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(Circulation. 1997;95:133-139.)
© 1997 American Heart Association, Inc.

Articles

Viral Infection of the Myocardium in Endocardial Fibroelastosis

Molecular Evidence for the Role of Mumps Virus as an Etiologic Agent

Jiyuan Ni, MD; Neil E. Bowles, PhD; Young-Hwue Kim, MD; Gail Demmler, MD; Debra Kearney, MD; J. Timothy Bricker, MD; Jeffrey A. Towbin, MD

the Departments of Pediatrics (J.N., N.E.B., Y.-H.K., G.D., J.T.B., J.A.T.), Pathology (G.D., D.K.), and Molecular and Human Genetics (J.A.T.), Baylor College of Medicine, Houston, Tex.

Correspondence to Jeffrey A. Towbin, MD, Department of Pediatrics (Cardiology), Baylor College of Medicine, One Baylor Plaza, Room 333E, Houston, TX 77030. E-mail jtowbin@bcm.tmc.edu.

	Abstract

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Background Endocardial fibroelastosis, previously a common disease of children, often resulted in congestive heart failure and death. Virus-induced myocarditis was the suspected first step in the pathogenesis of the disease, with enteroviruses and mumps virus considered potential causes. Direct evidence for their involvement was limited, however, and during the past two decades, a significant decline in the incidence of endocardial fibroelastosis occurred. Recently, we demonstrated polymerase chain reaction to be a rapid and sensitive method for identification of the viral genome in the myocardium of patients with myocarditis and dilated cardiomyopathy. The purpose of this study was to analyze myocardial samples of patients with endocardial fibroelastosis for the viral genome.

Methods and Results Myocardial samples from 29 patients with autopsy-proven endocardial fibroelastosis were analyzed for viral genome (enterovirus, adenovirus, mumps, cytomegalovirus, parvovirus, influenza, herpes simplex virus) by use of polymerase chain reaction or reverse transcriptase–polymerase chain reaction. In 90% of samples, the viral genome was amplified; >70% of the samples were positive for mumps viral RNA, while 28% amplified adenovirus. In contrast, only 1 of 65 control samples amplified a virus (enterovirus). Two regions of mumps virus were amplified: the nucleocapsid gene and the polymerase-associated protein gene. Interestingly, only 3 of the 21 samples that were positive for mumps RNA were positive with both sets of primers, indicating that the persistence of mumps virus in the myocardium may be related to the selection of defective virus mutants.

Conclusions These data suggest an etiologic role for viral infection in endocardial fibroelastosis, supporting the hypothesis that endocardial fibroelastosis is a sequela of a viral myocarditis, in particular of that due to mumps virus.

Key Words: myocarditis • viruses • endocardial fibroelastosis

	Introduction

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Endocardial fibroelastosis has been defined as a diffuse thickening of the left ventricular endocardium resulting from proliferation of fibrous and elastic tissue leading to decreased compliance and impaired diastolic function.^{1 2} Primary and secondary forms of EFE have been described. Most patients have a dilated left ventricular chamber (dilated form), although a small group of patients display ventricular hypoplasia (contracted form).¹ The primary forms of the disease are not associated with any other cardiac anomalies, whereas secondary EFE occurs with such disorders as critical aortic stenosis of the newborn. EFE usually occurs in infants and young children who present with signs of congestive heart failure, and although genetic forms of the disease have been described,³ most cases are of unknown etiology. In the past, the incidence of EFE in the United States was relatively high, 1 per 5000 live births. In recent decades, however, the incidence has declined significantly for unknown reasons.⁴

It was suggested that idiopathic cases of EFE resulted from increased endocardial mural tension produced by left ventricular dilatation, which in turn is secondary to myocarditis.⁵ Hutchins and Vie⁵ studied 64 children with either myocarditis or primary EFE; of these, 5 had myocarditis only, 18 had idiopathic EFE, and the remaining 41 had evidence of both myocarditis and EFE. With longer survival, the severity of myocarditis decreased but was replaced by an increase in EFE, and by 4 months of follow-up, no patient had histological evidence of myocarditis.

