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(Circulation. 1997;95:2434-2440.)
© 1997 American Heart Association, Inc.

Articles

Evidence for a Dystrophin Missense Mutation as a Cause of X-Linked Dilated Cardiomyopathy

Rocio Ortiz-Lopez, MS; Hua Li, PhD; Jason Su, DO; Veronica Goytia, BS; Jeffrey A. Towbin, MD

the Departments of Pediatrics (R.O.-L., H.L., J.S., V.G., J.A.T.) and Molecular and Human Genetics (J.A.T.), Baylor College of Medicine, Houston, Tex.

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Background X-linked dilated cardiomyopathy (XLCM) has previously been shown to be due to mutations in the dystrophin gene, which is located at Xp21. Mutations in the 5' portion of the gene, including the muscle promoter, exon 1, and the exon 1–intron 1 splice site, have been reported previously. The purpose of this study was to analyze the originally described family with XLCM (and others) for dystrophin mutations.

Methods and Results Polymerase chain reaction (PCR) was used to amplify genomic DNA, and reverse-transcriptase PCR amplified cDNA from RNA obtained from heart and lymphoblastoid cell lines. Primers to the muscle promoter, brain promoter, and Purkinje cell promoter were designed, in addition to the exon 1 to exon 14 regions of dystrophin. Single-strand conformation polymorphism analysis was used for mutation detection, and DNA sequencing defined the mutation. Protein modeling was used for amino acid and secondary structure analysis. A missense mutation in exon 9 at nucleotide 1043 was identified that causes an alanine to be substituted for threonine, a highly conserved amino acid, at position 279 (T279A). This mutation results in a change in polarity in the evolutionarily conserved first hinge region (H1) of the protein and substitution of a ß-sheet for -helix in this portion of the protein, destabilizing the protein.

Conclusions A novel missense mutation in exon 9 of dystrophin causing an abnormality at H1 leads to the cardiospecific phenotype of XLCM.

Key Words: cardiomyopathy • genetics • dystrophin • mutation

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Dilated cardiomyopathy, the most common form of primary myocardial disease, is a major cause of morbidity and mortality and a leading indication for cardiac transplantation.^{1 2 3} Approximately 20% to 30% of all cases of DCM are inherited,^{4 5} with autosomal dominant transmission being most common; autosomal recessive, X-linked, and mitochondrial inheritance patterns have also been described.^{6 7}

XLCM is a severe and rapidly progressive myocardial disease that affects young men in their teens or early twenties.⁸ Typically, affected males present with severe congestive heart failure that results in death or cardiac transplantation within 1 to 2 years of diagnosis. Clinically, XLCM appears to be identical to other causes of DCM except for its X-linked transmission^{6 8 9} and elevated serum CK-MM without evidence of clinical skeletal myopathy. Female carriers with XLCM may manifest symptoms later in life, usually in the fifth decade, but the disease is mild and progresses slowly.⁸

Molecular genetic linkage to the 5' end of the dystrophin gene at Xp21 was previously reported by Towbin et al.⁹ In addition, N-terminal dystrophin antibody against cardiac tissue protein extracts showed decreased abundance (or absence) of dystrophin, whereas C-terminal antibody immunoblots were normal.⁹ Dystrophin antibody against the rod domain also demonstrated low abundance of protein.¹⁰ Immunoblots using N-terminal, C-terminal, and rod domain dystrophin antibodies against skeletal muscle protein demonstrated normal dystrophin, however.^{9 10}

The dystrophin gene, which causes DMD and the milder allelic form, BMD, is the largest gene identified in humans thus far, covering >2.5 Mb and having 79 exons.^{11 12} The corresponding 14-kb dystrophin mRNA is expressed predominantly in skeletal, cardiac, and smooth muscle, although lower levels also appear in brain. Transcription of dystrophin in different tissues is regulated from either the P_B,^{13 14 15} which is active predominantly in neuronal cells; the P_M,¹⁶ which is active in differentiated myogenic cells and glial cells; and the P_P,^{17 18} which is active in cerebellum. The resultant protein, dystrophin,^{12 19} is a 427-kD protein localized to the cytoplasmic face of the sarcolemma, colocalizing with ß-spectrin and vinculin at the sarcolemma.²⁰ The C-terminal region of dystrophin is bound to the protoplasmic half of the plasmalemma. Thus, dystrophin forms an intricate part of the muscle cytoskeleton and may function to link the normal contractile apparatus to the sarcolemma. In skeletal and cardiac muscle, the C-terminal domain is bound to a large oligomeric glycoprotein complex of six novel proteins localized to the sarcolemma^{21 22 23 24} ; the N-terminus attaches to actin.

