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Advances in Genetics, Proteomics, and Metabolomics
Multiple Mutations in Genetic Cardiovascular Disease
A Marker of Disease Severity?
Matthew Kelly, BMedSc; Christopher Semsarian, MBBS, PhD
O
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phy, progression to heart failure, and sudden death (for
review, see reference 3). However, most recently, there is an
emerging recognition that a proportion of patients carry 2
(multiple) independent disease-causing gene mutations (ie,
not polymorphisms), leading to more severe clinical disease.
These mutations can occur in the same gene (compound
mutation) or in 2 different genes (double mutation), as
indicated in Figure 2. This challenges the well-accepted
paradigm in autosomal dominant monogenic medical diseases that 1 mutation in a single gene is the direct cause of
disease and has major implications on the clinical evaluation,
diagnosis, and management of families with a genetic cardiovascular disease.
This review will focus on the role of multiple mutations in
patients with genetic cardiovascular disease. Specifically,
HCM will be the focus of this review, given that it is the
disease most thoroughly studied in terms of both genetic
causes and clinical outcomes. The impact of multiple mutations both in our understanding of underlying pathogenic
mechanisms, as well as in diagnosis and counseling in
families with genetic heart disease will also be discussed.
ver the last 2 decades, major advances have been made
in our identification and understanding of the genetic
basis of cardiovascular disease. More than 40 cardiovascular
disorders have now been identified to be directly caused by
single-gene defects. These disorders span all aspects of
cardiovascular disease and affect all parts of the heart
structure. They include the inherited cardiomyopathies such
as hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy, and arrhythmogenic right ventricular dysplasia; primary arrhythmogenic disorders such as familial long-QT
syndrome (LQTS) and Brugada syndrome; congenital heart
diseases such as familial atrial septal defects; vascular diseases such as Marfan syndrome; and metabolic disorders such
as familial hypercholesterolemia (FH). Until recently, these
cardiac genetic disorders have been thought to involve only
single-gene defects (ie, in an individual patient, 1 mutation in
a single gene leads to a disease).
Editorial see p 95
A common feature of almost all genetic cardiovascular
diseases is the clinical or phenotype heterogeneity observed
in the affected individuals both within and between families.
Despite harboring the same gene mutation, affected individuals (eg, siblings) can often have marked clinical variability,
ranging from no symptoms to severe heart failure and
premature death. This widespread clinical heterogeneity suggests other factors apart from the gene mutation itself are
important in modifying the clinical phenotype, either by
exacerbating or protecting against the disease.1
These modifying factors are poorly understood and may
include a number of factors (Figure 1). These include environmental factors such as exercise and diet, age and genderrelated influences, and secondary genetic factors. To date, the
role of secondary genetic factors has focused largely on gene
variants or polymorphisms that do not directly cause disease
but may influence regulatory factors such as gene promoter
regions altering gene expression or influence the function of
key enzymes important in normal cardiovascular biology.2
An example is the insertion/deletion polymorphism of the
angiotensin-converting enzyme gene, which has been implicated as a modifying factor in a number of aspects of
cardiovascular disease, including extent of cardiac hypertro-
Multiple Mutations in HCM
Of all the inherited cardiovascular disorders, HCM was the
first in which a genetic basis was identified and, as such, has
acted as a paradigm in terms of how to study a genetic
cardiovascular disease. Although major advances have been
made in understanding the genetic causes of HCM, recent
reports suggest a significant proportion of patients with HCM
harbor multiple mutations.4 – 6
Clinical Basis of HCM
HCM remains the most common cardiovascular genetic
disorder, occurring in at least 1 in 500 people in the general
population.7 HCM is a primary inherited disorder of the
myocardium characterized by hypertrophy, usually of the left
ventricle, in the absence of other loading conditions such as
hypertension. Individuals with HCM exhibit marked diversity
in their morphological features and clinical manifestations,
ranging from no symptoms to heart failure and sudden death.8
This clinical heterogeneity reflects the complex pathophysiology underlying the disorder, which includes not only
From the Agnes Ginges Centre for Molecular Cardiology (M.K., C.S.), Centenary Institute, Sydney, Australia; Faculty of Medicine (M.K., C.S.),
University of Sydney, Australia; and Department of Cardiology, Royal Prince Alfred Hospital (C.S.), Sydney, Australia.
