Long-Term Outcomes in Hypertrophic Cardiomyopathy Caused by Mutations in the Cardiac...

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Long-Term Outcomes in Hypertrophic Cardiomyopathy Caused by

Mutations in the Cardiac Troponin T Gene

Running title: Pasquale et al.; Outcomes in troponin T mutations

Ferdinando Pasquale, MD, PhD1,2; Petros Syrris, PhD3; Juan Pablo Kaski, BSc, MBBS, MD4;

Jens Mogensen, MD, PhD5; William J. McKenna, MD, DSc, FRCP, FESC1; Perry Elliott MD1

1The Heart Hospital, Institute of Cardiovascular Sciences, University College London, London,

United Kingdom; 2Istituto di Cardiologia Ospedale S.Orsola Malpighi, Bologna, Italy; 3Institute

of Cardiovascular Science, University College London; 4Great Ormond Street Hospital for

Children, London, United Kingdom; 5Skejby University Hospital, Åarhus, Denmark

Address for correspondence:

Dr Perry Elliott

The Heart Hospital

16-18 Westmoreland Street

London, W1G 8PH, United Kingdom

Tel: +44-207-573-8888

Fax: +44-207-573-8838

E-mail: [email protected]

Journal Subject Codes: [109] Clinical genetics; [11] Other heart failure; [193] Clinical studies; [15] Hypertrophy; [16] Myocardial cardiomyopathy disease

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Abstract:

Background - Hypertrophic cardiomyopathy (HCM) caused by mutations in the cardiac troponin

T gene (TNNT2) has been associated with a high risk of sudden cardiac death (SCD) and mild

left ventricular hypertrophy (LVH). However, previous studies are limited by sample size, cross-

sectional design and few data in relatives.

Methods and Results - 552 unrelated HCM probands were screened for TNNT2 mutations. First-

degree relatives were invited for clinical and genetic evaluation. Ninety two individuals (20

probands and 72 relatives) carried TNNT2 mutations (51 (55%) male; 30±17 years). ECGs and

echo were available in 87 (95%) and 88 (96%) individuals, respectively. ECG was normal in 13

(68%) children (< 16 years) and 13 (19%) adults. Echo was normal in 18 (90%) children and 16

(24%) adults; 7 (10%) adults had a normal ECG and Echo.

Thirteen (65%) of 20 families had a history of SCD. Follow-up was available for 75 patients

(mean 9.9 ± 5.2 years); 2/16 adults and 2/18 children with normal echoes developed left

ventricular hypertrophy (LVH). Twenty three (22%) received an implantable cardioverter

defibrillator (ICD) (20 for primary prophylaxis). One child and three adults died from SCD and 2

adults were resuscitated from ventricular fibrillation. One patient had an appropriate ICD

discharge. The rate of cardiovascular death, transplant and ICD discharge was 1.6% (0.016

person/year; CI 0.83-2.79%) and SCD 0.93% (0.0093 person/year; CI 0.37-1.92%).

Conclusions - LVH is rare in children with TNNT2 mutations. LVH is absent in minority of

adults, but most have an abnormal ECG. In spite of adverse family histories, the rate of

cardiovascular death during follow-up was similar to that reported in large referral populations.

Key words: cardiomyopathy; gene mutation; hypertrophic cardiomyopathy; sudden death; troponin T

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Introduction

Hypertrophic cardiomyopathy (HCM), defined as left ventricular hypertrophy (LVH) in the

absence of abnormal loading conditions, occurs in approximately 1 in every 500 adults1. The

disease can present with cardiovascular symptoms at any age and is a frequent cause of sudden

cardiac death in young people2-4. In the long-term, many patients develop progressive symptoms

caused by a gradual deterioration in left ventricular function5,6.

HCM is usually inherited as an autosomal dominant trait caused by mutations in genes that

encode proteins of the cardiac sarcomere7-10. Based on the assumption that locus and allelic

heterogeneity have different effects on myocyte structure and function, it has been hypothesized

that individual mutations are associated with specific phenotypes10-15. If this is correct, then

mutation analysis could be used to guide therapy (e.g. prophylactic implantation of implantable

cardioverter defibrillators) and counselling strategies for families. One of the first reported

genotype-phenotype associations in HCM was a high prevalence of sudden cardiac death in

young carriers of mutations in the cardiac troponin T gene (TNNT2)9,16-18, paradoxically in the

presence of only mild left ventricular hypertrophy19,20. While, this finding has been used in

subsequent consensus management guidelines for HCM, studies examining the natural history of

troponin T gene mutations are limited by the small size of patient cohorts (often single families),

cross-sectional study design and a lack of data on disease expression in relatives. The aim of this

study was to examine clinical outcomes in a relatively large cohort of patients and relatives with

mutations in the cardiac troponin T gene.

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Methods

This study was approved by the local ethics committees of St George’s Hospital and University

College Hospitals London, United Kingdom. All study participants gave written informed

consent for venesection and DNA analysis. The study conformed to the principles of the Helsinki

declaration. All authors had full access to the data and take responsibility for its integrity. All

authors have read and agree to the manuscript as written.