For a number of years, it has been believed that most cases of myocarditis resulted from viral infection of the myocardium.⁶ However, there were few reports of successful virus isolation from the myocardium except during acute cases. Most of the evidence supporting an etiologic role for virus infection in myocarditis, especially the enteroviruses, originally came from retrospective serology.⁷ However, the application of the techniques of molecular virology to this issue has led to the demonstration of persistent virus infection of the myocardium in patients with myocarditis or even so-called IDCM.^{8 9 10 11 12} The finding of viral genetic material in the myocardium of patients with IDCM has strengthened the link between viral myocarditis and congestive heart failure.

The link between viral myocarditis and EFE, therefore, supports a role for chronic viral infection in the etiology of EFE.⁵ However, as with myocarditis, little direct evidence has been obtained for viral infection of the myocardium of patients with EFE by use of classic virological techniques. Fruhling et al¹³ reported that a significant proportion of myocardial samples from EFE patients had cultures that were positive for coxsackie B virus. However, there have been no other reports confirming this observation. It has also been proposed that EFE may develop in a particular subset of patients with viral myocarditis, those with mumps virus–induced disease. A link between mumps virus infection and EFE was established by positive skin-reactivity tests.¹⁴ Noren and colleagues¹⁴ showed that positive skin tests occurred in all nine children with EFE tested. In one case, the mother suffered a mumps infection during the first trimester of pregnancy, whereas two others were exposed to mumps. Therefore, it was suggested that intrauterine infection with the mumps virus may be involved in some cases of EFE. However, Gersony and colleagues¹⁵ and Nadas and Fyler¹⁶ discounted the concept that mumps and EFE were in some way related.

There have been a number of other reports linking mumps virus infection with myocarditis. It was first suggested in 1918 that myocarditis was a rare complication of mumps virus infection.¹⁷ During the 1940s and 1950s, other reports were published,^{18 19 20} but in 1984, a link between mumps myocarditis and subsequent cardiomyopathy was established.²¹

A mumps virus etiology for EFE may also explain the dramatic decline in incidence in the last few decades. In recent years, particularly since the introduction of the mumps vaccine, the prevalence of epidemic parotiditis has decreased significantly.²² Therefore, unlike the pattern of infection of the enteroviruses, which show periodic peaks in infection rates, the decline in incidence of EFE seems to reflect the decreased prevalence of mumps virus in the population.

We¹¹ have previously studied samples from a number of children with EFE for the presence of the genomic nucleic acid of a range of viruses, including enterovirus, adenovirus, HSV, and CMV, by RT-PCR or PCR. We found that of the 28 samples studied, 6 were positive for adenovirus DNA but were negative for all of the other viruses. The purpose of the present study was to extend this analysis by determining the frequency of mumps virus infections (as well as other viruses) of the myocardium in these patients by use of RT-PCR.

	Methods

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Patient Population
Twenty-nine children (aged 26 weeks' gestation to 7 years) with clinical and pathological (gross anatomic and histopathologic) evidence of EFE and congestive heart failure were analyzed. All patients were clinically evaluated at Texas Children's Hospital, Houston. Clinical criteria for study included gross histopathologic evidence of EFE. Twenty-six of the patients included in the study had evidence of primary EFE; 3 patients were included with critical aortic stenosis of the newborn (ie, secondary EFE).

Patient Samples
Formalin-fixed tissue samples (myocardium) were obtained from autopsies of all 29 of the patients with EFE; left ventricular specimens were obtained in each case. Gross and histopathologic analyses were performed in all cases by a pediatric cardiovascular pathologist. All autopsies were performed between 1955 and 1992.

Control and Other Samples
Formalin-fixed and fresh-frozen myocardial specimens (left and right ventricles) were obtained from 65 patients with no evidence of EFE or myocarditis. In 42 cases, specimens were obtained at the time of cardiac transplantation; the remaining cases (n=23) were obtained at autopsy. Diagnoses of these patients included congenital heart disease (n=53) and hypertrophic cardiomyopathy (n=12). Echocardiography, gross anatomy, and histopathology were prospectively performed in all cases. No patients had recent history of viral illness or clinical viral disease. Patients' ages ranged from 3 weeks to 17 years (mean, 5.5 years).

