Codon 102 of the cardiac troponin T gene is a putative hot spot for mutations in familial hypertrophic cardiomyopathy

BACKGROUND: Familial hypertrophic cardiomyopathy is a phenotypically and genetically heterogeneous disease. In some families, the disease is linked to the CMH2 locus on chromosome 1q3, in which the cardiac troponin T gene (TNNT2) has been identified as the disease gene. The mutations found in this gene appear to be associated with incomplete penetrance and poor prognosis. Because mutational hot spots offer unique possibilities for analysis of genotype-phenotype correlations, new missense mutations that could define such hot spots in TNNT2 were looked for in unrelated French families with familial hypertrophic cardiomyopathy. METHODS AND RESULTS: Family members were genotyped with microsatellite markers to detect linkage to the four known disease loci. In family 715, analyses showed linkage to CMH2 only. To accurately position potential mutations on TNNT2, its partial genomic organization was established. Screening for mutations was performed by single-strand conformation polymorphism analysis and sequencing. A new missense mutation, Arg102Leu, was identified in affected members of family 715 because of a G-->T transversion located in the 10th exon of the gene. Penetrance of this new mutation is complete; echocardiographic data show a wide range of hypertrophy; and there was no sudden cardiac death in this family. CONCLUSIONS: The codon 102 of the TNNT2 gene is a putative mutational hot spot in familial hypertrophic cardiomyopathy and is associated with phenotypic variability. Analysis of more pedigrees carrying mutations in this codon is necessary to better characterize the clinical and prognostic implications of TNNT2 mutations.     

Expression and functional assessment of a truncated cardiac troponin T that causes hypertrophic cardiomyopathy. Evidence for a dominant negative action

Mutations in the beta-myosin heavy chain gene are believed to cause hypertrophic cardiomyopathy (HCM) by acting as dominant negative alleles. In contrast, a truncated cardiac troponin T (TnT) that causes HCM implies that altered stoichiometry of contractile proteins may also cause cardiac hypertrophy. Wild-type and HCM-mutant (truncated) TnT were studied in a novel quail myotube expression system. Unexpectedly, antibody staining demonstrated incorporation of both forms of human cardiac TnT into the sarcomeres of quail myotubes. Functional studies of wild type and mutant transfected myotubes of normal appearance revealed that calcium-activated force of contraction was normal upon incorporation of wild type TnT, but greatly diminished for the mutant TnT. These findings indicate that HCM-causing mutations in TnT and beta-myosin heavy chain share abnormalities in common, acting as dominant negative alleles that impair contractile performance. This diminished force output is the likely stimulus for hypertrophy in the human heart.     

Paroxysmal atrial fibrillation: main cause of syncope in hypertrophic cardiomyopathy

The aim of this retrospective study was to determine the mechanism of syncope in idiopathic hypertrophic cardiomyopathy (HCM). An electrocardiographic study was undertaken in 43 patients with HCM: 27 (Group I) had a history of syncope and 16 (Group II) had no history of syncope but were investigated for conduction defects (n = 7) or unsustained ventricular tachycardia (VT) (n = 9). The stimulation protocol used programmed atrial pacing with 1 and 2 extrastimuli and ventricular pacing using up to 3 extrastimuli delivered at 2 sites. The following results were obtained: sustained atrial fibrillation (AF) (> 1 min) was induced in 21 patients in Group I (78%), 4 in Group II (25%); VT was induced in 3 patients in Group I (11%), and 3 in Group II (19%); infra-Hisian block was detected in 1 patient in Group I. The mechanism of syncope was elucidated in 23 patients in Group I (85%): one atrioventricular block 1 sinus node dysfunction, 18 atrial fibrillations, 2 associations of AF-VT and 1 VT. The authors conclude that the prevalence of inducible AF was higher in patients with HCA and syncope than in controls and HCM without syncope: this was the only detectable difference in 67% of patients with unexplained syncope. Paroxysmal AF could therefore explain malaise or syncope in up to 2/3 of cases of HCM.     