Clinical evaluation

The study cohort comprised apparently unrelated HCM probands referred to a dedicated

cardiomyopathy clinic over a 10 year period to St. George’s Hospital and then subsequently

followed (2003 to 2005) at The Heart Hospital, UCLH, London, United Kingdom) that were

tested for mutations in the TNNT2 gene. All relatives of mutation positive individuals were

invited for clinical and genetic evaluation.

Patients and consenting relatives underwent supine 12 lead electrocardiography (ECG), M-mode,

2-D and Doppler echocardiography, symptom limited upright exercise testing with simultaneous

respiratory gas analysis and 24-48 hour ambulatory ECG monitoring in accordance with

previously described methods 21. LVH on the 12 lead ECG was defined using the Romhilt-Estes

score system22; LVH was defined as a score 5. ECGs in infants and children were reviewed by

a pediatric cardiologist (JK) and were coded using standard criteria23.

HCM was diagnosed in probands and relatives when the maximum left ventricular wall thickness

(MLVWT) measured 13 mm in one or more myocardial segments on echocardiography or

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when left MLVWT exceeded two standard deviations corrected for age, size and gender in the

absence of other diseases that could explain the hypertrophy24. The pattern of hypertrophy was

classified as asymmetrical septal, concentric, or predominantly apical25. In probands with sudden

death as the first clinical manifestation, the diagnosis of HCM was, when possible, confirmed at

post-mortem. Relatives were considered clinically affected when they fulfilled extended familial

criteria for HCM26.

During ambulatory monitoring, supraventricular tachycardia was defined as three or more

consecutive supraventricular premature beats at a rate more than 100 beats/min and non-

sustained ventricular tachycardia (NSVT) as three or more consecutive ventricular ectopics at a

rate more than 120 beats/min. An abnormal blood-pressure response during symptom limited

upright exercise testing was defined as a failure of systolic blood pressure to rise by more than

25 mmHg from baseline values, or a fall of more than 10 mmHg from the maximum blood

pressure during upright exercise27.

Clinical Risk Stratification

Risk stratification was considered complete when patients had at least one echocardiogram,

ambulatory ECG recording and exercise test. Major risk factors for sudden cardiac death were

defined as: a family history of sudden cardiac death at an age 40 years; a syncopal episode

suggestive of arrhythmic origin; NSVT during ambulatory electrocardiography; MLVWT

30mm; and an abnormal exercise blood pressure response28.

Genetic evaluation

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Genomic DNA was extracted from whole blood and protein encoding exons of TNNT2 were

amplified using standard protocols (primer sequences and conditions for polymerase chain

reaction amplification are available upon request). Mutation analysis of TNNT2 was performed

by fluorescent SSCP (F-SSCP) and direct sequencing of abnormal conformers. As reported

previously, the sensitivity of F-SSCP to identify sequence variations in TNNT2 was 100%29,30.

DNA was screened for mutations in TNNT2 hot spot exons 8, 9 and 16. If these did not show

mutations, exons 10, 11, 13, 14 and 15 were also screened. When no mutation was identified the

TNNT2 gene was completely sequenced. When a disease causing mutation was found in

TNNT2, genetic analysis was interrupted.

A cohort of 200 healthy Caucasian volunteers served as controls for TNNT2 sequence variants

reported in this study. A sequence variant was defined as a pathogenic mutation if it was absent

in a cohort of 400 control chromosomes; it was predicted to result in a premature truncation,

frameshift or abnormal splicing or affected conserved amino acid residues; and cosegregated

with disease in the family (when information was available). Variants previously reported as

mutations were considered disease causing unless we could show otherwise. The likelihood of

pathogenic effect of all TNNT2 sequence variants was determined by in silico prediction

methods: PolyPhen-2 and SIFT for missense mutations and splice site prediction software Neural

Network and Genio/splice.

Statistics

SPSS (v13.0) was used for all statistical analyses. Normally distributed data are expressed as

mean (95% confidence intervals (CI). Differences between means were compared using the

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unpaired two tailed Student t-test. The chi-square test was used for comparison of categorical

data. The Mann-Whitney U test was used to analyze non-normally-distributed continuous data.

The predefined end-points in the survival analysis were: sudden cardiac death (witnessed death

with or without documented ventricular fibrillation, death within one hour of new symptoms, or

nocturnal deaths with no antecedent history of worsening symptoms); first appropriate ICD

shock; heart failure death (death preceded by signs and/or symptoms of heart failure, including

cardiogenic shock); orthotopic heart transplantation; and other cardiovascular death (stroke,

pulmonary or peripheral arterial embolization, procedure related and myocardial infarction).

Deaths of unknown cause were classified as non-cardiovascular deaths.

The cumulative probability for the occurrence of an outcome was estimated using the Kaplan-

Meier method. Survival from cardiovascular death, cardiac transplant and appropriate ICD shock

was modelled for all probands and relatives with an identified mutation in the TNNT2 gene from

their first evaluation at our institution.

To facilitate comparison with previously published data, survival was also modelled from birth

incorporating additional data on relatives (alive and dead) identified from pedigree analysis that

were known to have HCM (in life or at autopsy) or were obligate carriers of a TNNT2 mutation.

Analysis of survival from birth was also performed for individual mutations.

A p value <0.05 was considered significant in all analyses.

