Mutations in the human δ-sarcoglycan gene in familial and sporadic dilated cardiomyopathy Shinichi Tsubata, … , Neil E. Bowles, Jeffrey A. Towbin J Clin Invest. 2000;106(5):655-662. https://doi.org/10.1172/JCI9224. Article Dilated cardiomyopathy (DCM) is a major cause of morbidity and mortality. Two genes have been identified for the X- linked forms (dystrophin and tafazzin), whereas three other genes (actin, lamin A/C, and desmin) cause autosomal dominant DCM; seven other loci for autosomal dominant DCM have been mapped but the genes have not been identified. Hypothesizing that DCM is a disease of the cytoskeleton and sarcolemma, we have focused on candidate genes whose products are found in these structures. Here we report the screening of the human δ-sarcoglycan gene, a member of the dystrophin-associated protein complex, by single-stranded DNA conformation polymorphism analysis and by DNA sequencing in patients with DCM. Mutations affecting the secondary structure were identified in one family and two sporadic cases, whereas immunofluorescence analysis of myocardium from one of these patients demonstrated significant reduction in δ-sarcoglycan staining. No skeletal muscle disease occurred in any of these patients. These data suggest that δ-sarcoglycan is a disease-causing gene responsible for familial and idiopathic DCM and lend support to our “final common pathway” hypothesis that DCM is a cytoskeletalopathy. Find the latest version: https://jci.me/9224/pdf Mutations in the human δ-sarcoglycan gene in familial and sporadic dilated cardiomyopathy Shinichi Tsubata,1,2 Karla R. Bowles,3 Matteo Vatta,2 Carmelann Zintz,2 Jack Titus,4 Linda Muhonen,5 Neil E. Bowles,2 and Jeffrey A. Towbin2,3,6 1Department of Pediatrics, Toyama Medical and Pharmaceutical University, Toyama, Japan 2Department of Pediatrics, Section of Cardiology, and 3Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas, USA 4The Jesse Edwards Cardiac Registry, Minneapolis, Minnesota, USA 5Department of Pediatrics, Section of Cardiology, Orange County Children’s Hospital, Orange, California, USA 6Department of Cardiovascular Sciences, Baylor College of Medicine, Houston, Texas, USA Address correspondence to: Jeffrey A. Towbin, Department of Pediatrics, Section of Cardiology, Baylor College of Medicine, One Baylor Plaza, Room 333E, Houston, Texas 77030, USA. Phone: (713) 798-7342; Fax: (713) 798-8085; E-mail: [email protected]. Received for publication December 21, 1999, and accepted in revised form July 25, 2000. Dilated cardiomyopathy (DCM) is a major cause of morbidity and mortality. Two genes have been identified for the X-linked forms (dystrophin and tafazzin), whereas three other genes (actin, lamin A/C, and desmin) cause autosomal dominant DCM; seven other loci for autosomal dominant DCM have been mapped but the genes have not been identified. Hypothesizing that DCM is a disease of the cytoskeleton and sarcolemma, we have focused on candidate genes whose products are found in these structures. Here we report the screening of the human δ-sarcoglycan gene, a member of the dystrophin- associated protein complex, by single-stranded DNA conformation polymorphism analysis and by DNA sequencing in patients with DCM. Mutations affecting the secondary structure were identified in one family and two sporadic cases, whereas immunofluorescence analysis of myocardium from one of these patients demonstrated significant reduction in δ-sarcoglycan staining. No skeletal mus- cle disease occurred in any of these patients. These data suggest that δ-sarcoglycan is a disease-causing gene responsible for familial and idiopathic DCM and lend support to our “final common pathway” hypothesis that DCM is a cytoskeletalopathy. J. Clin. Invest. 106:655–662 (2000). Introduction syndrome) (11, 12) and isolated left ventricular non- Dilated cardiomyopathy (DCM) is a primary heart compaction (13, 14), and dystrophin in later-onset X- muscle disease characterized by left ventricular dilation linked cardiomyopathy (XLCM) (15–17). In the more and systolic dysfunction, with secondary diastolic dys- common autosomal dominant DCM, five loci have been function and occasionally associated right ventricular mapped for pure DCM (1q32 [ref. 18], 2q31 [ref. 19], disease (1). This disorder has an incidence of 9q13-q22 [ref. 20], 10q21-q23 [ref. 21], and 15q14 [ref. 3.5–8.5/100,000 population per year and a prevalence 22]), and four loci have been mapped in families with of approximately 36/100,000 population (2, 3), which DCM and associated with conduction disease (1p1-1q21 appears to be on the rise. In addition, DCM is the most [ref. 23], 2q14-q22 [ref. 24], 2q35 [ref. 25], 3p25-p22 [ref. common reason for heart failure and for cardiac trans- 26], and 6q23 [ref. 27]). Thus far, only the gene on chro- plantation in the US, with an estimated cost of $10 bil- mosome 15q14 encoding cardiac actin (22), the gene on lion to $40 billion yearly (4). chromosome 2q35 encoding desmin (25), and the gene The underlying causes of DCM are heterogeneous (5, on