Molecular basis of inherited factor XIII deficiency: identification of multiple mutations provides insights into protein function

Factor XIII (FXIII) is a zymogen essential for normal haemostasis. In inherited FXIII deficiency the majority of cases show absence of the FXIIIa subunit. Molecular analysis of PCR-amplified FXIIIa subunit exonic regions, and of RT-PCR amplified cDNA from six patients with FXIIIa subunit deficiency, from five unrelated families, has revealed 10 sequence changes: three mutations resulting in abnormal splicing of pre-mRNA, one nonsense mutation, one deletion/insertion change, three point mutations producing Val34Leu, Asn60Lys and Arg408Gln changes, and two silent mutations. In three families the patients are homozygous for a specific deficiency causing mutation, and patients from the remaining two families are compound heterozygotes. Understanding the molecular pathology of the disorder provides insights into the structure-function relationships of the various domains within the FXIII protein. From a clinical point of view, it enables direct diagnosis at the DNA level and may aid the development of FXIII analogues to promote wound healing.     

Murine MPS I: insights into the pathogenesis of Hurler syndrome

Mucopolysaccharidosis type I (MPS I) is an autosomal recessive disease resulting from deficiency of the lysosomal enzyme alpha-L-iduronidase. A murine model which shows complete deficiency in alpha-L-iduronidase activity has been developed and shows phenotypic features similar to severe MPS I in humans. Here we report on the long-term clinical, biochemical, and pathological course of MPS I in mice with emphasis on the skeletal and central nervous system (CNS) manifestations. Affected mice show a progressive clinical course with the development of coarse features, altered growth characteristics and a shortened life span. Progressive lysosomal accumulation is seen in all tissues. Skeletal manifestations represent the earliest clinical finding in MPS I mice with histologic analysis of growth plate and cortical bone revealing evidence that significant early pathology is present. Analysis of the CNS has revealed the novel finding of progressive neuronal loss within the cerebellum. In addition, brain tissue from MPS I mice shows increased levels of GM2 and GM3 gangliosides. This murine model clearly shows phenotypic and pathologic features which mimic those seen in severe human MPS I and should be an invaluable tool for the study of the pathogenesis of generalized storage disorders.     

New subtype of apical hypertrophic cardiomyopathy identified with nuclear magnetic resonance imaging as an underlying cause of markedly inverted T waves

OBJECTIVES: The aim of this study was to elucidate the clinical importance of a new subtype of apical hypertrophic cardiomyopathy that could not be diagnosed with the classical diagnostic criteria. BACKGROUND: Apical hypertrophic cardiomyopathy is recognized by a characteristic spade-shaped intraventricular cavity on the end-diastolic left ventriculogram in the right anterior oblique projection, often associated with giant negative T waves [negativity > or = 1.0 mV (10 mm)]. As an underlying cause of giant negative T waves, an additional new subtype of apical hypertrophic cardiomyopathy has been identified. METHODS: In 40 patients with inverted T waves (negativity > or = 0.5 mV), including 26 patients with giant negative T waves, nuclear magnetic resonance (NMR) long-axis images corresponding to the left ventriculogram in the right anterior oblique projection and short-axis images at various levels, including the apical level, were obtained to define the site of hypertrophied myocardium. RESULTS: Long-axis images indicated a spadelike configuration in 17 patients, whereas this diagnostic configuration was not present in the other 23 patients. Nine of these 23 patients had significantly hypertrophied myocardium at the basal level. In the 14 remaining patients, short-axis images indicated no hypertrophy at the basal level and proved that the area of hypertrophied myocardium was confined to a narrow region of the septum or the anterior or lateral wall at the apical level (nonspade apical hypertrophic cardiomyopathy). The hypertrophied myocardium of the nonspade type was so narrowly confined that the mass did not form a spadelike configuration or could not be detected on the long-axis image. CONCLUSIONS: Nonspade apical hypertrophic cardiomyopathy was newly identified on NMR short-axis images, and this could be an additional, important underlying cause of moderately to severely inverted T waves.     

Syncope and ventricular arrhythmias in hypertrophic cardiomyopathy are not related to the derangement of coronary microvascular function

