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Clinical Biochemistry xx (2009) xxx – xxx
Review
Familial hypertrophic cardiomyopathy:
Basic concepts and future molecular diagnostics
Jessica E. Rodríguez b , Christopher R. McCudden b , Monte S. Willis a,b,⁎
a
b
Carolina Cardiovascular Biology Center, University of North Carolina, Chapel Hill, NC, USA
Department of Pathology and Laboratory Medicine, University of North Carolina, Chapel Hill, NC, USA
Received 7 October 2008; received in revised form 24 January 2009; accepted 28 January 2009
Abstract
Familial hypertrophic cardiomyopathies (FHC) are the most common genetic heart diseases in the United States, affecting nearly 1 in 500
people. Manifesting as increased cardiac wall thickness, this autosomal dominant disease goes mainly unnoticed as most affected individuals are
asymptomatic. Up to 1–2% of children and adolescents and 0.5–1% adults with FHC die of sudden cardiac death, making it critical to quickly and
accurately diagnose FHC to institute therapy and potentially reduce mortality. However, due to the heterogeneity of the genetic defects in mainly
sarcomere proteins, this is a daunting task even with current diagnostic methods. Exciting new methods utilizing high-throughput microarray
technology to identify FHC mutations by a method known as array-based resequencing has recently been described. Additionally, next generation
sequencing methodologies may aid in improving FHC diagnosis. In this review, we discuss FHC pathophysiology, the rationale for testing, and
discuss the limitations and advantages of current and future diagnostics.
© 2009 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
Keywords: Familial hypertrophic cardiomyopathy; Sudden cardiac death; Cardiac hypertrophy; DNA resequencing; sequencing
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
FHC is most commonly due to gene mutations in sarcomeric proteins
Identification of FHC mutations may be able to risk stratify patients .
Cardiac myosin binding protein-C (cMyBP-C) . . . . . . . . . . .
Cardiac β-myosin heavy chain (β-MHC). . . . . . . . . . . . . .
Cardiac troponin T (cTnT) . . . . . . . . . . . . . . . . . . . . .
Modifier genes in FHC . . . . . . . . . . . . . . . . . . . . . . .
Therapeutic strategies in high risk FHC patients . . . . . . . . . .
Genetic testing methodologies . . . . . . . . . . . . . . . . . . . . .
Current methodologies of FHC diagnosis . . . . . . . . . . . . .
Resequencing methods of FHC diagnosis . . . . . . . . . . . . .
Advantages and disadvantages of resequencing applied to FHC . .
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Abbreviations: βMHC, beta myosin heavy chain; FHC, familial hypertrophic cardiomyopathy; LVH, left ventricular hypertrophy; cMyBP-C, myosin binding
protein-C; TnI/C/T, troponin I/C/T
⁎ Corresponding author. Department of Pathology and Laboratory Medicine, Carolina Cardiovascular Biology Center, University of North Carolina, Medical
Biomolecular Research Building, Rm 2340B, 103 Mason Farm Road, Chapel Hill, NC 27599-7525, USA. Fax: +1 919 843 4585.
E-mail address: [email protected] (M.S. Willis).
0009-9120/$ - see front matter © 2009 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
doi:10.1016/j.clinbiochem.2009.01.020
Please cite this article as: Rodríguez JE, et al., Familial hypertrophic cardiomyopathy: Basic concepts and future molecular diagnostics, Clin. Biochem. (2009),
doi:10.1016/j.clinbiochem.2009.01.020
ARTICLE IN PRESS
2
J.E. Rodríguez et al. / Clinical Biochemistry xx (2009) xxx–xxx
Challenges of genetic testing and
Challenges of genetic testing
Future diagnostics . . . . . .
Implications of genetic testing . .
Funding . . . . . . . . . . . . .
References . . . . . . . . . . . .
future diagnostics for FHC.
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Introduction
Familial hypertrophic cardiomyopathy (FHC) is the most
common genetic heart disease in the United States, affecting
nearly 1 in 500 people [6,7]. However, this autosomal dominant
disease goes mainly unnoticed because the majority of people
with it are asymptomatic [1–5]. FHC manifests as unexplained
increases in cardiac wall thickness and overall cardiac mass.
While most patients with FHC are asymptomatic, others can
present with chest pain, difficulty breathing, impaired consciousness, heart failure, and unexpected sudden death [8–10].
