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CLINICAL STUDIES

Prevalence and characteristics of dystrophin defects in adult male patients with dilated cardiomyopathy

Eloisa Arbustini, MD^*, Marta Diegoli, BD^*, Patrizia Morbini, MD^*, Barbara Dal Bello, MD^*, Nadia Banchieri, BD^*, Andrea Pilotto, TD^*, Filippo Magani, TD^*, Maurizia Grasso, PhD, Jagat Narula, MD, Antonello Gavazzi, MD, Mario Viganò, MD^|| and Luigi Tavazzi, MD

^* Pathology Department, IRCCS-Policlinico San Matteo, Pavia, Italy
Cardiology Department, IRCCS-Policlinico San Matteo, Pavia, Italy
^|| Cardiac Surgery Department Departments, IRCCS-Policlinico San Matteo, Pavia, Italy
Transplantation Experimental Laboratory, IRCCS-Policlinico San Matteo, Pavia, Italy
Allegheny University of Health Science, Philadelphia, Pennsylvania, USA

Manuscript received August 10, 1999; revised manuscript received December 30, 1999, accepted February 21, 2000.

Reprint requests and correspondence: Dr. Eloisa Arbustini, Istituto di Anatomia Patologica, Viale Forlanini 16, 27100 Pavia, Italy
e.arbustini{at}smatteo.pv.it

	Abstract

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OBJECTIVES

To assess the prevalence of dystrophin defects in dilated cardiomyopathy (DCM) in male patients and to formulate investigation strategies for their identification.

BACKGROUND

Dystrophin defects presenting with predominant or exclusive cardiac involvement may be clinically indistinguishable from "idiopathic" DCM. Diagnosis may be missed, unless specifically investigated.

METHODS

Clinical and biochemical evaluation, right ventricular endomyocardial biopsy (EMB), light and electron microscopic and immunohistochemical studies of biopsy samples, six multiplex and two single polymerase chain reactions for 38 exons and automated sequencing of exon 9 and muscle promoter-exon 1 were undertaken in 201 consecutive male patients presenting with DCM, with (n = 14) and without (n = 187) increased serum creatine phosphokinase (sCPK).

RESULTS

Dystrophin defects were identified in 13 of the 201 patients (6.5%, age 16–50). Family history was positive in four patients. Serum CPK levels were increased in 11 of 13 patients. Light microscopy examination of EMB was uninformative; ultrastructural study showed multiple membrane defects. Dystrophin immunostain was abnormal. Eight patients, all older than 20, had deletions affecting midrod domain, normal or mildly increased CPK and better outcome than the five remaining cases all younger than 20, with more than five-fold increase of sCPK. Two of these latter had proximal and rod-domain deletions. Sisters of two patients were diagnosed as noncarriers with microsatellite analysis.

CONCLUSIONS

Although the overall prevalence of dystrophin defects in our consecutive DCM male series is low (6.5%), immunohistochemical and molecular studies are essential to identify protein and gene defects; screening studies are justified to define prevalence, clinical profile and genotype-phenotype correlation.

Abbreviations and Acronyms   BMD = Becker muscular dystrophy   DAG = dystrophin-associated glycoprotein   DCM = dilated cardiomyopathy   ECG = electrocardiography   EMB = endomyocardial biopsy   LGMD = limb-girdle muscular dystrophy   MB = cardiac isoform   MM = muscle isoform   PCR = polymerase chain reaction   RT = reverse transcriptase   sCPK = serum creatine phosphokinase   STR = single tandem repeats

X-linked DCM was first described by Berko et al. (1) in 1987 in one large kindred with 11 affected young men. Since then, nine more families have been reported (Table 1) (2–10). When heart involvement is dominant and skeletal muscle impairment is minimal or null, these disorders are defined as X-linked DCM (1–10), even if the muscle isoform of serum creatine phosphokinase muscle isoform (sCPK-MM) is increased and muscle biopsy shows dystrophic changes and decreased dystrophin immunostain (2–10) (Table 2). At present, known gene defects associated with X-linked DCM seem to cluster in two different regions: 5' (the majority), including muscle promoter-exon 1 and hinge region defects (point mutations, deletions, inversions and transposable element insertions) (3,5–8,10) and midrod domain exons (Becker-type mutations) ([9,11,12]. X-linked DCMs with Becker type defects have been defined as Becker muscular dystrophy (BMD) with dominant cardiac involvement (11,12), especially before X-linked DCM was proposed as a distinct entity (2).

