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Mutation analysis in emery-dreifuss muscular dystrophy
Mutation Analysis in Emery-Dreifuss Muscular Dystrophy Yoram Nevo, MD, Mohammed Al-Lozi, MD, Alexander Sh. Parsadanian, PhD, Jeffrey L. Elliott, MD, Anne M. Connolly, MD, and Alan Pestronk, MD The purpose of this study was to search for STA gene defects in three families with clinically typical EmeryDreifuss muscular dystrophy. Emery-Dreifuss is an X-linked muscular dystrophy with humeroperoneal weakness and life-threatening, but treatable, cardiac abnormalities in male patients and in female carriers. The defect is in the gene coding for emerin, a 254 amino acid protein of unknown function. Complementary and genomic DNA from T lymphocytes from the reported patients and their family members were amplified, cloned, and sequenced. A novel mutation, a 26 base-pair deletion in three brothers and a carrier mother, was detected in one family. A splicing mutation with one base pair insertion and a five base-pair deletion, which have been described previously, were found in the second and third families, respectively. The additional novel mutation detected and the findings of three different mutations in these three families support the idea of genetic heterogeneity of EmeryDreifuss muscular dystrophy with different mutations in different families. © 1999 by Elsevier Science Inc. All rights reserved. Nevo Y, Al-Lozi M, Parsadanian A-Sh, Elliott JL, Connolly AM, Pestronk A. Mutation analysis in EmeryDreifuss muscular dystrophy. Pediatr Neurol 1999;21: 456-459.
Introduction Emery-Dreifuss muscular dystrophy (EDMD) is a rare, childhood-onset X-linked recessive muscular dystrophy. It is characterized by early contractures of the elbows, achilles tendons, and the posterior cervical muscles, initial humeroperoneal distribution of weakness and muscle atrophy, and cardiac arrhythmias that may lead to sudden
From the Department of Neurology; Washington University School of Medicine; St. Louis, Missouri 63110.
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death [1-4]. Slowly progressive weakness is common [5]. The disease is distinguished clinically from Duchenne’s and Becker’s muscular dystrophy by the humeroperoneal distribution of weakness and the appearance of contractures before significant weakness. The cardiomyopathy presents as conduction defects leading to atrioventricular block [6,7], with syncope or sudden death in young affected males and in female carriers [8]. Early recognition of the syndrome is essential because a cardiac pacemaker may be lifesaving. In most families, EDMD is linked to the terminal region of the long arm of the X chromosome (Xq28) [9], although sporadic [10] autosomal-dominant [11] and autosomalrecessive [12] variants have been described. Bione et al. [13,14] identified nonsense mutations in the STA gene coding for emerin, a 254 amino acid, serine-rich protein. The specific defects in the STA gene have been heterogeneous and different in most families. Approximately 60 different mutations have been identified [13-17]. The authors studied the STA gene in three families having clinically typical EDMD.
Patients Family 1. The parents of the three affected males are third cousins. BBRI is an 18-year-old white male with slowly progressive humeroperoneal weakness that was first observed at 6 years of age when he had difficulties running while playing baseball. He had flexion contractures of his elbows, wrists, and ankles, extension contracture of his cervical spine, and diffusely reduced deep tendon reflexes. His creatine kinase (CK) was moderately elevated at 830 U/L (normal 5 0-220 U/L). Muscle biopsy revealed variation in fiber size, with small type I fibers but no muscle fiber necrosis or inflammation. At 17 years of age, he developed a junctional cardiac rhythm of 28-70 beats per minute, absent P waves, and atrial standstill, with normal junctional and ventricular conduction. A permanent pacemaker was placed in the right ventricle. BDAR is a 16-year-old male with a pattern of weakness and contractures and a course similar to that of his elder brother. Cardiologic
Communications should be addressed to: Dr. Nevo; Institute for Child Development and Pediatric Neurology Unit; Tel Aviv Sourasky Medical Center; 14 Balfour Street; Tel Aviv 65211, Israel. Received October 17, 1998; accepted February 8, 1999.
