J Mol
Cell
Cardiol
24,
The
1471-1477
(1992)
Genetic
Basis
Eleanor
Elstein,
The Centre for Cardiovascular (Received 17 April E. ELSTEIN,
Choong-Chin
Cardiomyopathy Liew,
Research, Universi&
Michael
of Toronto,
1992, accepted in revisedform
J. Sole
Toronto, Ontario,
Canada
13 August 1992)
M. J. SOLE. The Genetic Basis of Hypertrophic Cardiomyopathy. Journal of (1992) 24, 1471-1477. In this article we review the techniques of molecular biology as they apply to the elucidation of the genetic basis of hypertrophic cardiomypathy. We review the evidence for linkage to chromosome 14 and the specific mutations described to date. The evidence for genetic heterogeneity is presented. We speculate on the pathophysiology of the disease from the perspective of the known molecular defects and review the clinical implications the evolving information may have.
Molecular
CHOONG-CHIN
of Hypertrophic
LIEW,
and Cellular Cardiology
KEY WORDS:
Hypertrophic
cardiomyopathy;
Genetics;
Introduction Hypertrophic cardiomyopathy (HCM) is characterized by abnormal left ventricular thickening in the absence of overt causes of hypertrophy. The predominant site of involvement is the left ventricular septum, however it is well recognized that right ventricular involvement may occur in apparent isolation or in association with left sided involvement. In addition other types of hypertrophy including midventricular, apical or concentric hypertrophy may also constitute the disease. Pathologically, the disease is characterized by myocardial fibre disarray. This myofiber disorganization may be fairly extensive with more than 5% of the ventricular septum being affected in more than 90% of HCM patients [I]. It is generally agreed that this feature is not specific for HCM and can be seen in other conditions resulting from pressure overload. Myofiber disarray can also be seen in normal hearts, but in such cases less than 1% of the septum is involved [I]. The myocardium is also characterized by extensive scarring and a marked increase in, and disorganization of, interstitial and intercellular matrix connective tissue [2]. This work Professorship Please Toronto
was supported and Dr Elstein,
address Hospital,
0022-2828/92/121471+07
by Heart a Research
all correspondence EN 13-208, 200 008.00/O
and
Stroke Fellowship
Molecular
biology;
Mutations.
The clinical presentation of HCM is variable and depends among other things on the extent and severity of the morphologic abnormalities, the presence or absence of a left ventricular outflow tract gradient and the age of the patient. The natural history of HCM is also extremely variable ranging from asymptomatic to rapidly progressive fatal disease. The annual mortality rate is 3-5% [3]. Most deaths are sudden; indeed, HCM is the most common cause of sudden death in young athletes. In this paper we will review current advances in the understanding of the genetic basis of HCM with particular emphasis on the use of molecular biological techniques to study the genetics of this disease. Genetic linkage HCM has an incidence of 3 per 10 000 [4]; The disease may present in a sporadic or hereditary form. The condition appears to affect males twice as often as females [5j. Despite this observation there is no evidence for x-linked inheritance [.5j. Early family studies using only M-mode echocardiography
Foundation of Ontario. Dr from the Heart and Stroke
to: Michael J. Sole, Director, The Centre Elizabeth Street, Toronto, Ontario, Canada,
Sole holds Foundation
a Distinguished of Ontario.
Research
for Cardiovascular M5G 2C4
Research,
0 1992
Press
Academic
The
Limited
1472
E. Elstein
suggested that most patients with HCM had a genetic defect that was transmitted as an autosomal dominant trait with a high degree of penetrance [6, 7]. Two larger studies using two dimensional echocardiography found that HCM was genetically transmitted in approximately 55% of families [5, 81. Based on these studies 45% of cases would appear to be sporadic. Some authors maintain that sporadic cases do not in fact exist and that these cases represent problems in case identification and family history documentation. One study has demonstrated that the frequency with which HCM was identified in first degree relatives of index cases increased with the number of first degree relatives studied [S]. It has also been argued that in some studies where only M-mode echocardiography was used the various phenotypic presentations of HCM were not appreciated due to technical limitations and that this led to false conclusions. The diagnosis of HCM by echocardiography can be influenced by age. Patients may require years to manifest overt hypertrophy and younger individuals may be misclassified. In family screening studies, more cases of HCM were detected in parents of probands than in siblings. More cases were also found in siblings than in offspring of index cases [!?I. Early studies into the genetics of HCM using microcytotoxicity methods addressed the possible association of the disease with the major histocompatibility genes found on chromosome 6. Matsumori et al. [9] showed no significant association between either the HLA-A or HLA-B antigens and HCM. They did however demonstrate an association between the HLA-DRW4 antigen and the obstructive form of HCM. They were unable to show any relation with the nonobstructive form of the disease. Other groups have not found an association between any of the HLA antigens and HCM [IQ]. Given the lack of consensus with regard to an association between HCM and the major histocompatibility loci, investigators began searching other chromosomes for linkage. Ferraro [II], studying the karyotypes of an Italian family with HCM reported that a fragile site on the long arm of chromosome 16 segregated with the disease. Nishi [12] et al. reported linkage to chromosome 18 in several Japanese
et al.
