DMD-Associated Dilated Cardiomyopathy: Genotypes, Phenotypes, and Phenocopies.

BACKGROUND: Variants in the DMD gene, that encodes the cytoskeletal protein, dystrophin, cause a severe form of dilated cardiomyopathy (DCM) associated with high rates of heart failure, heart transplantation, and ventricular arrhythmias. Improved early detection of individuals at risk is needed. METHODS: Genetic testing of 40 male probands with a potential X-linked genetic cause of primary DCM was undertaken using multi-gene panel sequencing, multiplex polymerase chain reaction, and array comparative genomic hybridization. Variant location was assessed with respect to dystrophin isoform patterns and exon usage. Telomere length was evaluated as a marker of myocardial dysfunction in left ventricular tissue and blood. RESULTS: Four pathogenic/likely pathogenic DMD variants were found in 5 probands (5/40: 12.5%). Only one rare variant was identified by gene panel testing with 3 additional multi-exon deletion/duplications found following targeted assays for structural variants. All of the pathogenic/likely pathogenic DMD variants involved dystrophin exons that had percent spliced-in scores >90, indicating high levels of constitutive expression in the human adult heart. Fifteen DMD variant-negative probands (15/40: 37.5%) had variants in autosomal genes including TTN, BAG3, LMNA, and RBM20. Myocardial telomere length was reduced in patients with DCM irrespective of genotype. No differences in blood telomere length were observed between genotype-positive family members with/without DCM and controls. CONCLUSIONS: Primary genetic testing using multi-gene panels has a low yield and specific assays for structural variants are required if DMD-associated cardiomyopathy is suspected. Distinguishing X-linked causes of DCM from autosomal genes that show sex differences in clinical presentation is crucial for informed family management.

Functional characterization of a novel mutation in NKX2-5 associated with congenital heart disease and adult-onset cardiomyopathy.

BACKGROUND: The transcription factor NKX2-5 is crucial for heart development, and mutations in this gene have been implicated in diverse congenital heart diseases and conduction defects in mouse models and humans. Whether NKX2-5 mutations have a role in adult-onset heart disease is unknown. METHODS AND RESULTS: Mutation screening was performed in 220 probands with adult-onset dilated cardiomyopathy. Six NKX2-5 coding sequence variants were identified, including 3 nonsynonymous variants. A novel heterozygous mutation, I184M, located within the NKX2-5 homeodomain, was identified in 1 family. A subset of family members had congenital heart disease, but there was an unexpectedly high prevalence of dilated cardiomyopathy. Functional analysis of I184M in vitro demonstrated a striking increase in protein expression when transfected into COS-7 cells or HL-1 cardiomyocytes because of reduced degradation by the Ubiquitin-proteasome system. In functional assays, DNA-binding activity of I184M was reduced, resulting in impaired activation of target genes despite increased expression levels of mutant protein. CONCLUSIONS: Certain NKX2-5 homeodomain mutations show abnormal protein degradation via the Ubiquitin-proteasome system and partially impaired transcriptional activity. We propose that this class of mutation can impair heart development and mature heart function and contribute to NKX2-5-related cardiomyopathies with graded severity.

Epistatic effects of potassium channel variation on cardiac repolarization and atrial fibrillation risk.

OBJECTIVES: The aim of this study was to evaluate the role of cardiac K(+) channel gene variants in families with atrial fibrillation (AF). BACKGROUND: The K(+) channels play a major role in atrial repolarization but single mutations in cardiac K(+) channel genes are infrequently present in AF families. The collective effect of background K(+) channel variants of varying prevalence and effect size on the atrial substrate for AF is largely unexplored. METHODS: Genes encoding the major cardiac K(+) channels were resequenced in 80 AF probands. Nonsynonymous coding sequence variants identified in AF probands were evaluated in 240 control subjects. Novel variants were characterized using patch-clamp techniques and in silico modeling was performed using the Courtemanche atrial cell model. RESULTS: Nineteen nonsynonymous variants in 9 genes were found, including 11 rare variants. Rare variants were more frequent in AF probands (18.8% vs. 4.2%, p < 0.001), and the mean number of variants was greater (0.21 vs. 0.04, p < 0.001). The majority of K(+) channel variants individually had modest functional effects. Modeling simulations to evaluate combinations of K(+) channel variants of varying population frequency indicated that simultaneous small perturbations of multiple current densities had nonlinear interactions and could result in substantial (>30 ms) shortening or lengthening of action potential duration as well as increased dispersion of repolarization. CONCLUSIONS: Families with AF show an excess of rare functional K(+) channel gene variants of varying phenotypic effect size that may contribute to an atrial arrhythmogenic substrate. Atrial cell modeling is a useful tool to assess epistatic interactions between multiple variants.

