Clinical Features and Outcome of Hypertrophic
Cardiomyopathy Associated with
Triple Sarcomere Protein Gene Mutations
Francesca Girolami BS°, Carolyn Y. Ho MD3, Christopher Semsarian MBBS PhD 1,2,
Massimo Baldi MD, Melissa L. Will BS¶, Katia Baldini Rn, Francesca Torricelli BS°,
Laura Yeates BSc 1, Franco Cecchi MD, Michael J Ackerman MD PhD¶, Iacopo Olivotto MD
From the Referral Center for Myocardial Diseases, (°) Unit for Genetic Diagnosis and Department of Physiology, Azienda Ospedaliera Universitaria Careggi and Università degli Studi, Florence, Italy, (#) Heart Science Center, Imperial College London, Harefield, UK and (¶) Departments of Medicine, Pediatrics, and Pharmacology/Divisions of Cardiovascular Diseases and Pediatric Cardiology, Mayo Clinic, Rochester, MN
1Agnes Ginges Centre for Molecular Cardiology, Centenary Institute, University of Sydney; 2Department of Cardiology, Royal Prince Alfred Hospital, Sydney, Australia.
3Cardiovascular Division, Brigham and Women’s Hospital, Boston, MA
Short title: Triple Mutations in Familial HCM Word count: (3862)
Address for correspondence: Dr. Iacopo Olivotto
Referral Center for Cardiomyopathies Cardiologia San Luca
Azienda Ospedaliera Universitaria Careggi Viale Pieraccini 19, 50134 Firenze
Tel. - Fax. ##39-055-7949335
e-mail: olivottoi@aou-careggi.toscana.it
Key words: Hypertrophic cardiomyopathy, genetics, complex genotypes, outcome.
Acknowledgments. MJA is supported by the Mayo Clinic Windland Smith Rice Comprehensive Sudden Cardiac Death Program. CS is the recipient of a National Health and Medical Research Council (NHMRC) Practitioner Fellowship.
ABSTRACT
Background. Double or compound sarcomere gene mutation heterozygosity have been described in 3-6% of hypertrophic cardiomyopathy (HCM) patients, and may be associated with earlier disease onset and more severe outcome. In the present study, we provide the first description of the clinical profile associated with triple sarcomere gene mutations in a large HCM cohort from three referral institutions. Methods. A total of 488 unrelated index HCM patients underwent screening for myofilament gene mutations by direct DNA sequencing of eight myofilament-encoding genes for myosin binding protein C (MYBPC3), beta-myosin heavy chain (MYH7), regulatory and essential light chains (MYL2,MYL3), troponin-T (TNNT2), troponin-I (TNNI3), alpha-tropomyosin (TPM1) and actin (ACTC). Results. Of the 488 index patients, 4 (0.8%) harboured triple sarcomere gene mutations, as follows: MYH7-R869H, MYBPC3- E258K and TNNI3-A86fs in a 32-year old female;
MYH7-R723C, MYH7-E1455X and MYBPC3-E165D in a 46-year old male; MYBPC3-insC1065, P371R and MYH7-R869H in a 45-year old female; and Q969X,
MYBPC3-R668H, MYH7-R1079Q in a 50-year old female. All patients had significant risk factors for sudden cardiac death (SCD) (n=3) or a history of resuscitated cardiac arrest (n=1), resulting in the insertion of implantable cardioverter-defibrillators (ICD) which appropriately intervened in two patients. Moreover, three out of four had a severe phenotype with progression to end-stage HCM by the fourth decade, requiring cardiac transplantation (n=1) or bivertricular pacing (n=2). The fourth patient, however, had clinically mild disease. Conclusions. HCM associated with triple sarcomere gene mutations is rare, and often associated with development of end stage disease and an increased risk of SCD. These findings reinforce the unfavourable significance of complex genotypes in patients with HCM.
Hypertrophic cardiomyopathy (HCM) is the most common genetic cardiac disease, characterized by heterogeneous morphologic expression and clinical course (1,2). Mutations in genes coding for myofilament contractile proteins of the cardiac sarcomere represent the most common genetic subtype of HCM, with a prevalence of 30–65% in cohort studies (3-7).
