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(Circulation. 2001;103:65.)
© 2001 American Heart Association, Inc.

Clinical Investigation and Reports

Hypertrophic Cardiomyopathy Caused by a Novel -Tropomyosin Mutation (V95A) Is Associated With Mild Cardiac Phenotype, Abnormal Calcium Binding to Troponin, Abnormal Myosin Cycling, and Poor Prognosis

Akihiko Karibe, MD, PhD; Larry S. Tobacman, MD, PhD; James Strand, BS; Carol Butters, MA; Nick Back, PhD; Linda L. Bachinski, PhD; Andrew E. Arai, MD; Anne Ortiz, MD; Robert Roberts, MD; Earl Homsher, PhD; Lameh Fananapazir, MD, FRCP

From the Department of Medicine, Baylor College of Medicine (A.K., L.L.B., R.R.), Houston, Tex; Departments of Internal Medicine (L.S.T., J.S., C.B.) and Biochemistry (L.S.T.), University of Iowa, Iowa City, Iowa; Department of Physiology (N.B., E.H.), University of California, Los Angeles; and National Heart, Lung, and Blood Institute (A.E.A., A.O., L.F.), National Institutes of Health, Bethesda, Md.

	Abstract

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Background—We report hypertrophic cardiomyopathy (HCM) in a Spanish-American family caused by a novel -tropomyosin (TPM1) mutation and examine the pathogenesis of the clinical disease by characterizing functional defects in the purified mutant protein.

Methods and Results—HCM was linked to the TPM1 gene (logarithm of the odds [LOD] score 3.17). Sequencing and restriction digestion analysis demonstrated a TPM1 mutation V95A that cosegregated with HCM. The mutation has been associated with 13 deaths in 26 affected members (11 sudden deaths and 2 related to heart failure), with a cumulative survival rate of 73±10% at the age of 40 years. Left ventricular wall thickness (mean 16±6 mm) and disease penetrance (53%) were similar to those for the ß-myosin mutations L908V and G256E previously associated with a benign prognosis. Left ventricular hypertrophy was milder than with the ß-myosin mutation R403Q, but the prognosis was similarly poor. With the use of recombinant tropomyosins, we identified several functional alterations at the protein level. The mutation caused a 40% to 50% increase in calcium affinity in regulated thin filament–myosin subfragment-1 (S1) MgATPase assays, a 20% decrease in MgATPase rates in the presence of saturating calcium, a 5% decrease in unloaded shortening velocity in in vitro motility assays, and no change in cooperative myosin S1 binding to regulated thin filaments.

Conclusions—In contrast to other reported TPM1 mutations, V95A-associated HCM exhibits unusual features of mild phenotype but poor prognosis. Both myosin cycling and calcium binding to troponin are abnormal in the presence of the mutant tropomyosin. The genetic diagnosis afforded by this mutation will be valuable in the management of HCM.

Key Words: cardiomyopathy • genetics • death, sudden • prognosis

	Introduction

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Hypertrophic cardiomyopathy (HCM) is characterized by left ventricular (LV) hypertrophy (LVH) and myocyte disarray. Clinical characteristics of HCM vary from a benign asymptomatic course to severe heart failure and sudden death. Molecular genetic studies^{1 2 3 4 5 6 7} have shown that HCM may be caused by mutations in several sarcomeric genes, including TPM1, that may determine clinical outcome. Most ß-myosin heavy chain gene (MYH7) mutations, such as R403Q, are associated with high disease penetrance and a poor prognosis.⁴ In contrast, the few MYH7 mutations that have a benign prognosis, such as L908V and G256E, are associated with a low disease penetrance.⁵ HCM caused by myosin-binding protein-C gene (MYBPC3) is characterized by low disease penetrance (60%), mild cardiac phenotype in young subjects, and a favorable prognosis.^{1 2 3} HCM due to cardiac troponin-T gene (TNNT2) is usually associated with relatively low disease penetrance (80%) and mild LVH but a high incidence of sudden death.⁶

