Author + information
- Received January 11, 2002
- Revision received June 10, 2002
- Accepted June 24, 2002
- Published online September 18, 2002.
- Jinong Feng, MD*,
- Jin Yan, MD*,
- Carolyn H Buzin, PhD*,
- Steve S Sommer, MD, PhD*,* ( and )
- Jeffrey A Towbin, MD†
- ↵*Reprint requests and correspondence:
Dr. Steve S. Sommer, Departments of Molecular Genetics and Molecular Diagnosis, City of Hope National Medical Center, 1500 East Duarte Road, Duarte, California 91010-3000, USA.
Objectives The goal of this study was to perform comprehensive mutation analysis of the dystrophin gene in patients with X-linked dilated cardiomyopathy (XLCM).
Background X-linked dilated cardiomyopathy is a familial disease that is characterized by congestive heart failure without clinical signs of skeletal myopathy. Mutations in the dystrophin gene have been associated with the X-linked form of dilated cardiomyopathy. However, the fraction of XLCM with dystrophin mutations and the distribution of those mutations is not clear. Technical difficulties previously limited comprehensive mutation analysis of this very large gene.
Methods The Detection Of Virtually All Mutations-Single Strand Conformation Polymorphism (SSCP) (DOVAM-S), a robotically enhanced multiplexed scanning method that is a highly sensitive modification of SSCP, has successfully detected all of 240 mutations and polymorphisms in three blinded analyses of the factor VIII, factor IX, and ATM genes. Utilizing this method all 79 coding exons and splice junctions for the muscle dystrophin gene, along with six alternative exon 1 sequences, were scanned in eight patients with XLCM.
Results This is the first comprehensive scanning of the dystrophin gene in XLCM. Three of eight patients have putative mutations, including two splicing mutations and a missense mutation at a highly conserved amino acid.
Conclusions Mutations within the coding regions and splice junctions in the dystrophin gene only account for some cases of XLCM. Genetic heterogeneity and/or undetected mutations in auxiliary regulatory regions or deep within introns may occur in XLCM.
Dilated cardiomyopathy (DCM) is the most common cardiomyopathy and the most common indication for heart transplantation. The etiology of most cases of DCM is unknown (sporadic DCM), while ∼30% of cases are thought to be inherited, including autosomal dominant (∼23%), X-linked (∼5%), autosomal recessive, and mitochondrial transmission (1). X-linked dilated cardiomyopathy (XLCM) typically affects young men in the second or early third decade of life and is characterized by severe congestive heart failure that results in death or cardiac transplantation (2–4). No skeletal myopathy is clinically evident, but elevation of the muscle isoform of creatine kinase (CK-MM) can occur.
Three genes have been associated with X-linked forms of cardiomyopathy and skeletal myopathy, including the tafazzin (also known as G4.5) gene in cases of infantile-onset DCM and the dystrophin gene in later-onset XLCM. The G4.5 gene is a small, single-copy gene that is localized in distal Xq28. Direct sequencing of genomic DNA from four unrelated male patients with Barth syndrome (an X-linked inherited disorder characterized by cardiac and skeletal myopathy, short stature, organic aciduria, and neutropenia) revealed nonsense mutations in the G4.5 gene. All the mutations were shown to segregate with the disease and were not found in normal controls (5). Four additional reports validated that G4.5 is responsible for infantile DCMs (6–9). Mutations in another gene at Xq28, emerin, result in Emery-Dreifuss muscular dystrophy (EDMD). Emery-Dreifuss muscular dystrophy is also characterized by skeletal myopathy and, in some patients, cardiac conduction defects with or without DCM (10–12). Typically, EDMD patients present with conduction system disturbance well in advance of evidence of DCM. Serum CK-MM is usually mildly elevated, and DCM severity is not correlated with the degree of skeletal muscle involvement. The risk of cardiac arrhythmia increases with age and usually affects the atria and right heart initially, progressing later to ventricular dilation and failure. The rhythm and conduction disturbances include junctional escape rhythms associated with bradycardia with heart rates at 40 to 50 beats/min without obvious atrial activity due to atrial standstill. In addition, atrial tachyarrhythmias, initially atrial fibrillation, and then atrial flutter are also common (10–12).
