Diagnosis of Cardiomyopathies and Rare Diseases: From "Phenocopy" to "Genocopy" Era Part 2

One Disease or Many Diseases? From " Phenocopy" to " Genocopy"

Genomic medicine has entered clinical practice, and the recognition of the diagnostic utility of genetic testing for cardiomyopathies (particularly, hypertrophic cardiomyopathy) is growing. With expanding knowledge of the genetic background of these diseases, primary cardiomyopathies have recently been subclassified into genetic, mixed, and acquired cardiomyopathies (AHA 2006) or familial and non familial disease (ESC 2007), shifting the general view of cardiomyopathies from a "phenocopy" to a "genocopy" model.

However, although a number of cardiomyopathies susceptibility genes involving different pathways have been identified, the search for novel mutations in new genes continues. As a result of the increasing genetic heterogeneity of HCM, a classification based on functional genetics might seem very helpful, but in the light of the low yield of mutations in a large number of these genes as well as the commercial availability of just a small number of these genes, a phenotypic classification might be a more useful tool in looking at this disease from a clinical-practice vantage point. With the growing number of cardiomyopathy-associated genes discovered, strategic choices have to be made in clinical practice.

Diagnosis of Cardiomyopathy

The utility of an accurate diagnosis and distinction from a phenocopy state is well illustrated in certain circumstances, such as Fabry disease, which could be clinically indistinguishable from HCM caused by mutations in sarcomeric proteins [9]. Enzyme replacement therapy with alpha-galactosidase, the enzyme responsible for Fabry disease, has been shown to impart considerable clinical benefit in management of patients with Fabry disease, while the conventional treatment offered for true HCM would render no significant benefit in such patients [20].

The age of onset (infancy, childhood, adolescency, or adulthood), the pattern of inheritance (autosomic, X linked or matrilinear), symptoms/signs at onset, physical abnormalities (dysmorphic features, myopathy, mental retardation), ECG abnormalities (i.e. short PR in metabolic or mitochondrial disorders), the echocardiography pattern (i.e. left ventricular non-compaction cardiomyopathy associated with specific neuromuscular disorders), and other biochemical or functional tests (i.e. premature lactic acidosis and flat oxigen pulse during metabolic stress test in mitochondrial disorders) may be relevant to discriminate between different causes of cardiomyopathies. Detailed clinical evaluation and mutation analysis are, therefore, important to provide an accurate diagnosis in order to enable genetic counselling, prognostic evaluation and appropriate clinical management [21].

Physical Examination

Physical abnormalities can be characteristics of specific disorders and lead to the final clinical diagnosis. Macroglossia, carpal tunnel syndrome, reticular lung infiltrates and Bence-Jones proteinuria may be hallmarks of plasma-cell-dyscrasia-related systemic amyloidosis. Metabolic disorders and syndromes are associated with characteristic physical abnormalities (dysmorphic features, myopathy, mental retardation) and symptoms at onset. Patients with inborn errors of metabolism involving impaired energy production or the accumulation of toxic metabolites often have signs and symptoms of multiple organ dysfunction. Dysmorphic features may characterize malformation syndromes as well as storage diseases, and therefore other minor and major malformations should also be sought. In patients with primary neuromuscular disorder, skeletal muscle weakness usually precedes the cardiomyopathy and dominates the clinical picture. Occasionally, however, skeletal myopathy is subtle, and the first symptom of disease may be cardiac failure. Encephalopathy is characteristically seen in the mitochondrial syndromes MELAS (Mitochondrial Encephalopathy, Lactic Acidosis, and Strokelike episodes), MERRF (Myoclonic Epilepsy, Ragged Red Fibers), Kearns-Sayre syndrome, and Leigh disease. Acute worsening can occur in these syndromes in association with intercurrent illness or metabolic stressors. In general, the neurological features of these syndromes (epilepsy, strokelike episodes, dementia, and ophthalmoplegia) predominate, and the cardiomyopathy typically occurs later in the clinical course [21,22].