The TPM1 gene consists of 14 exons and 4 isoforms (- and ß-tropomyosins, tropomyosin-4, and tropomyosin-30).^{1 8} The cardiac isoform is generated from 10 exons, is expressed in both myocardium and fast skeletal muscle fibers, and consists of 284 amino acids.⁸ Tropomyosin, actin, and troponin complex (troponins T, C, and I) make up most of the thin filament in striated muscle. Tropomyosin is a rigid, rodlike protein that binds along the length of the actin filament and is intimately associated with troponin complex.⁹ It adds to the rigidity and stability of the thin filament. In the absence of Ca²⁺ binding to troponin C, -tropomyosin inhibits the binding of myosin to actin. Binding of Ca²⁺ to troponin C results in the release of myosin-binding site of actin by the tropomyosin-troponin complex. The interaction of actin and myosin heads then generates contractile force.^{9 10}

Mutations in TPM1 account for 3% of cases of HCM.^{11 12 13 14} Four TPM1 mutations have been described: A63V, K70T, D175N, and E180G. The first 2 mutations are located in exon 2b in an area that may alter the binding of -tropomyosin to actin, and the latter 2 mutations are located in exon 5 near a Ca²⁺-dependent troponin-T binding domain.

We report here a novel TPM1 missense mutation with the unique features of mild cardiac hypertrophy but a high mortality rate. We also describe the properties of the isolated mutant protein, which exhibits fundamental defects in regulatory function.

	Methods

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Research Subjects
We identified HCM in a large Spanish-American family (Figure 1). Seventy-four family members were enrolled in the studies. Informed consent for the genetic studies was obtained under a protocol approved by the Institutional Review Board of the National Heart, Lung, and Blood Institute.

View larger version (26K): [in this window] [in a new window]   Figure 1. Kindred No. 001. Squares indicate males; circles, females; filled symbols, affected individuals; unfilled symbols, unaffected family members; and hatched symbols, mutation present but normal echocardiogram. Deceased family members are indicated by diagonal slash.

Genetic Studies
Linkage analysis was performed with markers from the ABI screening set 2¹⁵ and MLINK module of the LINKAGE 5.2 program.¹⁶ Sequences from exon 1 to exon 9a,b of TPM1 were amplified from genomic DNA with primers as previously described¹⁷ for 2 affected and 2 control individuals. To confirm segregation of the mutation in the family and absence of the mutation in normal subjects, amplified exon 3 fragments were digested with restriction endonuclease DdeI (Boehringer-Mannheim) and analyzed by electrophoresis through a 2% agarose gel.

Clinical Studies
Studies included 2D echocardiography, 12-lead ECG, and in selected cases, treadmill exercise test, exercise thallium scintigraphy, MRI, cardiac catheterization, and angiography. We also reviewed the clinical records, death certificates, and autopsy reports of 6 family members who died suddenly.

HCM was defined as LV wall thickness >13 mm in the absence of another cause for the LVH. Disease penetrance was defined as number of family members with HCM divided by the number of family members with the mutation.

The findings were compared with those previously reported for the MYH7 mutations L908V, G256E, and R403Q with well-defined clinical characteristics.^{4 5}

Biochemical Studies
Polymerase chain reaction–based mutagenesis of Ala-Ser rat striated muscle -tropomyosin¹⁸ was performed as described previously¹⁹ to produce cDNA encoding the V95A mutation. The fidelity of the entire coding sequence was confirmed by automated DNA sequencing. As previously for other recombinant tropomyosins, wild-type and mutant tropomyosins were expressed in DE3 cells with the pET3d expression vector²⁰ and then purified to homogeneity.²⁰ Purified actin,²¹ myosin subfragment-1 (S1),²² and troponin²³ were obtained as described previously.

Thin filament–myosin S1 MgATPase rates were determined by release of ³²P-P_i from ATP,^{24 25} and the free calcium concentration was manipulated with mixtures of CaCl₂ and di-bromo BAPTA.²⁶ MgATPase data as a function of the free calcium concentration were analyzed according to equation 12 in Tobacman and Sawyer,²⁶ using Scientist for nonlinear least squares fitting. We determined in vitro motility by labeling reconstituted regulated thin filaments with rhodamine-phalloidin and monitoring their movement over a rabbit skeletal heavy meromyosin–coated surface using epifluorescence microscopy and quantitative analysis of motion.^{27 28} Actin-myosin S1 binding was monitored by steady-state fluorescence with pyrene-labeled actin as described by Criddle et al²⁹ and Rosol et al.³⁰ These data were analyzed according to the model of Hill et al³¹ for cooperative myosin binding to the thin filament, using Scientist for nonlinear least squares curve fitting.

