beta-MHC Gene Research Articles

Sudden cardiac death in hypertrophic cardiomyopathy. Variability in phenotypic expression of beta-myosin heavy chain mutations.

Recent identification of mutations in the beta-myosin heavy chain gene (MYH7), a major responsible gene for HCM, has provided the opportunity to characterize genotype-phenotype correlation in HCM families. In this study we analysed the phenotypic expression of two beta-myosin heavy chain (beta MHC) mutations in three unrelated HCM families. Living individuals from three unrelated HCM families (Families 1, 2, and 3) were screened by history, physical examination, electrocardiography, and two-dimensional echocardiography. Blood was collected from all individuals for DNA extraction. Polymerase chain reaction (PCR), restriction endonuclease digestion and chemical cleavage were utilized for detection of mutations. All mutations were confirmed by sequence analysis. Identification of mutations: A missense mutation in exon 13 of the beta MHC gene (Arg403 Gln) was detected in HCM patients from Families 1 and 2. PCR amplification of the exon 13 DNA, followed by Ddel digestion of the PCR product and gel electrophoresis, showed two fragments of 84 and 70 bp in normal individuals and four fragments of 84, 70, 52 and 32 bp in HCM patients. Sequence analysis showed substitution of an adenine for guanine at coding position 1208. In Family 3, a missense mutation in exon 16 of the beta MHC gene (Val606 Met) was detected in HCM patients. Chemical cleavage of the PCR products showed an uncleaved product of 337 bp in the normal individuals, while in the affected individuals, in addition to the uncleaved product, a 90 bp cleaved product was also detected, indicating the presence of a mismatch in one allele. Sequence analysis showed substitution of an adenine for guanine in coding position 1817. Seven members of Family 1 had HCM, of whom five are alive. One patient died from sudden cardiac death (SCD) and another from recurrent cerebral emboli. In Family 2, 15 individuals had HCM of whom nine have died, seven from SCD. The mean age at the time of SCD was 33 years. The third family is comprised of 11 affected individuals and one obligate carrier, of whom one patient died at age 17 from progressive heart failure. Two additional individuals in this family have also succumbed to SCD to age 60. A variety of clinical and echocardiographic manifestations of HCM were present in each family. Logrank test of Kaplan-Meier survival curves indicates that Arg403 Gln mutation was associated with a poor prognosis in HCM families as compared to Val606 Met (P = 0.034). beta MHC mutations despite showing variable clinical and echocardiographic manifestations of HCM are predictors of survival in HCM families.

Alternatively Processed Isoforms of Cellular Nucleic Acid-binding Protein Interact with a Suppressor Region of the Human β-Myosin Heavy Chain Gene

Analysis of a series of human beta-myosin heavy chain (MHC) constructs with progressive deletions in the 5'-flanking region has localized a strong positive element at positions -298/277 with a repressor region located immediately upstream at -332/-300 (Flink, I. L., Edwards, J. G., Bahl, J. J., Liew, C.-C., Sole, M., and Morkin, E. (1992) J. Biol. Chem. 267, 9917-9924). A 49-base pair restriction fragment containing the suppressor element was used to screen a cardiac expression library. The 0.65-kilobase pair cDNA identified by this procedure was similar in sequence, except for the absence of a 21-base pair region encoding seven amino acids, to cellular nucleic acid-binding protein (CNBP), a 19-kDa zinc finger DNA-binding protein isolated earlier from liver, which may be involved in negative regulation of cholesterol biosynthesis (Rajavashisth, T. B., Taylor, A. K., Andalibi, A., Svenson, K. L., and Lusis, A. J. (1989) Science 245, 640-643). An additional clone identical to the one originally found in liver, referred to as CNBP alpha, was isolated from the cardiac library by hybridization screening. Gel mobility shift analysis indicated that CNBP alpha and CNBP beta isoforms preferentially interact with single-stranded DNA corresponding to the proximal and distal regions of the suppressor. When cotransfected with a beta-MHC reporter construct, CNBP alpha repressed activity in a dosage-dependent manner, whereas repression was not observed with the shorter construct (CNBP beta). Cotransfection of a combination of CNBP alpha and CNBP beta repressed reporter activity to an extent similar to cotransfection with CNBP alpha alone, suggesting that CNBP beta is not translationally active under these conditions. The results of RNase protection assays and genomic sequencing indicated that the alpha and beta isoforms are formed by alternative use of 5' donor sites within a single exon. These results suggest that CNBP isoforms may modulate the activity of the beta-MHC gene by interaction with a repressor region.