Investigation of association between PFO complicated by cryptogenic stroke and a common variant of the cardiac transcription factor GATA4.

Patent foramen ovale (PFO) is associated with clinical conditions including cryptogenic stroke, migraine and varicose veins. Data from studies in humans and mouse suggest that PFO and the secundum form of atrial septal defect (ASDII) exist in an anatomical continuum of septal dysmorphogenesis with a common genetic basis. Mutations in multiple members of the evolutionarily conserved cardiac transcription factor network, including GATA4, cause or predispose to ASDII and PFO. Here, we assessed whether the most prevalent variant of the GATA4 gene, S377G, was significantly associated with PFO or ASD. Our analysis of world indigenous populations showed that GATA4 S377G was largely Caucasian-specific, and so subjects were restricted to those of Caucasian descent. To select for patients with larger PFO, we limited our analysis to those with cryptogenic stroke in which PFO was a subsequent finding. In an initial study of Australian subjects, we observed a weak association between GATA4 S377G and PFO/Stroke relative to Caucasian controls in whom ASD and PFO had been excluded (OR = 2.16; p = 0.02). However, in a follow up study of German Caucasians no association was found with either PFO or ASD. Analysis of combined Australian and German data confirmed the lack of a significant association. Thus, the common GATA4 variant S377G is likely to be relatively benign in terms of its participation in CHD and PFO/Stroke.

Tinman/Nkx2-5 acts via miR-1 and upstream of Cdc42 to regulate heart function across species.

Unraveling the gene regulatory networks that govern development and function of the mammalian heart is critical for the rational design of therapeutic interventions in human heart disease. Using the Drosophila heart as a platform for identifying novel gene interactions leading to heart disease, we found that the Rho-GTPase Cdc42 cooperates with the cardiac transcription factor Tinman/Nkx2-5. Compound Cdc42, tinman heterozygous mutant flies exhibited impaired cardiac output and altered myofibrillar architecture, and adult heart-specific interference with Cdc42 function is sufficient to cause these same defects. We also identified K(+) channels, encoded by dSUR and slowpoke, as potential effectors of the Cdc42-Tinman interaction. To determine whether a Cdc42-Nkx2-5 interaction is conserved in the mammalian heart, we examined compound heterozygous mutant mice and found conduction system and cardiac output defects. In exploring the mechanism of Nkx2-5 interaction with Cdc42, we demonstrated that mouse Cdc42 was a target of, and negatively regulated by miR-1, which itself was negatively regulated by Nkx2-5 in the mouse heart and by Tinman in the fly heart. We conclude that Cdc42 plays a conserved role in regulating heart function and is an indirect target of Tinman/Nkx2-5 via miR-1.

Genetics of dilated cardiomyopathy.

Dilated cardiomyopathy (DCM) is a myocardial disorder defined by ventricular chamber enlargement and systolic dysfunction. DCM can result in progressive heart failure, arrhythmias, thromboembolism, and premature death, and contributes significantly to health care costs. In many cases, DCM results from acquired factors that affect cardiomyocyte function or survival. Inherited genetic variants are also now recognized to have an important role in the etiology of DCM. Despite substantial progress over the past decade, our understanding of familial DCM remains incomplete. Current concepts of the molecular pathogenesis, clinical presentation, natural history, and management of familial DCM are outlined in this review.

Identification and functional characterization of cardiac troponin I as a novel disease gene in autosomal dominant dilated cardiomyopathy.

RATIONALE: Idiopathic dilated cardiomyopathy (DCM) is inherited in approximately one third of cases, usually as an autosomal dominant trait. More than 30 loci have been identified, several of which encode sarcomeric proteins which can also be mutated to cause hypertrophic cardiomyopathy. One contractile protein gene well known as a hypertrophic cardiomyopathy disease gene, but with no reported mutation in autosomal dominant DCM, is TNNI3 which encodes cardiac troponin I. OBJECTIVE: To test TNNI3 as a candidate gene, a panel of 96 probands with DCM was analyzed. METHODS AND RESULTS: Genomic DNA was isolated and TNNI3 exons screened by heteroduplex analysis. Exons with aberrant profiles were sequenced and variants evaluated by segregation analysis and study of normal controls. We report 2 novel TNNI3 missense mutations, Lys36Gln and Asn185Lys, each associated with severe and early onset familial DCM. Of the 5 mutation carriers, cardiac transplantation was required in 3, at ages 6, 15, and 24 years. Analysis of Ca(2+) regulation of actin-tropomyosin-activated myosin ATPase by troponin revealed that troponin reconstituted with either mutant troponin I gave lower maximum ATPase rates and lower Ca(2+) sensitivity than wild type. Furthermore, mutant thin filaments had reduced Ca(2+) affinity compared with normal. CONCLUSIONS: The functional alterations mirror closely a consistent phenotype found in proven DCM mutations in other thin filament proteins, thus supporting the interpretation that these mutations are disease-causing. These are the first reported autosomal dominant DCM-causing mutations in TNNI3, and so the findings expand the spectrum of disease-causing genes that lead to either hypertrophic cardiomyopathy or DCM depending on the specific mutation.