The third gene, dystrophin, codes for a protein of 427kDa with 3,685 amino acids and spans nearly 2,400 kb on the X-chromosome (Xp21) (13). Mutations in the dystrophin gene cause Duchenne muscular dystrophy and the milder Becker muscular dystrophy (BMD), X-linked skeletal myopathies associated with DCM in most affected male patients as well as a significant proportion of female carriers.
The above genes cause X-linked cardiomyopathy in the context of more generalized neuromuscular disease. There are also multiple reports implicating dystrophin in nonsyndromic XLCM. Variants of the dystrophin gene that have been associated with XLCM fall into two classes: 1) mutations in the 5′ end of the gene, particularly in the promoter region or exon 1 of the muscle transcript; (14–17); and 2) mutations elsewhere in the gene (4,18,19). The mutations within the promoter region and exon 1 of the major muscle transcript are associated with upregulation of alternative dystrophin transcripts and the presence of brain and Purkinje cell dystrophin isoforms in skeletal muscle (15).
Several mutations elsewhere in the gene have been described. Two deletions in the deletion hot spot region normally associated with BMD were described in two patients with DCM (19). The reason that these patients did not have BMD is unclear. Other patients with deletions in this region also have been reported to have DCM associated with BMD (17). In addition, an Alu-like sequence rearrangement has been found 2.4 kb into intron 11, resulting in activation of a cryptic splice site and producing an alternative transcript with numerous in-frame stop codons (18). Only the mutant messenger ribonucleic acid was detected in heart muscle, but some normal transcript also was found in skeletal muscle. Finally, a missense mutation was identified in exon 9, which disrupts the first hinge region of dystrophin between the N-terminus and triple helical repeat region (4).
Based on these reports, mutations in the promoter region and exon 1 can cause DCM, and there is some evidence that mutations elsewhere in the gene are etiologically relevant for X-linked cardiomyopathy. However, the frequency and contribution of mutations in the dystrophin gene in XLCM are not known because technical difficulty due to the extreme size of the gene, including huge introns, has previously limited the gene scanning. Here we report a comprehensive scanning of the dystrophin gene in eight patients with XLCM.
The patients were ascertained, and samples were analyzed according to a protocol approved by the Institutional Review Board. All patients were evaluated by physical examination, chest X-ray, electrocardiography, and echocardiography. Holter monitoring for 24 h and cardiac catheterization with endomyocardial biopsy was performed when clinically indicated. The diagnostic features required for DCM diagnosis were as previously described (3,4). Left ventricular end-diastolic dimension was measured in all patients (in centimeters) and transformed into standard deviation (z score) based on body surface area. z scores >+2 demonstrate a dilated left ventricle. Systolic function was measured by shortening fraction in percent and also transformed into z scores as well, with z score <−2 demonstrating reduced systolic function. These patients did not have the conduction system disturbances associated with EDMD. In addition, all patients were evaluated clinically for neurologic and skeletal muscle function.
detection of virtually all mutations-SSCP (DOVAM-S).
Mutation scanning was performed by DOVAM-S (20,21). Genomic deoxyribonucleic acid and standard polymerase chain reaction (PCR) conditions (22) were used to generate 90 separate PCR segments labeled with [α33P]dATP (Amersham, Boston, Massachusetts), which included all 79 coding sequences and splice junctions for the muscle dystrophin gene, along with six alternative exon 1 (Dp427l, Dp427c, Dp427p, Dp260, Dp140, and Dp116 dystrophin isoforms). The segments were amplified robotically on the ABI PRISM 877 integrated thermal cycler (Applied Biosystems, Inc., Foster City, California). The 90 segments were divided into four groups for analysis. The PCR primer sequences are available on request. After PCR amplification the products were electrophoresed on nondenaturing acrylamide gels (45 cm × 37.5 cm × 0.4 mm) using a Poker Face SE 1500 Sequencing Apparatus (Hoefer Pharmacia Biotech, San Francisco, California) at 15 W constant power for approximately 12 h to 16 h. The five different nondenaturing gel conditions (matrix/buffer/temperature) are: Pageplus/Capso/4°C, Pageplus/Tri/Tri/20°C, Pageplus/TBE/5% glycerol/20°C, HR1000/TBE/2.5% glycerol/4°C, HR1000/Tri/Tri/4°C (20). The composition of the buffers are: Capso: 30 mM Capso/ethanolamine, pH 9.6; Tri/Tri: 30 mM tricine/triethanolamine, pH 7.9; TBE: 50 mM Tris/boric acid, pH 8.3. The analysis conditions for hemizygote or heterozygote were the same. Segments with mobility shifts were reamplified and sequenced in both directions by ABI 377 Automated Sequencer (Applied Biosystems, Inc.). Sequence chromatograms were analyzed with Sequencher software (Gene Codes, Ann Arbor, Michigan).