© 1999 by Elsevier Science Inc. All rights reserved. PII S0887-8994(99)00023-5 ● 0887-8994/99/$20.00
evaluation at 14 years of age revealed some junctional premature beats and a borderline dilated heart. BBRA is a 14-year-old male. He started having running difficulties and weakness of the upper extremities at 6 years of age. The weakness was slowly progressive and involved the lower extremities as well. Flexion contractures of the elbows were observed. No cardiac abnormalities have been found on evaluation. Family 2. FM is a 40-year-old white male with mild progressive weakness and elbow contractures initially evident in his teens. At 30 years of age, he developed atrial fibrillation and was treated with digoxin. Shortly thereafter, his heart rate slowed to 38 beats per minute, requiring a cardiac pacemaker. He remains independent in his activities of daily life. On examination, he had contractures of his elbows (flexion) to 45 degrees, his neck (extension), and his achilles tendons. The distribution of weakness was mainly humeroperoneal. His CK was 2,627 U/L. A muscle biopsy demonstrated variation in fiber size, small type I fibers, and occasional degenerating and regenerating muscle fibers. His brother, who had more severe weakness and contractures, died in his third decade. Family 3. JR is a 12-year-old male who presented with muscle weakness. He was born at term, and his neonatal course was unremarkable. His mother noticed that he had difficulty walking at 2-3 years of age (as if he “never outgrew” the clumsy stage of a toddler). He continued to have weakness in his arms and legs. He also developed contractures in his neck, elbows, and ankles. He had multiple surgeries for the ankle contractures. The patient is currently in the sixth grade and is doing well at school. JR’s 61-year-old maternal grandfather has bilateral elbow and ankle and posterior neck contractures. He had a cardiac pacemaker placed at 31 years of age. The maternal grandfather’s brother died at 40 years of age; his medical condition was not exactly known. JR’s mother is 31 years of age and healthy, and her brother is 34 years of age and unaffected. Motor examination was remarkable for severe contractures in the neck, elbows, and ankles. There was 4/5 strength in the biceps and triceps. Strength in the deltoids and the intermediate and distal muscles of the upper limbs were normal. Only mild weakness was detected in the distal leg muscles. Tendon reflexes were difficult to elicit because of the contractures. Gait was noteworthy for toe walking. His CK was 1,300 U/L. The electrocardiogram was normal, with no evidence for conduction block. A left quadriceps biopsy at 6 years of age demonstrated variation in fiber size, with prominent smallness of type I fibers. A mild increase in the endomysial connective tissue was evident. The biopsy was interpreted as consistent with muscular dystrophy. Staining for dystrophin was normal. Genetic study of the dystrophin gene revealed no mutations.
Methods Isolation of RNA. T cells were isolated from whole blood by Histopaque 1077 (Fisher Scientific, Pittsburgh, PA). The isolation of RNA was performed with the RNA STAT 30 kit (Tel-Test “B” Inc., Friendswood, TX) using the Chomczynski method [18]. RT-Polymerase Chain Reaction (PCR). Two mg of RNA were transcribed with 200 units Superscript ll Rtase (GibcoRBL, Grand Island, NY), and 7.5 ml of the RT-PCR products were amplified using the primers EM1 and EM2 (Table 1), as described by Bione et al. [13]. The PCR conditions were 94°C for 4 minutes followed by 40 cycles at 94°C for 1 minute, 62°C for 1.5 minutes, and 72°C for 2 minutes. The termination time at 72°C was 7 minutes. Isolation of Genomic DNA. Isolation of genomic DNA from T lymphocytes was performed by the QIAamp Blood Kit (Qiagen, Valencia, CA) for DNA purification from lymphocytes.
Table 1.