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Results

Genetic investigations

Five hundred and fifty two probands were screened for TNNT2 mutations. Twenty (3.6%) had

pathogenic mutations. Two hundred and thirty six relatives consented to clinical and genetic

testing; of these, 72 (30.5%) had a mutation. Ethnicity data was confirmed in 18 of the 20

families with TNNT2 mutations: 1 family of Indian ethnicity; 1 Caribbean; 1 Pakistani and the

remaining 15 families White British.

Twelve different mutations in exons 8, 9, 11, 15 and 16 were identified in 20 families:

Arg278Cys in 3 families; Arg92Leu in 2 families; Arg92Trp in 3 families; Glu163del in 3

families; IVS15+1G>A in 2 families; and Ala104Val, Arg278His, Arg92Gln, Arg94Leu,

Glu163Lys, Glu83Lys, Ile79Asn in single families. Of the 12 sequence variants in this study, 11

have already been reported (supplemental table 1) as disease causing in HCM.9,16,18,20,29,31,32

A summary of the bioinformatics analysis of the missense mutations (i.e. 9 known and 1 novel)

is provided as a supplemental data file (supplemental table 2).

Analysis of the IVS15+1G>A mutation by splice site prediction software (Neural Network and

Genio/splice) showed that this sequence change completely abolishes the splice donor site of

exon 15 of TNNT2 and is predicted to result in abnormal splicing of the troponin T protein.

The Arg278His sequence variant has not been reported before and was considered pathogenic on

the basis of definition described in the Methods section.

Clinical investigations

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ECG and echocardiogram at first evaluation were available for review in 87 (95%) and 88 (96%)

of the 92 mutation carriers. At least one ambulatory ECG recording was available in 68 (74%)

patients.

The ECG characteristics of adult and pediatric mutation carriers are shown in table 1 Thirteen

adults and 13 children had a normal ECG. In the pediatric population, the echo was normal in 18

individuals (90%). Two children aged 14 and 15 years had an abnormal echo with asymmetric

septal hypertrophy (ASH) and concentric hypertrophy, respectively; the ECG was abnormal in

both children. In adults, the echo was normal in 16 individuals (24%). The echo features of the

adult population are shown in Table 2. The relation between echo and ECG findings in adults are

shown in figure 1. The relation between age and echo findings at initial evaluation is shown in

figure 2.

Risk stratification at the initial clinical evaluation was complete for 54 (76%) adults and 10

(47%) children (table 3). A history of sudden cardiac death at any age was present in 13 (65%) of

the 20 families analyzed. Eight of these 20 families (40%) had a family history of multiple

sudden cardiac deaths; in 5 families (25%) there was only one sudden cardiac death.

Pedigree Analysis

In addition to the 92 proven carriers of a TNNT2 mutation, 29 cases of sudden cardiac death

were identified from pedigree analysis. Of these, five (age of death: 17, 21, 26, 22, and 51 yrs)

had a post-mortem report consistent with HCM; five (age of death: 38, 42, 59, 53, 50 yrs) were

obligate carriers of a mutation; and two were known to be affected with HCM before death but

were followed at other centers. A further 17 relatives (mean age 24 years; range: 5 to 59 years)

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died suddenly in circumstances suggesting a cardiac origin, but as the diagnosis of HCM could

not be confirmed they were excluded from further analysis.

Changes in echocardiographic parameters during follow-up

Follow up echoes were available in 45 adults and 14 children (mean intervals between echoes 9.3

± 5 years and 6.9 ± 2.9 years, respectively). There were no significant changes in mean cardiac

dimensions or systolic function, but 3 patients (MLVWT: 23, 15 and 11 mm) aged 26, 60 and 58

years at first evaluation (follow up 16 ± 2yrs (13 to 17 years)) developed left ventricular

dilatation and impaired systolic function.

Follow-up echoes were available in 11 of 16 adults with a normal echo at presentation (interval

between echoes 8.7 ± 5.2 years (range 1 to 18 years). Two developed hypertrophy: one with a

normal echo at 27 years had concentric hypertrophy with a maximal wall thickness of 17mm

when 42 years old; the second had a normal echo (MLVWT: 9mm) aged 65 and ASH (13 mm)

by the age of 74 years.

Thirteen of the 18 children with normal echoes at first evaluation had follow-up echoes (range 4

to 15 years, mean 6.7 ± 3 years): two developed asymmetric left ventricular hypertrophy at the

age of 19 and 29 years, respectively.

Events during follow-up

Clinical follow-up was available for 75 patients (81%) (mean 9.9 ± 5.2 years). The duration of

follow-up in adults (n=57) was 10.3 ± 5.4 years and in children (n=18) 8.2 ± 3.5 years.

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Twenty three individuals (22%) received an ICD: 20 for primary prophylaxis; 2 for secondary

prophylaxis; and one biventricular device for congestive cardiac failure. Three persons received

a dual chamber pacemaker for AV nodal disease and one for left ventricular outflow tract

obstruction. One individual underwent radiofrequency ablation for atrial fibrillation and later

required atrio-ventricular node ablation and biventricular pacemaker insertion. One individual

underwent septal myomyectomy for left ventricular outflow tract obstruction.

