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A possible missense mutation detected in the dystrophin gene by double strand conformation analysis (DSCA)
Neuromusc. Disord., Vol. 4, No. 4, pp. 335-341, 1994 Elsevier Science Ltd Printed in Great Britain 096~8966/94 $7.00 + .00
Pergamon
09 .60--8966(94)E0020-9
A POSSIBLE MISSENSE MUTATION DETECTED IN THE DYSTROPHIN GENE BY DOUBLE STRAND CONFORMATION ANALYSIS (DSCA) F. A. SAAD,*~"G. VITA,:~ L. TOFFOLATTI* and G. A. DANIELI* *Department of Biology, Universityof Padua, Italy; :~Departmentof Neurology, Universityof Messina, Italy
Abstract--A new and simple method for detecting point mutations is presented. The method, based on Double-Strand Conformation Analysis (DSCA) of PCR amplification products in polyacrylamide gel electrophoresis, was applied to 78 unrelated subjects affected with Duchenne or Becker muscular dystrophy and to 9 subjects suspected to be affected with an atypical dystrophinopathy. An A--*G substitution in the nucleotide 2525, which changes the codon for lysine to a codon for glutamic acid was detected in an 8-year-old boy, with normal neurological examination, but showing increased CK level and an abnormal EMG. The muscle biopsy was normal, without features of necrosis or regeneration. Immunoreactions with anti-dystrophin antibodies showed a normal distribution and intensity of the staining. A review of the dystrophin mutations detected so far is included. Key words: Dystrophin, mutations, DMD, BMD, missense, DSCA, nomenclature.
analysis involves the detection of mobility shifts of PCR amplification products in polyacrylamide gel electrophoresis, due to distortions in the double-strand conformation of DNA [6] introduced by single base changes. The method already proved to be effective in detecting a nonsense mutation and two single-base substitutions in intronic sequences in the dystrophin gene [7].
INTRODUCTION Approximately 60% of Duchenne (DMD) and Becker (BMD) patients carry a deletion in the dystrophin gene and 7% an intragenic duplication [1]. The detection of this type of mutations is routinely performed in several laboratories, by multiplex PCR amplification [2, 3] or by semiquantitative multiplex PCR [4, 5]. On the contrary, detection of point mutations is generally time consuming and technically demanding. On the other hand the identification of point mutations is particularly important to establish the functional role of the different segments of the dystrophin protein, deduced from the characteristics of the clinical phenotype. We report here a successful attempt to detect a point mutation by a new and simple screening method based on Double-Strand Conformation Analysis (DSCA). Double-strand conformation
MATERIALS AND METHODS
~:Author to whom all correspondenceshould be addressed at: Departmentof Biology,Universityof Padua, via Trieste75, 35121 Padova, Italy.
Patients affected with D M D or BMD or suspected to have an atypical dystrophinopathy were diagnosed at Neurology Departments of different Italian Universities. D N A was extracted by salting out procedure. Genomic D N A was amplified by three multiplex PCR reactions for deletion screening (including exons 2, 5, 17, 42, 43, 44, 48, 50, 51, 52; 6, 7, 12, 16, 19, 21, 32, 45, 47, 49 and 3, 4, 8, 13, 29, 34, 41,46, 53, 60, respectively) and two semi-quantative PCR reactions (involving exons 7, 8, 16, 21, 32, 42, 60 and 4, 12, 13, 19, 50, 53) for the duplication screening.
335
336
F.A. SAADel
The PCR conditions were: 80 ng genomic DNA in l0/11 of 1.5 mM MgC12, 67 mM Tris HCI pH 8.8, 16.6 mM ammonium sulphate, 0.01% Tween-20, 120/~M of each dNTP, 2.6 pM of each primer and 1 unit of RTB DNA polymerase (AMED, Italy). The cycling parameters were: a first denaturing step at 94°C for 2 rains, followed by 21 cycles (94°C for 30 s, 65°C for 2 min). PCR amplification products were run on vertical slab electrophoresis (8% polyacrylamide gel, 29:1 acrylamide:bis-acrylamide) at room temperature, 7.5 V/cm for 2-12 h, according to the different requirements. After electrophoresis, gel were stained either with ethidium bromide or by silver stain. Amplification products showing an altered electrophoretic mobility were singly amplified in a new PCR reaction and purified on Quiagen mini-columns. TA-cloning was performed by the TA-cloning kit (Invitrogen), according to the standard protocol. DNA from five different clones containing a given insert were pooled togehter in order to minimize the possibility of errors due to PCR amplification. The DNA sequences were produced by an ABI automatic sequencer, model 373A. Immunohistochemistry was performed on the muscle biopsy cryostat sections, using antibodies against three different portions of the dystrophin molecule, respectively, against 50-kD peptide in the N-terminal domain, against the 60- and the 30-kD peptide in the rod domain and against 75kD peptide in the C-terminal domain. Antibodies were provided by Novocastra (U.K). RESULTS
DSCA was applied on DNAs extracted from 78 unrelated subjects affected with Duchenne (50) or Becker (28) muscular dystrophy and from 9 subjects suspected to be affected with an atypical dystrophinopathy. None of these patients showed the presence of intragenic deletions and duplications. The amplification product of exon 19 in patient No. 1857 showed a shift in the electrophoretic mobility. Such a shift was never detected among 344 unrelated subjects (1032 independent electrophoretic separations of multiplex PCR reaction products). The sequence of
al.
