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.

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.

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.

[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.

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.

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.

[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.

The polymerase chain reaction: an improved method for the analysis of nucleic acids.

The polymerase chain reaction (PCR) is a method for the selective amplification of DNA or RNA segments of up to 2 kilobase-pairs (kb) or more in length. Synthetic oligonucleotides flanking sequences of interest are used in repeated cycles of enzymatic primer extension in opposite and overlapping directions. The essential steps in each cycle are thermal denaturation of double-stranded target molecules, primer annealing to both strands and enzymatic synthesis of DNA. The use of the heat-stable DNA polymerase from the archebacterium Thermus aquaticus (Taq polymerase) makes the reaction amenable to automation. Since both strands of a given DNA segment are used as templates, the number of target sequences increases exponentially. The reaction is simple, fast and extremely sensitive. The DNA or RNA content of a single cell is sufficient to detect a specific sequence. This method greatly facilitates the diagnosis of mutations or sequence polymorphisms of various types in human genetics, and the detection of pathogenic components and conditions in the context of clinical research and diagnostics; it is also useful in simplifying complex analytical or synthetic protocols in basic molecular biology. This article describes the principles of the reaction and discusses the applications in different areas of biomedical research.

Relaxation of supercoiled phosphorothioate DNA by mammalian topoisomerases is inhibited in a base-specific manner.

The nucleotide preferences of calf thymus topoisomerases I and II for recognition of supercoiled DNA have been assessed by the relaxation and cleavage of DNA containing base-specific phosphorothioate substitutions in one strand. The type I enzyme is inhibited to varying degrees by all modified DNAs, but most effectively (by approximately 60%) if deoxyguanosine 5'-O-(1-thiomonophosphate) (dGMP alpha S) is incorporated into negatively supercoiled DNA. A DNA in which all internucleotide linkages of one strand are phosphorothionate is relaxed, most probably via the unsubstituted strand. The type II enzyme is inhibited when deoxyadenosine 5'-O-(1-thiomonophosphate) (dAMP alpha S) or deoxyribosylthymine 5'-O-(1-thiomonophosphate) is incorporated into the DNA substrate, and the course of the relaxation reaction changes from a distributive mode to a predominantly processive mode. A fully substituted DNA is very poorly relaxed by the type II enzyme, illustrating the strict commitment of the enzyme to relaxation via double-strand cleavage. The sense of supercoiling does not affect the inhibition profile of either enzyme. DNA strand breaks introduced by type II topoisomerase in a normal control DNA or deoxycytidine 5'-O-(1-thiomonophosphate)-substituted DNA on treatment with sodium dodecyl sulfate at low ionic strength are prevented by pretreatment with 0.2 M NaCl. In contrast, breaks in DNA having either dAMP alpha S or all four phosphorothioate nucleotides incorporated in one strand are prevented only with higher NaCl concentrations. Thus indicating activity at the phosphorothioate linkage 5' to dA but not 5' to dC. We conclude that topoisomerase II activity occurs preferentially at sites possessing dAMP or dTMP, and that dGMP is involved in DNA recognition by topoisomerase I.

Enzymatic amplification of myosin heavy-chain mRNA sequences in vitro.

We have developed a procedure that detects the presence of mRNA coding for human beta-myosin heavy chain in small amounts of total, unfractionated RNA isolated from heart or skeletal muscle. The protocol is based on the enzymatic amplification in vitro of a selected 106-bp myosin isotype-specific subregion of this mRNA. The method, which is a modification of the so-called "polymerase chain reaction," requires two synthetic oligonucleotide primers (20-mers), reverse transcriptase, and DNA polymerase I (Klenow fragment). Two principle steps are involved: (i) the selected mRNA subregion is converted into a double-stranded cDNA, and (ii) this cDNA is amplified in 22 synthetic cycles. After gel electrophoresis and blotting the amplification product is identified by hybridization with a third oligonucleotide recognizing the region between the two primer annealing sites, and by restriction mapping. Only mRNA from muscle tissue promoted formation of the amplified 106-bp fragment. We estimate that less than 30,000 beta-myosin heavy-chain mRNA molecules are sufficient to produce a signal. The procedure is fast, specific, and very sensitive. It may be used in muscle gene expression studies with small numbers of cells or even in single muscle fibers.

Incorporation of phosphorothioate groups into fd and phi X174 DNA.

We have synthesized fd and phi X174DNA in the presence of 2'-deoxyadenosine 5'-O-(1-thiotriphosphate) (dATP alpha S) and the corresponding phosphorothioate derivatives of dCTP and dTTP using ether-permeabilized E. coli cells or crude cell extracts of E. coli DNA polymerase I. Reaction rates of enzymes involved in the formation or breakdown of DNA are decreased in the presence of phosphorothioates. The amount of label incorporated with [35S]dATP alpha S suggests that the dAMP has been completely substituted by 2'-deoxyadenosine 5'-0-phosphorothioate (dAMPS). The substituted DNAs have the same sedimentation coefficients, similar buoyant density, infectivity, and thermal stability as the unsubstituted DNAs. The procedure therefore allows specific modification at the 5' position of dA, dC, or dT in the DNA. In view of the recent demonstration of specific binding of Pt2+ complexes to the phosphorothioate analogue of poly[r(A-U)] (Strothkamp, K.G., and Lippard, S.J. (1976), Proc. Natl. Acad. Sci. U.S.A. 73, 2536), the synthesis of phosphorothioate containing DNA may be of use for DNA sequencing by electron microscopy.