Atrial fibrillation--a new cardiac channelopathy.

Atrial fibrillation (AF) is a common complication of a wide range of cardiac and systemic diseases and is regarded generally as a sporadic, acquired disorder. Familial clustering of AF has been reported but definitive links of genetic factors with AF pathogenesis have been lacking. Genome-wide linkage studies and the discovery of mutations in families with AF have provided compelling evidence that genetic factors can have a role in the development of AF. Although relatively few disease genes have been identified, current data indicate that inherited defects in cardiac ion channel genes can predispose to AF by altering ion channel activation and atrial conduction properties. Mutations in the reported disease genes account for only a minority of all familial AF cases and further gene discovery studies are required. Characterisation of the genetic variants that cause AF in families provides a framework for elucidation of key disease pathways that underlie the more commonly-occurring complex forms of AF. A better understanding of the molecular and electrophysiological defects that promote AF in families and in the general population will facilitate new approaches to the diagnosis, prevention and treatment of AF.

Mutations in cardiac T-box factor gene TBX20 are associated with diverse cardiac pathologies, including defects of septation and valvulogenesis and cardiomyopathy.

The T-box family transcription factor gene TBX20 acts in a conserved regulatory network, guiding heart formation and patterning in diverse species. Mouse Tbx20 is expressed in cardiac progenitor cells, differentiating cardiomyocytes, and developing valvular tissue, and its deletion or RNA interference-mediated knockdown is catastrophic for heart development. TBX20 interacts physically, functionally, and genetically with other cardiac transcription factors, including NKX2-5, GATA4, and TBX5, mutations of which cause congenital heart disease (CHD). Here, we report nonsense (Q195X) and missense (I152M) germline mutations within the T-box DNA-binding domain of human TBX20 that were associated with a family history of CHD and a complex spectrum of developmental anomalies, including defects in septation, chamber growth, and valvulogenesis. Biophysical characterization of wild-type and mutant proteins indicated how the missense mutation disrupts the structure and function of the TBX20 T-box. Dilated cardiomyopathy was a feature of the TBX20 mutant phenotype in humans and mice, suggesting that mutations in developmental transcription factors can provide a sensitized template for adult-onset heart disease. Our findings are the first to link TBX20 mutations to human pathology. They provide insights into how mutation of different genes in an interactive regulatory circuit lead to diverse clinical phenotypes, with implications for diagnosis, genetic screening, and patient follow-up.

Stretch-sensitive KCNQ1 mutation A link between genetic and environmental factors in the pathogenesis of atrial fibrillation?

OBJECTIVES: This study sought to evaluate mutations in genes encoding the slow component of the cardiac delayed rectifier K+ current (I(Ks)) channel in familial atrial fibrillation (AF). BACKGROUND: Although AF can have a genetic etiology, links between inherited gene defects and acquired factors such as atrial stretch have not been explored. METHODS: Mutation screening of the KCNQ1, KCNE1, KCNE2, and KCNE3 genes was performed in 50 families with AF. The effects of mutant protein on cardiac I(Ks) activation were evaluated using electrophysiological studies and human atrial action potential modeling. RESULTS: One missense KCNQ1 mutation, R14C, was identified in 1 family with a high prevalence of hypertension. Atrial fibrillation was present only in older individuals who had developed atrial dilation and who were genotype positive. Patch-clamp studies of wild-type or R14C KCNQ1 expressed with KCNE1 in CHO cells showed no statistically significant differences between wild-type and mutant channel kinetics at baseline, or after activation of adenylate cyclase with forskolin. After exposure to hypotonic solution to elicit cell swelling/stretch, mutant channels showed a marked increase in current, a leftward shift in the voltage dependence of activation, altered channel kinetics, and shortening of the modeled atrial action potential duration. CONCLUSIONS: These data suggest that the R14C KCNQ1 mutation alone is insufficient to cause AF. Rather, we suggest a model in which a "second hit", such as an environmental factor like hypertension, which promotes atrial stretch and thereby unmasks an inherited defect in ion channel kinetics (the "first hit"), is required for AF to be manifested. Such a model would also account for the age-related increase in AF development.

Molecular characterization of the spectrum of genomic deletions in the mismatch repair genes MSH2, MLH1, MSH6, and PMS2 responsible for hereditary nonpolyposis colorectal cancer (HNPCC).

