Phenotypic and Genotypic Heterogeneity in Transthyretin-Related Cardiac Amyloidosis: Towards Tailoring of Therapeutic Strategies?

By Rapezzi, Claudio; Perugini, Enrica; Salvi, Fabrizio; Grigioni, Francesco; Et al

Keywords: Amyloidosis, transthyretin, cardiomyopaihy, prognosis, gene mutations

Abbreviations: TTR = transthyretin; ATTR = transthyretin-related hereditary (amyloidosis); FAP = familial amyloid polyncuropathy; OLT – onhowpic liver transplantation; ECG = electrocardiogram; LV = left ventricular; MACE= major cardiovascular events

Abstract

Transthyretin-related hereditary amyloidosis (ATTR) is genotypically/phenotypically heterogeneous. We investigated myocardial involvement in ATTR in a cohort of patients with a wide range of mutations. Clinical echocardiographic follow-up of 41 consecutive symptomatic ATTR patients from a single referral center was analyzed according to TTR mutation. Diagnosis was based on histology, immunohistochemistry and genotyping. Median follow up was 40 months (range 8-120). Among the 12 different mutations identified, Val30Met was found in 10 patients and Glu89Gln in seven. Compared with Val30Met, Glu89Gln was associated with higher LV mass index, lower left ventricular ejection fraction and shorter E-wave deceleration time. All Glu89Gln carriers had cardiomynpathy, which was more severe (for left ventricular thickness, left ventricular mass and restrictive pathophysiolugy) than in the six affected Val30Met patients. Glu89Gln was independently associated with higher risk of major cardiovascular events among cardiomyopathy patients. This follow-up study of ATTR patients carrying a wide range of mutations indicates that (1) cardiac involvement is a very important component of phenotypic expression; and (2) genotype is an important source of heterogeneity in myocardial involvement, with Glu89Gln being associated with a severe, heart-driven prognosis. We think that combined heart liver transplantation could be considered for Glu89Gln carriers with established, morphologically severe cardiomyopathy.

Introduction

Hereditary transthyreting (TTR)-related amyloidosis (ATTR amyloidosis; also referred to as familial amyloidotic polyneuropathy, or FAP) is a relatively rare autosomal dominant disease resulting from the productin of an unstable variant of the TTR serum protein [1]. ATTR amyloidoses are phenotypically heterogeneous, and the TTR genotype shows high alletic gentic heterogeneity. Over 80 TTR point mutations have been identified, which predominantly result in neurological and/or cardiac dysfunction [2,3]. Prevalence of different mutations varies according to ethnicity and geographical region. In each of the three main reported geographical foci of ATTR amyloidosis (in Portugal [4] northern Sweden [5], and Japan [6]), Val30Met is the predominant mutation. cases of ATTR amyloidosis are increasingly being reported throughout Europe [7]. In the USA [8], the disease is thought to be widely underdiagnosed [1]. The likelihood that almost 4% of the black American population are heterozygote carriers of the Val122Ile TTR mutation (a documented cause of cardiac amyloidosis in old age) could partially explain the elevated prevalence of heart failure among older blacks [8].

Phenotypic variability and severity are related to the type and extent of multiorgan involvement. The course of disease commonly begins with sensorimotor polyneuropathy in early adulthood, and rapidly progresses to incapacitate the patient within a few years. Death tends to occur 5-15 years after onset of symptoms, and in many populations the median survival is below 10 years [3]. Clinically relevant cardiac involvement, which is generally a late phenomenon among Val30Met carriers, appears to be the predominant feature in many rarer mutations [1,2,9,10].

Since the liver is responsible for over 98% of the protein production, the treatment of choice is orthotopic liver transplantation (OLT) – which can be considered a form of ‘surgical gene therapy’ – at an early stage of the symptomatic course of the disease, when neurological progression can still be halted [1,2,10). Cardiac involvement has major clinical and prognostic implications in ATTR amyloidosis, since it can limit both the short- and long- term outcome of OLT and influence the decision to perform combined heart-liver transplantation [11-13]. Knowledge about genotype- phenotype correlations is limited, especially as regards cardiac involvement, where the available data mainly derives from case reports/series |2|, autopsy studies [14] and analyses of patients undergoing OLT [13]. Information on associations between genotype and cardiac involvement could be important for therapeutic decision making.

We studied a cohort of patients affected by AlTR amyloidosis (with different mutations) who were recruited at a single, large referral center, focusing on severity of myocardial involvement.

