Instability of short-sequence DNA repeats of pear pathogenic Erwinia strains from Japan and Erwinia amylovora fruit tree and raspberry strains.

An array of short-sequence DNA repeats (SSRs) occurs in the plasmid pEA29 of the fire blight pathogen Erwinia amylovora. A large number of "fruit tree" strains, mainly from Central and Western Europe, were screened for their SSR numbers, and the analyses were extended to five raspberry strains from North America and six pear pathogenic Erwinia strains from Japan. The repeat ATTACAGA present in all E. amylovorastrains was found to be reiterated 3 to 15 times. The Japanese strains contained the major repeat sequence GGATTCTG, which was reiterated 16 to 24 times. ATTACAGG, which resembles the SSR of E. amylovora, was reiterated two or three times. In a novel approach, sequencing gels were used to visualize the rare occurrence of shorter arrays (down to three repeats) in E. amylovoraand the Japanese Erwinia strains. Changes in the repeat numbers in E. amylovora were observed repeatedly when the bacteria had been exposed to stress conditions. The repeat structures of homo- and heteroduplices of PCR-amplified repeats were also analyzed by cleavage of annealed molecules with the single-strand-specific endonuclease from bacteriophage T4. Not only heteroduplexes, but also homoduplexes showed non-matching regions in the SSRs, which could arise from transient formation of loops due to strand slippage during the assays.

Genetic polymorphisms in the renin-angiotensin-aldosterone system associated with expression of left ventricular hypertrophy in hypertrophic cardiomyopathy: a study of five polymorphic genes in a family with a disease causing mutation in the myosin binding protein C gene.

BACKGROUND: Hypertrophic cardiomyopathy (HCM) is an inherited disease of the sarcomere characterised clinically by myocardial hypertrophy and its consequences. Phenotypic expression is heterogeneous even within families with the same aetiological mutation and may be influenced by additional genetic factors. OBJECTIVE: To determine the influence of genetic polymorphisms of the renin-angiotensin-aldosterone system (RAAS) on ECG and two dimensional echocardiographic left ventricular hypertrophy (LVH) in genetically identical patients with HCM. PATIENTS AND METHODS: Polymorphisms of five RAAS components were determined in 26 gene carriers from a single family with HCM caused by a previously identified myosin binding protein C mutation. Genotypes associated with a higher activation status of the RAAS were labelled "pro-LVH genotypes". RESULTS: There was a non-biased distribution of pro-LVH genotypes in the gene carriers. Those without pro-LVH genotypes did not manifest cardiac hypertrophy whereas gene carriers with pro-LVH genotypes did (mean (SD) left ventricular muscle mass 190 (48) v 320 (113), p = 0.002; interventricular septal thickness 11.5 (2.0) v 16.4 (6.7), p = 0.01; pathological ECG 0% (0 of 10) v 63% (10 of 16), respectively). Multivariate analysis controlling for age, sex, and hypertension confirmed an independent association between the presence of pro-LVH polymorphisms and left ventricular mass. When each polymorphism was assessed individually, carriers of each pro-LVH genotype had a significantly greater left ventricular mass than those with no pro-LVH mutation; these associations, with the exception of cardiac chymase A AA polymorphism (p = 0.06), remained significant in multivariate analysis. CONCLUSION: Genetic polymorphisms of the RAAS influence penetrance and degree of LVH in 26 gene carriers from one family with HCM caused by a myosin binding protein C mutation.

Apical hypertrophic cardiomyopathy due to a de novo mutation Arg719Trp of the beta-myosin heavy chain gene and cardiac arrest in childhood. A case report and family study.

Hypertrophic cardiomyopathy (HCM) is a myocardial disease with variable phenotpye and genotype. To demonstrate that the mutation Arg719Trp in the cardiac beta-myosin heavy chain (beta MHC) gene is a high risk factor for sudden death and can be associated with an unusual apical non-obstructive HCM, we report the case of a 6 1/2 year old boy, who suffered cardiac arrest. The proband had a de novo mutation of the beta MHC gene (Arg719Trp) on the paternal beta MHC allele and a second maternally transmitted mutation (Met349Thr), as was shown previously (Jeschke et al. 1998 (11)). Here we report the clinical phenotype of the proband and of his relatives in detail. The proband had a marked apical and midventricular hypertrophy of the left and right ventricle without obstruction. There was an abnormal relaxation of both ventricles. Holter monitoring detected no arrhythmia. Ventricular fibrillation was inducible only by aggressive programmed stimulation. The boy died 3 1/2 years later after another cardiac arrest due to arrhythmia. Five carriers of the Met349Thr mutation in the family were asymptomatic and had no echocardiographic changes in the heart, suggesting a neutral inherited polymorphism or a recessive mutation. It is concluded that there is an association of the mutation Arg719Trp in the beta-myosin heavy chain with sudden cardiac death in a young child. Disease history in conjunction with the genetic analysis suggests that the implantation of a defibrillator converter would have been a beneficial and probably life saving measure.