Three adults died from sudden cardiac death and 2 were resuscitated from ventricular fibrillation

(VF); of these, three (age at death or VF 20, 29, and 63 years) were males of Indian ethnicity

from the same family with the Arg92Gln mutation. The maximal wall thickness in these

individuals was 27mm, 15mm and 20mm, respectively. The third sudden death occurred in a

female carrier of Glu163del, aged 62 years with NSVT, a family history of multiple sudden

deaths and MVLWT 18mm. No post-mortem data were available. One female carrying the

Arg278Cys mutation was resuscitated from VF arrest aged 26. At that time she had a concentric

hypertrophy and the maximal wall thickness was 23 mm. She is still alive 18 years later, but has

left ventricular dilatation and impaired left ventricular systolic function. She has a monozygotic

twin who had an ICD implanted for primary prophylaxis, but during follow up she did not

receive any ICD therapies.

Of the remaining adults, one (MLVWT 23mm) received an appropriate ICD shock for

symptomatic VT (IVS15+1G>A) and four (Glu163del; Arg94Cys; Arg278Cys; IVS15+1G>A)

died from heart failure aged 29, 65, 65 and 68 years. In the pediatric cohort, one patient with the

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Ile79Asn mutation died suddenly aged 16 with no echo features of HCM (maximal wall

thickness was 9 mm); his cousin died from complications during an ICD lead extraction at

another center.

Kaplan-Meier survival curves for the study population are shown in figures 3 A and B. When

modelled from the time of first evaluation at our institution, (including two individuals who

presented with cardiac arrest as a first event), the annual rate of cardiovascular death, transplant

and ICD discharge in probands and relatives with a TNNT2 mutation was 1.6% (0.016

person/year; CI 0.83-2.79%) and the rate of sudden cardiac death 0.93% (0.0093 person/year; CI

0.37-1.92%).

When survival was modelled from birth for definite TNNT2 carriers and additional relatives

identified from pedigree analysis that were known to have HCM or were obligate gene carriers

the annual rate of cardiovascular death, transplant and ICD discharge was 0.64% (0.0064

person/year; CI 0.41-0.96%) and the rate of sudden cardiac death 0.51% (0.0051 person/year; CI

0.31-0.79%).

Figure 4 shows survival from birth for individual mutations. Two mutations (Ile79Asn and

Arg92Gln on exon 8 and 9, respectively) appeared to be associated with a higher rate of sudden

death at a juvenile age, at least in comparison with IVS15+1 G>A and Glu163del mutations. The

small number of SCDs (n=7) means that it is very difficult to perform sub-group analyses

(supplemental table 3). However, comparing probands with relatives there was no difference in

cardiac mortality (Kaplan Meier log rank test p=0.62). Similarly, there was no difference in

h 0.93% (0.009399393939999999999

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cardiac mortality in patients with echocardiographic expression of disease versus carriers without

LVH (Kaplan Meier log rank test: p=0.21).

Discussion

This is the largest study of patients with mutations in the TNNT2 gene since the first clinical

report suggesting that this form of hypertrophic cardiomyopathy is characterized by a high

incidence of sudden cardiac death16. The findings confirm that a history of young sudden cardiac

death is common in many families and that mild or absent hypertrophy is present in a minority of

adult mutation carriers.

Disease penetrance

In common with most other reports, this study shows that disease penetrance (defined by

echocardiographic evidence for HCM) is low in children. A substantial minority of adults with

TNNT2 mutations also have normal echoes, although most of these had an abnormal ECG,

emphasizing the role of both tests in clinical screening. In those adults with an abnormal echo,

the distribution and severity of myocardial hypertrophy was very similar to that reported in other

sarcomere protein mutations33. The one major difference with larger studies was a lower

prevalence of LVOTO, which was present in less than 10% of patients. The reasons for this are

not clear from this study, but we cannot exclude the possibility that some individuals had

provocable obstruction.

Survival analyses

A recent meta-analysis of large cohort studies indicates that the rate of sudden death in patients

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fulfilling diagnostic criteria for HCM is less than 1% per annum34. However, prospective data on

outcomes in genotyped patients are scarce and studies rarely consider both probands and

relatives that carry the same mutation.

In the case of TNNT2, we identified five studies that have reported on survival16;18;32;35;36.

Overall, 166 carriers of TNNT2 mutations identified by genetic and pedigree analysis are

reported. Of these, 61 died from an apparent cardiac cause, the majority (n=50) of whom died

suddenly. In two studies, however, the number of sudden deaths was low, in accord with the

prospective data in this report (Table 4).

In this study, while we adopted a conservative approach to the coding of deaths in relatives,

pedigree analysis still demonstrated a high prevalence of sudden cardiac death in affected

families. However, when we examined survival in the prospectively followed cohort, the annual

rate of sudden death was less than 1% and in line with the average seen in large follow-up

studies34. One possible explanation for this apparent paradox is the use of prophylactic ICDs in

patients and relatives deemed to be at high risk, but this seems unlikely given that only a single

patient (out of 23 with a device) had an appropriate ICD therapy for sustained ventricular

arrhythmia during follow-up. Another possibility is the inclusion of relatives with a mild or

absent clinical phenotype, which could have resulted in better outcomes. This study was too

small to conduct adequately powered sub-group analyses, but the annual rate of sudden death

was similar in probands and relatives suggesting that other mechanisms Including genetic,

epigenetic and environmental factors were at least partially responsible for the better outcomes in

individuals with a phenotype.