Methods

Clinical setting and patient recruitment/selection

Since 1994, our cardiology, neurology and genetics departments have operated in conjunction to provide one of the two main national referral centers for diagnosis and treatment of hereditary’ amyloidosis, serving patients from throughout Italy. All subjects on the center’s database are prospectively analyzed by genotyping and a predefined set of clinical/ instrumental examinations. Familial (clinical and genotype) screening is performed whenever applicable and feasible. All symptomatic ATTR amyloidosis patients who were evaluated between 1994 and March 2005 were included in the present analysis. Diagnosis of amyloidosis was defined by histological documentation of Congo-red staining and apple-green birefringence under cross-polarized light in at least one involved organ [1,10]. Diagnosis of familial TTR-related amyloidosis was denned by a documented TTR mutation at DNA analysis. All participants provided informed consent for anonymous data publication.

Genotyping

Genomic DNA was isolated from whole peripheral blood by standard techniques. Exons 2, 3 and 4 of the TTR gene (accession number ml 1844) were amplified by PCR (Takara ExTaq polymerase) using primers previously described [15]. Amplified DNA fragments were directly sequenced using ABI Prism 3100 automated sequencer.

Data collection and definitions

At the first (baseline) evaluation and at each follow-up visit (scheduled according to clinical needs), a comprehensive set of neurological, cardiological and other relevant clinical/ instrumental data were collected. Details of the history of neurological disease were elicited from the patients and their families, and completed with information from the patients* medical records. Decisions for familial referral were based on clinical and feasibility considerations. For assessment of autonomie neuropathy, onset of the following manifestations was recorded: impotence, symptomatic orthostatic hypotension, diarrhea and constipation, urinary incontinence, and sweating abnormalities. Neurosensory impairment was staged from 0 to 3 (0 – no impairment; 1 – impairment limited to the lower limbs without impeding autonomous deambulation, but including extensively impaired pain/temperature perception; 2 – steppage with distal amyotrophies of lower limbs and wasting/ weakness of hand muscles, plus extensively impaired pain/ temperature sensation in the upper limbs; 3 generalized muscular weakness, atrophy and areflexia leading to wheelchair/bed, with pain and temperature felt only in head/neck) [16],

In all patients, cardiac investigations included accurate search for signs and symptoms of heart failure, standard electrocardiogram (ECG), and transthoracic echocardiography. For descriptive purposes, we considered signs of cardiac involvement in terms of both standard ECG and echocardiographic abnormalities. “Abnormal ECG’ was defined as presence of one or more of the following features: conduction disturbances (right bundle branch block, left bundle branch block, atrioventricular block, left anterior hemiblock), low QRS voltage (QRS amplitude ≤0.5 mV in all limb leads or ≤1 mV in all precordial leads), ST and T wave abnormalities, and ‘pseudoinfarction’ pattern. Echocardiographically, LV mass, which was calculated according to Devereux et al. [17] was ‘increased’ when >130 g/m^sup 2^ in men and > 110 g/m^sup 2^ in women. Restrictive LV filling pattern was defined by an E to A wave ratio > 2 and E wave deceleration time < 150 cm/s [18]. Presence of amyloidotic cardiomyopathy was defined as end-diastolic thickness of the intcrvcntricular septum > 1.2 cm (in the absence of any other cause of ventricular hypertrophy) plus two or more of the following: (a) homogeneous a trio ventricular valve thickening; (b) atrial septal thickening; and (c) sparkling appearance of the ventricular septum (19]. Availability of cardiac catheterization data and right ventricular endomyocardial biopsies depended on patients’ clinical needs.

Analysis

Genotype suhgrouping was based on frequency considerations: direct comparisons were performed between the two most represented mutation subgroups. Events considered during follow-up were death (any cause), major cardiovascular events (MACE; defined as cardiovascular death, severe heart failure [NYHA III-IV], symptomatic complete atrioventricular block requiring pacemaker implantation), and severe neurosensory progression (onset of stage >2). Organ transplantations were not considered endpoints, due to their prophylactic intent [2]. In the overall p\opulation, survival analyses were performed both with and without censoring at transplantation, since the disease (and amyloid deposition) can progress despite interruption of mutant protein production by OLT.