families. It appeared even from this early data that HCM would likely be polygenic. With the advances made in molecular biology, the study of the genetics of HCM took on a different approach. Mapping of the disease locus to a specific region of the human genome using cosegregation analysis became possible using Southern blot analysis of restriction fragment length polymorphisms (RFLP) . Restriction enzymes recognize and cleave specific base pair sequences on DNA. Since differences in human DNA sequences exist between homologous chromosomes every 50-200 base pairs, restriction enzyme sites will vary. These variations represent the RFLP. The resulting DNA fragments of restriction enzyme digestion will thus be of variable length. Radiolabelled chromosomal markers called probes are hybridized with homologous regions on the cleaved DNA and after autoradiography yield a pattern of visible bands making up the different alleles of a RFLP. Each allele of a RFLP is inherited in an autosomal codominant manner and when an individual carries two different alleles of that marker he is said to be heterozygous and the marker is considered to be informative. The alleles can be analyzed in relation to the presence or absence of disease and their inheritance followed through generations in a family. If the RFLP allele and the disease locus segregate independently of each other, one would expect coinheritance approximately 50% of the time. If the observed frequency of coinheritance in a given family is greater than expected, linkage is suggested. Statistical analysis is then performed to determine the likelihood that the suggested linkage is real. The odds of linkage is the ratio of the likelihood that the observed coinheritance of the RFLP and disease would occur if the marker and disease were linked, to the likelihood that the coinheritance would occur if the marker and disease were not linked. The logarithm of this ratio represents the lod score. A lod score above + 3 indicates that the observed data are lOOO-fold more likely to occur if the loci are linked and is generally accepted as evidence for linkage. A lod score less than - 2 is generally accepted as proof of no linkage for a pair of loci. The lod score is related to the accuracy of the diagnosis of the disease, the number of individuals studied and
The
Genetic
Basis
of Hypertrophic
the informativeness of the probes selected. Using these techniques, Jarcho et al. [13] reported in one pedigree of a French Canadian family, a DNA locus (D14S26) on the long arm of chromosome 14 (14q) to be coinherited with HCM (FHC-I ) yielding a lod score of +9.37. This data indicated that the odds were greater than 1 x 10’: 1 that the gene responsible for HCM was located on chromosome 14 in the family studied. Using chromosomal in situ hybridization, wherein the same probe for locus D14S26 was hybridized to normal human chromosomes isolated from peripheral lymphocytes in metaphase, the investigators were able to physically map the locus D14S26 to chromosome 14 at 14qll [14]. Other genes assigned to this chromosomal location include the a and /I cardiac myosin heavy chain (MyHC) genes. Indeed, finer mapping using linkage analysis showed that locus D14S26 and the locus for the B cardiac MyHC gene are only 2.2 CM apart. As myosin is a protein of major structural and functional importance to the myocyte, it was a natural extension of the initial work to see if the disease locus might also link to the cardiac MyHC genes. Linkage analysis with a cardiac p MyHC gene probe yielded a lod score of 4.62 favouring linkage of HCM and the cardiac MyHC genes [14]. As logical as it would appear that a cardiac muscle disease might be linked to the loci of cardiac MyHC genes, these loci did not appear to be associated with the disease in all families. Solomon et al. [15] reported that only two of four families showed linkage to cardiac myosin. Epstein et al. [1q have recently shown that three of five families they tested do not link to chromosome 14 and the /3 MyHC gene. Schwartz et al. [17] reported on nine affected families of which two showed definite non linkage to chromosome 14 and while the other seven proved non informative. Ko et al. [18] has reported no linkage with chromosome 14 in a Chinese family with HCM. Approximately one half of cases of HCM studied to date are not accounted for by abnormalities in cardiac myosin heavy chain genes. The case for genetic heterogeneity seems to be well established. Studies are ongoing, but to date no other linkage has been identified.
Cardiomyopathy
1473
SpeciJic mutations Given the linkage of HCM to the cardiac MyHC genes in at least some families a direct investigation of the coding sequence of these genes was undertaken to identify any mutations which could account for HCM. Work from our laboratory had already provided detailed sequence information on the normal human CI and /3 cardiac MyHC genes [19]. Each cardiac MyHC gene consists of 40 exons encompassing 25 kb of DNA. There is an additional 4.1 kb which separates the two genes. Direct sequencing and comparison of the a and p MyHC gene sequences in both normal and affected individuals would clearly be painstaking. If however a mutation alters a restriction enzyme site one could identify this segment of DNA by Southern blot analysis and then sequence only this segment. Using this latter approach, Tanigawa [ZO] and Geisterfer-Lowrance [21] working in the same laboratory, each identified a mutation in the p MyHC gene of HCM. Tanigawa identified a unique 2.8 kb BamHl restriction enzyme fragment which always segregated with the disease in an American family of Northern European descent. Using other restriction enzymes this group also identified several rearrangements and duplications in affected individuals of this family. Analysis of these fragments revealed that the FHC-1 allele consisted of a hybrid gene of the a and fi cardiac MyHC genes. The transition zone occurred in exon 27. Geisterfer-Lowrance, working on the same French Canadian family of Jarcho’s initial linkage studies, identified another polymorphic allele of 385bp present in Ddel restriction enzyme digests of genomic DNA. The nucleotide sequence of this allele was determined and revealed a one base pair change from a guanine to an adenine residue. This mutation in exon 13 of the /? MyHC gene converts an arginine amino acid encoded by CGG to a glutamine amino acid encoded by CAG at amino acid residue 403 of the myosin heavy chain. Other laboratories including ours have undertaken studies to see if these mutations are responsible for HCM in other families. We and others have used the method of polymerase chain reaction (PCR) gene amplification to study this problem. The method
1474
E. Elstein
involves the use of a thermostable TAQDNA polymerase enzyme which catalyses the synthesis of a segment of DNA complementary to a target strand of interest in the presence of a short complementary DNA sequence primer to produce millions of copies in just a few hours. Using specifically designed primers surrounding exon 13 we used PCR to amplify the exon 13 DNA sequence in affected individuals. We subsequently sequenced this fragment and compared it to that known for the normal p MyHC gene. We have demonstrated that the exon 13 point mutation as well as the fusion gene defect previously described are not responsible for HCM in a large Canadian kindred [ZZ]. To date we have studied another eight smaller affected Canadian families unrelated to the family studied by Geisterfer-Lowrance and have shown that the exon 13 base pair change is absent in these families. Perryman et al. [13] have also shown that the exon 13 defect is uncommon having been found in only one of 39 families. It has subsequently been suggested that the a//? cardiac MyHC fusion gene defect is likely not responsible for HCM. Affected members of the family reported by Tanigawa to harbor the fusion gene also possessed a missense mutation in Exon 14 [24]. This missense mutation, resulting in a single amino acid substitution, appeared sufficient to account for HCM in another unrelated family with phenotypically similar disease. This substitution therefore was probably the significant defect. Most mutations of clinical significance occur within exons. Those occurring within introns do not usually cause disease. Therefore to screen the /3 MyHC gene for other mutations would require an independent analysis of each of its forty exons. ~Direct use of messenger RNA (mRNA) species would be more efficient than use of DNA, as the intron sequences have been spliced. This would permit larger fragments containing a number of exons to be analyzed at one time and allow more efficient gene amplification by PCR than does genomic DNA. The largest quantity of cardiac /3 MyHC mRNA is found in the heart and slow twitch skeletal muscles. It has been shown however that cardiac MyHC mRNA also exists in small quantities in, and can be isolated from, peripheral blood lym-
et al.