The dystrophin gene was scanned in nine patients with XLCM. After the analysis two of the patients (DCM-58 and DCM-59) were discovered to be related. Thus, eight independent families were examined. A total of four missense polymorphisms and three different putative mutations were identified (Table 1, including footnotes labeled with symbols *, ‡, and §).
Three of eight patients with XLCM have putative mutations including two splicing mutations and a missense mutation at a highly conserved amino acid. DNA samples were available from the mother of DCM-7 and an affected son of DCM-59. These family members contained their respective mutations.
Screening of 141 control individuals failed to identify these three putative mutations. The clinical features and mutations identified in these four individuals are described below and summarized in Table 2.
This 14-year-old white male presented at 12 years of age with acute-onset congestive heart failure requiring inotropic therapy and diuretics intravenously, as well as mechanical ventilation. He was extubated after three days, weaned off intravenous therapy, and placed on an oral anticongestive regime. In addition, ventricular tachycardia was observed on the second hospital day, which was successfully treated with lidocaine. He was discharged home on the 12th hospital day on oral therapy. This young man died suddenly at age 14 years. He never had clinical evidence of skeletal muscle involvement, but his CK-MM was >2,000. He was neurologically intact clinically. His family history was pertinent for a maternal uncle with DCM. DOVAM-S screening identified a missense mutation in exon 35 causing a substitution of a highly conserved asparagine by lysine at codon 1672 (N1672K).
An 11-year-old black male presented with respiratory difficulty to his local emergency room after a one week history of symptoms. Chest X-ray demonstrated cardiomegaly and pulmonary edema, and his echocardiogram identified an extremely dilated, poorly functioning left ventricle with moderate mitral regurgitation. Before this presentation he was active and competitively involved in basketball. Neurological exam of the patient was normal. He was treated briefly with intravenous inotropes and diuretics before converting to oral therapy. He continued to have signs and symptoms of heart failure despite maximal therapy and was listed for transplantation. He died suddenly while participating in a school physical education class. His family history was pertinent for six members with DCM consistent with X-linked inheritance. All affected members and carriers had elevated CK-MM between 1,000 to 2,500 but no evidence of skeletal muscle disease. The DOVAM-S screening of DCM-27 identified a splice mutation in the muscle intron 1 (IVS1+1 G>T), which is a causative mutation also reported in a study of a family with a severe form of XLCM (16). This mutation cosegregated with the disease in the analyzed family, and the expression of the major dystrophin messenger ribonucleic acid isoforms was completely abolished in the myocardium (16).
DCM-58 is a 30-year-old African American female obligate carrier with no cardiac symptoms. Her son developed DCM at 12 years of age, dying suddenly. Measurement of CK-MM demonstrated mild elevation. No skeletal muscle features or neurologic abnormalities are notable. Molecular analysis identified a novel splicing mutation in intron 1 of the muscle transcript (IVS1+6T>C). DCM-59, a 41-year-old African American female cousin of DCM-58 with two affected sons with DCM, presenting at ages 12 years and 14 years, developed congestive heart failure and DCM at age 39. She is asymptomatic on anticongestive medication and has no evidence of skeletal myopathy or neurologic impairment. Molecular analysis identified the identical intron 1 splicing mutation (IVS1+6T>C) found in DCM-58. The splicing mutation disrupts the +6 consensus nucleotide. When the existing donor sequence matches the consensus at five or six of the eight nucleotides, mutations at the T at position +6 disrupt splicing in multiple genes (23).