Primers
EM1-(91-109) 59-CGC CTG AGC CCG CAC CCG C EM2-(1895-1875) 59-CCC ACT GCT AAG GCA GTC AGC INT1-(640-623) 59-CTC TGG TAG AGT AAA GCG INT2-(957-974) 59-GAC TTC ATT CCC AGA TGC INT3-(1386-1379) 1 (993-984) 59-TCA TGC ACC TGG TGA TGG INT4-(1558-1539) 59-TAG TGC GTG ATG CTC TGG TA GEN1-(514-494) 59-TTC TCC AGT GCC GCT CTC GAC GEN2-(457-476) 59-GGG AGG ATG GGG TCG CGA GG GEN3-(1089-1069) 59-CCA CCA TTT GTA CCC AGT GCC
Genomic DNA PCR. One mg of genomic DNA was amplified with primers EM1-GEN1 and GEN2-GEN3 (Table 1). The PCR conditions were 95°C for 3 minutes followed by 35 cycles at 94°C for 45 seconds, 60°C for 30 seconds, and 72°C for 1.5 minutes. The termination time at 72°C was 10 minutes. Ligation, Transformation, and Sequencing. Both cDNA and genomic DNA products were ligated to PCR II plasmid and transformed into 5Ha Escherichia coli. Sequencing was performed with the Taq Dye Deoxy Terminator Cycle Sequencing Kit (Perkin Elmer/ABI, Norwalk, CT) under the conditions suggested by the supplier. Immunohistochemistry. Antibody E16 is a polyclonal antibody raised in New Zealand rabbits and purified by affinity chromatography. The E16 peptide (amino acids 173-188 of the emerin protein) was synthesized in the Washington University chemistry laboratory. The peptide was conjugated to Keyhole Limpet Hemocyanin with 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide as a linker (Pierce Kit #77102, Fisher Scientific). On day 0, 100 ml of the conjugate (prepared in complete Freud’s adjuvant) were injected subcutaneously. The animals were boosted on days 14 and 21 (the conjugate was prepared with incomplete Freud’s adjuvant). Antibodies in the animals’ serum were detected by dot blot methodology. The antibodies were purified from the serums by affinity chromatography (Pierce Kit #44895, Fisher Scientific). Immunohistochemistry was performed as previously described [19]. Antibodies were tested extensively and found to be specific for emerin. Results Family 1 Direct sequencing of the cDNA from the three brothers revealed a 26 base-pair deletion starting in genomic base pair 371(Fig 1A) with shifting of the reading frame and a stop codon after 6 amino acids. This result predicts a truncated 51 amino acid protein. Both normal and mutated cDNA were found in the asymptomatic heterozygote mother. The presence of the deletion was confirmed in the brothers’ and the mother’s genomic DNA. Family 2 In the patient FM an A to G substitution in genomic base pair 858 that caused a splicing mutation with one
Nevo et al: Mutations in Emery-Dreifuss Muscular Dystrophy 457
Figure 1. Chromatograms of a part of the sequencing gel demonstrating the mutations in Family 1 (A) and Family 2 (B). In figure A the arrow indicates the localization of the 26 base pairs deletion starting at base pair 371. In figure B the arrow points to an additional C in the reverse sequencing of the cDNA, indicating one base pair insertion (G) at the onset of exon 3.
base pair insertion at the onset of exon 3 was found (Fig 1B). This insertion resulted in shifting of the reading frame and a stop codon after three amino acids. The predicted protein is truncated, containing the first 92 amino acids of emerin. Family 3 A five base-pair deletion in exon 3 was detected in patient JR. In this patient the following sequence was found at base pairs 624-641 of the genomic emerin DNA: g c t t t a c c a g a g c a a g g g. This sequence can be produced by the following three different deletions: (1) deletion of t a c t c starting at base pair 628, (2) deletion of a c t c t starting at base pair 629, or (3) deletion of t c t a c starting at base pair 631. Immunohistochemistry Immunohistochemistry demonstrated an absence of emerin staining in all of the patients in the three families. Discussion Early diagnosis of EDMD is essential because of life-threatening, but potentially treatable, cardiac abnormalities in patients and carriers [8]. The diagnosis in this disease is usually clinical and based on early contractures, initial humeroperoneal distribution of weakness and atrophy, and the cardiac involvement [1-4]. Muscle histologic changes are often nonspecific [20]. However, recently, molecular biology and immunohistochemistry techniques enable specific diagnosis and genetic consultation for the X-linked EDMD. The EDMD gene STA is about 2,100 base pairs in size. It is organized in six small exons and five small introns