Correspondence to Christopher Semsarian, MBBS, PhD, Agnes Ginges Centre for Molecular Cardiology, Centenary Institute, Locked Bag 6, Newtown,
New South Wales 2042, Australia. E-mail [email protected]
(Circ Cardiovasc Genet. 2009;2:182-190.)
© 2009 American Heart Association, Inc.
Circ Cardiovasc Genet is available at http://circgenetics.ahajournals.org
182
DOI: 10.1161/CIRCGENETICS.108.836478
Kelly and Semsarian
Multiple Mutations in Genetic Heart Disease
Table 1.
Gene Name
␤MHC
Encoded Protein
Disease %
30 to 35
Myosin binding protein C
20 to 30
cTnT
Cardiac troponin T
10 to 15
␣TM
␣-Tropomyosin
5 to 15
cTnI
Cardiac troponin I
⬍5
Cardiac muscle LIM protein
⬍5
TCAP
Telethonin
⬍2
MYL2
Regulatory light chain
⬍1
MYL3
Essential light chain
⬍1
ACTC
Actin
⬍0.5
Titin
⬍0.5
CSRP3
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diastolic dysfunction, but also arrhythmogenic substrates
leading to ventricular arrhythmias, small-vessel disease leading to subendocardial ischemia, and left ventricular outflow
tract obstruction.9,10 Therefore, HCM is a disease with a
multitude of potential cardiac pathologies, resulting in a
diverse range of clinical outcomes.
Although relatively uncommon, sudden cardiac death remains the most devastating complication of HCM. The
prevalence of sudden death ranges from 0.5% to 5% in
various reported studies, most of which are derived from
tertiary referral centers and therefore have an inherent tertiary
referral bias of more severe cases.10,11 Sudden cardiac death
in HCM is often associated with exercise and, in some cases,
related to high-level, high-profile competitive sports.11,12
Over recent years, a number of risk stratification factors have
been identified in patients with HCM. Specifically, a positive
family history of HCM, a previous resuscitated cardiac arrest,
a left ventricular wall thickness greater than 30 mm, syncope,
Genetic Basis of HCM
␤-Myosin heavy chain
MyBP-C
Figure 1. Clinical and genetic heterogeneity in genetic cardiovascular disease. A number of potential modifying factors are
likely to be involved in disease pathogenesis.
183
TTN
␣MHC
␣-Myosin heavy chain
⬍0.5
cTnC
Cardiac troponin C
⬍0.5
and nonsustained ventricular tachycardia on 24-hour ambulatory ECG monitoring are all considered important risk
factors for sudden death in HCM.13 Other factors associated
with an increased risk of sudden death in HCM include an
abnormal blood pressure response to exercise, significant left
ventricular outflow tract obstruction, and the presence of
specific “malignant” gene mutations.14,15
Although important advances have been made in identifying factors that place patients with HCM at highest risk of
developing the 2 most serious complications of disease (ie,
progressive heart failure and sudden cardiac death), there are
likely to be other key factors that may help to stratify risk
more precisely.
Genetic Basis of HCM
Since 1990, more than 400 mutations in at least 13 genes have
been identified in patients with HCM (summarized in Table
1).9,16 Approximately 70% of all mutations are identified in
the 2 most common genes: ␤-myosin heavy chain (␤MHC)
and myosin-binding protein C (MyBP-C).5 All mutations are
inherited in an autosomal dominant pattern and occur in
genes that encode proteins of the sarcomere or are associated
with sarcomere-related structures. As with the clinical variability of disease, genetic heterogeneity is an important
characteristic of HCM. Many other disease genes have been
implicated in HCM, but these more likely represent diseases
which clinically mimic HCM but actually represent a different pathological disease, eg, glycogen storage diseases caused
by mutations in the PRKAG2 gene.17,18
Multiple Mutations in HCM Patients
Figure 2. Zygosity of disease-causing mutations. Individuals
carrying a single disease-causing mutation are heterozygous at
that allele, whereas individuals carrying multiple disease-causing
mutations can be trans or cis compound heterozygous or
homozygous if the mutations are in the same allele, or double
heterozygous if the mutations are in different alleles. Crosses
indicate disease-causing mutations; boxes, alleles.