A newly created splice donor site in exon 25 of the MyBP-C gene is responsible for inherited hypertrophic cardiomyopathy with incomplete disease penetrance.

BACKGROUND: Hypertrophic cardiomyopathy is a myocardial disorder resulting from inherited sarcomeric dysfunction. We report a mutation in the myosin-binding protein-C (MyBP-C) gene, its clinical consequences in a large family, and myocardial tissue findings that may provide insight into the mechanism of disease. METHODS AND RESULTS: History and clinical status (examination, ECG, and echocardiography) were assessed in 49 members of a multigeneration family. Linkage analysis implicated the MyBP-C gene on chromosome 11. Myocardial mRNA, genomic MyBP-C DNA, and the myocardial proteins of patients and healthy relatives were analyzed. A single guanine nucleotide insertion in exon 25 of the MyBP-C gene resulted in the loss of 40 bases in abnormally processed mRNA. A 30-kDa truncation at the C-terminus of the protein was predicted, but a polypeptide of the expected size ( approximately 95 kDa) was not detected by immunoblot testing. The disease phenotype in this family was characterized in detail: only 10 of 27 gene carriers fulfilled diagnostic criteria. Five carriers showed borderline hypertrophic cardiomyopathy, and 12 carriers were asymptomatic, with normal ECG and echocardiograms. The age of onset in symptomatic patients was late (29 to 68 years). In 2 patients, outflow obstruction required surgery. Two family members experienced premature sudden cardiac death, but survival at 50 years was 95%. CONCLUSIONS: Penetrance of this mutation was incomplete and age-dependent. The large number of asymptomatic carriers and the good prognosis support the interpretation of benign disease.

Genomic structure and fine mapping of the two human filamin gene paralogues FLNB and FLNC and comparative analysis of the filamin gene family.

The genomic structure of the filamin gene paralogues FLNB and FLNC was determined and related to FLNA. FLNB consists of 45 exons and 44 introns and spans approximately 80 kb of genomic DNA. FLNC is divided into 48 exons and 47 introns and covers approximately 29.5 kb of genomic DNA. A previously unknown intron was found in FLNA. The comparison of all three filamin gene paralogues revealed a highly conserved exon-intron structure with significant differences in the exons 32 of all paralogues encoding the hinge I region, as well as the insertion of a novel exon 40A in FLNC only. Gene organization does not correlate with the domain structures of the respective proteins. To improve candidate gene cloning approaches, FLNB was precisely mapped at 3p14 in an interval of 0.81 cM between WI3771 and WI6691 and FLNC at 7q32 in an interval of 2.07 cM between D7S530 and D7S649.

[Genetic causes of hypertrophic cardiomyopathy]. / Die genetischen Ursachen der hypertrophischen Kardiomyopathie.

Hypertrophic cardiomyopathy is a dominantly inherited disease of the heart. Heterogeneous sets of mutations responsible for this condition have been identified in seven genes coding for proteins involved in the contraction mechanism or in the control of contraction of the myocardium. Known mutations imply structural and functional changes in the following proteins: in ventricle specific beta-myosin heavy chain, in essential and regulatory myosin light chains, in troponin subunits T and I, in alpha-tropomyosin and in myosin binding protein-C. The gene of one additional genomic HCM-locus is not known. Since two thirds or more of all cases can be traced to one of the respective genes, HCM has been classified as a disease of the cardiac sarcomere. Heterogeneity does not only exist between genes, but also within genes. At least 84 different mutations have been identified to date. More than half of them have been detected in the beta-myosin heavy chain gene. Thus, mutations in this gene account for most of the cases of HCM. The extent of data about causes is in contrast to the lack of definite knowledge about pathogenic mechanisms. Since the disorder is in many cases mild with symptoms developing frequently not before the end of the second decade, myocardial dysfunctions can presumably not directly be traced to altered contractility, but rather to effects which accumulate with a long asymptomatic lag period and which gradually lead to hypertrophy, conduction problems and ultimately to cardiac failure. The disease may be considered as an indirect and secondary response to a mildly distorted contraction process. The rapid progress in the analysis of causes suggests that the study of genes will assume a role in the context of the clinical management of HCM, in particular regarding diagnosis, prognosis, counselling of patients and families and--possibly--therapy.

A high risk phenotype of hypertrophic cardiomyopathy associated with a compound genotype of two mutated beta-myosin heavy chain genes.