Abnormal contractile properties of muscle fibers expressing beta-myosin heavy chain gene mutations in patients with hypertrophic cardiomyopathy.

Missense mutations in the beta-myosin heavy chain (beta-MHC) gene cause hypertrophic cardiomyopathy (HCM). As normal and mutant beta-MHCs are expressed in slow-twitch skeletal muscle of HCM patients, we compared the contractile properties of single slow-twitch muscle fibers from patients with three distinct beta-MHC gene mutations and normal controls. Fibers with the 741Gly-->Arg mutation (near the binding site of essential light chain) demonstrated decreased maximum velocity of shortening (39% of normal) and decreased isometric force generation (42% of normal). Fibers with the 403Arg-->Gln mutation (at the actin interface of myosin) showed lowered force/stiffness ratio (56% of normal) and depressed velocity of shortening (50% of normal). Both the 741Gly-->Arg and 403Arg-->Gln mutation-containing fibers displayed abnormal force-velocity relationships and reduced power output. Fibers with the 256Gly-->Glu mutation (end of ATP-binding pocket) had contractile properties that were indistinguishable from normal. Thus there is variability in the nature and extent of functional impairments in skeletal fibers containing different beta-MHC gene mutations, which may correlate with the severity and penetrance of the disease that results from each mutation. These functional alterations likely constitute the primary stimulus for the cardiac hypertrophy that is characteristic of this disease.

Molecular and clinical aspects of inherited cardiomyopathies.

Hypertrophic cardiomyopathy (HCM) is phenotypically and genotypically a heterogeneous disease. Since 1989, four chromosomal loci have been identified for HCM and the genes residing on three of these have been identified as beta-myosin heavy chain (beta-MHC), cardiac troponin-T and alpha-tropomyosin. These genes code for sarcomeric proteins and exhibit the same phenotype, suggesting that HCM is a disease of the sarcomere. Over 40 missense mutations and one deletion of the beta-MHC gene have been identified. Similarly, missense mutations in the alpha-tropomyosin gene and the cardiac troponin-T gene have been identified. From genetic studies, including de novo mutations, it is established that these mutations are indeed responsible for HCM. The molecular basis of the pathogenesis of the cardiac hypertrophy appears to be a compensatory response to the primary defect. In addition to providing a definitive presymptomatic diagnosis, studies correlating beta-MHC mutations with clinical prognosis suggest they have significant predictive value and can be helpful in genetic counselling and medical management. Dilated cardiomiopathies (DCM), the most common form of cardiomyopathies, have an estimated prevalence of nearly 40 per 100,000 individuals, and are the most common cause for cardiac transplantation in the United States. Familial dilated cardiomyopathy is thought to account for approximately 20% of the so-called cases of idiopathic DCM.

Effect of chronic energy deprivation on cardiac thyroid hormone receptor and myosin isoform expression

Thyroid hormone (3,5,3'-triiodothyronine; T3) and its receptor (TR) play an important regulatory role for in vivo and in vitro cardiac myosin heavy chain (MHC) isoform gene expression by activating the alpha- and inhibiting the beta-MHC genes. Previous studies have shown that chronic energy deprivation (CED; 50% of normal caloric intake) in the rat impacts cardiac MHC protein expression and hemodynamic parameters in a pattern typically seen with hypothyroidism; yet, unlike hypothyroidism, circulating T3 levels are not depressed. Therefore, the goal of this study was to determine if the altered MHC isoform expression observed in CED is associated with altered TR expression, both at the mRNA and protein levels. Female rats weighing approximately 250 g were allocated into two groups, designated as normal control (NC) and CED. After 5 wk, the relative content of alpha-MHC protein and mRNA levels decreased in CED ventricles by 20% (P < 0.05). In contrast, the relative content of both beta-MHC protein and mRNA levels increased five- to sixfold in CED (P < 0.05). Although there were no changes in TR mRNA levels relative to 18S rRNA in CED, the total number of nuclear TRs decreased 3.5-fold in the CED group (P < 0.05), from a maximum binding capacity of 840 +/- 130 fmol/mg DNA in NC to 241 +/- 118 fmol/mg DNA in CED, with no change in the affinity of the receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

An evaluation of ribonuclease protection assays for the detection of beta-cardiac myosin heavy chain gene mutations.