phocytes. This enables one to obtain cardiac MyHC mRNA by simple venipuncture and obviates the need for tissue. The technique of RNase protection can be applied to mRNA obtained in this fashion to identify mutations of MyHC genes in HCM [24]. The technique involves the reverse ‘transcription of cardiac MyHC mRNA into complementary DNA which is then amplified by PCR to obtain larger quantities of double stranded DNA. These fragments are then hybridized with RNA probes of cardiac MyHC derived from normal individuals and subjected to RNAse treatment. This enzyme cleaves non-hybridized sequences and thus cleaves areas of mismatch between the normal and affected sequences. These cleaved products can then be visualized on a polyacrylamide gel and subsequently sequenced. RNase does not detect all nucleotide mismatches with equal affinity however and thus some mutations may be missed by this technique. In addition mutations that do not affect the coding sequence and mutations in genes other than the one targeted would not be identified. Using all the above techniques other point mutations have been identified in the p MyHC gene of individuals with HCM [251. These mutations are in exons 9, 14, 16, 17 and 23 which are all clustered in the globular head region of the MyHC molecule. It is in this region that the ATPase activity and major actin binding sites reside. Molecular
pathophysiology
It is not yet known how the mutations in the cardiac B MyHC gene produce cardiac hypertrophy in HCM. Two experimental models of myosin gene defects are known which might shed light on the pathogenesis of HCM. It is possible that a critical number of a and /I MyHC peptides are required for normal muscle function. The mutations in the /I MyHC gene may cause a relative increase in the a MyHC molecule and result in abnormal muscle architecture and dysfunction. This is known to occur in an analogous situation in Drosophila wherein a heterozygous mutation in either an actin or a MyHC gene results in an imbalance in the amounts of actin and myosin produced resulting in a flightless fly [26J. Double heterozygotes on the other hand
The Genetic
Basis of Hypertrophic
have nearly normal flight muscles since the ratio of the two muscle proteins is preserved. This model must be viewed with caution, in that although hypertrophy in the animal model results in a shift from LX to p MyHC predominance [27], altered expression of the CI and p cardiac MyHC genes has not to date been demonstrated in humans [28, ,291. In the nematode Caenorhabditis elegans, mutations in the uric-54 gene which code for myosin heavy chains result in abnormal myofibrillar assembly and result in impaired mobility of the worm [30]. It is possible in HCM that molecular interactions of b MyHC molecules is interfered with by the mutant condition resulting in abnormal muscle function. Furthermore ATPase activity and the interaction of actin with myosin may be impaired. To date however no mutations in humans with HCM have been described in the ATPase or the MyHC binding domains. One can postulate that the abnormal myosin does not itself cause hypertrophy but rather that the hypertrophy may be a compensatory attempt to normalize abnormal wall tensions resulting from the abnormal myosin. It is known that most of the mutations thus far described in the p MyHC gene cause an electrical charge change in the resulting peptide. One can speculate that the resulting charge change may influence nearby molecules and cause charge changes in the surrounding microenvironment leading to abnormal alignment of fibres. This could perhaps account for the patchy myofibrillar disarray seen in HCM. The charge change could also lead to abnormal ion movement and hence abnormal muscle function and perhaps contribute to the electrical abnormalities seen in this disease. Clinical implications The advances made in the molecular genetic identification of HCM will likely help in dealing with the limitations of the clinical and echocardiographic diagnosis of HCM. It is clear that clinical manifestations of the disease may be variable and clinical findings may be difficult to elicit. Greaves et al. [S] have shown that approximately 25% of first degree relatives of proband cases have normal clinical and ECG findings despite having an echocardio-
Cardiomyopathy
1475
graphic diagnosis of HCM. The diagnosis of HCM may only become manifest in the late teens or early adulthood, thus children may initially be misdiagnosed. We have shown in a recent study of 128 family members of a proband case of HCM that 8.6% of individuals at risk could not be clearly classified as affected or unaffected and hence fall into a borderline category [31]. The identification of an abnormal genotype in these situations would remove the ambiguity. In our large family study we also identified an affected individual whose parents did not have criteria for HCM. This suggests an obligate carrier state. Epstein et al. [32] have also reported obligate carrier states in four families they studied, including an identical twin of an affected individual. Most of these carriers demonstrated abnormal signal averaged ECG’s. The question of the dissociation between the electrical instability and echocardiographically demonstrable LVH in HCM has been raised. The ability to trace genotypes in families should help clarify such issues as non-penetrance, incomplete penetrance and somatic mutations. There is some preliminary data suggesting that the mutations causing a charge change in the myosin heavy chain peptide are associated with a disease with worse prognosis than are mutations not associated with a charge change. The identification of genotype will permit the study of the natural history of various mutations and will allow the evaluation of varied therapeutic interventions. Conclusion The application of molecular biology to cardiology has allowed major advances to be made in the understanding of the genetic basis of HCM. To date linkage to chromosome 14qll and specific mutations in the cardiac a MyHC gene have been shown in some affected families. In other families the responsible mutation lies elsewhere in the genome. The first steps towards understanding the molecular pathophysiology of this protean disease have been taken. We anticipate that advances made over the next few years will have substantive impact upon diagnosis, assessment of prognosis and ultimately on the treatment of the disease.
1476
E. Elstein
et
al.