To clinically assess the prevalence of dystrophin defects in a DCM male population and characterize the disease, we performed a prospective molecular and immunohistochemical screening in 201 consecutive men referred to our hospital for evaluation and management of DCM in the last four years.

	Methods

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From January 1995 to June 1999, 201 consecutive DCM men (ages 15 to 66 years; mean ± SD = 42.4 ± 12.8) were evaluated at the IRCCS Policlinico San Matteo of Pavia, Italy. Dilated cardiomyopathy was diagnosed on the basis of World Health Organization (WHO) criteria (17). Patients with specific heart muscle diseases, such as amyloidosis, hemochromatosis, glycogenosis, etc., patients with familial DCM with autosomal inheritance, as well as patients with prior proven diagnosis of dystrophin defects were excluded. Patients with suspected BMD, not proven with immunochemical or molecular studies, patients with a family history suggestive of an X-linked disease, and patients diagnosed with limb-girdle muscular dystrophy (LGMD) in other centers were not excluded. All patients entered a diagnostic protocol that consisted of clinical examination, biochemical investigation (total sCPK, sCPK cardiac isoform [MB] and lactic dehydrogenase), electrocardiography (ECG), chest-X-ray, two-dimensional and Doppler echocardiography, right and left heart catheterization, coronary angiography and right ventricular EMB. Informed and consenting relatives were screened for DCM with clinical examination, biochemical blood tests, ECG, signal averaged ECG and echocardiogram, and the disease was defined as familial or nonfamilial (18).

Right ventricular endomyocardial biopsy.   Biopsy was performed in all patients as per standard protocol (19). Biopsy samples were processed for light microscopy and ultrastructural study (19–21). One frozen sample was used for dystrophin immunostaining and one for molecular study (see following text).

All biopsy specimens from the 201 patients were evaluated with light and electron microscopy, and routine immunohistochemical studies were performed to diagnose or exclude myocarditis (19). Screening for enteroviral RNA was undertaken with reverse transcriptase (RT)-PCR and nested RT-PCR as well (22). Amyloid and iron deposits were excluded by Congo red and Pearls’ stains as well as with ultrastructural study (23,24). Desmin accumulation was excluded by ultrastructural examination (20). In addition, mitochondrial oxidative phosphorylation defects were analyzed with specific histoenzymatic stains (21,25).

Immunohistochemical study of dystrophin and dystrophin-associated proteins.   Four monoclonal antibodies that recognize N-terminal, rod, midrod (exons 49–51) and C-terminal domain epitopes of the dystrophin molecule (Novocastra Laboratories, Newcastle Upon Tyne, United Kingdom) were used to immunostain frozen myocardial sections. Dystrophin-associated glycoproteins (DAGs) were evaluated with monoclonal antibodies that recognize alpha, beta, gamma and delta sarcoglycan, beta dystroglycan, merosin and spectrin (Novocastra Laboratories, New Castle Upon Tyne, United Kingdom). Negative control studies were performed with the substitution of the primary antibodies either with normal swine serum or with unrelated primary antibody from the same species as the primary antibody. Immunohistochemical staining was performed with the avidin-biotin peroxidase complex (Amersham Life Science, Bucks, United Kingdom), using diaminobenzidine tetrahydrochloride as chromogen substrate or with fluorescein-conjugated secondary antibodies (Boehringer Mannheim, Germany) (19,20).