Double or compound heterozygosity (i.e. patients with two distinct mutations in the same or in different sarcomere genes) have been described in 3-6% of consecutively screened cohorts, and has been associated with earlier onset and more severe clinical profile, compared to HCM in single mutation carriers (8-12). To our knowledge, HCM associated with the occurrence of triple mutations has not previously been reported. In the present study, we describe four pedigrees from three HCM referral centers, in which each index patient harboured three distinct mutations in sarcomere genes.
METHODS Patient selection
A total of 488 unrelated index patients with a confirmed clinical diagnosis of HCM from three referral centers (Florence, Boston, Sydney), underwent a systematic screening for myofilament gene mutations in the protein-coding exons and splice sites of 8 candidate myofilament genes, in the period January 2002- February 2009) (4). The diagnosis was based on the standard accepted definition for clinically diagnosed HCM consisting of the two-dimensional echocardiographic identification of a hypertrophied, non-dilated left ventricle, in the absence of another cardiac or systemic disease capable of producing the magnitude of ventricular hypertrophy (1,2).
Echocardiography
Echocardiographic studies were performed with standard clinical equipment. Left ventricular (LV) hypertrophy was assessed with 2-dimensional echocardiography, and the site of maximum wall thickness was identified. Left ventricular outflow obstruction was considered present when a peak instantaneous outflow gradient ≥30 mmHg was estimated with continuous wave Doppler echocardiography under basal conditions (13).
Mutational Analysis
DNA extraction and PCR reaction
Following informed consent and complete family history, genomic DNA was extracted from peripheral blood using the QIAamp DNA Blood kit (QIAGEN GmbH, Germany). In vitro amplification of all candidate exons was performed by PCR (Polymerase Chain Reaction) using primers described previously (4).
Mutational analysis
Patients were screened for mutations in the protein-coding exons and splice sites of 8 candidate myofilament genes encoding myosin binding protein C (MYBPC3), thick filament proteins (beta-myosin heavy chain [MYH7] and the regulatory and essential light chains [MYL2 and
MYL3]), and thin filament proteins (troponin-T [TNNT2], troponin-I [TNNI3] alpha-tropomyosin
[TPM1], and alpha-actin [ACTC]). These HCM-susceptibility genes comprise the commercially available genetic test for HCM (http://genetics.med.harvard.edu/seidman/cg3). Direct DNA sequencing was employed using either Abi-Prism 3730 (Applied Biosystem, Foster City KA) or the HCM CardioChip platform (Laboratory for Molecular Medicine, Cambridge MA). This test consists of a combination of direct sequencing of MYPC3 and oligonucleotide hybridization-based DNA sequencing of the coding regions and splice sites of the MYH7, MYBPC3, TNNT2, TNNI3, TPM1, ACTC, MYL2, MYL3,
LAMP2, PRKAG2, and GLA genes using a custom designed Affymetrix GeneChip platform. Every mutation identified was confirmed by a direct sequencing, and, whenever possible, by restriction enzyme digestion.
Novel mutations were considered as potentially disease-causing only if they were absent in at least 300 unrelated chromosomes from adult, ethnically matched healthy controls, and produced a change in a highly conserved residue among species and isoforms. In all cases, co-segregation of the mutation with the disease was confirmed in the probands’ families. All patients and relatives received pre- and post- test genetic counselling (12,14).
RESULTS
Of the 488 index patients, 251 had at least one sarcomere gene mutation (51%). Four patients (0.8%) were found to harbour three distinct mutations, involving MYBPC3, MYH7, and
TNNI3. Three of the four index patients with triple mutations showed early onset of severe HCM
phenotype associated with marked symptoms, ventricular arrhythmias, and a progressive clinical course culminating in an end-stage HCM phenotype.