Most recently, a number of genetic studies in HCM have
suggested that up to 5% of families carry 2 distinct diseasecausing gene mutations.4 – 6 Compared with individuals with
single-gene mutations, HCM patients with homozygous and
double or compound heterozygous mutations typically present with more severe left ventricular hypertrophy and a higher
incidence of sudden cardiac death events, including resuscitated cardiac arrest, among family members. Collectively,
patients with multiple mutations are also significantly
184
Circ Cardiovasc Genet
Table 2.
April 2009
Homozygous HCM Mutations
Gene 1
Mutation 1
Gene 2
Mutation 2
SD/HTx
IVS, mm (Age, y)
Reference
␤MHC
Lys207Gln
␤MHC
Lys207Gln
No
21 (64)
Mohiddin et al19
Alpert et al20
␤MHC
Arg403Trp
␤MHC
Arg403Trp
HTx
20 (38)
Keller et al21
␤MHC
Asp778Glu
␤MHC
Asp778Glu
SD
19 (NA)
Richard et al5
␤MHC
Arg869Gly
␤MHC
Arg869Gly
No
35 (29)
Richard et al22
Richard et al5
␤MHC
Glu935Lys
␤MHC
Glu935Lys
No
13 (25)
Nishi et al23
MyBP-C
Gln76Ter
MyBP-C
Gln76Ter
No
16 (⬍1)
Richard et al5
MyBP-C
Ala627Val
MyBP-C
Ala627Val
No
28 (47)
Garcia-Castro et al24
MyBP-C
Arg810His
MyBP-C
Arg810His
No
32 (32)
Nanni et al25
MyBP-C
Pro873His
MyBP-C
Pro873His
No
27 (27)
Nanni et al25
MyBP-C
Asp1064fsX38
MyBP-C
Asp1064fsX38
SD
NA
Xin et al26
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cTnT
Phe110Ile
cTnT
Phe110Ile
No
21 (49)
Lin et al27
cTnT
Ser179Phe
cTnT
Ser179Phe
SD
25 (17)
Ho et al28
IVS indicates interventricular septum; SD, sudden death event; HTx, heart transplant; and NA, not available.
younger at diagnosis and more commonly present with
childhood-onset hypertrophy. In HCM, multiple mutations
have most commonly been reported to involve the ␤MHC and
MyBP-C genes, and include homozygote, double heterozygote, and compound heterozygote carriers (Figure 2).4 – 6
Homozygous HCM Mutations
A number of HCM cases have been reported over the last
decade in which homozygous mutations have been identified,
most frequently associated with both more severe clinical
disease and, in some cases, presentation in childhood (Table
2). Nishi et al23 first reported 2 brothers homozygous for the
Lys935Glu mutation in the ␤MHC gene. They presented with
severe left ventricular hypertrophy and a clinical course
culminating in severe progressive heart failure and sudden
death in their fourth decade of life. Both parents were
heterozygous for the Lys935Glu mutation, and although both
had left ventricular hypertrophy, both were asymptomatic.
Four additional homozygous mutations in ␤MHC have since
been reported, all demonstrating a severe clinical course
resulting in a dilated form of cardiomyopathy, associated with
heart failure, sudden death, and heart transplantation.5,19 –22,29
Homozygous mutations have also been described in the
MyBP-C gene in patients with HCM. Although single-gene
mutations in MyBP-C tend to cause later onset and less severe
disease, individuals homozygous for Arg810His and
Pro873His substitutions have more severe disease than heterozygous individuals.25 Furthermore, whereas HCM presenting in early childhood is relatively uncommon, 2 homozygous mutations that severely truncate the MyBP-C protein
have been reported as causing neonatal HCM, resulting in
sudden cardiac death within the first year of life.5 In 1 report,
a single mutation segregated in 3 Amish families with
heterozygous members showing mild HCM. However, each
family had a homozygous child who died of heart failure in
the first year of life.26 The intronic mutation identified was
shown to cause skipping of exon 30, leading to a premature
translation stop codon 211 amino acid residues from the
carboxyl-terminal end, thus removing the major myosinbinding domain of MyBP-C.
Fewer homozygous HCM cases have been reported in the
less commonly associated HCM genes. Homozygous
Phe110Ile and Ser179Phe mutations in the cardiac troponin T
(cTnT) gene have been reported in 2 large HCM families,
associated with significant biventricular hypertrophy and a
high incidence of sudden death.27,28 Each mutation was not
fully penetrant as some heterozygous family members were
asymptomatic, whereas others had mild left ventricular hypertrophy. The left ventricular septal wall thickness for 2
homozygous Phe110Ile patients was greater than family
members heterozygous for this mutation, suggesting that 1
normal cTnT allele can largely compensate for the disruption
caused by the Phe110Ile allele.