the DNA of the exon 19 in subject No. 1857 showed an A > G substitution in the nucleotide 2525, which changes the codon for lysine in a codon for glutamic acid (Fig. 1). Patient No. 1857 is one of the 9 subjects suspected to be affected with an atypical dystrophinopathy. The boy, 8 years old, was referred to the Neurologist because of pain in his heels after running and fatigue after moderate physical exercise. The neurological examination was normal, but serum creatine kinase (CK) level was found to be three-fold higher than the upper normal limit. Quantitative electromyography (EMG) on right vastus lateralis showed no spontaneous activity and a 25% reduced mean duration of the motor unit potentials (8.4 m s L). There were no polyphasic potentials. Electrocardiogram and echocardiogram were normal. The open muscle biopsy, performed on left vastus lateralis, revealed a normal muscle tissue with no features of necrosis or regeneration. Histochemical reactions for oxidative enzymes and phosphorylase were normal. The intracellular content of glycogen and lipids was normal. Immunoreactions with anti-dystrophin antibodies showed a normal distribution and intensity of the staining (Fig. 2) The boy was seen by the same Neurologist 22 months later. The neurological examination was still normal and the subject reported disappearance of symptoms he was previously complaining about. However, his serum CK level remained 4.5 times higher than the upper normal limit. The family history was negative for neuromuscular disorders. His mother and a 13-year-old sister had normal CK levels. DISCUSSION The screening method for point mutations adopted in this study is very simple and relatively effective, although its detection rate is much lower than SSCP or heteroduplex (Table 1). Double Strand Conformation Analysis (DSCA) is based on the principle that the DNA double helix structure in solution is not straight and that almost every base pair may induce a curvature. Therefore, each DNA molecule may display an individual silhouette [8]. Since curvature of the
Dystrophin Missense Mutation
337
(a) A
B
C
D
E
F
G
(b)
~ c r a ^ ^c~ ~ c2%c ^G
C T G A A~0]~ Cq'CAG
FIc. 1. (a) DSCA analysis. The amplification product of e×on 19 in lane D shows a clear electrophoretic mobility shift. (b) DNA sequence of the antisense strand of exon 19. Upper: normal; lower: mutant.
DNA molecule may affect migration in polyacrylamide gel, a single base-pair substitution may be effective in altering the silhouette and hence the electrophoretic mobility of a DNA molecule. This applies particularly to the stacks AA/TT, AC/GT, AG/CT, CA/TG, CC/GG, GA/TC, AT/AT, CG/CG, GC/GC and TA/TA [9]. The A ~ G change in the nucleotide 2525 occurred in a AA doublet, which justify the observed mobility shift. Such a shift was never observed in the series of 344 unrelated subjects, therefore, the possibility o f a polymorphism may be'excluded. On the other hand, it is impossible to conclude that the A ~ G change in nucleotide 2525 is causative of the muscle affection observed in the patient, since the presence of a point mutation in a different exon, not included in the screening, cannot be ruled out. Moreover, no functional data are presently available. Only one year elapsed from the referral of the patient and the recent follow-up, therefore it is
impossible to deduce that his affection is nonprogressive. It may be concluded provisionally that the coincidence of finding a base substitution in one exon of the dystrophin gene and the occurrence of increased serum creatine kinase level and abnormal E M G in a male boy is highly suggestive of a causal relationship. The first missense mutation in the dystrophin gene (Leu--*Arg) was detected in codon 54 of exon 3 in an 8-year-old boy, showing diffuse muscle weakness, large calf muscles, weak neck flexor muscles and a positive Gower's sign. Variability in fiber size, some internal nuclei and features of regeneration were present in the muscle biopsy [10]. The second missense mutation ( C ~ T in the nucleotide 10470, exon 71) was reported in a BMD patient aged 22, still deambulating [11]. The nucleotide substitution reported in this paper, occurring in the codon 773 ofexon 19, if really causative of muscle affection, seems to have a much milder clinical effect on the
338
F.A. SAAD et al.
