Molecular genetics of familial hypertrophic cardiomyopathy

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Pedf”atric Cardiology ELSEVIER

Progress in Pediatric Cardiology 6 (1996) 63-70

Molecular genetics of familial hypertrophic cardiomyopathy Antoine B. Abchee, Robert Roberts* Section of Cardiology, Baylor College of Medicine, 6550 Fannin, MS SMTH 677, Houston, TX 77030-3498, USA

Abstract Familial hypertrophic cardiomyopathy (FHC) is a genetically and phenotypically heterogeneous disease with an autosomal dominant Mendelian inheritance. Mutations in four different genes coding for sarcomeric proteins have been identified to heavy chain ( /3 MHC) gene was identified to cause familial cause the disease. In 1990, a mutation in the P-myosin hypertrophic cardiomyopathy. Soon after, more than 30 missense mutations and one deletion in the PMHC gene were identified to be responsible for FHC. This was followed by mutations in troponin T, (Y tropomyosin and cardiac myosin binding protein-C genes. In patients with familial hypertrophic cardiomyopathy due to p MHC mutations, the mutant p MHC mRNA and protein have been isolated from cardiac and skeletal muscle of these individuals. Functional studies have shown that the mutant /? MHC protein has impaired actin-myosin interaction and expression of the mutant PMHC in feline cardiocytes disrupts the assembly of the sarcomere. Similarly, mutations in the other three genes lead to mutant proteins which interfere with the normal structure and function of the sarcomere, resulting in an identical phenotype of sarcomere disarray, the hallmark of FHC. Keywords: Familial

hypertrophic

cardiomyopathy;

/3Myosin heavy chain; Troponin

T, LYTropomyosin;

Myosin binding

protein C

1. Introduction Familial hypertrophic cardiomyopathy (FHC) is an autosomal dominant disease that is characterized by hypertrophy, mostly of the left ventricle, with predominant involvement of the interventricular septum. The ventricular systolic function is usually preserved with a greater than normal ejection fraction while diastolic function is usually impaired [l]. Sarcomere disarray, the hallmark of this disease, and myocyte hypertrophy which is found in many cardiac diseases, are the predominant pathologic findings in the heart of patients with FHC 121. The clinical manifestations of FHC range from a benign asymptomatic course to severe heart failure or sudden cardiac death (SCD). The annual incidence of SCD in patients with FHC is

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higher in the younger age group (6%) compared to the elderly (1%) in whom SCD is an unusual event. In young athletes with FHC, SCD is often the first manifestation of the disease [3-S]. In an autopsy study of 29 highly conditioned young athletes who died of SCD, 48% of the cases had hypertrophic cardiomyopathy and an additional 18% had idiopathic left ventricular hypertrophy [Sl. The prevalence of FHC in the general population has been estimated to be 0.1-l/1000 [6-81. This may not be an accurate reflection of the true prevalence for several reasons. First, FHC may be asymptomatic and never detected except incidentally. Second, the presence of concomitant diseases such as hypertension or valvular heart diseases may confound the diagnosis. Third, the phenotypic expression of the disease (i.e. development of hypertrophy) is age dependent and may not be detected at the time of the evaluation. Fourthly, the penetrance of the gene in some families is very low. reserved

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2. Genetic basis of familial hypertrophic cardiomyopathy