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[14]. It is a single copy gene [13] coding for a 254 amino acid protein named emerin. Although emerin is ubiquitously expressed [13] and the 59 region of the gene is rich in recognition sites for transcription factors usually found in promoters of housekeeping genes [14], the results of its absence are mainly confined to cardiac and skeletal muscles. Thus far, about 60 different mutations have been found [13-17,21-23]. The authors have detected an additional new mutation in Family 1, distinct from those previously described. In this family, all three males were affected, and the asymptomatic mother was identified as a carrier. The 26 base-pair deletion in genomic base pair 371 causes shifting of the reading frame and a stop codon after six amino acids, resulting in a truncated protein of 51 amino acids. In Family 2 the authors detected a previously described [14] mutation in a 40-year-old male whose brother died from a similar disease in his third decade. An A to G substitution in genomic base pair 858 was found. Bione et al. [13] has reported another A to G mutation in an adjacent location (genomic bp 857). In that previous patient the mutation (bp 857) abolished splicing, causing insertion of 214 intronic base pairs. In the mutation of the authors’ patient (genomic bp 858) the splicing site was shifted back one bp, which resulted in a one bp insertion, shifting of the reading frame, and a stop codon after three amino acids. In Family 3 the authors found a five bp deletion in exon 3 in a 12-year-old child. The sequence of the DNA of this patient can be produced by a five bp deletion starting at bp 628, 629, or 631 (see results). This mutation has not been published but is found in the EDMD database [17]. The different mutations found in Emery-Dreifuss muscular dystrophy have several implications. Emerin is a hydrophilic protein localized to the inner nuclear mem-
brane [21], with a hydrophobic domain near the C terminus, the function of which is currently unknown. The hydrophilic domain may serve as a membrane anchor, similar to that described for proteins of the secretory pathway involved in vesicular transport [13]. Most of the different mutations found thus far in this disease either abolish the synthesis of the protein entirely or result in a truncated protein. A mutation affecting only the last 27 amino acids of the protein [16] resulted in clinical disease. Truncation of emerin, which occurs in most mutations, probably interrupts its binding to the membrane and eliminates its activity [21]. If that is the case, theoretically there may be no correlation between the length or localization of most mutations (genotype) and clinical course or severity of the disease (phenotype), except for whether they do or do not disrupt the reading frame. The EDMD phenotype, however, has variable severity and inheritance [4,5]. It is commonly an X-linked recessive disorder, although other modes of inheritance have been described [10-12]. In most cases of the familial disease a mutation is detected in the STA gene coding for emerin. The many different private mutations indicate that there is no common founder in this disease. The new mutation in one of the reported families further supports these previous findings of genetic heterogeneity of EDMD. As additional mutations are found, specific vulnerable sites for mutations within the gene may be detected, but the mutations already described are widespread throughout the gene [17]. The finding of a mutation in a gene site previously reported for a large Georgia family [9,14] provides an opportunity to evaluate whether there is a common founder for this mutation or whether the identical deletion has occurred spontaneously in different families. The identification of the EDMD gene enables accurate and specific diagnosis of the disease by molecular biology techniques. However, the finding of numerous private mutations suggests that detection of the presence or absence of the emerin protein in tissues may be more practical as a diagnostic tool. Tissue-gene correlations will be needed to define the optimal tissues for study and whether partial protein expression confounds this diagnostic technique. The authors thank Sarit Vagenfeld, Dr. Ruth Shomrat, and Prof. Cyril Legum from the Genetic Institute, Tel-Aviv Sourasky Medical Center for their technical assistance with the third patient.
References [1] Dreifuss FE, Hogan GR. Survival in X-chromosomal muscular dystrophy. Neurology 1961;11:734-37. [2] Emery AE, Dreifuss FE. Unusual type of benign X-linked muscular dystrophy. J Neurol Neurosurg Psychiatry 1966;29:338-42.
