Jonathan G. Seidman

Missense mutations in the rod domain of the lamin A/C gene as causes of dilated cardiomyopathy and conduction-system disease

BACKGROUND Inherited mutations cause approximately 35 percent of cases of dilated cardiomyopathy; however, few genes associated with this disease have been identified. Previously, we located a gene defect that was responsible for autosomal dominant dilated cardiomyopathy and conduction-system disease on chromosome 1p1-q21, where nuclear-envelope proteins lamin A and lamin C are encoded by the LMNA (lamin A/C) gene. Mutations in the head or tail domain of this gene cause Emery-Dreifuss muscular dystrophy, a childhood-onset disease characterized by joint contractures and in some cases by abnormalities of cardiac conduction during adulthood. METHODS We evaluated 11 families with autosomal dominant dilated cardiomyopathy and conduction-system disease. Sequences of the lamin A/C exons were determined in probands from each family, and variants were confirmed by restriction-enzyme digestion. The genotypes of the family members were ascertained. RESULTS Five novel missense mutations were identified: four in the alpha-helical-rod domain of the lamin A/C gene, and one in the lamin C tail domain. Each mutation caused heritable, progressive conduction-system disease (sinus bradycardia, atrioventricular conduction block, or atrial arrhythmias) and dilated cardiomyopathy. Heart failure and sudden death occurred frequently within these families. No family members with mutations had either joint contractures or skeletal myopathy. Serum creatine kinase levels were normal in family members with mutations of the lamin rod but mildly elevated in some family members with a defect in the tail domain of lamin C. CONCLUSIONS Genetic defects in distinct domains of the nuclear-envelope proteins lamin A and lamin C selectively cause dilated cardiomyopathy with conduction-system disease or autosomal dominant Emery-Dreifuss muscular dystrophy. Missense mutations in the rod domain of the lamin A/C gene provide a genetic cause for dilated cardiomyopathy and indicate that this intermediate filament protein has an important role in cardiac conduction and contractility.

The Genetic Basis for Cardiomyopathy: from Mutation Identification to Mechanistic Paradigms

We are grateful to Steven DePalma and Susanne Bartlett for their invaluable assistance in preparation of figures for this review. This work was supported by grants from N.H.L.B.I. and the Howard Hughes Medical Institute.

A Murine Model of Holt-Oram Syndrome Defines Roles of the T-Box Transcription Factor Tbx5 in Cardiogenesis and Disease

Heterozygous Tbx5(del/+) mice were generated to study the mechanisms by which TBX5 haploinsufficiency causes cardiac and forelimb abnormalities seen in Holt-Oram syndrome. Tbx5 deficiency in homozygous mice (Tbx5(del/del)) decreased expression of multiple genes and caused severe hypoplasia of posterior domains in the developing heart. Surprisingly, Tbx5 haploinsufficiency also markedly decreased atrial natriuretic factor (ANF) and connexin 40 (cx40) transcription, implicating these as Tbx5 target genes and providing a mechanism by which 50% reduction of T-box transcription factors cause disease. Direct and cooperative transactivation of the ANF and cx40 promoters by Tbx5 and the homeodomain transcription factor Nkx2-5 was also demonstrated. These studies provide one potential explanation for Holt-Oram syndrome conduction system defects, suggest mechanisms for intrafamilial phenotypic variability, and account for related cardiac malformations caused by other transcription factor mutations.

Mutations in the Genes for Cardiac Troponin T and α-Tropomyosin in Hypertrophic Cardiomyopathy

BACKGROUND Familial hypertrophic cardiomyopathy can be caused by mutations in the genes for beta cardiac myosin heavy chain, alpha-tropomyosin, or cardiac troponin T. It is not known how often the disease is caused by mutations in the tropomyosin and troponin genes, and the associated clinical phenotypes have not been carefully studied. METHODS Linkage between polymorphisms of the alpha-tropomyosin gene or the cardiac troponin T gene and hypertrophic cardiomyopathy was assessed in 27 families. In addition, 100 probands were screened for mutations in the alpha-tropomyosin gene, and 26 were screened for mutations in the cardiac troponin T gene. Life expectancy, the incidence of sudden death, and the extent of left ventricular hypertrophy were compared in patients with different mutations. RESULTS Genetic analyses identified only one alpha-tropomyosin mutation, identical to one previously described. Five novel mutations in cardiac troponin were identified, as well as a further example of a previously described mutation. The clinical phenotype of four troponin T mutations in seven unrelated families was similar and was characterized by a poor prognosis (life expectancy, approximately 35 years) and a high incidence of sudden death. The mean (+/- SD) maximal thickness of the left ventricular wall in subjects with cardiac troponin T mutations (16.7 +/- 5.5 mm) was significantly less than that in subjects with beta cardiac myosin heavy-chain mutations (23.7 +/- 7.7 mm, P < 0.001). CONCLUSIONS Mutations in alpha-tropomyosin are a rare cause of familial hypertrophic cardiomyopathy, accounting for approximately 3 percent of cases. Mutations in cardiac troponin T account for approximately 15 percent of cases of familial hypertrophic cardiomyopathy in this referral-center population. These mutations are characterized by relatively mild and sometimes subclinical hypertrophy but a high incidence of sudden death. Genetic testing may therefore be especially important in this group.