Ribonuclease (RNase) protection has been used to identify beta-cardiac myosin heavy chain (MHC) gene mutations that cause familial hypertrophic cardiomyopathy (FHC). Since more than 10 different mutations within this gene have been demonstrated to cause FHC in unrelated individuals, the genetic diagnosis of this condition will involve screening the beta-MHC gene. The accuracy with which RNase protection identifies such mutations is critical to defining the utility of this methodology in detecting mutations that cause FHC. Twelve unrelated individuals with FHC were selected for further study because their beta-MHC genes had been screened for mutations by use of RNase protection, and no mutation was found. We performed linkage analysis of the families of these 12 probands using polymorphic short tandem repeats within the beta-MHC gene to determine whether FHC was genetically linked to the MHC locus on chromosome 14. FHC was not genetically linked to the MHC locus in 11 families whose beta-cardiac MHC gene did not contain mutations detectable by RNase protection. We conclude that RNase protection is a sensitive method for screening for mutations within the beta-cardiac MHC gene. Further, mutations in the noncoding regions of the beta-MHC gene and mutations in the alpha-cardiac MHC gene are not a common cause of FHC. Negative RNase protection assays of affected individuals suggest that their FHC is due to mutations at other loci.

Transcriptional enhancer factor-1 in cardiac myocytes interacts with an alpha 1-adrenergic- and beta-protein kinase C-inducible element in the rat beta-myosin heavy chain promoter.

In cultured rat cardiac myocytes, a 20-base pair sequence (-215/-196) of the rat beta-myosin heavy chain (MHC) promoter mediates induction by both alpha 1-adrenergic stimulation and a constitutively activated beta-protein kinase C (PKC), and binds cardiac myocyte nuclear factor(s) through an "enhancer core" element (5'-TGTGG-TATG-3') (Kariya, K., Karns, L. R., and Simpson, P. C. (1994) J. Biol. Chem. 269, in press). Here, we report identification of this enhancer core binding factor as the rat homologue of transcriptional enhancer factor-1 (TEF-1), a human transcription factor for viral enhancers. In gel mobility shift and immunoblot analyses, the myocyte factor and human TEF-1 were indistinguishable in terms of sequence recognition, mobility, and immunoreactivity. Furthermore, DNA binding activity for the beta-MHC enhancer core and TEF-1 immunoreactivity correlated closely. These results are the first to suggest a role for TEF-1 in transcriptional regulation by PKC. The data also provide direct evidence for interaction of TEF-1 with the beta-MHC promoter, supporting a function for TEF-1 in regulation of cellular gene expression, as well as viral, and outline a pathway for alpha 1-adrenergic regulation of beta-MHC gene transcription in cardiac myocytes.

Mapping of a novel gene for familial hypertrophic cardiomyopathy to chromosome 11.

Familial hypertrophic cardiomyopathy (FHC) is a cardiac disorder transmitted as an autosomal dominant trait. FHC has been shown to be genetically heterogeneous with less than 50% of published pedigrees being associated with mutations in the beta myosin heavy chain (beta-MHC) gene on chromosome 14q11-q12. A second locus has recently been reported on chromosome 1. We examined the segregation of microsatellite markers in a French pedigree for which the disease is not linked to beta-MHC gene. We found significant linkage of the disease locus to several (CA)n repeats located on chromosome 11 (lod scores between +3.3 and +4.98). The data suggest the localization of the novel FHC gene in a region spanning 17 centiMorgans.

Regulation of myosin heavy chain genes in the heart.