Statistics
Patient data are presented as mean±SD. Differences for mean values of maximum LV wall thickness were compared by Student’s t test. Cumulative survival was determined by product-limit survival analysis with sudden death as the time variable. The log-rank test was used to compare actuarial survival curves. A 2-tailed value of P<0.05 was considered significant.

	Results

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Genetic Results
The TPM1 marker D15S127 gave a 2-point LOD score of 3.17 in 24 members of the family. A heterozygous base change from T to C was observed at nucleotide position 340 (cDNA sequence of the TPM1, GenBank accession No. M19713) in exon 3 from 2 affected individuals (Figure 2A). A change from a valine to alanine in amino acid residue 95 of the protein is predicted as a result of this change. The mutation adds a recognition site for the restriction endonuclease DdeI and changes the restriction digestion pattern of amplified exon 3 fragments (Figure 2B). Restriction digestion was performed to detect the mutation in 56 family members. All family members who were diagnosed with HCM had the V95A mutation. The mutation was not detected in the 200 unrelated normal individuals.

View larger version (44K): [in this window] [in a new window]   Figure 2. A, Electropherogram showing region of TPM1 exon 3 that contains T-to-C mutated base that results in substitution of valine for alanine at codon 95. Sequence from normal individuals is shown in top panel and from affected individual in bottom panel. Mutated base (N) is heterozygous for wild-type and mutant C. B, Subsection of pedigree is shown above photo. Each lane contains digested product of exon 3 from 1 family member. Pedigree corresponds to gel lanes 1 through 5. Lane U contains undigested amplification product consisting of 252 base pairs (bp). Lane M contains 100-bp DNA ladder. Normal DdeI restriction enzyme site B produces fragments of 177 and 75 bp that are present in all family members. T-to-C substitution creates a second DdeI restriction enzyme site (A). This results in smaller 114- and 63-bp fragments. Abnormal 114-bp fragment is present in affected male 231 and affected daughter 327 and is absent in remaining unaffected family members 326, 328, and 233.

Cardiac Phenotype
The data of 26 affected family members were examined. The individuals included all 14 members positive for the mutation, 11 additional members who died of the disease, and an affected family member for whom no DNA was available. The area of maximum LV wall thickness was localized to posterior basal wall, anterolateral free wall, and/or apex in 9 patients. None of the patients had LV outflow obstruction.

Maximum LV wall thickness was 16±6 mm (range 8 to 27 mm). MRI-aided diagnosis of HCM as LVH was very localized in some patients.

The ECG was abnormal in 11 (79%) of 14 affected family members, but only 6 (43%) showed LVH by voltage criteria. Eight (57%) of the 14 affected members showed ST-T–wave abnormalities. Two subjects with LV wall thickness of 10 and 13 mm, respectively, developed significant ST-wave depression during treadmill test but had normal coronary angiograms.

Disease Penetrance
Figure 3 shows the relation between maximum LV wall thickness and age in the family members. The maximum LV wall thickness was <12 mm in all adult members without the mutation. LVH and disease penetrance (53%, 8 of a total of 15 affected members in whom echocardiograms were available) were similar to subjects with the MYH7 mutations G256E and L908V (Figure 4). LVH and penetrance were significantly less than in the MYH7 mutation R403Q.

View larger version (21K): [in this window] [in a new window]   Figure 3. Relation of maximum LV wall thickness and age. Only 2 patients with mutation had both normal ECG and echocardiograms. However, 6 older family members had minor T-wave ECG changes in absence of mutation.

View larger version (21K): [in this window] [in a new window]   Figure 4. Comparison of LV hypertrophy in subjects with TPM1 mutation V95A and MYH7 mutations G256E, L908V, and R403Q.

