Circulation. 1995;92:1336-1347
(Circulation. 1995;92:1336-1347.)
© 1995 American Heart Association, Inc.
Recent Advances in the Molecular Genetics of Hypertrophic Cardiomyopathy
A.J. Marian, MD;
R. Roberts, MD
From the Department of Medicine, Section of Cardiology, Baylor College of
Medicine, Houston, Tex.
Correspondence to Robert Roberts, MD, Section of Cardiology, Baylor
College of Medicine, 6535 Fannin, MS F905, Houston, TX 77030.
Key Words: genetics molecular biology hypertrophy cardiomyopathy
 |
Introduction
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The recent evolution of
molecular genetic techniques and their
application in deciphering the
molecular genetic basis of inherited
diseases have facilitated the
dawning of molecular medicine.
A complete genetic map of the human
genome, based on easily
identifiable highly polymorphic DNA
markers, has been developed.
1 2 3 This
achievement is a
milestone in that it provides the
foundation for genetic linkage
analysis, a technique essential
to the mapping of the
chromosomal locus responsible for a disease.
Before the development of
multiple informative markers, identification
of a disease-related
gene required a priori knowledge of the
defective protein, which was
known for only a few diseases.
The previous approach of "from a
defective protein to a defective
gene" has been replaced with the
approach of "from a defective
gene to a defective protein." The
recent availability of the
highly informative STRP markers compared
with previous markers
based on RFLP has greatly accelerated chromosomal
mapping by
linkage analysis.
4 Furthermore, the
RFLP markers detected by
Southern blotting required 5 to 7 days,
whereas STRP markers
are detected by PCR and require as few as 1 to 2
days.
4 5 The
loci for more than 400 disease-related
genes have been mapped,
and the responsible genes have been identified
for more than
40 of these diseases.
1 Theoretically, it is
possible to map
the chromosomal locus of any disease-related gene
if a family
with 10 or more living affected individuals spanning two or
more
generations is available.
4 5 Subsequent to the
chromosomal
mapping of the locus by genetic linkage analysis,
several techniques,
such as positional cloning, are used to identify
the responsible
gene.
6 7 Positional cloning refers to
cloning of a segment
of DNA with only its chromosomal position in
relation to a marker
known. This process of identifying the genes,
which may require
years, has been accelerated recently through the
development
of two techniques: YAC and PFGE.
8 Before the
availability of
YAC, one could clone fragments of DNA only as large as
45 000
bp, whereas with YAC, it is possible to clone fragments as
large
as 1 to 2 million bp. Separation of DNA fragments by agarose
gel
electrophoresis was limited to those of

10 000 bp, whereas
with
PFGE, fragments as large as 2 million bp can be separated.
HCM was the first primary cardiomyopathy that was
subjected to these techniques. During the short period of 4 years,
three genes and a fourth locus responsible for this disease have been
identified.9 10 11 12 In
addition, structure-function
analysis has shed significant light on the molecular basis of
this disease. It is hoped that within the next few years the
application of molecular genetic tools will not only facilitate the
ability to diagnose HCM but also help to stratify and develop more
definitive therapy.
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Clinical Epidemiology
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Familial HCM is inherited as an autosomal dominant disease that
is
characterized by hypertrophy, often of the left ventricle,
with
predominant involvement of the interventricular
septum in the
absence of other causes of hypertrophy, such
as hypertension
or valvular heart disease.
13 The
predominant cardiac pathology
is myocyte hypertrophy and
sarcomere disarray; the former is
found in most cardiac diseases,
whereas the latter is the hallmark
of HCM.
14 The
ventricular systolic function is usually normal
or
supernormal, with an average left ventricular ejection
fraction
of 65% to 70%. However, ventricular
diastolic function often
is impaired in patients with HCM.
The clinical manifestations
of HCM are diverse, ranging from a benign
asymptomatic course
to severe heart failure and
SCD.
13 SCD is a well-recognized
outcome of HCM. The
annual incidence of SCD is higher in younger
patients with HCM (6%)
than in the elderly (1%).
15 16 17 In
young,
apparently
healthy athletes with HCM, SCD often is the first
manifestation of the
disease.
15 16 17 In a study of 29 highly
conditioned
young
(<35 years old) athletes who died suddenly, HCM was
present at
autopsy in 48% of the cases, and idiopathic left
ventricular
hypertrophy (probably HCM) was
present in an additional 18%
of the cases.
17
The true prevalence of HCM remains unknown. Clinical
diagnostic criteria probably underestimate the prevalence
of the disease as the phenotypic expression of the disease (ie,
development of hypertrophy) is age
dependent.18 The presence of concomitant diseases such as
hypertension and valvular heart disease also confounds the
diagnosis of HCM.19 Several investigators have estimated
the prevalence of the disease to be approximately 0.1 to 1 per 1000
population.20 21 22 23 24
The prevalence of the disease is higher
in older individuals and in those with an abnormal
ECG.23 24 In a study of 3607 men (452 men with ECG
abnormalities and 3155 men with no ECG abnormalities), the overall
prevalence of HCM was 1.1% in the study group, 0.8% in those with no
ECG abnormalities, and 3.6% in those with ECG
abnormalities.23 This probably reflects the fact that HCM
is often associated with ECG abnormalities.
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Genetic Basis of HCM
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The disease was first described in the 19th
century,
25 but
it was not until 1958 that Teare described
the familial inheritance
of HCM.