Patient #1
The first index patient is a 32-year old female (Figure 1; II-1), diagnosed with HCM at the age of 18 years following presentation with dyspnea and exertional angina. At the time, she had no family history of cardiovascular disease or sudden cardiac death. An echocardiogram at presentation showed extreme LV hypertrophy (maximum LV wall thickness 32 mm) and severe LV outflow tract obstruction (peak gradient 85 mmHg) at rest. At age 24 years her maximum LV wall thickness had regressed to 27 mm, LV obstruction had spontaneously disappeared, and there were signs of severe diastolic dysfunction with left atrial dilatation. Cardiac magnetic resonance imaging (CMR) showed substantial late-gadolinium enhancement in the thinned portion of the basal septum, compatible with replacement fibrosis. During follow-up she developed progressive deterioration of systolic and diastolic function, with recurrent angina, episodes of acute congestive heart failure and recurrent atrial fibrillation(AF). A second CMR at age 30 years showed marked increase in delayed contrast enhancement (Figure 2), and positron emission tomography (PET) scan showed severe
microvascular dysfunction (average myocardial flow following dipyridamole infusion 1.31 ml/gr/min; normal values ≥2.0 ml/gr/min). She underwent successful transcatheter ablation of AF, and has remained in sinus rhythm with satisfactory hemodynamic balance on amiodarone, carvedilol, angiotensin converting enzyme-inhibitors and loop diuretics. A prophylactic ICD with bi-ventricular pacing capability was subsequently implanted.
Genetic analysis revealed heterozygous mutations in three distinct sarcomere genes, including a missense mutation in MYH7 (R869H), a splice site mutation in MYBPC3 (E258K) and a frameshift mutation in TNNI3 (A86fs). Both MYH7-R869H and MYBPC3-E258K have been previously described (http://cardiogenomics.med.harvard.edu/home). Family screening revealed the
MYBPC3-E258K and TNNI3-A86fs mutations in the proband’s father (I-2), a 58-year old
gentleman who had always been active and totally asymptomatic. His echocardiogram showed definite HCM, with LV hypertrophy prevalently localized at the apex and preserved LV function (Figure 1). The proband’s mother (I-1), a 52-year old woman with a history of mild dyspnea on effort, had mild asymmetric septal hypertrophy associated with the MYH7-R869H mutation. The proband’s brother, age 28 years (II-2), carried only the TNNI3-A86fs mutation, and was clinically unaffected, with normal ECG and echocardiographic findings.
Patient #2
The second index patient is a 46-year old male, diagnosed at the age of 29 years due to mild exertional dyspnea (Figure 3). At the time, he had nonobstructive HCM with initial LV chamber dilatation and systolic function at the lower limits of normal (ejection fraction 56%). In the following years he developed marked LV remodeling with progressive systolic dysfunction and wall thinning in conjunction with severe symptoms of congestive heart failure (Figure 4). A PET scan at age 33 years showed marked microvascular dysfunction (average myocardial flow following dipyridamole infusion 1.06 ml/gr/min). At age 39 years he received an ICD, which repeatedly intervened to terminate runs of sustained ventricular tachycardia. At age 43 years he required
cardiac transplantation due to refractory heart failure. His LV cavity measured 238 ml in diastole, and his ejection fraction was 28%. Following transplantation he rapidly recovered, and is now well. Genetic analysis revealed three missense mutations, of which two were in MYH7 (R723C and E1455X) and one in MYBPC3 (E165D). Of these mutations, MYH7-R273C has been previously described (5), whereas MYBPC3-E165D and MYH7-E1455X are novel. Notably, although nonsense mutations in MYH7 have not been demonstrated as pathogenic, the two MYH7 mutations present in this patient were inherited in trans, indicating that he had no normal beta-myosin heavy chain protein. Both novel mutations were absent in 300 unrelated chromosomes from healthy controls; in addition MYBPC3-E165D produced a change in a highly conserved residue among species. The proband’s mother (I-2) was found to be double heterozygote for MYBPC3-E165D and E1455X, associated with mild, non-obstructive HCM; the proband’s father (I-1) carried the MYH7-R723C mutation but was clinically unaffected at age 80 years. The proband’s sister (II-3), aged 57 years, carried the MYBPC3-E165D and was also clinically unaffected. His brother (II-2) was genotype-negative. The proband’s son, aged 19 years, carried both the MYBPC3-E165D and
MYH7-E1455X mutations. He was asymptomatic, and his echocardiogram showed mild asymmetric
hypertrophy of the basal septum and anterior papillary muscle, consistent with early phenotypic expression of HCM..
