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

The effects of two progestogen-only pills containing either 75 microgram desogestrel (DSG) or 30 microgram levonorgestrel (LNG) on hemostasis were investigated in a double-blind, randomized, controlled study of seven treatment cycles in 78 healthy women. DSG reduced factor VII activity (p < 0.05) and prothrombin fragment 1+2 (p < 0.05) and increased protein S (p < 0.001). LNG reduced factor VII activity (p < 0.01) and plasminogen activity (p < 0.01) and increased tissue-plasminogen activator (t-PA) (p < 0.05). At the end of the post-treatment cycle with DSG, protein S (p < 0.01) and t-PA (p < 0.05) were increased and plasminogen activity was decreased (p < 0.05), whereas with LNG, t-PA was increased (p < 0.001) and prothrombin fragment 1+2 (p < 0.05) and plasminogen activity (p < 0.001) were decreased. Between-group comparisons revealed higher values for DSG regarding the anticoagulatory parameter protein S at cycle 7 (p < 0.01) and post-treatment assessments (p < 0.05), and the fibrinolytic parameter plasmin-antiplasmin complex was higher with DSG at cycle 7 (p < 0.05) and at post-treatment (p < 0.05). Both preparations had comparable and potentially favorable effects of hemostasis, and may offer suitable hormonal contraception to women with a personal or family history of venous thromboembolic diseases.     

Identification of two novel mutations in the ventricular regulatory myosin light chain gene (MYL2) associated with familial and classical forms of hypertrophic cardiomyopathy

Five disease genes encoding sarcomeric proteins and associated with familial and classical forms of hypertrophic cardiomyopathy have been determined since 1989. In 1996 two other genes encoding ventricular regulatory and essential myosin light chains were shown to be associated with a particular phenotype of the disease characterized by mid left ventricular obstruction. The aim of the present study was to search for mutations in the ventricular regulatory myosin light chain gene (MYL2), located on chromosome 12q23q24.3, in a panel of 42 probands presenting a classical phenotype of familial hypertrophic cardiomyopathy. Single-strand conformation polymorphism analysis was used to search for mutations in the coding segments of the MYL2 gene, and the abnormal products were sequenced. Two novel missense mutations, Phe18Leu in exon 2 and Arg58Gln in exon 4 were identified in three unrelated families. None of the affected patients had hypertrophy localized only at the level of the papillary muscle with mid left ventricular obstruction. By analysis of genetic recombinations, one of these mutations identified in a large family allowed us to refine the localization of the MYL2 gene on the genetic map, in an interval of 6 cM containing six informative microsatellite markers. In conclusion, we show that mutations in the MYL2 gene may be involved in familial and classical forms of hypertrophic cardiomyopathy, and we provide new tools for the genetic analysis of patients with familial hypertrophic cardiomyopathy.     

Developmental transitions in the myosin heavy chain phenotype of human respiratory muscle

The renal lymphatic system plays an important role in removing excess fluid from the kidneys. Unfortunately, the factors influencing lymphatic flow are difficult to measure. We used a simple model to represent renal lymphatics as a single pressure source (PL) pushing lymph through a single resistance (RL). In anesthetized dogs, we cannulated renal lymphatics and measured lymph flow rate (QL) as we varied pressure (PO) at the outflow end of the lymphatics. There was no significant change in QL as we increased PO from -5 to 0 cm H2O. In other words, there was a plateau in the QL vs. PO relationship. At higher PO's, QL decreased linearly with increases in PO. From this linear relationship, we calculated RL as -delta PO/ delta QL and we took PL as the PO at which QL = 0 microliter/min. At baseline, RL = 0.34 +/- 0.14 (SD) cm H2O.min/microliter and PL = 8.2 +/- 4.4 cm H2O. When we increased renal venous pressure (PV) from baseline (3.5 +/- 3.0 cm H2O), the plateau in the QL vs. PO relationship extended to higher PO's, RL decreased, and PL increased. Renal interstitial fluid volume and interstitial pressure increased following elevation of PV. The extension of the QL vs. PO plateau with increasing PV suggests that renal interstitial pressure may partially collapse intrarenal collecting lymphatics which may compromise lymph flow.     