In 1958, Teare described the familial inheritance of hypertrophic cardiomyopathy [9], almost a century after the first description of the disease by Liouville in 1869 [lo]. Later, Braunwald et al. and Frank et al. delineated the familial nature of the disease in several families with FHC [11,12], Clark et al. and van Dorp et al. determined by performing echocardiography on family members of patients with FHC that the pattern of inheritance was autosomal dominant with variable penetrance, and that hypertrophy was present in many asymptomatic relatives of affected individuals [13,14]. At that time, identification of a disease related gene required a priori knowledge of the defective protein which was only known for a small number of diseases, but not for FHC. Since that time, a complete genetic map of the human genome, based on easily identifiable highly polymorphic DNA markers, has been developed 115-171 and this provides the foundation for genetic linkage analysis. This technique makes it possible to map a chromosomal locus responsible for a disease without prior knowledge of the defective protein. Once the chromosomal locus for a genetic disease has been identified by linkage analysis, several techniques can be used to identify the responsible gene. One approach is the candidate gene approach which takes advantage of information available in different databases, regarding the loci of known genes, especially the Human Genome Project. Genes assigned to the region of the mapped locus are assessed as candidates by determining if there are mutations present and whether they are coinherited with the disease, and thus responsible for the disease. Another approach is positional cloning which refers to cloning a segment of DNA knowing only its chromosomal position in relation to a marker. This process of identifying the genes which may require years has been accelerated recently through the development of two techniques, yeast artificial chromosomes (YAC) and pulse field gel electrophoresis (PFGE) [181. Prior to the availability of YAC, one could only clone fragments of DNA up to 45 000 bp, while with YACs, it is possible to clone fragments of up to l-2000 000 bp. Separation of DNA fragments by agarose gel electrophoresis was limited to those of 10000 bp, whereas with PFGE, one can separate fragments up to 2 000 000 bp. Familial hypertrophic cardiomyopathy (FHC) was the first primary cardiomyopathy that succumbed to the application of these techniques. In 1989, Jarcho et al. showed linkage of FHC to the chromosomal locus of 14ql in a large French/Canadian family [19]. Soon after, several families from North America were shown to be linked to the chromosome 14 locus [20]. In the year to follow,

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the pMHC gene located on chromosome 14 was identified as the responsible gene and several mutations were shown to co-segregate with inheritance of the disease suggesting the causality of these mutations for FHC in these families [21,22]. Since, at least 36 mutations in the /? MHC gene have been reported worldwide to cause FHC [21-271 (Table 1). The search is not yet over since many families with FHC are not genetically linked to any of the known loci [28-301. Three other genes have been identified, namely, cardiac troponin T on lq3, (Y tropomyosin on 15q2 and the latest cardiac myosin binding protein-C on llqll [31-331. The true incidence of the mutations in FHC is unknown but it appears that mutations in these genes account for more than 50% of the families with FHC [34]. There has been reports of FHC linked to loci on chromosome 16, and the prealbumin gene on chromosome 18 without identification of the responsible genes [35,36]. Thus, FHC is a genetically heterogeneous disease with regards to both the responsible genes as well as the mutations, but all the genes identified so far code for sarcomeric proteins, the basic contractile unit of the heart. 3. p MHC gene and protein

The pMHC gene is located on chromosome 14, spanning 23 kb and comprising 40 exons. The mRNA is 6008 bp and encodes for a 220 kDa protein [37-391. The PMHC protein is the major contractile protein of the sarcomere and represents approximately 30% of the myocardial protein [40,41]. It is divided into three functional segments referred to as the globular head, hinge region, and the rod segment. The major domains of the protein such as the actin binding site and the ATP binding site are located in the globular head of the myosin molecule. The hinge region flexes during cardiac contraction going from a 90” angle to a 45”. This motion pulls the actin filaments toward the center resulting in shortening (contraction). Thick filaments are formed by rod to rod interbinding of myosin molecules. The rod, a coiled (Y helix, also coils around the rod of another p MHC molecule with the two heads separated and folded backwards on each other to give it a globular shape. One regulatory and one essential myosin light chain molecule bind to each p MHC head to form a hexameric protein called the Sl fragment. Each thick filament of the sarcomere is formed from more than 400 /3MHC molecules bound together by their tails (Fig. 1). The three-dimensional structure of the Sl fragment has been evaluated by atomic resolution after crystallization of the protein [42]. ATP binds to a small groove in the globular head of the myosin molecule which results in detachment of the myosin molecule from actin [43]. The myosin protein, in the presence

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of actin, generates ATPase activity which results in the hydrolysis of ATP to ADP and Pi. Removal of the latter products causes flexion of the hinge region of the myosin molecule displacing the globular head