Almost all (95%) patients with hypertrophic cardiomyopathy have an abnormal ECG. The most frequent ECG changes are left atrial enlargement, repolarization abnormalities, and pathologic Q waves, most commonly in the inferolateral leads. Voltage criteria for left ventricular hypertrophy alone are non-specific and are often seen in normal young adults. Giant negative T waves in the mid-precordial leads are characteristic of hypertrophy confined to the left ventricular apex. Some patients have a short PR interval, including metabolic/storage (Danon, PRKAG2, Fabry disease) or mitochondrial disorders. Patients with amyloidosis often show low voltages in the precordial leads[7-10, 21-23].

Non Invasive Imaging Technology

Standard echocardiography, new echocardiographic technologies, and Cardiac magnetic resonance (CMR) provide information on myocardial structure and have been suggested as a potential tool to discriminate between different phenocopy states.

Standard Echo

Left ventricular hypertrophy associated to congenital heart defects is frequently seen in malformation syndromes (such as pulmonary valve abnormalities in Noonan and LEOPARD syndrome). An abnormal texture of the interventricular septum ("granular sparkling" aspect), especially if associated with biatrial dilation, pericardial effusion and restrictive phenotype, may be diagnostic of amyloid. However, other infiltrative diseases (i.e. metabolic myopathies, Gaucher, Hunter’s, and Hurler’s diseases) or storage cardiomyopathies (haemochromatosis, Fabry’s disease, glycogen storage, and Niemann-Pick disease) should be considered. In advanced haemochromatosis all cardiac chambers may be dilated. Mucopolysaccharidosis and Gaucher’s disease may lead to aortic and mitral stenosis. In hypothyroidism, other than amyloidosis, a pericardial effusion can be present. Pieroni et al. showed in 83% of Fabry’s cardiomyopathy patients (95% of FC patients with LVH) a binary appearance of endocardial border absent in all HCM, hypertensive, and healthy subjects (sensitivity 94%; specificity 100%), reflecting an endomyocardial glycosphingolipids compartmentalization, consisting of thickened glycolipid-rich endocardium, free glycosphingolipid subendocardial storage, and an inner severely affected myocardial layer with a clear subendocardial-midwall layer gradient of disease severity. On the other hand, Kounas et al. showed the binary sign in 8/28 patients with HCM (3 patients) and with Fabry’s cardiomyopathy (5 patients). The sensitivity and specificity of the binary sign as a discriminator of AFD from HCM were 35% and 79%, respectively. The authors suggest that the binary endocardial appearance lacks sufficient sensitivity and specificity to be used as an echocardiographic screening tool. In neuromuscular disorders like glycogenosis, mitochondriopathy and myotonic dystrophy, myocardial thickening, hypertrabeculation/noncomnpaction and systolic dysfunction are found. The coexistence of left ventricular non-compaction and localised inferobasal left ventricular akinesia are almost pathognomonic of dystrophinopathies. Finally, a diagnosis of neuromuscular/metabolic/mitochondrial cardiomyopathy is favored in presence of concentric/asymmetric/apical, non-obstructive hypertrophic cardiomyopathy, with or without hypertrabeculation of the apex, especially when associated with an early onset impairment of LV systolic function. However, metabolic and mitochondrial cardiomyopathy might also be presented with dilated type, and hypertrophy may become dilated in the later stage [7,9,1012, 24-26].

Recently, genotype-phenotype studies from the Majo Clinic Cardiomyopathy Group have discovered an important relationship between the morphology of the left ventricle, its underlying genetic substrate and the long-term outcome of this disease. They observed that the septal contour was the strongest predictor of the presence of a myofilament mutation, regardless of age. Intriguing conclusions can be drawn from these observations. Whereas in initial morphological studies, sigmoidal HCM seemed to be associated with older age, the underlying genotype rather than age appears to be the predominant determinant of septal morphology. Furthermore, Z-disc HCM seems to have a predilection for sigmoidal contour status These observations may facilitate echo-guided genetic testing by enabling informed genetic counseling about the a-priori probability of a positive genetic test based upon the patient’s expressed anatomical phenotype[27].