In DMD, the levels of all members of the oligomeric glycoprotein complex are reduced.²⁵ In BMD, however, there is only a general mild reduction in these proteins.²⁶ It is believed that dystrophin localizes or stabilizes this oligomeric protein complex.²⁷ Without dystrophin, the complex is unable to organize properly, and consequently the linkage between the extracellular matrix and sarcolemma is disrupted.²⁸

Muntoni et al²⁹ reported a deletion within the P_M and E1 of dystrophin in a family with DCM, X-linked inheritance, and mild abnormalities of skeletal muscle histology and dystrophin immunohistochemical staining. They showed this mutation to be the cause of disease in this family and speculated that this deletion was the responsible mutation in all patients with XLCM. More recently, these authors^{30 31} also confirmed the dystrophin protein findings of Towbin et al.^{9 10} Holder et al³² described a mutation in the muscle promoter that potentially affects cardiac-specific regulatory sequences and therefore could selectively cause DCM without significant skeletal myopathy. This mutation is quite uncommon in BMD and DMD patients screened for dystrophin deletions,³³ however. Since previous reports of P_M-E1 mutations in other unrelated patients were usually associated only with mild skeletal muscle disease without cardiac disease,^{13 34} doubt arose that this mutation was the only mutation leading to XLCM. In fact, only the patient reported by Yoshida et al³⁵ with an E1 deletion and normal P_M had DCM. In addition, Towbin and Ortiz-Lopez³⁶ reported on three families with documented XLCM in whom P_M-E1 was normal. More recently, Milasin et al³⁷ reported a point mutation in the 5' splice site of dystrophin E1-I1 boundary causing XLCM clinically and abolishing expression of dystrophin in cardiac tissue. Therefore, XLCM appears to be due to various different mutations in the dystrophin gene. In this report, we describe a novel missense mutation in the 5' end of dystrophin in the originally described family with XLCM,⁸ and we also provide further data that suggest that there is allelic heterogeneity for XLCM, because two other families with XLCM studied here have none of the reported dystrophin mutations.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Families and Linkage Analysis
Clinical evaluation and linkage analysis of the three families (XLCM-1, XLCM-2, and XLCM-3) were previously described.^{8 9}

Control Individuals
One hundred unrelated and unaffected individuals (50 male, 50 female; 50 black and 50 white), as determined by history, physical examination, and echocardiography, were analyzed. After informed consent was obtained, blood for lymphoblastoid cell line immortalization³⁸ was obtained.

Procedures
RT-PCR. RNA was prepared from lymphoblastoid cell lines³⁸ or cardiac tissue as previously described.³⁹ Briefly, 2 µg of total RNA was reverse transcribed in a 20-µL reaction with 5 µg of random hexamer primers (Gibco-BRL) and 100 U murine Moloney leukemia virus RT at 37°C in the buffer provided by the supplier (Gibco-BRL). Of the RT reaction, 2 µL was used for each PCR reaction in 25 µL of 1.5 mmol/L MgCl₂, 67 mmol/L Tris-HCl (pH 8.8), 16.6 mmol/L ammonium sulfate, 0.01% Tween-20, 200 µmol/L of each dNTP, 25 pmol of each primer, and 1 U Taq DNA polymerase (Promega). PCR conditions included 94°C for 3 minutes, 30 cycles of 92°C for 1 minute, 58°C for 1 minute, 72°C for 1 minute, and a final extension of 72°C for 5 minutes. The PCR products were visualized on 2% agarose gel. The primers used for the amplification of the first 14 exons of dystrophin cDNA were derived from the reported dystrophin sequence¹¹ ; primers used for RT-PCR were exon 1F, 5'-TGGGAAGAAGTAGAGGACTGTTATG-3'; exon 5R, 5'-TTGACCTGCCAGTGGAGGAT-3'; exon 4F, 5'-GCA CTGCGGGTTTTGCAGAA-3'; exon 7R, 5'-GAATGCATTCCAGTCGTTGTGT-3'; exon 6F, 5'-TGAATGCTCTC ATCCAGTCATAG-3'; exon 10R, 5'-CTCTCCATCA ATGAACTGCC-3'; exon 10F, 5'-CATTGCAAGCACAAG GAGAG-3'; and exon 13R, 5'-CAGTTGCGTGATCTCCACTA GATTC-3'.