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over the actin filaments (contraction and cardiac systale). Subsequently, ATP is again taken up into the groove and myosin released from the actin filaments. New techniques developed recently can accurately

1 Mutations in hem patients

Table

BMHC

gene exon

3 3 3 5 5 5 7 8 9 9 13 13 13 14 15 16 16 16 16 16 19 19 20 20 20 20 20 20 21 22 23 23 23 23 23 40 T gene

Exon 8 Exon 9 Exon 9 Exon 11 Exon 11 Exon 14 Intron 15 Exon 16 cu Tropomyosin

gene

Exon 5 Exon 5 Cardiac

Nucleotides

Amino

C,+T C, +T G,+A G,-+A A,-+G C, + G A,-+G G,+A G,+A G,-+A G+T C,+T C,+T T, + G G, + C A,+T A,-+G G’-+A G,+C G, +A C, -+T C, +T C, +T T, + G G, +C G, + A G, +T A,-rC G,+A C, +G G,+A G,+A G,+A G, +A Deletion

Alaz6 = Val Args4 - stop Val’” 3 Ile Thrlz4 = Ile Arg 143= Gin Try’62 - cys Asn’*’ * Lys Asn23” = Ser A% 24y* Gin GUY256- Glu Arg ‘“’ = Gin Arg403 =Lell Arg403 * Trp Arg4’” 3 Cys Phe513 * Cys GIY~*~ = Arg Asps” = Val Asn6”’ = Ser Val’“’ = Met Lys6’5 -Asn Gly’l” - Arg Arg’” a Trp Arg723 * Cys Pro73’ 3 Leu lle73h * Met GIY’~’ - Arg GIY’~’ =) Arg GIY’~’ -Trp Asp”’ * Gly Arg8”’ * His Leuyox =) Val GIu”*~ 3 Lys GluY3” =-aLys GIu”~~ ==aLys Gluy4’ = Lys Gly’y3’-Leu-Asn-Glu-Glu’Y35

Nucleotides

Amino

T-A G+A T-A A GAG G+A G+T G+A C-G

Ile” =) Asn Argy2 - Glu Phe”“Glu A Glu”” Glulh3 * Lys GIuz4j = Asp Loss of 28 terminal amin acids Arg2” = Cys

C2+T

Troponin

MyBP-C

Intron (3’ splice acceptor site) Intron MN (5’ splice donor site) Exon P

65

acids

acids

Nucleotides

Amino

G+A A+G

Asp”” =Asn Glu’*” =) Gly

Nucleotides

Protein

A+G G-C 18 bp tandem duplication

Truncated protein Truncated protein Truncated protein

acids

66

A. B. .4bchee, R. Robem

Sarcomere

e

0

9-

THICK -----FILAMENT

/Progress

0 /

Actin Tropomyosin THIN FILAMENT

SAUCOMEUE

k-

I-;and +A-band

+ M

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ocytes was associated with disarray of the sarcomeres [47]; (5) in vitro motility studies showed impaired contractile function and decreased actin dependent ATPase activity of the mutant protein 1481.

Structure

\

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I-band Z

MYOFIBRIL

Fig. 1. Structure of Sarcomeres. More than ten different proteins form the sarcomere which is the contractile unit of the cardiac muscle. It is composed of thin and thick filaments. The thin filaments contain actin, Troponin T, I and C complex, and atropomyosin whereas the thick filament is formed by tail-to-tail binding of several hundred myosin molecules. MHC, myosin heavy chain; MLC, myosin light chain; TnT, Troponin T TnC, Troponin C; TnI, Troponin I.