26 Braunwald et al in
1964
27 and Frank et al in 1968
28 described
several families with HCM, and they further delineated
its familial
nature. Clark et al
29 and van Dorp et al
30
performed
routine echocardiography on all family
members of patients with
HCM and showed that the pattern of inheritance
of HCM was autosomal
dominant with a high but variable degree of
penetrance. Echocardiographic
screening of the families
in their studies showed that cardiac
hypertrophy was
present in many asymptomatic relatives of the
affected
individuals.
29 The data, indicating that 55% of
the cases of
HCM are familial and the remainder are sporadic, may have
to
be reevaluated once genetic screening becomes routinely
available.
31 32 Essentially, HCM is always a genetic
disorder. When de novo
mutations occur, the disease may not be
transmitted to offspring
in a small family as each offspring has only a
50% chance of
inheriting the mutation. Thus, despite being an
underlying genetic
cause of HCM, the disease may not exhibit familial
inheritance.
It is clear that many ß-
MHC mutations occur
spontaneously
and have an independent origin.
33
In 1989, Jarcho et al34 applied linkage analysis
to a large French Canadian family and showed linkage of the disease to
the chromosomal locus of 14q1. Through similar studies, Hejtmancik et
al35 showed that the chromosome 14 locus was linked to HCM
in several families from North America. The ß-MHC gene was
identified as the responsible gene, and several mutations were shown to
co-segregate with inheritance of the disease, suggesting a causal
role for ß-MHC in these families as well as confirming an
autosomal dominant pattern of inheritance.36 37 The
first
mutation identified was that of a missense mutation in which A
substituted for G in exon 13, resulting in a change in the coding of
arginine for glutamine.36 Subsequently, at least 36
mutations in the ß-MHC gene have been shown to be
responsible for HCM, and these mutations have been reported
worldwide in families with
HCM5 36 37 38 39 40 41
(Table 1
).
Several families, however, have been identified who did not show
linkage to chromosome 14q1.42 43 44 Two
new chromosomal loci
responsible for HCM have been mapped9 10 to 1q3 and
15q2,
and the responsible genes are cardiac troponin T and
-tropomyosin,12 respectively (Table
2
). A fourth locus has been mapped, 11q11, but the gene
has not been identified.11 Mutations in the cardiac
troponin T and the
-tropomyosin genes have been
identified that co-segregate with inheritance of the
disease12 (Table 1
). It appears that the mutations
in the
ß-MHC gene occur in approximately 20% to 30% of the
families with HCM.45 However, the true incidence of
ß-MHC mutations and of mutations in cardiac troponin
T,
-tropomyosin, and additional genes remains
unknown. Furthermore, linkage to a fragile site on chromosome 16 and
linkage to the prealbumin gene on chromosome 18 have been
reported without identification of the responsible
genes.46 47 Thus, HCM exhibits genetic
heterogeneity with regard to both the number of
responsible genes and the number of mutations. It is highly likely that
many genes responsible for HCM remain to be identified.
 |
ß-MHC Protein and Its Role in the Sarcomere
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The ß-
MHC gene on chromosome 14 is separated from the

-
MHC gene by only 3.5 kb DNA.
48 It is
composed of 40 exons that
span approximately 23 kb of the
genome.
49 50 The mature ß-
MHC mRNA is
6008 bp
and encodes for a protein with a molecular weight
of 220 kD. The
ß-MHC is the contractile protein of the
heart with enzymatic activity
that in the presence of actin
hydrolyzes ATP to ADP and P
i.
The ß-MHC protein is divided
into three functional segments referred
to as the globular head,
hinge region, and rod segment. The first 23
exons of the ß-
MHC gene encode for the globular head and
hinge regions of the protein,
whereas the remaining exons encode for
the rod portion of the
ß-MHC protein. The globular head of the myosin
molecule
contains the major domains of the protein, such as actin and
ATP-binding
sites. The hinge region flexes during cardiac contraction,
contracting
from a 90° angle to a 45° angle, which, in turn, pulls
the
actin filaments toward the center, resulting in shortening
(contraction).
The rod is essential for tail-to-tail
interbinding of the myosin
molecules that form the thick filament. The
rod, a coiled

helix,
also coils around the rod of another ß-MHC
molecule, with
the two heads separated and folded backward onto each
other
to form a globular shape. Attached to each ß-MHC head
are one
regulatory and one essential myosin light chain molecule,
which
together form a hexameric protein. Each thick filament
of the sarcomere
is formed from more than 400 ß-MHC molecules
bound together by their
tails (Fig 1

).

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Figure 1. Structure of sarcomeres. The sarcomere is the
contractile unit of cardiac muscle, composed of thin and thick
filaments encompassed by Z bands. More than 10 different sarcomeric
proteins have been identified. Thick filaments are formed by
tail-to-tail binding of several hundred myosin molecules,
whereas thin filaments are composed of actin, troponin T, I and C
complex, and -tropomyosin.
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The ß-MHC protein is the major contractile protein of the sarcomere
and makes up approximately 30% of the myocardial
protein.51 52 It is the predominant protein in the
cardiac
ventricles of large mammals as it is in the adult human heart,
comprising more than 95% of the myosin in the human
ventricles.51 52 The three-dimensional structure of
the S1 fragment of the myosin molecule (globular head and myosin light
chains) has been determined from atomic resolution after the molecule
was crystallized.53 ATP binds to a small groove in the
globular head of the myosin molecule, which results in detachment of
the myosin molecule from actin.54 Enzymatic activity of
the myosin ATPase results in hydrolysis of ATP to ADP and
Pi, and with removal of the latter end products,
flexion of the hinge region of the myosin molecule occurs that
displaces the globular head over the actin filaments (contraction and
cardiac systole). Subsequently, ATP is again taken up into the groove,
and myosin is again released from the actin filaments (Fig 2
).