Patient #3
The third patient is a 45-year old female who was diagnosed with HCM at the age of 24 years following resuscitation from cardiac arrest due to documented ventricular fibrillation (Figure 5). An echocardiogram showed massive asymmetric LV hypertrophy with a maximum thickness of 36 mm at the basal septum (Figure 6), elongated mitral leaflets without evidence of outflow obstruction, and normal LV systolic function. During follow-up her echocardiograms showed progressive septal thinning and a decline in LV function, with a recent evaluation showing a basal septum of 18 mm (Figure 6), and an LV ejection fraction of 42%. This coincided with a clinical
deterioration and development of exertional dyspnea despite significant medical therapy with carvedilol, angiotensin receptor blockers and diuretics. An ICD was placed for primary prevention following her cardiac arrest, repeatedly replaced over the years and recently upgraded to perform biventricular pacing. Of note, cardiac resynchronization has lead to significant subjective improvement in quality of life and exercise tolerance. During follow up, however, she received two appropriate ICD discharges due to rapid sustained ventricular tachycardia.
Genetic screening showed two distinct mutations in MYBPC3 (insC1065 and P371R) and one in MYH7 (R869H). Both MYBPC3-insC1065 and MYH7-R869H had been previously described (http://cardiogenomics.med.harvard.edu/home). Conversely, MYBPC3-P371R was novel, but was absent in 300 unrelated chromosome from healthy controls and produced a change in a highly conserved residue among species . Cosegregation studies in this family showed that insC1065 and
P371R are pathogenetic when heredited together, as in the proband’s sister (Figure 5). Of note,
another index case in the Florence cohort has been found to harbour both insC1065 and P371R. The proband’s father (I-1) age 69 years, had a family history of premature sudden death, and was found to have a mild HCM phenotype, characterized by maximum septal thickness of 19 mm, with preserved LV function and excellent exercise tolerance (VO2 max was 32 ml/min/kg). He was a carrier of both MYBPC3-insC1065 and MYBPC3-P371R. The mother had died of non-cardiac causes before genetic screening was available. One of the proband’s sisters (II-2) died suddenly at the age of 31 years, possibly from cardioembolic stroke. Her genetic status is unknown. However, she was an obligatory carrier of both MYBPC3-insC1065 and MYBPC3-P371R, as these mutations were found in her only child. The latter, age 21 years (III-1), has a mild form of nonobstructive HCM (maximum LV wall thickness 16 mm), with preserved LV function, and is asymptomatic.
Patient #4
The fourth patient is a 50-year old female (II:3, Figure 7), who was diagnosed with HCM at the age of 48 years as part of clinical screening of her family. On presentation, the patient had mild
symptoms of atypical chest pain for several years. Her echocardiogram showed moderate non-obstructive HCM with a maximum LV wall thickness of 19 mm and normal LV dimensions and systolic function. While she had no history of syncope, 24-hour ambulatory ECG monitoring identified several runs of non-sustained VT (HR >140 bpm, longest run of 7 beats; Figure 7). She has had an ICD implanted and has had no shocks delivered at 6 months follow-up.
Genetic analysis identified three missense mutations, of which two were in MYBPC3 (Q969X and R668H) and one in MYH7 (R1079Q). All three mutations have been identified in isolation in previous families or probands as single, presumed pathogenic gene mutations (http://cardiogenomics.med.harvard.edu/home). Subsequent clinical and genetic screening of first degree relatives revealed the three daughters of the patient (III;5, III:7, III:8), who are aged 33, 30, and 29 years respectively, and who all have normal ECG and echocardiograms, although all have inherited either one or two of the mutations identified in their mother (Figure 7). The patient’s brother (the family proband, II:2) is aged 54 years, and was diagnosed previously with mild non-obstructive HCM (maximum LV wall thickness 16mm) and is currently asymptomatic. Genetic screening shows that he carries two of the brother’s three mutations (Figure 7).