quantitate the movement of a single myosin head over an actin filament during contraction [43-451. These measurements indicate that each myosin molecule is capable of generating a force of about 10 pN that result in the displacement of the globular head by approximately 12 nm over the actin filament during each contraction [44,45]. Except for the deletion in exon 40 which codes for the tail of the rod region, all of the mutations in the j3MHC gene are missense mutations located in the first 23 exons which code for the head and hinge region. No single mutation has been found to be predominant in FHC but codon 403 has been considered a hot spot for mutations [25] with three different mutations occurring there, among them the Arg403Gln mutation which is the most commonly reported mutation in FHC. Haplotype analysis performed on different families with the same mutation showed an independent origin of these mutations indicating that they did not arise from a single founder, and that this region in the gene is very susceptible to mutagenesis. A de novo missense mutation was detected in the /3 MHC gene of a patient with sporadic HCM which was transmitted to two subsequent generations in an autosomal dominant pattern [461. This finding provides strong proof of the causality of PMHC mutations for the disease. Other evidence that the pMHC mutations cause FHC include: (1) these mutations are not found in the general population; (2) each affected individual within a single family carry the same mutation which is not present in the normals; (3) the mutation is expressed in the mRNA and protein of the heart of affected patients [461; (4) expression of the mutant human /3 MHC gene with the Arg403Gln mutation in feline adult cardiac my-

4. Troponin T gene and protein

The cardiac troponin T gene is located on chromosome lq3 1311. It is formed of 15 exons and transcribes a mature mRNA of approximately 1.2 kb 1491. There are several isoforms of the cardiac troponin T protein because the gene undergoes alternative splicing [5Ol. Troponin T represents approximately 5% of the cardiac myofibrillar protein, and in association with troponin I and troponin C regulates Ca2+ [51] and binds to tropomyosin to position the troponin complex on the thin filaments. It is estimated that cardiac troponin T gene mutations account for 15% of FHC [52]. Several missense mutations and a deletion have been reported in the cardiac troponin T gene (Table 1). The deletion results from a G + A transition at a 5’ splice donor site in residue 1 of intron 15 and results in a truncated form of troponin T which is 28 amino acids shorter than the full length protein [31]. This truncated protein degrades rapidly and causes a reduced amount of the normal protein in the sarcomere thus altering the stoichiometry of the sarcomeric protein and possibly leading to the phenotype of myofibril disarray and hypertrophy. This is the second deletion reported to cause FHC. The first reported deletion was in the 3’ region of the p MHC gene and is postulated to result in the expression of a truncated unstable message

LN. 5. (Y Tropomyosin gene and protein

(Y Tropomyosin is a rod shaped (Y helix protein which is coiled around the a helix tropomyosin of another molecule to form a dimer, the functional form of the protein. (Y Tropomyosin binds the troponin complex to the thin actin filament. The (Y tropomyosin gene is located on chromosome 15q2. It is formed of 15 exons which are transcribed into a 1 kb mature mRNA [51]. Two missense mutations in exon 5 have been reported (Table 1). Mutations in this gene are responsible for less than 5% of FHC

WI. 6. Cardiac myosin binding protein-C gene and protein

The most recent locus for FHC was identified on chromosome llqll in 1993 [53] and the gene was identified to be human cardiac myosin binding protein-C (MyBP-C). In four unrelated families with FHC linked to the cardiac MyBP-C locus, three mutations

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in the MyBP-C gene were found to cosegregate with the disease [32,33]. Two of these mutations were point mutations in intronic sequences leading to the deactivation of splice sites and resulting in aberrant transcripts. The third mutation was an 18-bp tandem duplication of residues. All these mutations create a frame shift which results in premature termination of translation and as a final product, a truncated cardiac MyBP-C (Table 1) [32,33]. The functions of MyBP-C include but are not limited to: (1) binding to myosin heavy chain and titin; (2) interaction with F-actin and the myosin head to modulate myosin ATPase; and (3) participation in the adrenergic regulation of cardiac contraction. The complete structure-function relationship of MyBP-C has not been established yet but it is known that the myosin heavy chain binds to the highly conserved C terminal of cardiac MyBP-C which is absent in the mutant protein due to its premature termination. 7. Genotype-phenotype correlation hypertrophic cardiomyopathy