Advances in our knowledge of the regulation of cardiac myosin isoforms made possible by molecular cloning of the alpha- and beta-MHCs genes are reviewed. Expression of these genes in heart does not seem to require MyoD or related proteins of the skeletal muscle myogenic program. Cardiac MHC genes are under the control of T3, which stimulates transcription of the alpha-MHC gene and inhibits beta-MHC mRNA production both in vivo and in cultured heart cells. The responsiveness of the genes to T3 varies in different mammals, however. The genes are most responsive in rat and rabbit, intermediate in sensitivity in calf and subhuman primate (baboon), and very resistant in the dog. The human alpha-MHC gene is T3-inducible in ventricle, but the degree of response has not been quantified. Introduction of chimeric plasmids containing 5' flanking sequences of cardiac MHC genes fused to the CAT gene into cultured heart cells and transgenic animals has permitted identification of regulatory elements. Although the genes are closely linked in genomic DNA, they are controlled independently. The element within the alpha-MHC promoter responsible for induction by T3 is located approximately 160 base pairs from the transcription initiation site. Additional transcriptional activators located 5' upstream amplify the response to T3, probably by looping out intervening DNA sequences. The proximal region of the beta-MHC gene contains important regulatory elements, including those required for repression by T3, muscle-specific expression, a MyoD-independent positive element, and a hormone-independent repressor.(ABSTRACT TRUNCATED AT 250 WORDS)

In vivo analysis of the murine beta-myosin heavy chain gene promoter.

The 5' upstream region of the murine beta-myosin heavy chain (MHC) gene has been isolated and tested for its ability to drive gene expression in transgenic mice. Three classes of transgenic mice were generated. The constructs contained approximately 5000, 2500, and 600 base pairs of beta-MHC upstream sequence fused to the chloramphenicol acetyltransferase gene and were termed beta 5, beta 2.5, and beta .6, respectively. Muscle-specific expression was observed with all three constructs. However, only the beta 5 lines directed high levels of muscle-specific transgene expression in both pre- and postbirth mice. Expression driven by the two shorter constructs was two to three orders of magnitude lower when the chloramphenicol acetyltransferase specific activities were compared. These data suggest that a distal-positive element directs high levels of gene expression in the ventricle and in slow skeletal muscles. Analyses of transgene expression during heart maturation revealed that some of the beta 5 lines were not able to respond in an appropriate manner to developmental transcriptional cues. Unlike the endogenous beta-MHC gene, which is down regulated in the ventricles around the time of birth, reporter gene expression in the majority of the lines generated was not shut off in the ventricles of the adult animals. These data indicate that high levels of muscle-specific beta-MHC gene expression are dependent upon the combinatorial interactions of a number of sequence elements that are distributed over a large region of the gene's upstream sequence.

Gene injection into canine myocardium as a useful model for studying gene expression in the heart of large mammals.

We have investigated the regulated expression of genes injected into the heart of large mammals in situ. Reporter constructs using the chloramphenicol acetyltransferase gene under the control of muscle-specific beta-myosin heavy chain (beta-MHC) or promiscuous (mouse sarcoma virus) promoters were injected into the canine myocardium. There was a linear dose-response relation between the level of gene expression and the quantity of plasmid DNA injected between 10 and 200 micrograms per injection site. The level of reporter gene expression did not correlate with the amount of injury imposed on the cardiac tissue. There was no regional variation in expression of injected reporter genes throughout the left ventricular wall. By use of both the mouse sarcoma virus and a muscle-specific beta-MHC promoter, reporter gene expression was one to two orders of magnitude greater in the heart than in skeletal muscle. Expression in the left ventricle was threefold higher than in the right ventricle. Chloramphenicol acetyltransferase activity was detected at 3, 7, 14, and 21 days after injection, with maximal expression at 7 days after injection. Statistical analysis of coinjection experiments revealed that coinjection of a second gene construct (Rous sarcoma virus-luciferase) is useful in the control of transfection efficiency in vivo. Furthermore, using reporter constructs containing serial deletions of the 5' flanking region of the beta-MHC gene, we performed a series of experiments that demonstrate the utility of this model in mapping promoter regions and identifying important regulatory gene sequences in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)

Hypertrophic cardiomyopathy mutation is expressed in messenger RNA of skeletal as well as cardiac muscle.