References 1 MARON, B., ROBERTS W. Quantitative analysis of cardiac muscle cell disorganization in the ventricular septum of patients with hypertrophic cardiomyopathy. Circulation 59, 689-706 (1979). 2 FACTOR, S. M., BUTANY, J., SOLE, M. J,, WIGLE, E. D., WILLIAMS, WM. C., ROJKIND, M. Pathologic fibrosis and matrix connective tissue in the subaortic myocardium of patients with hypertrophic cardiomyopathy. J Am Co11 Cardiol 17, 43-51 (1991). 3 WIGLE, E. D. Hypertrophic cardiomyopathy. Modem Concepts of Cardiovascular Disease 57, 1-6 (1988). 4 BJARNSON, I., JONSSON, S., HARDARSON, T. Mode of inheritance of hypcrtrophic cardiomyopathy in Iceland. Echocardiographic study. Br Heart J 47, 122-129 (1982). 5 MARON, B., NICHOLS, P., PICKLE, L., WESLEY, Y., MULVIHILL, J, Patterns of inheritance in hypertrophic cardiomyopathy: Assessment by M-mode and 2-Dimensional echocardiography. Am J Cardiol 53, 1087-1094 (1984). 6 CLARK, C., HENRY, W., EPSTEIN, S. Familial prevalence and genetic transmission of idiopathic hypertrophic subaortic stenosis. NEJM 289, 709-714 (1973). 7 VAN DORP, W., TENGATE, F., VLETTER, W., DOHMAN, H., ROELANDT, J. Familial prevalence of asymmetric septal hypertrophy. Eur J Cardiol 4, 349-357 (1976). 8 GREAVES, S., ROCHE, A., NEUTZE, J., WHITLOCK, R., VEALE, A. Inheritance of hypertrophic cardiomyopathy: A cross sectional and M-mode echocardiographic study of 50 families. Br Heart J 58, 259-266 (1987). 9 MATSUMORI, A., KAWAI, C., WAKABAYSHI, A., et al. HLA-DRW4 antigen linkage in patients with hypertrophic obstructive cardiomyopathy. Am Heart J 101, 14-16 (1981). 10 ZEZULKA, A., MACKINTOSH, P., JOBSON, S., LOWRY, P., SHAPIRO, L. Human lymphocyte antigens in hypertrophic cardiomyopathy. Int J Cardiol 12, 193-202 (1986). 11 FERRARO, M., SCARTON, G., AMBROSINI, M. Cosegregation of hypertrophic cardiomyopathy and a fragile site on chromosome 16 in a large Italian family. J Med Genet 27, 363-366 (1990). 12 NISHI, H., KIMURA, A., SASAKI, M., et al. Localization of the gene for hypertrophic cardiomyopathy to chromosome 18q. (Abstract). Circ 80 (Suppl II), II-457 (1989). 13 JARCHO, J., MCKENNA, W., PARE, P. et al. Mapping a gene for familial hypertrophic cardiomyopathy to chromosome 14ql. NEJM 321, 1372-1378 (1989). 14 SOLOMON S., GEISTERFER-LOWARANCE, A., VOSBERG, H. et al. A locus for familial hypertrophic cardiomypathy is closely linked to the cardiac MHC genes CRI-L436 and CRI-329 on chromosome 14 at ql l-q12. Am J Hum Genet 47, 389-394 (1990). 15 SOLOMON, S., JARCHO, J., MCKENNA, W. et al. Familial hypertrophic cardiomyopathy is a genetically heterogeneous disease. J Clin Invest 86, 993-999. (1990). 16 EPSTEIN, N., FANANAZAPIR, L., LIN, H. et al. Evidence of genetic heterogeneity in five kindreds with hypertrophic cardiomypathy. Circ ~0182, 6355642 (1992). 17 SCHWARTZ, K., DUFOUR, C., FAURE, L. et al. Exclusion of myosin heavy chain and cardiac actin gene involvement in hypertrophic cardiomyopathy of several French families, (Abstract). Circ 84, Suppl II, II-398 (1991). 18 Ko, Y. L., LIEN, W. P., CHEN, J. J., et al. No evidence for linkage between a Chinese family with familial hypertrophic cardiomyopathy and chromosome 14q 1 locus Dl4S26: Evidence for genetic heterogeneity. Human Genetics 1992 (in press). 19 LIEW, C. C., SOLE, M., YAMAUCHI-TAKIHARA et al. Complete sequence and organization of the human cardiac myosin heavy chain gene. Nucl Acids Res 18, 3647-3651 (1990). 20 TANIGAWA, G., JARCHO, J., KASS, S. et al. A molecular basis for familial hypertrophic cardiomyopathy: An a/B cardiac myosin heavy chain hybrid gene. Cell 62, 991-998 (1990). 21 GEISTERFER-LOWRANGE, A., KASS, S., TANIGAWA, G. sl al. A Molecular basis for familial hypertrophic cardiomyopathy: A /? cardiac myosin heavy chain gene missense mutation. Cell 62 99991006 (1990). 22 ELSTEIN, E., PERRYMAN, B., RAKOWSKI, H. et al. Genetic heterogeity for familial hypertrophic cardiomyopathy in Canada. (Abstract). Molecular and Cellular Biologv of the Cardiac Mvocvte. November 1991. 23 PERRYMA;, B., MARES, A., HEJTMANCIK, F., Gooc~y’G., ROBERTS, R. The fimyosin heavy chain missense mutation in Exon 13, a putative defect for hypertrophic cardiomyopathy is present in only one of thirty-nine affected families. (Abstract). Circ 84 (Suppl II), II-418 (1991). A., WATKINS, H., HWANG, D. S. etal. Preclinical diagnosis of familial hypertrophic cardiomyopathy 24 ROSENZWEIG, by genetic analysis of blood lymphocytes. NEJM 325, 175363 (1991). 25 WATKINS, H., HWANG, D., SEIDMAN, J., SEIDMAN, C., MCKENNA, W. Diversity of myosin heavy chain mutations which cause familial hypertrophic cardiomyopathy. (Abstract). Eur J Cardiol I2 (Suppl), (1991). 26 BEALL, C., SEPANSKI, M., FYRBERG, E. Genetic dissection of drosophila myofibril formation: Effects of actin and myosin heavy chain null alleles. Genes Dev 3 131-140 (1989). A., MATSUOKA, R. et al. Myosin heavy chain mRNA and protein isoform transitions during 27 IZUMO, S., LOMPRE, cardiac hypertrophy. J Clin Invest 79, 970-977 (1987). 28 MERCADIER, J., BOUVERET, P., GORZA. etal. Myosin isoenzymes in normal and hypertrophied human ventricular myocardium. Circ Res 53, 52262 ( 1983). R. Structural and enzymatic comparison of human cardiac muscle myosins isolated from 29 SCHIER, J., ADELSTEIN, infants, adults and patients with hypertrophic cardiomyopathy. J Clin Invest 69, 816-825 (1982).