Hypertrophic cardiomyopathy (HCM) is a genetically and clinically heterogeneous myocardial disease that is in most cases familial and transmitted in a dominant fashion. The most frequently affected gene codes for the cardiac (ventricular) beta-myosin heavy chain. We have investigated the genetic cause of an isolated case of HCM, which was marked by an extremely severe phenotype and a very early age of onset. HCM is normally not a disease of small children. The proband was a boy who had suffered cardiac arrest at the age of 6.5 years (resuscitation by cardioconversion). Upon screening of the beta-myosin heavy chain gene as a candidate, two missense mutations, one in exon 19 (Arg719Trp) and a second in exon 12 (Met349Thr), were identified. The Arg719Trp mutation was de novo, as it was not found in the parents. In contrast, the Met349Thr mutation was inherited through the maternal grandmother. Six family members were carriers of this mutation but only the proband was clinically affected. Segregation and molecular analysis allowed us to assign the Met349Thr mutation to the maternal and the Arg719Trp de novo mutation to the paternal beta-myosin allele. Thus, the patient has no normal myosin. We interpret these findings in terms of compound heterozygosity of a dominant (Arg719Trp) and a recessive (Met349Thr) mutation. Whereas a single mutated Arg719Trp allele would be sufficient to cause HCM, the concurrent Met349Thr mutation alone does not apparently induce the disease. Nevertheless, it conceivably contributes to the particularly severe phenotype.

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.

Cardiac myosin binding protein-C gene splice acceptor site mutation is associated with familial hypertrophic cardiomyopathy.

Familial hypertrophic cardiomyopathy (FHC) is an autosomal dominant disease characterized by a ventricular hypertrophy predominantly affecting the interventricular septum and associated with a large extent of myocardial and myofibrillar disarray. It is the most common cause of sudden death in the young. In the four disease loci found, three genes have been identified which code for beta-myosin heavy chain, cardiac troponin T and alpha-tropomyosin. Recently the human cardiac myosin binding protein-C (MyBP-C) gene was mapped to chromosome 11p11.2 (ref. 8), making this gene a good candidate for the fourth locus, CMH4 (ref. 5). Indeed, MyBP-C is a substantial component of the myofibrils that interacts with several proteins of the thick filament of the sarcomere. In two unrelated French families linked to CMH4, we found a mutation in a splice acceptor site of the MyBP-C gene, which causes the skipping of the associated exon and could produce truncated cardiac MyBP-Cs. Mutations in the cardiac MyBP-C gene likely cause chromosome 11-linked hypertrophic cardiomyopathy, further supporting the hypothesis that hypertrophic cardiomyopathy results from mutations in genes encoding contractile proteins.

Identification of gene defects by linkage analysis: use in inherited cardiomyopathies.

One of the central activities of current medical (including cardiological) research is identification of the causes of inherited diseases. The goals are the determination of genes and risk factors, introduction of new diagnostic standards and ultimately refinement of therapies. In cardiac disorders, molecular causes have been detected for certain types of hypertrophic cardiomyopathy (HCM), a disease characterized by increased ventricular wall thickness, a high risk of arrhythmias and an increased frequency of sudden cardiac death. The first known cause of HCM was a point mutation in the cardiac beta-myosin heavy chain gene on chromosome 14, detected using a genetic mapping procedure based on linkage of the clinical phenotype with genomic marker sequences. Additional missense mutations have been located in the globular head of beta-myosin, and other disease loci have been identified on chromosomes 1, 11, and 15; the disease genes in these loci have not yet been determined, however.

Identification of a mutation near a functional site of the beta cardiac myosin heavy chain gene in a family with hypertrophic cardiomyopathy.

Several mutations within the gene coding for the cardiac beta myosin heavy chain (designed MYH7) have been shown to be responsible for Familial Hypertrophic Cardiomyopathy (FHC) in several families, and evidence of genetic heterogeneity has been reported. To investigate the MYH7 gene as the cause of the disease in a small family with FHC, inheritance of the disease and chromosome 14 q11-q12 markers haplotype were studied, exons coding for the head domain of the cardiac beta myosin heavy chain (beta MHC) were analysed for mutations by MDE gel electrophoresis, and sequenced. We report a mutation within exon eight of the MYH7 gene at a very conserved amino acid at position 232, which results in the conversion of an asparagine to serine. This residue Asn-232 is located in a MHC area that has been recently identified as a critical site for ATPase activity. According to recent results on the three-dimensional structure of the myosin head or subfragment-1 (S1), Asn-232 is located in an alpha-helix which forms part of the nucleotide binding pocket. Although this mutation affects an active site, it seems to be associated with a favourable prognosis and a weak penetrance in this family.

Alpha-tropomyosin and cardiac troponin T mutations cause familial hypertrophic cardiomyopathy: a disease of the sarcomere.