The most publicized example of this is in young healthy athletes
who die of sudden cardiac death [10]. Sudden death occurs in
1–2% of children and adolescents and 0.5–1% of adults with
FHC disease causing mutations [8,9]. The onset and progression
of FHC is variable and has a wide range of clinical presentations
and prognoses. Due to the reduced penetrance of FHC, diagnosis
is usually made incidentally, or after the diagnosis of sudden
cardiac death in a family member. Clinically, the diagnosis of
FHC involves identification of unexplained cardiac hypertrophy
by electrocardiographic (ECG) and echocardiographic studies
[11]. Histologically, FHC can be identified by myocyte disarray,
sarcomere disorganization, and fibrosis. However, even with the
extensive knowledge of the underlying gene mutations that
cause FHC, there are many pitfalls and caveats to diagnosing
FHC in the laboratory. There is a high degree of intra- and intergenetic heterogeneity in FHC and the predominance of “private”
mutations. In contrast to other genetic diseases, which have
mutational hot-spots where the diagnostic workup can be
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focused, FHC mutations can be found in most parts of the
gene, including the introns. This review summarizes the
underlying pathophysiology of FHC in the context to which it
is diagnosed, and highlights exciting new developments
utilizing array-based assays designed to improve the performance, cost, and speed of FHC diagnosis.
FHC is most commonly due to gene mutations in
sarcomeric proteins
Autosomal dominant mutations in sarcomere proteins underlie most cases of FHC. The sarcomere is the fundamental
contractile apparatus found in both skeletal and cardiac muscles,
and is comprised of two fundamental components: the myosin
heavy chain and the actin light filaments (Fig. 1). The actin light
filaments, composed of actin, α-tropomyosin, and the troponin
complex (T/C/I), slide past the stationary thick filaments
composed of the β-myosin heavy chain (β-MHC) and its two
components, a regulatory myosin light chain and an essential
myosin light chain. Thick filaments are also anchored by myosin
binding protein C (MyBP-C), which, in addition to its structural
properties, regulates sarcomere contractility. Contraction occurs
as the thin and thick filaments slide past one another, and is
rapidly followed by sarcomere relaxation. These continuous
cycles are all regulated by fluxes in calcium and available energy
reserves (ATP).
To date, more than 450 FHC-causing mutations have been
identified in 20 genes that encode cardiac isoforms of sarcomeric
and sarcomere-related gene products (recently reviewed by
Fig. 1. Schematic of cardiac sarcomeric proteins. The most commonly mutated proteins in familial hypertrophic cardiomyopathy include MyBP-C, β-MHC, and
troponin T.
Please cite this article as: Rodríguez JE, et al., Familial hypertrophic cardiomyopathy: Basic concepts and future molecular diagnostics, Clin. Biochem. (2009),
doi:10.1016/j.clinbiochem.2009.01.020
ARTICLE IN PRESS
J.E. Rodríguez et al. / Clinical Biochemistry xx (2009) xxx–xxx
[12]). The prevalence of FHC-causing mutations varies by
population, with cardiac-myosin binding protein-C being the
most commonly affected gene (cMyBP-C, ∼ 15–50%) [13–15],
β-MHC being the next most common (∼13–25%) [13,14,16],
followed by mutations in cardiac troponin isoforms I and T
(4–15%) [2,12,13,15,17–20]. Two hypotheses have been proposed to explain why mutations in sarcomeric proteins induce the
disease spectrum of FHC. The first is the “poison polypeptide”
hypothesis, which proposes that a single mutant protein within
the sarcomere can disrupt the function of the entire ‘wild type’
sarcomere muscle unit [21]. The other hypothesis is that
mutations in sarcomeric proteins lead to ‘haploinsufficiency’.
In haploinsufficiency, mutations disrupt one copy of the gene,
leaving insufficient amounts of the remaining wildtype protein to
make up for the paucity of protein. Since the sarcomere structure
is stoichiometrically balanced, haploinsufficiency of a given
sarcomeric protein results in poor performance of the remaining
sarcomere [12,21]. Defects in the turnover of these key
sarcomeric proteins may also account for both hypertrophic
and functional defects [22,23]. Currently the basic mechanisms
of sarcomeric protein turnover are not completely understood
[24]. However, it is clear that protein turnover is essential for
sarcomere maintenance and the degradation of specific mutant
sarcomere proteins (i.e. cMyBP-C) by the proteasome is
fundamentally important for protein turnover and maintenance
of quality control [25,26]. Mutations in sarcomere proteins have
also been reported to effect cardiomyocyte calcium signaling
[27] and cause metabolic defects [28], which may underlie
patients' increased susceptibility to sudden cardiac death.
The frequency of FHC-causing mutations varies from study
to study, ranging from 30–61% in patients with clinically
defined disease [13,14,16]. This variability in mutation
prevalence is likely the result of differences in where these
studies were performed, the ages of the patients investigated in
the studies, and the nature of the patients referred to the studies.