DISCUSSION
In this report, genetic testing in a large cohort with HCM allowed identification of triple sarcomere gene mutations in 4 out of 488 probands, representing a prevalence of less than 1%. In comparison, a 3-6% prevalence has consistently been reported for double sarcomere mutations in HCM (8-12). The genotype of these four patients was variable: one had double MYH7 and a single
MYBPC3 mutation, two had double MYBPC3 and a single MYH7 mutation, and one had single
mutations in MYH7, MYBPC3 and TNNI3. To our knowledge, this is the first report of HCM caused by three independent genetic defects.
In our patients, complex genotypes were compatible with survival to adult life, but were generally associated with severe clinical features. Specifically, all four probands had significant ventricular
arrhythmias, prompting ICD placement in all, and associated with resuscitated cardiac arrest in one patient and multiple appropriate ICD interventions in another (15). Even more striking is the marked phenotypic progression and development of end stage HCM seen in three of four triple mutation carriers (16). Although all three initially presented with massive LV hypertrophy and severe symptoms by age 25 years, marked end-stage remodelling ensued in the subsequent 10 to 20 years. Over this period, these three patients uniformly developed restrictive physiology, marked atrial dilation and systolic dysfunction associated with progressive LV wall thinning; one patient required cardiac transplantation. Thus, 75% of patients with triple mutations progressed to end stage HCM. This is remarkable because end stage remodelling in HCM is quite rare, previously reported to occur in approximately 2-5% of the population (17,18), and also in our centers, only a minority of the patients developed this phenotype (4).
A possible pathophysiologic mechanism to explain the adverse consequences of complex genotypes is that multiple abnormal myofilament proteins may result in more profound derangement of sarcomere mechanics, and transition towards multifactorial cardiomyocyte dysfunction (19). Of note, in the TNI-203/MHC-403 double-mutant mouse model recently reported by Tsoutsman et al (10),while each mutation by itself was linked to a hypertrophic phenotype, the presence of both mutations rapidly led to LV dilatation, severe heart failure, and premature death. Other potentially relevant processes may be represented by adverse remodeling of the coronary arterioles, leading to microvascular ischemia and replacement fibrosis (20,21) Consistent with this hypothesis, our patients with triple mutations as a group showed a markedly increased prevalence of systolic dysfunction, abnormal microvascular function and extensive cardiac fibrosis. Of note, in two of these patients there was demonstrable evidence of severe microvascular dysfunction and blunted myocardial perfusion; factors which may predispose the myocardium to recurrent ischemia (20,21). In one of the two, we documented a marked increase in late gadolinium enhancement within the LV wall over time, suggesting that both thinning and functional impairment were secondary to widespread fibrotic replacement of the myocardium, possibly mediated by ischemia (22)
Our results reinforce the more general view that complex genotypes bear an unfavourable prognostic significance in HCM patients, and may therefore have relevant implications for management (8-12, 23,24). Young patients with multiple mutations should be followed up closely for timely recognition of pending end-stage progression and implementation of aggressive prevention strategies (22). Specifically, in the presence of an initial decline in systolic function, LV wall thinning or progression of intramyocardial fibrosis, treatment with ACE-inhibition or angiotensin receptor blockade may be beneficial to mitigate further adverse remodeling, and may be considered before overt systolic dysfunction has ensued (1,2). In addition, our results suggest the need for greater vigilance for ventricular arrhythmias in patients with more than one HCM-causing mutation (15).