New techniques have been developed that can
accurately quantify the movement of a single myosin head over an actin
filament during contraction.54 55 56
These data indicate that
each myosin molecule is capable of generating a force of
10 pN,
which results in approximately 12-nm displacement of the globular head
over the actin filament during each contraction.55 56

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Figure 2. Diagram illustrating the actin-myosin
interaction of cardiac contractility through one cycle of systole and
diastole. Globular head of the myosin molecule contains a
nucleotide-binding cleft, where an ATP molecule binds to a myosin.
The latter results in conformational changes in the ATP binding site,
which is transmitted to a stiff "level arm." The level arm of the
myosin is composed of a long helix surrounded by regulatory and
essential myosin light chains. Enzymatic activity of the myosin
hydrolyzes ATP to ADP and Pi, which results in
rebounding of the globular head and tight binding to actin filament.
Strong binding of the myosin to actin results in reopening of the ATP
binding site and release of ADP, which induces conformational changes
in the level arm and displaces the globular head over actin filament
(systole). A new ATP molecule occupies the now reopened ATP binding
site and detaches the myosin molecule from the actin filament
(diastole).
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ß-MHC Gene Mutations
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A large number of mutations in the ß-
MHC gene have
been
identified and shown to co-segregate with inheritance of the
disease.
5 36 37 38 39 40 41
All of the mutations of the
ß-
MHC gene
except one are missense mutations located
within the first 23
exons that encode for the globular head of the
ß-MHC protein.
Although Arg
403Gln (G1208A in exon 13) is
the most commonly
described mutation in families with HCM and the 403
codon is
considered a hot spot for mutations,
40 no single
mutation appears
to be predominant in HCM. The majority of the
ß-
MHC mutations
involve replacement of guanine or
cytosine nucleotides, as expected,
and involve an
evolutionary conserved amino acid. Only one mutation
encoding for the
rod region has been described
39 ; this was
a deletion
mutation that deletes part of intron 39, exon 40,
and the entire 3'
untranslated region.
39 In the ß-MHC
protein, the
deletion mutation results in deletion of the last
five amino acids in
the C-terminus. The C-terminus of the ß-MHC
protein is essential for
tail-to-tail interbinding of the myosin
molecules to form the
thick filament of the sarcomere. Haplotype
analysis of families
with HCM who have identical mutations has
indicated an independent
origin of the ß-
MHC mutations
in these
families
33 rather than arising from a single founder.
This
indicates that the ß-
MHC gene is highly vulnerable
to
mutagenesis, and one would expect to find many new mutations
arising
sporadically.
 |
Cardiac Troponin T and -Tropomyosin
Genes and Proteins
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The
cardiac troponin T gene, which is located on
chromosome
1q3,
12 is composed of 15 exons and transcribes
a mature mRNA
of approximately 1.2 kb.
57 The
cardiac
troponin T gene undergoes
multiple alternative splicing, resulting
in several isoforms
of
cardiac troponin T.
58
Expression of
cardiac troponin T is
developmentally
regulated.
58 Cardiac troponin T comprises
approximately
5% of the total myofibrillar protein and, along with
troponin I and
troponin C, is involved in
Ca
2+ regulation.
59 Troponin
T binds to tropomyosin and is responsible for positioning of the
troponin
complex on the thin filaments.
Thus far, seven missense mutations and a deletion mutation in the
cardiac troponin T gene have been identified in patients
with HCM.12 60 The missense mutations are located in
exons
8, 9, 11, 14, and 16. The deletion mutation is a 5' splice donor site
G
A transition in residue 1 of intron 15, which is expected to
produce a truncated cardiac troponin T with loss of the
terminal 28 amino acid residues. The 5' splice donor mutation is likely
to function as a null allele and result in synthesis of a truncated
troponin T that degrades rapidly. A decreased quantity of
stable cardiac troponin T is likely to alter the
stoichiometry of the sarcomeric proteins, which could be responsible
for the phenotype of HCM. The truncated cardiac troponin
T mutation is the second deletion mutation described in families
with HCM.12 39 The first deletion mutation described
in a
family with HCM was a deletion mutation in the 3' region of the
ß-MHC gene, which is also postulated to result in
expression of a truncated unstable message.39 These
mutations in the cardiac troponin T gene appear to be
uncommon mutations in families with HCM. In addition, screening for
cardiac troponin T gene mutations in families with HCM
indicates that such mutations account for HCM in 15% of affected
families.60
-Tropomyosin protein is a rod-shaped sarcomeric protein that
comprises approximately 5% of the total myofibrillar
protein.59 Each
-tropomyosin binds to another
molecule in an
helix coil, forming a dimer, which is the functional
form of the protein. The function of
-tropomyosin is to
bridge the binding of the troponin protein complex to thin actin
filament.59 The gene for
-tropomyosin is
located on chromosome 15q2 and is composed of 15 exons with the
corresponding mature mRNA of
1 kb.59 Two missense
mutations have been described in exon 5 of the
-tropomyosin gene in affected individuals from families
with HCM. The first mutation is due to substitution of adenine for
guanine at position 579, which alters the amino acid sequence from
aspartic acid to asparagine at position 175
(Asp175Asn).12 The second missense mutation is
an A
G substitution at position 595, which changes the glutamic acid
residue at amino acid 180 to a glycine residue. Fewer than 5% of cases
of HCM are caused by mutations in
-tropomyosin
gene.60
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Evidence That ß-MHC Mutations Are Responsible
for HCM
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The evidence that mutations in the ß-
MHC gene are
responsible
for the disease in families with the mutation is
conclusive:
(1) multiple families have been shown to be genetically
linked
to the ß-
MHC locus on chromosome 14; (2) DNA
analysis
shows that each affected individual within a single
family has
the same mutation, which is not present in the
unaffected individuals;
(3) ß-
MHC mutations are absent in
the general population;
and (4) ß-
MHC mutations are
expressed in the cardiac mRNA
and protein of individuals affected with
HCM. Perryman et al
61 showed that the
Arg
403Gln mutation was expressed in the mRNA
extracted from
the myocardium of a patient with HCM, and Greve
et
al
62 showed expression of Arg
741Lys in the
mRNA and protein
in the explanted heart of a patient with sporadic HCM.