PCR amplification of genomic DNA. The primers used to amplify sequences of the P_B, P_M, P_P, and exon-intron junctions were designed with the previously reported sequences^{13 14 15 16 17} and conditions. The primers used for promoter amplification included the following: P_M, 5'-GAAGATCTAGACAGTGGAT ACATAACAAATGCATG-3'; E1_R, 5'-TTCTCCGAAG GTAATTGCCTCCCAGATCTGAGTCC-3'; P_BF, 5'-GAA GATCTATATTTTACAACGCAGAAATGTGG-3'; P_BR, 5'-CTTCCATGCCAGCTGTTTTTCCTGTCACTC-3'; P_pF, 5'-CAGCCTCCGCAGAATTTGAAATG-3'; and exon 2R, 5'-CTTAGAAAATTGTGCATTTACCCA-3'.

Multiplex PCR. Multiplex PCR was performed with the five primer pairs described by Beggs et al⁴⁰ and nine primer pairs described by Chamberlain et al⁴¹ ; an additional set of primers designed to amplify exon 9 (F, 5'-GAATTCCTCTCGCAGAT CACG-3'; R, 5'-GTAAATGTTGACAGACCTGTG-3') was also added to each. This multiplex PCR was performed under the following conditions: 94°C for 6 minutes, 94°C for 30 seconds, 54°C for 30 seconds, 65°C for 4 minutes (25 cycles), and 65°C for 7 minutes. Amplification products were visualized on a 3% agarose gel with ethidium bromide under ultraviolet light.

SSCP. Mutation analysis was performed by the method of Orita et al.⁴² PCR primers were designed to amplify a region including the 3' portion of intron 8 and the 3' end of exon 9. This region flanked the normal, polymorphic, or mutated sequence of exon 9 (Fig 1A, primers a through d). Radioactive PCR was performed with 100 ng genomic DNA in a 10-µL reaction containing 2.5 mmol/L MgCl₂, 10 pmol of each primer, 0.05 µCi [-³²P]dCTP, 0.5 U Taq polymerase, and 30 rounds of amplification (92°C for 45 seconds, 60°C for 45 seconds, and 72°C for 1 minute). After PCR amplification, the samples were denatured by addition of 5 µL formamide dye (95% formamide, 20 mmol/L EDTA, 0.05% bromophenol blue, and 0.05% xylene cyanol) at 85°C for 5 minutes. After 10 minutes of cooling on ice, 3 µL of sample was electrophoresed on a nondenaturing 10% polyacrylamide:bisacrylamide (50:1) gel at 8 W over 24 hours in a 4°C cold room. Bands were visualized by exposure of the dried gels to Kodak X-AR film.

View larger version (35K): [in this window] [in a new window]   Figure 1. A, PCR analysis of dystrophin exon 9. Top, Position of primers (a through d) relative to intron 8 and exon 9 and position of mutation (denoted by ) found in affected and carrier members of XLCM-1. Bottom, Sequences of primers a through d and expected PCR products. Primers a/d were used to PCR-amplify normal exon 9 sequences; primers a/c were used to amplify mutated exon 9 sequences. Primers a/d were used for SSCP analysis. B, PCR analysis of eight members of XLCM-1 family. XLCM-1 family members analyzed (top) and resultant PCR products (bottom) are shown. With primers (a/b) designed to amplify normal dystrophin exon 9 sequence (top band) and primers (a/c) designed to amplify mutated sequence (lower band), PCR products were electrophoresed on a 2% agarose gel. Note that affected males amplify only mutated sequences; carrier females are heterozygous. All members of this family were evaluated with both primer pairs (data not shown), with expected results (based on clinical status) occurring in all cases. Genomic DNA was used as template for this experiment.

Sequencing. Normal and aberrant SSCP conformers were cut directly from dried gels and eluted in 100 µL distilled water (65°C for 30 minutes), and the eluted DNA (10 µL) was used as the template in a second PCR using the original primer pair. The PCR products were sequenced directly after fractionation in 2% low-melting agarose gel (FMC Corp) and purified by Qiaquick columns (Qiagen). Purified PCR products (200 ng) were used for each sequencing reaction with cycle sequencing.⁴³ Alternatively, purified PCR products were cloned into pBluescript 11 SK(+) (Stratagene) by the T-vector method as described previously.⁴⁴ Manual sequencing was performed according to conditions suggested by the supplier (United States Biochemical), and automated sequences were performed on an ABI Automated Sequencer (ABI model 373).