Collectively, individuals and families with homozygous
HCM mutations have been described. Their clinical profile is
highlighted by earlier onset of disease, more significant
cardiac hypertrophy, and more frequent progression to heart
failure and sudden cardiac death events when compared with
family relatives who carry single heterozygous mutations.
These observations support the notion of a mutation “dosage
effect,” whereby a larger amount of defective protein leads to
greater disruption to normal sarcomere function and resulting
in a more severe clinical course.
Double Heterozygous HCM Mutations
Double heterozygous mutations, whereby single diseasecausing mutations in 2 different genes exist, have been
reported in several HCM families (Table 3). Analogous to
families with homozygous mutations, double heterozygous
HCM patients also collectively display more severe clinical
disease. In the first such report, in a family in which both the
Glu1096Ter nonsense mutation in MyBP-C and the
Glu483Lys mutation in the ␤MHC genes coexisted, affected
family members who carried both mutations had more severe
left ventricular hypertrophy compared with those who carried
only 1 of the 2 mutations, suggesting an additive effect.5,30
Kelly and Semsarian
Table 3.
Multiple Mutations in Genetic Heart Disease
185
Double Heterozygous HCM Mutations
Gene 1
Mutation 1
Gene 2
Mutation 2
SD/HTx
IVS, mm (Age, y)
Reference
␤MHC
Ala355Thr
MyBP-C
Val896Met
No
NA
Richard et al5
Richard et al30
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␤MHC
Glu483Lys
MyBP-C
Glu1096Ter
No
30 (NA)
Richard et al5
␤MHC
Arg694Cys
MyBP-C
Gln791fsX40
NA
NA
van Driest et al4
␤MHC
Arg719Gln
MyBP-C
Arg273His
HTx
17 (35)
Ingles et al6
␤MHC
Glu894Gly
MyBP-C
Asp605Asn
NA
NA
van Driest et al4
␤MHC
Arg453Cys
cTnT
Gln191del
NA
NA
van Driest et al4
␤MHC
Arg453Ser
cTnI
Pro82Ser
No
15 (19)
Frazier et al31
␤MHC
Cys905Phe
cTnI
Ser166Phe
NA
14 (39)
Erdmann et al32
␤MHC
Arg787Cys
ACTC
Arg97Cys
No
NA
Morita et al33
MyBP-C
Val256Ile
cTnT
Arg92Trp
NA
NA
van Driest et al4
MyBP-C
Ala833Thr
cTnT
Arg286His
NA
NA
van Driest et al4
MyBP-C
Arg495Gln
cTnI
Arg141Gln
No
NA
Morita et al33
MyBP-C
Arg943Ter
cTnI
Ser166Phe
NA
NA
van Driest et al4
MyBP-C
Phe1113Ile
␣TM
Ile172Thr
NA
NA
van Driest et al4
IVS indicates interventricular septum; SD, sudden death event; HTx, heart transplant; and NA, not available.
Ingles et al6 recently reported on a double heterozygous
proband having both Arg719Gln and Arg273His mutations in
the ␤MHC and MyBP-C genes, respectively. The family
pedigree is shown in Figure 3A. The proband (IV:4) passed
on both mutations to her son (V:1), who is clinically affected
at age 15 years, whereas his brother, aged 13 years (V:2), who
inherited only the MyBP-C mutation, is clinically normal.
Double heterozygous mutations in HCM seem to occur
most commonly in the ␤MHC and MyBP-C genes, which is
most likely due to mutations in these genes being the most
common causes of HCM. Apart from the MyBP-C gene,
mutations in the ␤MHC gene have also been reported to occur
together with mutations in the cTnT, cardiac troponin I
(cTnI), and cardiac actin (ACTC) genes. Similarly, mutations
in the MyBP-C gene have been reported to coexist with
mutations in the cTnT, cTnI, and the ␣-tropomyosin (␣TM)
genes. Double mutations affecting ␤MHC with ACTC, and
MyBP-C with cTnI have been reported to cause severe
childhood-onset hypertrophy, whereas each mutation in the
single heterozygous state causes only mild HCM.33 This
supports the collective data that, like homozygous HCM
patients, double heterozygous patients display more clinically
severe disease in HCM compared with single heterozygous
patients.