FIG. 2. lmmunofluorescent staining of dystrophin in patient's muscle (A C) and in normal control (D). (A) N-terminal domain; (B) mid-rod domain; (C, D) C-terminal domain, x 450. Table 1. Results from some revelevant screenings for point mutations in the dystrophin gene Author Kilimann Lenk Nigro Prior Roberts Saad
No. of cases
Exons screened
Coding sequence screened (%)
Mutations detected
Method
60 26 29 110 7 78
14 20 Il 16 79 30
t3.85 34.38 l 1.72 18.57 100 31.95
2 7 1 5 7 3*
CC SSCP SSCP HT RT + CC DSCA
c c = Chemical Cleavage, DSCA = Double Strand Conformation Analysis, HT = Heteroduplex, SSCP = Single Strand Conformation Polymorphism, RT = Reverse Transcriptase. *Including the mutation reported in this paper.
p h e n o t y p e . I n d e e d , it is e x p e c t e d t h a t m u t a t i o n in t h e r o d h a v e little c o n s e q u e n c e s a t t h e c l i n i c a l level, s i n c e t h e d e s c r i b e d c a s e s o f m i l d d y s t r o p h i nopathies involve in-frame deletions or dupli-
c a t i o n s in t h e r o d [12, 13]. P o i n t m u t a t i o n s d e t e c t e d so f a r in t h e d y s t r o p h i n g e n e a r e r e p o r t e d in T a b l e 2. T h e l a r g e m a j o r i t y is r e p r e s e n t e d b y n o n s e n s e m u t a t i o n s d u e to C - + T
Dystrophin Missense Mutation
339
Table 2. Point mutations in the dystrophin gene Exon
Intron
Mutation
Position
Name*
Ref.
Nonsense mutations
8 19 19 21 26 40 41 44 48 51 60 70 70
CoT CoT GoT GoT GoT C---,T C--*T CoT CoT GoT C--*T C--*T CoT
932 2510 2522 2999 3714 5759 6107 6572 7163 7610 9152 10316 10349
Q242X R768X E772X E931X E1169X Q1851X RI967X Q2182X Q2319X E2467X R2982X R3370X R3381X
[16] [20] [20] [ 17] [14] [17] [ 7] [19] [15] [18] [ 17] [17] [18]
Indirect generation of a stop codon
12 19 48 74 74 74 74
+T -C -G -T -T -T -7nt
1551 2567 7286 10662 10662 10691 10694
I551insT 2567de1C 7286delG 10662delT 10662delT 10691delT 10694de17
[21 [20] [21] [17] [11] [11] [11]
AoC - 5nt GoC G--*A ToA GoT GoA
2588+3 6643 8599- 1 9771 + 1 10182+2 10431+5 10431 + 1
2588+ 3 A o C 6643de15 8599- 1GoC 9771 + 1G--,A 10182+2ToA 10431+ 5 G O T 10431+ 1 G o A
[24] [22] [23] [11] [17] [11] [11]
T~G AoG C--,T
368 2525 10470
L54R K773E A3421V
[16] [ps] [11]
Splicing site mutations 44
Missense mutations
3 19 71
19 44 56 65 68 70 70
Note: the numbering is according to the dystrophin coding sequence available in GenBank and names are according to the new nomenclature [27]; [ps] = present study.
transversions, which are apparently distributed at random over 11 different exons [7, l 1, 14-20]. A smaller number of mutations cause the formation of stop codons by single base deletions or insertions [11, 17, 20, 21] or by exon skipping following splicing site mutations [l 1, 17, 22-24]. All mutations but three were found in subjects clinically diagnosed as affected with Duchenne muscular dystrophy. Among these three, one was detected in a Becker patient (acceptor splicing site mutation in intron 56), while the other two were missense mutations with very mild clinical manifestations ( T ~ G 368) or with sub-clinical manifestation ( A ~ G 2525). Point mutations were detected by different approaches (Table 1). If we exclude RT-PCR followed by chemical cleavage, all the methods applied so far were able to screen only a part of
the coding sequence, with a rather poor detection rate. The dystrophin gene is more than 2500 kb long. Errors in D N A replication occur in vitro at a frequency between 10 -9 and l0 -~ per incorporated nucleotide [25], therefore mutations due to errors of incorporation, should occur in the dystrophin gene at a rate of about 2.5/10,000. The most recent estimate of its mutation rate calculated from D M D and B M D incidence gives a value of about 99.3 per million gametes [26]. Since we know that about 67% of mutations are intragenic deletions or duplications, the rate of point mutation according to this estimate should be about 30/1,000,000, i.e. about eight times less than theoretically expected. This might be due, at least in part, to a strong bias in clinical detection, since very mild dystrophinopathies are likely to