Mutations in the gene for cardiac myosin-binding protein C and late-onset familial hypertrophic cardiomyopathy

BACKGROUND Mutations in the gene for cardiac myosin-binding protein C account for approximately 15 percent of cases of familial hypertrophic cardiomyopathy. The spectrum of disease-causing mutations and the associated clinical features of these gene defects are unknown. METHODS DNA sequences encoding cardiac myosin-binding protein C were determined in unrelated patients with familial hypertrophic cardiomyopathy. Mutations were found in 16 probands, who had 574 family members at risk of inheriting these defects. The genotypes of these family members were determined, and the clinical status of 212 family members with mutations in the gene for cardiac myosin-binding protein C was assessed. RESULTS Twelve novel mutations were identified in probands from 16 families. Four were missense mutations; eight defects (insertions, deletions, and splice mutations) were predicted to truncate cardiac myosin-binding protein C. The clinical expression of either missense or truncation mutations was similar to that observed for other genetic causes of hypertrophic cardiomyopathy, but the age at onset of the disease differed markedly. Only 58 percent of adults under the age of 50 years who had a mutation in the cardiac myosin-binding protein C gene (68 of 117 patients) had cardiac hypertrophy; disease penetrance remained incomplete through the age of 60 years. Survival was generally better than that observed among patients with hypertrophic cardiomyopathy caused by other mutations in the genes for sarcomere proteins. Most deaths due to cardiac causes in these families occurred suddenly. CONCLUSIONS The clinical expression of mutations in the gene for cardiac myosin-binding protein C is often delayed until middle age or old age. Delayed expression of cardiac hypertrophy and a favorable clinical course may hinder recognition of the heritable nature of mutations in the cardiac myosin-binding protein C gene. Clinical screening in adult life may be warranted for members of families characterized by hypertrophic cardiomyopathy.

Characteristics and prognostic implications of myosin missense mutations in familial hypertrophic cardiomyopathy.

BACKGROUND Familial hypertrophic cardiomyopathy is characterized by a variable degree of myocardial hypertrophy and a wide range of symptoms. Different mutations in the beta cardiac myosin heavy-chain gene have been identified in three affected families. However, neither the proportion of cases attributable to myosin mutations nor the effects of different mutations on clinical outcome are known. METHODS Using a ribonuclease protection assay, we screened the beta cardiac myosin heavy-chain genes of probands from 25 unrelated families with familial hypertrophic cardiomyopathy; this assay is a sensitive method for detecting the presence and location of mutations. We further defined the mutations by analyzing their nucleotide sequences. The clinical features of the disease were compared in families with various myosin mutations. RESULTS Seven mutations in the beta cardiac myosin heavy-chain gene were identified in 12 of the 25 families. All were missense mutations (i.e., causing the substitution of a single amino acid) clustered in the head and head-rod junction regions of the molecule. Six mutations resulted in a change in the charge of the amino acid. Patients with mutations that changed the charge of the altered amino acid (such as that from arginine to glutamine at nucleotide 403 or from arginine to cysteine at nucleotide 453) had a significantly shorter life expectancy (mean age at death, 33 years), whereas patients with the one mutation that did not produce a change in charge (Val606Met) had nearly normal survival. However, patients with different mutations did not differ appreciably in their clinical manifestations of familial hypertrophic cardiomyopathy. CONCLUSIONS Different missense mutations in the beta cardiac myosin heavy-chain gene can be identified in approximately 50 percent of families with hypertrophic cardiomyopathy. In those families, a definite genetic diagnosis can be made in all members. Since the location of a mutation or its DNA-sequence alteration (or both) appears to influence survival, we suggest that the precise definition of the disease-causing mutation can provide important prognostic information about affected members.