Altered expression of myosin heavy chain in human skeletal muscle in chronic heart failure

To explore further alterations in skeletal muscle in chronic heart failure (CHF), we examined myosin heavy chain (MHC) isoforms from biopsies of the vastus lateralis in nine male patients with class II-III (CHF) (left ventricular ejection fraction (LVEF) 26 +/- 11%, peak oxygen consumption (peak VO2) 12.6 +/- 2 mL.kg-1.min-1) and nine age-matched sedentary normal males (NL). The relative content of MHC isoforms I, IIa, and IIx was determined by gel electrophoresis as follows: The normal sedentary group (NL) had a higher percent of MHC type I when compared with the patients (NL 48.4 +/- 7% vs CHF patients 24 +/- 21.6%, P < 0.05, no difference between MCH IIa (NL 45.1 +/- 10.5% vs CHF 56.0 +/- 12.5%), and CHF patients had a higher relative content of MHC type IIx than did the normal group (NL 6.5 +/- 9.6% vs CHF 20.0 +/- 12.9%, P < 0.05. Three of nine patients had no detectable MHC type I. In patients relative expression of MHC type I (%) was related to peak VO2 (r = 0.70, P < 0.05). Our results indicate that major alterations in MHC isoform expression are present in skeletal muscle in CHF. These alterations parallel previously reported changes in fiber typing that may affect contractile function i skeletal muscle and possibly exercise performance. The absence of MHC type I in some CHF patients suggests that skeletal muscle changes in this disorder are not solely a result of deconditioning, buy may reflect a specific skeletal muscle myopathy in this disorder.     

The effect of angiotensin II on myosin heavy chain expression in cultured myocardial cells

Angiotensin II (AII), the principal mediator of the renin-angiotensin system, is an important regulator of vascular and cardiac homeostasis. AII has also been shown to be a regulator of cardiac hypertrophy and of the corresponding changes in amount and composition of certain tissue proteins. We examined the trophic effects of AII on cultured myocytes derived from neonatal rat ventricles and followed, by Northern blot analysis and polyacrylamide gel electrophoresis, the expression of alpha- and beta-myosin heavy chain iso-mRNAs and isoproteins. Our findings show that a single administration of AII is sufficient to induce a trophic response in cultured beating myocytes and to enhance the expression of beta-myosin heavy chain iso-mRNA and isoprotein, having no effect on alpha-myosin heavy chain. Induction of alpha-myosin heavy chain expression by thyroid hormone before AII was administered showed that AII could not potentiate a shift from alpha- to beta-myosin heavy chain predominance. We suggest that the potency of AII to regulate the expression of myosin heavy chain isogenes is restricted to the beta isoform and is overridden by thyroid hormone.     

A point mutation associated with a severe phenotype of neurofibromatosis 2

Neurofibromatosis 2 (NF2) is an autosomal dominant disease characterized by bilateral vestibular schwannomas and other nonmalignant tumors of the brain, spinal cord, and peripheral nerves. Although the average age of onset of NF2 is 20 years, some individuals may become symptomatic in childhood. We studied 5 unrelated NF2 patients who became symptomatic before age 13. All 5 had multiple tumors in addition to vestibular schwannoma, and none had a positive family history. Sequence analysis of the NF2 gene revealed identical nonsense mutation of exon 6 in 3 patients. Because this mutation destroys a restriction enzyme recognition site, genomic DNA from the 2 other children was directly tested for this change and identical alterations were detected. Although the work of our laboratory and others has not, in general, detected identical mutations in unrelated patients, this mutation seems to occur particularly frequently in the pediatric population and thus may be associated with an especially severe phenotype. Restriction analysis in children with NF2 may be a cost effective way of identifying their mutation. Further work is needed to characterize the effects of this change on the NF2 protein product and its relationship to this severe phenotype.     