Protein modeling. Dystrophin sequences were analyzed for species conservation by use of GenBank. Analysis of amino acid sequences was performed by the MOSAIC computer program.^{45 46} Hydrophobicity and plot structure predictions were performed according to the Garnier-Osguthorpe-Robson prediction method.⁴⁷

	Results

Top Abstract Introduction Methods Results Discussion References

 
Phenotypic Analysis of XLCM Families
To identify the disease-causing mutations in XLCM, three previously described multigenerational families with familial DCM, X-linked inheritance, and no clinical evidence of skeletal disease were studied.^{8 9 10} These families were not related, and all were of North American descent. Two of the families were black (XLCM-1, XLCM-3) and one family was white (XLCM-2). All three families were previously found to have linkage to the 5' portion of the dystrophin gene at Xp21, with maximal logarithm-of-the-odds scores at intron 7 within DXS206.^{9 10}

PCR Amplification of Promoter-Specific Transcripts
With oligonucleotide primers designed to amplify sequences of the P_B,¹³ P_M,^{16 29} P_P,¹⁷ and E1-I1^{11 29 37} from DNA and RNA isolated from lymphoblastoid cell lines and cardiac tissue, normal amplification of all promoters and E1-I1 was seen in all individuals. DNA sequencing revealed normal sequences of all amplified products.

5' Dystrophin Mutation Analysis
Primers designed to individually amplify exons 2 through 14 of dystrophin¹¹ were used to analyze the region with tightest linkage in XLCM-1, XLCM-2, and XLCM-3. In XLCM-2 and XLCM-3, all exons were able to be amplified, and the resultant amplimers were the predicted sizes. SSCP analysis was also normal (ie, no abnormal conformers) in XLCM-2 and XLCM-3, as were the sequences of all amplimers. In XLCM-1, however, exon 9 could not be amplified with normal sequence primers (ie, primers a/b). SSCP analysis of affected and carrier individuals from XLCM-1 demonstrated an abnormal conformer at exon 9 in all affected individuals (hemizygous); a heterozygous result (an abnormal conformer and a normal conformer) was found in all carrier females (Fig 2). Evaluation of exon 9 sequence (GenBank reference No. X14298) identified a mutation at nucleotide 1043, with substitution of AG only in affected and carrier individuals (Fig 3). The AG mutation changes the conserved amino acid threonine at amino acid position 279 to alanine (T279A). This amino acid, which is within the H1 of dystrophin,^{48 49} is conserved across species^{50 51 52 53 54} (Table). Oligonucleotide primers containing the mutated sequence within the primers (primers a/c, Fig 1A) were able to amplify a PCR product in affected and carrier XLCM-1 patients but not in unaffected individuals (Fig 1B), whereas PCR primers designed to the normal sequence (primers a/b, Fig 1A) were able to amplify the normal exon 9 sequence in unaffected individuals but were unable to amplify this portion of exon 9 in affected patients in XLCM-1 (Fig 1B). This selective amplification of the mutated sequence in affected individuals by use of mutation-specific primers occurred because of the use of stringent PCR conditions and sequence-specific oligonucleotides. Multiplex PCR^{40 41} using 6 primer pairs (P_M, exon 9, exon 13, exon 43, exon 50, exon 52) and 10 primer pairs (exon 4, exon 8, exon 9, exon 12, exon 17, exon 19, exon 44, exon 45, exon 48, exon 51) identified the mutation in all affected and carrier individuals from XLCM-1 at exon 9. Primers designed to flank the mutated region amplified appropriately in all individuals; sequence analysis in the affected patients in pedigree XLCM-1 identified this same mutation. Hence, the missense mutation segregates with disease status within family XLCM-1. No other individuals in XLCM-1, XLCM-2, or XLCM-3 had the AG mutation at nucleotide 1043; none of 100 unrelated control individuals analyzed had the mutation as seen by PCR, SSCP, and mutation analysis. However, a GA polymorphism in the third nucleotide of this codon was commonly detected at nucleotide 1045, consistent with codon redundancy (degenerate code). In fact, the GA polymorphism at 1045 was seen in 8% of individuals in the three families as well as in 8% of the 100 unrelated normal control patients analyzed.

