Elizabeth Sparks

Truncations of Titin Causing Dilated Cardiomyopathy

BACKGROUND Dilated cardiomyopathy and hypertrophic cardiomyopathy arise from mutations in many genes. TTN, the gene encoding the sarcomere protein titin, has been insufficiently analyzed for cardiomyopathy mutations because of its enormous size. METHODS We analyzed TTN in 312 subjects with dilated cardiomyopathy, 231 subjects with hypertrophic cardiomyopathy, and 249 controls by using next-generation or dideoxy sequencing. We evaluated deleterious variants for cosegregation in families and assessed clinical characteristics. RESULTS We identified 72 unique mutations (25 nonsense, 23 frameshift, 23 splicing, and 1 large tandem insertion) that altered full-length titin. Among subjects studied by means of next-generation sequencing, the frequency of TTN mutations was significantly higher among subjects with dilated cardiomyopathy (54 of 203 [27%]) than among subjects with hypertrophic cardiomyopathy (3 of 231 [1%], P=3×10(-16)) or controls (7 of 249 [3%], P=9×10(-14)). TTN mutations cosegregated with dilated cardiomyopathy in families (combined lod score, 11.1) with high (>95%) observed penetrance after the age of 40 years. Mutations associated with dilated cardiomyopathy were overrepresented in the titin A-band but were absent from the Z-disk and M-band regions of titin (P≤0.01 for all comparisons). Overall, the rates of cardiac outcomes were similar in subjects with and those without TTN mutations, but adverse events occurred earlier in male mutation carriers than in female carriers (P=4×10(-5)). CONCLUSIONS TTN truncating mutations are a common cause of dilated cardiomyopathy, occurring in approximately 25% of familial cases of idiopathic dilated cardiomyopathy and in 18% of sporadic cases. Incorporation of sequencing approaches that detect TTN truncations into genetic testing for dilated cardiomyopathy should substantially increase test sensitivity, thereby allowing earlier diagnosis and therapeutic intervention for many patients with dilated cardiomyopathy. Defining the functional effects of TTN truncating mutations should improve our understanding of the pathophysiology of dilated cardiomyopathy. (Funded by the Howard Hughes Medical Institute and others.).

Constitutively active AMP kinase mutations cause glycogen storage disease mimicking hypertrophic cardiomyopathy

Mutations in PRKAG2, the gene for the gamma 2 regulatory subunit of AMP-activated protein kinase, cause cardiac hypertrophy and electrophysiologic abnormalities, particularly preexcitation (Wolff-Parkinson-White syndrome) and atrioventricular conduction block. To understand the mechanisms by which PRKAG2 defects cause disease, we defined novel mutations, characterized the associated cardiac histopathology, and studied the consequences of introducing these mutations into the yeast homologue of PRKAG2, Snf4. Although the cardiac pathology caused by PRKAG2 mutations Arg302Gln, Thr400Asn, and Asn488Ile include myocyte enlargement and minimal interstitial fibrosis, these mutations were not associated with myocyte and myofibrillar disarray, the pathognomonic features of hypertrophic cardiomyopathy caused by sarcomere protein mutations. Instead PRKAG2 mutations caused pronounced vacuole formation within myocytes. Several lines of evidence indicated these vacuoles were filled with glycogen-associated granules. Analyses of the effects of human PRKAG2 mutations on Snf1/Snf4 kinase function demonstrated constitutive activity, which could foster glycogen accumulation. Taken together, our data indicate that PRKAG2 mutations do not cause hypertrophic cardiomyopathy but rather lead to a novel myocardial metabolic storage disease, in which hypertrophy, ventricular pre-excitation and conduction system defects coexist.

Mutations in Transforming Growth Factor-β Receptor Type II Cause Familial Thoracic Aortic Aneurysms and Dissections