Prominent sensory and autonomic disturbances in familial amyotrophic lateral sclerosis with a Gly93Ser mutation in the SOD1 gene

Sudden death at night is known to occur in young patients with insulin-dependent (Type 1) diabetes mellitus (IDDM) but the aetiology is uncertain. A cardiac arrhythmia has been postulated, but there has been little evidence to support this. We present the case of a 31-year-old man with IDDM of 17 years duration, who died suddenly while asleep. Over preceding months, he had had strict glycaemic control (HbA1 8.9%), normal 24 h blood pressure (mean 131 +/- 2.1/76 +/- 2.2 mmHg), no evidence of microangiopathy or endothelial dysfunction and normal standard clinical tests of autonomic function. An electrocardiogram was similarly unremarkable, with a QTc interval of 0.414 s, and an echocardiogram had demonstrated normal left ventricular mass index (96.4 g m-2). However, there was no nocturnal dip in heart rate (daytime 74 +/- 2.7, and nocturnal 68 +/- 1.6 beats min-1), and he had grossly impaired baroreflex sensitivity during Phase 4 of the valsalva manoeuvre (0.5 ms mmHg-1), with power spectral analysis studies suggesting an abnormality of parasympathetic function. The coroner's autopsy demonstrated no structural abnormalities. We hypothesize that abnormal baroreflex sensitivity could either predict a risk of or account for some of the unexplained deaths in IDDM, in that relative overactivity of the sympathetic nervous system could cause ventricular arrhythmias.     

Identification of a genomic locus containing three slow myosin heavy chain genes in the chicken

Gene technology using polymerase chain reaction (PCR) has markedly advanced in recent year and has been introduced in clinical laboratories. In this paper, the genotypes of genomic DNAs of subjects with cisAB blood group were analysed using three methods, polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP), and the PCR-direct sequencing method, and directly determined using the polymerase chain reaction (PCR) amplification of specific alleles (PASA)-method. The differences among the methods were as follows, PCR-RFLP and PCR-direct sequencing method require 2-step procedures, and are complicated for clinical laboratories. The PASA method is based on the fact that PCR amplification occurs only when the 3' endbase of the primer is matched to sites of the nucleotide substitution of ABO allelic cDNA. Three of five regions of allelic DNAs were co-amplified in a single PCR (multiplex-PCR) in this study. ABO and cisAB blood group genotypes were directly determined, based on the molecular size of allele-specific amplification products. The PASA method requires only about 4 hours from starting PCR to results, making it rapid, simple and useful for detecting the genotype of ABO and cisAB blood groups in comparison with PCR-RFLP and the direct sequencing methods and will allow this procedure to be very versatile and widely used throughout the research and clinical diagnostic communities. The analyses of the nucleotide sequence at nucleotides No. 261, 526, 703, 796 and 803 in 3 major subjects in the cisAB blood group (cisA2B3, cisA1B3 and cisA2B) revealed chimeric structures of the A allele and B allele on the same gene.     

Variable phenotype of familial adenomatous polyposis in pedigrees with 3'' mutation in the APC gene

Traumatic rupture of the corpus cavernosum is relatively frequent in the authors' experience. Based on the study of a series of 80 cases and a review of the literature, the authors analyse the diagnostic and therapeutic aspects and outcome of this disease. The patients in this series had a mean age of 30 years. Meticulous and intimate clinical interview demonstrated that the commonest mechanism is forced manipulation of the erect penis (68%). Clinical examination localized the site of the fracture (proximal: 57%, distal 43%). The fracture was unilateral (78 cases), rarely bilateral (2 cases) and associated with complete rupture of the urethra (1 case). Treatment was surgical in 79 patients. A distal semicircumferential incision was used in the case of bilateral rupture, distal rupture and associated urethral lesion (34 cases). A favourable course was observed in 86% of cases. However, 9 postoperative complications (12.5%) were observed (6 cases of fibrous plaques, 3 cases of chordee of the penis), due either to the extent of the haematoma or to the delay in treatment. Traumatic rupture of the corpus cavernosum is a disease of young adults, which requires early surgical treatment with an approach adapted to the type of lesions.     

