Elisa Carturan

Postmortem genetic testing for conventional autopsy-negative sudden unexplained death: an evaluation of different DNA extraction protocols and the feasibility of mutational analysis from archival paraffin-embedded heart tissue.

One third of autopsy-negative sudden unexplained deaths (SUDs) can be attributed to a cardiac channelopathy. Typically, paraffin-embedded tissue (PET) is the only source of DNA available for genetic analyses. We examined different DNA extraction procedures, involving 2 deparaffinization methods, 2 digestion methods, 4 laboratory-based purification methods, and 5 commercial kits. Mutational analysis involving 25 RYR2 exons was performed on PET DNA from 35 SUD cases to evaluate the feasibility of using PET DNA for genetic testing. With the best PET-DNA extraction method, an average of only two thirds of the region of interest could be evaluated. Although we initially identified 5 missense mutations in 5 of 35 SUD cases, repeated analysis failed to confirm these mutations. DNA from PET should be considered error prone and unreliable in comprehensive surveillance of SUD-associated genes. Given these shortcomings, the standard autopsy for SUD should include archiving EDTA-preserved blood or frozen tissue to facilitate postmortem genetic testing.

Overexpression of tumor necrosis factor (TNF)α and TNFα receptor I in human viral myocarditis: clinicopathologic correlations

Proinflammatory cytokines, including tumor necrosis factor (TNF)α, have been recognized as important physiopathogenetic factors in the initiation and continuation of inflammatory cardiomyopathies. Experimental and preliminary human studies have demonstrated that TNFα plays a crucial role in enteroviral-induced myocarditis. In this study, we investigated the expression of TNFα and both its receptors (TNFRI and TNFRII) in both viral and nonviral myocarditis. Myocardial expression of TNFα was then correlated with different clinical and pathologic findings. TNFα expression was investigated in endomyocardial biopsies obtained from 38 patients with myocarditis and from eight control subjects by using reverse transcriptase-polymerase chain reaction (PCR) and immunohistochemistry. Viral etiology was diagnosed by PCR in 20 cases: enterovirus in seven, Epstein–Barr virus in four, hepatitis C virus in three, adenovirus in two, influenza virus in two, cytomegalovirus in one, and double infection adenovirus and enterovirus in one. Immunohistochemistry was also used to analyze both TNFα receptors (RI and RII). A semiquantitative analysis was employed (score 0–3) for necrosis, inflammation, fibrosis and immunohistochemical findings. TNFα mRNA and TNFα protein were significantly more present in viral myocarditis than in nonviral myocarditis (16/20 vs 3/18, P=0.001). Remarkable immunostaining was observed for both receptors, particularly TNFRI. Histological analysis revealed that myocardial necrosis (mean score 1.89 vs 1.15, P=0.01) and cellular infiltration (mean score 2.26 vs 1.78, P=0.05) were more prominent in TNFα-positive cases. Among TNFα-positive cases, the greater TNFα mRNAs, the more impaired was cardiac function. Our findings suggest that the expression of TNFα may play an important role in the pathogenesis of viral myocarditis of any etiology and may influence the severity of cardiac dysfunction. Cytokine effects are more strictly linked to overexpression of TNFRI.

Endomyocardial fibrosis : pathological and molecular findings of surgically resected ventricular endomyocardium

Endomyocardial fibrosis (EMF) is a restrictive cardiomyopathy of unknown etiology prevalent in tropical regions affecting the inflow tract and apex of one or both ventricles, which show fibrous thickening of the endocardium and adjacent myocardium. Surgical treatment is recommended for patients in functional classes III or IV (New York Heart Association). The gross and histological features of the heart have been comprehensively studied in autopsies, but studies in surgical samples are still lacking. Histological and immunohistochemical features of EMF in surgical samples collected from 32 patients were described and correlated with clinical data. Polymerase chain reaction (PCR) and reverse transcription-PCR, performed on formalin fixed endomyocardial samples, were used retrospectively to detect genomes of certain cardiotropic viruses and Toxoplasma gondii. Ventricular endocardium was thickened by superficial acellular hyaline collagen fibers type I and III, with predominance of the former type. Besides fibrosis, a chronic inflammatory process and an anomalous lymphatic rich vascular pattern were observed in the deep endocardium, connected to the terminal coronary circulation of the myocardium, which might be an important pathological finding concerning EMF pathogenesis. Molecular analysis of the endomyocardium revealed high incidence of cardiotropic infective agents (6/12, 50%); however, their role in the disease pathogenesis is still controversial.