The
Genetic
Basis
of Hypertrophic
Cardiomyopathy
1477
30 DIBB, N., BROWN, D., KARN, J. et al. Sequence analysis of mutations that affect the synthesis, assembly and enzymatic activity of the uric-54 Myosin heavy chain gene ofcaenorhabditis elegans. J Mel Bial183,543-5 1 ( 1985). 3 1 ELSTEIN, E., WIGLE, E. D., RAKOWSKI, H., SOLE, M. The incidence of borderline hypertrophy in a large Canadian family with hypertrophic cardiomyopathy. Can J Cardiol 7 (Suppl A) 11, 115A (1991). 32 EPSTEIN, N., LIN, H., FANANAPAZIR, L. Genetic evidence of dissociation (generational skips) of electrical from morphologic forms of hypertrophic cardiomyopathy. Am J Cardiol 66, 627-63 1 (1990).
Cell
Cardiol
24,
The
1471-1477
(1992)
Genetic
Basis
Eleanor
Elstein,
The Centre for Cardiovascular (Received 17 April E. ELSTEIN,
Choong-Chin
Cardiomyopathy Liew,
Research, Universi&
Michael
of Toronto,
1992, accepted in revisedform
J. Sole
Toronto, Ontario,
Canada
13 August 1992)
M. J. SOLE. The Genetic Basis of Hypertrophic Cardiomyopathy. Journal of (1992) 24, 1471-1477. In this article we review the techniques of molecular biology as they apply to the elucidation of the genetic basis of hypertrophic cardiomypathy. We review the evidence for linkage to chromosome 14 and the specific mutations described to date. The evidence for genetic heterogeneity is presented. We speculate on the pathophysiology of the disease from the perspective of the known molecular defects and review the clinical implications the evolving information may have.
Molecular
CHOONG-CHIN
of Hypertrophic
LIEW,
and Cellular Cardiology
KEY WORDS:
Hypertrophic
cardiomyopathy;
Genetics;
Introduction Hypertrophic cardiomyopathy (HCM) is characterized by abnormal left ventricular thickening in the absence of overt causes of hypertrophy. The predominant site of involvement is the left ventricular septum, however it is well recognized that right ventricular involvement may occur in apparent isolation or in association with left sided involvement. In addition other types of hypertrophy including midventricular, apical or concentric hypertrophy may also constitute the disease. Pathologically, the disease is characterized by myocardial fibre disarray. This myofiber disorganization may be fairly extensive with more than 5% of the ventricular septum being affected in more than 90% of HCM patients [I]. It is generally agreed that this feature is not specific for HCM and can be seen in other conditions resulting from pressure overload. Myofiber disarray can also be seen in normal hearts, but in such cases less than 1% of the septum is involved [I]. The myocardium is also characterized by extensive scarring and a marked increase in, and disorganization of, interstitial and intercellular matrix connective tissue [2]. This work Professorship Please Toronto
was supported and Dr Elstein,
address Hospital,
0022-2828/92/121471+07
by Heart a Research
all correspondence EN 13-208, 200 008.00/O
and
Stroke Fellowship
Molecular
biology;
Mutations.
The clinical presentation of HCM is variable and depends among other things on the extent and severity of the morphologic abnormalities, the presence or absence of a left ventricular outflow tract gradient and the age of the patient. The natural history of HCM is also extremely variable ranging from asymptomatic to rapidly progressive fatal disease. The annual mortality rate is 3-5% [3]. Most deaths are sudden; indeed, HCM is the most common cause of sudden death in young athletes. In this paper we will review current advances in the understanding of the genetic basis of HCM with particular emphasis on the use of molecular biological techniques to study the genetics of this disease. Genetic linkage HCM has an incidence of 3 per 10 000 [4]; The disease may present in a sporadic or hereditary form. The condition appears to affect males twice as often as females [5j. Despite this observation there is no evidence for x-linked inheritance [.5j. Early family studies using only M-mode echocardiography
Foundation of Ontario. Dr from the Heart and Stroke
to: Michael J. Sole, Director, The Centre Elizabeth Street, Toronto, Ontario, Canada,
Sole holds Foundation
a Distinguished of Ontario.
Research
for Cardiovascular M5G 2C4
Research,
0 1992
Press
Academic
The
Limited
1472
E. Elstein
suggested that most patients with HCM had a genetic defect that was transmitted as an autosomal dominant trait with a high degree of penetrance [6, 7]. Two larger studies using two dimensional echocardiography found that HCM was genetically transmitted in approximately 55% of families [5, 81. Based on these studies 45% of cases would appear to be sporadic. Some authors maintain that sporadic cases do not in fact exist and that these cases represent problems in case identification and family history documentation. One study has demonstrated that the frequency with which HCM was identified in first degree relatives of index cases increased with the number of first degree relatives studied [S]. It has also been argued that in some studies where only M-mode echocardiography was used the various phenotypic presentations of HCM were not appreciated due to technical limitations and that this led to false conclusions. The diagnosis of HCM by echocardiography can be influenced by age. Patients may require years to manifest overt hypertrophy and younger individuals may be misclassified. In family screening studies, more cases of HCM were detected in parents of probands than in siblings. More cases were also found in siblings than in offspring of index cases [!?I. Early studies into the genetics of HCM using microcytotoxicity methods addressed the possible association of the disease with the major histocompatibility genes found on chromosome 6. Matsumori et al. [9] showed no significant association between either the HLA-A or HLA-B antigens and HCM. They did however demonstrate an association between the HLA-DRW4 antigen and the obstructive form of HCM. They were unable to show any relation with the nonobstructive form of the disease. Other groups have not found an association between any of the HLA antigens and HCM [IQ]. Given the lack of consensus with regard to an association between HCM and the major histocompatibility loci, investigators began searching other chromosomes for linkage. Ferraro [II], studying the karyotypes of an Italian family with HCM reported that a fragile site on the long arm of chromosome 16 segregated with the disease. Nishi [12] et al. reported linkage to chromosome 18 in several Japanese
et al.
