X-chromosome inactivation in female patients with Fabry disease.

Fabry disease (FD) is an X-linked genetic disorder caused by the deficient activity of lysosomal α-galactosidase (α-Gal). While males are usually severely affected, clinical presentation in female patients may be more variable ranging from asymptomatic to, occasionally, as severely affected as male patients. The aim of this study was to evaluate the existence of skewed X-chromosome inactivation (XCI) in females with FD, its concordance between tissues, and its contribution to the phenotype. Fifty-six females with FD were enrolled. Clinical and biological work-up included two global scores [Mainz Severity Score Index (MSSI) and DS3], cardiac magnetic resonance imaging, measured glomerular filtration rate, and measurement of α-Gal activity. XCI was analyzed in four tissues using DNA methylation studies. Skewed XCI was found in 29% of the study population. A correlation was found in XCI patterns between blood and the other analyzed tissues although some punctual variability was detected. Significant differences in residual α-Gal levels, severity scores, progression of cardiomyopathy and deterioration of kidney function, depending on the direction and degree of skewing of XCI were evidenced. XCI significantly impacts the phenotype and natural history of FD in females.

[Myoblast transplantation for heart failure: where are we heading?]. / Thérapie cellulaire en cardiologie: rêve ou réalité?

Cell therapy in cardiology is already a reality, as evidenced by the number of ongoing clinical trials. These studies entail administration of either skeletal myoblasts in patients with severe ischemic left ventricular dysfunction or of bone marrow-derived cells in patients with acute myocardial infarction and in whom cell therapy is an adjunct to a percutaneous revascularization procedure. The techniques of preparation, expansion and storage of myoblasts are now quite effective. The problem is simpler for bone marrow cells as in most studies, the procedure is limited to an iliac crest biopsy followed by reinjection of the crude, unfractionated bone marrow, as routinely done in clinical haematology since many years. The results of these studies are not yet fully available. Some of them have been enthusiastically reported to be positive but should be interpreted cautiously because of the usually small sample sizes and the common lack of randomisation and double-blind assessment of outcomes. Thus, the fact that cell therapy has now become a reality should not lead to underscore the yet unsettled fundamental issue, i.e., the ability of this novel mode of therapy to truly regenerate areas of necrotic myocardium and restore function in once akinetic territories. From this standpoint, cell therapy is still a dream. Since the beginning, it has been clear that myoblasts were exclusively committed to differentiate into myotubes, without any evidence for a phenotypic conversion into cardiomyocytes. Although the debate is more controversial for bone marrow cells, the reliance on accurate genetic methods of cell tracking has led to increasingly challenge the purported plasticity of these cells. This by no means implies that cell therapy does not exert beneficial effects that could be mediated by alternate mechanisms like limitation of remodelling of paracrine effects. The basic point is that neither skeletal myoblasts nor bone marrow cells fulfill the major criteria required for a true cardiac regeneration: a coupling of the grafted cells with those of the recipient myocardium and the subsequent generation of a contractile force. It is therefore critical to go on exploring other paths, among which embryonic stem cells are particularly attractive.

[Cardiac cellular therapy: from cells to the first clinical uses]. / Thérapie cellulaire cardiaque: des cellules aux premières indications cliniques.

Despite the improvement in revascularisation techniques, coronary artery disease remains the principal aetiology of cardiac failure in developed countries. The therapeutic management of cardiac failure has been improved over recent years, yet cardiac failure is still associated with significant morbidity and mortality. As cardiac transplantation lacks donors, techniques that allow myocardial regeneration represent an attractive alternative. To date, several types of cells are under study and are suitable for implantation into infarcted myocardium (myoblasts, medullary stem cells...). Following good preclinical study results, the first human cell therapy trials, using the intramyocardial route, have begun, in the course of aorto-coronary bypass surgery in patients with chronic ischaemic cardiopathy and little altered left ventricular function, and then in those with ventricular dysfunction. Different modes of administration of these cell therapy products are under study and could be envisaged in clinical situations such as just after infarction in order to improve ventricular remodelling with an intracoronary injection technique. As for every new treatment, there are numerous problems to resolve, from understanding the relevant mechanisms of cellular transplantation, to the secondary effects that it could entail. Nevertheless, cardiac cellular transplantation is expanding rapidly and with the evolution of techniques it allows a glimpse of a new field of treatment for cardiac failure.

Survival, haemodynamics and cardiac remodelling follow up in mice after myocardial infarction.