Yu et
al
63 showed expression of the ß-
MHC
mutation Arg
403Gln
in mRNA extracted from skeletal muscle,
and Cuda et al
64 65 isolated several different mutant
ß-MHC proteins from
skeletal muscle of patients with HCM. (5) De novo
mutations
occurring in the ß-
MHC gene have been shown to
result
in transmittal of the disease to offspring. Greve et
al
62 showed
that the mutation Arg
741Lys, which
was proved by haplotyping
to occur de novo, was transmitted to two
subsequent generations
and induced the disease in an autosomal dominant
manner. Furthermore,
the explanted heart from the proband showed
expression of the
mutation in the cardiac mRNA and protein. These
studies provide
conclusive evidence that ß-
MHC mutations
are responsible
for HCM in these families. In addition, a de novo
mutation in

-tropomyosin that causes HCM has also been
reported.
66 (6)
In vitro motility studies (discussed
later) show the mutant
ß-MHC protein to have impaired contractile
function and
decreased actin-dependent ATPase
activity.
67 (7) Expression
of the mutant human
ß-
MHC gene with the Arg
403Gln mutation
in
feline adult cardiac myocytes was associated with disarray
of the
sarcomeres
68 (discussed later).
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Genotype-Phenotype Correlation in HCM
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The consistent phenotype of patients with HCM is
cardiac hypertrophy,
which predominantly affects the
interventricular septum. However,
the degree of
hypertrophy, its distribution, patient age at
onset, and
type and severity of clinical manifestations vary
markedly. The natural
course of HCM in certain families is riddled
with SCD, whereas in
others SCD is almost absent, and the life
span is essentially
normal.
69 Identification of the underlying
genetic defect
provides the opportunity to relate phenotype
to specific
genotypes. Results of these studies indicate
70
that
in the majority of families described, the ß-
MHC
mutations
Arg
403Gln, Arg
453Cys, and
Arg
719Trp are associated with a poor
prognosis and a high
incidence of
SCD.
38 71 72 73 74 75
In
contrast, the
ß-
MHC mutation Leu
908Val is associated with
a
near-normal life expectancy.
74 In addition, the
mutations
Glu
930Lys and Arg
249Gln are
associated with an intermediary
risk of SCD.
38 75
The phenotypes associated with Arg403Gln have been
described in several families with HCM and are characterized by high
penetrance, a high incidence of SCD, and severe
hypertrophy.38 72 73 In the majority of
families described, the incidence of premature death in affected
individuals with Arg403Gln mutations is approximately
50%.75 We identified two families with the
Arg403Gln mutation in whom 11 of the 20 affected
individuals died prematurely9 from SCD.75 The mean age
at the time of SCD was 33 years. There also was an early onset of
symptoms, a high penetrance, and a high prevalence of ECG and
echocardiographic abnormalities. Watkins et
al38 observed a high incidence of SCD in two families with
the Arg403Gln mutation in whom 21 of 44 affected
individuals died prematurely9 from SCD, at a mean age of 33±15
years. A third group of investigators reported two families with HCM
who had the Arg403Gln mutation.73 74 In
both
families, there was a high penetrance and a high incidence of
myocardial ischemia, but the survival rates of affected
individuals in these two families were different. In the white family,
SCD occurred in 6 of the 15 affected individuals between the ages of 19
and 45 years, resulting in a cumulative SCD rate at 40 years of age of
45%. In contrast, in the Korean family with the Arg403Gln
mutation, none of the 6 affected individuals had died. The variability
in the incidence of SCD in two families with the identical mutation may
in part reflect the different genetic background of these families or
the small size of the Korean family, which precludes meaningful
assessment of survival rate.
A second ß-MHC mutation associated with a high incidence
of SCD is Arg719Gln, which has been described in four
families composed of 61 individuals.76 Thirty-five of
the 61 affected individuals with Arg719Gln mutation have
died22 from SCD.71 The life expectancy of the affected
individuals in these four families was 38 years. The
Arg453Cys mutation is another malignant mutation that has
been described in one family in whom 9 of the 13 affected individuals
have died6 from SCD.38 The mean age at the time of death
of the affected individuals was 30±12 years.