Non-sustained ventricular tachycardia on Holter and syncope have been considered risk factors for sudden death in hypertrophic cardiomyopathy. AIMS: In these patients the coronary vasodilator reserve is impaired despite normal coronaries, so we evaluated the correlation between the severity of coronary vasodilator reserve impairment and the occurrence of syncope and non-sustained ventricular tachycardia. METHODS AND RESULTS: Eighty-four patients with hypertrophic cardiomyopathy (62 males, age 43 +/- 12 years) had a two-dimensional echocardiographic study and a 48-h Holter. Myocardial blood flow was measured by positron emission tomography, at baseline and after dipyridamole, and the coronary vasodilator reserve was computed as dipyridamole myocardial blood flow/baseline myocardial blood flow. In 27 patients, subendocardial and subepicardial myocardial blood flow was measured in the septum and the subendocardial/subepicardial ratio was computed. Twenty of 84 patients had at least one syncopal episode, and 26 had at least one run of non-sustained ventricular tachycardia on Holter. Baseline and dipyridamole myocardial blood flow, coronary vasodilator reserve, and baseline and dipyridamole subendocardial/subepicardial myocardial blood flow ratio were similar in patients with and without syncope and with and without non-sustained ventricular tachycardia on Holter. However, patients with non-sustained ventricular tachycardia had larger left ventricular end-diastolic (47 +/- 6 vs 44 +/- 5 mm, P < 0.05) and end-systolic diameters (30 +/- 6 vs 27 +/- 4 mm, P < 0.05). CONCLUSIONS: (1) Coronary vasodilation is not more severely impaired in patients with hypertrophic cardiomyopathy and syncope or non-sustained ventricular tachycardia. (2) The left ventricle is more dilated in hypertrophic cardiomyopathy with non-sustained ventricular tachycardia.     

Transgenic and knockout analyses of the role of TNF in immune regulation and disease pathogenesis

Transgenic mutagenesis in whole animals has become without doubt the most rewarding approach to analyse gene structure, expression, and function. In the TNF field, much of what we now question about TNF/TNF receptor function is based, to a large extent, on what we have already learned by overexpressing these molecules in transgenic mice or by ablating their expression in knockout systems. In addition, a clearer view of the involvement of these molecules in disease pathogenesis has emerged, and useful models for human disease have been generated. In this overview, we summarise our experience with TNF transgenic and knockout systems, and highlight advances made in our understanding of the role played by TNF and its receptors in immune regulation and in the pathogenesis of infectious, inflammatory, and autoimmune disease.     

Alterations of the architecture of subendocardial arterioles in patients with hypertrophic cardiomyopathy and impaired coronary vasodilator reserve: a possible cause for myocardial ischemia

OBJECTIVES: The study was designed to investigate the architecture of subendocardial arterioles of patients with hypertrophic cardiomyopathy (HCM) and angina pectoris with respect to coronary vasodilator reserve. BACKGROUND: There is growing evidence that the coronary microvasculature is abnormal in HCM. Arterioles, which mainly regulate intramyocardial blood flow, are especially suspect. METHODS: Thirteen patients with HCM (50.1+/-12.6 years old, mean value +/- SD) were studied after exclusion of any relevant coronary stenoses. Subendocardial arterioles (density [n/mm2], wall area [microm2], percent lumen area [%lumen], periarteriolar collagen area [microm2]), myocyte diameter (microm) and interstitial collagen fraction (Vv%) were evaluated by means of stereologic morphometry of transvenous biopsy samples. Coronary blood flow was measured quantitatively with the inert chromatographic argon method at basal conditions and after dipyridamole (0.5 mg/kg body weight over 4 min intravenously), and coronary vasodilator reserve was calculated as the ratio of coronary resistance at basal conditions and after pharmacologic vasodilation. Data from five normotensive subjects (45.4+/-11 years old, p = NS) served as control data. RESULTS: Arteriolar density was diminished by 38% (p = 0.004) and %lumen by 13% (p = 0.009) in patients with HCM compared with control subjects. Coronary reserve was impaired in patients with HCM (2.28+/-0.6 vs. 5.34+/-1.49, p = 0.003) because of higher coronary resistance after vasodilation (0.48+/-0.14 vs. 0.22+/-0.06 mm Hg x min x 100 g/ml, p = 0.004). Coronary vasodilator reserve correlated with arteriolar density (r = +0.47, p = 0.045) and with %lumen (r = 0.65, p = 0.003). CONCLUSIONS: In HCM, the architecture of preterminal subendocardial arterioles is altered by a reduced total cross-sectional lumen area, corresponding to an impaired coronary vasodilator capacity that may predispose to myocardial ischemia.     