chromosome 1p1-1q21 encoding lamin A/C (28) have 6), including myocarditis, drug toxicity (adriamycin), been identified and mutations characterized. and ischemia-induced, metabolic, mitochondrial, and We have proposed a “final common pathway” genetic abnormalities. A genetic cause of DCM is identi- hypothesis (29, 30), which states that hereditary car- fied in approximately 30% of cases (7–9), with autosomal diovascular diseases with similar phenotypes and dominant inheritance being the most common (6). X- genetic heterogeneity will occur due to abnormalities linked, autosomal recessive, and mitochondrial inheri- in genes encoding proteins of similar function or genes tance have also been reported, albeit less frequently (10). encoding proteins participating in a common pathway In the past several years, the genetic basis of DCM has cascade. In the case of DCM, we have proposed that been sought, resulting in the identification of multiple mutations affect elements of the cell cytoarchitecture, genetic loci and five genes causing DCM to date. For X- based around the cytoskeleton (i.e., a cytoskeletalopa- linked DCM, two genes have been identified, including thy) and sarcolemma, but also including elements that tafazzin (G4.5) in cases of the infantile-onset DCM (Barth interact with the cytoskeleton including dystrophin, The Journal of Clinical Investigation | September 2000 | Volume 106 | Number 5 655 the dystrophin-associated glycoprotein complex (DAG; exons containing the coding region. PCR was per- i.e., the sarcoglycans and dystroglycans), and interme- formed in a 10-µL reaction containing 1.5 mM MgCl2, diate filaments. Other final common pathways for spe- 10 pmol of each primer, 0.5 U Taq DNA polymerase cific cardiac phenotypes include the sarcomere in (Life Technologies Inc., Rockville, Maryland, USA), hypertrophic cardiomyopathy (31) and ion channels in using a Stratagene Robocyler (La Jolla, California, cardiac rhythm disorders, such as long QT syndromes USA). After a 5-minute denaturation step at 94°C, 35 and Brugada syndrome (32). rounds of amplification (94°C for 30 seconds, Animal models of cardiomyopathy have also been 54–60°C for 30 seconds, and 72°C for 20 seconds) described, including the BIO14.6 hypertrophic car- were performed. This was followed by a 72°C incuba- diomyopathy and the TO-2 DCM hamsters (33, 34), tion for 2 minutes. which are both due to mutations in the δ-sarcoglycan The exons of δ-sarcoglycan were subcloned into the gene (35–37). These animals also exhibit histological vector pZero (Invitrogen, Carlsbad, California, USA). features of muscular dystrophy (38). Mutations in Clones were identified by whole cell PCR (20 µL reac- this gene in humans cause autosomal recessive limb- tion) and sequenced using an ABI373 (Perkin-Elmer girdle muscular dystrophy, LGMD2F (39), a disorder Applied Biosystems, Foster City, California, USA) and in which skeletal muscle disease can vary from mild Big Dye Terminator chemistry, according to the man- (i.e., ambulatory beyond 15 years, similar to Becker ufacturer’s instructions. muscular dystrophy [BMD]) to severe (i.e., similar to Single-strand conformational polymorphism analysis. Duchenne muscular dystrophy [DMD] with early Mutation analysis was performed using a modifica- requirement of a wheelchair), and cardiac involve- tion of the method of Orita et al. (42). PCR primers ment has been documented in some patients (40). In were designed to amplify across the entire human δ- addition, other LGMD2 disorders (LGMD2C, 2D, 2E) sarcoglycan gene in an exon-by-exon manner using the are caused by sarcoglycan mutations (γ-, α-, and β- primers in Table 1. Radioactive PCR was performed sarcoglycan, respectively), suggesting that some with 100 ng genomic DNA in a 10 µL reaction con- LGMD phenotypes are sarcoglycanopathies. LGMDs taining 1.5 mM MgCl2, 10 pmol of each primer, 0.05 may also include cardiac involvement associated with µCi [α-32P] dCTP (Amersham Life Sciences Inc., skeletal myopathy (41). Arlington Heights, Illinois, USA), 0.5 U Taq DNA poly- For these reasons, we have begun to analyze merase, and 30 rounds of amplification (92°C for 30 patients with DCM for mutations in genes encoding seconds, Y°C for 30 seconds, 72°C for 30 seconds), these proteins. In this report, we describe the identi- where Y represents the annealing temperature shown fication of mutations in the δ-sarcoglycan gene in in Table 1. After PCR amplification, the samples were familial and sporadic cases of pure DCM. This denatured and then electrophoresed for 24 hours in a extends the spectrum of phenotypes seen with sarco- denaturing 10% polyacrylamide:bisacrylamide (50:1; glycanopathies, now adding pure DCM to the poten- Bio-Rad Laboratories Inc., Hercules, California, USA) tial clinical presentation. gel with and without 12% glycerol, at 8 W in a 4°C cold room. Bands were visualized by exposure of the dried Methods gels to Kodak BioMax MS-1 film (Eastman Kodak Co., Patient evaluation. All patients were evaluated by physi- Rochester, New York, USA).