Cardiac infections: focus on molecular diagnosis.

The role of different types of infections in heart diseases is more important than commonly thought, with new and re-emerging infections (i.e., Mycobacterium tuberculosis). This review addresses the pathology of infective pericarditis, myocarditis, and endocarditis, mainly focusing on the significance of molecular techniques in the detection of infective agents. Molecular investigations represent important ancillary diagnostic tools and combined with other conventional approaches provide a more precise final diagnosis. A close collaboration and communication among cardiologists, cardiac surgeons, pathologists, and microbiologists is essential to ensure optimal diagnoses and management as well as a favorable impact on patient outcome.

Viral detection and tumor necrosis factor alpha profile in tracheal aspirates from children with suspicion of myocarditis.

Pediatric myocarditis is a serious disease resulting in significant morbidity and mortality. Tracheal aspirate (TA) has been demonstrated to be a sensitive diagnostic tool to detect viral agents responsible for respiratory disorders and myocardial dysfunction. Tumor necrosis factor alpha (TNFα) is thought to play an important role in the pathogenesis of these disorders. The aim of the present study was to investigate the presence of different viruses and the expression of TNFα in children with clinical suspicion of myocarditis. Forty-five TAs from children (20 males/25 females, mean age 4.4±5.0 y) with myocardial dysfunction and respiratory symptoms were analyzed for detection of viral genomes by using molecular techniques. In 10 cases endomyocardial biopsy was also performed due to a severe and rapid progression of heart failure. TNFα mRNAs of TA and TNFα protein plasma levels were quantified. Viral etiology was detected in 25/45 (56%) cases: the most frequent etiology was enterovirus (19 cases, 59%). Polymerase chain reaction viral concordance was found in TA and endomyocardial biopsy. TNFα mRNA and TNFα serum levels were significantly more expressed in viral cases than nonviral cases (1.26±0.76 vs. 0.56±0.76, P=0.001). More impaired cardiac function (particularly ejection fraction) was detected in viral positive than in viral negative cases (39.91±20.09 vs. 55.61±20.36, P=0.04). TA seems to be an excellent tool for viral investigation in pediatric patients with suspicion of myocarditis. The analysis of TNFα in TA may represent an important marker to better define patient status.

Nonischemic Left Ventricular Scar Sporadic or Familial? Screen the Genes, Scan the Mutation Carriers

A 20-year-old soccer player died suddenly while watching a game with friends at home. At annual preparticipation screening, ECG was normal with consequent sport eligibility (Figure 1A). History for juvenile sudden death and hypertrophic cardiomyopathy was reported on the mother’s side of the family. Postmortem examination of the heart showed normal dimensions (weight, 347 g; wall thicknesses of left ventricle [LV] and septum 13 mm and right ventricle [RV], 3 mm), in the absence of aneurysms or chamber dilatation; a subepicardial scar-like grey rim was evident in the anterolateral and posterior LV free wall and in the septum (Figure 1B). Coronary arteries had a normal origin and course, with patent lumen. Histological examination revealed extensive subepicardial and intramural fibrous replacement with scarce fatty tissue infiltration, involving the entire LV circumference and the septum (Figure 1C). Right ventricular involvement was only focally detected, in the anterior wall. The features were in keeping with either chronic myocarditis or left-dominant arrhythmogenic cardiomyopathy (AC). Figure 1. A , Basal 12-lead ECG at annual preparticipation screening showing normal findings. B , Transverse section of the heart showing a subepicardial scar-like grey rim in the anterolateral and posterior LV free wall and in the septum, in the absence of wall thinning and aneurysm formation. …

Myocarditis and Inflammatory Cardiomyopathy: Histomorphological Diagnosis

Myocarditis is a non-ischemic inflammatory disease of the myocardium associated with cardiac dysfunction. It most often results from infectious agents, hypersensitivity responses, or immune-related injury. In spite of the development of various diagnostic modalities, early and definite diagnosis of myocarditis still depends on the detection of inflammatory infiltrates in endomyocardial biopsy specimens according to Dallas criteria. Routine application of immunohistochemistry (for characterization of inflammatory cell infiltration) and Polymerase Chain Reaction PCR analysis (for identification of infective agents) has become an essential part of the diagnostic armamentarium for a more precise biopsy report. A new morphological classification is advanced to overcome the limits of Dallas criteria. A semiquantitative assessment of myocyte damage/inflammation (grading) as well as of fibrosis (staging) is indicated, thus providing histopathological diagnosis useful to the clinician for more appropriate patient risk stratification and for the application of new therapies. Consequently, the final diagnosis of myocarditis should be mainly based on three features: etiology, grade, and stage of the disease.