1. In the present study, the time-course, over a 1 year period, of postischaemic dilated cardiomyopathy and/or development of congestive heart failure was investigated in mice in terms of survival and cardiac functional and structural characteristics. 2. C57BL/6 mice with myocardial infarction (MI mice; coronary ligation n = 78) or sham-operated animals (n = 45) were used and echocardiographic, haemodynamic and histomorphometric parameters were assessed at 3, 6 and 12 months post-MI. 3. At 12 months, the survival rate was 70% in MI mice. Left ventricular dysfunction was evidenced by a strong decrease in ejection fraction (EF; -48 and -53% at 6 and 12 months, respectively; both P < 0.05) and an increase in left ventricular end-diastolic pressure (+100% at both 6 and 12 months; both P < 0.05). There was no major worsening in cardiac function between 6 and 12 months, suggesting strong compensatory mechanisms. Cardiac remodelling was observed, characterized by strong left ventricular hypertrophy (+38 and +62% at 6 and 12 months, respectively; both P < 0.05) and dilatation (+53% at 6 months; P < 0.05), but collagen was not significantly increased. Significant correlations were found between EF (echocardiography) and dP/dtmax, between end-diastolic volume (echocardiography) and left ventricular internal perimeter (histomorphometry) and between left ventricular mass (echocardiography) and weight. 4. In conclusion, despite a high survival rate, the MI mouse model displays most of the hallmarks of postischaemic dilated cardiomyopathy and/or congestive heart failure, thus affording the necessary background for the subsequent evaluation of gene manipulation and/or drug effects. In addition, two-dimensional echocardiography appears to be a suitable tool for the long-term follow up of cardiac function and remodelling in this model.

Regeneration of the myocardium: a new role in the treatment of ischemic heart disease?

Intramyocardial cell grafting aims to limit the consequences of the loss of contractile function of a damaged left ventricle. Its functional efficacy is suggested by a wealth of experimental data using multiple evaluation techniques in different animal species. Intramyocardial injections of cultured fetal cardiomyocytes after infarction increase the ejection fraction. Cultured autologous skeletal myoblasts, which do not raise immunologic, ethical, tumorigenesis, or donor availability problems, improve ventricular function to a similar extent. The presence of connexin-43 is demonstrated between fetal (but not myoblast) grafted cells and host myocytes. Thus, the mechanisms of this beneficial effect (direct systolic effect, paracrine factors, passive girdling effect, and decrease in wall stress) remain controversial. These encouraging results have opened the way to the first clinical trial in patients with low ejection fractions, akinetic and nonviable postinfarction scars, and indications for coronary artery bypasses in remote, viable, and ischemic areas. Large-scale cell expansion allows a yield of >10(9) myoblasts from a single human muscular biopsy. Cultured autologous myoblasts are directly administered by multiple injections within and around the infarcted area during open-chest surgery. Preliminary postoperative observations show an improvement in ejection fraction, reappearance of a systolic thickening of the grafted scars, and a new-onset metabolic viability within this area. Thus, this new procedure might become a useful adjunct to current treatments of severe ischemic heart failure.

[Transplantation of autologous skeletal myoblasts in ischemic cardiac insufficiency]. / Transplantation de myoblastes squelettiques autologues dans l'insuffisance cardiaque ischémique.

Despite medical therapeutic advances, congestive heart failure (CHF), which is the common ultimate consequence of many primary cardiovascular diseases, remains a major and growing public health problem. Although orthotopic heart transplantation is the gold standard, there is now growing evidence that one therapeutic option could be cellular cardiomyoplasty. Autologous adult skeletal myoblast transplantation seems to be the most clinically relevant, compared with other cell types, in that it avoids immunosuppression therapy, availability and ethical issues. Previous experimental studies have documented the efficacy of myoblast transplantation in improving function of infarcted myocardium. Although the mechanisms involved in this improvement are not elucidated, it has been demonstrated convincingly enough to consider ripping to clinical trials.

Is skeletal myoblast transplantation clinically relevant in the era of angiotensin-converting enzyme inhibitors?