Gene dosage effects have been postulated to play a role in HCM, resulting in more severe clinical phenotypes and disease course in individuals harbouring multiple gene mutations (11,12,25,26) However, the true impact of multiple mutations is difficult to evaluate, owing to the marked phenotypic and genotypic variability in this disease, with hundreds of sequence variants, delayed penetrance, and variable expression (12). Indeed, one of our patients with three mutations (MYBPC3-Q969X, MYBPC3-R668H, MYH7-R1079Q) demonstrated only modest hypertrophy and mild symptoms at age 50 years. Moreover, several adult relatives of these patients had mild or no disease expression despite harbouring double mutations. Thus, an inherent, and often unavoidable limitation to genetic studies in HCM remains in proving that newly identified sequence variants are truly pathogenic (12,14). Some novel DNA variants may not be capable of causing disease in isolation, but may exert important modifying effects on disease expression in combination with other mutations (10,12,27,28). Nevertheless, all the novel variants found in our triple mutation carriers impact evolutionarily conserved residues and are absent in normal controls. In addition, triple mutation carriers uniformly demonstrated a more severe phenotype than can be observed in most HCM patients, with a clinical pattern resembling the course exhibited by patients and animal models with double sarcomere gene mutations (8,10). Thus, is it plausible to support the
view that these novel variants substantially contributed to disease pathogenesis by influencing the phenotype and clinical course in our patients.
In conclusion, HCM associated with triple sarcomere gene mutations is rare but often associated with adverse outcomes, including high prevalence of ventricular arrhythmias and remarkably increased risk of developing an end-stage HCM phenotype. These findings highlight the need for further longitudinal investigation and strengthen the hypothesis that complex genotypes may be unfavourable in patients with HCM, raising issues relevant to clinical management.
REFERENCES
1.Maron BJ. Hypertrophic cardiomyopathy: a systematic review. JAMA 2002;287:1308–1320. 2.Maron BJ, McKenna WJ, Danielson GK, et al. American College of Cardiology/European Soci-ety of Cardiology clinical expert consensus document on hypertrophic cardiomyopathy. J Am Coll Cardiol 2003;42:1687–1713.
3.Van Driest SL, Ommen SR, Tajik AJ, Gersh BJ, Ackerman MJ. Yield of genetic testing in hyper-trophic cardiomyopathy. Mayo Clin Proc. 2005;80:739-44.
4.Olivotto I, Girolami F, Ackerman MJ, Nistri S, Bos JM, Zachara E, Ommen SR, Theis JL, Vaubel RA, Re F, Armentano C, Poggesi C, Torricelli F, Cecchi F. Myofilament protein gene mutation screening and outcome of patients with hypertrophic cardiomyopathy. Mayo Clin Proc. 2008;83:630-8.
5.Richard PP, Charron P, Carrier L, Ledeuil C, Cheav T, Pichereau C,Benaiche A, Isnard R, Dubourg O, Burban M, Gueffet J-P, Millaire A,Desnos M, Schwartz K, Hainque B, Komajda M; for the EUROGENEHeart Failure Project. Hypertrophic cardiomyopathy: distribution of disease genes, spectrum of mutations, and implications for a molecular diagnosis strategy. Circulation. 2003;107:2227– 2232.
6. Erdmann J Clin Genet 2000
7. Van Driest SL Circulation 2003
8. Van Driest SL, Vasile VC, Ommen SR, Will ML, Tajik AJ, Gersh BJ, Ackerman MJ. Myosin binding protein C mutations and compound heterozygosity in hypertrophic cardiomyopathy. J Am Coll Cardiol. 2004; 44:1903–1910.
9. Alpert NR, Mohiddin SA, Tripodi D, Jacobson-Hatzell J, Vaughn-Whitley K, Brosseau C, Warshaw DM, Fananapazir L. Molecular and phenotypic effects of heterozygous, homozygous, and compound heterozygote myosin heavy-chain mutations. Am J Physiol Heart Circ Physiol. 2005;288:H1097–H1102.
10. Tsoutsman T, Kelly M, Ng DCH, Tan J-E, Tu E, Lam L, Bogoyevitch M, Seidman CE, Seidman JG, Semsarian C. Severe heart failure and early mortality in a double-mutation mouse model of familial hypertrophic cardiomyopathy. Circulation. 2008;117:1820 –1831.