Analysis was performed using SPSS statistical software 12.0 (Chicago, IL, USA) with significance set to P < 0.05. Variables are expressed as mean + standard deviation, or numbers (percentages). Differences between subgroups were determined using the Mann- Whitney U-test, Fisher's exact test or contingency tables, as appropriate. Multiple regression analysis was used to identify within the overall population variables associated with left ventricular (LV) mass, considered as a dependent, continuous variable. The Kolmogonov Smirnoff test was used to verify normal distribution of LV mass, and variance inflation factor was used to test the absence of colinearity among independent variables. For follow-up analysis, probabilities of events were calculated according to Kaplan Meier, and curves were compared using die log- rank test. Univariate and multivariable Cox's proportional hazard regression models were used to identify predictors of death and events. Variables reaching P≤0.1 at the risk-ratio test were forced into the multivariate model using a forward stepwise procedure. The assumption of proportionality was assessed for the selected variables and colinearity was tested. Risks associated with variables were expressed as risk ratios with 95% confidence intervals.

Results

Study population and genotype distribution

Forty-one patients were eligible for analysis (including some described in case reports [20-26]). Definitive diagnosis of ATTR amyloidosis was ultimately generated by initial neurological symptoms in 26 (63%) patients, initial cardiac symptoms in three (7%), and family screening in 12 (30%). Sequence analysis of the TTR gene coding region (exons 2-4) identified the following mutations: Val30Met (n = 10), Glu89Gln (n = 7), Thr49Ala (n = 5), Phe64Leu (n = 3), Ile68Leu (n = 4), Glu54Lys (n = 3), Ala36Pro (n = 2), Arg34Thr (n = 2), Ser50Arg (n = 2), Gly47Ala (n = 1), Val30Ala (n = 1), and Phe33Val (n = 1). All patients were hetero/ygous for the identified mutation. No other TTR mutation was found on exons 2, 3 or 4.

Baseline disease profile and genotype

Main baseline characteristics of the overall study population are summarized in Table I according to the two most prevalent genotype subgroups (Val30Met, Glu89Gln) and the rest of the population; data regarding individual mutations other than Val30Met and Glu89Gln are detailed in Table II. All patients were Caucasian. The 41 patients belonged to 26 distinct families originating from southern, central and northern parts of Italy, including two evident areas of geographical aggregation (both near the coast): seven families in the southern part of Lazio (south of Rome); six families in the southeastern portion of Sicily. Remarkably, all six families carrying Val30Met came from Lazio, while four of the six families with Glu89Gln came from Sicily. No difference in gender/age was apparent between genotype subgroups. Severity of sensory/motor involvement and frequency of autonomie impairment both appeared similar in the genotype subgroups. Neither of the two patients with (moderate) impairment of renal function required treatment.

Cardiac involvement was assessed in terms of standard ECG, echocardiographic and clinical abnormalities. Forty of 41 patients were in sinus rhythm (one patient has had permanent atrial fibrillation since first evaluation). As can be seen from Figure 1, the frequency of different clinical/ HCG/echocardiographic signs of cardiac involvement within tile overall study population ranged from 12 to 80%. Of note, mutations other than Vl30Met by far the largest subgroup – accounted for the majority of instances of each type of abnormality (Figure 1). When compared with carriers of Val30Met, patients with Glu89Gln had higher LV mass index, lower left ventricular ejection fraction and shorter E-wave deceleration time and showed a trend toward larger left atrial diameter (Table I).

Table I. Characteristics of the study population according to genotype.

Thirty patients had amyloidotic cardiomyopathy. Endomyocardial biopsies were available for 19 of these patients, and in all cases confirmed moderate to severe interstitial amyloidotic infiltration (in five cases accompanied by vascular involvement). In 11 of the 30 cardiomyopathic patients, heart disease was isolated or predominant with respect to neurological manifestations.

To assess possible relations between genotype and myocardial involvement, we first analyzed the contribution of the mutation and the other main basal features to the amount of LV mass (Table III). At univariate analysis, variables associated with the increase of LV mass were Glu89Gln mutation and male gender. At multiple regression, the role of the mutation remained highly significant, and sex almost reached significance.

We then compared patients with (n = 30) and without (n = 11) cardiomyopathy with respect to mutation and the other main basal features. No significant difference was apparent in terms of male gender (70% [n = 21] vs. 55% [6]; P = 0.58), age at first presentation (47 12 vs. 44 17 years; P = 0.54), age at onset of disease (45 11 vs. 44 12 years P = 0.74), Glu89Gln (23% [7] vs. 0% [0]; P = 0.19), Val30Met (20% [6] vs. 36% [4]; P = 0.50). History of carpal tunnel syndrome was more than 4 times more frequent among the patients with CMP (90% [27] vs. 20% [2]; P = 0.0001).

Table II. Characteristics of patients carrying mutations other than Val30Met or Glu89Gln.

