American Journal of Medical Genetics 80:99–102 (1998) Brief Clinical Report Double Missense Mutation in Exon 41 of the Human Dystrophin Gene Detected by Double Strand Conformation Analysis Fawzy A. Saad,1,2* Luciano Merlini,3 Maria Luisa Mostacciuolo,1 and Gian Antonio Danieli1 1 Department of Biology, University of Padua, Padua, Italy Department of Genetics, University of Tanta, Kafr El Sheikh, Egypt 3 Neuromuscular Laboratory, Rizzoli Institute, University of Bologna, Bologna, Italy 2 Development of late-onset Becker muscular dystrophy is reported in a patient whose two healthy brothers showed high serum creatine kinase level. No cases of neuromuscular disorders had been previously reported in this family. The analysis of the dystrophin gene showed that the three brothers had A → C transversion at nucleotide 6092 in exon 41, a missense mutation which converts lysine into glutamine. The symptomatic patient showed an additional mutation in the same exon, a T → C transition at nucleotide 6119, converting a phenylalanine to leucine. The possible pathogenic role of this mutation is discussed. Am. J. Med. Genet. 80:99–102, 1998. © 1998 Wiley-Liss, Inc. KEY WORDS: dystrophin; double mutation; missense; DSCA INTRODUCTION Screening for point mutations of the dystrophin gene is widely used for Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) patients who show no intragenic alteration of the gene. However, the occurrence of dystrophinopathies with variant clinical expression [Gospe et al., 1989; Bushby et al., 1991; Servidei et al., 1993; Heald et al., 1994; Palmucci et al., 1994] suggests that screening of all Contract grant sponsor: Telethon; Contract grant sponsor: Italian Ministry of Foreign Affairs. *Current address of F.A.S. is Department of Medicine, University of Toronto, 67 College Street, Toronto, Ontario M5G 2M1 Canada. E-mail: firstname.lastname@example.org *Correspondence to: Fawzy A. Saad, Ph.D., University of Padua, Via Trieste 75, I-35121 Padova, Italy. Received 5 October 1996; Accepted 21 July 1998 © 1998 Wiley-Liss, Inc. dystrophinopathies for dystrophin gene point mutations may be of considerable clinical value. The present report deals with a patient manifesting late-onset Becker muscular dystrophy, in whom mutation screening was motivated by detection of unusually high serum creatine kinase (CK) levels in the patient’s two apparently healthy brothers. CLINICAL REPORT A 62-year-old Italian man was admitted with marked muscle impairment of limb girdle motor function. Lower limb weakness began at age 55. He experienced increasing difficulty in running, raising himself from the floor, and climbing stairs. Six years later he was admitted to the local hospital and found to have an abnormally high serum CK level. The patient was treated with steroids for a few months, without any noticeable improvement of his clinical conditions. Therefore, at age 62 the patient was referred to a specialized center for further investigation. On examination, he was slender without calf hypertrophy. Manual muscle testing demonstrated very mild weakness of the deltoids (MRC 5−), overt involvement of the biceps (MRC 4−), and of the finger extensors (MRC 3−). Pelvic girdle muscles were also involved (MRC 4−), while quadricep and abdominal muscle strength was graded at MRC 4. The quadriceps peak torque values, recorded at 30 degrees/sec [Merlini et al., 1992], were 65 Nm on the right and 90 Nm on the left (normal mean value, 200 Nm). The hamstring peak torque values were 68 and 78 Nm respectively (normal mean value, 119 Nm). Achilles tendon reflexes were absent. Muscle ultrasound imaging showed strikingly diffuse echo increase in the quadriceps. Electromyography (EMG) demonstrated a myopathic pattern in the biceps with many brief (and occasionally polyphasic) motor unit potentials. Muscle computed tomographic scan showed mild and diffuse muscle hypotrophy with focal rounded areas of low density in the thigh muscles and in the glutei. 