BACKGROUND: There is compelling experimental evidence that autologous skeletal muscle (SM) cell transplantation improves postinfarction cardiac function. This study assessed whether this benefit is still manifested in the clinically relevant setting of a treatment by ACE inhibitors. METHODS AND RESULTS: A myocardial infarction was created in 99 rats by coronary artery ligation. They were divided into 4 groups. Two groups did not receive any drug and were intramyocardially injected 7 days after the infarct with either culture medium alone (control rats, n=16) or autologous SM cells (2.3x10(6) myoblasts) previously expanded ex vivo for 7 days (myoblasts, n=24). Two other groups received the ACE inhibitor perindoprilat (1 mg. kg(-1). d(-1)), started the day of the infarct and continued uninterruptedly thereafter, and underwent time-matched procedures, that is, they were intramyocardially injected at 7 days after infarction with either culture medium alone (ACE inhibitors, n=22) or autologous SM cells (2.5x10(6) myoblasts) previously expanded ex vivo for 7 days (ACE inhibitors+myoblasts, n=37). Left ventricular function was assessed by 2D echocardiography. At the end of the 2-month study, left ventricular ejection fraction (%, mean+/-SEM) was increased in all groups (myoblasts, 37.4+/-1.2; ACE inhibitors, 31.6+/-1.7; ACE inhibitors+myoblasts, 43.9+/-1.4) compared with that in control rats (19.8+/-0.7) (P<0.0001). The improvement in ejection fraction was similar in the ACE inhibitor and the myoblast groups (31.6+/-1.7 versus 37.4+/-1.2, P=0.0636). However, in the ACE inhibitor+myoblast group, this improvement was greater than that seen in hearts receiving either treatment alone (43.9+/-1.4 versus 31.6+/-1.7 in the ACE inhibitor group and 43.9+/-1.4. versus 37.4+/-1.2 in the myoblast group, P<0.0001 and P=0.0084, respectively). CONCLUSIONS: These data provide further support for the clinical relevance of autologous SM cell transplantation in that its cardioprotective effects are additive to those observed with ACE inhibitors.

Factors affecting functional outcome after autologous skeletal myoblast transplantation.

BACKGROUND: This study assessed the extent to which the initial degree of functional impairment and the number of injected cells may influence the functional improvement provided by autologous skeletal myoblast transplantation into infarcted myocardium. METHODS: One week after left coronary artery ligation, 44 rats received into the infarcted scar, autologous skeletal myoblasts expanded in vitro for 7 days (mean, 3.5 x 10(6), n = 21), or culture medium alone (controls, n = 23). Left ventricular function was assessed by two-dimensional echocardiography. RESULTS: When transplanted hearts were stratified according to their baseline ejection fraction, a significant improvement occurred at 2 months in the less than 25% (from 21.4% to 37%), 25% to 35% (from 29% to 43.8%), and in the 35% to 40% (from 37.2% to 41.7%) groups, compared to controls (p = 0.048, 0.0057, and 0.034, respectively), but not in the more than 40% stratum. A significant linear relationship was found between the improvement in ejection fraction and the number of injected myoblasts, both at 1 and 2 months after transplantation (p < 0.0001). CONCLUSIONS: Autologous myoblast transplantation is functionally effective over a wide range of postinfarct ejection fractions, including in the sickest hearts provided that they are injected with a sufficiently high number of cells.

Myoblast transplantation for heart failure.

Intramyocardial skeletal muscle transplantation has been shown experimentally to improve heart function after infarction. We report success with this procedure in a patient with severe ischaemic heart failure. We implanted autologous skeletal myoblasts into the postinfarction scar during coronary artery bypass grafting of remote myocardial areas. 5 months later, there was evidence of contraction and viability in the grafted scar on echocardiography and positron emission tomography. Although this result is encouraging, it requires validation by additional studies.

Cardiovascular abnormalities with normal blood pressure in tissue kallikrein-deficient mice.

Tissue kallikrein is a serine protease thought to be involved in the generation of bioactive peptide kinins in many organs like the kidneys, colon, salivary glands, pancreas, and blood vessels. Low renal synthesis and urinary excretion of tissue kallikrein have been repeatedly linked to hypertension in animals and humans, but the exact role of the protease in cardiovascular function has not been established largely because of the lack of specific inhibitors. This study demonstrates that mice lacking tissue kallikrein are unable to generate significant levels of kinins in most tissues and develop cardiovascular abnormalities early in adulthood despite normal blood pressure. The heart exhibits septum and posterior wall thinning and a tendency to dilatation resulting in reduced left ventricular mass. Cardiac function estimated in vivo and in vitro is decreased both under basal conditions and in response to beta-adrenergic stimulation. Furthermore, flow-induced vasodilatation is impaired in isolated perfused carotid arteries, which express, like the heart, low levels of the protease. These data show that tissue kallikrein is the main kinin-generating enzyme in vivo and that a functional kallikrein-kinin system is necessary for normal cardiac and arterial function in the mouse. They suggest that the kallikrein-kinin system could be involved in the development or progression of cardiovascular diseases.