11. Ingles J, Doolan A, Chiu C, Seidman J, Seidman C, Semsarian C. Compound and double mutations in patients with hypertrophic cardiomyopathy: implications for genetic testing and counselling. J Med Genet. 2005;42:e59.
12. Kelly M, Semsarian C. Multiple mutations in genetic cardiovascular disease: a marker of dis-ease severity. Circulation. Cardiovasc. Genet. 2009 (in press; accepted 15.01.09).
13. Maron MS, Olivotto I, Betocchi S, et al. Effect of left ventricular outflow tract obstruction on clinical outcome in hypertrophic cardiomyopathy. N Engl J Med 2003;348:295–303.
14. HGVS2008
15. Maron BJ, Spirito P, Shen WK, Haas TS, Formisano F, Link MS, Epstein AE,Almquist AK, Daubert JP, Lawrenz T, Boriani G, Estes NA 3rd, Favale S, Piccininno M, Winters SL, Santini M, Betocchi S, Arribas F, Sherrid MV, Buja G, Semsarian C, Bruzzi P. Implantable cardioverter-defib-rillators and prevention of sudden cardiac death in hypertrophic cardiomyopathy. JAMA. 2007;298:405-12.)
16. Yacoub MH, Olivotto I, Cecchi F. 'End-stage' hypertrophic cardiomyopathy: from mystery to model. Nat Clin Pract Cardiovasc Med. 2007;4:232-3.
17. Harris KM, Spirito P, Maron MS, Zenovich AG, Formisano F, Lesser JR, Mackey-Bojack S, Manning WJ, Udelson JE, Maron BJ. Prevalence, clinical profile, and significance of left ventricu-lar remodeling in the end-stage phase of hypertrophic cardiomyopathy. Circulation. 2006;114:216-225;
18. Biagini E, Coccolo F, Ferlito M, Perugini E, Rocchi G, Bacchi-Reggiani L, Lofiego C, Boriani G, Prandstraller D, Picchio FM, Branzi A, Rapezzi C. Dilated-Hypokinetic Evolution of Hyper-trophic Cardiomyopathy: Prevalence, Incidence, Risk Factors, and Prognostic Implications in Pedi-atric and Adult Patients. J Am Coll Cardiol. 2005;46:1543-1550)
19. Belus A, Piroddi N, Scellini B, Tesi C, Amati GD, Girolami F, Yacoub M, Cecchi F, Olivotto I, Poggesi C. The familial hypertrophic cardiomyopathy-associated myosin mutation R403Q acceler-ates tension generation and relaxation of human cardiac myofibrils. J Physiol. 2008;586:3639-44.
20. Olivotto I, Cecchi F, Gistri R, Lorenzoni R, Chiriatti G, Girolami F, Torricelli F, Camici PG. Relevance of coronary microvascular flow impairment to long-term remodeling and systolic dys-function in hypertrophic cardiomyopathy. J Am Coll Cardiol. 2006;47:1043-8.
21. Sotgia B, Sciagrà R, Olivotto I, Casolo G, Rega L, Betti I, Pupi A, Camici PG,Cecchi F. Spatial relationship between coronary microvascular dysfunction and delayed contrast enhancement in patients with hypertrophic cardiomyopathy. J Nucl Med. 2008;49:1090-6.
22. Harris, Olivotto, Sotgia, Adabag AS, Maron BJ, Appelbaum E, Harrigan CJ, Buros JL, Gibson CM, Lesser JR, Hanna CA, Udelson JE, Manning WJ, Maron MS. Occurrence and frequency of arrhythmias in hypertrophic cardiomyopathy in relation to delayed enhancement on cardiovascular magnetic resonance. J Am Coll Cardiol. 2008;51:1369-74).
23. Semsarian editorial (Hilfiker-Kleiner D; Knöll D. Disease-modifying mutations in familial hypertrophic cardiomyopathy: complexity from simplicity. Circulation. 2008;117:1775-1777. 24. Ashrafian H, Watkins H. Reviews of translational medicine and genomics in cardiovascular dis-ease: new disease taxonomy and therapeutic implications cardiomyopathies: therapeutics based on molecular phenotype. J Am Coll Cardiol. 2007;49:1251-64.