families. It appeared even from this early data that HCM would likely be polygenic. With the advances made in molecular biology, the study of the genetics of HCM took on a different approach. Mapping of the disease locus to a specific region of the human genome using cosegregation analysis became possible using Southern blot analysis of restriction fragment length polymorphisms (RFLP) . Restriction enzymes recognize and cleave specific base pair sequences on DNA. Since differences in human DNA sequences exist between homologous chromosomes every 50-200 base pairs, restriction enzyme sites will vary. These variations represent the RFLP. The resulting DNA fragments of restriction enzyme digestion will thus be of variable length. Radiolabelled chromosomal markers called probes are hybridized with homologous regions on the cleaved DNA and after autoradiography yield a pattern of visible bands making up the different alleles of a RFLP. Each allele of a RFLP is inherited in an autosomal codominant manner and when an individual carries two different alleles of that marker he is said to be heterozygous and the marker is considered to be informative. The alleles can be analyzed in relation to the presence or absence of disease and their inheritance followed through generations in a family. If the RFLP allele and the disease locus segregate independently of each other, one would expect coinheritance approximately 50% of the time. If the observed frequency of coinheritance in a given family is greater than expected, linkage is suggested. Statistical analysis is then performed to determine the likelihood that the suggested linkage is real. The odds of linkage is the ratio of the likelihood that the observed coinheritance of the RFLP and disease would occur if the marker and disease were linked, to the likelihood that the coinheritance would occur if the marker and disease were not linked. The logarithm of this ratio represents the lod score. A lod score above + 3 indicates that the observed data are lOOO-fold more likely to occur if the loci are linked and is generally accepted as evidence for linkage. A lod score less than - 2 is generally accepted as proof of no linkage for a pair of loci. The lod score is related to the accuracy of the diagnosis of the disease, the number of individuals studied and
The
Genetic
Basis
of Hypertrophic
the informativeness of the probes selected. Using these techniques, Jarcho et al. [13] reported in one pedigree of a French Canadian family, a DNA locus (D14S26) on the long arm of chromosome 14 (14q) to be coinherited with HCM (FHC-I ) yielding a lod score of +9.37. This data indicated that the odds were greater than 1 x 10’: 1 that the gene responsible for HCM was located on chromosome 14 in the family studied. Using chromosomal in situ hybridization, wherein the same probe for locus D14S26 was hybridized to normal human chromosomes isolated from peripheral lymphocytes in metaphase, the investigators were able to physically map the locus D14S26 to chromosome 14 at 14qll [14]. Other genes assigned to this chromosomal location include the a and /I cardiac myosin heavy chain (MyHC) genes. Indeed, finer mapping using linkage analysis showed that locus D14S26 and the locus for the B cardiac MyHC gene are only 2.2 CM apart. As myosin is a protein of major structural and functional importance to the myocyte, it was a natural extension of the initial work to see if the disease locus might also link to the cardiac MyHC genes. Linkage analysis with a cardiac p MyHC gene probe yielded a lod score of 4.62 favouring linkage of HCM and the cardiac MyHC genes [14]. As logical as it would appear that a cardiac muscle disease might be linked to the loci of cardiac MyHC genes, these loci did not appear to be associated with the disease in all families. Solomon et al. [15] reported that only two of four families showed linkage to cardiac myosin. Epstein et al. [1q have recently shown that three of five families they tested do not link to chromosome 14 and the /3 MyHC gene. Schwartz et al. [17] reported on nine affected families of which two showed definite non linkage to chromosome 14 and while the other seven proved non informative. Ko et al. [18] has reported no linkage with chromosome 14 in a Chinese family with HCM. Approximately one half of cases of HCM studied to date are not accounted for by abnormalities in cardiac myosin heavy chain genes. The case for genetic heterogeneity seems to be well established. Studies are ongoing, but to date no other linkage has been identified.
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SpeciJic mutations Given the linkage of HCM to the cardiac MyHC genes in at least some families a direct investigation of the coding sequence of these genes was undertaken to identify any mutations which could account for HCM. Work from our laboratory had already provided detailed sequence information on the normal human CI and /3 cardiac MyHC genes [19]. Each cardiac MyHC gene consists of 40 exons encompassing 25 kb of DNA. There is an additional 4.1 kb which separates the two genes. Direct sequencing and comparison of the a and p MyHC gene sequences in both normal and affected individuals would clearly be painstaking. If however a mutation alters a restriction enzyme site one could identify this segment of DNA by Southern blot analysis and then sequence only this segment. Using this latter approach, Tanigawa [ZO] and Geisterfer-Lowrance [21] working in the same laboratory, each identified a mutation in the p MyHC gene of HCM. Tanigawa identified a unique 2.8 kb BamHl restriction enzyme fragment which always segregated with the disease in an American family of Northern European descent. Using other restriction enzymes this group also identified several rearrangements and duplications in affected individuals of this family. Analysis of these fragments revealed that the FHC-1 allele consisted of a hybrid gene of the a and fi cardiac MyHC genes. The transition zone occurred in exon 27. Geisterfer-Lowrance, working on the same French Canadian family of Jarcho’s initial linkage studies, identified another polymorphic allele of 385bp present in Ddel restriction enzyme digests of genomic DNA. The nucleotide sequence of this allele was determined and revealed a one base pair change from a guanine to an adenine residue. This mutation in exon 13 of the /? MyHC gene converts an arginine amino acid encoded by CGG to a glutamine amino acid encoded by CAG at amino acid residue 403 of the myosin heavy chain. Other laboratories including ours have undertaken studies to see if these mutations are responsible for HCM in other families. We and others have used the method of polymerase chain reaction (PCR) gene amplification to study this problem. The method
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involves the use of a thermostable TAQDNA polymerase enzyme which catalyses the synthesis of a segment of DNA complementary to a target strand of interest in the presence of a short complementary DNA sequence primer to produce millions of copies in just a few hours. Using specifically designed primers surrounding exon 13 we used PCR to amplify the exon 13 DNA sequence in affected individuals. We subsequently sequenced this fragment and compared it to that known for the normal p MyHC gene. We have demonstrated that the exon 13 point mutation as well as the fusion gene defect previously described are not responsible for HCM in a large Canadian kindred [ZZ]. To date we have studied another eight smaller affected Canadian families unrelated to the family studied by Geisterfer-Lowrance and have shown that the exon 13 base pair change is absent in these families. Perryman et al. [13] have also shown that the exon 13 defect is uncommon having been found in only one of 39 families. It has subsequently been suggested that the a//? cardiac MyHC fusion gene defect is likely not responsible for HCM. Affected members of the family reported by Tanigawa to harbor the fusion gene also possessed a missense mutation in Exon 14 [24]. This missense mutation, resulting in a single amino acid substitution, appeared sufficient to account for HCM in another unrelated family with phenotypically similar disease. This substitution therefore was probably the significant defect. Most mutations of clinical significance occur within exons. Those occurring within introns do not usually cause disease. Therefore to screen the /3 MyHC gene for other mutations would require an independent analysis of each of its forty exons. ~Direct use of messenger RNA (mRNA) species would be more efficient than use of DNA, as the intron sequences have been spliced. This would permit larger fragments containing a number of exons to be analyzed at one time and allow more efficient gene amplification by PCR than does genomic DNA. The largest quantity of cardiac /3 MyHC mRNA is found in the heart and slow twitch skeletal muscles. It has been shown however that cardiac MyHC mRNA also exists in small quantities in, and can be isolated from, peripheral blood lym-
et al.