New Imaging Techonologies

Weidemann et al. have investigated in a prospective study whether regional non-ischaemic fibrosis in hypertrophic myocardium can also be detected by ultrasonic strain-rate imaging based on specific visual features of the myocardial deformation traces. This diagnostic study aimed to define left ventricular fibrotic segments in 30 patients with hypertrophic cardiomyopathy (n = 10), severe aortic valve stenosis (n = 10), Fabry disease cardiomyopathy (n = 10), and 10 healthy controls. In total, 42 segments showed late enhancement by magnetic resonance imaging. Using strain-rate imaging, all late enhancement positive segments displayed a characteristic pattern consisting of a first peak in early systole followed by a rapid fall in strain rate close to zero and a second peak during isovolumetric relaxation. This ‘double peak sign’ was never seen in segments of healthy controls. However, it was detected in 10 segments without late enhancement. These ‘false-positive’ segments belonged to Fabry patients who often develop a fast progressing fibrosis. In a follow-up magnetic resonance imaging study after 2 years, all these segments had developed late enhancement. Therefore, the ‘double peak sign’ in strain-rate imaging tracings seems to be a reliable tool to diagnose regional fibrosis[26].


Moon JC et al. have shown that late gadolinium enhancement cardiovascular magnetic resonance can visualize myocardial interstitial abnormalities. Late enhancement was demonstrated in nine patients with different specific cardiomyopathies, with a mean signal intensity of 390 +/- 220% compared with normal regions. The distribution pattern of late enhancement was unlike the subendocardial late enhancement related to coronary territories found in myocardial infarction. The affected areas included papillary muscles (sarcoid), the mid-myocardium (Anderson-Fabry disease, glycogen storage disease, myocarditis, Becker muscular dystrophy) and the global sub-endocardium (systemic sclerosis, Loeffler’s endocarditis, amyloid, Churg-Strauss). Focal myocardial late gadolinium enhancement have been found in these specific cardiomyopathies, and the pattern is distinct from that seen in infarction. CMR hyperenhancement pattern is very characteristic for cardiac involvement of amyloidosis and can therefore be used to discriminate this disease from other forms of restrictive or hypertrophic cardiomyopathies. Although most profound in the subendocardial layer of myocardium, amyloid deposition occurs throughout the entire myocardium, causing the entire myocardium to have a higher signal on delayed contrast enhancement images than normal myocardium [28].

Biochemical/Metabolic Tests

Biochemical analysis represents an step for the diagnosis of mitochondrial, metabolic and neuromuscular cardiomyopathies. The presence of hypoglycemia, primary metabolic acidosis with an increased anion gap, or hyperammonemia should alert the physician to the possibility of a metabolic disorder. The insulin-excess states of Beckwith-Wiedemann syndrome and the infant of a diabetic mother can produce hypoketotic hypoglycemia but are distinguished by low free fatty acid levels by characteristic clinical features. Disorders in fatty acid metabolism can be identified as defects of fatty acid B-oxidation or of carnitine-dependent transport depending on quantitative carnitine levels in blood, urine, and tissue; acylcarnitine profile in blood; and urine organic acids (fatty acids, dicarboxylic, and hydroxydicarboxylic acids). In Fabry disease, electrolyte imbalances and proteinuria reflecting renal failure may be seen. Level of globotriaosylceramide (Gb3 or GL-3) a glycosphingolipid may be elevated. Enzymatic analysis performed by using plasma or leukocytes may show a deficiency of alpha-galactosidase A. However, levels of Gb3 and alpha-galactosidase A may be normal in female heterozygote Fabry patients. Therefore, genetic and/or molecular diagnosis is necessary to confirm Fabry disease if suspected based on clinical features of proteinuria and acroparesthesias that were invariably present in both men and women with Fabry mutation and cryptogenic stroke. Elevated serum creatine kinase levels can be associated with diagnosis of a neuromuscular disease. Although clinical signs and laboratory tests are useful for identifying and classifying diseases of the lower motor unit, in isolation, they rarely lead to a specific diagnosis. However, a markedly elevated serum creatine kinase level (10 to 100 times higher than normal) is invariably found early in the clinical course of Duchenne muscular dystrophy and almost always in its milder allelic form, Becker-type muscular dystrophy, whereas the serum creatine kinase level is usually lower in other muscular dystrophies and myopathies (1 to 10 times higher than normal). Because creatine kinase levels can vary markedly among different patients with the same disease and may fluctuate in a given patient over time, clinical judgment is necessary to interpret these values. A premature lactic acidosis, a very low VO2 and a flat oxygen pulse may represent markers of metabolic/mitochondrial diseases. The diagnosis can be confirmed on measurement of blood and cerebrospinal fluid lactate and pyruvate levels, histological analysis of skeletal muscle, assay of respiratory chain enzymes, and/or mitochondrial DNA analysis[21, 29].