Prognosis
LV systolic dysfunction was noted in 5 patients; 3 were complicated by symptomatic bradycardia or cardiac arrest, and 2 are asymptomatic. There have been 13 deaths: 11 sudden deaths and 2 related to heart failure. Four of the sudden deaths were related to physical activity; 2 occurred in subjects with mild or no LVH. An additional patient has presyncope with exertion. Cumulative survival rates have been 73±10% and 32±13% at 40 and 60 years of age, respectively. The survival rate was worse than that for the MYH7 mutations L908V and G256E and similar to that for the MYH7 mutation R403Q (Figure 5).

View larger version (23K): [in this window] [in a new window]   Figure 5. Comparison of cumulative survival rates of members with TPM1 mutation V95A and MYH7 mutations G256E, L908V, and R403Q.

Effect of the Mutation on Protein Function
Effect of the Mutation on Calcium-Sensitive Regulation of Acto-Myosin S1
To understand how the mutation acts to produce the observed clinical phenotype, control and mutant tropomyosins were expressed in bacteria and their properties studied in detail. Previous studies of HCM tropomyosin mutants 175 and 180 suggested that these alterations increased the calcium sensitivity of muscle contraction in fibers or cells containing mixtures of normal and mutant molecules.^{32 33} This effect has not been demonstrated in a purified reconstituted system for any HCM tropomyosin mutation. Thin filaments were reconstituted with actin, troponin, and recombinant tropomyosin, and a biochemical correlate of muscle regulation was evaluated: calcium-sensitive regulation of myosin S1 MgATPase activity. Representative data are shown in Figure 6. The V95A mutation resulted in abnormal regulation, characterized most notably by increased calcium affinity. To examine this more precisely, multiple experiments performed as in Figure 6 showed a 40% to 50% increase in apparent calcium affinity, from 3.2±0.5 to 5.0±0.7x10⁵ M^-1, a 0.2 pCa shift. Also, Figure 6 suggests and additional determinations confirmed that the maximum MgATPase rate in the presence of saturating calcium was diminished from 2.95±0.20 to 2.28±0.18 s^-1, a 22±6% decrease in paired studies (n=5). This small but reproducible effect implies an effect of the mutation on myosin cycling. There were no statistically significant effects on the cooperativity of activation (Y=13±4 for control and 8±3 for V95A where Yn_H^{19 26} ) or on the ATPase rate in the absence of calcium (0.24±0.05 versus 0.25±0.02 s^-1) (Figure 7).

View larger version (14K): [in this window] [in a new window]   Figure 6. Effects of V95A TPM1 mutation on thin filament–myosin S1 MgATPase rate regulation with purified recombinant proteins. This representative experiment shows calcium-sensitive regulation of thin filament–activated myosin S1 ATP hydrolysis in presence of either normal (circles) or mutant (triangles) thin filaments in presence of calcium. Note left shift in curve when thin filaments contain mutant tropomyosin, showing increased apparent calcium affinity, from 3.4±0.2x105 mol/L-1 to 4.7±0.3x105 mol/L-1 in this experiment. Also, maximum rate in presence of saturating calcium was 10% less when mutation was present. Conditions: 25°C, 7 µmol/L F-actin, 1 µmol/L tropomyosin, 1 µmol/L troponin, 0.3 µmol/L myosin S1, 20 mmol/L imidazole (pH 7.5), 3.5 mmol/L MgCl2, 7 mmol/L KCl, 1 mmol/L dithiothreitol. Plotted rates are moles of Pi per mole of myosin S1 per second.