phocytes. This enables one to obtain cardiac MyHC mRNA by simple venipuncture and obviates the need for tissue. The technique of RNase protection can be applied to mRNA obtained in this fashion to identify mutations of MyHC genes in HCM [24]. The technique involves the reverse ‘transcription of cardiac MyHC mRNA into complementary DNA which is then amplified by PCR to obtain larger quantities of double stranded DNA. These fragments are then hybridized with RNA probes of cardiac MyHC derived from normal individuals and subjected to RNAse treatment. This enzyme cleaves non-hybridized sequences and thus cleaves areas of mismatch between the normal and affected sequences. These cleaved products can then be visualized on a polyacrylamide gel and subsequently sequenced. RNase does not detect all nucleotide mismatches with equal affinity however and thus some mutations may be missed by this technique. In addition mutations that do not affect the coding sequence and mutations in genes other than the one targeted would not be identified. Using all the above techniques other point mutations have been identified in the p MyHC gene of individuals with HCM [251. These mutations are in exons 9, 14, 16, 17 and 23 which are all clustered in the globular head region of the MyHC molecule. It is in this region that the ATPase activity and major actin binding sites reside. Molecular
pathophysiology
It is not yet known how the mutations in the cardiac B MyHC gene produce cardiac hypertrophy in HCM. Two experimental models of myosin gene defects are known which might shed light on the pathogenesis of HCM. It is possible that a critical number of a and /I MyHC peptides are required for normal muscle function. The mutations in the /I MyHC gene may cause a relative increase in the a MyHC molecule and result in abnormal muscle architecture and dysfunction. This is known to occur in an analogous situation in Drosophila wherein a heterozygous mutation in either an actin or a MyHC gene results in an imbalance in the amounts of actin and myosin produced resulting in a flightless fly [26J. Double heterozygotes on the other hand
The Genetic
Basis of Hypertrophic
have nearly normal flight muscles since the ratio of the two muscle proteins is preserved. This model must be viewed with caution, in that although hypertrophy in the animal model results in a shift from LX to p MyHC predominance [27], altered expression of the CI and p cardiac MyHC genes has not to date been demonstrated in humans [28, ,291. In the nematode Caenorhabditis elegans, mutations in the uric-54 gene which code for myosin heavy chains result in abnormal myofibrillar assembly and result in impaired mobility of the worm [30]. It is possible in HCM that molecular interactions of b MyHC molecules is interfered with by the mutant condition resulting in abnormal muscle function. Furthermore ATPase activity and the interaction of actin with myosin may be impaired. To date however no mutations in humans with HCM have been described in the ATPase or the MyHC binding domains. One can postulate that the abnormal myosin does not itself cause hypertrophy but rather that the hypertrophy may be a compensatory attempt to normalize abnormal wall tensions resulting from the abnormal myosin. It is known that most of the mutations thus far described in the p MyHC gene cause an electrical charge change in the resulting peptide. One can speculate that the resulting charge change may influence nearby molecules and cause charge changes in the surrounding microenvironment leading to abnormal alignment of fibres. This could perhaps account for the patchy myofibrillar disarray seen in HCM. The charge change could also lead to abnormal ion movement and hence abnormal muscle function and perhaps contribute to the electrical abnormalities seen in this disease. Clinical implications The advances made in the molecular genetic identification of HCM will likely help in dealing with the limitations of the clinical and echocardiographic diagnosis of HCM. It is clear that clinical manifestations of the disease may be variable and clinical findings may be difficult to elicit. Greaves et al. [S] have shown that approximately 25% of first degree relatives of proband cases have normal clinical and ECG findings despite having an echocardio-
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graphic diagnosis of HCM. The diagnosis of HCM may only become manifest in the late teens or early adulthood, thus children may initially be misdiagnosed. We have shown in a recent study of 128 family members of a proband case of HCM that 8.6% of individuals at risk could not be clearly classified as affected or unaffected and hence fall into a borderline category [31]. The identification of an abnormal genotype in these situations would remove the ambiguity. In our large family study we also identified an affected individual whose parents did not have criteria for HCM. This suggests an obligate carrier state. Epstein et al. [32] have also reported obligate carrier states in four families they studied, including an identical twin of an affected individual. Most of these carriers demonstrated abnormal signal averaged ECG’s. The question of the dissociation between the electrical instability and echocardiographically demonstrable LVH in HCM has been raised. The ability to trace genotypes in families should help clarify such issues as non-penetrance, incomplete penetrance and somatic mutations. There is some preliminary data suggesting that the mutations causing a charge change in the myosin heavy chain peptide are associated with a disease with worse prognosis than are mutations not associated with a charge change. The identification of genotype will permit the study of the natural history of various mutations and will allow the evaluation of varied therapeutic interventions. Conclusion The application of molecular biology to cardiology has allowed major advances to be made in the understanding of the genetic basis of HCM. To date linkage to chromosome 14qll and specific mutations in the cardiac a MyHC gene have been shown in some affected families. In other families the responsible mutation lies elsewhere in the genome. The first steps towards understanding the molecular pathophysiology of this protean disease have been taken. We anticipate that advances made over the next few years will have substantive impact upon diagnosis, assessment of prognosis and ultimately on the treatment of the disease.