Figure 1. Frequency of clinical, electrocardiographic and echocardiographic signs of cardiac involvement.

Table III. Multiple regression analysis of baseline characteristics with LV mass (g/m^sup 2^) as the dependent variable.

Finally, within the subset of patients with cardiomyopathy we compared the genotype subgroups as regards basal findings (Table IV). Patients with Glu89 Gln showed more severe myocardial disease in terms of LV thickness, LV mass, and restrictive pathophysiology, Overall, a pattern of increasing echocardiographic severity can be discerned in Table IV, with Val30Met showing the mildest alterations and Glu89Gln the most severe changes (and the miscellaneous group occupying an intermediate position).

Disease course and prognosis

Overall study population. Duration of follow-up ranged from 8 to 120 months (median, 40). All living patients were monitored until within 2 months of the close of the study. OLT was performed in 15 patients (according to international recommendations [11,13]). Combined heart-liver transplantation was planned in seven patients (in one of whom only heart transplantation was eventually performed, due to intraoperativc complications); this strategy was adopted due to coexistence of morphologically severe cardiomyopathy (symptomatic in two patients). No patient acquired echocardiographic evidence of amyloidotic cardiomyopathy during follow up. The following events occurred during the follow-up period: eight deaths from any cause (three due to end-stage progression of neurological impairment; two sudden death; three transplant-related); six severe neurosensorial impairments (stage 3); four severe heart failures (NYHA III-IV); three symptomatic complete atrioventricular blocks requiring pacemaker implantation. Of note, one of the combined heart-liver transplantation recipients died from end-stage neurological progression (after being operated at an advanced stage).

To provide information on both the natural course of the disease and the effects of a transplantation strategy we constructed survival curves with and without censoring at the time of OLT (including combined transplantation) (Figure 2). Overall uncensored and censored mortality rates (per 100 patient-years) were 4.3 and 1,9, respectively (a difference explained by transplant-related mortality and post-transplant disease progression). Cox’s proportional hazard regression analysis was feasible only for uncensored data. At univariate analysis, variables that reached significance were NYHA class III-IV (RR 9.32, 95% confidence interval [95%CI] 1.84-47.2, P= 0.007) and LV mass index (RR 1.009 for each g/m^sup 2^ increment, 95%CI 1.001-1.017, P = 0.026); no variable reached significance at multivariable analysis.

Table IV. Baseline characteristics of patients with cardiomyopathy according to genotype.

Event-free survival curves are shown in Figure 2. Uncensored and censored event rates (including death) per 100 patient-years were 9.6 and 9.3, respectively. At univuriate Cox’s proportional hazard regression analysis of uncensored data (Table V), LV mass, E-wave deceleration time, NYHA class and type of mutation were significantly associated with risk of death. At multivariable analysis, Val30Met mutation almost reached significance (P- 0.06) as a ‘protective’ factor.

Patients with cardiomyopathy. Events recorded within this subset of patients were: eight deaths (three end-stage neurological progression; two sudden; three transplant-related); six severe neurosensory impairments (stage 3); four severe heart failures (NYHA III-IV); three symptomatic complete atrioventricular blocks requiring pacemaker implantation. The mortality rate was 5.4 per 100 patientyears. Figure 3 depicts event-free and MACE-free survival by genotype subgroup; outcome appears to have been mainly heart driven. Rates (per 100 patient-years) of major (cardiological neurological) events and MACE were 12.0 and 10.3, respectively. Significantly worse outcomes were associated with Glu89Gln with respect to Val30Met (Figure 3). Table VI reports univariate/multivariable analysis of risk of MACE: the only independent predictor was Glu89Gln.

Discussion

The results of this study confirm that outside the context of endemic Val30Met geographic foci – the c\ontext of most previous studies – cardiac involvement can be a very important component of the phenotypic expression of familial TTR-related amyloidosis. Our findings regarding Glu89Gln are clinically relevant and could have implications for appropriate selection of organ transplantation candidates.

Figure 2. Kaplan-Meier curves (uncensored and censored data) for the entire study sample. Top: overall survival. Bottom: event-free survival according to genotype.

Table V. Cox proportional hazard regression analysis for determinants of death from any cause within the entire study population.