Genetic Testing

The clinical application of mutation analysis is technically possible, but has been hindered by logistics and high cost. Given the cost of mutation analysis, however, a strategic approach based on probabilities should be employed where possible. Careful phenotyping should identify the most common phenocopies of cardiomyopathies.

The major goals of genetic testing in patients with cardiomyopathies are:

- -to contribute to diagnosis;

- -to provide prognostic and therapeutic benefits;

- -most important, to detect relatives affected or at risk to develop the disease (carriers).

Once a mutation has been detected in a proband, the possibility of genetic testing should be suggested to first-degree relatives (who have a 50% probability of being gene-positive in autosomal disorders: ‘cascade’ screening). This type of screening enables close clinical management of mutation carriers, and identifies genetically normal family members, obviating the need for them to undergo clinical screening and repeat follow-up examinations. Appropriate genetic counselling, performed by a well trained physician (clinicians, geneticist) or genetic counsellor, should precede and follow genetic testing to help the patient and his/her family to comprehend the reasons to perform the test and the clinical significance and impact of a positive/negative diagnosis. A specially trained and experienced nurse may serve as coordinator of the investigations and as contact person for the family [3,4,7,15,22].

Organ-Specific and Skeletal Muscle Biopsy

Biopsy with Congo red staining and immunostaining is the procedure of choice for the diagnosis of amyloidosis. Stain the tissue with an alkaline solution of Congo red, and examine it under polarized light, where positive (green) birefringence is detectable in the presence of amyloidosis of any type. The nature of the fibril precursor can be established by immunohistochemical staining with antibodies specific for the major amyloid precursors (Amyloid A, immunoglobulin L chains of k or l type, antitransthyretin). In Amyloid A amyloidosis, only the Amyloid A is positive. The amyloid nature of the deposit can by confirmed by staining with an antiserum specific for serum amyloid P-component. In amyloidosis, the tissue with the highest yield, particularly in the presence of proteinuria or renal failure, is the kidney (technically adequate samples have a diagnostic yield close to 100%). If renal biopsy is deemed too risky for a specific patient or if amyloidosis without renal disease is suspected, 2 sites have been shown to be useful in obtaining tissue for histologic and immunochemical analysis. Subcutaneous fat aspiration is positive in approximately 60% of individuals with Amyloid A amyloidosis, except in the case of familial Mediterranean fever, when it rarely, if ever, is positive. Rectal biopsy is more useful than subcutaneous fat aspiration in Amyloid A amyloidosis. It has been found to produce positive results (assuming that submucosa is included in the biopsy specimen) in 80-85% of patients ultimately found to have tissue amyloid at a clinically relevant site. Samples from either the subcutaneous fat aspirate or the rectal biopsy can be stained as conventional tissue biopsies to determine the presence and nature of the amyloid precursor. Occasionally, patients have positive results on subcutaneous fat aspirates in the presence of a negative result on rectal biopsy, while others may have deposits in the rectal tissue and not in the aspirate. Use of both procedures may increase the yield to 90%. Abdominal subcutaneous fat biopsy results are not very sensitive in Amyloid A caused by familial Mediterranean fever and in dialysis-related amyloidosis. The results are usually negative, probably because beta2-microglobulin does not accumulate in this tissue.