Intramyocardial transplantation of autologous myoblasts: can tissue processing be optimized?

BACKGROUND: Autologous skeletal myoblast (SM) transplantation improves function of infarcted myocardium, but pretransplantation cultures remain a complex process. This study assessed whether it could be optimized by muscle preconditioning with the local anesthetic bupivacaine or even bypassed with the use of the so-called mince technique. METHODS AND RESULTS: Muscle preconditioning consisted of intramuscular injections of the tibialis anterior of rats, 2 days before harvest. After 7 days of culture, the number of available myoblasts was significantly increased compared with nonconditioned controls (1 683 147 versus 85 300, P:=0.0013). The mince technique was then assessed. A myocardial infarction was created in 66 rats by coronary artery ligation. One week later, rats were reoperated on and intramyocardially injected with culture medium alone (controls, n=23), autologous cultured SM (3.5 x 10(6), n=21), or autologous muscle minced into a fine slurry, which was immediately transplanted (n=22). All muscles had been preconditioned. Left ventricular function was assessed by 2D echocardiography. Whereas end-diastolic volumes expanded over time in all groups, left ventricular ejection fraction (%, mean+/-SEM) was increased only in the cultured SM-transplanted group at 1 (P:=0. 0006) and 2 months (P:=0.0008) versus baseline (37.52+/-1.92 and 40. 92+/-2.17 versus 30.34+/-1.74), with a significant additional benefit between 1 and 2 months (P:=0.0069). CONCLUSIONS: Cell culture remains mandatory for SM transplantation to be successful but, in a clinical perspective, this process can be made more expeditious by preharvest muscle conditioning with bupivacaine, which greatly enhances the baseline cell yield.

Cardiac functional improvement by a human Bcl-2 transgene in a mouse model of ischemia/reperfusion injury.

BACKGROUND: Apoptosis has been shown to contribute to myocardial reperfusion injury. It has been suggested that, in reducing the apoptotic component within the ischemic area at risk, Bcl-2 overexpression could lead to a ventricular function improvement. METHODS: Transgenic mice overexpressing the anti-apoptotic human Bcl-2 cDNA in heart were subjected to a 1-h left coronary artery occlusion followed by a 24-h reperfusion. At the end of the experiment, left ventricular function was assessed by two-dimensional echocardiography. After sacrifice, the area at risk (AR) and the infarct area (IA) were determined by Evans blue and triphenyltetrazolium chloride staining, respectively. The extent of apoptosis was assessed by the TUNEL method. Non-transgenic littermates served as controls. RESULTS: Baseline AR was not different between Bcl-2 transgenic mice and their wild-type littermates. In contrast, left ventricular ejection fraction was significantly improved in the transgenic mice line (61.25 +/- 4.0%) compared to non-transgenic littermates (43.2 +/- 5.0%, p < 0.01). This functional amelioration was correlated with a significant reduction of infarct size in transgenic animals (IA/AR 18.51 +/- 3.4% vs 50.83 +/- 8.4% in non-transgenic littermates). Finally, apoptotic nuclei were less numerous in transgenic mice than in controls as quantified by TUNEL analysis (8.1 +/- 2.2% vs 20.6 +/- 4.4%). CONCLUSIONS: Bcl-2 overexpression is effective in reducing myocardial reperfusion injury and improving heart function. This benefit correlates with a reduction of cardiomyocyte apoptosis. The apoptotic component of ischemia/reperfusion injury could therefore constitute a new therapeutic target in the acute phase of myocardial infarction.

Can cellular transplantation improve function in doxorubicin-induced heart failure?

BACKGROUND: Transplantation of fetal cardiomyocytes has been shown to improve function of regionally infarcted myocardium, but its effects on global heart failure are still unknown. METHODS AND RESULTS: Heart failure was induced in female mice by intraperitoneal injection of doxorubicin (2 mg/kg twice per week over 2 cycles of 2 weeks separated by a 2-week drug-free period). One week after the end of treatment, left ventricular function was assessed by transthoracic echocardiography (baseline). Animals were then randomized into 3 groups: The treated group (n = 12) received an intramyocardial injection of fetal cardiomyocytes (1 x 10(6) in 10 microL) harvested from transgenic mice expressing the gene of beta-galactosidase, the control group (n = 15) received an equivalent volume of culture medium alone, and 10 sham mice had no surgery. Two weeks and 1 month after transplantation, function was again assessed echocardiographically. At baseline, fractional shortening was not significantly different between the 3 groups. It then significantly increased in cell-treated mice at 2 weeks and 1 month after transplantation (P < 0.002 and P < 0.03 versus baseline, respectively), whereas it did not change in untreated animals. Transplanted cells could not be identified by beta-galactosidase activity or presence of Y chromosome (with 1 exception). CONCLUSIONS: Cellular transplantation can improve function of globally failing hearts by a mechanism that might not necessarily involve the sustained presence of transplanted cells but rather the effects of cardioprotective factors released by them.