View larger version (16K): [in this window] [in a new window]   Figure 7. Myosin S1 binding to thin filaments containing normal and mutant tropomyosins. Thin filaments containing actin, troponin, and either wild-type (circles) or V95A (triangles) tropomyosin were examined in either presence (A) or absence (B) of calcium. In both panels, titrations are indistinguishable for the 2 tropomyosins, indicating that V95A tropomyosin mutation does not alter myosin binding to thin filament. Binding is more cooperative in absence of calcium (B). Conditions: 25°C, 20 mmol/L imidazole (pH 7.5), 125 mmol/L KCl, 5 mmol/L MgCl2, 1 mmol/L dithiothreitol, 2 mmol/L ADP, 1 µmol/L actin, 0.5 µmol/L troponin, 0.5 µmol/L tropomyosin, 0.2 mg/mL bovine serum albumin, 14 U/mL hexokinase, 1 mmol/L glucose, 20 µmol/L Ap5A, 0.5 mmol/L EGTA, and (B only) 0.6 mmol/L CaCl2. Lines are based on best fit parameters, with affinity constant K=2.31±0.06 and 2.30±0.08x106 mol/L-1 for control and mutant tropomyosins in presence of calcium and 2.38±0.06 and 2.29±0.03 in absence of calcium.

Effect on In Vitro Motility
To determine whether the mutation altered mechanical actin-myosin function, the in vitro motility of control and mutant filaments was examined. Wild-type or mutant tropomyosin-containing actin-troponin-tropomyosin filaments were combined with rhodamine-phalloidin, and their unloaded sliding over a heavy meromyosin–coated surface was measured.²⁷ The data were analyzed 2 ways: for all of the observed filaments, and for the subset of filaments that moved over the surface at a continuous, smooth speed. In either case, the effect of the mutations was a small decrease (5%) in the maximum sliding speed in the presence of saturating calcium (Table). The large values of n (range 179 to 232) resulted in low standard errors for these measurements. (Standard deviation values are shown in the Table). Both control and mutant filaments were highly regulated by calcium: in the presence of calcium, 90% of the filaments moved smoothly and rapidly, whereas in the absence of calcium, <2% moved smoothly and did so at speeds that were decreased by 90%. These in vitro motility data are consistent with the MgATPase data; the mutation produces subtle alterations in myosin cycling but does not prevent regulation.

View this table: [in this window] [in a new window]   Table 1. Effect of TPM1 V95A Mutation on Myosin-Propelled, Calcium-Regulated, In Vitro Sliding of Thin Filaments

Effect on Myosin Cross-Bridge Binding to the Filament
The above effects could in principle be due to altered myosin binding to thin filaments containing the mutant tropomyosin. To evaluate this, actin was labeled on Cys374 with n-(1-pyrenyl)iodoacetamide, and binding of myosin S1-ADP to thin filaments was monitored by steady-state fluorescence. Thin filaments containing pyrene actin, troponin, and either wild-type or mutant tropomyosin were examined in the presence or absence of calcium. In both cases, tropomyosin had no effect on the myosin-binding isotherm (Figures 7A and 7B), indicating that the V95A mutation does not alter myosin binding to the thin filament.

	Discussion

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We identified a Spanish-American family with HCM caused by a novel TPM1 mutation V95A that was associated with several abnormalities of tropomyosin function, a low disease penetrance, and a mild and unusual distribution of LVH, yet a poor prognosis.

V95A Mutation in TPM1 and -Tropomyosin Function
That the TPM1 mutation V95A causes HCM is supported by several observations. First, linkage analysis showed a statistically significant linkage of the disease locus to chromosome 15q22. Second, the mutation was present in all clinically affected family members but not in 200 normal individuals (400 chromosomes; population frequency <0.25%). In addition, the mutated valine residue has been highly conserved in vertebrates throughout evolution.

In a series of recent articles,^{18 19 30} a region of tropomyosin that includes the site of the V95A mutation has been implicated as critical for thin filament activation. Preceding structural work^{34 35 36} suggested that the thin filament has 3 conformations: (1) without Ca²⁺ bound to troponin, and with tropomyosin blocking the myosin binding site on actin; (2) with Ca²⁺ bound to the troponin, causing tropomyosin movement on actin to partially expose the binding site for myosin; and (3) a third (fully active) conformation in which myosin is bound and the tropomyosin moves further.³⁷ Tropomyosin residues 89 to 207 appear to be critical for stabilizing this final state of the thin filament, which is required for thin filament activation. The V95A mutation may alter 1 or more transitions among these states.