View larger version (38K): [in this window] [in a new window]   Figure 2. SSCP analysis of dystrophin exon 9 in genomic DNA from normal male and female individuals and individuals from XLCM-1 (proband) and XLCM-3 (mother and proband) with primers a through d. In XLCM-1 (lane 3), note aberrant conformer (ie, "mutation") that differs from normal and polymorphic bands seen in normal individuals (lanes 1 and 2) and XLCM-3 patients (lanes 4 and 5). All XLCM-1 affected and carrier individuals were found to have "mutation"; none of 100 normal control subjects or XLCM-2 or XLCM-3 patients had this abnormal conformer.

View larger version (89K): [in this window] [in a new window]   Figure 3. Manual (A) and automated (B) sequencing of normal, polymorphic, and mutated conformers obtained by SSCP. Mutated sequence AG at nucleotide 1043, which occurred in proband and all affected and carrier individuals from XLCM-1, changes conserved amino acid threonine (at position 279) to alanine. Polymorphism seen in normal population and XLCM-2 and XLCM-3 patients is also shown.

View this table: [in this window] [in a new window]   Table 1.

Dystrophin Protein Analysis
Analysis of amino acid sequences was performed by the MOSAIC computer program^{45 46} ; prediction of -helix and ß-sheet was performed by a segment-oriented method designed to locate secondary structure elements. In addition, hydrophobicity and plot structure predictions were performed by use of the Garnier-Osguthorpe-Robson prediction.⁴⁷ The T279A amino acid substitution induces a change in the polarity in this critical region of the dystrophin protein because threonine is a neutral-polar amino acid and alanine is neutral-nonpolar. This, in turn, changes the secondary and tertiary structure of dystrophin by substituting a ß-sheet for -helix at the H1 between the N-terminal domain and rod domain of dystrophin^{49 55} (Fig 4).

View larger version (38K): [in this window] [in a new window]   Figure 4. Plot structure prediction of mutant dystrophin protein compared with normal dystrophin. A, Normal dystrophin protein and mutant dystrophin (inset). Note structural change between amino acids 250 and 300 (arrow), thus causing a change in secondary structure. B, Idealized dystrophin protein structure. Change in structure due to T279A mutation at H1 is shown. This change is predicted to functionally destabilize the protein.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
XLCM is the result of mutations within the dystrophin gene at the Xp21 locus. The missense mutation in dystrophin reported here in the originally described family with XLCM initially described by Berko and Swift⁸ is found within exon 9 (AG at position 1043) and is within a critical structural region (H1) of this cytoskeletal protein.^{19 47 48 49 55 56} The resultant amino acid substitution (threoninealanine) at amino acid 279 is within a highly conserved portion of the protein,^{50 51 52 53 54} causing a change in polarity in this part of the protein and leading to a change in the dystrophin secondary and tertiary structure. The end result is conversion of a segment of -helix to a ß-sheet in the H1 between the N-terminal domain and the rod domain of dystrophin⁴⁹ ; this abnormality could potentially destabilize the protein. This mutation was not found in the other two XLCM families (XLCM-2, XLCM-3) studied.

Dystrophin is predicted to fold into four domains,^{19 49 56} including the N-terminal domain, a long repeat domain (rod domain), a cysteine-rich domain, and a C-terminal domain. The N-terminal domain has significant similarity to the N-terminal domain of -actinin and ß-spectrin,^{57 58} possibly reflecting a common actin-binding function. The large midportion of the protein is formed by repeat elements predicted to adopt a triple-helical conformation,⁵⁹ and this region is quite similar to that seen in -actinin and spectrin as well. In dystrophin, this repeat domain is composed of 24 repeat units (109 amino acid repeats) and shows evolutionary conservation.⁶⁰ In addition, Koenig and Kunkel⁴⁹ showed that four hinge segments are interspersed along the dystrophin molecule, and these hinges appear to confer flexibility to the membrane-associated network of dystrophin, resulting in membrane resilience. The existence of these flexible hinges at precise positions within the elongated dystrophin molecule is thought to be important for the mechanical properties of the membrane cytoskeleton. It is possible that flexibility is needed during contraction-relaxation of the muscle fibers. These hinge regions are found at positions within the protein encoded by exon 9 and part of exon 8 (hinge 1, amino acid 253 to 327), exon 17 (hinge 2, amino acid 667 to 717), and exon 50 to 51 (hinge 3, amino acid 2424 to 2470); the hinge 4 sequence (amino acid 3041 to 3112) has not been definitively identified.⁴⁹ The mutation identified in family XLCM-1 is in exon 9, within the H1.