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A number of studies have reported sudden deaths in TNNT2 mutation carriers with little or no

hypertrophy. The same phenomenon was seen in a few patients in this study, but the relative

paucity of events in the prospectively followed cohort means that we can not determine the

relative risk of sudden death in mutation carriers with normal echoes. Similarly, although most

deaths occurred in families with mutations in exons 8 and 9 this study was not powered to detect

differences in outcome between specific mutations.

Comparison with other sarcomere protein gene mutations

A lack of prospective survival data in patients with other sarcomere protein gene mutations

makes direct comparison with this study challenging. A recent study37 evaluating not only

probands, but also asymptomatic relatives carrying MyBPC3 mutations (a total of 235 mutations

carriers) reported an annual rate of sudden cardiac death of only 0.1%. However, approximately

two thirds of the population carried a well recognized Dutch founder mutation, limiting

comparison with our own population in which there was a greater spectrum of genetic

abnormalities including single base substitutions, deletions and splice-donor site mutations.

Disease progression

Progressive myocardial thinning and contractile impairment is a well recognized phenomenon in

HCM5-6. In this study, the rate of disease progression appeared slow in that only three patients

developed ventricular dilatation after more than a decade of follow-up. However, five patients

already had ventricular dilatation at presentation (age 48 to 64 yrs) and four patients died from

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heart failure during follow-up, suggesting that progression to heart failure may be relatively

common in patients with TNNT2 mutations.

Limitations

The population in this study is inevitably subject to referral and ascertainment bias. The selection

of the population is also different from some previous reports in that we included only patients

with genetic, clinical or pathologic confirmation of the disease, whereas in previous outcome

studies all relatives who died suddenly were presumed affected with disease. This means that the

burden of sudden death caused by TNNT2 mutations may have been underestimated in some

families.

The extreme phenotypic variability observed in this study shows that outcomes in individuals

carrying mutations in TNNT2 is influenced by many factors other than the position and nature of

the mutation itself. Due to financial and other inevitable constraints in a study performed over

two decades, we were unable to exclude the possibility that some patients harbored additional

sarcomere protein gene mutations. However, contemporary genetic studies indicate that this

occurs in only 5% of patients38. While double mutations could account for a high mortality in

individual families, it is unlikely that the presence of additional mutations explains for the

relatively low annual incidence of sudden death observed in the prospective cohort.

Conclusion

Investigation of genotype-phenotype relations in a diverse and complex disorder such as HCM is

challenging. While this study shares many of the limitations of previous studies, it is reasonable

been underestiimmmmmmmmmmmmmmmmm

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to conclude the following about TNNT2 mutations: ventricular hypertrophy is uncommon in

children and adolescents; ventricular hypertrophy on echo is absent in an important minority of

adult patients; irrespective of echocardiographic findings, most adult carriers have an abnormal

ECG; sudden cardiac death is an important complication, but at least in this prospectively

managed cohort, its incidence is no greater than in most contemporary cohort studies; and

finally, although rare, sudden cardiac death can occur in individuals with little or no hypertrophy.

Acknowledgements: We are grateful to Dr. Letizia Bacchi Reggiani (biostatistician at

Cardiology Department, S.Orsola-Malpighi Hospital, Bologna) for the assistance with the

statistical analysis. Professor McKenna, Dr Elliott and Dr Syrris acknowledge support from the

UCLH/UCL NIHR Comprehensive Biomedical Research Centre.

Conflict of Interest Disclosures: None

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Committee for Practice Guidelines. European Society of Cardiology. American College of Cardiology/European Society of Cardiology clinical expert consensus document on hypertrophic cardiomyopathy. A report of the American College of Cardiology Foundation Task Force on Clinical Expert Consensus Documents and the European Society of Cardiology Committee for Practice Guidelines. J Am Coll Cardiol. 2003;42:1687-1713. 29. Mogensen J, Bahl A, Kubo T, Elanko N, Taylor R, McKenna WJ. Comparison of fluorescent SSCP and denaturing HPLC analysis with direct sequencing for mutation screening in hypertrophic cardiomyopathy. J Med Genet. 2003;40:e59. 30. Mogensen J, Murphy RT, Shaw T, Bahl A, Redwood C, Watkins H, et al. Severe disease expression of cardiac troponin C and T mutations in patients with idiopathic dilated cardiomyopathy. J Am Coll Cardiol. 2004;44:2033-2040. 31. Forissier JF, Carrier L, Farza H, Bonne G, Bercovici J, Richard P, et al. Codon 102 of the cardiac troponin T gene is a putative hot spot for mutations in familial hypertrophic cardiomyopathy. Circulation. 1996;94:3069-3073. 32. Nakajima-Taniguchi C, Matsui H, Fujio Y, Nagata S, Kishimoto T, Yamauchi-Takihara K. Novel missense mutation in cardiac troponin T gene found in Japanese patient with hypertrophic cardiomyopathy. J Mol Cell Cardiol. 1997;29:839-843. 33. Van Driest SL, Ommen SR, Tajik AJ, Gersh BJ, Ackerman MJ. Yield of genetic testing in hypertrophic cardiomyopathy. Mayo Clin Proc. 2005;80:739-744. 34. Elliott PM, Gimeno JR, Thaman R, Shah J, Ward D, Dickie S, et al. Historical trends in reported survival rates in patients with hypertrophic cardiomyopathy. Heart. 2006;92:785-791. 35. Anan R, Shono H, Kisanuki A, Arima S, Nakao S, Tanaka H. Patients with familial hypertrophic cardiomyopathy caused by a Phe110Ile missense mutation in the cardiac troponin T gene have variable cardiac morphologies and a favorable prognosis. Circulation. 1998;98:391-397. 36. Torricelli F, Girolami F, Olivotto I, Passerini I, Frusconi S, Vargiu D, et al. Prevalence and clinical profile of troponin T mutations among patients with hypertrophic cardiomyopathy in tuscany. Am J Cardiol. 2003;92:1358-1362. 37. Christiaans I, Birnie E, van Langen IM, van Spaendonck-Zwarts KY, van Tintelen JP, van den Berg MP, et al. The yield of risk stratification for sudden cardiac death in hypertrophic cardiomyopathy myosin-binding protein C gene mutation carriers: focus on predictive screening. Eur Heart J. 2010;31:842-848. 38. Richard P, Charron P, Carrier L, Ledeuil C, Cheav T, Pichereau C, et al. EUROGENE Heart Failure Project. Hypertrophic cardiomyopathy: distribution of disease genes, spectrum of mutations, and implications for a molecular diagnosis strategy. Circulation. 2003;107:2227-2232.