A skeletal muscle biopsy is often necessary, especially in infants, when the clinical and laboratory findings are nonspecific. If a muscular dystrophy is suspected, particularly in a boy, molecular analysis of the dystrophin gene and/or protein is indicated. Dystrophin, a cytoskeletal protein normally found in all muscle cell types, is thought to stabilize the plasma membrane of the muscle cell and may be important in the regulation of intracellular calcium. Approximately 65% of patients with Duchenne muscular dystrophy or Becker-type muscular dystrophy have deletions of the dystrophin gene that can be detected by PCR in blood lymphocytes. In the other 35% of patients, including manifesting female carriers for whom PCR results are difficult to interpret, a muscle biopsy is required to detect a reduced amount of the dystrophin protein or abnormalities of its size. The presence of dystrophic changes in a skeletal muscle biopsy specimen is also an indication for molecular analysis of dystrophin [22,30,31].

Endomyocardial Biopsy

Although the role of endomyocardial biopsy (EMB) in the diagnosis and treatment of adult and pediatric cardiovascular disease remains controversial, a recent joint AHA/ACC/ESC statement recommends endomyocardial biopsy (class I, evidence B) in patients with suspected myocarditis, including 1) new onset heart failure of less than two weeks duration associated with a normal sized or dilated left ventricle and haemodynamic compromise; 2) new onset heart failure of 2 weeks to 3 months duration associated with a dilated left ventricle and new ventricular arrhythmias, second- or third-degree heart block, or failure to respond to usual care within 1 to 2 weeks. In addition, endomyocardial biopsy is reasonable (IIA; evidence C):

- in the clinical setting of unexplained heart failure of >3 months’ duration associated with a dilated left ventricle and new ventricular arrhythmias, Mobitz type II second-or third-degree AV heart block, or failure to respond to usual care within 1 to 2 weeks;

- in the setting of unexplained heart failure associated with suspected anthracycline cardiomyopathy;

- in the setting of heart failure associated with unexplained restrictive cardiomyopathy;

- in the setting of unexplained heart failure associated with a DCM of any duration that is associated with suspected allergic reaction in addition to eosinophilia.

- in the setting of suspected cardiac tumors, with the exception of typical myxomas whereas adenovirus is most commonly associated with histological [32].


In 1968, the World Health Organization defined cardiomyopathies as "diseases of different and often unknown etiology in which the dominant feature is cardiomegaly and heart failure". This statement was updated in 1980 and defined cardiomyopathies as "heart muscle diseases of unknown cause", thereby differentiating them from specific identified heart muscle diseases of known cause such as myocarditis. In 1995, a World Health Organization/International Society and Federation of Cardiology Task Force on cardiomyopathies classified the different cardiomyopathies by the dominant pathophysiology or by etiological/pathogenetic factors (Phenocopy era). Over the last two decades, the importance of gene defects in the etiology of cardiomyopathies has been recognized, and several new disease entities have been identified with the introduction of molecular biology into clinical medicine (Genocopy era), rendering previous classifications and formal cardiomyopathies concepts obsolete, and leading to different reclassification of cardiomyopathies by the AHA and the Working Group of the ESC.

However, given the extreme heterogeneity of cardiomyopathies, there probably is no single classification or "model" that can be regarded as generally acceptable to all the interested parties from diverse disciplines (researchers, clinicians, epidemiologists, geneticists). Nevertheless, cardiologists and cardiomyopathy specialists need to become familiar with the basic principles of molecular biology and clinical genetics, in order to generally understand the basis of the disease, to provide a correct characterization of the clinical phenotype and to eventually guide the genotype, to understand and manage the implications of a positive genetic diagnosis for the proband and his/her family.

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