DNA analysis.   The DNA was isolated from peripheral blood leukocytes by standard methods (26). In all DCM patients, analysis of the dystrophin gene was performed with different multiplex and single PCR assays; 100 ng of genomic DNA was amplified with Taq Gold Polymerase (Perkin Elmer–Applied Biosystems, Foster City, California) in 50 µl reaction solution. The assays tested exons 1-muscle promoter, 3, 6, 13, 43, 47, 52 and 60 in one reaction, exons 49 and 50 in a second (14); exons 4, 8, 12, 17, 19, 44, 45, 48 and 51 in a third (15) and exons 16, 32, 34, 41, 42, 53 in a fourth reaction (16). Two multiplex PCR assays were standardized for exons 7, 46, 66, 68, 70 and exons 29, 54, 58, 61, 69, and 76 at the following conditions: denaturation 10' at 95°C; denaturation 30'' at 94°C, annealing 30'' at 53°C, elongation 4' at 65°C for 23 cycles; final elongation 6' at 65°C. Exons 9 and 64 were amplified in single PCR reactions. Primers were designed according to the Leiden Muscular Dystrophy Pages. All deletions identified in multiplex PCR assays were confirmed using single exon PCR. The nucleotide sequence of exon 9 (10) was determined by direct automated sequencing analysis. Exons 1 through 25 were sequenced in the three patients in whom no defects were found with the above screening techniques, and immunohistochemistry had documented the absence of immunostain.

Microsatellite analysis.   A short tandem repeat (STR) analysis was performed with a 377 DNA Sequencer and software Genescan 377 (ABI Prism, Santa Clara, California), using a panel of previously reported microsatellites (heterozygosity 35% to 93%) located on the dystrophin gene: DYS I, DYS II, DYS MSA, IVS44 SK21, DMD 44, DMD 45, DMA 49, DMD 50, DYS CA (27–31).

	Results

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Of the 201 patients, 13 (6.5%) were diagnosed with dystrophin defects (Table 3). In 10 of the 13 cases, the molecular analysis identified the gene defects. Before admission, of these 13 patients, four (30.7%) were unsuspected, three (23.1%) had been misdiagnosed as limb-girdle myopathy and six (46.2%) were clinically suspected as BMD, mostly based on high sCPK levels, plus family history in two. Total sCPK levels were raised in 10 patients; sCPK MB levels were normal in all patients. Of the 188 patients with nondystrophin-related DCM, four patients had high sCPK levels: two were affected by non-X-linked familial DCM and two by nonfamilial DCM.

View larger version (35K): [in this window] [in a new window]   Figure 1 Graph showing the prevalence of dystrophin defects-associated dilated cardiomyopathies in the overall series of 201 consecutive male patients, grouped by decades of age. Gray bar = DCM; Black bar = dystrophin defect DCM. DCM = dilated cardiomyopathy.

Clinical outcome was better for seven patients with exclusive rod domain defects: five are alive, all stable in New York Heart Association classes II and III, after a mean follow-up of 3.4 years (range from six months [the last diagnosed 50-year-old patient] to six years), one underwent transplantation four years after onset of cardiac symptoms and one died of congestive heart failure two years after the onset of symptoms while awaiting heart transplantation. Two remaining patients with N-terminal defects, as well as those in whom molecular defects were not identified, had relatively poor outcomes; one died a year after onset of symptoms and four underwent heart transplantation with a mean interval of 1.9 years after onset (range 11 months to four years). All transplanted patients are alive 20, 13, 10, 10 and 3 months after surgery. Overall, the patients with nonrod domain defects had worse pathophysiological parameters at the time of their initial evaluation (Table 3).

Familial disease.   Family history was positive in four cases. In two of these patients, the diagnosis had been suspected based on affected male cousins. Relatives of two other families with no prior history of dystrophin defect accepted both clinical evaluation and molecular analysis. The extension of family studies to other patients was either refused or impossible. In one of the two families that accepted evaluation, the proband had a younger brother and sister; both were shown to be respectively not affected and noncarrier by STR analysis of dystrophin gene markers (Fig. 2). In the other family, although only four microsatellites were informative, a younger sister of the proband did not carry the affected maternal X (Fig. 2).