in Familial

FHC is phenotypically characterized by cardiac hypertrophy, predominantly in the interventricular septum. However, in different patients the hypertrophy varies markedly in extent and distribution as does the severity of clinical symptoms, the age of onset, and the natural course of the disease not only among families, but within the same family with the same mutation. In some families the onset of disease is late in adulthood, the hypertrophy is minimal and they have a normal lifespan, while other families have a very early onset, massive hypertrophy associated with severe symptoms, and a very short lifespan due to sudden cardiac death. Now, with knowledge of the responsible genes and the ability to detect the underlying genetic defect, we are able to determine whether specific genotypes lead to different phenotypes. In at least five families with the /3 MHC Arg403Gln mutation, there is high penetrance, severe hypertrophy and a high incidence of SCD (up to 50% of affected individuals) [23,54-561. The mean age at the time of SCD was 33 + 15 years. However, a small Korean family with the Arg 4”3Gln mutation has been reported to have a more benign clinical course, where none of the six affected family members had SCD 1571. The variability in the incidence of SCD among families with the same mutation may in part be due to the different genetic background of these families. Two other PMHC mutations, Arg’“Gln and Arg4s3Cys have been associated with a high incidence of SCD. In four families, thirty five of the 61 affected individuals with the Arg”’ Gln mutation have died, 22 from SCD [58]. The average life expectancy in the affected individuals in these four families was 38

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years. In one family with the Arg453Cys mutation, nine of the 13 affected individuals have died, six from SCD [23]. The mean age at the time of death was 30 * 12. On the other hand, some mutations have been associated with a low penetrance, a benign course and a low incidence of SCD. In a large family with FHC, 46 individuals had the LeuyO*Val mutation and only two died [55]. The cumulative survival rate at age 60 was 96%. Similarly, a large family with 39 affected individuals carrying the Gly 256Glu mutation showed that this mutation has a benign prognosis 1571. The cumulative sudden death rate at age 50 in the affected individuals was only 2%. In four families with FHC, the Va1606Met mutation which caused the disease was associated with a benign prognosis [23,54]. However, Fananapazir et al. reported a family with the Va1606Met mutation which is associated with a severe form of the disease and a high incidence of SCD [57]. Genotype phenotype correlations must be interpreted with caution because the number of families having the same mutation is not large enough and because we know that the genetic background of each patient affects the phenotypic expression of the disease. Even though different individuals carrying the same mutation develop variable degrees of hypertrophy, mutations associated with poor prognosis tend to also associate with more left ventricular involvement and greater septal thickness than the more benign mutations. In our preliminary studies of 14 patients with FHC carrying the Arg403Gln mutation which is associated with a poor prognosis, the mean septal thickness measured on echocardiograms was 18.1 mm ). 6.4, whereas the mean septal thickness in nine patients with the benign Va1606Met mutation was 13.3 mm + 3.9 (P = 0.04). Similarly, we found that the left ventricular mass index of patients with the Arg’l’Trp mutation was greater than that of patients with Valm6Met mutation. A larger number of patients need to be studied to verify these preliminary findings and to characterize the diagnostic and prognostic implications of echocardiographic findings in the context of each mutation. 8. Effect of environmental factors and genetic background on the phenotypic expression of fhc mutations

Within the same family, different patients carrying the same mutation have a different phenotype, emphasizing the role of environmental and other genetic factors on the phenotypic expression of FHC. One example of environmental effect on the phenotype is the fact that the p MHC gene is expressed equally in the right and left ventricle but the hypertrophy is

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evident only in the left ventricle, mostly in the septum. This differential expressivity of the gene is suspected to be secondary to the higher systemic pressures on the left side of the heart. As we mentioned earlier, the genetic defect, i.e. the mutation, may predict the phenotype, i.e. degree of hypertrophy and incidence of SCD. But siblings carrying the same mutation have different outcomes, and this may not be due to environmental differences alone. Studies have shown that other genetic factors play a role in the phenotypic expression of FHC. One of these factors is the Angiotensin-1 converting enzyme (ACE) gene. The DD genotype, as opposed to the ID or II genotypes, was associated with increased left ventricular mass. In a study of 120 patients with FHC, we have shown that a left ventricular mass index of 2 100 g/m2 was six times more likely to occur in DD genotype patients as opposed to II genotype patients [59]. The ACE genotypes DD, II and ID, are independent of the genes causing FHC, but in individuals having the same FHC mutation, those who carry the ACE genotype DD are more likely to develop more extensive left ventricular hypertrophy. It is very likely that other genes, like the ACE gene, influence the expressivity of the FHC mutations because the phenotypic expression of hypertrophy requires the coordination of many other genes which control cellular growth and development. 9. Why search for the molecular

defects?