Compound Heterozygous FHC Mutations
Most reports of multiple mutation carriers in HCM to date
have involved compound heterozygous mutations (ie, when 2
mutations are identified in the same gene) (Table 4). This was
first reported in a proband with a nonsense mutation and
missense mutation on different ␤MHC alleles, but only those
family members heterozygous for the missense mutation
were clinically affected.34 The nonsense mutation did not
show a dominant phenotype. One possibility is that the
mutation might not be translated and incorporated into the
sarcomere. In a second report, a boy aged 6 years, who had
marked left ventricular hypertrophy and suffered a cardiac
arrest, had a de novo Arg719Trp mutation on the paternally
inherited ␤MHC allele and a Met349Thr mutation on the
maternally inherited ␤MHC allele.35 The Arg719Trp mutation has been widely reported as causing severe HCM, with a
mortality rate of 50% by age 38 years.37 The Met349Thr
mutation was present in 5 asymptomatic family members and
has been reported as a polymorphic variant in the normal
population, making its pathogenecity in this family unclear.
Compound heterozygous mutations in the MyBP-C gene
have also been reported and seem to also correlate with more
severe disease. In one report, a baby with severe hypertrophy
who died suddenly at age 5 weeks was found to carry a
compound heterozygous mutation in the MyBP-C gene.36 The
normal mother had a single base insertion causing a reading
frame shift, leading to a premature translation stop, whereas
the father, who had mild HCM, had an intronic mutation in an
invariant splice donor (AG) signal sequence, which predicts
skipping of exon 15 in the mRNA. The same authors further
reported on a second unrelated baby who died at age 6 weeks
and also had 2 MyBP-C mutations that caused premature
termination of translation. In both of these cases, the babies
lacked an intact MyBP-C protein, accounting for the neonatal
HCM observed, similar to the situation seen in patients with
homozygous MyBP-C nonsense mutations discussed previously. The parents, who are heterozygous for the mutations,
have 1 intact functional allele, which presumably largely
compensates for the MyBP-C haploinsufficiency.
Multiple Mutations in Other Genetic
Cardiovascular Disorders
Although the number of reports are limited to date, it seems
that up to 5% of families with HCM have multiple mutations,
which include homozygous, double heterozygous, and compound heterozygous carriers. Collectively, these multiple
mutation HCM patients have clinically more severe disease,
including earlier age of disease onset, more severe left
ventricular hypertrophy, and more frequent and rapid progression to the most significant complications of HCM (ie,
186
Circ Cardiovasc Genet
April 2009
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Figure 3. Double heterozygous mutations in HCM. A, HCM family with double heterozygous mutations in the ␤MHC (Arg719Gln) and
MyBP-C (Arg273His) genes. The proband, IV:4 (arrowed), carries both mutations. Squares indicate males; circles, females; filled-in symbols, clinically affected individuals; crossed symbols, deceased individuals; N, clinically unaffected; ⫹, carries mutation; and ⫺, does
not carry mutation. B, Double-mutant TnI-203/MHC-403 mice with severe disease. Kaplan–Meier survival curves show 100% mortality
by age 21 days due to a severe dilated phenotype, marked interstitial myocardial fibrosis, heart failure, and, in some cases, inducibility
of ventricular tachycardia. Arrow indicates onset of ventricular tachycardia. Data derived from References 6 and 49.
heart failure and sudden death. This suggests identification of
2 disease-causing mutations in an affected individual may be
an additional marker in terms of risk stratification and may
identify a subgroup of patients who may require more
aggressive treatment and initiation of prevention strategies.
Although multiple mutations have been most frequently
reported in HCM, it seems the notion of 2 disease-causing
gene mutations causing more severe clinical disease is not
restricted to only HCM. Recent reports in a number of other
genetic cardiovascular disorders have implicated a role of
multiple mutations in disease severity. Two examples are
briefly mentioned here.
Multiple Mutations in FH
FH is a common inherited disease caused by dominant single
mutations in any one of several genes that affect receptormediated uptake of low-density lipoproteins, such as the
low-density lipoproteins receptor, apolipoprotein B100, and
neural apoptosis-regulated convertase 1.38 The prevalence of
FH in the general population is estimated to be between 0.2%
Kelly and Semsarian
Table 4.