The Leu908Val mutation has been associated with a low
penetrance, a benign course, and a low incidence of SCD.73
The genotype-phenotype correlation was described in
a large family with HCM composed of 46 genotype-positive
individuals, only 2 of whom have died.73 The cumulative
survival rate at 60 years of age was 92%. Similarly, the
Gly256Glu mutation has been associated with a benign
prognosis in a large family with HCM composed of 39 affected
individuals.74 The cumulative SCD rate at age 50 in the
affected individuals with the Gly256Glu mutation was only
2%. The Val606Met mutation has also been associated with a
benign prognosis in four families with HCM.38 72
Watkins
et al38 reported the Val606Met mutation in
three small families composed of 18 affected individuals, 1 of whom has
died. We identified the Val606Met mutation in a family with
HCM composed of 12 affected individuals, only 1 of whom has
died.72 However, Fananapazir et al74 reported
a family with the Val606Met mutation in whom there is a
severe form of the disease and a high incidence of SCD. These
correlations must be interpreted with caution as the number of families
studied remains too small for definitive conclusions to be made.
Two mutations have been characterized as being associated with an
intermediary prognosis: the Glu930Lys and
Arg249Gln mutations.38 75 We described a
family composed of 16 affected individuals with Glu930Lys
mutation 2 of whom have died at ages 14 and 16 and 1 had undergone
cardiac transplantation due to progressive heart failure at age 58. The
ß-MHC mutation Arg249Gln has been described in
a family composed of 26 affected individuals, 10 of whom have died4
from SCD.38 The average age of the affected individuals at
the time of death was 49 years.38
Echocardiography is the most sensitive and specific
clinical method of diagnosing HCM. The diagnosis is based on
echocardiographic detection of cardiac
hypertrophy in the absence of other causes for
hypertrophy, such as hypertension or valvular
disease.35 However, hypertrophy is not usually
evident before puberty and, when the penetrance is low, may not develop
until middle age.18 20 39 In individuals
with other causes
for hypertrophy or in the elderly, the
echocardiographic detection of hypertrophy
is inconclusive, and only a genetic diagnosis is definitive. Mutations
with low penetrance will characteristically provide families with many
individuals who have the defective genotype but no disease that
can be detected with the use of echocardiography.
In a study by Solomon et al,32 a considerable overlap was
present in the left ventricular wall thickness between
genotype-positive individuals and
genotype-negative individuals. Thus, in these situations,
echocardiography is inadequate for the diagnosis of
HCM.32 Individuals with the same HCM genotype have
a broad spectrum of echocardiographic findings, but the
presence and extent of hypertrophy on the echocardiogram
are reflective of clinical severity. Mutations that are associated with
high penetrance and poor prognosis are more likely to show a greater
degree of left ventricular hypertrophy or
septal thickness than are those associated with low penetrance and good
prognosis.32 72 73 74 75
In our preliminary research, the mean
septal thickness as measured with echocardiography
for 14 patients with HCM due to the Arg403Gln mutation
(associated with a poor prognosis) was 18.1±6.4 mm, whereas in 9
patients with the benign Val606Met mutation,72
the mean septal thickness was 13.3±3.9 mm (P=.04).
Similarly, we found that the left ventricular mass index
was greater in patients with the Arg719Trp mutation than in
those with the Val606Met mutation (unpublished data).
Large-scale analysis is required to verify these
preliminary findings and to characterize the diagnostic and
prognostic implications of echocardiographic findings
in the context of each mutation.
Two additional genes for HCM have been describedcardiac
troponin T and
-tropomyosin, as well as a fourth
locus, to be discussed. The spectrum of clinical manifestations
observed in these families is similar to that caused by the
ß-MHC gene.9 10 11
Genotype-phenotype correlations of cardiac troponin
T mutations show the presence of relatively mild and sometimes
subclinical hypertrophy but a high incidence of SCD.
 |
Influence of Environmental Factors and Genetic Background on
Phenotypic Expression of HCM Mutations
|
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Even within the same family, the phenotypic expression of HCM
among
affected individuals sharing the same mutation varies markedly,
indicating
a role for environmental factors and possibly other genetic
factors.
The most striking example to indicate an environmental effect
is
the observation that despite ß-MHC being expressed similarly
in
the right and left ventricles, HCM is primarily a disease
of the left
ventricle. Hypertrophy presumably occurs in response
to the
higher systolic pressure in the left ventricle.
There also is evidence suggesting that other genetic factors play a
role in the expression of cardiac hypertrophy associated
with HCM. ACE genotype DD (rather than
ID or II) was found to be more common in patients
from families with HCM, showing a malignant phenotype
characterized by a high incidence of SCD.77 We have shown
in a study of 120 patients with HCM that a left ventricular
mass index of
100 g/m2 (defined as an increased mass
index) was six times more likely to occur in patients with the
ACE genotype DD than in those with the
ACE genotype II.78
Furthermore, the ACE genotype DD was
associated with more extensive left ventricular
hypertrophy characterized by involvement of the entire
interventricular septum, apex, and lateral
wall.78 The genotypes of the ACE gene
DD, II, and ID, are independent of the genes
responsible for HCM, but in an individual with HCM
genotype who also has the ACE genotype
DD, hypertrophy is more likely to be manifested
and to be more extensive than if the genotype is II.
It appears that genotypes such as DD may have a
significant role in the degree of penetrance and the variation in
phenotype (expressivity) observed in patients with HCM.