We demonstrate that missense mutations (Asp175Asn; Glu180Gly) in the alpha-tropomyosin gene cause familial hypertrophic cardiomyopathy (FHC) linked to chromosome 15q2. These findings implicated components of the troponin complex as candidate genes at other FHC loci, particularly cardiac troponin T, which was mapped in this study to chromosome 1q. Missense mutations (Ile79Asn; Arg92Gln) and a mutation in the splice donor sequence of intron 15 of the cardiac troponin T gene are also shown to cause FHC. Because alpha-tropomyosin and cardiac troponin T as well as beta myosin heavy chain mutations cause the same phenotype, we conclude that FHC is a disease of the sarcomere. Further, because the splice site mutation is predicted to function as a null allele, we suggest that abnormal stoichiometry of sarcomeric proteins can cause cardiac hypertrophy.

Myosin mutations in hypertrophic cardiomyopathy and functional implications.

Hypertrophic cardiomyopathy (HCM) can be an inherited disorder. Typically, the inheritance is dominant and genetic cases account for about 50% of all patients with this pathology. Four different HCM loci have been mapped to different chromosomes (no. 1, 11, 14 and 15), yet, only one responsible gene has been identified. It is the beta myosin heavy chain gene on chromosome 14, which is expressed in ventricles and in slow skeletal muscle fibers. A large number of missense mutations has been reported which are predominantly located in the globular head region of the beta myosin. An apparent hot spot of mutation has been detected within exon 13 of the gene, corresponding to amino acid position 403. Although the functional consequences of the various mutations for the activity of beta myosin are not known, by inference and on the basis of published data, it may be suggested that a mutation in position 403 affects the myosin-actin dissociation in the contractile cycle. Despite our knowledge of mutations in the myosin gene, and of many of the pathological sequelas, there still is insufficient information which precludes unequivocal conclusions on the molecular mechanisms by which the pathogenesis of the myosin deficient heart develops. Molecular biology and genetics should help to define the determinants of this disease.

Mapping of the actomyosin interfaces.

Recombinant DNA methods were used to obtain soluble, undenatured fragments of the heavy chain of myosin subfragment 1 (S-1). These fragments were of preselected lengths and could include protease-sensitive segments that are destroyed when other preparation methods are used. Actin binding by each of the three contiguous segments (residues 1-248, 249-524, and 518-722, essentially spanning the entire S-1 heavy chain) was demonstrated. ATP binding, comparable to that of native S-1, was obtained only with a segment consisting of residues 1-524. Competition among the various fragments for actin was also studied. The data are discussed in relation to the recently reported resolved structure of S-1 [Rayment, I., Rypnieski, R. W., Schmidt-Bäse, K., Smith, R., Tomchick, D. R., Benning, M. M., Winkelmann, D. A., Wesenberg, G. & Holden, H. M. (1993) Science 261, 50-58].

[Research in gene therapy--its status in Germany]. / Gentherapieforschung--ihr Stand in Deutschland.

Progress in mammalian molecular biology and in the analysis of the human genome has allowed to identify the causes of an increasing number of human diseases in recent years. Newly developed gene transfer techniques were reason to implement new therapeutic concepts. By means of viral vectors or other transfer vehicles genes can be introduced into cells of the human body in order to replace a deficient function (in inherited diseases) or to play a role in defending the body (against cancer or, in the cardiovascular field, eventually by preventing restenosis). Despite considerable achievements of current DNA transfer technologies it seems premature to qualify gene therapy already as a new medical practice. The development in Germany is characterized by a late start in this field of research. The number of projects is correspondingly small. However, it may be expected that newly initiated governmental support of gene therapy research will lead to an expansion of the activities in this area.

Exclusion of cardiac myosin heavy chain and actin gene involvement in hypertrophic cardiomyopathy of several French families.

Familial hypertrophic cardiomyopathy (FHC) is characterized by idiopathic myocardial hypertrophy, which often and predominantly involves the interventricular septum. The disease is transmitted as an autosomal dominant trait, and its major risk is sudden death. It was recently demonstrated that this disease is genetically heterogeneous and that in 13 of 18 unrelated families the morbid locus, termed FHC-1, maps to chromosome 14q11-12 in and/or very near the cardiac beta-myosin heavy chain gene. We have performed linkage analysis with five chromosomal markers detecting polymorphisms in either the cardiac beta-myosin heavy chain gene or the cardiac actin gene (located on chromosome 15q) on eight families from different regions of France. We show that 1) it is possible to analyze medium-sized families by using highly informative microsatellite markers located in these genes and 2) the disease is not linked to the two contractile protein genes in any of these families. Moreover, 10-20% of chromosome 14 and 20-40% of chromosome 15 in the vicinity of the respective markers were excluded as possible locations for the morbid locus. These results provide new insights into the identification of the genes responsible for FHC.