340
F. A, SAAD et al.
be undetected or misdiagnosed. Most point mutations discovered so far are C - * T or G - * T , as predicted by the higher mutability of CpG. Seven mutations of the splice junction site were identified so far, but the creation of novel splice sites in introns was never reported. Similarly, no point mutations affecting R N A processing and translation were ever described. This lack of observations may be due to the methods chosen for detection of point mutations. Actually, only in one case the adopted protocol was able to detect splice sites mutations in introns or in intron/exon boundaries as well [17]. Therefore, in order to identify point mutations a two-step strategy should be devised: a preliminary screening by simple methods (heteroduplex, SSCP, DSCA) followed by an analysis by R T - P C R and chemical cleavage on those D N A s in which the first procedure failed to detect point mutations. The increasing number of described mutations for several human genes claims for the adoption of a defined system of nomenclature as suggested recently [27] and for a thorough check of the sequences used as reference. As a matter of fact, the dystrophin c D N A sequence submitted to GenBank [28] lacks the first 37 nt to the transcription site [29] and contains two differences from the real sequence: a T insertion (nucleotide No. 6) and an A ~ C substitution in nucleotide No. 8. Acknowledgements--The financial support of Telethon (grants to Dr G. Vita and to Dr S. Oliviero, respectively) is gratefully acknowledged. Dr F. A. Saad is recipient of a fellowship from the Italian Ministry of Foreign Affairs. The technical suggestions of Dr R. Tombolini and Dr C. Bonghi were very helpful for performing TA cloning and automatic DNA sequencing.
REFERENCES
1. Den Dunnen J T, Grootscholten P M, Bakker E, Blonden L A J, Ginjaar H B, Wapenaar M C, van Paassen H M B, van Broeckhoven C, Pearson P K, van Ommen G J B. Topography of the Duchenne muscular dystrophy (DMD) gene: FIGE and cDNA Analysis of 194 cases reveals 115 deletions and 13 duplications. Am J Hum Genet 1989; 34 835 847. 2. Chamberlain J, Gibbs R, Ranier J, Nguyen P, Caskey T. Dcetection screening of the Ducheene muscular dystrophy locus via multiplex DNA amplification. Nucleic Acids Res 1989; 16:11,141 11,156. 3. Beggs A, Koenig M, Boyce F, Kunkel L. Detection of
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98% of DMD/BMD gene deletions by polymerase chain reaction. J Hum Genet 1990; 86: 45~48. Abbs S, Bobrow M. Analysis of quantitavie PCR for diagnosis of deletion and duplication carriers in the dystrophin gene. J Med Genet 1992; 29:191 196. Galvagni F, Saad F, Danieli G A, Miorin M, Vitiello L, Mostacciuolo M L, Angelini C. A study of duplications of the dystrophin gene. Evidence of a geographical difference in the distributions by intron. Hum Genet (in press). Ulanovsky L E, Trilbnov E A N. Estimation of wedge components in curved DNA. Nature 1987; 326:720 722. Saad F A, Vita G, Mora M, Morandi L, Vitiello L, Oliviero S, Danieli G A. A novel nonsense mutation in the human dystrophin gene. Hum Mutation 1993: 2: 314 316. Trifonov E N. DNA in profile. T1BS 1991; 16: 467~470. Bolshoy A, McNamara P, Harrington R E, Trifonove E N. Curved DNA without A-A: experimental estimation of all 16 DNA wedge angles. Proc Natl Acad Sci USA 1991; 88:2312 2316. Prior T W, Papp A C, Snyder P J, Burghes A H M, Bartolo C, Sedra M S, Western L M, Mendell J R. A missense mutation in the dystrophin gene in a Duchenne muscular dystrophy patient. Nature Genetics 1993; 4:357 360. Lenk U, Hanke R, Thiele H, Speer A. Point mutations at the carboxy terminus of the human dystrophin gene: implications for an association with mental retardation in MD patients. Hum Molec Genet 1993; 2:1877 1881. Fischbeck K H. Mild myopathic syndromes. In: C Angelini, G A Danieli, D Fontanari, eds. Muscular Dystrophy Research. Amsterdam: Excerpta Medica Int. Congress Series 934, 1991: 61-65. Angelini C, Beggs A H, Hoffman E B, Fanin M, Kunkel L M. Enormous dystrophin in a patient with Becker muscular dystrophy. Neurology 1990; 40:808 812. Bulman D E, Gangopadhyay S B, Bebchuck K G, Worton R G, Ray P N. Point mutation in the human dystrophin gene: identification through western blot analysis. Genomics 1991; 10:457 460. Clemens P R, Ward P A, Caskey C T, Bulman D E, Fenwick R G. Premature chain termination mutation causing Duchenne muscular