Multiple Mutations in Genetic Heart Disease
187
Compound Heterozygous HCM Mutations
Gene 1
Mutation 1
Gene 2
Mutation 2
SD/HTx
IVS, mm (Age, y)
Reference
␤MHC
Val39Met
␤MHC
Arg723Cys
NA
20 (NA)
Richard et al5
␤MHC
Arg54Ter
␤MHC
Arg870His
No
20 (16)
Nishi et al34
␤MHC
Pro211Leu
␤MHC
Arg663His
No
20 (65)
Mohiddin et al19
␤MHC
Met349Thr
␤MHC
Arg719Trp
SD
18 (8)
Jeschke et al35
␤MHC
Arg663His
␤MHC
Val763Met
No
NA
Morita et al33
␤MHC
Arg719Gln
␤MHC
Thr1513Ser
NA
NA
van Driest et al4
␤MHC
Asp906Gly
␤MHC
Leu908Val
No
NA
Alpert et al20
Gly5Arg
MyBP-C
Arg502Trp
NA
NA
van Driest et al4
MyBP-C
Gln76Ter
MyBP-C
His257Pro
No
18 (NA)
Richard et al5
MyBP-C
Ile154Thr
MyBP-C
Asp605del
No
NA
Morita et al33
MyBP-C
Glu258Lys
MyBP-C
Ala954fsX94
NA
NA
van Driest et al4
MyBP-C
Arg502Trp
MyBP-C
Ser858Asn
No
NA
Morita et al33
MyBP-C
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MyBP-C
Glu542Gln
MyBP-C
Ala851Val
No
34 (34)
Ingles et al6
MyBP-C
Asp745Gly
MyBP-C
Pro873His
No
30 (29)
Ingles et al6
MyBP-C
Trp792fsX17
MyBP-C
IVS15⫹1G⬎A
SD
11 (⬍1)
Lekanne Deprez et al36
MyBP-C
Arg810His
MyBP-C
Arg820Gln
No
23 (53)
Nanni et al25
MyBP-C
Arg943Ter
MyBP-C
Glu1096fsX92
SD
NA
Lekanne Deprez et al36
MyBP-C
Thr1028Ser
MyBP-C
IVS31⫹2T⬎G
No
NA
Morita et al33
MyBP-C
Gln1233Ter
MyBP-C
Arg326Gln
No
28 (53)
Ingles et al6
IVS indicates interventricular septum; SD, sudden death event; HTx, heart transplant; and NA, not available.
and 0.5%. FH heterozygotes have elevated low-density lipoproteins particle concentrations in plasma from birth up to
3 times greater than unaffected individuals and increasing
with age.39 From approximately the third decade of life these
patients also develop xanthomatous lesions and have a greater
risk of myocardial infarction.
The prevalence of patients with multiple FH diseasecausing mutations is estimated at 1 in 1 million people in the
general population.39 This condition is commonly referred to
as homozygous FH, although the term compound heterozygote is more fitting as many of these individuals inherit a
different disease-causing mutation from each parent. The
inheritance of multiple FH-causing mutations results in a
more severe phenotype and is consistent with the findings in
patients with multiple mutations causing HCM. Specifically,
patients with multiple FH-causing mutations develop disease
earlier in life, with plasma low-density lipoprotein particle
concentrations being elevated 6- to 8-fold during intrauterine
life.40 Furthermore, multiple mutation FH patients develop
the characteristic xanthomatous lesions in the first years of
life. It is rare for these FH patients to live past the age of 30
years because of the high risk of myocardial infarction due to
extensive coronary atherosclerosis.
Familial LQTS
Familial LQTS is an autosomal dominantly inherited disease
with a reported prevalence of ⬇1 in 5000, although it is likely
that this is an underestimate.41 Mutations in at least 10 genes
affecting Na⫹, K⫹, and Ca2⫹ ion currents in cardiomyocytes
are known to cause familial LQTS. Symptoms can include
syncope and sudden death due to ventricular fibrillation,
however, ⬇30% of LQTS single mutants are asymptomatic
gene mutation carriers with a normal QT interval.41,42
Recent case reports of both compound and double heterozygous LQTS patients show they have more severe
disease, with an earlier age of disease onset and a more
prolonged QT interval in double-mutant patients compared
with single-mutant family members.43 A recent study by
Tester et al42 screened 5 cardiac channel genes in 541
unrelated LQTS patients and found the prevalence of doublemutations to be 5% and that these double-mutant patients
were significantly younger at diagnosis.