[3] Rowland LP, Fetell M, Olarte M, Hays A, Singh N, Wanat FE. Emery-Dreifuss muscular dystrophy. Ann Neurol 1979;5:111-7. [4] Emery AE. X-linked muscular dystrophy with early contractures and cardiomyopathy (Emery-Dreifusstype). Clin Genet 1987;32:360-7. [5] Emery AE. Emery-Dreifuss syndrome. J Med Genet 1989;26: 637-41. [6] Emery AE. Emery-Dreifuss muscular dystrophy and other related disorders. Br Med Bull 1989;45:772-87. [7] Yoshioka M, Saida K, Itagaki Y, Kamiya T. Follow-up study of cardiac involvement in Emery-Dreifuss muscular dystrophy. Arch Dis Child 1989;64:713-5. [8] Fishbein MC, Siegel RJ, Thompson CE, Hopkins LC. Sudden death of a carrier of X-linked Emery-Dreifuss muscular dystrophy. Ann Intern Med 1993;119:900-5. [9] Consalez GG, Thomas NST, Stayton CL, Knight SJL, Johnson M, Hopkins LC, Harper PS, Elsas LJ, Warren ST. Assignment of Emery-Dreifuss muscular dystrophy to the distal region of Xq28: The results of a collaborative study. Am J Hum Genet 1991;48:468-80. [10] Rudenskaya GE, Ginter EK, Petrin AN, Djomina NA. EmeryDreifuss syndrome: Genetic and clinical varieties. Am J Med Genet 1994;50:228-33. [11] Miller RG, Layzer RB, Mellenthin MA, Golabi M, Francoz RA, Mall JC. Emery-Dreifuss muscular dystrophy with autosomaldominant transmission. Neurology 1985;35:1230-3. [12] Takamoto K, Hirose K, Uono M, Nonaka I. A genetic variant of Emery-Dreifuss disease. Muscular dystrophy with humeropelvic distribution, early joint contracture, and permanent atrial paralysis. Arch Neurol 1984;41:1292-3. [13] Bione S, Maestrini E, Rivella S, et al. Identification of a novel X-linked gene responsible for Emery-Dreifuss muscular dystrophy. Nat Genet 1994;8:323-7. [14] Bione S, Small K, Aksmanovic VMA, et al. Identification of new mutations in the Emery-Dreifuss muscular dystrophy gene and evidence for genetic heterogeneity of the disease. Hum Mol Genet 1995;4:1859-63. [15] Klauck SM, Wilgenbus P, Yates JRW, Muller CR, Poustka A. Identification of novel mutations in three families with Emery-Dreifuss muscular dystrophy. Hum Mol Genet 1995;4:1853-7. [16] Nigro V, Bruni P, Ciccodicola A, et al. SSCP detection of novel mutations in patients with Emery-Dreifuss muscular dystrophy: Definition of a small C-terminal region required for emerin function. Hum Mol Genet 1995;4:2003-4. [17] Yates J. Emery-Dreifuss muscular dystrophy mutation database, 1998. Available at ,http://www.path.cam.ac.uk/emd/.. [18] Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 1987;162:156-9. [19] Al-Lozi MT, Hemelt VB, Pestronk A. Steroid responsive myopathy with deficient chondroitin sulfate C in skeletal muscle connective tissue. Neurology 1998;50:526-9. [20] Voit T, Krogmann O, Lenard HG, Neuen-Jacob E. EmeryDreifuss muscular dystrophy: Disease spectrum and differential diagnosis. Neuropediatrics 1988;19:62-71. [21] Nagano A, Koga R, Ogawa M, et al. Emerin deficiency at the nuclear membrane in patients with Emery-Dreifuss muscular dystrophy. Nat Genet 1996;12:254-9. [22] Ichikawa Y, Watanabe M, Kowa H, et al. A Japanese family carrying a novel mutation in the Emery-Dreifuss muscular dystrophy. Ann Neurol 1997;41:399-402. [23] Small K, Iber J, Warren ST. Emerin deletion reveals a common X-chromosome inversion mediated by inverted repeats. Nat Genet 1997;16:96-100.
Nevo et al: Mutations in Emery-Dreifuss Muscular Dystrophy 459
Introduction Emery-Dreifuss muscular dystrophy (EDMD) is a rare, childhood-onset X-linked recessive muscular dystrophy. It is characterized by early contractures of the elbows, achilles tendons, and the posterior cervical muscles, initial humeroperoneal distribution of weakness and muscle atrophy, and cardiac arrhythmias that may lead to sudden
From the Department of Neurology; Washington University School of Medicine; St. Louis, Missouri 63110.
456
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death [1-4]. Slowly progressive weakness is common [5]. The disease is distinguished clinically from Duchenne’s and Becker’s muscular dystrophy by the humeroperoneal distribution of weakness and the appearance of contractures before significant weakness. The cardiomyopathy presents as conduction defects leading to atrioventricular block [6,7], with syncope or sudden death in young affected males and in female carriers [8]. Early recognition of the syndrome is essential because a cardiac pacemaker may be lifesaving. In most families, EDMD is linked to the terminal region of the long arm of the X chromosome (Xq28) [9], although sporadic [10] autosomal-dominant [11] and autosomalrecessive [12] variants have been described. Bione et al. [13,14] identified nonsense mutations in the STA gene coding for emerin, a 254 amino acid, serine-rich protein. The specific defects in the STA gene have been heterogeneous and different in most families. Approximately 60 different mutations have been identified [13-17]. The authors studied the STA gene in three families having clinically typical EDMD.