100 Saad et al. The two brothers of the patient reported no history of muscle weakness and were able to climb stairs, stand from floor, and rise from a chair without any difficulty. On clinical examination, manual muscle test was normal. Muscle ultrasound study of the quadriceps showed a normal muscle and bone echo; and serum CK level was consistently elevated (2 to 3× normal). Serum CK level of the four sisters was normal (Fig. 1). No cases of neuromuscular disorders had been previously reported in this family. METHODS Leukocyte genomic DNA from 50 DMD and 28 BMD patients in which no intragenic alterations were detected, and from 20 DNAs from subjects showing high serum CK level, were extracted using a salting out procedure [Miller et al., 1988] or DNA microextraction. To this end, 25 l of whole blood was added to 100 l of NaCl (0.75%), spun for 1 min at 12,000 rpm, and the leukocyte pellets resuspended in 15 l of distilled water and incubated at 94°C for 3 min. Clear supernatants containing DNA were then transferred to a new Eppendorf tube. Screening for point mutations was performed by Double Strand Conformation Analysis (DSCA) [Saad et al., 1994]. Briefly, leukocyte genomic DNA was amplified by polymerase chain reaction (PCR), a fraction of PCR products loaded onto vertical slab polyacrylamide gel, and then gels were silver stained according to procedures described elsewhere [Saad et al., 1997]. For each amplicon showing electrophoretic mobility shift, the products of five independent PCR amplifications were pooled and an aliquot of this mixture was used for cloning the sequence in pCRII vector (Invitrogen). The insert orientation deduction and generation of biotinylated PCR products were as described [Saad et al., 1997]. The biotinylated PCR products from five independent colonies were pooled and directly immobilized onto streptavidin-coated paramagnetic beads. After denaturation of the double-stranded DNA bound to the beads, and elution of the nonbiotinylated strand, single-stranded DNA for sequencing was obtained. The immobilized strand was solid phase DNA sequenced using the forward primer specific for the inserted sequence, whereas the nonbiotinylated strand was DNA sequenced by using the reverse primer specific for the inserted sequence. DNA sequencing was also performed on PCR products from genomic DNA by T7 DNA polymerase (USA Biolabs). All family members and 100 healthy male subjects were tested for both point mutations by Allele Specific Oligonucleotides (ASO). For haplotype identification, Fig. 1. The pedigree of the family. Serum CK values are indicated for each individual. five different polymorphic markers were used: PERT87.8 TaqI, PERT87.15 XmnI, STR44, STR50, and 3⬘CA repeat according to the methods described elsewhere [Mostacciuolo et al., 1994; Clemens et al., 1991]. RESULTS DSCA screening for point mutations detected a mobility shift of exon 41 in the propositus and his two brothers, but no in other individuals among the 98 subjects under study (Fig. 2). Sequence analysis of exon 41 amplified segments from the three brothers revealed the mobility shift to reflect an A → C transversion at nucleotide 6092 (lysine substitution to glutamine). The myopathic patient showed an additional T → C transition at nucleotide 6119, which converts a phenylalanine to a leucine residue (Fig. 3). These mutations were not detected in any of the other 98 subjects under study, nor in 100 healthy male subjects, an observation which essentially precludes the presence of a polymorphism at each of the substituted nucleotides. The three brothers shared the same haplotype, as demonstrated by the presence of the same alleles at each of the polymorphic markers mapping within the dystrophin gene. However, none of the sisters was found to be a carrier of the mutations. DISCUSSION An A → C transversion of nucleotide 6092, which converts lysine to glutamine, was detected in the myopathic patient studied here and in his two healthy brothers, who showed only high serum CK level. Fig. 2. Electrophoresis separation under nondenaturing 8% polyacrylamide gel of some exons of dystrophin gene. Lanes A and C: controls. Lane B: mutations A6092C and T6119C in exon 41. Dystrophin Double Missense Mutation Fig. 3. Partial nucleotide sequence analysis of exon 41 of dystrophin gene for mutations A6092C and T6119C. Among the missense mutations of dystrophin reported in the literature [Prior et al., 1993, 1994; Lenk et al., 1993; Saad et al., 1994], only one was associated with high serum CK level: A mutation in a codon 773 of exon 19 was described associated with abnormally high serum CK level and a 25% reduced mean duration of the motor