Primer Design and Synthesis
All oligonucleotide primers were synthesized on an Applied Biosystems model 380 DNA synthesizer with the use of published viral sequences. Primer pairs to amplify the genomic sequences of adenoviruses, enteroviruses, CMV, and HSV have been described previously¹² (Table 1); these primers were designed to amplify most virus types within their genus. Two sets of primer pairs were synthesized to amplify mumps viral RNA sequences corresponding to either the NP gene²⁴ or the P gene²⁵ (Table 1). An additional set of primers was prepared to amplify the region of segment eight encoding the nonstructural proteins of the influenza A virus.³⁰ Primers corresponding to sequences in the 12th codon of K-ras were used as a positive control for the isolation of intact DNA.¹²

View this table: [in this window] [in a new window]   Table 1. PCR Primers Used to Detect Viral Sequences in Cardiac Samples

RNA and DNA Template Preparation
Tissue samples were first homogenized in RNAzol by use of a Brinkman Polytron homogenizer. Total RNA and genomic/viral DNA were isolated simultaneously from patient specimens by use of Tris-saturated phenol (pH 6.6) RNAzol in a modification of the RNAzol method,³¹ as previously described.^{11 12} The quality of the extraction was determined by gel electrophoresis without nucleic acid quantitation. Contamination was controlled between homogenization of samples as follows: the homogenizer was washed with distilled water treated with diethyl pyrocarbonate for 1 minute then washed with 1% SDS for 10 minutes before being placed in 100% ethanol for 10 minutes. All washing was performed with the homogenizer at medium speed. The homogenizer was autoclaved for 20 minutes and cooled before reuse. Adenovirus type 5, coxsackievirus B4, CMV strain AD169, HSV type 1, influenza type A, and mumps virus were used as positive viral controls for the PCR analysis after nucleic acid extraction.

RT-PCR
For the detection of genomic nucleic acid of the RNA viruses, RT-PCR was used.³² First-strand cDNA, for the detection of enterovirus, influenza A virus, or mumps virus, was synthesized from 3 µg extracted total nucleic acid in the presence of 20 U RNasin (Amersham International), an RNase inhibitor. This mixture was heated to 95°C for 5 minutes then snap-cooled on ice. After the addition of 5 pmol of the reverse-transcription primer (No. 195 for enterovirus, Nos. 110 or 112 for mumps, or No. cDNA for influenza A; see Table 1), 4 µL of 5 mmol/L each dNTPs, 4 µL of 5x reverse-transcriptase buffer (5x buffer=15 mmol/L MgCl₂, 50 mmol/L dithiothreitol, 250 mmol/L Tris pH 8.3, and 375 mmol/L KCl), 200 U of Moloney-murine leukemia virus reverse transcriptase (BRL-Gibco), and diethyl pyrocarbonate–treated dH₂O to 20 µL, the samples were incubated at 37°C for 1 hour. Two microliters of this first-strand cDNA or 500 ng of control human genomic DNA was combined with 25 pmol of each primer pair, 5 µL of 10x PCR buffer (100 mmol/L Tris pH 8.3, 500 mmol/L KCl, and 15 mmol/L MgCl₂), and 5 µL of 2 mmol/L dNTPs in a final volume of 50 µL. Two and a half units of Taq DNA polymerase (Promega) was added after an initial 5-minute incubation at 95°C, and then 35 rounds of amplification were performed with the use of an MJ Research Thermocycler. In the case of enterovirus amplifications, the conditions were as follows: 95°C for 30 seconds, 57°C for 30 seconds, and 72°C for 1 minute, whereas for mumps virus and influenza A virus, the conditions were 95°C for 1 minute, 50°C for 1.5 minutes, and 72°C for 1.5 minutes.

For adenovirus, CMV, or HSV (ie, DNA viruses), 3 µL or 1000 ng of extracted total DNA was combined with 25 pmol of the appropriate primers, 5 µL of 10x PCR buffer, and 5 µL of 2 mmol/L dNTPs in a final volume of 50 µL. Taq DNA polymerase (2.5 U) was added after an initial 5-minute incubation at 95°C. In the case of adenovirus or CMV, 40 rounds of amplification were performed under the following conditions: 95°C for 30 seconds, 57°C for 30 seconds, and 72°C for 1 minute. For HSV, the conditions were 95°C for 1 minute, 61°C for 1 minute, and 72°C for 1 minute, with 32 rounds of amplification used.