View larger version (23K): [in this window] [in a new window]   Figure 2 Microsatellite analysis of families of probands 2 and 13. In the family of proband 2, four microsatellites were informative. In the family of proband 13, six microsatellites were informative. Sisters of both probands were shown to be noncarriers.

View larger version (100K): [in this window] [in a new window]   Figure 3 Light micrographs from frozen sections of endomyocardial biopsy from patient #13, with deletions of exons 6–8, 12, 13, 16, 17, 19 and 44. The immunostain of rod domain (a) and N-terminus (c) is almost absent: only sporadic, possibly revertant fibers retain immunoreactivity; the expression of C-terminus is discontinuous or absent in some cells (b). Alpha (d) and gamma (e) sarcoglycan immunostains show focal interruptions and irregular intensity; beta dystroglycan (f) only shows minimal decrease. Merosin (g) is normally expressed. (Fluorescein-conjugated rabbit antimouse antibodies; a–g 280x, reduced by 60%).

	Discussion

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Prevalence of dystrophin-related genetic abnormalities in DCM.   Screening a large cohort of consecutive male patients with DCM, we have identified dystrophin gene defects in 6.5%. All patients presented with dominant or exclusive cardiac involvement. Nine of 13 had some clinical evidence of mild myopathy, mostly in the form of raised sCPK; two patients had normal sCPK. None developed or showed a progression of skeletal myopathy during follow-up. We started this program when a heart recipient with pretransplantation diagnosis of idiopathic DCM developed increased sCPK levels one year after surgery. Initially, the increased sCPK was attributed to simvastatin toxicity, but its persistence after simvastatin withdrawal led to reevaluation of the patient. A revised family history documented an X-linked recessive inheritance. The Western blot analysis of a skeletal muscle sample and the molecular analysis of the gene showed a truncated protein and an in-frame deletion spanning exons 45 to 47 (12). After 1994, we have undertaken a combined immunohistochemical study of EMB samples and molecular DNA screening of most common dystrophin gene defects associated with heart involvement in men presenting with DCM.

Genotype-phenotype correlation.   Dystrophin gene deletions spanning exons 45 to 53, recurrently involving exons 46 and 47, have been reported in most DCM associated with dystrophin defects (9,11,32–34). The integrity of the rod domain (in particular the midrod domain) of the protein plays a major role in structural and functional integrity of the myocyte sarcolemma; intronic segments from this region could include sequences relevant to the function of dystrophin in cardiac muscle (9,33). Eight of our patients had deletions exclusively affecting rod domain exons; all were older than 20 and had slowly progressive DCM. None had overt myopathy. Four of the five remaining cases were younger than 20; all had increased sCPK. Two of these patients, in whom the molecular defect has been identified, had multiple and noncontiguous deletions of N-terminal and midrod domain exons. Although rare, these defects may occur (35). Although all patients presented with dominant cardiac involvement, rod domain exon deletions seem to be associated with normal to moderately increased sCPK and more benign clinical outcome, while earlier defects are associated with a faster evolution and high sCPK levels.

Identification of dystrophin defects in DCM patients.   Light microscopy study of EMB samples does not contribute to the diagnosis. With conventional histopathology, we did not identify any specific morphological marker useful to distinguish dystrophin gene defect-related cardiomyopathy from "idiopathic" DCM. Ultrastructural study raised some suspicion, but the diagnosis cannot rely on this type of study. Immunohistochemical study of the different dystrophin domains plays a diagnostic role and should be routinely performed in all young or adult males to obtain a systematic screening. Given that EMB provides a limited amount of tissue, it is helpful to undertake parallel tissue studies with molecular analyses on peripheral blood DNA at least for those disorders with known genetic background, such as X-linked DCM. However, with both multiplex and single exon PCR assays, as well as with direct sequencing of those dystrophin gene regions in which single base mutations causing cardiomyopathy have been detected (4,5), deletions in other gene regions or unusual mutations (6–8) are not investigated. This is likely the case of our three patients in whom gene defects were not identified. Therefore, the entire gene should theoretically be analyzed. This implies heavy and expensive workloads. Among policies of analyzing the entire gene in one patient and screening most frequent mutations in the entire patient population, we adopted the latter approach.