However, it is consistently reported that not all patients with
clinical FHC have identifiable mutations. There may be several
reasons that FHC mutations cannot be identified in these
patients. First, a reduced sensitivity in testing can result from
not investigating the known disease genes for deep intronic
mutations, large insertions and deletions, and gross genomic
rearrangements. Secondly, there may be mutations in yet to be
identified sarcomeric genes. Evidence for this comes from
recent studies identifying disease causing mutations in sarcomere-associated proteins such as myosin light chain kinase and
telethonin, among others (reviewed in [12]). In this case, if
testing for mutations is not conducted in these other sarcomere
genes, they will not be identified. Lastly, there are several
diseases that present phenotypically in a similar manner to FHC,
yet do not involve mutations in sarcomere or sarcomereassociated proteins. These so-called phenocopy disorders
include metabolic disorders that present with unexplained left
ventricular hypertrophy [29,30]. In some of these diseases, such
as Friedrich's ataxia, Noonan Disease, and LEOPARD
Syndrome, there is a constellation of additional symptoms
that the observant clinician may use to differentiate them
clinically from the isolated left ventricular hypertrophy of FHC
3
(see Table 1). However, other phenocopy disorders, such as
Wolff–Parkinson–White syndrome can have few if any extracardiac symptoms making it clinically indistinguishable from
FHC. Therefore, accurate genetic diagnosis is essential to
differentiate it from FHC, as therapy for these two diseases
differs greatly. Therefore, in patients presenting with unexplained left ventricular hypertrophy, it is important to test for
other genetic diseases in addition to FHC-causing mutations to
accurately determine the underlying etiology.
Identification of FHC mutations may be able to risk
stratify patients
One of the main reasons for identifying underlying mutations
in FHC is to help determine if patients have a higher or lower
possibility of complications, including sudden death. In
children, several mutations have been identified that do not
cause physical symptoms, despite impressive clinical presentation (significant left ventricular hypertrophy) [31]. Other
mutations are associated with survival comparable to control
patients [32–36]. However, even in these apparently benign
mutations there is some discrepancy with patient outcomes [37–
39]. It seems that the distinction of benign or malignant
mutations can only be applied to immediate family members
[39]. The following sections provide specific examples of the
genotype–phenotype associations that have been made for the
most commonly affected proteins, cMyBP-C, β-MHC, and
cardiac troponin T.
Cardiac myosin binding protein-C (cMyBP-C)
Identification of specific cMyBP-C mutations may give
clinicians insightful information on the clinical course of
disease. Mutations in cMyBP-C are the most common type
found in patients with FHC; various reports indicate that
cMyBP-C mutations are present in ∼15–50% of patients
[13–15]. cMyBP-C is both a structural and regulatory protein
found in the sarcomere (Fig. 1). During cardiac development,
cMyBP-C plays a role in assembly of the sarcomere [40]. In the
adult heart, cMyBP-C phosphorylation regulates cardiac contraction in response to catecholamine stimulation [41]. Initial
studies reported that cMyBP-C mutations might be comparatively mild clinically compared to mutations in βMHC and TnT
[36]. Correspondingly, these studies reported that disease onset
occurred in the middle decades and patients generally had better
outcomes [35,42]. However, more recent studies have identified
cMyBP-C mutations in children with either severe disease [15]
or mild to moderate hypertrophy and good prognosis [43,44]. In
addition, increased sudden death has been reported with at least
one cMyBP-C mutation and death from cardiac causes occurs
in 13% of individuals with cMyBP-C mutations [35]. The later
onset of disease expression may account for better survival of
patients with cMyBP-C, but once disease is expressed, there are
recognized cardiac complications that can occur. The severity
of FHC caused by cMyBP-C mutations depends on the
interplay of the disease causing mutation(s), genetic modifiers,
and exogenic factors.
Please cite this article as: Rodríguez JE, et al., Familial hypertrophic cardiomyopathy: Basic concepts and future molecular diagnostics, Clin. Biochem. (2009),
doi:10.1016/j.clinbiochem.2009.01.020
ARTICLE IN PRESS
4
J.E. Rodríguez et al. / Clinical Biochemistry xx (2009) xxx–xxx
Table 1
Phenocopy diseases and known cardiac phenotypes
Disease
Cardiac phenotype
Gene mutation
Wolff–Parkinson–White
Ventricular pre-excitation, supraventricular arrhythmias, progressive
conductive disease, abnormal cardiac glycogen storage, and
unexplained cardiac hypertrophy
Other clinical signs/symptoms: may have tachycardia, palpitations,
dizziness, lightheadedness, syncope, or no symptoms.
Unexplained LVH, myocardial fibrosis (mid-myocardial layers),
accumulation of glycosphingolipids in the tissues and vasculature,
conduction abnormalities, ischemic heart disease.54,55
Other clinical signs/symptoms: severe limb pain, telangiectasia,
angiokeratomas, lip thickening, and bulbous nose.
Vacuolar glycogen accumulation leading thickening of the ventricular
walls and intraventricular septum (Unexplained LVH).56
Other clinical signs/symptoms: in classic infantile form, severe generalized
skeletal muscle hypotonia is pronounced.
Glycogen deposition in cardiac muscle leading thickening of the
ventricular walls and intraventricular septum. (Unexplained LVH).
Conduction defects.57,58
Other clinical signs/symptoms (depends on subtype) may include:
muscle weakness, hepatomegaly and hypoglycemia due to liver disease,
hyperlipidemia, and growth retardation.