The K-ras primers (K-ras-2 and K-ras-3, 2 mmol/L each) were combined with 5 µL 10x PCR buffer, 8 µL of 1.25 mmol/L dNTPs, 5 µL of sample, and dH₂O to 50 µL. The mixture was incubated at 95°C for 5 minutes, Taq DNA polymerase (2.5 U) was added, and 40 rounds of amplification were performed under the following conditions: 95°C for 30 seconds, 57°C for 30 seconds, and 72°C for 60 seconds.²⁹

Analysis of PCR Products
Ten microliters of each reaction was analyzed on a 2% agarose gel (FMC Biochemicals) containing 0.5 µg/mL ethidium bromide (Sigma), and the DNA product was visualized by ultraviolet transillumination.

In all cases, positive (purified viral nucleic acid) and negative (water or nucleic acid from a tissue sample known to be negative) control reactions were performed simultaneously with the test samples. If a band was detected in the negative control lane, the PCR sample was considered contaminated, and the sample was either reanalyzed or reextracted and analyzed. All samples were analyzed without prior knowledge of clinical or culture/serological data for each patient, and all PCR-positive samples were required to have duplicate results. Control PCR amplifications to verify the presence of amplifiable nucleic acid extracted from each sample were performed with the use of K-ras primers.²⁹ If the K-ras primers failed to amplify the appropriate 135-bp amplimer, the sample was reextracted or excluded. Southern blotting and hybridization³³ as well as direct sequencing of the PCR product³⁴ were used to confirm positive results. Radioactive labeling of previously described probes and positive control PCR products were as described previously.^{27 29 33 35}

Statistical Analysis
The statistical significance of the PCR results obtained from samples in children with EFE versus the negative control samples (ie, comparison between groups) was analyzed by use of Fisher's exact test. Statistical significance was defined by a probability value <.05.

	Results

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Clinical Presentation
In all 28 living children, the initial clinical presentation was consistent with signs and symptoms of congestive heart failure (Table 2). One fetus (patient 28 in Table 2) succumbed at 26 weeks' gestation and was stillborn. In all patients, left ventricular EFE was identified at autopsy; in 3 children (patients 21, 22, and 28 in Table 2), neonatal critical aortic stenosis was also seen. Only 5 of the 28 living children had a prior history of viral infection, in all cases during the third trimester of their mothers' pregnancies (patients 3, 5, 10, 19, and 25 in Table 2); 12 children had viral prodrome. In 10 patients, sudden cardiac death occurred; the remaining children succumbed to chronic congestive heart failure (n=13) or postoperative complications (n=5).

View this table: [in this window] [in a new window]   Table 2. Clinical Data and Corresponding PCR Results

Serology and Virus Cultures
Serological evaluation was performed in 11 of 29 patients, and viral cultures were obtained in 15 of 29 cases. No positive serological results or cultures were reported for mumps virus, but positive serological results were identified in 3 patients (1 CMV, 2 enterovirus).

Histopathology
In all patients, gross and histological evidences of EFE were described. No viral inclusions were evident in any cases. Only 2 patients (patients 9 and 12 in Table 2) had evidence of inflammatory infiltrate, and on retrospective analysis, both of these samples were consistent with "borderline" myocarditis according to the Dallas criteria.³⁶ All patients had evidence of EFE; 3 children (patients 21, 22, and 28 in Table 2) had thickened, sclerotic aortic valves consistent with critical aortic stenosis of the newborn. Two other children had aortic abnormalities, 1 with bicuspid aortic valve and mild aortic stenosis (patient 24 in Table 2) and 1 with supravalvar aortic stenosis (patient 20). Hepatic necrosis was notable in 3 patients (patients 1, 3, and 21 in Table 2), while cerebral infarction (patient 7, Table 2) and renal infarction (patient 25, Table 2) occurred in 1 child each. None of the 65 control patients had evidence of EFE or myocarditis.

PCR Analysis
RT-PCR or PCR was performed on samples from 29 patients with EFE and 65 control samples (from patients with heart disease of known etiology). RT-PCR was performed to detect either enteroviral, mumps, or influenza A viral RNA, whereas PCR was performed to detect adenoviral, CMV, or HSV genomic DNA.