unit potentials at the EMG in an 8-year-oldboy [Saad et al., 1994]. However, the age of the patient made it impossible to draw any conclusion about the future development of his condition. To date, only one mutation of exon 41 has been described in the literature, this being a C → T transition at nucleotide 6107 in two DMD patients [Saad et al., 1993; Nigro et al., 1994]. This mutation converts an arginine codon at position 1967 to a stop codon. In the myopathic patient described in the present report, an additional mutation in exon 41 (a T → C transition in nucleotide 6119, converting phenylalanine to leucine) was discovered. However, the finding of two different mutations in the same exon is not exceptional [Rodrigues et al., 1987; Savov et al., 1995], especially in the dystrophin gene [Lenk et al., 1993; Kilimann et al., 1992; Winnard et al., 1992]. It is impossible to establish if the mutation at nucleotide 6119 is itself sufficient to induce the shift from high serum CK level without clinical signs to myopathy, since the presence of additional mutation(s) in different exons, not considered by the screening, cannot be excluded. However, the fact that two brothers, 55 and 69 years old, showed only high serum CK level, with no clinical and muscle ultrasound signs of myopathy, whereas the patient showed an overt muscular impairment, is highly suggestive of a difference in the origin of the two conditions. On the other hand, the possibility that high serum CK level is due to the effect of a different gene cannot be ruled out. Idiopathic high serum CK level has been reported as a genetic-clinical entity. This conditions may be inherited as an autosomal dominant trait (OMIM 123320), but is genetically very heterogeneous. Exacerbation of the clinical condition of a DMD case 101 due to the inheritance of a paternal gene causing high serum CK level has also been previously reported [Frydman et al., 1995]. The two mutations detected in the case reported here produced nonconservative modifications of the protein structure: One exchanged a basic amino acid for an amide and the other substituted an aromatic amino acid for an aliphatic. The induced conformational change could result in local disruption of the threedimensional structure of the protein and in a consequent functional alteration. Moreover, it must be considered that the first mutation (shared by the three brothers) is localized in the 3⬘ end of the repeat 15, whereas the second mutation (detected only in the patient DNA) falls in a segment, 18 amino acids long, placed between repeats 15 and 16 [Koenig and Kunkel, 1990]. Since this segment corresponds to a unique sequence placed within a series of several repeats, its functional relevance is highly probable. Therefore, the hypothesis that the late-onset myopathy observed in the patient might be due to a double-mutant 41 exon should be considered seriously. Double mutations of the dystrophin gene may be more common than has been anticipated. The occurrence of two independent mutations in the same exon or in different exons could account for variability in clinical phenotype [Kilimann et al., 1992]. This situation might apply in particular to intrafamilial variability, seldom reported in BMD [Jackson et al., 1974; Hausmanova-Petrusewicz and Borkowska, 1974; Mostacciuolo et al., 1987]. ACKNOWLEDGMENTS The financial support of Telethon, the comments of M. Miorin, the technical assistance of U. Arezzini and C. Friso, and the critical reading of the manuscript by Dr. K. Siminovitch (Samuel Lunenfeld Research Institute, Mount Sinai Hospital, University of Toronto, Toronto, Canada) are gratefully acknowledged. Dr. F.A. Saad is a research fellow of the Italian Ministry of Foreign Affairs. REFERENCES Bushby KMD, Cleghorn NJ, Curtis A, Haggerty ID, Nicholson LVB, Johnson MA, Harris JB, Bhattacharya SS (1991): Identification of a mutation in the promoter region of the dystrophin gene in a patient with atypical Becker muscular dystrophy. Hum Genet 88:195–199. Clemens PR, Fenwick RG, Chamberlain JS, Gibbs RA, de Andrade M, Chakraborty R, Caskey CT (1991): Carrier detection and prenatal diagnosis in Duchenne and Becker Muscular Dystrophy families using dinucleotide repeat polymorphisms. Am J Hum Genet 49:951–960. Frydman M, Straussberg R, Shomart R, Goebel H, Legum C, Shiloh Y (1995): Duchenne muscular dystrophy and idiopathic hyperCKemia segregating in a family. Am J Med Genet 58:209–212. Gospe SM, Lazaro RP, Lava NS, Grootsholten PM, Scott MO, Fischbeck KH (1989): Familial X-linked myalgia and cramps: A non-progressive myopathy associated with a deletion in the dystrophin gene. Neurology 39:1277–1280. Hausmanova-Petrusewicz I, Borkowska J (1974): Intrafamilial variability of X-liked progressive muscular dystrophy. J Neurol 218:43–50. Heald A, Anderson LVB, Bushby KMD, Shaw PJ (1994): Becker muscular dystrophy with onset after 60 years. Neurology 44:2388–2390. Jackson RC, Taylor BD, Zellweger H, Bianchine JW (1974): Muscular dys- 102 Saad et al. trophy: Duchenne type and Becker type within a kindred. Am J Hum Genet 26:44A. Western LM, Mendell RA (1993): Missense mutation in the dystrophin gene in a Duchenne muscular dystrophy patient. Nat Genet 4:357–360. Kilimann MW, Pizzuti A, Grompe M, Caskey CT (1992): Point mutations and polymorphisms in the human dystrophin gene identified in genomic DNA sequences amplified by multiplex PCR. Hum Genet 89:253– 258. Prior TW, Bartolo C, Papp AC, Snyder PJ, Sedra MS, Burghes AHM, Mendell JR (1994): Identification of a missense mutation, single base deletion and a polymorphism in the dystrophin exon 16. Hum Mol Genet 3:1173–1174. Koenig M, Kunkel LM (1990): Detailed analysis of the repeat domain of dystrophin reveals four potential hinge segments that may confer flexibility. J Biol Chem 265:4560–4566. Rodrigues NR, Dunham I, Yu CY, Carroll MC, Porter RR, Campbell RD (1987): Molecular characterization of the HLA-linked steroid 21hydroxylase B gene from individual with congenital adrenal hyperplasia. EMBO J 6:1653–1661. Lenk U, Hanke R, Thiele H, Speer A (1993): Point mutations at the carboxy terminus of the dystrophin gene: Implications for an association with mental retardation in DMD patients. Hum Mol Genet 2:1877–1881. Merlini L, Dell’Accio D, Granata C (1992): Isokinetic muscle testing (IMT) in neuromuscular diseases: Preliminary report. Neuromuscul Disord 2:210–207. Miller SA, Dykes DD, Polesky HF (1988): A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res 16:1215. Mostacciuolo ML, Lombardi A, Cambissa V, Danieli GA, Angelini C (1987): Population data on benign and severe forms of X-linked muscular dystrophy. Hum Genet 75:217–220. Mostacciuolo ML, Miorin M, Vitiello L, Rampazzo A, Fanin M, Angelini C, Danieli GA (1994): Occurrence of two different intragenic deletions in two male relatives affected with Duchenne Muscular Dystrophy. Am J Med Genet 50:84–86. Nigro V, Nigro G, Esposito MG, Comi LI, Molinari AM, Puca GA, Politano L (1994): Novel small mutations along the DMD/BMD gene associated with different phenotypes. Hum Mol Genet 3:1907–1908. Palmucci L, Doriguzzi C, Mongini T, Restagno C, Chiado-Piat L, Maniscalco M (1994): Unusual expression and very mild course of Xp21 muscular dystrophy (Becker type) in a 60 years old man with 26 percent deletion of the dystrophin gene. Neurology 44:541–543. Prior TW, Papp AC, Snyder PJ, Burghes AHM, Bartolo C, Sedra MS, Saad FA, Vita G, Mora M, Morandi L, Vitiello L, Oliviero S, Danieli GA (1993): A novel nonsense mutation in the human dystrophin gene. Hum Mutat 2:314–316. Saad FA, Halligar B, Müller CR, Roberts RG, Danieli GA (1994a): Single base substitutions are detected by double strand conformation analysis. Nucleic Acids Res 22:4352–4353. Saad FA, Vita G, Toffolatti L, Danieli GA (1994b): A possible missense mutation detected in the dystrophin gene by double strand conformation analysis. Neuromuscul Disord 4:335–341. Saad FA, Mostacciuolo ML, Trevisan CP, Tomelleri G, Angelini C, Abedel Salam E, Danieli GA (1997): Novel mutations and polymorphisms in the human dystrophin gene detected by double strand conformation analysis. Hum Mutat 9:188–190. Savov A, Angeliheva D, Balassopoulou A, Jordanova A, NoussiaArvanitakis S, Kalaydjieva L (1995): Double mutant alleles: are they rare? Hum Mol Genet 4:1169–1171. Servidei S, Manfredi G, Mirabella M, Galluzzi G, Bertini E, Ricci E, Tonali P (1993): Familial hyperCKemia can be a variant of Becker muscular dystrophy. Neurology 43:293. Winnard AV, Jia-Hsu Y, Gibbs RA, Mendell JR, Burghes AHM (1992): Identification of a 2 base pair nonsense mutation causing a cryptic site in a DMD patient. Hum Mol Genet 1:645–646.