Hypertrophy requires the coordination of a large number of
genes, and thus, it is highly likely that many other genes such as the
ACE genotypes influence expressivity of the primary
genetic defect. It should not be concluded that the DD
allele is necessarily causative of the hypertrophy as
it may simply reflect a more important causative gene that is located
in close proximity. However, the finding that ACE
genotype DD influences the phenotypic expression of
HCM, probably through the mitogenic effect of
angiotensin II,79 raises the intriguing
question of the role of ACE inhibitors in HCM. It is now
well established that ACE inhibitors induce regression of
hypertrophy due to pressure overload, independent of
afterload.80 It is intriguing whether ACE
inhibitors could also induce regression of
hypertrophy in patients with HCM. The data are inadequate
to recommend such therapy for HCM at this time, and should such therapy
be used in the future, it might be contraindicated in patients with
outflow tract obstruction. Nevertheless, ACE inhibitors
have recently been shown to improve diastolic function in
patients with aortic stenosis.81 However, the
knowledge that angiotensin II is a mitogen that induces
cardiac hypertrophy and that ACE inhibitors
have been shown to induce regression of hypertrophy makes
this observation worth pursuing, since ACE inhibitors may
be beneficial.
 |
Structure-Function Analysis of ß-MHC
Mutations to Identify the Primary Defect
|
|---|
Studies are under way to determine the primary defect initiated
by
the mutations that lead to the phenotype of HCM. It is most
appropriate
to concentrate on the myocardial lesion of
hypertrophy since
the heart is the only organ responsible
for the phenotype. Hypertrophy,
along with myocyte
and sarcomere disarray, is associated with
normal systolic but impaired
diastolic function. Three of these
studies have used a
recently developed in vitro motility assay
to assess the effect of the
mutation on the contractile properties
of the ß-MHC protein. In this
assay, microscopic beads
are coated with actin, and the myosin globular
head is fixed
to a nitrocellulose membrane. The extent to which myosin
moves
the beads on binding with actin is detected with a laser beam.
The
displacement of the bead for each individual myosin molecule
averages
10 nm with an average force of 10
pN.
54 55 56 Cuda
et al
64 isolated the ß-MHC protein from skeletal muscle
of patients
with HCM and showed that the velocity of contraction was
significantly
less than that of ß-MHC isolated from skeletal muscle
of
unaffected individuals. Furthermore, Cuda et al
65
demonstrated
that the degree of impairment of velocity of the
contraction
varied with the site of mutation. The results of these
studies
were corroborated by Sweeney et al
67 with in vitro
expression
of rat cardiac

-myosin that was mutated in a region
homologous
to that of the human mutation (Arg
403Gln) in the
ß-
MHC gene. A normal and a mutant (Arg
403Gln)
full-length rat

-cardiac
myosin were expressed in Sf9 cells,
and the heavy meromyosin
fragment was isolated for in vitro motility
studies. The mutant
fragment showed an approximately fivefold reduction
in the rate
of displacement of actin filaments compared with the normal
fragment.
67 Furthermore, by mixing different ratios of
normal and mutant
fragments, they showed that the mutant myosin
disproportionately
reduced the velocity of actin displacement. In these
experiments,
an increase in the ratio of mutant to normal myosin to
50% reduced
the rate of actin filament displacement to 20% of
control. The
actin-activated ATPase activity of the mutant
(Arg
403Gln) fragment
was also significantly reduced
compared with control.
Lankford et al82 compared the force-velocity
relationship and power output of single slow-twitch muscle fibers
isolated from the soleus muscle of patients with three distinct
ß-MHC gene mutations. They showed that fibers with
Arg403Gln and Gly741Arg exhibited a
significantly reduced maximum velocity of shortening and isometric
force generation. In contrast, fibers containing the
Gly256Gln mutation displayed essentially normal contractile
properties.
Straceski et al83 studied the formation of microfilaments
in COS cells after expression of normal and mutant rat
-MHC
constructs. COS cells normally do not form filamentous structure;
however, expression of the normal rat cardiac
-myosin in COS
cells resulted in formation of structures similar to thick
filaments84 in 25% of the transfected cells, whereas the
mutant
-MHC showed filament-like structures in only
2% of the transfected cells.83
To determine whether the sarcomere disarray observed in the
myocardium of patients affected with HCM was the primary
lesion resulting from ß-MHC mutations, we selected feline
cardiac myocytes because they normally form sarcomeres as their
contractile unit and have ß-MHC as their adult cardiac myosin, as in
humans. Furthermore, HCM is a common disease in cats.68 A
full-length human ß-MHC cDNA was incorporated into an adenoviral
vector with a cytomegalovirus promoter and was expressed in adult
feline cardiac myocytes. Electron microscopic examination of myocytes
48 hours after infection showed only minor changes in the structure of
sarcomeres in cardiac myocytes. However, 120 hours after infection,
approximately 50% of the myocytes infected with the mutant ß-MHC
construct showed severe disruption of the sarcomere. In contrast, the
structure of the sarcomere remained largely intact in myocytes
transfected with normal ß-MHC construct.68
These studies have important implications for understanding the primary
defect and the pathogenesis of the phenotype. The results of
the in vitro studies are very compelling in that the mutant protein is
inherently defective as a contractile molecule. This is in accord with
our postulated hypothesis that the hypertrophy is
compensatory.61 63 This suggests that the
hypertrophy is similar to that occurring in response to
pressure or volume overload or, more appropriately, that of myocardial
infarction. The studies in COS cells and, more specifically, in feline
adult cardiac myocytes suggest sarcomere disarray to be an early
lesion. To further understand and confirm the significance of these
studies will require confirmation by other investigators and, more
important, documentation in the intact animal, as with overexpression
in transgenic animals, or with targeted mutations, such as with
replacement of genes by homologous recombination. A major obstacle
delaying both studies is the presence of
-MHC in the mouse adult
heart compared with ß-MHC in human myocardium.