Vacuolation and degeneration of cardiomyocytes, myofibrillar disruption,
lipofuscin accumulation, conduction abnormalities, LVH, Cardiac failure.59,60
Other clinical signs/symptoms may include: short stature, dysmorphic
feathers, pulmonic stenosis, webbed neck, chest deformity, cryptorchidism,
mental retardation, and bleeding diatheses.
LS: Conduction abnormalities, LVH, valve abnormalities, coronary artery
abnormalities, ventricular septal defects. (9) NS-Pulmonary Valve stenosis,
atrial septal defects, ventricular septal defects, LVH, persistent ductus
arteriosus, conduction defects.61
Other clinical signs/symptoms possibly present: LS: characteristic freckling
(over 10,000+ covering most of skin), EEG abnormalities, including bundle
branch block, wide set eyes, pulmonary stenosis, abnormal genitalia, missing
testicle/ovary, retarded growth, and deafness.
NS: short stature, cervical spine fusion, scoliosis, joint contractures, growth
retardation, and hypotonia.
Increased iron resulting from reduced mitochondria respiration, decreased
cardiac bioenergetics, and LVH.62–64
Other clinical signs/symptoms: neurologic dysfunction, specifically ataxia
(incoordination) of all four limbs, and diabetes.
γ-2 Regulatory subunit of
AMP-activated protein
kinase; PRKAG2
Anderson–Fabry Disease
Pompe Disease
Glycogen Storage Disease Type III
(Forbes Disease, Cori's Disease)
Danon's Disease
LEOPARD⁎ Syndrome/
Noonan Syndrome
Friedreich's Ataxia
X-linked enzyme,
α-galactosidase A; GLA
α-Glucosidase; GAA
Glycogen debranching enzyme,
4-Glucanotransferase, AGL
Lysosome-associated
membrane protein 2, LAMP2
Protein tyrosine phosphatase
SHP-2, PTPN11
(LS—dominant negative effect;
NS—gain of function)
GAA triplet repeats in
Frataxin, FRDA
⁎ Mnemonic for: Lentigines, Electrocardiographic conduction abnormalities, Ocular hypertelorism, Pulmonary stenosis, Abnormal genitalia, Deafness.
Cardiac β-myosin heavy chain (β-MHC)
While initial studies reported that mutations in β-MHC were
responsible for up to 50% of cases, more recent analyses have
identified that these mutations occur in 13–25% of patients with
FHC [13,14,16]. This apparent discrepancy in prevalence may
be due the consequence of sampling in different parts of the
world, and that β-MHC mutations are associated with disease at
an earlier age and more severe hypertrophy and are therefore
recognized more often [16]. As many as 50 missense mutations
have been identified in the β-MHC gene. Those mutations
found in the globular head of β-MHC show a high penetrance
where most individuals develop significant hypertrophy [45].
Specific mutations in Arg403Gln and Arg453Cys have been
associated with severe hypertrophy and the highest rates of
sudden death [32]. In comparison, other mutations, such as
Val606Met, are associated with a moderate left ventricular
hypertrophy and good prognosis. As with mutations in cMyBP-
C, investigators have reported genotype–phenotype associations that may affect treatment strategies in patients with the
intention of improving outcomes. In most cases, however, it is
not possible to predict the individual outcome of a specific
mutation. In the few mutations for which a specific course of
disease is highly likely (i.e. specific β-MHC gene mutations),
the management of the patient may be modified according to the
likely outcome. Cases where a modification of the treatment
strategy based on a known genotype/phenotype correlation is
justified are extremely rare.
Cardiac troponin T (cTnT)
Cardiac troponin T mutations account for only 4–15% of
FHC cases. Generally these cTnT mutations are associated with
less severe hypertrophy and fibrosis than mutations in β-MHC
[13,17,18]. However, a higher incidence of premature death has
been reported in cTnT mutations, and a higher proportion of
Please cite this article as: Rodríguez JE, et al., Familial hypertrophic cardiomyopathy: Basic concepts and future molecular diagnostics, Clin. Biochem. (2009),
doi:10.1016/j.clinbiochem.2009.01.020
ARTICLE IN PRESS
J.E. Rodríguez et al. / Clinical Biochemistry xx (2009) xxx–xxx
these patients die suddenly with mild or absent left ventricular
hypertrophy [18,46,47]. This makes identification of family
members with these mutations important, as their clinical
phenotype may not reflect the severity of their disease [46].
Identifying patients with cTnT mutations that are at risk for
reduced survival may have implications on therapy and long
term care of these patients.
