13 and 20 probands, respectively (n=33; 10.6% overall). One MYH6 variant was classified as unlikely to be disease causing. Conclusion—Rare variants in these 5 genes likely or possibly caused 10.6% of DCM in this cohort. When combined with our prior resequencing reports, ≈27% of DCM probands had possible or likely disease-causing variants identified.

Lamin A/C mutation analysis in a cohort of 324 unrelated patients with idiopathic or familial dilated cardiomyopathy

BACKGROUND Lamin A/C mutations are a well-established cause of dilated cardiomyopathy (DCM), although their frequency has not been examined in a large cohort of patients. We sought to examine the frequency of mutations in LMNA, the gene encoding lamin A/C, in patients with idiopathic (IDC) or familial dilated cardiomyopathy (FDC). METHODS Clinical cardiovascular data, family histories, and blood samples were collected from 324 unrelated IDC probands, of whom 187 had FDC. DNA samples were sequenced for nucleotide alterations in LMNA. Likely protein-altering mutations were followed up by evaluating additional family members, when possible. RESULTS We identified 18 protein-altering LMNA variants in 19 probands or 5.9% of all cases (7.5% of FDC; 3.6% of IDC). Of the 18 alterations, 11 were missense (one present in 2 kindreds), 3 were nonsense, 3 were insertion/deletions, and 1 was a splice site alteration. Conduction system disease and DCM were common in carriers of LMNA variants. Unexpectedly, in 6 of the 19 kindreds with a protein-altering LMNA variant (32%), at least one affected family member was negative for the LMNA variant. CONCLUSIONS Lamin A/C variants were observed with a frequency of 5.9% in probands with DCM. The novel observation of FDC pedigrees in which not all affected individuals carry the putative disease-causing LMNA mutation suggests that some protein-altering LMNA variants are not causative or that some proportion of FDC may be because of multiple causative factors. These findings warrant increased caution in FDC research and molecular diagnostics.

Coding sequence mutations identified in MYH7, TNNT2, SCN5A, CSRP3, LBD3, and TCAP from 313 patients with familial or idiopathic dilated cardiomyopathy.

Background: More than 20 genes have been reported to cause idiopathic and familial dilated cardiomyopathy (IDC/FDC), but the frequency of genetic causation remains poorly understood.

Rare Variant Mutations in Pregnancy-Associated or Peripartum Cardiomyopathy

Background— The term peripartum cardiomyopathy (PPCM) describes dilated cardiomyopathy (DCM) without known cause that occurs during the last month of pregnancy to 5 months postpartum. A related term, pregnancy-associated cardiomyopathy (PACM), refers to DCM onset earlier in pregnancy. Multiple studies have focused on inflammatory, immunologic, and environmental causes. An alternative hypothesis is that PPCM and PACM result, in part, from a genetic cause. In this study, we sought to test the hypothesis that rare DCM-associated mutations underlie a proportion of PACM or PPCM cases. Methods and Results— A systematic search of our DCM database designed for family-based genetic studies was undertaken for cases associated with pregnancy and the postpartum period; in the identified cases, clinical and molecular genetic data, including exonic and near intron/exon boundaries of DCM genes, were analyzed. Of 4110 women from 520 pedigrees in the Familial Dilated Cardiomyopathy Research Project database, we identified 45 cases of PPCM/PACM. Evidence of familial clustering with DCM was present in 23 unrelated cases. Of the 45 cases, 19 had been resequenced for known DCM genes, and 6 carried mutations. Five had PPCM, of which 3 were familial with mutations found in MYH7, SCN5A, and PSEN2, and 2 were sporadic with mutations in MYH6 and TNNT2. One case had PACM and carried a mutation in MYBPC3. Conclusions— These findings suggest that a proportion of PPCM/PACM cases results from a genetic cause.

Identification of novel mutations in RBM20 in patients with dilated cardiomyopathy.

The genetic basis of most of dilated cardiomyopathy (DCM) cases remains unknown. A recent study indicated that mutations in a highly localized five amino acid hotspot in exon 9 of RBM20, a gene encoding a ribonucleic acid‐binding protein, caused aggressive DCM. We undertook this study to confi rm and extend the nature of RBM20 mutations in another DCM cohort. Clinical cardiovascular data, family histories, and blood samples were collected from patients with idiopathic DCM. DNA from 312 DCM probands was sequenced for nucleotide alterations in exons 6 through 9 of RBM20, and additional family members as possible. We found six unique RBM20 rare variants in six unrelated probands (1.9%). Four mutations, two of which were novel (R634W and R636C) and two previously identified (R634Q and R636H), were identified in a five amino acid hotspot in exon 6. Two other novel variants (V535I in exon 6 and R716Q in exon 9) were outside of this hotspot. Age of onset and severity of heart failure were variable, as were arrhythmias and conduction system defects, but many subjects suffered severe heart failure resulting in early death or cardiac transplantation. This article concludes that DCM in patients with RBM20 mutations is associated with advanced disease. Clin Trans Sci 2010; Volume 3: 90–97