Nevertheless, if one assumes the mutant ß-MHC protein exhibits
impaired contractility and disrupts formation of the
normal sarcomeres, this would lead to increased stress encountered by
the normal contractile units and fibers, a stimulus known to induce
cardiac hypertrophy.
 |
Evidence That Hypertrophy Is Compensatory in
Familial HCM
|
|---|
Hypertrophy is the common response of the heart to
pressure
overload. The mutant ß-MHC protein, with impaired
contractility,
may result in increased fiber stress
that leads to compensatory
hypertrophy. Evidence favoring
the hypothesis that the hypertrophy
is
compensatory
61 in response to the primary defect is as
follows.
(1) The phenotypes of HCM resulting from several
distinct genes
and a variety of mutations are similar, suggesting a
common
mechanism for the hypertrophy. (2) The disease
occurs almost
exclusively in the left ventricle, despite ß-MHC
accounting
for 35% of the myosin in the right and left ventricles.
This
suggests that the hypertrophy is secondary to an
environmental
stimulus such as the higher pressure and work load of the
left
ventricle. (3) Skeletal muscle, which also expresses the mutant
ß-
MHC,
appears to have normal function in patients with
HCM. Although
this may reflect the differences in the quantity of the
mutant
myosin present in the heart and the skeletal muscles, it may
also
reflect the different physiological properties
of these two
organs. (4) The age-dependent manifestation of the
cardiac hypertrophy
of HCM indicates that the
phenotype is secondary to environmental
or other genetic
factors. (5) The genes upregulated in
pressure-overloadinduced
cardiac hypertrophy are
also upregulated in the hypertrophied
myocardium of
patients with HCM, indicating a common hypertrophic
response.
Oncogenes, such as c-
fos, c-
jun, and
c-
myc, are upregulated,
as are ANP and BNP in the
hypertrophied myocardium of patients
with
HCM.
85 86 87 (6) The phenotypic
expression of
hypertrophy
in patients with HCM is influenced by the
underlying genetic
background in which mutations occur. Patients with
HCM and
ACE genotype
DD are more likely
to develop severe hypertrophy and
involvement of the entire
interventricular septum, apex, and
lateral wall than
are patients with
ACE genotype
II. These
data
indicate that the mutant protein harbinger of an impaired function
triggers
a cascade that induces the final phenotype of
hypertrophy in
patients with HCM.
 |
Response of the Heart to Injury Is Limited to
Hypertrophy, Dilatation, or a Combination
|
|---|
It is an important caveat to appreciate that the heart responds
to
injury, whether it is pressure overload, volume overload,
loss of
contractile mass (myocardial infarction), or inherited
defects, by one
of two mechanisms: hypertrophy, dilation (or
a
combination).
41 This is why familial HCM, being a disease
in
which the heart responds only through hypertrophy, is
important
to an ultimate understanding of the hypertrophic response of
the
myocardium. The reciprocal disease is familial DCM, in
which
the heart responds predominantly by dilatation.
88
Many inherited
defects induce hypertrophy. Several
mutations in the mitochondrial
genome have been associated with cardiac
hypertrophy.
89 90 In mitochondrial DNA
mutations, HCM is part of a general phenotypic
expression of a systemic
disease that is characterized by metabolic
disorders and
usually involves the central nervous and skeletal
muscle systems. A
characteristic feature of mitochondrial DNA
mutation is the maternal
inheritance of the disease, due to
the inheritance of mitochondrial DNA
only from the ovum. A confounding
problem is establishing the causal
relation of mitochondrial
DNA mutations to HCM, due to the presence of
enormous polymorphism
in the mitochondrial DNA and a gradual
increase in mitochondrial
DNA mutations over time. Furthermore, the
mitochondrial DNA
content of different cells varies (heteroplasmy),
with some
cells showing mutations and others lacking that particular
mutation.
No chromosomal locus has been identified for familial
DCM90 ; however, DCM occurring in association with
conduction defects91 has been mapped to chromosome 1p-1q,
but the gene has not yet been identified. Several diseases of muscle,
such as Duchenne's muscular
dystrophy92 93 94 or myotonic
dystrophy,95 96 that affect the heart secondarily
induce
DCM, as does the X-linked cardiomyopathy
syndrome.97
The hypertrophy observed with acquired disorders (eg, from
hypertension or from other inherited defects) does not show the myocyte
or sarcomere disarray that is the hallmark of HCM previously referred
to as IHSS. HCM as defined by the phenotype of myocardial
hypertrophy in the absence of an increased load is also
seen in overexpression studies in transgenic animals. Overexpression of
calmodulin in the transgenic mouse induced a hypertrophic
response in the myocardium.98 Similarly,
overexpression of insulin-like growth factor1, Ras,
and Myf5 genes in transgenic mice also produced a
phenotype of hypertrophy.99 100
However, no disruption of sarcomeric structure, a characteristic
finding in HCM, was reported in these studies. Soon, through
overexpression of transgenes or elimination of defective genes through
homologous recombination, one can expect to identify the molecular
basis for why cardiac hypertrophy occurs in one disease and
dilatation occurs in another.
 |
Perspective on the Pathogenesis of HCM
|
|---|
Results from the in vitro studies strongly indicate that the
primary
defect is inherent in the mutant ß-
MHC and is
manifested
as impaired contractility. Because there are
so many different
mutations in the ß-
MHC gene, it is
likely that mechanism(s)
by which the mutations lead to impaired
contractility will also
be multiple. Until such
hypotheses are confirmed in vivo, however,
it is reasonable to assume
that impaired contractility is the
primary defect,
regardless of the mechanism. How does such a
defect lead to the
hallmark of this disease as seen in the pathology
of the
myocardium, ie, hypertrophy, myocyte and
sarcomere disarray,
increased systolic function, and
diastolic dysfunction?