The beta-myosin heavy chain (beta-MHC) gene has been identified as a major locus for familial hypertrophic cardiomyopathy (FHCM). We recently showed that one of the common mutations associated with FHCM is expressed in the cardiac muscle messenger RNA (mRNA) of an affected individual. Since beta-MHC is a major sarcomeric protein of cardiac and skeletal muscle, studies were performed to determine whether the mutation is also expressed in skeletal muscle. Biopsies were obtained of skeletal muscle (biceps brachii) from a proband with FHCM known to have the missense mutation in exon 13 of the beta-MHC gene. RNA was extracted from skeletal muscle and lymphocytes by the RNAzol method. First-strand complementary DNA was synthesized by reverse transcription using an antisense primer to exon 16. Polymerase chain reaction (PCR) was performed using primers to exons 12 and 14 to amplify the segment encompassing exon 13. The PCR products were digested with Ddel restriction endonuclease. Undigested PCR product in the control and the proband was 321 base-pairs (bp). Ddel digestion of the PCR product from normal skeletal and lymphocytes showed two DNA fragments of 181 and 140 bp as expected, whereas digestion of the PCR product from the proband's skeletal muscle and lymphocytes showed four DNA fragments of 181, 149, 140, and 32 bp due to the mutation in exon 13. This indicates that the mutation in affected individuals is also expressed in the mRNA of skeletal muscle and lymphocytes. To our knowledge, this is the first documentation of a beta-MHC gene mutation expressed in skeletal muscle. This finding is provocative. Does it impair skeletal muscle function? If so, how? If not, why not? Is the impairment, or lack of it, a clue to the molecular defect of cardiac muscle? Furthermore, skeletal muscle provides a readily accessible source of mRNA for expression studies and for purification of the beta-MHC protein, which is probably essential to future investigation designed to unravel the molecular basis of this disorder.

Contractile protein gene expression in serum-free cultured adult rat cardiac myocytes.

The effects of two adhesion substrates (serum and laminin) and time in culture on the expression of genes encoding myosin heavy chain (MHC) isoforms and alpha-skeletal actin were analysed in myocytes isolated from adult rat heart and maintained in serum-free culture. Relative messenger ribonucleic acid (mRNA) abundances were quantitated by dot-blot analysis. Gene expression was not influenced by the substrate used. Time in culture induced a decrease in total mRNA abundance and an up-regulation of beta-MHC and alpha-skeletal actin genes. It is proposed that atrophy of adult myocytes is associated with a pattern of gene expression similar to the fetal program.

Effect of caffeine on expression of cardiac myosin heavy chain gene in adult hypothyroid and fetal rats.

Changes in cardiac myosin heavy chain (MHC) gene expression and isozyme transitions have been shown to be caused by developmental changes, hemodynamic overload, or the activity of various hormones. In this study, to examine whether caffeine, which has teratogenic effects on the fetal cardiovascular system, causes the distribution of cardiac MHC phenotype and, if so, to evaluate the mechanisms of the distribution of cardiac MHC phenotype by caffeine, we examined the effects of caffeine, theophylline, and cAMP on the cardiac MHC isoform transitions at the gene and protein levels using hypothyroid adult rats. Furthermore, we examined the expression of alpha- and beta-MHC gene in cardiac muscles of fetuses whose dams had received caffeine. The results showed that caffeine, theophylline, and cAMP caused accumulations of alpha-MHC mRNA and MHC isozyme V1. Furthermore, in the fetal hearts, it was recognized that caffeine induced an accumulation of alpha-MHC gene expression, as was also seen in the dams. However, this effect of caffeine on the heart was stronger in the fetus than in the dam. Intracellular cAMP concentration was increased by the administration of caffeine, theophylline, or cAMP, and the levels showed a positive correlation with those of alpha-MHC mRNA. These results suggest that the induction of alpha-MHC mRNA expression by the administration of caffeine may be induced by an increase in intracellular cAMP concentration.