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Table 1: Baseline clinical characteristics in adults and children with TNNT2 mutations Adults 16 years p value n 71 21 Male 36 (51%) 15 (71%) 0.44 Age (yrs) 37 ± 14 9 ± 5 na NYHA I-II (assessed in 62 adults and 18 children)

59 (95%) 18 (100%) 0.9

Chest Pain 18 (29%) 0 (0%) 0.06 Palpitations 10 (17%) 0 (0%) 0.21 Obstruction 6 (9%) 0 (0%) 0.48 ECG available 68 19 PR msec 157 ± 30 123 ± 15 0.001 QRS msec 95 ± 21 89 ± 11 0.3 QTc msec 427 ± 29 412 ± 23 0.23 QRS axis deviation 10 (15%) 2 (10%) 0.86 LVH 35 (51%) 4 (21%) 0.76 Q wave 25 (37%) 3 (16%) 0.07 Repolarization abnormalities 42 (61%) 4 (21%) 0.91 LBBB 1 (1%) 0 (0%) na RBBB 4 (6%) 0 (0%) na LAH 4 (6%) 0 (0%) na Normal ECG 13 (19%) * 13 (68%) 0.01 Key: LVH: left ventricular hypertrophy; LBBB: Left Bundle Branch Block; RBBB: Right Bundle Branch Block; LAH: Left Anterior Hemiblock; na: not applicable. Yates correction was employed when appropriate. *related to 68 available ECGs

Table 2: Echocardiographic characteristics at baseline in all adult carriers of a TNNT2 mutation compared to those observed in adults with a mutation and an abnormal echocardiogram. All Adults TNNT2+ Adults with abnormal echo N 68 52 Male 35 (51%) 27 (52%) Age yrs 38 ± 15 37 ± 15 LVEDd mm 45 ± 6 45 ± 6 LVESd mm 28 ± 6 28 ± 6 LVMWT mm 17 ± 6 19 ± 5 FS% 38 ± 8 37 ± 8 LA mm 41 ± 10 42 ± 9 ASH 37 (54%) 37 (71%) Concentric Hypertrophy 12 (18%) 12 (23%) Apical Hypertrophy 3 (4%) 3 (6%) Left ventricular dilatation and/or low FS* 5 (7%) 5 (10%) Biventricular Hypertrophy 3 (4%) 3 (6%) Normal Echo 16 (24%) --- Key: Echo+: phenotypic expression of disease on Echocardiography; LVEDd: left ventricular end diastolic diameter; LVESd: left ventricular end systolic diameter; LVMWT: maximal left ventricular wall thickness: FS: fractional shortening; LA: left atrium diameter on M-Mode; ASH: asymmetrical septal hypertrophy *end-diastolic cavity dimension measured by M-mode echocardiography exceeding the reference upper 95% confidence interval (CI) for body surface area or a FS 25%

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Table 3: Prevalence of risk factors for sudden cardiac death in adults and children with TNNT2 mutations. Risk profile Adults 16 years Complete stratification (5 RF) 54 10 Number of risk factors 1.9 ± 0.9 1.6 ± 0.8 Number of patients with 2 RF 35 (65%) 6 (60%) Family History SD age 40yrs 45 (83%) 9 (90%) Multiple SD in the family 35 (65%) 8 (80%) SD in 1st degree relative 27 (50%) 4 (40%) NSVT 13 (24%) 0 - recorded in first ECG Holter 8 (61%) 0 - recorded in subsequent ECG Holter 5 (39%) 0 Maximal wall thickness 30 mm 3 (5%) 0 Syncope 17 (31%) 3 (30%) Abnormal blood pressure response 26 (48%) 4 (40%) Key: SD: sudden cardiac death; RF: risk factors; NSVT: non sustained ventricular tachycardia.