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.     

Delayed somite formation in a quail line exhibiting myofiber hyperplasia is accompanied by delayed expression of myogenic regulatory factors and myosin heavy chain

A myofiber hyperplastic quail line P has been developed through selection for heavy body weight. Since the number of muscle fibers is determined early in development and skeletal muscle originates from somites, we compared somite formation and muscle-specific gene expression in P- and control C-line quail embryos. At 47 hours of incubation, C embryos had 18 somite pairs and P embryos had 14.3. By 72 and 120 hours, both lines appeared to be at the same stage of somite development. To determine whether the delay in the formation of the brachial somites was accompanied by alterations in muscle-specific gene expression, we conducted whole-mount in situ hybridization and immunofluorescence studies. At 47 hours of incubation, C embryos were expressing qmf1 in the first 12 somites, while in P embryos only the first 7 somites showed qmf1 activation. Delays in expression were also observed for qmf3 at 43 hours and for all three myogenic factors (qmf1, qmf2 and qmf3) at 60 hours. At 65 hours, C embryos expressed myosin heavy chain in the first 15 somite pairs and P embryos in the first 7. At 72 hours, the transient delay in somite formation had disappeared and there was no lag in myosin heavy chain expression between the lines. The phase delay in brachial somite formation, myogenic factors and myosin heavy chain expression may be associated with the observed myofiber hyperplasia in P-line quail by allowing an increase in the muscle stem cell population.     

Clinical features of hypertrophic cardiomyopathy caused by mutation of a "hot spot" in the alpha-tropomyosin gene

OBJECTIVES: We studied the clinical and genetic features of familial hypertrophic cardiomyopathy (FHC) caused by an Asp175Asn mutation in the alpha-tropomyosin gene in affected subjects from three unrelated families. BACKGROUND: Correlation of genotype and phenotype has provided important information in FHC caused by beta-cardiac myosin and cardiac troponin T mutations. Comparable analyses of hypertrophic cardiomyopathy caused by alpha-tropomyosin mutations have been hampered by the rarity of these genetic defects. METHODS: The haplotypes of three kindreds with FHC due to an alpha-tropomyosin gene mutation, Asp175Asn, were analyzed. The cardiac histopathologic findings of this mutation are reported. Distribution of left ventricular hypertrophy in affected members was assessed by two-dimensional echocardiography, and patient survival rates were compared. RESULTS: Genetic studies defined unique haplotypes in the three families, demonstrating that independent mutations caused the disease in each. The Asp175Asn mutation caused cardiac histopathologic findings of myocyte hypertrophy, disarray and replacement fibrosis. The severity and distribution of left ventricular hypertrophy varied considerably in affected members from the three families (mean maximal wall thickness +/- SD: 24 +/- 4.5 mm in anterior septum of Family DT; 15 +/- 2.7 mm in anterior septum and free wall of Family DB; 18 +/- 2.1 mm in posterior septum of Family MI), but survival was comparable and favorable. CONCLUSIONS: Nucleotide residue 579 in the alpha-tropomyosin gene may have increased susceptibility to mutation. On cardiac histopathologic study, defects in this sarcomere thin filament component are indistinguishable from other genetic etiologies of hypertrophic cardiomyopathy. The Asp175Asn mutation can elicit different morphologic responses, suggesting that the hypertrophic phenotype is modulated not by genetic etiologic factors alone. In contrast, prognosis reflected genotype; near normal life expectancy is found in hypertrophic cardiomyopathy caused by the alpha-tropomyosin mutation Asp175Asn.     