25.Ho CY, Seidman CE. A contemporary approach to hypertrophic cardiomyopathy. Circulation. 2006;113:e858-62.
26. Marian AJ. Genetic determinants of cardiac hypertrophy. Curr Opin Cardiol. 2008;23:199-205. 27. Ho CY, Lever HM, DeSanctis R, Farver CF, Seidman JG, Seidman CE. Homozygous mutation in cardiac troponin T: implications for hypertrophic cardiomyopathy. Circulation. 2000;102:1950 –1955.
28. Keren A, Syrris P, McKenna WJ. Hypertrophic cardiomyopathy: the genetic determinants of clinical disease expression. Nat Clin Pract Cardiovasc Med. 2008;5:747.
29. Olivotto I, Cecchi F, Poggesi C, Yacoub MH. Developmental origins of hypertrophic cardiomy-opathy phenotypes: a unifying hypothesis. Nat Rev Cardiovasc Med, in press.
FIGURE LEGENDS
Figure 1. Pedigree of patient #1. The index patient is indicated by the black arrow. The electro-pherograms show the identified mutations. Echocardiographic still-frames in the apical end-dias-tolic 4-chamber view are shown for the three affected individuals. LA= left atrium; LV=left ventri-cle; RA=right atrium, RV=right atrium.
Figure 2. Evidence of microvascular dysfunction and severe myocardial fibrosis in patient #1. (A) NH3 PET short-axis slice at level of basal LV segments. Color scale shows highest values of flow in red and lowest in green. (B) First-pass MRI short-axis slice at base of left ventricle, showing diffuse septal hypertrophy. (C) After gadolinium infusion, extensive delayed contrast enhancement (DCE) is evident (white signal), involving interventricular septum and extending into an-terior wall. DCE and areas of reduced flow at PET closely agree. (D) Diagram il-lustrating nomenclature used for classification of myocardial segments with ex-tent and proximity of DCE as follows: (1) transmural DCE, (2) nontransmural DCE, (3) without DCE but contiguous to DCE segments, (4) without DCE and re-mote from DCE. Numbers in italic indicate hMBF (mL/min/g) in segment. RV = right ventricle. Reproduced from Sotgia et al J Nucl Med 2008; 49:1090–1096.
Figure 3. Pedigree of patient #2. The electropherograms show the identified mutations.
Figure 4. Evidence of disease progression in patient #2. Stop frames of echocardiograms obtained at the time of the positron emission tomography (PET) scan (age 33 years, panels A, C, and E) and at final evaluation (age 42 years, panels B, D, and F) in a HCM patient with missense mutations of the myosin beta-heavy chain and of the myosin binding protein C genes
(Arg723-Cys and Glu165Asp, respectively). Individual measurements are provided in Ta-ble 2 (Patient #1). Comparison of the two echocardiograms shows progression of LV cavity enlargement and systolic impairment, with regression of septal hy-pertrophy. (A to D) Parasternal long-axis view. (E and F) Apical four-chamber view. *= Interventricular septum. LA = left atrium, LV = left ventricle. From Olivotto et al, J Am Coll Cardiol 2006.
Figure 5. Pedigree of patient #3. The electropherograms on the right side of the figure show the identified mutations.
Figure 6. Evidence of disease progression in patient #3. Stop frames of echocar-diographic 4-chamber view at end-diastole at age 28 (panel A) and 45 (panel B). Comparison of the two echocardiograms shows progression of LV cavity en-largement with marked regression of septal hypertrophy. LA= left atrium; LV=left ventricle; RA=right atrium, RV=right atrium.
Figure 7. Pedigree of patient #4. The electropherograms show the identified mutations. Arrow = proband, “N” = clinically normal on screening, “+/-“ = genotype posi-tive, number after gene name = exon. (A) Ambulatory ECG tracing showing a 7 beat run of nonsustained ventricular tachycardia at 142 bpm.