Patients Family 1. The parents of the three affected males are third cousins. BBRI is an 18-year-old white male with slowly progressive humeroperoneal weakness that was first observed at 6 years of age when he had difficulties running while playing baseball. He had flexion contractures of his elbows, wrists, and ankles, extension contracture of his cervical spine, and diffusely reduced deep tendon reflexes. His creatine kinase (CK) was moderately elevated at 830 U/L (normal 5 0-220 U/L). Muscle biopsy revealed variation in fiber size, with small type I fibers but no muscle fiber necrosis or inflammation. At 17 years of age, he developed a junctional cardiac rhythm of 28-70 beats per minute, absent P waves, and atrial standstill, with normal junctional and ventricular conduction. A permanent pacemaker was placed in the right ventricle. BDAR is a 16-year-old male with a pattern of weakness and contractures and a course similar to that of his elder brother. Cardiologic
Communications should be addressed to: Dr. Nevo; Institute for Child Development and Pediatric Neurology Unit; Tel Aviv Sourasky Medical Center; 14 Balfour Street; Tel Aviv 65211, Israel. Received October 17, 1998; accepted February 8, 1999.
© 1999 by Elsevier Science Inc. All rights reserved. PII S0887-8994(99)00023-5 ● 0887-8994/99/$20.00
evaluation at 14 years of age revealed some junctional premature beats and a borderline dilated heart. BBRA is a 14-year-old male. He started having running difficulties and weakness of the upper extremities at 6 years of age. The weakness was slowly progressive and involved the lower extremities as well. Flexion contractures of the elbows were observed. No cardiac abnormalities have been found on evaluation. Family 2. FM is a 40-year-old white male with mild progressive weakness and elbow contractures initially evident in his teens. At 30 years of age, he developed atrial fibrillation and was treated with digoxin. Shortly thereafter, his heart rate slowed to 38 beats per minute, requiring a cardiac pacemaker. He remains independent in his activities of daily life. On examination, he had contractures of his elbows (flexion) to 45 degrees, his neck (extension), and his achilles tendons. The distribution of weakness was mainly humeroperoneal. His CK was 2,627 U/L. A muscle biopsy demonstrated variation in fiber size, small type I fibers, and occasional degenerating and regenerating muscle fibers. His brother, who had more severe weakness and contractures, died in his third decade. Family 3. JR is a 12-year-old male who presented with muscle weakness. He was born at term, and his neonatal course was unremarkable. His mother noticed that he had difficulty walking at 2-3 years of age (as if he “never outgrew” the clumsy stage of a toddler). He continued to have weakness in his arms and legs. He also developed contractures in his neck, elbows, and ankles. He had multiple surgeries for the ankle contractures. The patient is currently in the sixth grade and is doing well at school. JR’s 61-year-old maternal grandfather has bilateral elbow and ankle and posterior neck contractures. He had a cardiac pacemaker placed at 31 years of age. The maternal grandfather’s brother died at 40 years of age; his medical condition was not exactly known. JR’s mother is 31 years of age and healthy, and her brother is 34 years of age and unaffected. Motor examination was remarkable for severe contractures in the neck, elbows, and ankles. There was 4/5 strength in the biceps and triceps. Strength in the deltoids and the intermediate and distal muscles of the upper limbs were normal. Only mild weakness was detected in the distal leg muscles. Tendon reflexes were difficult to elicit because of the contractures. Gait was noteworthy for toe walking. His CK was 1,300 U/L. The electrocardiogram was normal, with no evidence for conduction block. A left quadriceps biopsy at 6 years of age demonstrated variation in fiber size, with prominent smallness of type I fibers. A mild increase in the endomysial connective tissue was evident. The biopsy was interpreted as consistent with muscular dystrophy. Staining for dystrophin was normal. Genetic study of the dystrophin gene revealed no mutations.