To better target screening, useful clinical or biochemical markers should be identified. Raised sCPK levels may potentially indicate dystrophin abnormalities; however, in our series, two patients had normal total sCPK and four had less than a five-fold increase in sCPK levels, as required by BMD diagnostic criteria. Furthermore, five-fold increased sCPK was also reported in patients with X-linked DCM (3,4,6,8); therefore, it does not constitute, by itself, a useful marker to assign patients with DCM and proven dystrophin defect to BMD with mild muscular impairment and dominant cardiomyopathy, rather than to the X-linked DCM subgroup. The distinction only seems to rely on the absence of clinically overt myopathy. However, given that none of the reported patients (3–9) had normal skeletal muscle morphology, the distinction seems to be, in some cases, purely semantic. Moreover, because patients with congestive heart failure may develop a secondary peripheral myopathy (36), a mild sCPK increase may not be sufficiently informative. Finally, in our experience, a prior clinical diagnosis of LGMD, which should by itself exclude a dystrophin defect and which is only exceptionally associated with heart involvement (37), does not exclude dystrophin defects.

Because, at present, we do not have reliable tools to properly assign EMB material to the different types of investigations, our strategy is to screen all DCM male patients for dystrophin gene defects. However, the information obtained from present research has provided the basis for a more critical approach. We developed and are adopting a better targeted diagnostic protocol for evaluation of male patients with DCM. Given that 14 of the overall 201 patients had raised levels of sCPK with normal sCPK-MB and that 10 of them (71.4%) belong to the group of patients affected by dystrophin defects, we routinely perform molecular screening on peripheral blood DNA, even before EMB, in all patients with increased sCPK levels. The same approach is used for patients with clinical suspicion of BMD and LGMD. If molecular analysis identifies dystrophin defects, EMB or skeletal muscle biopsy is not an obligate diagnostic step. On the other hand, if routine molecular analysis does not identify gene defects, tissue studies are necessary. For men with normal or minimally raised sCPK who are younger than 30, EMB is essential; when dystrophin immunostain is normal, no further molecular analysis is necessary. In cases of abnormal immunostain, gene defects should be investigated. However, a recent finding complicates our approach: enteroviruses, which may be causally linked to DCM, have been shown to synthesize enzymes that are able to cleave dystrophin, thus modifying the integrity of membrane immunostain (38). More complex is the approach to the screening of patients older than 30 years with normal sCPK levels. The likelihood of finding a dystrophin defect in these patients is, in our experience, very low (2 of 161, 1.2%) but not null, and, in any case, all patients should be given equal chances for diagnosis. Therefore, costs and efforts have to be balanced with beneficial effects deriving from the correct diagnosis. Benefits are for relatives more than for the probands themselves; namely brothers for potential preclinical diagnosis, sisters and maternal aunts as potential carriers and, for older patients, daughters as obliged carriers, given that fertility is not affected by the disease. Immunostaining for dystrophin routine EMB samples is the first screening approach in unsuspected adult patients with normal CPK; molecular analysis will be limited to cases with irregular or unclear immunolabeling results.

In conclusion, routine clinical and laboratory evaluation may not distinguish dystrophin-defect-associated cardiomyopathy from idiopathic DCM. In particular, sCPK levels may be normal or lower than those reported in BMD. Immunohistochemical and molecular studies are essential to identify protein and gene defects. The observed prevalence of dystrophin-related DCM may not represent a real estimate of the disease in the overall group of DCM. In particular, our results may be biased (especially for the relatively high proportion of patients younger than 30 years [17.4%]) by the characteristics of our institution, which is a tertiary academic hospital for chronic heart failure and cardiac transplantation, and by the ethnic origin of the tested population. Screening studies performed in different settings and ethnically distinct populations might give different results. In this regard, further data are needed.

	Footnotes

 
This study was supported by grants: "Ricerche Finalizzate IRCCS Policlinico San Matteo—Health Ministry," Italy.

	References

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