Table 4: Studies reporting on survival of TNNT2 mutations Study No of

Patients No of Cardiac Deaths

No of Sudden Deaths

No of Families

Mutation

Watkins H, et al. 199516 112 50 39 11 Ile79Asn Arg92Gln Phe110Ile �Glu160 Glu163Lys Glu244Asp Intron 15 G�A Arg278Cys

Nakajima-Taniguchi C, et al. 199732

4 2 2 1 Ala104Val

Moolman JC, et al. 199718 22 7 7 2 Arg92Trp Anan R, et al 199835 18 2 2 6 Phe110Ile Torricelli F, et al 200336 10 0 0 5 Phe110Leu

Arg130Cys Glu160del Arg92Gln Arg278Cys

Total 166 61 50 25

Figure Legends:

Figure 1: Relation between ECG and echocardiographic findings in adult patients with TNNT2

mutations. Echo+: phenotypic expression of disease on Echo; ECG+: phenotypic expression of

orti rviv l f TNNT2 t ti

5

NNNNNo o f f ff fPaaattiennttts

NNNNNo oo ooof f f f fCaCardiaiiac DeDeDeDeDeatatatatathshshshshs

NNNNNo ooooof f f fSuSuSuSS dddden DeDeDeDeDeatatattthshshshshs

ortititititingngngngng o o o o on nnnn sususususurvival of TNNT2 m m mmmutations NNNNNo o oof f f ff Faaammimm llies

516 1122222 5 5 5 550 0000 3939393939 1 1 11 11



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23

disease on ECG; Echo-: no phenotypic expression of disease on Echo; ECG-: no phenotypic

expression of disease on ECG.

Figure 2: Prevalence of normal echocardiogram at different ages.

Figure 3: Kaplan Meier survival curves for survival from birth (A) and from first evaluation (B).

The cohort evaluated from birth includes 12 cases of SD identified from pedigrees (obligate

mutation carriers, post-mortem evidence for HCM and known HCM prior to death).

Figure 4: Kaplan Meier survival curves showing survival from cardiovascular death and sudden

cardiac death from diagnosis and from birth for individual mutations. The cohort evaluated from

birth includes 12 cases of SD identified from pedigrees (obligate mutation carriers, post-mortem

evidence for HCM and known HCM prior to death).

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t

e

n

iaaaaagggngg osisss a a aandndnndn fffffrororororom mmmm bibbibib rtrtrtrtrth h h hh fofofor rrr iindn ivviddddduauauauaual lll l mumumumum tatatataatititititionooo s.s.s.s.s. TT TT Theheheheh cocococoohohohohohort

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ndnnd k knononownwnwn HH HCMCMCM p p pppririororor tt to o o dedeatatath)h)))).



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1,0

0,8

0,6

0,4

0,2

0,0

0 5 10 15 20 25 years

Arg92Gln

Arg92Trp

Glu163del

Ile79Asn

IVS15+1G>A

Other mutationsCu

mul

ativ

e su

rviv

al

Follow up

SD

1,0

0,8

0,6

0,4

0,2

0,0

0 5 10 15 20 25 years

Arg92Gln

Arg92Trp

Glu163del

Ile79Asn

IVS15+1G>A

Other mutations

Cum

ulat

ive

surv

ival

Follow up

CV death

1,0

0,8

0,6

0,4

0,2

0,0

0 10 20 30 40 50 60 70 80 years

Arg92Gln

Arg92Trp

Glu163del

Ile79Asn

IVS15+1G>A

Other mutations

Cum

ulat

ive

surv

ival

Age

SD

1,0

0,8

0,6

0,4

0,2

0,0

0 10 20 30 40 50 60 70 80 years

Arg92Gln

Arg92Trp

Glu163del

Ile79Asn

IVS15+1G>A

Other mutations

Cum

ulat

ive

surv

ival

Age

CV death

10101010101010000011111111 20222222220 10 20ears 0 10 20ears



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Perry ElliottFerdinando Pasquale, Petros Syrris, Juan Pablo Kaski, Jens Mogensen, William J. McKenna and

Troponin T GeneLong-Term Outcomes in Hypertrophic Cardiomyopathy Caused by Mutations in the Cardiac

Print ISSN: 1942-325X. Online ISSN: 1942-3268 Copyright © 2011 American Heart Association, Inc. All rights reserved.

TX 75231is published by the American Heart Association, 7272 Greenville Avenue, Dallas,Circulation: Cardiovascular Genetics

published online December 5, 2011;Circ Cardiovasc Genet.

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SUPPLEMENTALMATERIAL

SupplementalTable1:Numberofcontrolstestedformutationsinpreviousstudies

Mutation Reference Controls

(normalchromosomes)

ThierfelderLetal.Alpha‐tropomyosinandcardiactroponinTmutations

causefamilialhypertrophiccardiomyopathy:adiseaseofthesarcomere.

Cell1994;77(5):701‐12.

200

WatkinsHetal.MutationsinthegenesforcardiactroponinTandalpha‐

tropomyosininhypertrophiccardiomyopathy.NEnglJMed

1995;332(16):1058‐64.

200

Ile79Asn

VarnavaAMetal.Hypertrophiccardiomyopathy:histopathological

featuresofsuddendeathincardiactroponinTdisease.Circulation.

2001;104(12):1380‐4.

180

Glu83Lys MogensenJetal.ComparisonoffluorescentSSCPanddenaturingHPLC

analysiswithdirectsequencingformutationscreeninginhypertrophic

cardiomyopathy.JMedGenet2003;40:e59

na

Varnava AM et al. Hypertrophic cardiomyopathy: histopathological

features of sudden death in cardiac troponin T disease. Circulation.