Evaluating pathogenicity of rare variants from dilated cardiomyopathy in the exome era

Background— Human exome sequencing is a recently developed tool to aid in the discovery of novel coding variants. Now broadly applied, exome sequencing data sets provide a novel opportunity to evaluate the allele frequencies of previously published pathogenic rare variants. Methods and Results— We examined the exome data set from the National Heart, Lung and Blood Institute Exome Sequencing Project and compared this data set with a catalog of 197 previously published rare variants reported as causative of dilated cardiomyopathy (DCM) from familial and sporadic cases. Of these 197, 33 (16.8%) were also present in the Exome Sequencing Project database, raising the question of whether they were uncommon polymorphisms. Supporting functional data has been published for 14 of the 33 (42%), suggesting they are unlikely to be false-positives. The frequencies of these functional variants in the Exome Sequencing Project data set ranged from 0.02 to 1.33% (median 0.04%), which when applied as a cutoff to filter variants in a DCM pedigree identified an additional DCM candidate gene. A greater proportion of sporadic DCM cases had variants that were present in the Exome Sequencing Project data set versus novel variants (ie, not in the Exome Sequencing Project; 44% versus 21%; P=0.002), suggesting some of the variants identified as disease causing in sporadic DCM are either false-positives or low penetrance alleles in human populations. Conclusions— Rare nonsynonymous variants identified in DCM subjects also present at very low frequencies in public databases are likely relevant for DCM. Allele frequencies >0.04% are of less certain pathogenicity, especially if identified in sporadic cases, although this cutoff should be viewed as preliminary.

Exome sequencing and genome-wide linkage analysis in 17 families illustrate the complex contribution of TTN truncating variants to dilated cardiomyopathy.

Background—Familial dilated cardiomyopathy (DCM) is a genetically heterogeneous disease with >30 known genes. TTN truncating variants were recently implicated in a candidate gene study to cause 25% of familial and 18% of sporadic DCM cases. Methods and Results—We used an unbiased genome-wide approach using both linkage analysis and variant filtering across the exome sequences of 48 individuals affected with DCM from 17 families to identify genetic cause. Linkage analysis ranked the TTN region as falling under the second highest genome-wide multipoint linkage peak, multipoint logarithm of odds, 1.59. We identified 6 TTN truncating variants carried by individuals affected with DCM in 7 of 17 DCM families (logarithm of odds, 2.99); 2 of these 7 families also had novel missense variants that segregated with disease. Two additional novel truncating TTN variants did not segregate with DCM. Nucleotide diversity at the TTN locus, including missense variants, was comparable with 5 other known DCM genes. The average number of missense variants in the exome sequences from the DCM cases or the ≈5400 cases from the Exome Sequencing Project was ≈23 per individual. The average number of TTN truncating variants in the Exome Sequencing Project was 0.014 per individual. We also identified a region (chr9q21.11-q22.31) with no known DCM genes with a maximum heterogeneity logarithm of odds score of 1.74. Conclusions—These data suggest that TTN truncating variants contribute to DCM cause. However, the lack of segregation of all identified TTN truncating variants illustrates the challenge of determining variant pathogenicity even with full exome sequencing.

Clinical and functional Characterization of TNNT2 mutations identified in patients with dilated cardiomyopathy

Background—A key issue for cardiovascular genetic medicine is ascertaining if a putative mutation indeed causes dilated cardiomyopathy (DCM). This is critically important as genetic DCM, usually presenting with advanced, life-threatening disease, may be preventable with early intervention in relatives known to carry the mutation. Methods and Results—We recently undertook bidirectional resequencing of TNNT2, the cardiac troponin T gene, in 313 probands with DCM. We identified 6 TNNT2 protein-altering variants in 9 probands, all who had early onset, aggressive disease. Additional family members of mutation carriers were then studied when available. Four of the 9 probands had DCM without a family history, and 5 probands had familial DCM. Only 1 mutation (Lys210del) could be attributed as definitively causative from previous reports. Four of the 5 missense mutations were novel (Arg134Gly, Arg151Cys, Arg159Gln, and Arg205Trp), and one was previously reported with hypertrophic cardiomyopathy (Glu244Asp). Based on the clinical, pedigree, and molecular genetic data, these 5 mutations were considered possibly or likely disease causing. To further clarify their potential pathophysiologic impact, we undertook functional studies of these mutations in cardiac myocytes reconstituted with mutant troponin T proteins. We observed decreased Ca2+ sensitivity of force development, a hallmark of DCM, in support of the conclusion that these mutations are disease causing. Conclusions—We conclude that the combination of clinical, pedigree, molecular genetic, and functional data strengthen the interpretation of TNNT2 mutations in DCM.