Molecular Pathogenesis of Multiple
Mutation Phenotypes
The presence of multiple mutations in a number of different
genetic cardiovascular disorders, and the association of multiple mutations with a more severe clinical phenotype, suggests the presence of 2 mutations has an additive effect in
terms of molecular pathogenesis. As more genotype-phenotype correlation studies are performed in these multiple
mutation cohorts, an important step is to understand the
underlying mechanisms of how mutations interact to cause
more profound disease and how these interactions could be
modulated to improve clinical outcomes. To this end, studies
in animal models are beginning to shed light on the role of
multiple mutations in cardiovascular disease.
In HCM, a number of murine models have been developed
over the last decade that support the clinical observation that
multiple mutations lead to more severe HCM, associated with
rapid progression to heart failure and premature death. Until
recently, these models have predominantly involved homozygous mutations involving key cardiac sarcomere proteins.
Two separate models of homozygote HCM mutations have
been reported. In the first model, in which the ␣MHC
188
Table 5.
Circ Cardiovasc Genet
April 2009
Comparison of Phenotype in Humans and Mice
Clinical Feature
Survival
HCM Patients With
Multiple Mutations
Double-Mutant
TnI-203/MHC-403
Mice
Decreased (frequently
⬍40 y)
Decreased
(21 d)
Early onset
Yes
Yes
Left ventricular hypertrophy
Yes
Minimal
Progression to dilation
Yes
Yes
Ventricular tachycardia
Present
Present
Increased sudden death
Yes
Yes
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Arg403Gln mutation is introduced by homologous recombination, heterozygote mice develop mild HCM by age 30
weeks with a normal lifespan.44 – 46 However, when bred to
homozygosity, these mice develop a severe neonatal dilated
cardiomyopathy, with death within 8 days of birth.47 In the
second model, in which a truncated form of MyBP-C is
introduced, heterozygote mice develop mild HCM by age 30
to 50 weeks with a normal lifespan.44 However, when bred to
homozygosity, these mice develop a dilated cardiomyopathy
early in life, although there is cardiac compensation and
lifespan is normal.48
Most recently, the first mouse model of double heterozygous HCM was reported in Circulation.49 In brief, 2 previously reported mouse models of HCM (ie, the cTnI
Gly203Ser and ␣MHC Arg403Gln models) were crossed to
develop a double-mutant genotype (designated TnI-203/
MHC-403). By age 14 days, the TnI-203/MHC-403 doublemutant mice develop a severe phenotype characterized by
cardiac hypertrophy followed by 4-chamber cardiac dilatation, heart failure, severe myocyte disarray and interstitial
fibrosis, and inducible ventricular tachycardia (Figure 3B).
By age 21 days, mortality is 100%. In beginning to understand the underlying molecular mechanisms in this severe
model of double-mutant HCM, preliminary results suggest a
significant downregulation of key Ca2⫹ regulation genes
(ryanodine receptor, L-type Ca2⫹ channel, SERCA2a, and
phospholamban) as well as a cardiac-specific upregulation of
the phosphorylated (active) form of the latent transcription
factor signal and activator of transcription-3 (STAT-3) with
onset of disease.49
This double-mutant model replicates the subgroup of
human HCM patients (Table 5) who have severe heart failure
requiring aggressive treatment, including transplantation.
Further investigation of key animal models of HCM caused
by multiple mutations will be important to investigate the
mechanisms involved in HCM and to help elucidate possible
mechanistic links between HCM and dilated cardiomyopathy.
Development of cellular and animal models of other genetic
cardiovascular disorders caused by multiple mutations will be
an important step in our understanding of the role of genegene interactions and how this translates to the clinical
phenotype.
Impact of Multiple Mutations on
Clinical Practice
The identification of multiple mutations in individual patients
with a number of different inherited cardiovascular disorders
has a major impact on genetic diagnosis, highlights the
importance of genetic counseling in families, and raises the
possibility that such multiple mutation carriers may require
more aggressive and targeted treatment.
The role of genetic testing and counseling is clearly an
important component of the evaluation, diagnosis, and management of individuals and families with genetic cardiovascular diseases. The identification of multiple mutations
among individual patients directly impacts on the genetic
testing strategy.6 For example, whole panels of genes rather
than single-gene testing should be carried out in new families
presenting with disease. The implications to a family of not
identifying a second gene mutation could be devastating. For
example, an individual with a family history of HCM who
receives a negative predictive gene test is released from
clinical screening and believe their children are no longer at
risk of developing HCM.