Background—A genetic predisposition for progressive enlargement of thoracic aortic aneurysms leading to type A dissection (TAAD) is inherited in an autosomal-dominant manner in up to 19% of patients, and a number of chromosomal loci have been identified for the condition. Having mapped a TAAD locus to 3p24–25, we sequenced the gene for transforming growth factor-&bgr; receptor type II (TGFBR2) to determine whether mutations in this gene resulted in familial TAAD. Methods and Results—We sequenced all 8 coding exons of TGFBR2 by using genomic DNA from 80 unrelated familial TAAD cases. We found TGFBR2 mutations in 4 unrelated families with familial TAAD who did not have Marfan syndrome. Affected family members also had descending aortic disease and aneurysms of other arteries. Strikingly, all 4 mutations affected an arginine residue at position 460 in the intracellular domain, suggesting a mutation “hot spot” for familial TAAD. Despite identical mutations in the families, assessment of linked polymorphisms suggested that these families were not distantly related. Structural analysis of the TGFBR2 serine/threonine kinase domain revealed that R460 is strategically located within a highly conserved region of this domain and that the amino acid substitutions resulting from these mutations will interfere with the receptor’s ability to transduce signals. Conclusion—Germline TGFBR2 mutations are responsible for the inherited predisposition to familial TAAD in 5% of these cases. Our results have broad implications for understanding the role of TGF-&bgr; signaling in the pathophysiology of TAAD.

Mutations in Smooth Muscle Alpha-Actin (ACTA2) Cause Coronary Artery Disease, Stroke, and Moyamoya Disease, Along with Thoracic Aortic Disease

The vascular smooth muscle cell (SMC)-specific isoform of alpha-actin (ACTA2) is a major component of the contractile apparatus in SMCs located throughout the arterial system. Heterozygous ACTA2 mutations cause familial thoracic aortic aneurysms and dissections (TAAD), but only half of mutation carriers have aortic disease. Linkage analysis and association studies of individuals in 20 families with ACTA2 mutations indicate that mutation carriers can have a diversity of vascular diseases, including premature onset of coronary artery disease (CAD) and premature ischemic strokes (including Moyamoya disease [MMD]), as well as previously defined TAAD. Sequencing of DNA from patients with nonfamilial TAAD and from premature-onset CAD patients independently identified ACTA2 mutations in these patients and premature onset strokes in family members with ACTA2 mutations. Vascular pathology and analysis of explanted SMCs and myofibroblasts from patients harboring ACTA2 suggested that increased proliferation of SMCs contributed to occlusive diseases. These results indicate that heterozygous ACTA2 mutations predispose patients to a variety of diffuse and diverse vascular diseases, including TAAD, premature CAD, ischemic strokes, and MMD. These data demonstrate that diffuse vascular diseases resulting from either occluded or enlarged arteries can be caused by mutations in a single gene and have direct implications for clinical management and research on familial vascular diseases.

THE MARFAN SYNDROME : ABNORMAL AORTIC ELASTIC PROPERTIES

Aortic distensibility and aortic stiffness index were measured at the ascending aorta (3 cm above the aortic valve) and the mid-portion of the abdominal aorta from the changes in echocardiographic diameters and pulse pressure in 14 patients with the Marfan syndrome and 15 age- and gender-matched normal control subjects. The following formulas were used: 1) Aortic distensibility = 2(Changes in aortic diameter)/(Diastolic aortic diameter) (Pulse pressure); and 2) Aortic stiffness index = ln(Systolic blood pressure)/(Diastolic blood pressure)(Changes in aortic diameter)/Diastolic aortic diameter. Pulse wave velocity was also measured. Compared with normal subjects, patients with the Marfan syndrome had decreased aortic distensibility in the ascending and the abdominal aorta (2.9 +/- 1.3 vs. 5.6 +/- 1.4 cm2 dynes-1, p less than 0.001 and 4.5 +/- 2.1, vs. 7.7 +/- 2.5, cm2 dynes-1, p less than 0.001, respectively) and had an increased aortic stiffness index in the ascending and the abdominal aorta (10.9 +/- 5.6 vs. 5.9 +/- 2.2, p less than 0.005 and 7.1 +/- 3.1 vs. 3.9 +/- 1.2, p less than 0.005, respectively). Aortic diameters in the ascending aorta were larger in these patients than in normal subjects, but those in the abdominal aorta were similar in the two groups. Linear correlations for both aortic distensibility and stiffness index were found between the ascending and the abdominal aorta (r = 0.85 and 0.71, respectively). Pulse wave velocity was more rapid in the patients than in the normal subjects (11.6 +/- 2.5 vs. 9.5 +/- 1.4 m/s, respectively, p less than 0.01). Thus, aortic elastic properties are abnormal in patients with the Marfan syndrome irrespective of the aortic diameter, which suggests an intrinsic abnormality of the aortic arterial wall.