dystrophy. Neurology 1992; 42: 1775-1782. Nigro V, Politano L, Nigro G, Romano S C, Molinari A M, Puca G A. Detection of nonsense mutation in the dystrophin gene by multiplex SSCP. Hum Molec Genet 1992; 1:517 520. Roberts R G, Bobrow M, Bentley D R. Point mutations in the dystrophin gene. Proc Natl Acad Sci USA 1992; 89: 2331-2335. Winnard A V, Jia-Hsu Y, Gibbs R A, Mendell J R, Burghes A H M. Identification ofa 2 base pair nonsense mutation causing a cryptic site in a DMD patient. Hum Molec Genet 1992; I: 645-646. Prior T W, Papp A C, Snyder P J, Burges A H M, Sedra M S, Western L M, Bantolo C, Mendel J R. Exon 44 nonsense mutation in two-Duchenne muscular dystrophy brothers detected by heteroduplex analysis. Hum Mutation 1993; 2:192 195. Prior T W, Papp A C, Snyder P J, Burghes A H M, Sedra M S, Western L M, Bartolo C, Mendell J R. Identification of two point mutations and a one base deletion in exon 19 of the dystrophin gene by heteroduplex formation. Hum Molec Genet 1993; 2:
Dystrophin Missense Mutation 311-313. Kilimann M W, Pizzuti A, Grompe M, Caskey C T. Point mutations and polymorphisms in the human dystrophin gene identified in genomic DNA sequences amplified by multiplex PCR. Hum Genet 1992; 89: 253258. 22. Saad F A, Vitiello L, Merlini L, Mostacciuolo M L, Oliviero S, Danieli G A. A 3' consensus splice mutation in the human dystrophin gene detected by a screening for intra-exonic deletions. Hum Mole Genet 1992; 1: 345- 346. 23. Roberts R G, Passos-Bueno M R, Bobrow M, Vainzof M, Zatz M. Point mutation in a Becker muscular dystrophy patient. Hum Molec Genet 1993; 2: 7%77. 24. Wilton S D, Johnsen R D, Pedretti J R, Laiug N G. Two distinct mutations in a single dystrophin gene: identification of an altered splice site as the primary Becket muscular dystrophy mutation. Am J Med Genet 1993; 46: 563-569. 21.
25.
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Thacker J. The molecular nature of mutations in cultured mammalian cells: a review. Mutat Res 1985; 150: 431-442. 26. Mostacciuolo M L, Miorin M, Pegoraro E, Fanin M, Schiavon F, Vitiello L, Saad F A, Angelini C, Danieli G A. A reappraisal of the incidence rate of Duchenne and Becker muscular dystrophy, based on molecular diagnosis. Neuroepiderniology 1993; 12: 32(~330. 27. Beaduet A L, Tsui L C. A suggested nomenclature for designating mutations. Hum Murat 1993; 2: 245-248. 28. Koenig M, Hoffman E P, Bertelson C J, Monaco A P, Feener C, Kunkel L M. Complete cloning of the Duchenne muscular dystrophy (DMD) cDNA and preliminary genomic organization of the DMD gene in normal and affected individuals. Cell 1987; 50:509 517. 29. Klamut H J, Gangopadhyay S B, Worton R G, Ray P N. Molecular and functional analysis of the muscle specific promoter region of the Duchenne muscular dystrophy gene. Molec Cell Bio 1990; 10: 193-205.
Pergamon
09 .60--8966(94)E0020-9
A POSSIBLE MISSENSE MUTATION DETECTED IN THE DYSTROPHIN GENE BY DOUBLE STRAND CONFORMATION ANALYSIS (DSCA) F. A. SAAD,*~"G. VITA,:~ L. TOFFOLATTI* and G. A. DANIELI* *Department of Biology, Universityof Padua, Italy; :~Departmentof Neurology, Universityof Messina, Italy
Abstract--A new and simple method for detecting point mutations is presented. The method, based on Double-Strand Conformation Analysis (DSCA) of PCR amplification products in polyacrylamide gel electrophoresis, was applied to 78 unrelated subjects affected with Duchenne or Becker muscular dystrophy and to 9 subjects suspected to be affected with an atypical dystrophinopathy. An A--*G substitution in the nucleotide 2525, which changes the codon for lysine to a codon for glutamic acid was detected in an 8-year-old boy, with normal neurological examination, but showing increased CK level and an abnormal EMG. The muscle biopsy was normal, without features of necrosis or regeneration. Immunoreactions with anti-dystrophin antibodies showed a normal distribution and intensity of the staining. A review of the dystrophin mutations detected so far is included. Key words: Dystrophin, mutations, DMD, BMD, missense, DSCA, nomenclature.
analysis involves the detection of mobility shifts of PCR amplification products in polyacrylamide gel electrophoresis, due to distortions in the double-strand conformation of DNA [6] introduced by single base changes. The method already proved to be effective in detecting a nonsense mutation and two single-base substitutions in intronic sequences in the dystrophin gene [7].