Clinical diagnosis of FHC may come too late for some patients, especially the young athletes, since their initial presentation can be sudden cardiac death. In families known to be affected with FHC, early clinical screening may give the patient a false sense of security because the expression of hypertrophy is age dependent and the hypertrophy may not be evident at the time of testing. With genetic screening, a genetic diagnosis can be made from a blood sample at birth, or even before. Screening for mutations is a tedious and lengthy procedure, but with development of more rapid molecular techniques, genetic screening will become a routine. Once a mutation is detected in a family, the family members can be easily screened. Familial hypertrophic cardiomyopathy is an autosoma1 dominant disease, so half of the offspring will inherit the mutation and therefore will be at risk for developing the disease, while the other half will be normal. Those who do not carry the mutation would be expected to lead a normal life and do not need to refrain from physical activities, while those who are detected with the mutation will require closer medical follow up and lifestyle adjustment. Also knowing the type of mutation and the co-existing genetic background will help to risk-stratify patients for more

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appropriate therapeutic interventions. Patients who carry mutations which place them at higher risk for sudden cardiac death will be evaluated and treated accordingly, while those with low risk of death and normal lifespan will be treated less aggressively. The exact pathogenesis of FHC is not clear, but it is thought that hypertrophy is a compensatory phenomenon to the defective protein. The mutant protein acts as a dominant negative molecule or poison peptide which leads to impaired contractility providing the stimulants for compensatory growth. Each individual carries a normal allele and a mutant allele from the culprit gene, and since the heart renews itself every few weeks, compensatory growth intercedes. So if one could inhibit the defective allele from producing the poison protein, the normal allele will produce the normal protein which could potentially replace the defective protein in the heart and thus regenerate a new and normally functioning heart. This therapeutic approach, while speculative at present is likely to be accomplished in the next few years. For all the reasons stated earlier, it is imperative that the search for all possible culprit genes responsible for FHC continue as well as efforts to screen for all possible mutations and study their clinical significance. Meanwhile, while waiting for such diagnosis and therapy to become routinely available, we must apply our knowledge as best we can to treat our patients and to avoid complications of FHC such as sudden cardiac death. Acknowledgements

We greatly appreciate the secretarial assistance of Esther Yeager in the preparation of this manuscript and figures. This work is supported in part by grants from the National Heart, Lung, and Blood Institute, Specialized Centers of Research (P50-HU2267-00, the National Institutes of Health Training Center in Molecular Cardiology (T32-HLO77061, and the American Heart Association, Bugher Foundation Center for Molecular Biology (86-2216). References 111 Towbin

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[41

J, Roberts R. Cardiovascular diseases due to genetic abnormalities. In: Schlant RC and Alexander RW, eds. Hurst’s The Heart: Arteries and Veins. 8th ed. New York, NY: McGraw Hill, 1994:1725-1759. Roberts WC, Ferrans VJ. In: Kaltenbach M, Epstein SW, eds. Hypertrophic Cardiomyopathy: The Therapeutic Role of Calcium Antagonists. New York, NY: Springer-Verlag. 1982:59 Maron BJ, Roberts WC, Epstein SE. Sudden death in hypertrophic cardiomyopathy: a profile of 78 patients. Circulation 1982;65:1388-1394. McKenna W, Deanfield J, Farugui A, England D, Oakley C, Goodwin J. Prognosis in hypertrophic cardiomyopathy: Role