The conservation of the mutated amino acids was analyzed. N1672K is highly conserved in dystrophins, including chicken and dogfish, and in utrophin, which is very similar in overall structure to dystrophin. However, D1715G and F1388V are not highly conserved (only in dog and mouse dystrophins, respectively), hinting that these two missense alterations could be polymorphisms.
This first comprehensive mutation scanning of the dystrophin gene in patients with nonsyndromic DCM reveals candidate mutations in three of eight families. DOVAM-S, a form of multiconditional SSCP utilizing a generic set of five conditions, has successfully detected all of 240 mutations and polymorphisms in three blinded analyses of the factor VIII, factor IX, and ATM genes previously reported (20,21). Our data herein regarding dystrophin scanning confirm that mutations in the dystrophin gene are associated with XLCM, and provide further evidence that the N-terminal region of dystrophin is most commonly involved.
Abnormalities in dystrophin in patients with XLCM were first reported in the early 1990s (3,14) and were followed by confirmatory reports subsequently. The majority of abnormalities reported have affected the N-terminus of the dystrophin protein via mutations in the 5′ portion of the dystrophin gene, including mutations in the muscle-specific promoter (14–17). However, due to the extreme size of the dystrophin gene, only a small number of patients have had identifiable mutations. In this report we confirm the consistent finding of 5′ mutations in the patients, as well as identifying a novel missense mutation in exon 35 (N1672K). Further, the clinical phenotype of affected males continues to be severe, with rapidly progressive symptoms and sudden death common despite maximal medical management. Unlike Barth syndrome, where no manifesting female carriers with G4.5 mutations have been identified, female carriers of dystrophin mutations sometimes demonstrate late-onset DCM, often with slow disease progression. Therefore, identification of mutations in these families can play an important role in management of both affected males and female carriers.
Because sudden death appears to be relatively common in males shortly after presentation despite maximal therapy, early transplant listing and consideration for implantation of cardioverter defibrillators is appropriate and could be facilitated by early, presymptomatic diagnosis. Additionally, measurement of CK-MM, a biochemical marker of skeletal muscle dysfunction, should be considered in male patients with DCM in order to identify at-risk patients with probable XLCM, as well as obligate carriers. The issue of sudden death is of significant importance. As 300,000 to 400,000 individuals succumb to sudden death yearly in the U.S., and in most cases this is unexpected and without known etiology, developing the capacity to identify genetically at-risk individuals would be important. Furthermore, our understanding of the process leading to sudden death is relatively naive. We (J. A. T.) have recently demonstrated that patients with all forms of DCM (ischemic acquired, genetic) have reduction or loss of N-terminal dystrophin, while C-terminal dystrophin is normal by immunoblotting and immunohistochemistry of the heart. Implantation of a left ventricular assist device to reduce mechanical stress causes reverse remodeling with normalization of left ventricular size and function associated with normalization of N-terminal dystrophin (24), suggesting that dystrophin is important in the mechanism of DCM and sudden death and that novel therapies may be able to be designed once these abnormalities are determined. This pathogenetic concept was also supported by a further example that dystrophin cleavage and cytoskeletal disruption have been observed in enterovirus-induced cardiomyopathy, a specific acquired etiology of DCM (25).
Importantly, no putative mutation was detected in five patients. This suggests that undetected mutations may lie in auxiliary regulatory regions or deep within introns, or that genetic heterogeneity may play a role in XLCM. However, it is becoming increasingly clear that dystrophin plays a central role in the development of DCM.
☆ Dr. Sommer and Dr. Towbin contributed equally to the work. Supported, in part, by grants from the City of Hope National Medical Center (Dr. Sommer) and the National Institutes of Health, National Heart, Lung, and Blood Institute (R01HL62570; Dr. Towbin), the John Patrick Albright Foundation (Dr. Towbin), and the Texas Children’s Hospital Foundation Chair in Pediatric Molecular Cardiology Research (Dr. Towbin).
- Becker muscular dystrophy
- creatine kinase-all muscle isoform
- dilated cardiomyopathy
- Detection Of Virtually All Mutations-Single Strand Conformation Polymorphism
- Emery-Dreifuss muscular dystrophy
- polymerase chain reaction
- X-linked dilated cardiomyopathy
- Received January 11, 2002.
- Revision received June 10, 2002.
- Accepted June 24, 2002.
- American College of Cardiology Foundation
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