Modifier genes in FHC
As with other cardiac diseases, multiple factors can modify
FHC including gender, blood pressure, and lifestyle factors such
as physical exercise. Additionally, severe FHC phenotypes can
result from polymorphisms in modifier genes or the presence of
multiple mutations. The presence of modifier genes explains
why patients with the same mutation, even in the same family,
can exhibit variable severity of disease [13,15]. Polymorphisms
in ACE (angiotensin converting enzyme), a major component of
the rennin–angiotensin system, were initially identified to be
associated with increases in cardiac mass and left ventricular
hypertrophy score in FHC families with a high incidence of
cardiac death compared to unaffected members of the same
families [48]. Further analysis identified that approximately
10% of the variability in cardiac hypertrophy in FHC was related
to the ACE genotypes [49]. Recently, a second angiotensinconverting enzyme (ACE2) has been identified, which may be
involved in cardiac structure and function. The minor alleles of 4
single nucleotide polymorphisms (SNPs) have been found to be
associated with a higher left ventricular mass index in men, with
similar, but less pronounced trends in women. Interestingly, no
association between these SNPS and left ventricular systolic or
diastolic function or blood pressure levels was observed in these
studies, suggesting a more local effect on the heart [50]. In the
context of FHC, recent studies have investigated the role of ACE
gene polymorphisms in FHC, identifying that the higher risk
ACE2 alleles were associated with increased interventricular
septal thickness in male patients [51]. Other candidate modifier
genes that have recently been identified in patients with the same
mutation in MyBP-C include ITGA8, C10orf97 (CARP), and
PTER, with the former 2 implicated in cardiac fibrosis and
apoptosis, respectively [52]. Multiple disease-causing mutations
occur in up to 5% of FHC cases, generally in patients with more
severe phenotypes compared to single mutation carriers [53,54].
These multiple mutations generally involve βMHC and MyBPC most often, and to a lesser extent TnI [53]. Recent studies have
reported mouse models of multiple mutation FHC [54], allowing
for further understanding of its unique disease pathogenesis and
severity. The identification of disease modifying genes may one
day be used to direct therapy and lower the risk of sudden
cardiac death in specific patient populations with higher
morbidity and mortality.
Therapeutic strategies in high risk FHC patients
Once patients have been identified with FHC through genetic
testing, several therapeutic strategies have been implemented to
improve patient outcomes by reducing the risk of sudden
5
cardiac death. Medical therapy is based mainly on clinical
experience as randomized trials have not been performed for
FHC [55]. Therapies include negative inotropes, including beta
adrenergic antagonists and calcium channel blockers (verapamil), and also the anti-arrhythmic drug disopyramide [56]. The
present study discusses the identification of patients at higher
risk for sudden cardiac death by genetic diagnosis of specific
mutations. Patients at higher risk for sudden cardiac death are
also identified clinically by family history, abnormal blood
pressure response to exercise, and nonsustained ventricular
tachycardia [57]. This has led to studies investigating the merits
of implantable cardioverter-defibrillators (ICD) to prevent
sudden death in high risk FHC patients [58]. In FHC patients
with as little as one risk factor, the use of implantable ICDs has
proven effective in restoring normal rhythms to high risk FHC
patients with life-threatening ventricular tachyarrhythmias [58].
With the prospect of both medical and ICD therapies for
treatment of FHC, the identification of patients with disease
causing and high risk mutations has a tremendous potential for
improving survival.
Genetic testing methodologies
Current methodologies of FHC diagnosis
Since the first FHC-related mutation was recognized, there
has been interest in developing DNA-based tests to screen for
FHC in patient families to identify others with disease. There are
several dozen laboratories that currently offer referral testing for
FHC in suspected patients and their families (www.genetests.
com). The most common methodology used by these laboratories is sequence analysis of the entire coding region specifically
for cardiac α-actin (ACTC1), cMyBP-C (MYBPC3), β-MYH
(MYH7), myosin light chain (Regulatory, MYL2; Essential,
MYL3), cardiac troponin I (TNNI3), cardiac troponin T (TNNT2),
and α-tropomyosin (TPM1). While analyzing the whole coding
region can be time consuming, some laboratories focus their
efforts on analyzing select exons of particular genes (MYBPC3,
MYH7, TNNI3, and TNNT2). Alternatively, laboratories may
analyze the entire coding region by mutation scanning of genes
(MYBPC3, MYH7, TNNI3, TNNT2, and TPM1).
Mutation scanning for FHC genes can be performed using a
variety of similar techniques. For simplicity, we describe here
the single-stranded conformation polymorphism (SSCP) technique as one technique for mutation scanning, using the MyBPC gene as an example (see Fig. 2A). SSCP analysis can be used
to detect mutations by comparing the electrophoretic mobility
of patient DNA to known normal DNA. This methodology is
based on the finding that under non-denaturing conditions and
reduced temperature, single-stranded DNA assumes unique
conformations based on its sequence. These sequence differences result in mobility shifts that can be detected by
electrophoresis. SSCP involves first PCR amplifying the region
of interest in DNA containing potential mutations with primers
flanking these regions (shown in Fig. 2B). The resulting doublestranded DNA is then denatured (to generate single stranded
DNA), and rapidly chilled to inhibit re-annealing of
Please cite this article as: Rodríguez JE, et al., Familial hypertrophic cardiomyopathy: Basic concepts and future molecular diagnostics, Clin. Biochem. (2009),
doi:10.1016/j.clinbiochem.2009.01.020
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Fig. 2. Schematic of the human MYBPC3 gene and PCR scanning for gene mutations. MYCP3 is composed of 35 exons spanning 21 kb on chromosome 11p11.2. PCR
scanning involves amplification of target sequences to generate amplicons for mutation analysis. Amplicons are analyzed by electrophoresis to identify unique
migration patterns that reflect the presence of mutations. Specific mutations are then identified by direct DNA sequencing. At the bottom of the figure, region 2 of
patient ‘A’ represents a mutant MYCP3 exon. Adapted from [77].