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Familial prevalence and genetic transmission of idiopathic hypertrophic subaortic stenosis. NEJM 289, 709-714 (1973). 7 VAN DORP, W., TENGATE, F., VLETTER, W., DOHMAN, H., ROELANDT, J. Familial prevalence of asymmetric septal hypertrophy. Eur J Cardiol 4, 349-357 (1976). 8 GREAVES, S., ROCHE, A., NEUTZE, J., WHITLOCK, R., VEALE, A. Inheritance of hypertrophic cardiomyopathy: A cross sectional and M-mode echocardiographic study of 50 families. Br Heart J 58, 259-266 (1987). 9 MATSUMORI, A., KAWAI, C., WAKABAYSHI, A., et al. HLA-DRW4 antigen linkage in patients with hypertrophic obstructive cardiomyopathy. Am Heart J 101, 14-16 (1981). 10 ZEZULKA, A., MACKINTOSH, P., JOBSON, S., LOWRY, P., SHAPIRO, L. Human lymphocyte antigens in hypertrophic cardiomyopathy. Int J Cardiol 12, 193-202 (1986). 11 FERRARO, M., SCARTON, G., AMBROSINI, M. Cosegregation of hypertrophic cardiomyopathy and a fragile site on chromosome 16 in a large Italian family. 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Exclusion of myosin heavy chain and cardiac actin gene involvement in hypertrophic cardiomyopathy of several French families, (Abstract). Circ 84, Suppl II, II-398 (1991). 18 Ko, Y. L., LIEN, W. P., CHEN, J. J., et al. No evidence for linkage between a Chinese family with familial hypertrophic cardiomyopathy and chromosome 14q 1 locus Dl4S26: Evidence for genetic heterogeneity. Human Genetics 1992 (in press). 19 LIEW, C. C., SOLE, M., YAMAUCHI-TAKIHARA et al. Complete sequence and organization of the human cardiac myosin heavy chain gene. Nucl Acids Res 18, 3647-3651 (1990). 20 TANIGAWA, G., JARCHO, J., KASS, S. et al. A molecular basis for familial hypertrophic cardiomyopathy: An a/B cardiac myosin heavy chain hybrid gene. Cell 62, 991-998 (1990). 21 GEISTERFER-LOWRANGE, A., KASS, S., TANIGAWA, G. sl al. A Molecular basis for familial hypertrophic cardiomyopathy: A /? cardiac myosin heavy chain gene missense mutation. Cell 62 99991006 (1990). 22 ELSTEIN, E., PERRYMAN, B., RAKOWSKI, H. et al. Genetic heterogeity for familial hypertrophic cardiomyopathy in Canada. (Abstract). Molecular and Cellular Biologv of the Cardiac Mvocvte. November 1991. 23 PERRYMA;, B., MARES, A., HEJTMANCIK, F., Gooc~y’G., ROBERTS, R. The fimyosin heavy chain missense mutation in Exon 13, a putative defect for hypertrophic cardiomyopathy is present in only one of thirty-nine affected families. (Abstract). Circ 84 (Suppl II), II-418 (1991). A., WATKINS, H., HWANG, D. S. etal. Preclinical diagnosis of familial hypertrophic cardiomyopathy 24 ROSENZWEIG, by genetic analysis of blood lymphocytes. NEJM 325, 175363 (1991). 25 WATKINS, H., HWANG, D., SEIDMAN, J., SEIDMAN, C., MCKENNA, W. Diversity of myosin heavy chain mutations which cause familial hypertrophic cardiomyopathy. (Abstract). Eur J Cardiol I2 (Suppl), (1991). 26 BEALL, C., SEPANSKI, M., FYRBERG, E. Genetic dissection of drosophila myofibril formation: Effects of actin and myosin heavy chain null alleles. Genes Dev 3 131-140 (1989). A., MATSUOKA, R. et al. Myosin heavy chain mRNA and protein isoform transitions during 27 IZUMO, S., LOMPRE, cardiac hypertrophy. J Clin Invest 79, 970-977 (1987). 28 MERCADIER, J., BOUVERET, P., GORZA. etal. Myosin isoenzymes in normal and hypertrophied human ventricular myocardium. Circ Res 53, 52262 ( 1983). R. Structural and enzymatic comparison of human cardiac muscle myosins isolated from 29 SCHIER, J., ADELSTEIN, infants, adults and patients with hypertrophic cardiomyopathy. J Clin Invest 69, 816-825 (1982).
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30 DIBB, N., BROWN, D., KARN, J. et al. Sequence analysis of mutations that affect the synthesis, assembly and enzymatic activity of the uric-54 Myosin heavy chain gene ofcaenorhabditis elegans. J Mel Bial183,543-5 1 ( 1985). 3 1 ELSTEIN, E., WIGLE, E. D., RAKOWSKI, H., SOLE, M. The incidence of borderline hypertrophy in a large Canadian family with hypertrophic cardiomyopathy. Can J Cardiol 7 (Suppl A) 11, 115A (1991). 32 EPSTEIN, N., LIN, H., FANANAPAZIR, L. Genetic evidence of dissociation (generational skips) of electrical from morphologic forms of hypertrophic cardiomyopathy. Am J Cardiol 66, 627-63 1 (1990).