INTRODUCTION Approximately 60% of Duchenne (DMD) and Becker (BMD) patients carry a deletion in the dystrophin gene and 7% an intragenic duplication [1]. The detection of this type of mutations is routinely performed in several laboratories, by multiplex PCR amplification [2, 3] or by semiquantitative multiplex PCR [4, 5]. On the contrary, detection of point mutations is generally time consuming and technically demanding. On the other hand the identification of point mutations is particularly important to establish the functional role of the different segments of the dystrophin protein, deduced from the characteristics of the clinical phenotype. We report here a successful attempt to detect a point mutation by a new and simple screening method based on Double-Strand Conformation Analysis (DSCA). Double-strand conformation
MATERIALS AND METHODS
~:Author to whom all correspondenceshould be addressed at: Departmentof Biology,Universityof Padua, via Trieste75, 35121 Padova, Italy.
Patients affected with D M D or BMD or suspected to have an atypical dystrophinopathy were diagnosed at Neurology Departments of different Italian Universities. D N A was extracted by salting out procedure. Genomic D N A was amplified by three multiplex PCR reactions for deletion screening (including exons 2, 5, 17, 42, 43, 44, 48, 50, 51, 52; 6, 7, 12, 16, 19, 21, 32, 45, 47, 49 and 3, 4, 8, 13, 29, 34, 41,46, 53, 60, respectively) and two semi-quantative PCR reactions (involving exons 7, 8, 16, 21, 32, 42, 60 and 4, 12, 13, 19, 50, 53) for the duplication screening.
335
336
F.A. SAADel
The PCR conditions were: 80 ng genomic DNA in l0/11 of 1.5 mM MgC12, 67 mM Tris HCI pH 8.8, 16.6 mM ammonium sulphate, 0.01% Tween-20, 120/~M of each dNTP, 2.6 pM of each primer and 1 unit of RTB DNA polymerase (AMED, Italy). The cycling parameters were: a first denaturing step at 94°C for 2 rains, followed by 21 cycles (94°C for 30 s, 65°C for 2 min). PCR amplification products were run on vertical slab electrophoresis (8% polyacrylamide gel, 29:1 acrylamide:bis-acrylamide) at room temperature, 7.5 V/cm for 2-12 h, according to the different requirements. After electrophoresis, gel were stained either with ethidium bromide or by silver stain. Amplification products showing an altered electrophoretic mobility were singly amplified in a new PCR reaction and purified on Quiagen mini-columns. TA-cloning was performed by the TA-cloning kit (Invitrogen), according to the standard protocol. DNA from five different clones containing a given insert were pooled togehter in order to minimize the possibility of errors due to PCR amplification. The DNA sequences were produced by an ABI automatic sequencer, model 373A. Immunohistochemistry was performed on the muscle biopsy cryostat sections, using antibodies against three different portions of the dystrophin molecule, respectively, against 50-kD peptide in the N-terminal domain, against the 60- and the 30-kD peptide in the rod domain and against 75kD peptide in the C-terminal domain. Antibodies were provided by Novocastra (U.K). RESULTS
DSCA was applied on DNAs extracted from 78 unrelated subjects affected with Duchenne (50) or Becker (28) muscular dystrophy and from 9 subjects suspected to be affected with an atypical dystrophinopathy. None of these patients showed the presence of intragenic deletions and duplications. The amplification product of exon 19 in patient No. 1857 showed a shift in the electrophoretic mobility. Such a shift was never detected among 344 unrelated subjects (1032 independent electrophoretic separations of multiplex PCR reaction products). The sequence of
al.
