Hugh Watkins

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.

Sudden Death due to Troponin T Mutations

OBJECTIVESnThis study was designed to verify initial observations of the clinical and prognostic features of hypertrophic cardiomyopathy caused by cardiac tropnin T gene mutations.nnnBACKGROUNDnThe most common cause of sudden cardiac death in the young is hypertrophic cardiomyopathy, which is usually familial. Mutations causing familial hypertrophic cardiomyopathy have been identified in a number of contractile protein genes, raising the possibility of genetic screening for subjects at risk. A previous report suggested that mutations in the cardiac troponin T gene were notable because they were associated with a particularly poor prognosis but only mild hypertrophy. Given the variability of some genotype:phenotype correlations, further analysis of cardiac troponin T mutations has been a priority.nnnMETHODSnDeoxyribonucleic acid from subjects with hypertrophic cardiomyopathy was screened for cardiac troponin T mutations using a ribonuclease protection assay. Polymerase chain reaction-based detection of a novel mutation was used to genotype members of two affected pedigrees. Gene carriers were examined by echocardiography and electrocardiology, and a family history was obtained.nnnRESULTSnA novel cardiac troponin T gene mutation, arginine 92 tryptophan, was identified in 19 of 48 members of two affected pedigrees. The clinical phenotype was characterized by minimal hypertrophy (mean [+/-SD] maximal ventricular wall thickness 11.3 +/- 5.4 mm) and low disease penetrance by clinical criteria (40% by echocardiography) but a high incidence of sudden cardiac death (mean age 17 +/- 9 years).nnnCONCLUSIONSnThese data support the observation that apparently diverse cardiac troponin T gene mutations produce a consistent disease phenotype. Because this is one of poor prognosis, despite deceptively mild or undetectable hypertrophy, genotyping at this locus may be particularly informative in patient management and counselling.

Prognostic implications of novel beta cardiac myosin heavy chain gene mutations that cause familial hypertrophic cardiomyopathy.

Three novel beta cardiac myosin heavy chain (MHC) gene missense mutations, Phe513Cys, Gly716Arg, and Arg719Trp, which cause familial hypertrophic cardiomyopathy (FHC) are described. One mutation in exon 15 (Phe513Cys) does not alter the charge of the encoded amino acid, and affected family members have a near normal life expectancy. The Gly716Arg mutation (exon 19; charge change of +1) causes FHC in three family members, one of whom underwent transplantation for heart failure. The Arg719Trp mutation (exon 19; charge change of -1) was found in four unrelated FHC families with a high incidence of premature death and an average life expectancy in affected individuals of 38 yr. A comparable high frequency of disease-related deaths in four families with the Arg719Trp mutation suggests that this specific gene defect directly accounts for the observed malignant phenotype. Further, the significantly different life expectancies associated with the Arg719Trp vs. Phe513Cys mutation (P < 0.001) support the hypothesis that mutations which alter the charge of the encoded amino acid affect survival more significantly than those that produce a conservative amino acid change.

Preclinical Diagnosis of Familial Hypertrophic Cardiomyopathy by Genetic Analysis of Blood Lymphocytes

BACKGROUNDnThe clinical diagnosis of familial hypertrophic cardiomyopathy is usually made on the basis of the physical examination, electrocardiogram, and echocardiogram. Making an accurate diagnosis can be particularly difficult in children, who may not have cardiac hypertrophy until adulthood. Recently, we demonstrated that mutations in the cardiac myosin heavy-chain genes cause familial hypertrophic cardiomyopathy in some families. We report a diagnostic test for familial hypertrophic cardiomyopathy that relies on the detection of mutations in the beta myosin heavy-chain gene in circulating lymphocytes that we used to evaluate three generations of a family, including the children.nnnMETHODS AND RESULTSnUsing the polymerase chain reaction, we found that normal and mutant beta cardiac myosin heavy-chain genes are transcribed in circulating lymphocytes. This allowed us to examine beta cardiac myosin heavy-chain messenger RNA from blood lymphocytes, even though ordinary expression of the gene is virtually restricted to the heart. Base sequences amplified from this messenger RNA were analyzed with a ribonuclease protection assay to identify small deletions, abnormal splicing, or missense mutations. Using this technique we identified a novel missense mutation in a patient with familial hypertrophic cardiomyopathy. We evaluated 15 of the patients adult relatives and found perfect agreement with the clinical diagnosis (8 affected and 7 not affected). Clinical analysis of 14 of the children (age, 1 to 20 years) of these affected family members revealed 1 child with echocardiographic findings diagnostic of familial hypertrophic cardiomyopathy. However, genetic analyses showed that six other children had also inherited the missense mutation and might later manifest the disease.nnnCONCLUSIONSnTranscripts of beta cardiac myosin heavy-chain gene can be detected in blood lymphocytes and used to screen for mutations that cause familial hypertrophic cardiomyopathy. This approach makes practical the identification of mutations responsible for this disorder and may be applicable to other diseases in which direct analysis is difficult because the mutated gene is expressed only in certain tissues. Preclinical or prenatal screening in an affected family will make it possible to study the disease longitudinally and to develop preventive interventions.