complementary strands. These ssDNA fragments are then
separated by electrophoresis in a non-denaturing medium
where the mobilities compared (see Fig. 2C). When alterations
are identified, indicating differences in DNA sequence,
sequencing of the specific region is performed to identify
which nucleotide(s) are altered. In addition to mutation
scanning and sequencing methodologies to diagnose FHC in
suspected patients, pre-implantation genetic diagnosis for
MYH7 is available, as is pre-natal diagnosis for the genes
named in the previous paragraph (see www.genetests.com).
Testing is also available for diseases that mimic FHC clinically,
briefly summarized in Table 1.
Resequencing methods of FHC diagnosis
Alternative methodologies to those methods described above
for FHC mutations have recently been described, which use
microarray technology to accelerate existing sequence based
analysis. While these have mainly been ‘proof-of-concept’
studies, they demonstrate a viable alternative to the long and
expensive process of sequence-based diagnostics applied to
FHC. Two groups have recently reported the use of DNA
resequencing technology to assist in the complex diagnosis of
FHC. This technology uses 25mer probes that recognize single
nucleotide substitutions to determine the sequence of a short
strand of complementary DNA (Fig. 3). The process of
resequencing occurs in several steps. First, DNA is isolated
from peripheral blood lymphocytes and the gene of interest is
amplified by short- or long-range PCR, using primers for
specific gene regions of interest (Fig. 3A). DNA is then digested
to generate 50 bp fragments, which are then labeled at each end
with biotinylated terminal nucleotides (Fig. 3B). This target
DNA is then hybridized overnight and washed stringently
in the morning, after which target DNA fragments remain
Please cite this article as: Rodríguez JE, et al., Familial hypertrophic cardiomyopathy: Basic concepts and future molecular diagnostics, Clin. Biochem. (2009),
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7
Fig. 3. DNA resequencing methodology for detection of point mutations in large DNA fragments. The principle of DNA resequencing is that groups of 4
complimentary probes are used to determine the identity of a single base pair in the sense strand of the gene. These 25mers differ only at one base (A, T, G, or C),
allowing the identity of that base to be made according to the labeled DNA that hybridizes to it. 25mers representing exons and flanking introns are represented by
thousands of these 25mers, each set of 4 allowing the sequence determination of a single nucleotide. The location of the signal on the array reveals the sequence of the
patient's gene. With the capability to interrogate hundreds of thousands of 25mers at one time, microarray technology allows the determination of sequences faster than
traditional sequencing. The process involves (A) isolation and PCR amplification of patient DNA, (B) digest and labeling of DNA probes, and (C) hybridization of the
DNA probe to a microarray. Each nucleotide is represented on the chip for every base pair.
preferentially bound to sequences with all 25 complementary
base pairs (Fig. 3C). The principle of array-based sequencing is
that hundreds of complementary 25mers representing the
different regions of the sequences of interest are allowed to
hybridize with the labeled sequences from patients (see Fig. 3C
legend). These sequences have single nucleotide alterations
at different specified points, enabling the binding of labeled
complementary DNA to determine the sequence of patient DNA
at specific regions of a gene. After hybridization, DNA is
stained using a combination of streptavidin phycoerythrin and a
biotinylated anti-streptavidin antibody for fluorescence visualization. Analysis of the hybridization patterns of the DNA label
(fluorescence) enables indirect determination of the sequence of
specific gene regions. Since each chip can have hundreds of
Please cite this article as: Rodríguez JE, et al., Familial hypertrophic cardiomyopathy: Basic concepts and future molecular diagnostics, Clin. Biochem. (2009),
doi:10.1016/j.clinbiochem.2009.01.020
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thousands (up to 2.1 million) of printed oligonucleotides, a high
throughput screen can identify an array of specific single
nucleotide changes in a large number of specific DNA
sequences of interest rapidly.
The proof of concept that the microarray “resequencing”
could be applied to FHC was reported as an oral communication
at the European Society of Human Genetics in 2005 by Fokstuen
et al. [59]. In this study, Fokstuen et al. reported the development
of a “CustomSeq Resequencing Array” (Affymetrix) with
30,000 probes enabling the rapid molecular diagnosis of FHC.