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of age and clinical electrocardiographic and hemodynamic features. Am J Cardiol 1981;47:532-538. [5] Maron BJ, Epstein SE, Roberts WC. Causes of sudden death in competitive athletes. J Am Coil Cardiol 1986;7:204-214. [6] Maron BJ, Gardin JM, Flack JM, Gidding SS, Bild DE. How common is hypertrophic cardiomyopathy? Echocardiography identified prevalence in a general population of young adults (The CARDIA Study) (abstract). Circulation 1993;88:1-452. [7] Hada Y, Sakamoto T, Amano K et al. Prevalence of hypertrophic cardiomyopathy in a population of adult Japanese workers as detected by echocardiographic screening. Am J Cardiol 1987;59:183-184. [8] Codd MB, Sugrue DD, Gersh BJ, Melton W, III. Epidemiology of idiopathic dilated and hypertrophic cardiomyopathy: A population-based study in Olmsted County, Minnesota, 1975-1984. Circulation 1989;80:564-572. hypertrophy of the heart in young [91 Teare RD. Asymmetrical adults. Br Heart J 1958;20:1-10. cardiaque sous aortique. Gazette [lOI Liouville H. Retrecessment Med Paris 1869;24:161-165. [ill Braunwald E, Lambrew CT, Rockoff SD, Ross J, Jr., Morrow AC. Idiopathic hypertrophic subaortic stenosis: A description of the disease based upon an analysis of 64 patients. Circulation 1964;IV3-IV119. E. Idiopathic hypertrophic subaortic [121 Frank S, Braunwald stenosis. Clinical analysis of 126 patients with emphasis on the natural history. Circulation 1968;37:759-788. prevalence and [131 Clark CE, Henry WL, Epstein SE. Familial genetic transmission of idiopathic hypertrophic subaortic stenosis. N Engl J Med 1973;289:709-714. WC, Ten Cate FJ, Vletter WB, Dohmen H, [141 van Dorp Roelandt J. Familial prevalence of asymmetric septal hypertrophy. Eur J Cardiol 1976;4:349-357. Human Linkage Center (CHLC). A comprehen[ISI Cooperative sive human linkage map with centimorgan density. Science 1994;265:2049-2054. [161 Weissenbach J, Gyapay G, Dib C et al. A second generation linkage map of the human genome. Nature 1992;359:794-801. J. A second generation linkage map of the I171 Weissenbach human genome based on highly informative microsatellite loci. Gene 1994;135:275-278. [I81 Marian AJ, Roberts R. Molecular genetics of hypertrophic cardiomyopathy. Annu Rev Med 1995;46:213-222. W, Pare JAP et al. Mapping a gene for [191 Jarcho JA, McKenna familial hypertrophic cardiomyopathy to chromosome 14ql. N Engl J Med 1989;321:1372-1378. D.01 Hejtmancik JF, Brink PA, Towbin J et al. Localization of the gene for familial hypertrophic cardiomyopathy to chromosome 14ql in a diverse U.S. population. Circulation 1991;83:1592-1597. AA, Kass S, Tanigawa G et al. A molecD.11 Geisterfer-Lawrance ular basis for familial hypertrophic cardiomyopathy: A betacardiac myosin heavy chain gene missense mutation, Cell 1990;62:999-1006. P21 Rosenzweig A, Watkins H, Hwang D et al. Pre-clinical diagnosis of familial hypertrophic cardiomyopathy by genetic analysis of blood lymphocytes. N Engl J Med 1991;325:1753-1760. P31 Watkins H, Rosenzweig A, Hwang D et al. Characteristics and prognostic implications of myosin missense mutations in familial hypertrophic cardiomyopathy. N Engl J Med 1992;326:1108-1114. AJ, Yu QT, Mares A. Jr., Hill R, Roberts R, Perry[241 Marian man MB. Detection of a new mutation in the P-myosin heavy chain gene in an individual with hypertrophic cardiomyopathy. J Clin Invest 1992;90:2156-2165. [251 Dausse E. Komajda M, Fetler L et al. Familial hypertrophic

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[27]

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[31]

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]40]

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