Differences in clinical expression of hypertrophic cardiomyopathy associated with two distinct mutations in the beta-myosin heavy chain gene. A 908Leu----Val mutation and a 403Arg----Gln mutation.

The disease gene for hypertrophic cardiomyopathy (HCM) has been identified as the beta-myosin heavy chain (beta-MHC) gene in some HCM families. We describe extensive clinical evaluations in two kindreds with two distinct point mutations in the beta-MHC gene. We used single-strand confirmation polymorphism (SSCP) gel analysis of polymerase chain reaction-amplified products capturing each of the 40 beta-MHC gene exons to identify distinct missense mutations in two HCM kindreds. Clinical, ECG, and echocardiographic studies were performed in the two kindreds: kindred 2755 with amino acid 908Leu----Val mutation and kindred 2002 with amino acid 403Arg----Gln mutation. The morphological appearances of HCM were similar in these two kindreds. However, the two kindreds differed with respect to disease penetrance, age of onset of disease, and incidence of premature sudden death. Twelve of 31 adults (greater than or equal to 17 years) with the disease gene in kindred 2755 did not have left ventricular hypertrophy (LVH), and only five of these had ECG abnormalities. Thus, the disease penetrance in adults with this mutation was only 61%. None of 11 children aged less than 16 years had LVH. The 908 mutation was associated with a low incidence of cardiac events: Only two sudden deaths and one syncope occurred in 46 individuals with the mutant allele. In contrast, LVH was present in all 11 adults in kindred 2002 with the 403 mutation (100% disease penetrance). In addition, three of four affected children were symptomatic and had clinical evidence of HCM. The disease in this kindred was severe and resulted in six premature sudden deaths. Seven additional patients had syncope or presyncope. In some kindreds, the HCM disease gene is more prevalent than indicated by echocardiography and ECG. Some point mutations may be associated with a more malignant prognosis. Preclinical identification of children with mutations associated with a high incidence of sudden death and syncope provides the opportunity to evaluate efficacy of early therapeutic interventions.

Expression of a missense mutation in the messenger RNA for beta-myosin heavy chain in myocardial tissue in hypertrophic cardiomyopathy.

We have determined that a missense mutation in exon 13 of the beta-myosin heavy chain (beta MHC) gene is expressed in the messenger RNA (mRNA) isolated from a right ventricular endomyocardial biopsy obtained from the proband of a family with hypertrophic cardiomyopathy. The mutation is the result of a substitution of an adenine for a guanine residue in one allele of the beta MHC gene and creates a second recognition site for the restriction endonuclease Ddel in exon 13. The mutation is inherited in a Mendelian fashion and co-segregates with hypertrophic cardiomyopathy in this family. Complementary DNAs synthesized from RNA isolated from the endomyocardial biopsy were cloned into a plasmid vector and sequenced to confirm the expression of both the normal and mutant allele in mRNA of myocardial tissue. This is the first report of the transcription of a mutant beta MHC gene allele into mRNA of the myocardium.

Cardiac expressions of alpha- and beta-myosin heavy chains and sarcomeric alpha-actins are regulated through transcriptional mechanisms. Results from nuclear run-on assays in isolated rat cardiac nuclei.

In the heart, mRNA accumulations for sarcomeric actins and myosin heavy chains (MHC) are subject to diverse regulatorial processes. To study cardiac contractile protein transcriptional regulations, an in vitro transcription system using nonenzymatically isolated rat cardiac nuclei was characterized. Transcription was shown to be rapid and continuous during the first 20 min of incubation and 5.4-fold less than that seen from comparably isolated hepatocyte nuclei. Neither RNase nor DNase activities were detectable. Direct transcriptional analyses of the alpha- and beta-MHC and cardiac and skeletal alpha-actin genes from cardiac nuclei were performed. In 23-24-day-old rats, significant levels of transcription were seen for alpha-MHC and for the sarcomeric alpha-actins. beta-MHC was just detectable, and no positive signals were ever seen for fibronectin. We then compared the perecentages of MHC and sarcomeric alpha-actin expressions determined from 1) the transcriptional assays and 2) total isolated RNA (alpha-MHC: 90.1 +/- 4.8% (transcription), 93.0 +/- 4.7% (accumulation); beta-MHC: 9.9 +/- 4.8%, 7.0 +/- 4.7%; cardiac alpha-actin: 84.0 +/- 2.5%, 84.9 +/- 2.5%; skeletal alpha-actin: 16.1 +/- 2.5%, 15.0 +/- 2.5%). The results support the conclusion that the primary mechanisms controlling the accumulations of these gene products are transcriptional. Additionally, we show that an anti-sense mRNA showing strong homology or identity with the 5' end of the beta-MHC gene is transcribed in cardiac nuclei but not in hepatocyte nuclei.