It appears that dystrophin performs a number of distinct cytoskeletal functions. Three related but conceptually distinct roles of dystrophin function have been suggested: (1) membrane stability (dystrophin may stabilize the membrane during repeated cycles of muscle contraction), (2) force transduction (dystrophin may link the contractile force produced in the intracellular domain to the extracellular environment), and (3) organization of membrane specializations. The biochemistry and function of the cardiac membrane and T tubules appear to be different from those of skeletal muscle. In the heart, this exon 9 mutation affecting the H1 portion of the protein is likely to result in loss of membrane integrity and eventual loss of function, probably due to the continual stress placed on the beating pump. In addition, this portion of the protein has been shown to have significant sequence similarity to troponin I⁶¹ in the region that functionally enables binding with calcium binding proteins. It has also been speculated that dystrophin binds to calmodulin in this region, thereby modulating interaction of dystrophin with actin or other proteins associated with the cytoskeleton. It is possible that disruption of this region of dystrophin leads to the development of dilated cardiomyopathy, in part because of the inability of the mutated protein to modulate this interaction. Conversely, skeletal muscle appears to maintain normal clinical function significantly longer in XLCM patients, with the only evidence of skeletal disease being elevated serum CK-MM.

The mutation identified in family XLCM-1 reported here differs from the P_M-E1 deletion mutation reported by Muntoni et al²⁹ and the E1-I1 splice site mutation reported by Milasin et al.³⁷ Because two other families with XLCM (XLCM-2, XLCM-3) that were also analyzed for the P_M mutation, E1-I1 boundary mutation, and exon 9 mutation have normal sequences in these portions of the dystrophin gene, one can speculate that the XLCM phenotype results from multiple different dystrophin mutations that are within important functional regions of this huge protein. This possibility provides a different mechanism for the development of differential dystrophinopathic phenotypes than does the "frame-shift" hypothesis,^{62 63} which is believed to explain the phenotypic differences between the allelic diseases DMD and BMD when deletions occur in >95% of cases.

Mutations and polymorphisms in exon 9 have rarely been reported.^{64 65 66 67 68} Roberts et al⁶⁵ analyzed 70 small mutations and Prior et al⁶⁶ reviewed 29 mutations, and in no case was exon 9 mutated. Reiss and Rininsland⁶⁴ described a constitutive exon 9 cassette-splicing phenomenon found in peripheral blood lymphocyte studies. These authors found that in approximately half of the transcripts of the dystrophin gene isolated from lymphocytes, exon 9 was omitted. This exon skipping was found to occur to a variable extent in all tissues not specifically expressing dystrophin but was rarely seen in muscle, heart, or brain. When seen, omission of exon 9 did not disrupt the reading frame of the mRNA. They also suggested that when exon 9 mutations occur, BMD results (as long as the correct reading frame is maintained), consistent with the observations of Koenig et al.⁶³

Once the mutations are identified in other families with XLCM (ie, XLCM-2 and XLCM-3), more insight will be gained as to the important cardiac functional regions of dystrophin. Animal models (such as transgenic mice) with these "cardiospecific mutations" could open doors to our knowledge of the function of the dystrophin protein as well as its relationship to other interrelated proteins.

	Selected Abbreviations and Acronyms

 

BMD = Becker muscular dystrophy C-terminal = carboxy-terminal CK-MM = creatine kinase muscle isoforms DCM = dilated cardiomyopathy DMD = Duchenne muscular dystrophy E1 = exon 1 H1 = first hinge region I1 = intron 1 N-terminal = amino-terminal PB = brain promoter PCR = polymerase chain reaction PM = muscle promoter PP = Purkinje-cell promoter RT = reverse-transcriptase SSCP = single-strand conformation polymorphism XLCM = X-linked dilated cardiomyopathy

	Acknowledgments

 
This work was supported in part by the National Institutes of Health (K08-HL-02485-01 and R01-HL-53392-01) and was performed in the Phoebe Willingham Muzzy Pediatric Molecular Cardiology Laboratory. The authors wish to acknowledge the contribution of Valerie R. Price in the preparation of this manuscript.

	Footnotes

 
Reprint requests to Jeffrey A. Towbin, MD, Pediatric Cardiology, Baylor College of Medicine, One Baylor Plaza, Room 333E, Houston, TX 77030.

Guest editor for this article was Christine Seidman, MD, Harvard Medical School, Boston, Mass.

Received June 3, 1996; revision received November 12, 1996; accepted December 13, 1996.

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