Mutations in the cardiac transcription factor NKX2.5 affect diverse cardiac developmental pathways

Heterozygous mutations in NKX2.5, a homeobox transcription factor, were reported to cause secundum atrial septal defects and result in atrioventricular (AV) conduction block during postnatal life. To further characterize the role of NKX2.5 in cardiac morphogenesis, we sought additional mutations in groups of probands with cardiac anomalies and first-degree AV block, idiopathic AV block, or tetralogy of Fallot. We identified 7 novel mutations by sequence analysis of the NKX2.5-coding region in 26 individuals. Associated phenotypes included AV block, which was the primary manifestation of cardiac disease in nearly a quarter of affected individuals, as well as atrial septal defect and ventricular septal defect. Ventricular septal defect was associated with tetralogy of Fallot or double-outlet right ventricle in 3 individuals. Ebsteins anomaly and other tricuspid valve abnormalities were also present. Mutations in human NKX2.5 cause a variety of cardiac anomalies and may account for a clinically significant portion of tetralogy of Fallot and idiopathic AV block. The coinheritance of NKX2.5 mutations with various congenital heart defects suggests that this transcription factor contributes to diverse cardiac developmental pathways.

A Mouse Model of Familial Hypertrophic Cardiomyopathy

A mouse model of familial hypertrophic cardiomyopathy (FHC) was generated by the introduction of an Arg403 → Gln mutation into the α cardiac myosin heavy chain (MHC) gene. Homozygous αMHC403/403 mice died 7 days after birth, and sedentary heterozygous αMHC403/+ mice survived for 1 year. Cardiac histopathology and dysfunction in the αMHC403/+ mice resembled human FHC. Cardiac dysfunction preceded histopathologic changes, and myocyte disarray, hypertrophy, and fibrosis increased with age. Young male αMHC403/+ mice showed more evidence of disease than did their female counterparts. Preliminary results suggested that exercise capacity may have been compromised in the αMHC403/+ mice. This mouse model may help to define the natural history of FHC.

De novo mutations in histone-modifying genes in congenital heart disease.

Congenital heart disease (CHD) is the most frequent birth defect, affecting 0.8% of live births. Many cases occur sporadically and impair reproductive fitness, suggesting a role for de novo mutations. Here we compare the incidence of de novo mutations in 362 severe CHD cases and 264 controls by analysing exome sequencing of parent–offspring trios. CHD cases show a significant excess of protein-altering de novo mutations in genes expressed in the developing heart, with an odds ratio of 7.5 for damaging (premature termination, frameshift, splice site) mutations. Similar odds ratios are seen across the main classes of severe CHD. We find a marked excess of de novo mutations in genes involved in the production, removal or reading of histone 3 lysine 4 (H3K4) methylation, or ubiquitination of H2BK120, which is required for H3K4 methylation. There are also two de novo mutations in SMAD2, which regulates H3K27 methylation in the embryonic left–right organizer. The combination of both activating (H3K4 methylation) and inactivating (H3K27 methylation) chromatin marks characterizes ‘poised’ promoters and enhancers, which regulate expression of key developmental genes. These findings implicate de novo point mutations in several hundreds of genes that collectively contribute to approximately 10% of severe CHD.

Mutations of TTN , encoding the giant muscle filament titin, cause familial dilated cardiomyopathy

Congestive heart failure (CHF) can result from various disease states with inadequate cardiac output. CHF due to dilated cardiomyopathy (DCM) is a familial disease in 20–30% of cases and is associated with mutations in genes encoding cytoskeletal, contractile or inner–nuclear membrane proteins. We show that mutations in the gene encoding giant-muscle filament titin (TTN) cause autosomal dominant DCM linked to chromosome 2q31 (CMD1G; MIM 604145). Titin molecules extend from sarcomeric Z-discs to M-lines, provide an extensible scaffold for the contractile machinery and are crucial for myofibrillar elasticity and integrity. In a large DCM kindred, a segregating 2-bp insertion mutation in TTN exon 326 causes a frameshift, truncating A-band titin. The truncated protein of approximately 2 mD is expressed in skeletal muscle, but western blot studies with epitope-specific anti-titin antibodies suggest that the mutant protein is truncated to a 1.14-mD subfragment by site-specific cleavage. In another large family with DCM linked to CMD1G, a TTN missense mutation (Trp930Arg) is predicted to disrupt a highly conserved hydrophobic core sequence of an immunoglobulin fold located in the Z-disc–I-band transition zone. The identification of TTN mutations in individuals with CMD1G should provide further insights into the pathogenesis of familial forms of CHF and myofibrillar titin turnover.