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

OBJECTIVESnWe 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.nnnBACKGROUNDnCorrelation 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.nnnMETHODSnThe 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.nnnRESULTSnGenetic 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.nnnCONCLUSIONSnNucleotide 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.

Left ventricular hypertrophy and morphology in familial hypertrophic cardiomyopathy associated with mutations of the beta-myosin heavy chain gene

OBJECTIVESnThe purpose of this study was to determine the spectrum of left ventricular hypertrophy and ventricular morphology in adults with hypertrophic cardiomyopathy due to mutations of the beta-myosin heavy-chain gene.nnnBACKGROUNDnAlthough echocardiography is an important test in diagnosing hypertrophic cardiomyopathy, the lack of an independent diagnostic criterion has been an obstacle in determining the full echocardiographic spectrum of this disease. Mutations in the beta-myosin heavy chain gene occur in approximately 50% of familial cases; in members of families with a known mutation, the diagnosis can be made with certainty.nnnMETHODSnEchocardiograms from 39 genetically affected and 30 genetically unaffected adult family members over age 16 years from 10 families were analyzed. Left ventricular wall thickness was measured at 10 separate locations, and the presence of systolic anterior motion of the mitral valve, right ventricular hypertrophy and left ventricular morphology was evaluated independently by three separate observers without knowledge of the genetic diagnosis.nnnRESULTSnThe mean maximal wall thickness in the genetically affected group was 24 +/- 8 mm (range 11 to 40), compared with 11 +/- 2 mm (range 7 to 16) in the unaffected group (p < 0.0001). Systolic anterior motion of the mitral valve or chordae tendineae with or without leaflet-septal contact was present in 62% of the affected group and in none of the unaffected group. The morphologic finding of reversed septal curvature was present in 79% of the affected group and in none of the unaffected group. Seventy-seven percent of patients in the affected group had a septal/free wall ratio > or = 1.3 compared with 6% in the unaffected group, with a septal/posterior wall ratio > or = 1.3 associated with only a 55% probability of being affected.nnnCONCLUSIONSnThe two-dimensional echocardiographic spectrum of hypertrophic cardiomyopathy in a genetically defined adult population is broad. Previous echocardiographic criteria may be too strict to diagnose the disease in some patients who are genetically affected and therefore at risk for adverse events related to the disease. Ultimately, genetic testing may supersede echocardiography in diagnosing hypertrophic cardiomyopathy.

Amplification of Sequences from Affected Individuals

This unit describes methods for obtaining DNA sequences from an individual affected by a genetic disorder. Target sequences that may be present at very low copy number in patient samples are amplified by the polymerase chain reaction (PCR). The first Basic Protocol 1 describes harvesting of mRNA from peripheral lymphocytes, which can even be utilized for genes with tissue-specific patterns of expression. This technique has the advantage of ease of access to appropriate patient samples and also may provide a more efficient means of screening coding sequences than would analysis of individual exons. An alternate protocol describes modifications in PCR conditions that facilitate mutation analysis and sequencing. When the genomic sequence for a given candidate gene is known, the second Basic Protocol 2 may be used to obtain appropriate sequences for analysis of individual exons or noncoding regions of interest.

Molecular Genetics: New Mutations that Cause Familial Hypertrophic Cardiomyopathy

Since the description of the first genetic locus for familial hypertrophic cardiomyopathy (FHC; cardiomyopathy, hypertrophic, or CMH1) on chromosome 14, our molecular understanding of the syndrome has extended greatly [1] Mutations in the cardiac β myosin heavy chain (PMHC) gene have been shown to cause FHC at this locus [2,3]. The α cardiac myosin heavy chain gene does not appear to cause any FHC [4]. Over 30 discrete βMHC gene missense mutations have now been documented, and individual mutations have been shown to be associated with disparate risks of sudden cardiac death [5]. The precise mechanisms by which myosin mutations cause FHC is not yet known, but abnormalities of myofibrillar assembly in vitro have been demonstrated [6]. Homologous recombination is being used to generate mouse models for β myosin missense mutations that will help to answer these questions definitively.