Methods Isolation of RNA. T cells were isolated from whole blood by Histopaque 1077 (Fisher Scientific, Pittsburgh, PA). The isolation of RNA was performed with the RNA STAT 30 kit (Tel-Test “B” Inc., Friendswood, TX) using the Chomczynski method [18]. RT-Polymerase Chain Reaction (PCR). Two mg of RNA were transcribed with 200 units Superscript ll Rtase (GibcoRBL, Grand Island, NY), and 7.5 ml of the RT-PCR products were amplified using the primers EM1 and EM2 (Table 1), as described by Bione et al. [13]. The PCR conditions were 94°C for 4 minutes followed by 40 cycles at 94°C for 1 minute, 62°C for 1.5 minutes, and 72°C for 2 minutes. The termination time at 72°C was 7 minutes. Isolation of Genomic DNA. Isolation of genomic DNA from T lymphocytes was performed by the QIAamp Blood Kit (Qiagen, Valencia, CA) for DNA purification from lymphocytes.
Table 1.
Primers
EM1-(91-109) 59-CGC CTG AGC CCG CAC CCG C EM2-(1895-1875) 59-CCC ACT GCT AAG GCA GTC AGC INT1-(640-623) 59-CTC TGG TAG AGT AAA GCG INT2-(957-974) 59-GAC TTC ATT CCC AGA TGC INT3-(1386-1379) 1 (993-984) 59-TCA TGC ACC TGG TGA TGG INT4-(1558-1539) 59-TAG TGC GTG ATG CTC TGG TA GEN1-(514-494) 59-TTC TCC AGT GCC GCT CTC GAC GEN2-(457-476) 59-GGG AGG ATG GGG TCG CGA GG GEN3-(1089-1069) 59-CCA CCA TTT GTA CCC AGT GCC
Genomic DNA PCR. One mg of genomic DNA was amplified with primers EM1-GEN1 and GEN2-GEN3 (Table 1). The PCR conditions were 95°C for 3 minutes followed by 35 cycles at 94°C for 45 seconds, 60°C for 30 seconds, and 72°C for 1.5 minutes. The termination time at 72°C was 10 minutes. Ligation, Transformation, and Sequencing. Both cDNA and genomic DNA products were ligated to PCR II plasmid and transformed into 5Ha Escherichia coli. Sequencing was performed with the Taq Dye Deoxy Terminator Cycle Sequencing Kit (Perkin Elmer/ABI, Norwalk, CT) under the conditions suggested by the supplier. Immunohistochemistry. Antibody E16 is a polyclonal antibody raised in New Zealand rabbits and purified by affinity chromatography. The E16 peptide (amino acids 173-188 of the emerin protein) was synthesized in the Washington University chemistry laboratory. The peptide was conjugated to Keyhole Limpet Hemocyanin with 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide as a linker (Pierce Kit #77102, Fisher Scientific). On day 0, 100 ml of the conjugate (prepared in complete Freud’s adjuvant) were injected subcutaneously. The animals were boosted on days 14 and 21 (the conjugate was prepared with incomplete Freud’s adjuvant). Antibodies in the animals’ serum were detected by dot blot methodology. The antibodies were purified from the serums by affinity chromatography (Pierce Kit #44895, Fisher Scientific). Immunohistochemistry was performed as previously described [19]. Antibodies were tested extensively and found to be specific for emerin. Results Family 1 Direct sequencing of the cDNA from the three brothers revealed a 26 base-pair deletion starting in genomic base pair 371(Fig 1A) with shifting of the reading frame and a stop codon after 6 amino acids. This result predicts a truncated 51 amino acid protein. Both normal and mutated cDNA were found in the asymptomatic heterozygote mother. The presence of the deletion was confirmed in the brothers’ and the mother’s genomic DNA. Family 2 In the patient FM an A to G substitution in genomic base pair 858 that caused a splicing mutation with one
Nevo et al: Mutations in Emery-Dreifuss Muscular Dystrophy 457
Figure 1. Chromatograms of a part of the sequencing gel demonstrating the mutations in Family 1 (A) and Family 2 (B). In figure A the arrow indicates the localization of the 26 base pairs deletion starting at base pair 371. In figure B the arrow points to an additional C in the reverse sequencing of the cDNA, indicating one base pair insertion (G) at the onset of exon 3.