A systematic search by Southern blot analysis in a cohort of 439 hereditary nonpolyposis colorectal cancer (HNPCC) families for genomic rearrangements in the main mismatch repair (MMR) genes, namely, MSH2, MLH1, MSH6, and PMS2, identified 48 genomic rearrangements causative of this inherited predisposition to colorectal cancer in 68 unrelated kindreds. Twenty-nine of the 48 rearrangements were found in MSH2, 13 in MLH1, 2 in MSH6, and 4 in PMS2. The vast majority were deletions, although one previously described large inversion, an intronic insertion, and a more complex rearrangement also were found. Twenty-four deletion breakpoints have been identified and sequenced in order to determine the underlying recombination mechanisms. Most fall within repetitive sequences, mainly Alu repeats, in agreement with the differential distribution of deletions between the MSH2 and MLH1 genes: the higher number and density of Alu repeats in MSH2 corresponded with a higher incidence of genomic rearrangement at this disease locus when compared with other MMR genes. Long interspersed nuclear element (LINE) repeats, relatively abundant in, for example, MLH1, did not seem to contribute to the genesis of the deletions, presumably because of their older evolutionary age and divergence among individual repeat units when compared with short interspersed nuclear element (SINE) repeats, including Alu repeats. Moreover, Southern blot analysis of the introns and the genomic regions flanking the MMR genes allowed us to detect 6 novel genomic rearrangements that left the coding region of the disease-causing gene intact. These rearrangements comprised 4 deletions upstream of the coding region of MSH2 (3 cases) and MSH6 (1 case), a 2-kb insertion in intron 7 of PMS2, and a small (459-bp) deletion in intron 13 of MLH1. The characterization of these genomic rearrangements underlines the importance of genomic deletions in the etiology of HNPCC and will facilitate the development of PCR-based tests for their detection in diagnostic laboratories.

Defects in nuclear structure and function promote dilated cardiomyopathy in lamin A/C-deficient mice.

Laminopathies are a group of disorders caused by mutations in the LMNA gene that encodes the nuclear lamina proteins, lamin A and lamin C; their pathophysiological basis is unknown. We report that lamin A/C-deficient (Lmna(-/-)) mice develop rapidly progressive dilated cardiomyopathy (DCM) characterized by left ventricular (LV) dilation and reduced systolic contraction. Isolated Lmna(-/-) myocytes show reduced shortening with normal baseline and peak amplitude of Ca(2+) transients. Lmna(-/-) LV myocyte nuclei have marked alterations of shape and size with central displacement and fragmentation of heterochromatin; these changes are present but less severe in left atrial nuclei. Electron microscopy of Lmna(-/-) cardiomyocytes shows disorganization and detachment of desmin filaments from the nuclear surface with progressive disruption of the cytoskeletal desmin network. Alterations in nuclear architecture are associated with defective nuclear function evidenced by decreased SREBP1 import, reduced PPARgamma expression, and a lack of hypertrophic gene activation. These findings suggest a model in which the primary pathophysiological mechanism in Lmna(-/-) mice is defective force transmission resulting from disruption of lamin interactions with the muscle-specific desmin network and loss of cytoskeletal tension. Despite severe DCM, defects in nuclear function prevent Lmna(-/-) cardiomyocytes from developing compensatory hypertrophy and accelerate disease progression.

Two mismatch repair gene mutations found in a colon cancer patient--which one is pathogenic?

Hereditary nonpolyposis colorectal cancer (HNPCC) is a dominantly inherited cancer syndrome. Germline mutations in five different mismatch repair (MMR) genes, MSH2, MSH6, MLH1, MLH3, and PMS2 are linked to HNPCC. Here, we describe two colon cancer families in which the index patients carry missense mutations in both MSH2 and MSH6. The MSH2 mutation, I145M, is the same in both families, whereas the MSH6 mutations are different (R1095H and L1354Q). The families do not fulfil the international criteria for HNPCC, one family comprising two and the other family four colon cancer patients, all in one generation, resembling a recessive rather than dominant inheritance characteristic of HNPCC. The tumors of the index patients showed microsatellite instability. Functional analysis was performed to determine which one of the mutations could primarily underlie the cancer susceptibility in the families. MSH2 and MSH6 are known to form a heterodimeric complex (MutSalpha) responsible for mismatch recognition. The interaction of each mutated protein with its wild-type partner and with its mutated partner present in the colon cancer patient, and the MMR function of the mutated MutSalpha complexes were determined. Since none of the three mutations affected the MSH2-MSH6 interaction or the function of MutSalpha in an in-vitro MMR assay, our results suggest that alone the mutations do not cause MMR deficiency typical of HNPCC. However, our results do not exclude the possible compound pathogenicity of the two mutations.