In addition, a key aspect of the genetic counseling process
is the explanation of the inheritance of the disease and risk to
children. The presence of multiple mutations in an autosomal
disorder such as HCM or familial LQTS, alters the chances of
inheritance in subsequent testing of offspring because there
are now 3 possibilities of what the offspring may inherit (ie,
no mutation, 1 mutation, or 2 mutations). This is illustrated in
the family described in Figure 3A. The proband in this family
is a double heterozygote carrying a gene mutation on 2
different genes inherited independently. Therefore, the risk
that her child will receive at least 1 disease-causing gene
mutation becomes 75%, with a 25% chance that the child will
inherit both gene mutations. These are important considerations for a family and highlight the importance of accurate
genetic testing of the proband. Of equal importance is
ensuring cardiologists dealing with families with an inherited
cardiovascular disorder are equipped with the skills to offer
appropriate genetic counseling, perhaps as part of a multidisciplinary cardiac genetic service.6,50
The impact on treatment and prevention strategies in those
who carry multiple mutations currently remains unclear.
Although there is a clear association of multiple mutations
with a more severe clinical phenotype in HCM, familial
LQTS, and FH, further genotype-based prospective studies
are required to fully evaluate the use of multiple mutation
information for risk stratification purposes. For example, the
presence of multiple mutations in some cardiovascular diseases which can lead to sudden death may become part of a
risk stratification algorithm, leading to the earlier initiation of
prevention therapies such as ␤-blockers or insertion of an
implantable cardioverter-defibrillator.
One of the key issues that needs to be resolved in the field
of multiple mutations in human disease is determining
whether the DNA variants identified are truly pathogenic,
rather than common silent polymorphisms of no functional
significance. It is possible that in some cases, including ones
already reported in the literature, both DNA variants are not
causative. Therefore, rigorous methods and approaches will
be required to determine the pathogenicity of each DNA
variant identified in these patients. Traditional factors which
may support the notion that a DNA variant is causative need
to be investigated, including coinheritance with disease,
Kelly and Semsarian
conservation of the altered amino acid across species, and
absence of the variant in normal populations. Furthermore,
assays and methods to demonstrate functional significance,
including both cellular and animal models, will all assist in
determining the pathogenicity of the DNA variants identified
and their role in clinical disease.
Multiple Mutations in Genetic Heart Disease
8.
9.
10.
Conclusions
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The occurrence of multiple gene mutations in patients with
inherited cardiovascular diseases has changed the longstanding paradigm that a single mutation in a single gene is
the cause of disease. Patients with multiple mutations have
been found to develop more severe clinical disease, including
earlier onset of disease, and more rapid development of
disease complications including progressive heart failure and
premature death. This suggests an additive effect of these
multiple gene mutations on cardiovascular pathology and
function and highlights the importance of basic molecular,
cellular, and animal model studies to elucidate the key
pathogenic pathways. The presence of multiple mutations has
a direct impact on genetic diagnosis strategies as well as
genetic counseling approaches whereby the chances of inheritance in offspring are altered depending on the type of
multiple mutations. Although not established currently, the
presence of multiple gene mutations may play a role as a
marker of disease severity and therefore identify patients who
would most benefit from earlier treatment and prevention
strategies.
11.
12.
13.
14.
15.
16.
17.
18.
19.
Acknowledgments
We thank Drs Richard Bagnall and Tatiana Tsoutsman for assistance
with literature searches and intellectual input.
20.
Disclosures
Dr Semsarian is the recipient of a cofunded National Health and
Medical Research Council (NHMRC) and National Heart Foundation of Australia (NHFA) Practitioner Fellowship in addition to
research grants from the NHMRC, NHFA, and RT Hall Foundation
(Sydney, Australia).
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KEY WORDS: cardiomyopathy 䡲 diagnosis 䡲 genetics 䡲 genetic heart
disease 䡲 gene 䡲 cardiovascular 䡲 multiple mutation 䡲 disease severity
Multiple Mutations in Genetic Cardiovascular Disease: A Marker of Disease Severity?
Matthew Kelly and Christopher Semsarian
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Circ Cardiovasc Genet. 2009;2:182-190
doi: 10.1161/CIRCGENETICS.108.836478
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