the DNA of the exon 19 in subject No. 1857 showed an A > G substitution in the nucleotide 2525, which changes the codon for lysine in a codon for glutamic acid (Fig. 1). Patient No. 1857 is one of the 9 subjects suspected to be affected with an atypical dystrophinopathy. The boy, 8 years old, was referred to the Neurologist because of pain in his heels after running and fatigue after moderate physical exercise. The neurological examination was normal, but serum creatine kinase (CK) level was found to be three-fold higher than the upper normal limit. Quantitative electromyography (EMG) on right vastus lateralis showed no spontaneous activity and a 25% reduced mean duration of the motor unit potentials (8.4 m s L). There were no polyphasic potentials. Electrocardiogram and echocardiogram were normal. The open muscle biopsy, performed on left vastus lateralis, revealed a normal muscle tissue with no features of necrosis or regeneration. Histochemical reactions for oxidative enzymes and phosphorylase were normal. The intracellular content of glycogen and lipids was normal. Immunoreactions with anti-dystrophin antibodies showed a normal distribution and intensity of the staining (Fig. 2) The boy was seen by the same Neurologist 22 months later. The neurological examination was still normal and the subject reported disappearance of symptoms he was previously complaining about. However, his serum CK level remained 4.5 times higher than the upper normal limit. The family history was negative for neuromuscular disorders. His mother and a 13-year-old sister had normal CK levels. DISCUSSION The screening method for point mutations adopted in this study is very simple and relatively effective, although its detection rate is much lower than SSCP or heteroduplex (Table 1). Double Strand Conformation Analysis (DSCA) is based on the principle that the DNA double helix structure in solution is not straight and that almost every base pair may induce a curvature. Therefore, each DNA molecule may display an individual silhouette [8]. Since curvature of the
Dystrophin Missense Mutation
337
(a) A
B
C
D
E
F
G
(b)
~ c r a ^ ^c~ ~ c2%c ^G
C T G A A~0]~ Cq'CAG
FIc. 1. (a) DSCA analysis. The amplification product of e×on 19 in lane D shows a clear electrophoretic mobility shift. (b) DNA sequence of the antisense strand of exon 19. Upper: normal; lower: mutant.
DNA molecule may affect migration in polyacrylamide gel, a single base-pair substitution may be effective in altering the silhouette and hence the electrophoretic mobility of a DNA molecule. This applies particularly to the stacks AA/TT, AC/GT, AG/CT, CA/TG, CC/GG, GA/TC, AT/AT, CG/CG, GC/GC and TA/TA [9]. The A ~ G change in the nucleotide 2525 occurred in a AA doublet, which justify the observed mobility shift. Such a shift was never observed in the series of 344 unrelated subjects, therefore, the possibility o f a polymorphism may be'excluded. On the other hand, it is impossible to conclude that the A ~ G change in nucleotide 2525 is causative of the muscle affection observed in the patient, since the presence of a point mutation in a different exon, not included in the screening, cannot be ruled out. Moreover, no functional data are presently available. Only one year elapsed from the referral of the patient and the recent follow-up, therefore it is
impossible to deduce that his affection is nonprogressive. It may be concluded provisionally that the coincidence of finding a base substitution in one exon of the dystrophin gene and the occurrence of increased serum creatine kinase level and abnormal E M G in a male boy is highly suggestive of a causal relationship. The first missense mutation in the dystrophin gene (Leu--*Arg) was detected in codon 54 of exon 3 in an 8-year-old boy, showing diffuse muscle weakness, large calf muscles, weak neck flexor muscles and a positive Gower's sign. Variability in fiber size, some internal nuclei and features of regeneration were present in the muscle biopsy [10]. The second missense mutation ( C ~ T in the nucleotide 10470, exon 71) was reported in a BMD patient aged 22, still deambulating [11]. The nucleotide substitution reported in this paper, occurring in the codon 773 ofexon 19, if really causative of muscle affection, seems to have a much milder clinical effect on the
338
F.A. SAAD et al.
FIG. 2. lmmunofluorescent staining of dystrophin in patient's muscle (A C) and in normal control (D). (A) N-terminal domain; (B) mid-rod domain; (C, D) C-terminal domain, x 450. Table 1. Results from some revelevant screenings for point mutations in the dystrophin gene Author Kilimann Lenk Nigro Prior Roberts Saad
No. of cases
Exons screened
Coding sequence screened (%)
Mutations detected
Method
60 26 29 110 7 78
14 20 Il 16 79 30
t3.85 34.38 l 1.72 18.57 100 31.95
2 7 1 5 7 3*
CC SSCP SSCP HT RT + CC DSCA
c c = Chemical Cleavage, DSCA = Double Strand Conformation Analysis, HT = Heteroduplex, SSCP = Single Strand Conformation Polymorphism, RT = Reverse Transcriptase. *Including the mutation reported in this paper.
p h e n o t y p e . I n d e e d , it is e x p e c t e d t h a t m u t a t i o n in t h e r o d h a v e little c o n s e q u e n c e s a t t h e c l i n i c a l level, s i n c e t h e d e s c r i b e d c a s e s o f m i l d d y s t r o p h i nopathies involve in-frame deletions or dupli-
c a t i o n s in t h e r o d [12, 13]. P o i n t m u t a t i o n s d e t e c t e d so f a r in t h e d y s t r o p h i n g e n e a r e r e p o r t e d in T a b l e 2. T h e l a r g e m a j o r i t y is r e p r e s e n t e d b y n o n s e n s e m u t a t i o n s d u e to C - + T
Dystrophin Missense Mutation
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Table 2. Point mutations in the dystrophin gene Exon
Intron
Mutation
Position
Name*
Ref.