base pair insertion at the onset of exon 3 was found (Fig 1B). This insertion resulted in shifting of the reading frame and a stop codon after three amino acids. The predicted protein is truncated, containing the first 92 amino acids of emerin. Family 3 A five base-pair deletion in exon 3 was detected in patient JR. In this patient the following sequence was found at base pairs 624-641 of the genomic emerin DNA: g c t t t a c c a g a g c a a g g g. This sequence can be produced by the following three different deletions: (1) deletion of t a c t c starting at base pair 628, (2) deletion of a c t c t starting at base pair 629, or (3) deletion of t c t a c starting at base pair 631. Immunohistochemistry Immunohistochemistry demonstrated an absence of emerin staining in all of the patients in the three families. Discussion Early diagnosis of EDMD is essential because of life-threatening, but potentially treatable, cardiac abnormalities in patients and carriers [8]. The diagnosis in this disease is usually clinical and based on early contractures, initial humeroperoneal distribution of weakness and atrophy, and the cardiac involvement [1-4]. Muscle histologic changes are often nonspecific [20]. However, recently, molecular biology and immunohistochemistry techniques enable specific diagnosis and genetic consultation for the X-linked EDMD. The EDMD gene STA is about 2,100 base pairs in size. It is organized in six small exons and five small introns
458
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[14]. It is a single copy gene [13] coding for a 254 amino acid protein named emerin. Although emerin is ubiquitously expressed [13] and the 59 region of the gene is rich in recognition sites for transcription factors usually found in promoters of housekeeping genes [14], the results of its absence are mainly confined to cardiac and skeletal muscles. Thus far, about 60 different mutations have been found [13-17,21-23]. The authors have detected an additional new mutation in Family 1, distinct from those previously described. In this family, all three males were affected, and the asymptomatic mother was identified as a carrier. The 26 base-pair deletion in genomic base pair 371 causes shifting of the reading frame and a stop codon after six amino acids, resulting in a truncated protein of 51 amino acids. In Family 2 the authors detected a previously described [14] mutation in a 40-year-old male whose brother died from a similar disease in his third decade. An A to G substitution in genomic base pair 858 was found. Bione et al. [13] has reported another A to G mutation in an adjacent location (genomic bp 857). In that previous patient the mutation (bp 857) abolished splicing, causing insertion of 214 intronic base pairs. In the mutation of the authors’ patient (genomic bp 858) the splicing site was shifted back one bp, which resulted in a one bp insertion, shifting of the reading frame, and a stop codon after three amino acids. In Family 3 the authors found a five bp deletion in exon 3 in a 12-year-old child. The sequence of the DNA of this patient can be produced by a five bp deletion starting at bp 628, 629, or 631 (see results). This mutation has not been published but is found in the EDMD database [17]. The different mutations found in Emery-Dreifuss muscular dystrophy have several implications. Emerin is a hydrophilic protein localized to the inner nuclear mem-
brane [21], with a hydrophobic domain near the C terminus, the function of which is currently unknown. The hydrophilic domain may serve as a membrane anchor, similar to that described for proteins of the secretory pathway involved in vesicular transport [13]. Most of the different mutations found thus far in this disease either abolish the synthesis of the protein entirely or result in a truncated protein. A mutation affecting only the last 27 amino acids of the protein [16] resulted in clinical disease. Truncation of emerin, which occurs in most mutations, probably interrupts its binding to the membrane and eliminates its activity [21]. If that is the case, theoretically there may be no correlation between the length or localization of most mutations (genotype) and clinical course or severity of the disease (phenotype), except for whether they do or do not disrupt the reading frame. The EDMD phenotype, however, has variable severity and inheritance [4,5]. It is commonly an X-linked recessive disorder, although other modes of inheritance have been described [10-12]. In most cases of the familial disease a mutation is detected in the STA gene coding for emerin. The many different private mutations indicate that there is no common founder in this disease. The new mutation in one of the reported families further supports these previous findings of genetic heterogeneity of EDMD. As additional mutations are found, specific vulnerable sites for mutations within the gene may be detected, but the mutations already described are widespread throughout the gene [17]. The finding of a mutation in a gene site previously reported for a large Georgia family [9,14] provides an opportunity to evaluate whether there is a common founder for this mutation or whether the identical deletion has occurred spontaneously in different families. The identification of the EDMD gene enables accurate and specific diagnosis of the disease by molecular biology techniques. However, the finding of numerous private mutations suggests that detection of the presence or absence of the emerin protein in tissues may be more practical as a diagnostic tool. Tissue-gene correlations will be needed to define the optimal tissues for study and whether partial protein expression confounds this diagnostic technique. The authors thank Sarit Vagenfeld, Dr. Ruth Shomrat, and Prof. Cyril Legum from the Genetic Institute, Tel-Aviv Sourasky Medical Center for their technical assistance with the third patient.
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