The Figure demonstrates the PCR amplification of mumps virus from EFE patient samples by use of the primers corresponding to the P gene of mumps virus.²⁵ Examples of the amplification products of viral sequences with the other primers have been published previously.^{11 12} The specificity of the amplification is demonstrated both by the correct size of the amplimer (top of Figure) and by hybridization with a mumps viral RNA-specific probe (bottom of Figure).

View larger version (77K): [in this window] [in a new window]   Figure 1. Top, Mumps PCR with the use of the primers designed for the polymerase-associated region. The products were detected by ethidium bromide staining of the 2% agarose gel. A 1-kb ladder (size marker) is shown in the first lane. Mumps positive control PCR is seen as a 223-bp amplimer in the second lane; the negative control (PCR reactants minus nucleic acid) is seen in the third lane. Note that lane 3 is devoid of a PCR amplimer, thus excluding contamination. Left ventricular (LV) autopsy specimens from several children with EFE (patients 2, 4, 5, and 15, corresponding to patients listed in Table 2) demonstrate the 223-bp band consistent with mumps virus infection (P-region primers). Bottom, Southern blot analysis of the gel shown in the top panel, using a 32P-labeled mumps-specific probe. Note the specificity of hybridization of the radiolabeled probe for the mumps positive control and LV autopsy samples. The negative control is devoid of hybridization signal.

The results of RT-PCR and PCR analyses are summarized in Table 3. Of the 29 EFE patients studied, all but 3 (90%) were positive for at least one virus type. In contrast, only 1 of the 65 control samples (1.5%) was positive (P<.001) for any viral sequence (enterovirus). Eight (28%) of the EFE patient samples were positive for adenoviral genomic DNA, although 3 of these were positive for mumps viral RNA, including the explant material. One sample was positive for CMV DNA and 1 for enteroviral RNA (both were positive by serological analysis); both of these samples were also positive for mumps viral RNA. None of the samples tested positive for either HSV or influenza A.

View this table: [in this window] [in a new window]   Table 3. Summary of Culture, Serological, and PCR Results

The most striking result, however, was the number of EFE patient samples that were PCR positive for mumps. In 21 (72%) of the patients, the PCR was positive for mumps viral RNA with at least one of the two sets of primers used. Of these 21 positive cases, 16 had viral RNA corresponding to the P gene, 8 had sequences of the NP gene, but only 3 had sequences from both genes.

	Discussion

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EFE was previously considered a significant cause of infant mortality. In recent years, however, the incidence of this disease has declined dramatically,²² probably because of the availability of the mumps vaccine. This decline in incidence, which corresponds chronologically to the decline in mumps-related disease, in addition to the positive serological studies of mumps in EFE,^{14 15 16 17 18 19 20 21} supported the proposal that mumps virus was an important etiologic agent for this disease. However, with the exception of a single report of the isolation of coxsackie B viruses from cardiac samples of EFE patients,¹³ a virus had not been cultured from these patients. Therefore, little direct evidence of a viral etiologic agent for EFE was available.

With the use of RT-PCR or PCR, autopsy material from a group of patients with EFE was analyzed previously for evidence of chronic infection by a range of viruses, including the enteroviruses, adenovirus, HSV, and CMV.¹¹ In that study, adenoviral genomic DNA was detected in the myocardium of 6 (21%) of 28 EFE patients without clinical or histological evidence of myocarditis; no other virus was detected. In the present study, 28 EFE patients plus 1 additional patient were reevaluated for evidence of virus infection with PCR used to detect not only those viruses listed above but also mumps virus and influenza A. DNA and RNA were reisolated from these samples together with 65 control samples.

In agreement with our previous study,¹¹ we found evidence of adenovirus infection in 8 (28%) of 29 EFE patients, including all 6 who were previously found to be positive, while 1 patient was positive for CMV and another was positive for enterovirus RNA. Both patients who were positive by PCR for either CMV or enterovirus also amplified a second viral genome (ie, mumps). However, with the mumps-specific primers, 21 (72%) of the 29 patients were positive for mumps virus. The specificity of these amplifications was confirmed in a number of ways. First, the size of the PCR product was as expected and was the same as that obtained after amplification of purified mumps viral genomic RNA (top of Figure). Second, the product hybridized with a mumps virus–specific probe (bottom of Figure). Third, a limited number of the positive samples were analyzed by DNA sequencing of the P region; in each case, a sequence similar or identical to that of the Enders strain²⁵ was identified. Fourth, all of the control samples from patients with heart disease of known etiology or normal autopsy material were negative for mumps sequences. Finally, in our laboratory, we have performed PCR on hundreds of cardiac samples, and in no other disease have we found evidence of mumps virus infection.