In the first published study, Waldmuller, et al. utilized an
Affymetrix resequencing array and a single long range PCR
protocol to cover commonly affected genes underlying FHC,
including MYH7, MYBPC3, and TNNI2 [60]. They compared
automated capillary sequencing to the array-based resequencing
method. In their first set of experiments, four disease causing
point mutations were detected by both methods, while a 3 bp
deletion (in MYH7) was identified only by sequencing (detection
rates of 50% and 40%, respectively) [60]. A second set of
experiments investigated 25 previously characterized FHC
samples, harboring a total of 24 point mutations, one compound
genotype, two deletions, and a single nucleotide insertion.
Array-based resequencing identified 92–96% of the known
single nucleotide variants/mutations, and revealed an additional
two mutations that had been overlooked in previous analyses.
Resequencing also provided evidence of mutations involving a
G-insertion and a 3 bp deletion, which were read as a single
uncalled nucleotide (N). In the last set of experiments, 18
uncharacterized FHC samples were run on the array-based
resequencing platform, and identified 7 presumptive causative
mutations (39% detection rate), three of which were previously
described. Overall, 13 novel putative mutations were identified
in this study. This study provided the first published report that
array-based resequencing can be used comparably to sequencing
to identify FHC mutations, with the substantial benefit of
reducing workload currently associated with sequencing
modalities, and allow large scale studies of the diversity of FHC.
In the next recently published paper applying resequencing
techniques to FHC, Fokstuen et al. designed a platform that
contained complementary strands for all coding exons of 12
genes, including sarcomeric genes and genes underlying
phenocopy diseases; βMHC (MYH7), cMyBP-C (MYBPC3),
troponin T (TNNT2), tropomyosin (TPM1), troponin I (TNNI3)
myosin light chains (MYL2, MYL3), cardiac muscle LIM protein
(CSRP3), phospholamban (PLM), actin (ACTC1), AMP kinase
(PRKAG2), and troponin C (TNNC2) [61]. The FHC resequencing array contains the complete sequence of all exons, splice
sites, and promoter regions of the 12 genes in which so far N 98%
of mutations have been identified [61]. Following the creation of
the resequencing platform, 38 unrelated patients with FHC (both
familial and sporadic cases) were analyzed. Nearly one million
base pairs across the 38 patients were determined and compared
to currently available sequencing methodologies. Pathogenic
mutations in MYH7, MYBPC3, TNNI3, and MYL3 (six known
and six novel) were identified in 69% of familial cases (10 of 17
patients) and 10% (2 of 21) of sporadic cases. Overall,
microarray-based resequencing performs at nearly the same
accuracy as conventional sequencing methods (99.99%), with the
advantage of being significantly cheaper [62]. Similar to other
sequencing methodologies, the underlying genetic defect may
not be detected in phenotypic FHC patients due to the presence of
FHC phenocopy diseases (Table 1), the presence of mutations in
areas not covered by the array, and the involvement of additional,
yet to be identified genes.
Advantages and disadvantages of resequencing applied
to FHC
There are both advantages and disadvantages to the application of these new resequencing methods applied to FHC. First,
some insertion/deletion mutations are missed using resequencing
methods, particularly missense mutations falling in G/C rich
regions [60,61]. However, the 39% detection rate in all patient
samples is comparable to meta-analyses of sequencing methodologies of patients from the United States as reported by
Waldmuller et al. [14,60]. In terms of throughput, the studies by
Fokstuen, et al., 2008, estimate 4–16 arrays could be simultaneously analyzed and less DNA was needed than conventional
analyses [61]. Waldmuller et al., 2008 estimates a sample
throughput of ∼100 patients per technician per month [60],
which contrasts to conventional methods which can take weeks,
depending on the extent of testing. Since resequencing takes less
time and is less labor intensive compared to conventional
methods (SSCA, DHPLC, and direct sequencing), it is predicted
to be cheaper. It is estimated the resequencing methods in FHC
diagnostics would cost ∼6 times less than corresponding direct
sequencing costs after the initial investment for the design and
production of the array [61]. Given the considerable savings in
time and money compared to sequencing, resequencing methodologies are attractive for routine mutation screening, triaging
negative tests for the more extensive workup by currently
established methods. In particular, large scale genetic studies and
initial screening in diagnostic laboratories may be a particularly
good use of this diagnostic platform.
Challenges of genetic testing and future diagnostics
for FHC
Challenges of genetic testing
Since the first disease-related mutation was recognized, there
has been an interest in developing DNA-based tests for FHC to
permit screening of patient families to identify other relatives at
risk for disease. While this appears a simple task, there are several
complicating issues. Only ∼60% of patients with FHC have
identifiable mutations, despite extensive interrogation by sequencing [63,64]. This relatively low sensitivity is a result of several
factors. First, not all potentially affected sarcomere proteins have
been identified. In addition to the most common sarcomere
proteins commonly interrogated by sequencing (i.e. cMyBP-C,
β-MHC, cTnT), mutations in alpha-myosin heavy chain, titin,
myosin light chain have also been reported to cause FHC. A
second challenge to genetic testing for FHC mutations is that not
all underlying mutations in the commonly affected sarcomere
Please cite this article as: Rodríguez JE, et al., Familial hypertrophic cardiomyopathy: Basic concepts and future molecular diagnostics, Clin. Biochem. (2009),
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9
proteins (i.e. cMyBP-C, β-MHC, cTnT) have been identified.