Effect of deoxynucleoside phosphorothioates incorporated in DNA on cleavage by restriction enzymes.

DNA synthesized in vitro using deoxynucleoside phosphorothioates as substrates is quite similar to normal DNA in its biochemical properties (Vosberg, H.P., and Eckstein, F. (1977) Biochemistry 16, 3633-3640). In order to investigate the effect of phosphorothioate groups in DNA on the cleavage pattern of restriction endonucleases phosphorothioate double-stranded, circular, replicative form of fd DNA was synthesized in vitro with Escherichia coli DNA polymerase I using native single-stranded DNA as template and mixtures of three normal nucleotides and one nucleoside phosphorothioate analogue as substrates. The double-stranded products were hybrids with respect to their phosphorothioate content. Restriction analysis of normal and phosphorothioate DNA with the restriction endonucleases Hae III, Bam HI, Hpa II, HindII, Alu I, and Taq I showed that the enzymes were inhibited to different degrees depending on which of the nucleotides was replaced by the phosphorothioate. Most significant, inhibition was seen throughout with those DNAs which contained a phosphorothioate exactly at the cleavage site. Phosphorothioate substitutions at other positions, but still within the recognition sequences, were, except for Alu I, not or weakly inhibitory. Phosphorothioate nucleotides not present in the recognition sequences did not affect at all the fragment patterns. The results show that recognition sequences of restriction endonucleases can be selectively protected against cleavage by base-specific introduction of phosphorothioate groups into DNA.

Chromosomal localization of a human myosin heavy-chain gene by in situ hybridization.

A cloned rabbit heart muscle myosin heavy-chain cDNA was hybridized in situ with human metaphase chromosomes. The probe was known to have sequence homology with human genomic heavy-chain DNA. Only one site in the human haploid karyotype was labeled with the cDNA, and this site was found on the short arm of chromosome 17. The localization of autoradiographic grains suggests a subregional assignment of the myosin heavy-chain locus to 17p 1,2-pter.

Characterization of a human genomic DNA fragment coding for a myosin heavy chain.

A DNA segment from the human genome with information for myosin heavy chain (MHC) was isolated from a human genomic DNA library cloned in lambda Charon 4A phages. The isolation was accomplished by a myosin cDNA probe obtained from rabbit heart muscle mRNA (Sinha et al. 1982). The selected human DNA clone, designated lambda gMHC1, contains a genomic DNA fragment of about 14 kilobase pairs. The transcriptional polarity of this DNA was determined. The 5'-end of the gene is missing from the cloned fragment. This human gene exhibits sequence homology to MHC DNA of rabbit and chicken, but not to an MHC sequence of nematode. The isolated gene fragment is a member of the human MHC multi-gene family, which is presumed to consist of probably more than ten separate sarcomeric MHC genes per haploid genome.

Characterisation of size variants of type I DNA topoisomerase isolated from calf thymus.

Calf thymus DNA topoisomerase I, which belongs to the eukaryotic type I topoisomerases, is in a typical preparation purified as a set of five major polypeptides with Mr between 70000 and 100000. At least four of these proteins have binding affinity for DNA as was shown by incubating them with radioactive single-stranded DNA after separation in dodecylsulfate polyacrylamide gels and blotting onto nitrocellulose filters. That these polypeptides have DNA relaxing activity was directly demonstrated with protein extracted from single bands of dodecylsulfate/polyacrylamide gels. We consider the 100000-Mr protein to be the native enzyme. The smaller components are catalytically active fragments of the native topoisomerase most probably arising from limited proteolysis either within the nucleus or during the purification of the enzyme. In two-dimensional non-equilibrium pH-gradient electrophoresis gels the topoisomerase size variants exhibit apparent pI values between 8.1 and 8.3, with small but distinct differences between the components. The calf thymus topoisomerase I, upon binding to phage fd-DNA, protects a stretch of 15-25 nucleotides against digestion with DNase I.

The regulation of the human beta myosin heavy-chain gene.

The human myosin heavy-chain (MHC) genes for cardiac and skeletal muscle exist as a multigene family with eight or more non-allelic genes. Two of them code for the cardiac alpha and beta myocin HCs. They are located on chromosome 14. The skeletal muscle myosin HC genes are on chromosome 17. The cardiac MHCs coexist in the heart, however, with a distinct distribution within cardiac tissue of the human adult. alpha-MHC is predominantly found in the atria and beta-MHC is found in the ventricles. Both genes are also expressed in certain types of skeletal muscle fibers. We have sequenced the beta-gene in its entire length and have further studied in detail its expression in muscle cells. Promoter activities were tested using DNA-mediated gene transfer in cultured chicken embryonic myoblasts. By deletion mapping of the 5' flanking region of the beta-gene a candidate signal sequence was identified in a region which stimulates the promoter in a tissue specific and differentiation dependent mode. The presumed signal was located about 210 bp 5' to the basic promoter which, by itself, is almost inactive, even in muscle cells. The sequence of the signal (CAGCTG) has homology to known E-box sequences. E-boxes (consensus sequence CANNTG) constitute a family of transcription control sites frequently found upstream of muscle genes. In nuclear extracts of cardiac and skeletal muscle (of rabbit) a protein was identified which binds to the region containing the E-box like motif of the beta-gene. Since this protein was present in both types of muscle, overlapping expression control patterns are assumed to operate in these tissues.