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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
Department of Biology, University of Padua, Padua, Italy
Department of Genetics, University of Tanta, Kafr El Sheikh, Egypt
Neuromuscular Laboratory, Rizzoli Institute, University of Bologna, Bologna, Italy
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
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:
*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.
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
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.
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.
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].
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.
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
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].
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.
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