Familial dilated cardiomyopathy caused by an alpha-tropomyosin mutation: the distinctive natural history of sarcomeric dilated cardiomyopathy.

OBJECTIVES We sought to further define the role of sarcomere mutations in dilated cardiomyopathy (DCM) and associated clinical phenotypes. BACKGROUND Mutations in several contractile proteins contribute to DCM, but definitive evidence for the roles of most sarcomere genes remains limited by the lack of robust genetic support. METHODS Direct sequencing of 6 sarcomere genes was performed on 334 probands with DCM. A novel D230N missense mutation in the gene encoding alpha-tropomyosin (TPM1) was identified. Functional assessment was performed by the use of an in vitro reconstituted sarcomere complex to evaluate ATPase regulation and Ca(2+) affinity as correlates of contractility. RESULTS TPM1 D230N segregated with DCM in 2 large unrelated families. This mutation altered an evolutionarily conserved residue and was absent in >1,000 control chromosomes. In vitro studies demonstrated major inhibitory effects on sarcomere function with reduced Ca(2+) sensitivity, maximum activation, and Ca(2+) affinity compared with wild-type TPM1. Clinical manifestations ranged from decompensated heart failure or sudden death in those presenting early in life to asymptomatic left ventricular dysfunction in those diagnosed during adulthood. Notably, several affected infants had remarkable improvement. CONCLUSIONS Genetic segregation in 2 unrelated families and functional analyses conclusively establish a pathogenic role for TPM1 mutations in DCM. In vitro results demonstrate contrasting effects of DCM and hypertrophic cardiomyopathy mutations in TPM1, suggesting that specific functional consequences shape cardiac remodeling. Along with previous reports, our data support a distinctive, age-dependent phenotype with sarcomere-associated DCM where presentation early in life is associated with severe, sometimes lethal, disease. These observations have implications for the management of familial DCM.

Mid-Myocardial Fibrosis by Cardiac Magnetic Resonance in Patients with Lamin A/C Cardiomyopathy: Possible Substrate for Diastolic Dysfunction

AIMS We sought to identify patterns of myocardial fibrosis in vivo in patients with lamin cardiomyopathy, and to determine its functional significance. METHODS AND RESULTS Eleven patients sharing the identical mutation in LMNA without contraindication to magnetic resonance were identified from a 1016-member pedigree. Eight autopsy hearts from deceased relatives were reviewed. Patients and age-matched controls underwent cardiac magnetic resonance that included measures of cardiac function and late gadolinium enhancement (LGE). LGE-CMR identified midmyocardial fibrosis of the basal interventricular septum in 5 of 11 LMNA patients that was identical to that seen in 6 autopsy specimens of related genotype-positive family members; this was not present in any of 11 controls. LGE-CMR was positive in the 5 oldest patients in the cohort, age 46 +/- 6 years compared to 24 +/- 10 years for LGE-negative subjects (p = 0.003). Systolic function was abnormal in 2 subjects, both with myocardial fibrosis. LGE-positivity distinguished patients with diastolic dysfunction by mitral inflow velocities from those with normal diastolic function; these patients also had significant left atrial enlargement compared to controls (p < 0.05). CONCLUSIONS LGE-CMR can identify myocardial fibrosis under genetic control in vivo in patients with heritable cardiomyopathy similar in distribution to that observed at autopsy. Mid-myocardial fibrosis may form the substrate for diastolic dysfunction in these patients.

Short Communication: The Cardiac Myosin Binding Protein C Arg502Trp Mutation A Common Cause of Hypertrophic Cardiomyopathy