Genotype-phenotype analysis in four families with mutations in beta-myosin heavy chain gene responsible for familial hypertrophic cardiomyopathy.

Familial hypertrophic cardiomyopathy is a genetically heterogeneous disease in which one of the most frequently implicated gene is the gene encoding the beta-myosin heavy chain. To date, more than 40 distinct mutations have been found within this gene. In order to progress on the determination of genotype-phenotype relationship, we have screened the beta-myosin heavy chain gene for mutations in 18 probands from unrelated families. We identified the mutation implicated in the disease in four families. Two of them, the Glu930 codon deletion and the Ile263Thr mutation, are reported here for the first time. The two other mutations are the Arg723Cys mutation, that was previously described in a proband as a de novo mutation, and the Arg719Trp mutation. A poor prognosis was associated with the Glu930codon deletion (mean maximal wall thickness (MWT) = 19.5 mm +/- 5) and the Arg719Trp mutation (mean MWT = 15.3 mm +/- 7), whereas a good prognosis was associated with the Arg723Cys mutation (mean MWT = 20.1 mm +/- 7). The combination of clinical and genetic characteristics of each family member suggests that prognosis is related neither to the degree of left ventricular wall thickness nor to a change in the net electrical charge of the protein. Additional family studies are needed to confirm these findings and to contribute to stratify the prognosis according to the mutation involved.

Familial hypertrophic cardiomyopathy. Cardiac ultrasonic abnormalities in genetically affected subjects without echocardiographic evidence of left ventricular hypertrophy.

AIMS: It is not known whether the apparent normality of echocardiographic examination results, in subjects bearing a mutation for hypertrophic cardiomyopathy but without ultrasonic left ventricular hypertrophy, is due to incomplete phenotypic expression, or inaccurate echocardiographic criteria. The aim of this study was to search for echocardiographic abnormalities in these patients. METHODS AND RESULTS: Echocardiography was performed in 100 subjects from two families with a mutation in the beta-MHC (720) or My-BPC (714) genes. We compared genetically affected subjects with an apparently normal left ventricle (thickness < 13 mm) (20 patients), and nonaffected first-degree relatives (61 normal subjects). (1) Patients had a thicker left ventricular wall (9.7 +/- 1.4 vs 8.9 +/- 1.4 mm, P = 0.03), a greater indexed mass (107 +/- 18 vs 97 +/- 17 g. m-2, P = 0.03), a larger left atrium (27 +/- 9 vs 23 +/- 10 mm3, P = 0.09) and lower wall stress (78 +/- 11 vs 89 +/- 15 10(3) dynes. cm-2, P = 0.002); these differences were highly significant after adjustment for height, age and systolic blood pressure either for wall thickness (P = 0.000003), mass (P = 0.005) or atrial volume (P = 0.001), and the ventricular systolic dimension appeared smaller (P = 0.01); (2) results remained significant (P < 0.01) when a lower cut-off value (< or = 11 mm) or only adults (> or = 18 years) were considered; (3) a subanalysis of Family 714 (13 patients, 25 normals matched for sex, age and height) showed the same trends. CONCLUSION: In familial hypertrophic cardiomyopathy, genetically affected subjects with an apparently normal heart by echocardiography show slight ultrasonic structural and functional left ventricular modifications, suggesting that the phenotype of the disease is a continuous spectrum from normal structure to typical hypertrophy.

Does transplantation of cardiomyocytes improve function of infarcted myocardium?