The use of two different primer sets for mumps viral RNA yielded very different results. With primers complementary to the P region, 16 of the 29 samples were PCR positive, whereas with NP gene primers, only 8 were PCR positive; only 3 of the 21 mumps virus–positive samples contained viral RNA corresponding to both of these regions. Three explanations could account for these observations. First, the primers may be capable of detecting some but not all strains of mumps virus. Several isolates of mumps virus have been sequenced in the P region, and all should be amplified by the primers used. Few sequence data are available for the NP region, but under the conditions used, the primers should amplify both strains reported thus far (Enders and Miyahara). Second, some of the samples were collected several years ago when EFE was more prevalent, and therefore, the viral RNA could be partially and differentially degraded, although there was no apparent correlation between the age of the samples and the ability of either band to be detected. Finally, different regions of the viral genomic RNA may have persisted in different samples and/or different cells. This last possibility is probably the most intriguing because it could offer an explanation for the mechanism of virus persistence and account for the failure to detect infectious virus in these samples.

The generation of DI particles during the infectious cycle has been described for a number of viruses in vitro,³⁷ including the paramyxoviruses.³⁸ If a cell has been infected by such a DI particle, viral gene expression may remain latent until the cell is superinfected with a virus encoding the complementary genes. Thus, the life cycle of the mumps virus may be controlled after the initial lytic stage (which could give rise to myocarditis), leading to a persistent infection without viral antigen expression.

The significance of the detection of two viruses in five of the samples from EFE patients is not clear, although two of these patients were among the four children in whom sudden death occurred, suggesting a link between the severity of cardiac symptoms and multiviral infection. There were no significant histological differences between any of the groups, as defined by type of virus detected, suggesting that there is not a correlation between the grade of disease or clinical outcome and viral isolate. We have demonstrated that infection with multiple virus types can occur in utero, and therefore, these observations may be related, because it is likely that a significant proportion of EFE patients were infected during pregnancy. All five of the patients with a history of viral infection (during the third trimester of their mothers' pregnancies) tested positive for virus. Two of the three patients with secondary EFE tested positive for virus (adenovirus), thus raising the possibility that fetal infection gives rise to aortic stenosis. The mechanism is not clear; however, one could speculate that reduced cardiac output could potentially result in abnormal growth of the aortic valve (as described by Clark³⁹ ), whereas valve scarring could potentially occur by direct action of the virus.

In conclusion, we have demonstrated that a significant proportion of patients with EFE have mumps virus genome in the myocardium. The very high proportion of positive samples compared with studies of so-called IDCM probably reflects the rapid progression to end stage, which is usually the death of the child. Alternatively, the data may reflect a more profound infection of the myocardium by mumps virus than occurs with the enteroviruses or adenoviruses, reducing sampling errors. The data presented here clearly support the hypothesis that EFE is a sequela of a mumps virus–induced myocarditis rather than an enterovirus-induced episode. More studies are required to delineate the role of DI viruses in disease pathogenesis and mechanisms of virus persistence.

	Selected Abbreviations and Acronyms

 

CMV = cytomegalovirus DI = defective interfering dNTP = deoxynucleotide triphosphate EFE = endocardial fibroelastosis HSV = herpes simplex virus IDCM = idiopathic cardiomyopathy NP gene = nucleocapsid gene P gene = polymerase-associated protein gene PCR = polymerase chain reaction RT-PCR = reverse transcription–polymerase chain reaction

	Acknowledgments

 
Funding for this study was provided in part by the Section of Cardiology, Texas Children's Hospital, Houston. The authors wish to thank Valerie R. Price for her expert secretarial support. This work was performed in the Phoebe Willingham Muzzy Pediatric Molecular Cardiology Laboratory.

	Footnotes

 
Guest editor for this article was J. David Bristow, MD, Oregon Health Sciences University, Portland.

Received February 19, 1996; revision received August 15, 1996; accepted August 24, 1996.

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