Follow-up with exercise test of effort-induced ventricular arrhythmias linked to ryanodine receptor type 2 gene mutations.

The aim of this study was to assess exercise test results and efficacy of therapy with a β blocker (acebutolol) in ryanodine receptor type 2 (RyR2) mutation carriers with documented ventricular arrhythmias (VAs) and long-term follow-up. Twenty RyR2 mutation carriers belonging to 8 families and regularly followed at our center were analyzed using a study protocol involving electrocardiography, exercise tests off and on β-blocker therapy, 2-dimensional echocardiography, and signal-averaged electrocardiography. Off-therapy exercise testing triggered the onset of VAs at different heart rates (mean 132 ± 13 beats/min) with various patterns that worsened while exercising and disappeared immediately after stopping. The most severe VAs detected were nonsustained ventricular tachycardia in 35% and ventricular couplets in 35%. In the remaining subjects single ventricular premature beats were recorded. In 15% of patients single monomorphic ventricular premature beats were detected and identified to be linked to RyR2 mutations owing to the presence of sudden deaths of their family members and subsequent family screening. Acebutolol made the VAs disappear completely in 20% of subjects and decreased their complexity in 50%, whereas it did not change VAs appreciably in 30% of patients with less complex VAs. After 11 ± 8 years of follow-up 2 patients developed syncope. In conclusion, exercise testing was a fundamental tool for assessing the clinical phenotype and efficacy of therapy in RyR2 mutation carriers and therapy with acebutolol led in most subjects to a decreased complexity of the arrhythmic pattern or to complete suppression.

Sudden cardiac death, borderline myocarditis and molecular diagnosis: evidence or assumption?

Purpose Sudden unexpected death autopsy is sometimes non-conclusive both from a macroscopic and from a histological point of view, even if carried out according to the guidelines for sudden cardiac death examination. Molecular biology techniques are required in this setting and may play a crucial role in reaching the final diagnosis. A case report The postmortem examination and toxicology findings of the body of a young monk found dead in his cell were negative. Rare focal myocardial lymphocytic infiltrates were seen microscopically, associated with interstitial oedema. The findings were not sufficient to diagnose a myocarditis as the certain final cause of cardiac arrest. According to the recent guidelines for sudden cardiac death, a molecular investigation by polymerase chain reaction analysis was performed on samples of myocardium and spleen, with detection of parvovirus B19 DNA in the myocardium. Accordingly, a diagnosis of parvovirus B19 borderline acute myocarditis was put forward as the possible cause of sudden cardiac death. Conclusion In sudden death cases in which there is lack of a cause-effect relationship with the postmortem findings, the final report should be expressed as a descriptive association of evidence, not providing unreliable certainty, as the Association for European Cardiovascular Pathology recommends.

Molecular Pathobiology of Myocarditis

Abstract According to the WHO classification, myocarditis is defined as an ‘inflammatory disease of the myocardium associated with cardiac dysfunction’ and is diagnosed on endomyocardial biopsy (EMB) by established histological, immunological and immunohistochemical criteria. A realistic diagnostic cut-off is a value of >14 leucocytes/mm2 and >7 cells/mm2 T lymphocytes by histology and immunohistochemistry. Infectious, autoimmune, and idiopathic forms of inflammatory cardiomyopathy are recognized that eventually may lead to dilated cardiomyopathy. Molecular techniques on EMB specimens are recommended to identify viral etiology. Positive PCR results obtained on EMB specimens should always be accompanied by a parallel investigation on blood samples collected at the time of the EMB. This chapter will focus on the diagnostic role of molecular tools as well as on the pathogenetic pathways involved in disease onset and progression, including host, viral genetics, and environmental factors. The role of key inflammatory immune effectors and regulators, such as chemokines and cytokines, will also be addressed.