Evidence of genetic heterogeneity in five kindreds with familial hypertrophic cardiomyopathy.

Recently, two families with hypertrophic cardiomyopathy have been shown to have mutations in the cardiac beta-myosin heavy chain gene (beta-MHC) located on the long arm of chromosome 14. We have performed linkage analysis of five newly ascertained pedigrees with more than 50 chromosomal markers detecting polymorphisms. Our findings confirm the linkage to beta-MHC gene locus on chromosome 14 in one family (LOD score, 4.50) and suggest linkage to the same gene in another kindred. Chromosome 14 markers were not linked to the disease gene in the other three kindreds, however, and a test for genetic heterogeneity was statistically significant. Moreover, markers for the beta-MHC gene identified affected individuals who were recombinants with respect to this gene and the disease phenotype in these three kindreds. These results provide conclusive evidence that hypertrophic cardiomyopathy in separate families is caused by mutations in disease genes at two or more locations in the genome.

Hypertrophic cardiomyopathy: failure to demonstrate mutations in exon 13 of the cardiac beta myosin heavy-chain gene.

Familial hypertrophic cardiomyopathy (FHCM) has been linked to the cardiac beta-myosin heavy-chain (MHC) genes on chromosome 14 (14q1), and a missense mutation within exon 13 of the beta MHC gene has been implicated in the pathogenesis of the disease. To test whether this constitutional mutation occurs somatically in the myocardium of the sporadic form of the disease, we studied seven patients with familial (n = 3) or sporadic (n = 4) hypertrophic cardiomyopathy (HCM). Amplification of exon 13 of the beta MHC from paraffin-embedded myocardium using the polymerase chain reaction (PCR) was performed and analysis of the amplified product for migration abnormalities using denaturing gradient gel electrophoresis (DGGE) and direct sequencing of the PCR product were used. Neither patients with HCM nor subjects with dilated cardiomyopathy (DCM) (n = 2) exhibited an aberration within exon 13 of the myocardial beta MHC. It is concluded that a specific beta MHC gene mutation is displayed only in a subset of patients with familial disease, thus further emphasizing the notion of genetic heterogeneity. In addition, in the sporadic form of the disease, somatically occurring mutations in this particular exon could not be demonstrated.

Tissue-specific regulation of the alpha-myosin heavy chain gene promoter in transgenic mice.

The intergenic region between the mouse alpha-myosin heavy chain (MHC) and beta-MHC genes was analyzed in terms of its ability to drive gene expression in transgenic mice. Earlier, we reported that the entire intergenic region was sufficient to direct expression of the bacterial chloramphenicol acetyl transferase reporter gene in a tissue-specific and developmental stage-specific manner. Additional transgenic lines have been generated which include two deletions. The first deletion, alpha-3, which lacks the distal 2.5 kilobase pairs of the upstream region, is competent to direct tissue- and developmental-specific expression of the transgene. A larger deletion, in which only 138 base pairs upstream of the transcriptional start site remain, shows no chloramphenicol acetyltransferase activity in either muscle or non-muscle tissue. Tissue surveys of transgene expression indicated low levels of activity in the lung, and analyses via the polymerase chain reaction confirmed the presence of the endogenous alpha-MHC gene transcripts in this tissue. Subsequently, an alpha-MHC gene-specific riboprobe was used to detect the cognate transcripts in lung sections by in situ hybridization. The data show that, in the lung, the transcripts are localized to the thick intimal wall of the veins and venules.