Rationale: The myosin-binding protein C isoform 3 (MYBPC3) variant Arg502Trp has been identified in multiple hypertrophic cardiomyopathy (HCM) cases, but compelling evidence to support or refute the pathogenicity of this variant is lacking. Objective: To determine the prevalence, origin and clinical significance of the MYBPC3 Arg502Trp variant. Methods and Results: The prevalence of MYBPC3 Arg502Trp was ascertained in 1414 sequential HCM patients of primarily European descent. MYBPC3 Arg502Trp was identified in 34 of these 1414 unrelated HCM patients. Segregation of MYBPC3 Arg502Trp with clinical status was assessed in family members. Disease haplotypes were examined in 17 families using two loci flanking MYBPC3. Family studies identified an additional 43 variant carriers, many with manifest disease, yielding a calculated odds ratio of 11 000:1 for segregation of MYBPC3 Arg502Trp with HCM. Analyses in 17 families showed at least 4 independent haplotypes flanked MYBPC3 Arg502Trp. Eight individuals (4 probands and 4 family members) also had another sarcomere protein gene mutation. Major adverse clinical events occurred in approximately 30% of MYBPC3 Arg502Trp carriers by age 50; these were significantly more likely (P<0.0001) when another sarcomere mutation was present. Conclusions: MYBPC3 Arg502Trp is the most common and recurrent pathogenic mutation in a diverse primarily European descent HCM cohort, occurring in 2.4% of patients. MYBPC3 Arg502Trp conveys a 340-fold increased risk for HCM by 45 years of age, when more than 50% of carriers have overt disease. HCM prognosis worsens when MYBPC3 Arg502Trp occurs in the setting of another sarcomere protein gene mutation.

Cardiovascular Effects of Type 1 Diabetes Mellitus in Children

Previous studies have shown that type 1 diabetes mellitus (DM) is associated with cardiovascular abnormalities. Early detection and treatment of these abnormalities may help to prevent the natural progression of the disease. The present study was undertaken to define early cardiovascular abnormalities in children with type 1 DM. Simultaneous evaluation of multiple cardiovascular parameters was performed in 14 children with type 1 DM and 14 age- and gender-matched normal subjects. Measurements of carotid artery intima-media thickness (cIMT, echocardiography), carotid and aortic (ascending and abdominal) distensibility (echocardiography, brachial artery blood pressure), aortic pulse wave velocity (carotid to femoral artery, Doppler), and left ventricular dimensions, mass, and function (echocardiography) were performed. Diabetic children demonstrated a greater cIMT (0.36 ±0.04 mm vs 0.31 ±0.03 mm, p=0.002) and decreased carotid artery distensibility (4.4 ±1.6 cm2·dynes-1·10-6 vs 6.0 ±1.9 cm2·dynes-1·10-6, p<0.01) compared to control. Aortic pulse wave velocity was increased in DM (6.70 ±0.39 vs 6.30 ±0.31, p=0.02) compared to control. Left ventricular diameters, mass, and systolic and diastolic function did not differ between the 2 groups. Simultaneous assessment of multiple cardiovascular parameters in children with type 1 DM revealed impaired carotid artery structure and function, and decreased elastic properties of the aorta, before demonstrable changes in left ventricular structure and function could be detected.

Subtle Abnormalities in Contractile Function Are an Early Manifestation of Sarcomere Mutations in Dilated Cardiomyopathy

Background—Sarcomere mutations cause both dilated cardiomyopathy (DCM) and hypertrophic cardiomyopathy (HCM); however, the steps leading from mutation to disease are not well described. By studying mutation carriers before a clinical diagnosis develops, we characterize the early manifestations of sarcomere mutations in DCM and investigate how these manifestations differ from sarcomere mutations associated with HCM. Methods and Results—Sixty-two genotyped individuals in families with sarcomeric DCM underwent clinical evaluation including strain echocardiography. The group included 12 subclinical DCM mutation carriers with normal cardiac dimensions and left ventricular ejection fraction (LVEF ≥55%), 21 overt DCM subjects, and 29 related mutation (-) normal controls. Results were compared with a previously characterized cohort of 60 subclinical HCM subjects (sarcomere mutation carriers without left ventricular hypertrophy). Systolic myocardial tissue velocity, longitudinal, circumferential, and radial strain, and longitudinal and radial strain rate were reduced by 10%–23% in subclinical DCM mutation carriers compared with controls (P<0.001 for all comparisons), after adjusting for age and family relations. No significant differences in diastolic parameters were identified comparing the subclinical and control cohorts. The opposite pattern of contractile abnormalities with reduced diastolic but preserved systolic function was seen in subclinical HCM. Conclusions—Subtle abnormalities in systolic function are present in subclinical DCM mutation carriers, despite normal left ventricular size and ejection fraction. In contrast, impaired relaxation and preserved systolic function appear to be the predominant early manifestations of sarcomere mutations that lead to HCM. These findings support the theory that the mutation’s intrinsic impact on sarcomere function influences whether a dilated or hypertrophic phenotype develops.