BACKGROUND: The feasibility of successfully grafting fetal cardiomyocytes into infarcted myocardium is now established, but the functional effects of such a procedure still remain elusive. METHODS AND RESULTS: Twenty-three female rats underwent 45 minutes of coronary artery occlusion followed by 30 minutes of reperfusion. At this time point, 13 animals received intramyocardial injections of fetal cardiomyocytes (6 x 10(6) cells in 60 microL of culture medium) in the once ischemic area, whereas the 10 control rats were injected with an equivalent volume of culture medium alone. One month after transplantation, left ventricular function was assessed by two-dimensional (2D) and Doppler echocardiography using a short focus 10- to 13-MHz transducer, and a numeric acquisition of 2D images up to 65.5 frames/second. Explanted hearts were then processed for histological assessment of infarct size. The presence of male donor cells into female recipient myocardium was detected by fluorescent in situ hybridization using a deoxyribonucleic acid probe specific for Y chromosome. Cellular transplantation resulted in an improved left ventricular function, as demonstrated by significantly higher 2D ejection fraction and cardiac output (P<.02 and P<.02 versus control hearts, respectively). The histological sections of female recipient myocardium were Y-positive in all but one heart, thereby suggesting that this improvement of function was causally related to the presence of transplanted cells. CONCLUSIONS: These data suggest that transplantation of cardiomyocytes might be an effective means of improving function of infarcted myocardium.

Organization and sequence of human cardiac myosin binding protein C gene (MYBPC3) and identification of mutations predicted to produce truncated proteins in familial hypertrophic cardiomyopathy.

Cardiac myosin binding protein C (MyBP-C) is a sarcomeric protein belonging to the intracellular immunoglobulin superfamily. Its function is uncertain, but for a decade evidence has existed for both structural and regulatory roles. The gene encoding cardiac MyBP-C (MYBPC3) in humans is located on chromosome 11p11.2, and mutations have been identified in this gene in unrelated families with familial hypertrophic cardiomyopathy (FHC). Detailed characterization of the MYBPC3 gene is essential for studies on gene regulation, analysis of the role of MyBP-C in cardiac contraction through the use of recombinant DNA technology, and mutational analyses of FHC. The organization of human MYBPC3 and screening for mutations in a panel of French families with FHC were established using polymerase chain reaction, single-strand conformation polymorphism, and sequencing. The MYBPC3 gene comprises > 21,000 base pairs and contains 35 exons. Two exons are unusually small in size, 3 bp each. We found six new mutations associated with FHC in seven unrelated French families. Four of these mutations are predicted to produce truncated cardiac MyBP-C polypeptides. The two others should each produce two aberrant proteins, one truncated and one mutated. The present study provides the first organization and sequence for an MyBP-C gene. The mutations reported here and previously in MYBPC3 result in aberrant transcripts that are predicted to encode significantly truncated cardiac MyBP-C polypeptides. This spectrum of mutations differs from the ones previously observed in other disease genes causing FHC. Our data strengthen the functional importance of MyBP-C in the regulation of cardiac work and provide the basis for further studies.

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.

"Crochetage" (notch) on R wave in inferior limb leads: a new independent electrocardiographic sign of atrial septal defect.

OBJECTIVES: This study sought to determine the clinical significance of a "crochetage" pattern--a notch near the apex of the R wave in electrocardiographic (ECG) inferior limb leads--in secundum atrial septal defect. BACKGROUND: Atrial septal defect is often overdiagnosed on the basis of classical clinical features. Thus, more specific signs on the ECG for screening are needed. Methods. We searched for a crochetage pattern in 1,560 older children and adults: 532 with secundum atrial septal defect, 266 with ventricular septal defect, 146 with pulmonary stenosis, 110 with mitral stenosis, 47 with cor pulmonale and 459 normal subjects. RESULTS: This pattern was observed respectively in 73.1%, 35.7%, 23.3%, 6.4%, 10.6% and 7.4% of these groups (p<0.001). In atrial septal defect, its incidence increased with larger anatomic defect (p<0.0001) or greater left-to-right shunt (p<0.0001), even in the presence of pulmonary hypertension. By multiple regression analysis, only shunt size (p<0.0006) and defect location (p<0.0001) were the determinants of its presence. In all groups, the specificity of this sign for the diagnosis was remarkably high when present in all three inferior limb leads (> or = to 92%), even when comparison was limited to patients with an incomplete right bundle branch block (> or = 95.2%). Early disappearance of this pattern was observed in 35.1% of the operated-on patients although the right bundle branch block pattern persisted. CONCLUSIONS: A crochetage pattern of the R wave in inferior limb leads is frequent in patients with atrial septal defect, correlates with shunt severity and is independent of the right bundle branch block pattern. Sensitivity and specificity of this sign are remarkably high when it is associated with an incomplete right bundle branch block or present in all inferior limb leads.