Genotype and phenotype heterogeneity

Our referral center attracts a widespread sample of amyloidosis patients from all Italy (mainly via the neurology branch). Compared with the main reported series of FAP patients – from Portugal, Sweden and Japan [4-6] – and also with postmortem findings from elderly Afro-Americans in the United States [R], our patients displayed a rather wide spectrum of TTR mutations other than Val30Met, providing a picture of genotypic and phenotypic heterogeneity. Val30Met and Glu89Gln were the two most common mutations (each accounting for six of the 26 affected families), and 10 other TTR mutations were represented. It is difficult to explain the frequency of non-Val30Met mutations; the families came from many parts of Italy (with just two areas of evident geographical aggregation). In our series of patients, the clinical picture was almost exclusively determined by neurological and cardiologic involvement.

Cardiac involvement and genotype

A broad spectrum of cardiac abnormalities was observable (Figure 1), with prevalence ranging from 24% for cardiomyopathies with restrictive physiology to 80% for any type of ECG abnormality. Remarkably, all but one of the mutations were associated with cardiomyopathy (of varying degrees of severity). Even severe morphological abnormalities (e.g. major increase in LV mass) did not invariably translate into overt LV dysfunction (Figure 1). However, subtle systolic and diastolic signs of LV dysfunction can be detected only utilizing more sophisticated echocardiographic techniques for exploration of myocardial tissue kinetics or longitudinal ventricular function [27].

Figure 3. Kaplan-Meier curves for patients with cardiomyopathy according to genotype. Top: event-free survival. Bottom: survival free from major cardiovascular events. (Data were censored at time of heart/OLT transplantations).

Table VI. Cox proportional hazard regression analysis of determinants of major cardiovascular events (including death) within the subpopulatiun of patients with cardiomyopathy.

Analysis of basal features (Tables III and IV) indicated that genotype can strongly influence severity of associated cardiomyopathy. Glu89Gln was strongly associated with increased LV mass (and the only independent variable). In comparison with the Val30Met subgroup, patients with Glu89Gln showed greater intervemricular septal thickness and LV mass and more frequent restrictive pathophysiology. On the other hand, the two subgroups appeared comparable for age, absence of cardiac comorbidities, and diagnostic route (symptom-driven versus family referral). Other potentially relevant factors were carpal tunnel syndrome and male gender. High frequency of carpal tunnel syndrome in patients who later develop cardiomyopathy has been reported in the context of VaBOMet in non-endemic areas [2,6] and Val122Ile in black Americans [8]. An association between male gender and increased LV mass was evident (at univariate analysis), but turned out (at multivariable analysis) to be partly mediated by Glu89Gln genotype. This intriguing association deserves further study (of note, another TTR- related form – senile amyloidosis – almost exclusively affects men [28]).

Comparison of our findings with reports from other geographic areas [4-6] is problematic due to different distributions of ITR mutations. In series regarding endemic foci of Val30Met mutations, conduction disturbances were the single most frequent form of cardiac involvement, whereas heart failure due to cardiomyopathy was a rare, age-related manifestation. Some reports regarding Val30Met in non-endemic areas highlight the possibility of earlier onset of cardiomyopathy. As in our series, cardiomyopathy (often not accompanied by polyneuropathy) has been reported as the predominant clinical feature in the context of other mutations (e.g. Vall22Ile in the USA, Leu111Met in Denmark and Thr60Ala in Japan [4-6]).

Prognosis and genotype

The relatively favorable course of our patients was influenced by frequent use of OLT or combined heart-liver transplantation. Both in the overall study population and the cardiomyopathic subset, eventfree survival appeared to be influenced by genotype. In particular, Glu89Gln conferred a poor prognosis with respect to Val30Met, Considering the patients’ overall history (before and after any OLT), Glu89Gln mutation appeared to be the only independent prognostic variable associated with the combined endpoint of death or major events. We tried to gain information on the natural history of cardiac involvement by censoring MACE-free survival of cardiomyopaihy patients at the time of any heart transplantation: the results (Figure 3) suggest that patients with Glu89Gln have a higher risk of dying or developing severe heart failure or complete atrioventricular block. Taken together, these observations confirm and extend findings from autopsy studies [14] and impressions gleaned from case reports/scries [3,4-7]. Moreover, our results are in line with the observation that after OLT carriers of mutations other than Val30Met have a worse prognosis and greater risk of progression of myocardial involvement [29].

The greater anatomic and functional severity of cardiomyopathy in Glu89Gln carriers could account for the unfavorable clinical course encountered by these patients. More generally, the phenotypic peculiarities are not necessarily causally related to the physicochemical characteristics of different mutant proteins, and epigenetic and environmental factors are also possible.