2001;104(12):1380‐4.(180normalchromosomes)

180Arg92Leu

RichardPetal.Hypertrophiccardiomyopathy:distributionofdisease

genes,spectrumofmutations,andimplicationsforamoleculardiagnosis

strategy.Circulation2003;107(17):2227‐32.

200

Moolman‐SmookJCetal.SuddendeathduetotroponinTmutations.J

AmCollCardiol1997;29(3):549‐55.

200Arg92Trp

VanDriestSLetal.Prevalenceandspectrumofthinfilamentmutationsin

anoutpatientreferralpopulationwithhypertrophiccardiomyopathy.

Circulation2003;108(4):445‐51.

400

ThierfelderLetal.Alpha‐tropomyosinandcardiactroponinTmutations


Cell1994;77(5):701‐12.

200



1995;332(16):1058‐64.

200

Arg92Gln

VanDriestSLetal.MyosinbindingproteinCmutationsandcompound

heterozygosityinhypertrophiccardiomyopathy.JAmCollCardiol

400

2

2004;44(9):1903‐10.

VarnavaAMetal.AnewmutationofthecardiactroponinTgenecausing

familialhypertrophiccardiomyopathywithoutleftventricular

hypertrophy.Heart1999;82(5):621‐4.

200Arg94Leu

VarnavaAMetal.Hypertrophiccardiomyopathy:histopathological

featuresofsuddendeathincardiactroponinTdisease.Circulation.

2001;104(12):1380‐4.

180

Ala104Val Nakajima‐TaniguchiCetal.Novelmissensemutationincardiactroponin

TgenefoundinJapanesepatientwithhypertrophiccardiomyopathy.J

MolCellCardiol1997;29(2):839‐43.

>50

Glu163Lys WatkinsHetal.MutationsinthegenesforcardiactroponinTandalpha‐


1995;332(16):1058‐64.

200



1995;332(16):1058‐64.

200Glu163del

RichardPetal.Hypertrophiccardiomyopathy:distributionofdisease

genes,spectrumofmutations,andimplicationsforamoleculardiagnosis

strategy.Circulation2003;107(17):2227‐32.

200

IVS15+1G>A ThierfelderLetal.Alpha‐tropomyosinandcardiactroponinTmutations


Cell1994;77(5):701‐12.

200



1995;332(16):1058‐64.

200

VanDriestSLetal.Prevalenceandspectrumofthinfilamentmutationsin

anoutpatientreferralpopulationwithhypertrophiccardiomyopathy.

Circulation2003;108(4):445‐51.

400

Garcia‐CastroMetal.Hypertrophiccardiomyopathy:lowfrequencyof

mutationsinthebeta‐myosinheavychain(MYH7)andcardiactroponinT

(TNNT2)genesamongSpanishpatients.ClinChem2003;49(8):1279‐85.

400

Arg278Cys

InglesJetal.Compoundanddoublemutationsinpatientswith

hypertrophiccardiomyopathy:implicationsforgenetictestingand

counselling.JMedGenet.2005;42(10):e59.

300

3

Supplemental Table 2: The following is a summary of the bioinformatics analysis of themissensemutations(i.e.9knownand1novel).Thisisprovidedasasupplementaldatafile.Sequencevariant PolyPhen‐2prediction SIFTprediction

Ile79Asn Probablydamaging NottoleratedGlu83Lys Probablydamaging NottoleratedArg92Leu Probablydamaging NottoleratedArg92Trp Probablydamaging NottoleratedArg92Gln Probablydamaging NottoleratedArg94Leu Probablydamaging NottoleratedAla104Val Benign NottoleratedGlu163Lys Probablydamaging ToleratedArg278Cys Probablydamaging ToleratedArg278His(novel)

Probablydamaging Tolerated

Supplemental Table3: numberof carriers for eachmutationgroup starting the followupfromdateofbirth.

Mutation Carriers Events FUP yrs Rate SD CI 95% Arg92Gln 6 3 242.9 0.21 0.04-0.06 Ile79Asn 6 1 166.8 0.1 0.00-0.56 Arg92Trp 13 2 361 0.04 0.01-0.15 Glu163del 27 6 1098.5 0.02 0.01-0.04 IVS15+1G->A 20 1 843.2 0.01 0.00-0.03 Other mutations 27 6 1019.4 0.02 0.01-0.05 Commentary: Ever since the first gene for hypertrophic cardiomyopathy was identified in the late 1980s, investigators have sought to link genotypes with clinical phenotypes. One of the best known putative associations is that between mutations in the troponin T gene (TNNT2) and a high risk of sudden cardiac death. In this study we present the first large-scale longitudinal cohort study in patients and genotyped relatives with TNNT2 mutations. Themostimportantfindingisthat,despiteahighprevalenceofhistoricdeathsinfamilies,theincidenceofsuddendeathinthisprospectivelymanagedcohortwassimilartothatobservedinothersarcomereproteingenemutations.Wealsodemonstratethatventricularhypertrophyisuncommoninchildrenandadolescentsandthatventricularhypertrophyisabsentinanimportantminorityofadultpatients.Together,thesefindingshavemajorimplicationsforclinicalandgeneticscreeningoffamilieswithTNNT2mutations.

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