Electrospray ionisation mass spectrometry facilitates detection of fibrinogen (Bbeta 14 Arg --> Cys) mutation in a family with thrombosis

We report the first direct detection of a fibrinogen mutation by electrospray ionisation mass spectrometry. The propositus, from a family with a history of thrombosis, came to attention after a pulmonary embolism subsequent to a spontaneous abortion. Prolonged thrombin (41 s) and reptilase times (26 s) together with an impairment of fibrinopeptide B release suggested a mutation at the thrombin cleavage site of the Bbeta chain. Direct mass analysis of purified fibrin chains from a thrombin induced clot showed that 50% of the Bbeta chains remained uncleaved. The measured mass of the mono sialo isoform of this uncleaved chain was 54150 Da, compared to a value of 54198 Da for normal Bbeta chains. This decrease of 48 Da in the intact protein is indicative of either a Bbeta 14 Arg to Cys, or Arg to Leu substitution. Heterozygosity for the Bbeta 14 Arg --> Cys mutation was verified by PCR amplification and DNA sequence analysis.     

Identification of Myo3, a second type-II myosin heavy chain in the fission yeast Schizosaccharomyces pombe

We cloned the myo3+ gene of Schizosaccharomyces pombe which encodes a type-II myosin heavy chain. myo3 null cells showed a defect in cytokinesis under certain conditions. Overproduction of Myo3 also showed a defect in cytokinesis. Double mutant analysis indicated that Myo3 genetically interacts with Cdc8 tropomyosin and actin. Myo3 may be implicated in cytokinesis and stabilization of F-actin cables. Moreover, the function of Myo2 can be replaced by overexpressed Myo3. We observed a modest synthetic interaction between Myo2 and Myo3. Thus, Myo2 and Myo3 seem to cooperate in the formation of the F-actin ring in S. pombe.     

Multiple adenomatous papillary colonic polyps in a family with frequent cases of stomach carcinoma--a new phenotype of familial colonic polyposis]

A kindred of "minor adenomatous polyposis" associated with a high incidence of gastric cancer is described. Five out of 14 mainly asymptomatic individuals in 2 generations of kindred examined clinically, by panendoscopy and coloscopy exhibited multiple polyps of colon or/and stomach and jejunum. Single or multiple papillary adenomas were detected mainly in the right or middle colon (3 times or in the jejunum (once). Hyperplastic polyps were found in the stomach (3 times) and in the colon (twice of these 5 individuals. Our findings suggest that "minor adenomatous polyposis" associated with gastric cancer may represent another, hitherto unrecognized, phenotype of familial multiple polyposis.     

Dissociation of left ventricular hypertrophy, beta-myosin heavy chain gene expression, and myosin isoform switch in rats after ascending aortic stenosis

The regulation of angiotensin II (Ang II) receptors and Ang II-induced modulation of intracellular Ca2+ concentration in cardiac cells from hearts of experimentally induced hypertensive deoxycorticosterone acetate (DOCA)-salt and control unilaterally nephrectomized (Uni-Nx) Sprague-Dawley rats was assessed. Ang II receptor density and intracellular Ca2+ concentration measurements were examined in adult ventricular myocytes and fibroblasts by radioligand binding assay and digital imaging using fura 2 methodology, respectively. Four-week DOCA-salt treatment induced hypertension associated with cardiac hypertrophy. Ang II binding studies demonstrated that adult ventricular myocytes and fibroblasts possess mainly the AT1 subtype receptor. Moreover, DOCA-salt hypertension was associated with a 1.8-fold increase in Ang II-specific binding compared with myocytes from Uni-Nx control rats. Intracellular Ca2+ responses induced by increasing Ang II concentrations (10[-12] to 10[-4] mol/L) were significantly enhanced in cardiomyocytes from DOCA-salt rats. The effects of Ang II on intracellular Ca2+ spike frequency were unaltered in cardiomyocytes from DOCA-salt-hypertensive rats. The density of AT1 subtype receptors was not modified in ventricular fibroblasts after DOCA-salt treatment. Ang II increased intracellular Ca2+ concentration similarly in ventricular fibroblasts from normal and hypertensive rats. In conclusion, DOCA-salt hypertension is characterized by an increased AT1 receptor density and intracellular calcium responses in ventricular myocytes, whereas in ventricular fibroblasts the AT1 receptor status is unaltered. These findings report for the first time the cardiac cell-specific implication of Ang II and the intracellular calcium signaling pathway stimulated by the AT1 receptor in cardiac hypertrophy in DOCA-salt-hypertensive rats.