Clinical implications

Confirmation that cardiac involvement is a major component of the phenotypic expression of TTR-related familial amyloidosis – particularly in patients with Glu89Gln and other non-Va!30Met mutations underlines the importance of a comprehensive cardiological work-up. Since the ominous prognosis of patients with Glu89Gln appears to be mainly due to cardiovascular events, irrespective of the degree of neurological impairment, combined heart liver transplantation (as opposed to OLT alone) could be considered with the aim to reduce operative risks and improve long-term outcome. The rationale for such a strategy is also supported by: (1) the promising results achieved with combined interventions performed in relatively early disease stages [5,24]; (2) incremental surgical risks of OLT in cardiomyopathic patients [12,13]; and (3) the poor prognostic impact of established severe cardiomyopathy in patients who survive OLT [13,28], The rapidity of the decline of patients carrying Glu89Gln provides a further argument for performing combined heart-liver transplantation when definite cardiomyopathy is present (perhaps even in the absence of cardiovascular symptoms at the time of scheduled OLT).

Study limitations

The restricted sample size must be considered in relation to the rarity of the disease. Subgroup analysis was based on prevalence considerations, and our results are not informative regarding the prognostic implications of individual mutations other than Glu89Gln and Val30Met. Although we screened as many family members as possible, systematic study of all family pedigrees was not appropriate. This study of symptomatic patients is not informative as regards penetrance data and may not fully reflect the clinical spectrum of the disease, possibly leading to a generalized overestimation of disease severity. Given the cohort’s Italian ancestry, caution is required when generalizing the results in terms of prevalence and severity of cardiac involvement.

Even though the families were apparently unrelated, it is possible that similarities in phenotypic expression within each of our two main genotype subgroups could be partially attributed to similar genetic backgrounds stemming from a common ancestor (founder effect). Our analysis was restricted to identification of TTR mutations and did not extend in polymorphisms (though presence of exonic nucleotide variation was excluded by extensive sequencing) or to analysis of the promoter region. These issues should be addressed in future studies.

Conclusions

Myocardial involvement seems to be a frequent and heterogeneous event in ATTR amyloidosis, and genotype appears to be an important cause of this variability. Although cardiomyopathy can occur irrespectively of the type of mutation, Val30Met appears to be associated with less severe disease, whereas Glu89Gln mutation showed particularly severe involvement and prognosis in our patients. Our findings imply that patients with Glu89Gln require especially close cardiological (as well as neurological) follow-up. We also think that combined heart-liver transplantation could be considered for Glu89Gln carriers with definite cardiomyopathy for whom OLT has already been scheduled, even if cardiovascular symptoms have yet to appear. Formal analysis of genotype-phenotype correlations in ‘endemic’ geographic settings (and eventually worldwide) could expand our knowledge of the pathogenetic and clinical role of the ITR mutation within this complex and rare disease and might provide a basis for tailoring of therapeutic strategies.

Acknowledgement

This study was supported by the Fanti Melloni Foundation.

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CLAUDIO RAPEZZI1, ENRICA PERUGINI1, FABRIZIO SALVI2, FRANCESCO GRIGIONI1, LETIZIA RIVA1, ROBIN M. T. COOKE1, ALESSANDRA FERLINI3, PAOLA RIMESSI3, LETIZIA BACCHI-REGGIANI1, PAOLO CILIBERTI1, FRANCESCA PASTORELLI2, ORNELLA LEONE4, ILARIA BARTOLOMEI2, ANTONIO D. PINNA6, GIORGIO ARPESELLA5, & ANGELO BRANZI1

Institute of Cardiology, University of Bologna and S. Orsola- Malpighi Hospital, Bologna, Italy, 2Department of Neurology,

Bellaria Hospital, Bologna, Bologna, Italy, 3 Department of Diagnostic and Experimental Medicine, Section of Medical

Genetics, University of Ferrara, Ferrara, Italy, 4 Department of Pathology, University of Bologna and S. Orsola-Malpighi

Hospital, Bologna, Italy, 5 Department of Cardiovascular Surgery, University of Bologna and S. Orsola-Malpighi Hospital,

Bologna, Italy, and 6 Liver and Multiorgan Transplant Center, University of Bologna and S. Orsola-Malpighi Hospital,

Bologna, Italy

Correspondence: Claudio Rapezzi, Istituto di Cardiologia, Policlinico S. Orsola-Malpighi, Via Massarenti 9, 40138 Bologna, Italy. Tel: +39 51349858. Fax: +39 51344859. E-mail: crapezzi@aosp.bo.it

Copyright Taylor & Francis Ltd. Sep 2006

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