The studies in the COS cells suggest filament assembly is impaired;
however, given that sarcomeres do not form in COS cells, even with
normal myosin, no implications for sarcomere formation can be deduced.
Results of studies in adult feline cardiac myocytes may reflect induced
breakdown of the formed sarcomeres, or the breakdown in sarcomere may
reflect the normal turnover of myosin, with the defect being the
inability to regenerate new sarcomeres. The defective protein may
destabilize the sarcomere through either increased turnover or impaired
binding of myosin to actin or through impaired interaction with other
proteins necessary for structural integrity. What is the stimulus for
increased protein synthesis (hypertrophy) by the myocyte?
We have shown previously that the ß-MHC protein synthesized by the
heart from an individual with HCM appears normal, as is the ratio of
ß-MHC to actin or to
-MHC.101
In the studies in adult cardiac myocytes, it was also evident that the
filament formation was normal. It is reasonable to assume that the
increased breakdown of ß-MHC protein provides the stimulus for
compensatory hypertrophy. Presumably, the growth stimulus
is highly localized and may be modulated by autocrine factor(s) given
that hypertrophy is localized, in most patients, primarily
to the interventricular septum. Despite the hallmark of
extensive sarcomere and myocyte disarray, there is no clue as to why
this occurs or why it is primarily localized to the
ventricular septum. Similarly, there is no obvious reason
why systolic ventricular function is hyperdynamic.
Elucidation of the molecular basis for the pathogenesis of this disease
must provide a rationale for three puzzling consistent features
of the pathology: (1) predominance of hypertrophy in the
septum, (2) sarcomere and myocyte disarray, and (3) the supernormal
systolic function. The diastolic stiffness or decreased
compliance is expected with hypertrophy whether primary or
compensatory, but these other features are not typically seen in
compensatory hypertrophy such as that observed after
pressure or volume overload.
 |
Significance of Identification and Characterization of HCM
Mutations and Their Impact on the Practice of
Cardiology
|
|---|
Identification of the mutations responsible for HCM will provide
an
important diagnostic armamentarium. The present
diagnosis of
HCM occurs through echocardiographic
detection of cardiac hypertrophy.
This usually is not
evident until puberty or later, whereas
a genetic diagnosis is possible
before or at any time after
birth and requires only a blood sample. A
characteristic of
HCM is that the development of
hypertrophy and its phenotypic
expression are age
dependent. This also is true for the diagnosis
of HCM late in life for
mutations that are associated with a
low penetrance. Also, diseases
such as hypertensive HCM in the
elderly are indistinguishable from HCM
by clinical criteria.
19 Because HCM is an autosomal
dominant disease, only half of
the offspring of an affected individual
will inherit the disease,
and the other half will be normal. Thus,
genetic screening through
identification of the mutation will identify
the individuals
at risk of developing the disease before and
independent of
the presence of symptoms or the development of
hypertrophy.
Genetic diagnosis will also provide prognostic information, as shown,
particularly for the risk of SCD. This is particularly important as SCD
often is the first manifestation of HCM and occurs frequently in
apparently healthy young individuals.16 17 HCM is the
most
common cause of death in young competitive athletes.17
Although the clinical parameters of lack of syncope, lack
of inducibility by electrophysiological
testing, and lack of ischemia (in children) carry a favorable
prognosis, none of the parameters when present alone or
in combination have the desirable positive predictability. The need to
determine whether an individual is at risk of subsequently developing
HCM and possible SCD is greatest in children before the development of
cardiac hypertrophy. The ability to identify and separate
individuals with the mutation who are at risk of developing the disease
from those without the mutation and therefore not at risk of developing
the disease provides additional information that will supplement
clinical ability to manage these patients. The indication for
interventions such as implantable defibrillators should be decided
after risk stratification based on many clinical as well as genetic
variables that influence the outcome in patients with HCM.
Therefore, genetic screening of members of a family affected with HCM
will determine who is normal and who is likely to develop the disease
as well as provide important prognostic data of particular importance
for the children and asymptomatic individuals.
Screening for known mutations in individuals from a family affected
with HCM is feasible but tedious, time consuming, and expensive.
However, this is only possible for known mutations, whereas in a family
in whom the disease is not due to a known mutation, chromosomal mapping
and subsequent identification of the gene are required. Our preliminary
studies indicate that the ß-MHC mutation is responsible
for the occurrence of HCM in fewer than 20% of families with
HCM.45 It is expected, however, that the techniques for
mass genetic screening to identify known mutations will soon be
automated. Thus, it is very likely that within the next few years,
physicians will be able to routinely screen and identify
genotype-positive individuals. Ultimately, if therapy such
as gene transfer becomes available, genotyping will, of course, be
essential.