This is evident as novel and new disease causing mutations in
cMyBP-C, β-MHC, and cTnT are discovered routinely. A third
challenge is the lack of functional assays that can prove the
pathogenic effect of a previously unknown mutation. There is
also a significant problem testing for FHC mutations in patients
that carry multiple disease causing mutations, whereby more
severe disease may be present and lead to complex phenotypes.
For example, patients have been identified that 1) carry two
different mutations in the same FHC gene; 2) carry two mutations
in different FHC affected genes; or 3) carry two copies of the
same FHC mutation [13]. These multiple mutations pose a
diagnostic challenge, requiring each patient to have all the known
affected genes sequenced to identify all the present mutations.
However, even with such an extensive analysis, yet-identified
mutations can still be missed in a patient with one identified gene
mutation. Lastly, current methods do not test known disease
genes for deep intronic mutations, large insertions and deletions,
or gross genomic rearrangements, which can lead to the reduced
sensitivity of genetic testing. This is important because intronic
mutations have been identified in intronic regions flanking
MyBP-C [65,66]. Introns are not routinely interrogated further
because of their large size, which would vastly increase the
number of regions to be tested. As with all genetic tests, there is
the potential for psychological hardship because there is no cure
for FHC. However, diagnosis allows for more vigilance for
problems as they arise for both the individual and family
members, and health care providers taking care of them.
[16,73–76]. For example, mutation screening of MYH7 covering
10 kb was performed on a total of 5700 amplicons, more than
6750 DHPLC injections, which were completed within 35 days
[73]. DHPLC sequencing does not appear as fast as resequencing,
but a more rigorous application of all of these applications will be
necessary to make this distinction. Much like “resequencing” on
microarray chips DHPLC has been applied to only a small
number of clinical applications published to date, and their role in
clinical diagnostics unproven.
Future diagnostics
Appendix A. Supplementary data
In addition to array-based resequencing (described in Genetic
testing methodologies) there are several other “next-generation”
sequencing technologies being developed as recently reviewed in
detail [67–69]. The methodologies being developed to supplant
dye-terminator sequencing with capillary electrophoresis
include: Pyrosequencing, Fluorescently labeled sequencing by
synthesis, and Sequencing by hybridization and ligation. These
next-generation sequencing technologies have short read lengths,
but very high densities, which allow the reconstruction of the full
sequence by massive parallel processing by computers, resulting
in a considerable improvement in throughput compared to current
sequencing methods. Pyrosequencing by the 454 Life Science
instrument by Roche yields 100 million base pairs in a 7.5 h run
with N99% accuracy. Fluorescently labeled sequencing by
synthesis utilized by the Solexa Genome Analyzer by Illumina
yields 3 Gb per 5 day run, again with N99% accuracy. Sequencing
using the SOLiD (Sequencing by Oligonucleotide Ligation and
Detection) system by Applied Biosystems (ABI) reads 6 Gb per
10 day run with N99% accuracy. In contrast, current Sanger
sequencing reads approximately 650–700 bp in 1–2 h, with 97–
99% accuracy [70–72]. The advantages and disadvantages of
each of these methodologies are application specific as recently
reviewed in detail [67–69]. These next-generation sequencing
methodologies have not been applied to FHC to date. There are,
however, a few reports of denaturing high pressure liquid
chromatography (DHPLC) being applied to FHC diagnostics
Supplementary data associated with this article can be found,
in the online version, at doi:10.1016/j.clinbiochem.2009.01.020.
Implications of genetic testing
With improving techniques to identify FHC and other
phenotypically similar causes of unexplained left ventricular
hypertrophy, better management plans for patients through
ongoing close monitoring of symptoms and cardiac physiology
can be designed. Based on their symptoms and mutation
associations, patients can be treated appropriately and first degree
relatives tested as clinically needed. Similarly, with the diagnosis of
phenocopy diseases, the underlying disease process can be treated.
Recent advances in microarray-based resequencing techniques
applied to FHC diagnostics appear to allow a faster diagnosis to be
made with similar efficacy as current sequencing diagnostics.
Funding
Children's Cardiomyopathy Foundation and American Heart
Association Scientist Development Grant (to M.W.).
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Please cite this article as: Rodríguez JE, et al., Familial hypertrophic cardiomyopathy: Basic concepts and future molecular diagnostics, Clin. Biochem. (2009),
doi:10.1016/j.clinbiochem.2009.01.020