Dominant-negative effect of a mutant cardiac troponin T on cardiac structure and function in transgenic mice

Hypertrophic cardiomyopathy (HCM) is a disease of sarcomeric proteins. The mechanism by which mutant sarcomeric proteins cause HCM is unknown. The leading hypothesis proposes that mutant sarcomeric proteins exert a dominant-negative effect on myocyte structure and function. To test this, we produced transgenic mice expressing low levels of normal or mutant human cardiac troponin T (cTnT). We constructed normal (cTnT-Arg92) and mutant (cTnT-Gln92) transgenes, driven by a murine cTnT promoter, and produced three normal and five mutant transgenic lines, which were identified by PCR and Southern blotting. Expression levels of the transgene proteins, detected using a specific antibody, ranged from 1 to 10% of the total cTnT pool. M-mode and Doppler echocardiography showed normal left ventricular dimensions and systolic function, but diastolic dysfunction in the mutant mice evidenced by a 50% reduction in the E/A ratio of mitral inflow velocities. Histological examination showed cardiac myocyte disarray in the mutant mice, which amounted to 1-15% of the total myocardium, and a twofold increase in the myocardial interstitial collagen content. Thus, the mutant cTnT-Gln92, responsible for human HCM, exerted a dominant-negative effect on cardiac structure and function leading to disarray, increased collagen synthesis, and diastolic dysfunction in transgenic mice.     

New insights into the regulation and function of brain estrogen synthase (aromatase)

In the brain, conversion of androgens into estrogens by the enzyme aromatase (estrogen synthase) is a key mechanism by which testosterone regulates many physiological and behavioral processes, including the activation of male sexual behavior, brain sexual differentiation and negative feedback effects of steroid hormones on gonadotropin secretion. Studies on the distribution and regulation of brain aromatase have led to a new perspective on the control and function of this enzyme. A growing body of evidence indicates that the estrogen regulation of aromatase is, at least in part, trans-synaptic. Afferent catecholamine pathways appear to regulate aromatase activity in some brain areas and thereby provide a way for environmental cues to modulate this enzyme. The localization of aromatase in pre-synaptic boutons suggests possible roles for estrogens at the synapse.     

Analysis of regulated exocytosis in adrenal chromaffin cells: insights into NSF/SNAP/SNARE function

In a duplicate study during 1987-1991, 478 24-h duplicate samples from 14 homes for elderly people and 10 homes for youth were investigated for their contents of selected harmful substances. The analyses covered 45 active substances of pesticides, 17 PCB-congeners as well as lead, cadmium, and nitrate contents. Pesticides could be detected only in 15% of the investigated samples. The pesticide contents reached max. 8% of the respective FAO/WHO-limits. As the mean intake of the three most important PCB-congeners (sum of the congeners 138, 153, and 180) values of 0.9 and 1.1 micrograms per day and ration or person, respectively, were found. Also in the worst case the daily PCB intake was below the recommended ADI value of the FDA of 1 microgram/kg of body weight. The daily nutritional intake of lead and cadmium via the investigated daily rations reached about 5.6% and 20% of the Provisionally Tolerably Weekly Intake values of the FAO/WHO. The mean nitrate content of the duplicate portions was 101 mg per day and person (median: 79 mg per day and person). Referred to the median value the WHO limit (3.65 mg/kg body weight and day) was exhausted to about 36%.     

Functional characterization of missense mutations in ATP7B: Wilson disease mutation or normal variant?

Wilson disease is an autosomal recessive disorder of copper transport that causes hepatic and/or neurological disease resulting from copper accumulation in the liver and brain. The protein defective in this disorder is a putative copper-transporting P-type ATPase, ATP7B. More than 100 mutations have been identified in the ATP7B gene of patients with Wilson disease. To determine the effect of Wilson disease missense mutations on ATP7B function, we have developed a yeast complementation assay based on the ability of ATP7B to complement the high-affinity iron-uptake deficiency of the yeast mutant ccc2. We characterized missense mutations found in the predicted membrane-spanning segments of ATP7B. Ten mutations have been made in the ATP7B cDNA by site-directed mutagenesis: five Wilson disease missense mutations, two mutations originally classified as possible disease-causing mutations, two putative ATP7B normal variants, and mutation of the cysteine-proline-cysteine (CPC) motif conserved in heavy-metal-transporting P-type ATPases. All seven putative Wilson disease mutants tested were able to at least partially complement ccc2 mutant yeast, indicating that they retain some ability to transport copper. One mutation was a temperature-sensitive mutation that was able to complement ccc2 mutant yeast at 30 degreesC but was unable to complement at 37 degreesC. Mutation of the CPC motif resulted in a nonfunctional protein, which demonstrates that this motif is essential for copper transport by ATP7B. Of the two putative ATP7B normal variants tested, one resulted in a nonfunctional protein, which suggests that it is a disease-causing mutation.