Nonsense mutations
8 19 19 21 26 40 41 44 48 51 60 70 70
CoT CoT GoT GoT GoT C---,T C--*T CoT CoT GoT C--*T C--*T CoT
932 2510 2522 2999 3714 5759 6107 6572 7163 7610 9152 10316 10349
Q242X R768X E772X E931X E1169X Q1851X RI967X Q2182X Q2319X E2467X R2982X R3370X R3381X
[16] [20] [20] [ 17] [14] [17] [ 7] [19] [15] [18] [ 17] [17] [18]
Indirect generation of a stop codon
12 19 48 74 74 74 74
+T -C -G -T -T -T -7nt
1551 2567 7286 10662 10662 10691 10694
I551insT 2567de1C 7286delG 10662delT 10662delT 10691delT 10694de17
[21 [20] [21] [17] [11] [11] [11]
AoC - 5nt GoC G--*A ToA GoT GoA
2588+3 6643 8599- 1 9771 + 1 10182+2 10431+5 10431 + 1
2588+ 3 A o C 6643de15 8599- 1GoC 9771 + 1G--,A 10182+2ToA 10431+ 5 G O T 10431+ 1 G o A
[24] [22] [23] [11] [17] [11] [11]
T~G AoG C--,T
368 2525 10470
L54R K773E A3421V
[16] [ps] [11]
Splicing site mutations 44
Missense mutations
3 19 71
19 44 56 65 68 70 70
Note: the numbering is according to the dystrophin coding sequence available in GenBank and names are according to the new nomenclature [27]; [ps] = present study.
transversions, which are apparently distributed at random over 11 different exons [7, l 1, 14-20]. A smaller number of mutations cause the formation of stop codons by single base deletions or insertions [11, 17, 20, 21] or by exon skipping following splicing site mutations [l 1, 17, 22-24]. All mutations but three were found in subjects clinically diagnosed as affected with Duchenne muscular dystrophy. Among these three, one was detected in a Becker patient (acceptor splicing site mutation in intron 56), while the other two were missense mutations with very mild clinical manifestations ( T ~ G 368) or with sub-clinical manifestation ( A ~ G 2525). Point mutations were detected by different approaches (Table 1). If we exclude RT-PCR followed by chemical cleavage, all the methods applied so far were able to screen only a part of
the coding sequence, with a rather poor detection rate. The dystrophin gene is more than 2500 kb long. Errors in D N A replication occur in vitro at a frequency between 10 -9 and l0 -~ per incorporated nucleotide [25], therefore mutations due to errors of incorporation, should occur in the dystrophin gene at a rate of about 2.5/10,000. The most recent estimate of its mutation rate calculated from D M D and B M D incidence gives a value of about 99.3 per million gametes [26]. Since we know that about 67% of mutations are intragenic deletions or duplications, the rate of point mutation according to this estimate should be about 30/1,000,000, i.e. about eight times less than theoretically expected. This might be due, at least in part, to a strong bias in clinical detection, since very mild dystrophinopathies are likely to
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F. A, SAAD et al.
be undetected or misdiagnosed. Most point mutations discovered so far are C - * T or G - * T , as predicted by the higher mutability of CpG. Seven mutations of the splice junction site were identified so far, but the creation of novel splice sites in introns was never reported. Similarly, no point mutations affecting R N A processing and translation were ever described. This lack of observations may be due to the methods chosen for detection of point mutations. Actually, only in one case the adopted protocol was able to detect splice sites mutations in introns or in intron/exon boundaries as well [17]. Therefore, in order to identify point mutations a two-step strategy should be devised: a preliminary screening by simple methods (heteroduplex, SSCP, DSCA) followed by an analysis by R T - P C R and chemical cleavage on those D N A s in which the first procedure failed to detect point mutations. The increasing number of described mutations for several human genes claims for the adoption of a defined system of nomenclature as suggested recently [27] and for a thorough check of the sequences used as reference. As a matter of fact, the dystrophin c D N A sequence submitted to GenBank [28] lacks the first 37 nt to the transcription site [29] and contains two differences from the real sequence: a T insertion (nucleotide No. 6) and an A ~ C substitution in nucleotide No. 8. Acknowledgements--The financial support of Telethon (grants to Dr G. Vita and to Dr S. Oliviero, respectively) is gratefully acknowledged. Dr F. A. Saad is recipient of a fellowship from the Italian Ministry of Foreign Affairs. The technical suggestions of Dr R. Tombolini and Dr C. Bonghi were very helpful for performing TA cloning and automatic DNA sequencing.
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