Similarly, tropomyosin HCM mutations at positions D175N and E180G have been proposed to alter thin filament activation,^{38 39} although neither myosin affinity nor Ca²⁺ affinity was examined for these mutants. Functional studies of TPM1 cDNA expressed in skeletal muscle and those performed on single skinned skeletal fibers from transgenic animals and affected patients demonstrated increased Ca²⁺ sensitivity.^{32 39 40} Furthermore, adenovirus-mediated expression of several HCM-causing tropomyosins at the 35% level in adult cardiac myocytes resulted in Ca²⁺-sensitizing effects on force,³³ with severity dependent on the mutation. Figures 6 and 7 in the present work show a similar effect under highly defined conditions using purified proteins and demonstrate that the effect is not due to increased myosin binding. By implication, it is calcium affinity per se that is altered for this mutation. Michele et al³³ suggested that the severity of the calcium-sensitizing effect may correlate with clinical severity. However, the clinical severity of the V95A patients is particularly high, yet the alteration in Ca²⁺ sensitivity for reconstituted 100% mutant filaments (Figure 6) is no greater than that observed for 35% mutant fibers.³³ Altered myosin cycling (Figure 6, the Table) may also be an important contributor to disease pathogenesis.

Cardiac Phenotype and Prognosis
Clinical characteristics and prognosis associated with mutations of TPM1 have not been well characterized owing to small size and composition of affected families. In previous reports, HCM caused by the TPM1 mutation D175N showed a variable phenotype with a good prognosis.¹⁴ The TPM1 mutation E180G has been associated with mild LVH, but the prognosis is unclear.¹⁷ Few HCM cases caused by the TPM1 mutations A63V and K70T have been reported: mild LVH associated with sudden death, congestive heart failure, and LV dilatation.^{12 13} In the present kindred, the mean maximum LV wall thickness was 16±6 mm in the 15 affected members. The presence of cardiomyopathy was indicated by abnormal ECG in several carriers of the mutation in the absence of LVH. Conversely, ECG criteria cannot be used to diagnose the V95A mutation, because a number of affected family members did not have classic ECG features associated with HCM. However, under the age of 50 years, an abnormal ECG often indicated disease in the absence of LVH. The distribution of LVH was unusual. Although the disease penetrance was only 53%, overall, clinical features of this TPM1 mutation V95A, including LVH, LV dysfunction, and sudden death, were observed in 85% of the affected members. Although clinical and genetic data were not available in several of the deceased family members, the history of sudden death at young ages provided compelling evidence that the events were related to the disease mutation. Notably, the clinical expression and calcium sensitivity of TPM1 mutation V95A are similar to HCM caused by TNNT2 mutations associated with similarly poor prognosis.⁴¹

Genotyping is important for definitive diagnosis, because the myopathy is associated with atypical findings. Detection of the TPM1 mutation V95A is important for early counseling and treatment of family members.⁴²

	Acknowledgments

 
This study was supported by NIH grant HL-63774 (to Dr Tobacman) and AR-30988 (to Dr Homsher). We would like to express our appreciation to Judith Winkler, BSc, Dorothy Tripodi, RN, and Denise McLaughlin, RN, for their assistance with the family study.

	Footnotes

 
Reprint requests to Lameh Fananapazir, MD, FRCP, Bldg 10, Room 7B-15, 10 Center Dr MSC 1650, Bethesda, MD 20892-1650. We report clinical characteristics and outcome and protein functional defects of a novel hypertrophic cardiomyopathy (HCM)-causing TPM1 mutation, V95A, in a large Spanish-American family. Mean left ventricular wall thickness was 16±6 mm (disease penetrance of 53% in adult patients). Thirteen deaths have occurred in 26 affected members (4 during physical activity). HCM was associated with left ventricular systolic dysfunction in 5 patients. Both myosin cycling and calcium binding to troponin are abnormal in the presence of the mutant tropomyosin. HCM caused by the V95A mutation is associated with mild cardiac phenotype but with several functional defects and a poor prognosis.

Guest Editor for this article was Ketty Schwartz, PhD, Group Hospitalier Pitie-Salpetriere, Paris, France.

Received April 19, 2000; revision received July 26, 2000; accepted August 14, 2000.

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